Accepted Manuscript Silk fibroin/hydroxyapatite engineering
composites
for
bone
tissue
Mehdi Farokhi, Fatemeh Mottaghitalab, Saeed Samani, Mohammad Ali Shokrgozar, Subhas C. Kundu, Rui L. Reis, Yousef Fattahi, David L. Kaplan PII: DOI: Reference:
S0734-9750(17)30121-0 doi:10.1016/j.biotechadv.2017.10.001 JBA 7159
To appear in:
Biotechnology Advances
Received date: Revised date: Accepted date:
12 June 2017 12 September 2017 4 October 2017
Please cite this article as: Mehdi Farokhi, Fatemeh Mottaghitalab, Saeed Samani, Mohammad Ali Shokrgozar, Subhas C. Kundu, Rui L. Reis, Yousef Fattahi, David L. Kaplan , Silk fibroin/hydroxyapatite composites for bone tissue engineering. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jba(2017), doi:10.1016/j.biotechadv.2017.10.001
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ACCEPTED MANUSCRIPT Silk fibroin/hydroxyapatite composites for bone tissue engineering Mehdi Farokhia,* , Fatemeh Mottaghitalab b, Saeed Samanic, Mohammad Ali Shokrgozara, Subhas C Kundud, Rui L. Reisd, Yousef Fattahie, David L. Kapland National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
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a
b
c
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Sciences, Tehran, Iran
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Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical
Department of Tissue Engineering & Applied Cell Sciences, School of Advanced Technologies
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in Medicine, Tehran University of Medical Sciences, Tehran, Iran d
3Bs Research Group, Headquarters of the European Institute of Excellence on Tissue
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Engineering and Regenerative Medicine, University of Minho, AvePark - 4805-017 Barco, Guimaraes, Portugal Department of pharmaceutical nanotechnology, Faculty of pharmacy, Tehran university of
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medical sciences
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USA
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Department of Biomedical Engineering, Tufts University, 4 Colby St, Medford, MA 02155,
Corresponding author:
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Mehdi Farokhi, E-mail address: [email protected]
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ACCEPTED MANUSCRIPT Abstract Silk fibroin (SF) is a natural fibrous polymer with strong potential for many biomedical applications. SF has attracted interest in the field of bone tissue engineering due to its extraordinary
characteristics
in
terms
of
elasticity,
flexibility,
biocompatibility
and
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biodegradability. However, low osteogenic capacity has limited applications for SF in the orthopedic arena unless suitably functionalized. Hydroxyapatite (HAp) is a well-established
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bioceramic with biocompatibility and appropriate for constructing orthopedic and dental
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substitutes. However, HAp ceramics tend to be brittle which can restrict applications in the repair of load-bearing tissues such as bones. Therefore, blending SF and HAp combines the useful
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properties of both materials as bone constructs for tissue engineering, the subject of this review.
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Keywords: Silk fibroin, Hydroxyapatite, Scaffold, Bone tissue engineering
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Contents 1. Introduction 2- Bioceramics for bone tissue engineering applications
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2.2. Signaling responses of osteoblast cells to hydroxyapatite
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2.1. Hydroxyapatite
3. Structure and characteristics of silk fibroin
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3.1. Osteogenic signaling responses of silk fibroin
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3.2. Silk fibroin scaffolds for bone tissue engineering
4. Silk fibroin/hydroxyapatite for bone tissue engineering
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4.1. Silk fibroin/hydroxyapatite composites for bone tissue engineering
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4.2. Silk fibroin/nano-hydroxyapatite composites for bone tissue engineering 4.3. Silk nanofibers containing hydroxyapatite for bone tissue engineering application
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4.4. Silk fibroin/hydroxyapatite for drug delivery in bone tissue engineering
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4.5. Silk fibroin/hydroxyapatite for stem cell differentiation 5. Conclusion and future perspective
References
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Acknowledgment
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ACCEPTED MANUSCRIPT 1. Introduction Bone has an intrinsic capacity for regeneration; however, strategies to improve the repair capacity of bone are still needed in many circumstances. Bone regeneration depends on the type of defect. For example, about 10% of fractures are non-union that do not heal spontaneously and
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lead to inappropriate bone tissue regeneration and delayed unions. The probability of normal bone healing in these fractures is influenced by age, metabolic condition, and severity of the
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trauma. Autologous bone or autografts are still considered the clinical “gold standard” and the
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most effective method for bone regeneration. This approach promotes bone formation by direct bone bonding (osteoconduction) and induces local stem cells to differentiate into bone cells
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(osteoinduction) without any immune responses (Zhang et al., 2014). About 2.2 million bone
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grafts are performed annually (Giannoudis et al., 2005) and are met some limitations due to the limited supply, morbidity of the donor site and are associated with more than 50% failure in
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specific regions (Bajaj et al., 2003; Clavero and Lundgren, 2003). Therefore, many attempts have
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been made to develop suitable bone constructs consisting of natural or synthetic biomaterials in order to increase bone regeneration capacity and avoid the above limitations. Some polymers are
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usually good candidates for this purpose due to their biocompatibility, biodegradability, tunable
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structural properties and ease of fabrication. Degradable polymers are important for these types of repairs to avoid barriers to full regeneration of bone, and are categorized to natural polymers such as alginate, chitosan, collagen, silk and synthetic polymers including polydioxanone (PDS), polycaprolactone (PCL), polyglycolic acid (PGA), polylactic acid (PLA), and poly (lactic-coglycolic acid) (PLGA) (Lee and Yuk, 2007; Rezwan et al., 2006). Recently, more attention has been paid to natural polymers in comparison to synthetic polymers for bone tissue engineering applications due to their unique biocompatibility, versatility and biodegradability. These types of
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ACCEPTED MANUSCRIPT polymers provide well-patterned structures consisting of ligands that can bind to cell surface receptors or provide accessible enzymatic degradation sites (Swetha et al., 2010). Silk fibroin (SF) as a natural protein polymer has potent characteristics for tissue engineering applications, as a biocompatible, biodegradable, and low immunogenic polymer (Farokhi et al., 2014a; Farokhi
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et al., 2016a; Mottaghitalab et al., 2013; Omenetto and Kaplan, 2010; Sugihara et al., 2000).
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Moreover, SF possesses extraordinary properties for stimulating bone repair; for example, the
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fibrous structure of SF is mostly similar to collagen type I (Col I). The amorphous links between the β-sheets in the structure of SF act as sites for deposition of HAp nanocrystals because it can
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mimic the anionic structure of noncollagenous proteins (NCPs) (Marelli et al., 2012). However,
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there are also some reports believed that β-sheets crystal can alone act as a nucleation site for deposition of HAp nanaocrystals (Vetsch et al., 2015). Furthermore, this polymer is introduced
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as a potential bone construct due to appropriate mechanical behaviors and structural integrity
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(Mandal et al., 2012; Sofia et al., 2001). The ultimate tensile strength of SF extracted from Bombyx mori (B. mori) is 300–740 MPa (Cunniff et al., 1994; Shao and Vollrath, 2002) and it
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also has great breaking strain and high toughness more than synthetic fibers such as Kevlar (Du
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et al., 2011; Gosline et al., 1999). Upon searching the Scopus database for studies related to tissue engineering and bone tissue engineering applications with SF from 2005 to Sep 2017, the
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number of peer-reviewed original articles per year and the first ten scientific study fields that attract the most attention and the highest numbers of published articles were assembled (Figure 1). Various studies have also recently focused on the applications of natural polymers as well as SF in bone repair. Although some of these polymers have intrinsic capacities for the bone regeneration, this is insufficient to repair large bone defects. For this reason, incorporating
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ACCEPTED MANUSCRIPT particles into the polymeric matrix can be advantageous to improve the mechanical behavior and tune the topographical features of the scaffold in order to mimic the structure of natural bone (Abadi et al., 2010; Aboudzadeh et al., 2010; Shokrgozar et al., 2010; Swetha et al., 2010). HAp with similar properties to bone tissue and suitable biocompatibility has been introduced as an
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appropriate bioceramic phase which can be incorporated in different polymeric phases (Ginebra
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et al., 2006a; Ginebra et al., 2006b). This is osteoconductive (Cai et al., 2009), non-toxic (He, X.
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et al., 2012), low in immunogenicity with the ability to directly bind to hard tissues (Azadi et al., 2016; Chen, Li et al., 2015; Li et al., 2011). HAp is the most stable calcium phosphates (CaPs)
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under neutral or basic pHs in the biological fluids and humid conditions (Aoki, 1991). Thus, this
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ceramic is usually formed in the fluids containing phosphates in the bodies of vertebrates that has the pH between 7.2 to 7.4. The chemical structure of HAp is mostly similar to inorganic
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compounds existed in the bone matrix. High-affinity of HAp to bone tissue makes it a good bone
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replacement in comparison to allografts and metallic implants (Bauer et al., 1991). There are two common structures of HAp including monoclinic and hexagonal constructs. In humans and
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animal bones, HAp is mainly in the hexagonal structure and thus synthetic HAp is the
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predominant form of CaP used in biomedical fields (Figueiredo et al., 2010). However, HAp is weakly dispersed in aqueous media which leads to aggregation. Thus, it is necessary to
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functionalize the surface of HAp (Kim, H.H. et al., 2014). In addition, poor fracture toughness of HAp (Azadi et al., 2016; Li et al., 2011; Wang et al., 2004b), migration of nanoparticles from implanted sites (Azadi et al., 2016; Wang et al., 2005) and challenging processability (Azadi et al., 2016) restrict the clinical applications of this biomaterial. In order to overcome these limitations, many efforts have been directed to develop hybrid structures containing HAp and biopolymers or organic molecules (Li et al., 2008; Li et al., 2011; Wang et al., 2004b; Wang et
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ACCEPTED MANUSCRIPT al., 2005). Several natural polymers, including collagen, gelatin, chitosan, silk, and chondroitin sulfate have been used in combination with HAp to enhance bone regeneration (He, J. et al., 2012). Many structures based on SF/HAp composites alone or in combination with bioactive molecules and stem cells for bone tissue regeneration are schematically represented in Figure 2.
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In this review, we focused on recent studies that used SF/HAp as bone constituents. This review starts with a brief description about bioceramics and CaPs, peruses structural properties of HAp
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and SF, and continues with descriptions of the signaling responses. Finally, the combination of
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SF/HAp for orthopedic applications will be discussed in detail.
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2- Bioceramics for bone tissue engineering applications
Ceramics are solid materials consisting of non-metallic, inorganic substances in two crystalline
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and non-crystalline (amorphous) forms (Hench and Jones, 2005). Ceramic and glasses have
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different applications in various fields of human health and in industry, such as diagnostic instruments, thermometers, eyeglasses, tissue culture flasks and fiber optics for endoscopy
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(Dorozhkin, 2010). During past decades, bioceramics have been used for repairing bone defects
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(Eshtiagh-Hosseini et al., 2007) and hard tissues (Hench and Jones, 2005). Bioceramics are included in the ceramic materials category that usually have non-crystalline structures (Baino
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and Vitale-Brovarone, 2014). The main classes of bioceramics and their subset groups and some applications are presented in Figure 3. Nontoxic and biologically inactive ceramics such as Al2 O 3 and ZrO 2 as bioinert bioceramics (Park, 2009) are usually used for joint prostheses because of their mechanical properties. Bioactive glasses and glass-ceramics directly bond to hard tissues and support new bone formation (Baino and Vitale-Brovarone, 2014). In the biomaterial industry, CaPs are used
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ACCEPTED MANUSCRIPT predominantly as bone replacements due to their biocompatibility, low density, chemical stability (Fathi et al., 2008), chemical and crystallographic similarity to bone mineral (Ding, ZZ et al., 2016), and integration into living tissue similar to the healthy bone (Dorozhkin, 2010). These inorganic materials include diverse types from bioresorbable tricalcium phosphate (TCP)
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to bioactive and biostable HAp (Fathi et al., 2008). Most CaP bioceramics are based on HAp,
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beta-tricalcium phosphate (β-TCP), alpha-tricalcium phosphate (α-TCP) or biphasic calcium
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phosphate (BCP) containing a mixture of α-TCP+HAp or β-TCP+HAp (Dorozhkin, 2010). The major physical and structural properties of CaPs and inorganic components of bone and tooth are
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listed in Table 1.
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ACCEPTED MANUSCRIPT Table 1. Major properties of calcium phosphates and inorganic phases of adult human calcified tissues (Dorozhkin, 2012; Ducheyne et al., 2015; Park, 2009; Ratner et al., 2004; Wnek et al., 2008).
Ca/P mola r ratio
Ca(H2 PO4 ) 2 .H2 O
0.5
Densit y (g/cm 3 )
Z1
Lattice structure
a= 5.6261(5), b= 11.889(2), c= 6.4731(8) Å,
2.23
2
T riclinic
a= 7.5577(5), b= 8.2531(6), c= 5.5504(3) Å,
~18 (1.14)
0.0–2.0
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0.5
pH stability range in aqueous solution s at 25 °C
2
T riclinic
~100
~17 (1.14)
2
2.32
4
Monoclinic
~80
~0.088 (6.59)
2.0–6.0
2.89
4
T riclinic
~100
~0.048 (6.90)
1
2.58
Dicalcium phosphate anhydrous (DCPA or DCP), mineral mone tite
1.0
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CaHPO4 .2H2 O
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Dicalcium phosphate dihydrate (DCPD), mine ral brushite
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Ca(H2 PO4 ) 2
~100
Solubilit y at 25°C, g/L (logKa )
transforms into MCPA
α= 98.633(6)°, β= 118.262(6)° ,γ= 83.344(6)° Monocalcium phosphate anhydrous (MCPA or MCP)
Transformatio n temperature (°C)
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Formula
Lattice parameter s
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Monocalcium phosphate monohydrate (MCPM)
Composition
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Che mical name
CaHPO4
α= 109.87(1)°, β= 93.68(1)°, γ = 109.15(1)° a= 5.812(2), b = 15.180(3), c= 6.239(2) Å, β= 116.42(3)°
1.0
a= 6.910(1), b = 6.627(2), c= 6.998(2) Å,
Sinter ~300
α =96.34(2)°, β= 103.82(2)°, γ= 88.33(2)°
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ACCEPTED MANUSCRIPT O ctacalcium phosphate (O CP)
Ca8 (HPO4) 2(PO4)4 .5H2 O
1.33
a= 19.692(4), b= 9.523(2), c = 6.835(2) Å,
2.61
1
T riclinic
2.86
24
Monoclinic
An unstable transient intermediate
~0.0081 (96.6)
5.5–7.0
~0.0025 (25.5)
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a= 12.887(2), b= 27.280(4), c= 15.219(2) Å, β= 126.20(1)°
β-Tricalcium phosphate (βTCP)
β-Ca3 (PO4 ) 2
Amorphous calcium phosphate s (ACP)
Cax Hy (PO4 ) z.nH2 O, n = 3–4.5, 15–20% H2O
1.22.2
Calciumde ficie nt hydroxyapatit e (CDHA or Ca-de f HA) 7
Ca10-x (HPO4 ) x (PO4 ) 6x (OH) 2-x (0 < x < 1)
1.51.67
Hydroxyapatit e (HA, HAp or O HAp)
Ca10 (PO4 ) 6 (OH) 2
a=b= 10.4183(5), c= 37.3464(23 ) Å, γ = 120°
3.08
21 4
Rhombohedr al
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1.5
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1.5
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α-Ca3 (PO4 ) 2
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α-Tricalcium phosphate (αTCP)
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α= 90.15(2)°, β = 92.54(2)°, γ = 108.65(1)°
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a= 9.84214(8), b = 2a, c = 6.8814(7) Å, γ= 120° (monoclinic )
~1125
~0.0005 (28.9)
3
transforms into α-T CP
-
-
-
-
-
-
-
~700
5
~5–12 6
~0.0094 (385)
6.5–9.5
~250, monoclinic to hexagonal Sinter ~1000
~0.0003 (116.8)
9.5–12
-
~0.0002
7–12
transforms into β-T CP
3.16
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Monoclinic
2
Hexagonal
a=b= 9.4302(5), c =6.8911(2) Å, γ = 120° (hexagonal) Fluorapatite
Ca10 (PO4 ) 6 F2
1.67
a=b=
3.20
10
2
Hexagonal
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(120)
9.367, c = 6.884 Å,
O xyapatite (O A, O Ap or O XA) 8
Ca10 (PO4 ) 6 O
1.67
a =b = 9.432, c = 6.881 Å, α = 90.3°, β = 90.0°,γ = 119.9°
~3.2
1
Hexagonal
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Te tracalcium phosphate (TTCP or Te tCP), mine ral hilgenstockite
Ca4 (PO4 ) 2 O
2.0
a= 7.023(1), b = 11.986(4), c= 9.473(2) Å, β= 90.90(1)°
3.05
4
Monoclinic
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γ = 120°
Ename l
-
1.63
a=9.441, c= 6.880 Å
-
De ntine
-
1.61
a= 9.421, c= 6.887 Å
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Bone
-
1.71
a= 9.41, c= 6.89 Å
-
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~0.0007 (38-44)
3
-
-
-
-
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3
-
-
Number of formula units per unit cell, 2Stable at temperatures above 100°C, 3Cannot be precipitated from aqueous solutions, Per the hexagonal unit cell, 5Cannot be measured precisely. However, the following values were found: 25.7± 0.1 (pH 7.40), 29.9 ± 0.1 (pH 6.00), 32.7± 0.1 (pH 5.28). The comparative extent of dissolution in acidic buffer is: ACP >> α-TCP >> β-TCP > CDHA >> HA > FA, 6Always metastable, 7Occasionally referred to as “precipitated HA” (PHA), 8The existence of OA remains questionable
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~0.087 (369)
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ACCEPTED MANUSCRIPT 2.1. Hydroxyapatite The term ‘apatite’, given by Wener in 1788, refers to inorganic compounds with similar structure but different compositions (Kokubo, 2008). These minerals have lattice parameters a=0.9432 nm and c=0.6881 nm and crystallize into hexagonal rhombic prisms (Park, 2009; Park and Lakes, or
pure
HAp
is
introduced
by
Ca10 PO4 6 OH 2 or
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Synthetic
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2007).
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Ca10z HPO4 z PO4 6z OH 2z [0 < z < 1] as a general formula. The chemical behavior of HAp depends on stoichiometry status, which is basic for stoichiometric apatite (z = 1) and acidic
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for the others (Faria et al., 2008).
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Inorganic materials coexist with organic molecules in a well-ordered manner in some tissues such as bone, cartilage and skin (Wang et al., 2004a). Natural bone is a nanocomposite
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(Andiappan et al., 2013; Bhattacharjee et al., 2016b) composed of organic components (~90%
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type I collagen, ~5% NCPs,~2% lipids by weight), water and inorganic CaP (Boskey, 2013;
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Šupová, 2015; Wu et al., 2014; Zhao et al., 2009). Recently, NCPs have attracted attention due to their effect on bone tissue behavior, including cell proliferation and attachment, HAp and Ca2+
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binding and degree of mineralization (Young et al., 1992). Osteocalcin, sialoprotein, alkaline
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phosphatase (ALP) and matrix Gla protein are the most abundant NCPs that are a main focus of research; however, it is not well-understood that which of these proteins may have a crucial role in bone behavior (Figure 4). It is reported that the combination of these proteins may influence bone functions synergistically more than each of them alone. It should be noted that some features such as gender, age, health status and ethnicity affect the proportion of these proteins in bone structure of an individual patient. Therefore, the quantity and quality of NCPs can influence the characteristics of bone tissue in terms of structure and function (Boskey, 1989, 2013).
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ACCEPTED MANUSCRIPT The inorganic phase of bone consists of HAp nanocrystals (about 70 wt% (Andiappan et al., 2013; Wang et al., 2004a)) dispersed in Col I (Andiappan et al., 2013; Wang et al., 2004a). This mineral phase is crystallized along the long axis of collagen fibrils (Hu et al., 2015) and integrates together in a tight hierarchical organization (Figure 4) [28]. The X-ray diffraction
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(XRD) spectrum of sintered bone and mineral apatite showed that HAp and fluorapatite are the
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main minerals in bone and teeth. For many years, it was assumed that the inorganic parts of
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calcified tissues were HAp. However, studies focused on synthetic carbonated apatite and biological apatite showed that the bone mineral does not consist of pure apatite but contains
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carbonated apatite (Kokubo, 2008) and some ions such as K +, Mg2+, Na+, F- and CO 3 2- (Li et al.,
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2008; Webster et al., 2004). Moreover, bioactivity of bone mineral is higher than synthetic HAp (Fathi et al., 2008). Therefore, non-stoichiometric HAp substituted with suitable ions during
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synthesis would be useful to mimic the function of bony apatite and osteoinductive activity when
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compared to pure HAp (Ren et al., 2009).
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Different methodologies have been used to prepare HAp-based products in micro- or nanoscale quantities. Co-precipitation (Cao et al., 2013; Ming et al., 2014a), chemical precipitation (He, X.
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et al., 2012; Kweon et al., 2011; Wang et al., 2002), solid-state reaction (Ming et al., 2014a;
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Wang et al., 2002), hydrothermal methods (Cao et al., 2013; Kweon et al., 2011; Ming et al., 2014a; Wang et al., 2002), sol-gel synthesis (Cao et al., 2013; Kweon et al., 2011; Ming et al., 2014a) and wet mechanochemical routes (Cao et al., 2013; Nemoto et al., 2001; Wang et al., 2002) are common methods used for HAp synthesis. Microemulsions, spray drying and plasma melting are also applied for synthesizing spherical HAp powders (Liu, J. et al., 2013). Structural features of the synthesized powders are mainly related to the corresponding preparation method (Kweon et al., 2011). Control of size distribution and morphology of nano-sized HAp powders
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synthesis of nHAps are summarized in Table 2.
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ACCEPTED MANUSCRIPT Table 2. Different methods for nHAps fabrication and main properties based on the materials used (El Briak-BenAbdeslam et al., 2008; Gedanken, 2004; Sadat-Shojai et al., 2013; Sasikumar and Vijayaraghavan, 2008, 2010; Tas, 2000). Raw materials
Advantage s
Disadvant ages
Synthetic conditions
Shape categori es 1
Solid-state reaction
Few Ca and PO4 containing chemicals
Relatively simple
Weak precise control over product features
900~1300° C, usually with water vapor flowing
1, 3
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Diverse morpholog y
Usually low purity Variable Ca/P
Irregular shape
e.g.: CaHPO4 .2H2 O + CaO
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(Mechanosynt hesis)
Few Ca and PO4 containing chemicals
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Mechanochem ical
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Low cost
Heterogene ous compositio n of powder
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Using organic templates to control morpholog y
Product features
Very high crystallinity
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Mass production
Usually micronWide
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Presynthesized CaP salts
Size-Size distribut ion
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Synthesis method
CaCO3 +NH4 H2 PO4 Ca3 (PO4 )2 +Ca( OH)2
Simplicity
Cannot be exploited for biomimetic synthesis -
Reproducib ility
Reaction activated
Production of nanocrystal line alloys and ceramics
Ca(OH)2 +P2 O5
Welldefined structure of powder
CaO+Ca(OH)2 +P2 O5
Mass production
Wet or dry medium
by mechanical milling Effective factors: reagents, milling medium, atmosphere , rotational speed, diameter of milling
Low cost
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1, 2, 3
NanoUsually wide
Diverse morpholog y Very high crystallinity Low purity Nonstoichiomet ric Ca/P
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Using organic templates and molecules to control morpholog y Low cost
Variable chemical reagents
Molecular level mixing of reactants
High cost of some raw materials
Improving chemical homogeneit y
Possible impurities (CaO, Ca2 P2 O7 , Ca3 (PO4 )2 , CaCO3 )
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Sol-gel
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Ca(OEt)2 , Ca(NO3 )2 , P(OEt)3 , P2 O5 , (NH4 )2 HPO4
Low temperature for various steps Stoichiome tric structure
Hydrothermal
Variable reagents Wet
Using organic modifiers to control
Long aging to complete reaction Thermal treatment to get pure HAp Expensive equipment for high temperatur
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Diverse morpholog y Frequently low crystallinity
Aqueous medium
Variable purity
pH 7-12
Usually high cost
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Method to modify a prepared HAp
Usually nanoVariable
Atmospher ic pressure
pH 6-7 for acidic phases (DCPD, DCPA)
1, 3, 5, 7
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Other CaP phase (DCPDc, DCPA TCP)
Distinct method to convert a CaP into HAp
1, 2, 3, 4, 5, 7, 8, 11
VariableVariable
Conversion depends on pH, T and other ions
Aqueous or organic media
Stoichiomet ric Ca/P 1, 2, 3
NanoNarrow
Diverse morpholog y Variable crystallinity Variable purity
Drying at ~100150°C
Stoichiomet ric Ca/P Higher bioresorbab ility than chemical precipitatio n
Heat treatment at ~300900°C
High
Diverse morpholog y
Usually high purity
Aging at room temperatur e
Aqueous solution
Usually nonstoichiomet ric Ca/P
Variable crystallinity
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Few chemical substances
Usually at room temperatur e
Phase impurities (DCPA a, OCPb )
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Hydrolysis
Precise control over conditions necessary to minimize defects
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The simplest route
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Frequently few Ca and PO4 containing reagents
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Chemical precipitation
1, 2, 3, 4, 5, 6, 8, 9
Nano and micronWide
Frequently needle-like Usually
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growth, and aging Simplicity
-
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Low temperatur e
Usually high purity Stoichiomet ric Ca/P
1, 2, 3, 5
NanoNarrow
Minimal agglomerati on
Frequently low crystallinity
Mild conditions
Variable purity
Aqueous environme nt
Heterogene ous
Using organic modifiers to control or change process
reactions between liquid and solid reactants
Usually low cost
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Frequently needle-like
Different surfactants
Chemical reactions activated by powerful ultrasound radiation
More uniform, smaller and purer crystals
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e.g.: Ca(OH)2 + Ca(H2 PO4 )2
Increasing rate of crystal growth
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Few Ca ad PO4 containing chemicals
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Agglomerat e-free
Sonochemical
100~200°C (1~2 MPa), 300~600°C (1~2 kbar)
High cost
More effective control over morpholog y and microstruct ure
Very high crystallinity
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Many raw materials
irregular
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Emulsion
Poor control over morpholog y and size distribution
temperatur e and pressure
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Regulating more predictable reactions as crystal nucleation,
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Nonstoichiomet ric Ca/P
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chemically prepared HAp, other calcium phosphates, seeding large crystals
1, 2, 3
NanoUsually narrow
Diverse morpholog y (usually needle-like) Variable crystallinity Usually high purity Variable Ca/P
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Inexpensiv e raw materials Relatively simple Well chemical homogeneit y of powder
front
used
e.g.: Ca(NO3 )2 , (NH4 )2 HPO4
combustio n of a fuel and oxidiser at elevated
Diverse morpholog y (usually irregular) Variable crystallinity Usually high purity
HAp decomposit ion into αTCP at high temperatur e of flame
Heat sources: flame or furnace
1, 2, 3, 5
Secondary aggregation s leading to decrease in specific surface area
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Usually low cost
1
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Easily scaled-up for continuous production
Usually nanoWide
Variable Ca/P
temperatur es
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Variable chemical materials
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Pyrolysis
1, 2, 3, 5
solution via spontaneou s
Particle morpholog y dependent on fuel
Fairly low temperature Usually low cost
Particles formed from aqueous
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Organic fuels (glycine, urea, sucrose, citric acid, succinic acid)
Single step operation
Mixed phases due to uncontrolla ble high temperatur e reaction
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Oxidants (Ca(NO3 )2 , (NH4 )2 HPO4 and HNO3 )
Quick production of powder
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Few raw materials
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Selfpropagating combustion synthesis
Nano particles embedde d in micron aggregate sVariable
Diverse morpholog y High crystallinity Variable purity Usually stoichiomet ric Ca/P
1-Irregular sphere, 2-(micro and nano)Sphere or ball-shaped, 3-rod- or needle-shaped or strip-like, 4-plate or sheet, 5-self-assembled or bundled nanorod, 6-dendelion or rosette, 7-bundled sheets or leaves, 8-Flower, 9-porous microsphere or mesoporous sphere, 10-Bowknot, 11-Dumbbell a
Anhydrous dicalcium phosphate, b Octacalcium phosphate, cDicalcium phosphate dihydrate
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ACCEPTED MANUSCRIPT 2.2. Signaling responses of osteoblast cells to hydroxyapatite Dynamic interactions between osteoblasts and their extracellular matrix (ECM) regulate their behavior and differentiation potential. Understanding the principal ECM cues promoting osteogenic differentiation can be pivotal for both bone tissue engineering and regenerative
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medicine. HAp can trigger signaling cascades; however, the mechanism of action is not well understood. The bioactivity of any material, here defined as the ability of osteoblasts to adhere
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and spread upon it, depends on these signaling mechanisms (Kokubo, 1991; Zambuzzi et al.,
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2011a). The signaling pathway involved in osteoblasts adhering on HAp scaffolds was recently evaluated (Gemini‐ Piperni et al., 2014). Adhesion of osteoblast cells on HAp substrates actives
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such signaling pathway that leads to activation of protein kinase C (PKC), protein kinase A
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(PKA), Adducin 1 (ADD1) and vascular endothelial growth factor (VEGF).
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Moreover, kinase activation leads to increased phosphorylation of Ser-421 in histone deacetylase 1 (HDAC1), a cyclin-dependent kinase 5 (CDK5) substrate. The surface chemistry of HAp is
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crucial for promoting the differentiation of osteoblasts via phosphorylation (Gemini‐ Piperni et
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al., 2014). In response to HAp, other signaling molecules such as extracellular regulated kinases (ERK) and SOX9 are also activated (Song et al., 2008b). The integrin receptors mediate the
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interaction between osteoblast cells cultured on HAp substrates with some domains of ECM proteins that can activate the cytoskeletal and intracellular signaling cascades like focal adhesion kinase (FAK). FAK is associated with Shc protein activating Ras that stimulates the ERK signaling cascade (Miyamoto et al., 1996). Activated ERK affects the expression level of different osteoblast genes such as osteocalcin and Col I (Figure 5) (Mimori et al., 2007; Xiao et al., 2002). Therefore, ERK signaling is essential for cytoskeletal rearrangement and intracellular signaling (Zambuzzi et al., 2011b). Various signaling cascades are also triggered upon the 19
ACCEPTED MANUSCRIPT interaction between HAp and osteoblast integrins such as BMP/Smad (Liu, H. et al., 2013), Wnt (Thorfve et al., 2014), TGF-β, MAPK, and Notch signaling pathways (Lü et al., 2014). 3. Structure and characteristic of silk fibroin
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Silk protein is found in the gland of silk producing arthropods such as spiders, silkworms, mites,
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scorpions, and bees. During metamorphosis, silk is spun to fibers. Cocoon silk, as a Food and
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Drug Administration (FDA) approved biomaterial for some medical applications, is produced by silkworm B. mori (Kaplan et al., 1994). B. mori silk is composed of two major types of proteins,
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silk fibroin (SF) and sericin. SF is located in the core of B. mori silk fibers and consists of light (~26 kDa) and heavy (~390kDa) chains; while, sericin coats the SF chains. Silk fibers contain
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about 70-75% SF filaments and 25-30% sericin. The primary sequence of sericin consists of
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some repeat motifs (Sutherland et al., 2010). The structure of the SF heavy chain is similar to
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amphiphilic block copolymers because it contains several hydrophobic and hydrophilic domains or blocks. The hydrophobic blocks of SF are comprised of highly conserved (GAGAGS) and less
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conserved (GAGAGX) repeats (where X is either valine or tyrosine) that are responsible for the
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crystalline structure and β-sheet conformation of SF. The hydrophilic part of SF core consists of short and non-repetitive segments in comparison with hydrophobic parts (Bini et al., 2004;
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Kaplan and McGrath, 2012). Silk fibers have useful mechanical properties, including a tensile strength of 0.5 GPa, breaking elongation of 15% and 62,104 J kg−1 toughness (Li et al., 2012). The Young’s modulus of silk obtained from forced silking of silkworm in the laboratory is about 12.4–17.9 GPa, with an ultimate tensile strength of 360–530 MPa and 18-21% breaking elongation (Pérez‐ Rigueiro et al., 2001). Silk fibers have been used for many years as sutures and for textile engineering due to their high toughness (Nova et al., 2010). Some studies have reported comparable mechanical behavior for silk filaments with nylons (Lucas, 1964). These 20
ACCEPTED MANUSCRIPT unique mechanical characteristics make SF a useful polymer candidate for load-bearing applications, such as for bone tissue engineering in composite systems with HAp. 3.1. Osteogenic signaling responses to silk fibroin
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While silk scaffolds do not inherently contain any cell-specific signaling epitopes in the primary
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sequence, silk-based scaffolds provide suitable substrates for supporting the proliferation and
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adhesion of mesenchymal stem cells (MSCs) (Mandal and Kundu, 2009; Mauney et al., 2007). The ability of hydrolyzed silk protein to improve the ALP activity of osteoblast cells and their
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osteogenic differentiation has also been reported (Seo et al., 2011). Moreover, B. mori silk induces mineral deposition from rat bone marrow stromal cells (BMSCs) during 12 days of
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culture in vitro (Nikbakht Dastjerdi, 2006). This study demonstrated that by using silk, no
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dexamethasone additives are required for pre-osteogenic factor, although the compound can
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accelerate the outcomes. Dexamethasone can trigger Wnt/β-catenin signaling dependent Runx2 expression (Langenbach and Handschel, 2013). Furthermore, other signaling factors like Wnt,
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Notch, fibroblast growth factor (FGF), bone morphogenetic protein/Transforming growth factor
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beta (BMP/TGFβ), insulin-like growth factor (IGF), and platelet derived growth factor (PDGF) can regulate the osteogenic differentiation of stem cells on silk-based substrates (Midha et al.,
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2016). Based on pharmacologic and molecular studies, Notch signaling was responsible for differentiation of mesenchymal progenitor cells into osteoblast cells. Silk protein provoked the upregulation of some osteoblast differentiation markers (e.g. ALP, osteorix and Runx2) by inhibiting Notch signaling (Jung et al., 2013). Additionally, the mass of trabecular bone in the skeletogenic mesenchyme of mice limbs was significantly increased by inhibiting the Notch signaling pathway in adolescent mice. Severe osteopenia was observed in mice during aging period when they were without mesenchymal progenitors in their bone marrow. The inhibition of 21
ACCEPTED MANUSCRIPT osteogenic differentiation by Notch signaling is triggered by Hes and Hey proteins, which can suppress Runx2 activity through physical interaction (Figure 6a) (Hilton et al., 2008). Thus, for optimal osteogenic induction of silk scaffolds, suppression of the Notch signaling pathway is advantageous (Figure 6b).
properties
of
SF
such
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outstanding
as
biocompatibility,
biodegradability,
low
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The
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3.2. Silk fibroin-based scaffolds for bone tissue engineering
immunogenicity, and suitable processability make it a useful scaffold system for various tissue
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engineering applications, including bone constructs. Another advantage of SF as a bone substitute is its high mechanical strength (Kundu et al., 2013; Mottaghitalab et al., 2015b;
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Nourmohammadi et al., 2017); it can bear the force produced in vivo (Nazarov et al., 2004).
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These properties of SF have attracted many researchers to use this biomaterial in bone tissue
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engineering applications. Recent investigations on the SF-based scaffold as a bone construct are summarized in Table 3.
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Table 3. SF based scaffolds indicating types of material used for blending, methods for
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applications.
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preparation of matrices, cell types used and their main findings in bone tissue engineering
Material
Processing method
Scaffold structure
Cell
Key findings
Ref.
TSF1 / PLA2
Electrospinning
Nanofibers
mMSCs 3
Inducing the proliferation and attachment of MSC, increasing the rate of ALP4 production, osteogenesis, and bone mineralization
(Shao et al., 2016a)
Electrospinning
Nanofibers
-
Stimulating the early bone
(Bhattacharjee
(90:10)
NSF5 /PCL6
22
ACCEPTED MANUSCRIPT et al., 2016a)
Increased proliferation, adhesion and differentiation of hMSCs confirmed by ALP production, transcription of RUNX2, and expressions of osteocalcin and type1 collagen
(Singh et al., 2016)
MG-63
Capability of the modified scaffold for alveolar bone repair due to outstanding biofunctionality
(Sangkert et al., 2016)
3D scaffold
MC3T3-E1
Increased wettability, suitable porosity, high mechanical strength, and good biocompatibility by incorporating diopside nanopowders into the SF matrix
(Ghorbanian et al., 2013)
3D scaffold
HGF12
Formation of a porous scaffold with appropriate pore size for cell infiltration, improved compressive strength and water uptake, and decreased porosity
(Teimouri et al., 2015)
Electrospinning
Nanofibers
SF/Decellularized pulp/Collagen/Fi bronectin
Freeze-drying
3D scaffold
SF/Diopside
Freeze-drying
hMSCs 9
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SF7 /CMC8
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formation, infiltration of implants and formation of strong bonds at the interface of boneimplant, activating the initial immune reaction which reaching to normal after 4 weeks postimplantation
SF/CS 10 /Nano ZrO2 11
Freeze-drying
23
ACCEPTED MANUSCRIPT after adding zirconia Electrospinning/ Freeze-drying
3D scaffold
SF
Casting
Film
hOBs 15
Providing an optimal microenvironment for the biological behavior of osteoblast cells due to acceptable porosity, chemical and physical properties with ability to sustain the release rate of VEGF16 within 28 days, stimulating the new bone formation at the site of injury 10 weeks after implantation
(Farokhi et al., 2014b)
BMSC17
More efficient delivery of BMP218 after immobilizing this biomolecule on the surface of biomaterial in comparison to soluble delivery, retained in vitro biological activity of BMP-2 based on its potential to induce the osteogenic markers of BMSCs
(Karageorgiou et al., 2004)
OEC19 /hOBs
Extracellular matrix deposition by hOBs during 4 weeks, enhancing expression of osteogenic markers, retained functionality of both cell types in provoking the formation of capillary like structures and
(Fuchs et al., 2009)
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SF/CaP13 /PLGA14
SF
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Mesh
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ACCEPTED MANUSCRIPT differentiation of hOBs Salt leaching
3D scaffold
hMSC
Increased bone formation in calvarial critical size defects in mice during 5 weeks by using tissueengineered bone implants, detecting less bone formation by using stem cell loaded SF scaffolds and scaffolds alone
(Meinel et al., 2005a)
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SF
Tussah silk fibroin, 2 Polylactic acid, 3 Mouse mesenchymal stem cells, 4 Alkaline phosphatase, 5 Nonmulberry silk fibroin, 6 Silk fibroin, 7 Poly(ε-caprolactone), 8 Carboxymethyl cellulose, 9 Human mesenchymal stem cells, 10 Chitosan, 11 Zirconia, 12 Human gingival fibroblast, 13 Calcium phosphate, 14 Poly (lactic-co-glycolic acid), 15 Human osteoblast cell, 16 Vascular endothelial growth factor, 17 Bone marrow mesenchymal stem cells, 18 Bone morphogenetic protein-2, 19 Outgrowth endothelial cells
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4. Silk fibroin/hydroxyapatite for bone tissue engineering
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4.1. Silk fibroin/hydroxyapatite composites for bone tissue engineering Bone tissue engineering offers suitable constructs for restoring and repairing bone defects (Ma et
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al., 2005). Mimicking the structure and composition of natural tissues by engineering biomaterials, such as developing inorganic-organic composites, has grown in interest and focus
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over the past decades. Some typical ceramics such as bioactive glass, HAp, β-TCP, and calcium
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citrate are suitable candidates for bone tissue engineering applications (Chouzouri and Xanthos, 2007; Maquet et al., 2004). The most common organic material used as bone constructs is collagen. This protein polymer can be obtained from some animals (e.g. skin of pig and cow) or generated through transgenic techniques. However, collagen is not cost-effective, provoke immune responses, and retains a risk for contamination (Du et al., 2009). For these limitations, some researchers have tried to use other natural biomaterials such as silk for bone tissue engineering. 25
ACCEPTED MANUSCRIPT Blending silk with HAp enhanced crystal formation of HAp along the c-axis and the coordinative effect on the structure and properties between SF and HAp was found during the composite film fabrication. The nucleation of HAp could enhance the molecular orientation and crystallinity of SF (Du et al., 2009). Recently, Jo et al. evaluated alginate/HAp/SF composites as a bone
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replacement (Jo et al., 2017). Four weeks after implantation, the rate of bone formation in the
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defected site was significantly higher using alginate/HAp/SF scaffold than alginate and control
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(unfilled defects) groups. The suitable biocompatibility of alginate/HAp/SF scaffold led to less immunogenic response or formation of giant cells around the degraded grafts. Significantly
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lower expression level of tumor necrosis factor-alpha (TNF-α) was also observed in
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alginate/HAp/SF group compared with alginate and alginate/HAp groups; while, the osteogenic markers such as Runx2, osteoprotegerin, and FGF-23 showed higher expression rate. The
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alginate/HAp/SF scaffold was introduced as a potential bone construct for repairing calvarial
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defects in rat model that was stably biodegraded without provoking the immunogenic responses
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(Jo et al., 2017).
The homogenous dispersion and interfacial bonding between polymeric matrix and HAp is still
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challenging in fabricating composites. The interfacial bonding between HAp and the organic
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phase is dependent to the density of contact numbers and the chemical interactions. In this context, pretreatment of SF fibrils with an alkali increased the exposed active sites on the surface that further improved the interactions between the organic and mineral phases. The alkali pretreatment effected the crystallographic characteristics of HAp in comparison to pretreatment with proteinase K, a protease that digests the SF. Applying both pretreatments enhanced the exposed active sites to interact with the mineral phases. It was suggested that SF surface fibrils were mostly disentangled by alkali, while the enzymatic pretreatment was effective in
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ACCEPTED MANUSCRIPT disentanglement of the SF blocks into smaller fragments (Wang et al., 2007). An increase in number of contact sites enhanced the interactions between organic and inorganic phases to improve microhardness (>50%) (Wang et al., 2007; Wang et al., 2004b; Wang et al., 2005). However, the remaining sodium ions from the alkali reagent may have unexpected side effects
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on crystallographic properties of HAp due to replacement of calcium with sodium ions. During the past decade, many efforts have been attempted to develop hybrid biomaterials based
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on organic and inorganic materials to mimic the structure and function of natural biomaterials.
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The existence of organic materials like polymers and proteins in the structure of hybrid materials regulate the nucleation of inorganic crystals, and enhance the physicochemical properties of the
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microstructure. As mentioned above, adding HAp to SF improve the biological and mechanical
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properties of SF. HAp/SF composites are so similar to the natural bone structure that mimic the inorganic and organic phases of native bone tissue. Thus, combination of inorganic and organic
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materials in a unique hybrid composite can improve the flexibility, mechanical strength, and the
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toughness of the bone tissue scaffolds. Some applications of SF/HAp composites for bone tissue
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engineering are listed in Table 4.
Table 4. The composites based on SF/HAp, methods for preparation. Types of cells, properties
Materials SF1 /HAp2
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of the matrices and findings are reported for bone tissue engineering. Processing method
Cell
Structural properties
In vitro/in vivo key findings
Ref.
MAPLE3
SaOs2
Exhibition of 1540 cm−1 amide II, 1654 cm−1 amide I, 1243 cm−1 amide III peaks in the FTIR spectrum of SF, and observing of
Improved proliferation of cells with normal morphology after 72h culture on SF and HAp/SF films
(Miroiu et al., 2010)
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ACCEPTED MANUSCRIPT 1027 cm−1 only for HAp and HAp/SF composite groups SF/HAp
Layer solvent casting/Freeze drying
BMSCs 4
Decreasing pore average size with increasing SF content from 5% (350 ± 67 μm) to 10% (112 ± 89 μm), more uniform pores in the scaffolds containing 5% HAp
SF/HAp
Casting
-
Exhibition of silk II conformation in the secondary structure of SF films, no impact of HAp content in the solution on the structure of SF films, Significant effect of HAp content on rheological behavior of SF solution
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(Ming et al., 2014b)
NSF5 /HAp
Electrospinning
Osteoblast
Higher crystallinity and thermal stability of electrospun NSF/HAp scaffold compared with pure NSF and NSF/HAp formed by soaking method
Higher cell viability on electrospun NSF/HAp scaffold than pure NSF and NSF/HAp formed by soaking method
(Sekar et al., 2016)
MG-63
Scaffolds exhibited 90.5– 96.1% porosity with 100–300 µm pore size, better mechanical
Suitable for increasing the proliferation and adhesion of cells, higher efficacy of CS/SF/nHA composite porous
(Qi et al., 2014)
(Gholipourmalekabadi et al., 2015)
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No variation in the proliferation rate of BMSCs on HAp/SF and no toxicity, good in vivo biocompatibility of SF containing 5% HAp, no significant increase in the average number of lymphocytes in long and short periods
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and soaking
CS 6 /SF/nHAp
Salt fractionation
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ACCEPTED MANUSCRIPT behavior of tricomponent scaffold compared with bi-component scaffolds
scaffold than other groups
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Coprecipitation
-
Characteristics of primary HAp crystals: needlelike shape, 20-50 nm long, and ~10nm wide, Chemical interactions between amino group of chitosan or amid bands of SF with Ca2+ ions
SF/COL7 /HAp
Particulate leaching
MG-63
Displaying porous structure with adjustable macropores with high interconnectivity, uniform dispersion of SF and collagen within the scaffold
Significantly higher biocompatibility at days 7 and 14 compared with day 3, suitable features of scaffold in inducing the cellular proliferation, and potential of the scaffold to stimulate the cellular infiltration and migration
(Mou et al., 2013)
PVA8 /SF/HAp
Freezing and thawing
No agglomeration and uniform distribution of silk in the composite hydrogel, high impact of silk on elastic modulus of PVA-HApSilk composite hydrogel
-
(Zhang et al., 2012)
5
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(Wang and Li, 2007)
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CS/SF/HAp
Silk fibroin, 2 Hydroxyapatite, 3 Matrix Assisted Pulsed Laser Evaporation, 4 Bone marrow stromal cells, Non-mulberry silk fibroin, 6 Chitosan, 7 Collagen, 8 Polyvinyl alcohol
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ACCEPTED MANUSCRIPT 4.2. Silk fibroin/nano-hydroxyapatite composites for bone tissue engineering Recently, nanoparticles have attracted attention in biomedical fields due to their exceptional properties such as high surface to volume ratio, tunable structural properties in terms of surface chemistry, size and shape (Brannon-Peppas and Blanchette, 2012). Today, different applications
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are considered for nanoparticles including theranostics therapy, molecular imaging, drug delivery and cell labeling (Hassani Besheli et al., 2017; Kim, E.-S. et al., 2014; Mottaghitalab et al.,
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2015a; Mottaghitalab et al., 2017). It is also observed that the alterations in nanoscale features
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can affect cellular responses. For example, nano-patterned structures were potent in directing stem cell differentiation without the using of exogenous biochemical agents (Kim et al., 2012a;
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Kim et al., 2012b). Controlling the cellular behavior by using nano-patterned structures is a key
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application for nanotechnology in biomedical fields. In the orthopedic field, HAp nanocrystals were sintered and improved densification because of the high surface area. The high surface area
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can also increase fracture toughness and mechanical strength of HAp nanocrystals. However,
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agglomeration is the most challenging issue in using nano-powders (LeGeros, 1993). nHAp showed better bioactivity in comparison to coarser crystals that broaden its applications in tissue-
have
high
bioresorption
properties
and
similar
crystallographic
and
chemical
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nHAp
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engineered implants when compared to other constructs (Stupp and Ciegler, 1992). Moreover,
characteristics to the natural apatite in bone (Murugan and Ramakrishna, 2005). Composites containing nHAp induced both osteogenesis and angiogenesis by stimulating the proliferation, adhesion, and differentiation of osteoblasts; therefore, these composites are potent for bone tissue engineering applications (Kilian et al., 2008; Nageeb et al., 2012; Wei and Ma, 2004). Besides, in many studies SF protein extracted from B.mori alone or in blended with CaP has been used for bone tissue engineering. However, there is no RGD motifs or sufficient arginine 30
ACCEPTED MANUSCRIPT amino acids in the structure of SF derived B.mori to support cellular adhesion (Sen and Babu, 2004). Some reports suggesting the presence of RGD epitopes and high arginine content in the structure of non-mulberry silk fibroins (NSF) that reduced the cytotoxicity and inflammatory responses (Datta et al., 2001; Mandal and Kundu, 2008). Moreover, presence of hydrophilic
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amino acids in the structure of NSF promotes cellular attachment to the surface of the materials
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(Meinel et al., 2005b; Sen and Babu, 2004; Zhang, Y.-Q. et al., 2005). The in situ reinforcement
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of NSF-nHAp scaffold and the NSF scaffold with external nHAp deposition showed higher mechanical properties and biocompatibility in comparison to NSF-nHAp reinforced fibroin
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scaffolds. Both composite scaffolds also showed less immunogenicity by using osteoblast-
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macrophage co-culture model (Behera et al., 2017). However, no significant anti-inflammatory differences were observed through the modified scaffold indicating the native anti-inflammatory
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properties of fibroin structures. It was also observed that by culturing the macrophages on the
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modified scaffolds, the level of interleukin 1 beta (IL1-β) increased that might be related to the role of HAp (Loi et al., 2016). Similarly, nHAp/SF powders were synthesized using a co-
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precipitation method, added to SF solution in order to fabricate nHAp/SF composite material
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(Liu et al., 2011). These composites showed improved compressive properties. The good compressive properties were due to the uniform dispersion of nHAp/SF in SF solution, the high
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surface area of needle-like nHAp/SF powders, and good molecular interaction between SF molecules in the structure of nHAp/SF powder and SF matrix. The good interfacial interactions between the nHAp/SF powders and the SF matrix promoted cellular proliferation, attachment and differentiation (Liu et al., 2011). The superior biological behavior of nHAp powders when compared to micro-scaled versions provides options for many biomedical applications. Recently, nHAp particles 30-100 nm were homogeneously dispersed in a collagen/silk fibroin (Col-SF)
31
ACCEPTED MANUSCRIPT matrix and the electrostatic interaction between Ca2+ of the inorganic phase and carboxyl or amino groups of organic phase generated nanocomposites (Col-SF/HAp) with suitable structural properties and
biocompatibility.
Additionally,
the existence of SF in the structure of
nanocomposites enhanced elastic modulus in comparison to Col/nHAp (Chen, Li et al., 2014). In
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another study, it was observed that SF affected the mineralization of HAp nanocrystals. SF was
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able to significantly increase the growth of HAp nanocrystals at pH 8 in room temperature. The
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strong chemical interaction between HAp and the SF protein shifted the amide II peak from 1517-1539 cm-1 in FTIR spectra, which indicated that HAp crystals were replaced by carbonated
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HAp (Kong et al., 2004). SF not only influenced the morphological aspects of HAp crystals but
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also affected the water dispersibility of HAp. Moreover, SF surrounded HAp nanocrystals to form a layer with a negative charge to prevent aggregation of nHAp particles in aqueous solution
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(Huang et al., 2014). Previous studies also confirmed the regulatory activity of SF on
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biomineralization of calcium salts (Takeuchi et al., 2005; Wang et al., 2012). However, the distance between SF and HAp nanocrystals played an important role in the intensity of their
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interactions. At longer distances, the interactions became weaker (Zhang et al., 2013). Thus, SF
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and nHAp particle ratios are important factors in controlling the growth of nanocrystals and crystal morphology. Accordingly, regular SF nanostructures were implemented in order to
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evaluate their effect on the formation and morphology of HAp nanocrystals. SF nanospheres induced the formation of rice-like nHAp, while SF nanofibers stimulated the formation of HAp nanofibers (He, X. et al., 2012). Therefore, it was suggested that different nanostructures of SF can be considered as templates for the formation of different morphologies of HAp. Silk fibroin/sodium alginate (SF/SA) hydrogels induced the formation of HAp nanorods with rectangular column morphologies at room temperature. The strong interaction between SF/SA
32
ACCEPTED MANUSCRIPT nanofibers and Ca2+ ions in HAp had significant effect on the morphology of nanocrystals (Ming et al., 2014a). Generally, the phosphorylated motifs on the surface of SF play an important role in triggering the nucleation and formation of apatite crystals. Each of apatite crystals grew in size to form a thick mineralized layer during mineralization. Adding foreign molecules may trigger
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the initial formation of HAp crystals; however, mineral proliferation may be delayed due to
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inhibition of the growth sites (Boskey, 1998; LeGeros, 1990). Furthermore, one of the main
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challenges of using biomaterials to induce the process of bone regeneration is the bacterial resistance before complete bone repair. Thus, the biomaterials should have the ability to
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stimulate the regeneration before the adhesion of bacterial species. For this purpose, SF/nHAp
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hydrogen was modified with gold nanoparticles (AuNPs) and AgNPs in order to induce the antimicrobial effect (Ribeiro et al., 2017). The obtained results revealed that the hydrogel
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containing high concentration of AgNPs had strong capability to reduce the number of different
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gram positive and gram negative bacterial strains such as S. aureus (MSSA), S. aureus (MRSA), S. epidermidis, Escherichia coli (E. coli), and P. aeruginosa. However, the hydrogen comprising
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AuNPs did not show a linear antimicrobial effect against S. epidermidis. The hydrogels
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containing AuNPs≥0.5% had suitable antibacterial activity toward MRSA and P. aeruginosa; while, those containing AuNPs≥0.1% reduced the number of MSSA and E. coli. It seems that the
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higher surface activity of AgNPs in comparison to AuNPs might be responsible for this observation (Ribeiro et al., 2017). Taken together, the biological HAp existed in the natural bone has nanometric thickness and length with nanoscopic plate-like or rod-like shape. Thus, applying nHAp would be a good bone substitute with high regeneration capacity that highly mimic the mineral phase of
natural bone
tissue (Vallet-Regi and González-Calbet, 2004). nHAp possess great characteristics compared
33
ACCEPTED MANUSCRIPT with HAp such as high sinterability and densification as a result of high surface area that lead to improved mechanical strength and toughness (LeGeros, 1993). Additionally, the appropriate biocompatibility, osteoconductivity, the ability to promote the angiogenesis, and inducing the attachment, migration, and differentiation of osteoblast make it useful for bone regeneration
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(Hassan et al., 2016). However, nHAp is fragile, less flexible with intrinsic hardness that make it
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difficult to shape into the desired form which limits its applications in repairing load-bearing
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bone defects. In order to hamper the aforementioned drawbacks, nHAp in blended with other polymers such as SF to form nanocomposites with high osteoconductivity suitable for orthopedic
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surgery (Sun et al., 2011).
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4.3. Silk nanofibers containing hydroxyapatite for bone tissue engineering application
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The high surface area of nanofiber scaffolds makes them useful for protein absorption and
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binding to cell membrane receptors. The absorbed proteins can change the structural conformation of the nanoscale scaffolds along with generating more binding sites that make
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them more suitable for tissue engineering. Electrospinning is one of the most useful techniques
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for fabricating nanofibrous scaffolds. Electrospun mats are 3D structures with appropriate porosity and high surface area to volume ratio that mimic to some extent the structure of the
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ECM. Therefore, electrospun nanofibers are considered as potential scaffolds for tissue engineering applications due to their ability to support cellular proliferation, adhesion, and differentiation (Agarwal et al., 2008; Liang et al., 2007). However, using electrospun SF nanofibers is still limited for bone tissue applications because of their low mechanical properties. In order to overcome this limitation, inorganic ceramics can be incorporated into the polymeric matrix. However, the aggregation of a ceramic particle in the SF matrix can interrupt the electrospinning process. Therefore, further investigations are needed to fabricate appropriate 34
ACCEPTED MANUSCRIPT SF/HAp nanofibers for bone tissue engineering purposes. In several studies, the aggregation of HAp particles in SF/HAp nanocomposites was reported (Kim et al., 2008; Li et al., 2006; Zhang et al., 2010). The aggregation can reduce the mechanical strength of the SF/HAp composites (Kim, 2007; Song et al., 2008a) and can result in non-uniform physical and chemical structures
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in these scaffolds. A useful strategy for enhancing the uniform dispersion of HAp particles in the SF matrix is the
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surface modification of the SF polymer by γ-glycidoxypropyltrimethoxysilane (GPTMS). The
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epoxide functional groups of GPTMS are able to attach to amino groups in the side chains of SF. Concomitantly, another side group of GPTMS can interact with the hydroxyl groups on the HAp
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surface (Arafat et al., 2011; Kim, H. et al., 2014). Electrospun SF nanofibers containing 20%) disrupted the polymeric chains
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that affect the mechanical behavior of the scaffolds (Kim, H. et al., 2014). Similarly, using more
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(Yang et al., 2016).
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than 20% HAp content showed decreased bending modulus due to local aggregation of nHAp
Electrospinning of SF/HAp blends faces another limitation such as immediate precipitation of
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nHAp before ejection. A three-way stopcock connector was developed for rapid mixing of various polymeric solutions and then rapidly injecting under the electric field to form blended nanofibers (Sheikh et al., 2013). Based on FTIR analysis, the chemical interaction between SF and nHAp changed the conformation of SF from random coil to β-sheet, which improved the mechanical strength of SF nanofibers (Sheikh et al., 2013). It was also showed that combining electrospun SF nanofibers with high pure HAp and silver nanoparticles (AgNPs) produced a suitable scaffold with antimicrobial properties for tissue engineering (Sheikh et al., 2014). 35
ACCEPTED MANUSCRIPT AgNPs can enhance the antimicrobial activity of nanofibrous structures. SF nanofibers containing 0.5% silver nitrate showed no toxicity toward NIH 3T3 fibroblast cells; however, using higher concentrations (1.0 or 1.5%) of silver nitrate revealed typical toxicity (Sheikh et al., 2014). New biocomposites based on electrospun SF nanofibers containing mesoporous bioactive
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glass/hydroxyapatite nanocomposite (MGHA) were reported (Liu et al., 2014). Recently,
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mesoporous bioactive glass (MBGs) has been introduced as potential candidates for drug
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delivery and bone tissue engineering applications. The ternary SiO2 –CaO–P2 O5 structure of MBG possess useful properties such as high surface area, degradability and large pore volume in
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comparison to non-mesoporous bioactive glasses that make them suitable for inducing bone
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formation (Wu et al., 2013; Xia and Chang, 2006). Moreover, adding MGHA particles into SF solution increased fiber diameter related to conductivity affecting fiber diameter and particle
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dispersion. The decrease in the conductivity of the blend solution was controlled by using higher
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MGHA content and less SF solution in the matrix. Using MGHA as a reinforcing component in the structure of SF-based nanocomposites improved swelling, surface hydrophilicity, and pore
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size, while tensile strength decreased. SF/MGHA scaffolds induced the formation of larger bones
CE
than pure SF after 4 and 8 weeks post-implantation. Additionally, in both implants, new bones containing osteocytes were directly formed near the scaffolds. The capability of SF/MGHA
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composites in bone regeneration may be related to the chemical interactions between the implant and host tissue, along with their surface bioactivity (Liu et al., 2014). In a recent study, coaxial electrospinning was applied in order to prepare a core-shell structure consisting of HAp and tussah silk fibroin (TSF) (Shao et al., 2016b). The crystalline region of TSF has a higher amount of alanine (Ala) in comparison to B. mori silk; therefore, Ala repeats are the main sequences in the structure of TSF, while domesticated silk is mostly comprised of
36
ACCEPTED MANUSCRIPT Gly-Ala-Gly-Ser repeats. In addition, the existence of an Arg-Gly-Asp motif in TSF structure induced specific cellular attachment, useful for biomedical purposes (He et al., 2013; Zhang et al., 2009). An increased content of nanoparticles in the core of prepared fibers increased the tensile strength. In other words, the content of core nanoparticles can be used to gradually
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change the mechanical properties of the composite from soft to rigid. A similar trend was also
tissue engineering applications
due to
potential in inducing cellular proliferation,
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bone
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seen in the Young's modulus of the composites. This scaffold showed promising properties for
attachment, ALP production, and biomineralization (Shao et al., 2016b). It was also found that
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the robust interaction between nHAp and SF nanofibrills induced the formation of flower-like
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structure that potentially facilitated the mineralization of HAp in situ. Moreover, the good mechanical properties and thixotropy with storage modulus (G’) make SF/HAp hydrogel useful
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to recover to over 85% in 50 seconds after applying a large shearing strain (5000%) (Mi et al.,
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2016).
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Generally, it is necessary to use nanofibrous structure for bone tissue engineering applications due to the nanometric nature of ECM components existed in the natural bone. The bone is a
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natural nanocomposite comprising a nanoscale HAp with about 20-80 nm in length that are
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dispersed in an organic collagenous matrix (Rho et al., 1998). The individual collagen chains have the length about 10 nm which reach to 500 nm after collagen fiber formation. Thus, using nanoscale bone constructs not only mimic the structure of natural bone but also can provoke the cellular activities in terms of adhesion, growth, differentiation, and expression of various proteins (Jiang et al., 2015). Despite the potential of electrospun mats in resembling the structure of collagenous fibers, the weak mechanical behavior compared with porous materials might be challenging for bone tissue engineering applications. Therefore, it is crucial to find a way for
37
ACCEPTED MANUSCRIPT increasing the mechanical strength of fibrous SF nanofibers to reach to adequate tensional or compressional stresses similar to skeletal bone (Fu et al., 2011; Wei and Ma, 2004). It seems that addition of ceramic phases in SF fibers are good strategies to improve the mechanical properties of fibrous scaffolds (Ito et al., 2005).
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4.4. Silk fibroin/Hydroxyapatite for drug delivery in bone tissue engineering
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To date, many attempts have been made to develop an appropriate carrier based on novel materials for drug delivery purposes. These systems control the release rate of drugs in a more
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sustained manner that can be helpful for decreasing side effects. Different structures such as membranes, micro- and nanoparticles, fibers and three-dimensional scaffolds have been
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introduced as carriers for different biomolecules (Balmayor, 2015; Zarrabi et al., 2014). Most of
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these structures are mainly comprised of polymers; however, using inorganic materials can be
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also useful.
Bioceramics are attractive inorganic materials to incorporate bioactive molecules or even cells,
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without denaturation or loss of bioactivity during preparation or implantation (Ginebra et al.,
CE
2012). Large surface area, narrow pore size distribution, and well-ordered pore systems make CaPs useful candidates for drug delivery (Bose and Tarafder, 2012; Vallet-Regí et al., 2007).
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Moreover, CaP is useful for controlling the local release rate of antibiotics (Arcos and ValletRegí, 2013; Verron et al., 2010), anticancer, anti-inflammatory, and analgesic drugs (Ginebra et al., 2006a) and growth factors (Bose and Tarafder, 2012; Ginebra et al., 2006b). Oral administration of CaPs is impossible because of rapid degradation at low pH of the gastric system. Therefore, drug delivery systems for the intestinal system (pH 8-9) with a controlled and targeted release profile are useful. However, current investigations have been focused more on drug delivery to locally or directly to bone tissue or as coatings (Kolmas et al., 2016). 38
ACCEPTED MANUSCRIPT CaPs also absorb many chemical molecules on their surface, which is useful for applications such as chromatography, and different biomolecules isolation and purification (Ginebra et al., 2006a; Tiselius et al., 1956). For instance, this criterion has been used for purification of bone growth factor through HAp chromatography (Urist et al., 1984). The high affinity of HAp to
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different biomolecules can provide options for developing novel matrixes for drug delivery
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systems. However, applying HAp-based carriers faces challenges due to their low capacity for
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loading drugs and the inability to control the release kinetics of such biomolecules. In contrast, silk is a useful biomaterial with high drug loading capacity and capability in controlling the
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release rate of drugs. Different drugs and biological macromolecules also can be loaded on silk-
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based structures without changing their bioactivity (Kundu et al., 2010; Shi et al., 2013a). For this reason, considerable efforts have been devoted to developing combinations of the intrinsic
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bone regeneration potential of HAp blended with SF, with the ability to incorporate drugs or
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other active molecules relevant for different therapeutic needs.
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Electrospun SF fibers containing nHAp/bone morphogenetic protein 2 (BMP-2) were used to induce bone formation in vitro using human bone marrow-derived mesenchymal stem cells
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(hMSCs) (Li et al., 2006). The authors found that it was impossible to electrspin the SF solution
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with the concentration more than 18% because the insufficient viscosity and surface tension of this solution. Higher concentrations of SF solution induced the gel formation during the electrospinning process. Furthermore, nHAp had a tendency to form aggregations after suspending in aqueous media and to overcome to this issue the SF and polyethylene glycol (PEG) solutions were prepared in 0.001M phosphate buffer (pH 6.8) that reduced the aggregation. It was observed that the nHAp were effectively embedded in the SF/ PEO fiber scaffold. The nanoparticles showed different fashions in dispersing in the polymeric matrix in
39
ACCEPTED MANUSCRIPT which they were well-oriented along the fiber axis in some regions while agglomerated in the other parts. It seems that high viscosity of SF/PEO blend solution led to non-homogenous dispersion of individual particles. Moreover, this study revealed that co-processed BMP-2 supported more calcium deposition and more upregulation of bone-specific markers (BMP-2,
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Col I and bone sialoprotein-II) in comparison to control groups. This observation suggested that
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silk nanofibers were useful for BMP-2 delivery, which broadened the application of this
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biomaterial in bone tissue engineering applications. Based on XRD spectra, the crystallinity of apatite on SF/BMP-2 scaffolds was higher than bulk SF scaffolds. Additionally, the
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incorporation of nHAp with SF/BMP-2 during electrospinning induced bone formation (Li et al.,
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2006). In another study, silk was used as a surface stabilizer and template in order to enhance surface properties of nHAp (Ding, ZZ et al., 2016). The core-shell structure of these composite
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materials facilitated the interaction between silk on the outer surface of nanoparticles with BMP-
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2 resulting increase the loading capacity of nHAp. This study showed that removing silk from nHAp had no effect on the morphological aspects of nHAp; however, aggregation particles was
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observed because of the elimination of repulsive forces from SF. Several studies confirmed that
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the high loading capacity of silk was related to electrostatic interactions with BMP-2 (Shi et al., 2013a; Shi et al., 2013b). Moreover, HAp particles alone showed 90% release of BMP-2 during
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2h, it was about 50% and 70% for silk alone and silk-nHAp composite groups after 21 days, respectively. Despite the faster release kinetics of BMP-2 from silk-nHAp compared to silk particles, the trend was linear. Moreover, BMP-2 loaded silk-nHAp showed more potential for inducing osteogenesis from bone mesenchymal stem cells (BMSCs) in comparison to BMP-2 loaded HAp particles or silk scaffolds (Ding, ZZ et al., 2016).
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ACCEPTED MANUSCRIPT In a study, a BCP consisting of HAp/β-TCP (60/40% ratio) and rhBMP-2/SF microsphere was developed which were then integrated into a BCP/rhBMP-2/SF composite (Chen, Liang et al., 2014). Based on SEM, HAp particles showed a needle-like structure, while β-TCP had hexahedral-like morphology. The rhBMP-2/SF had the mean diameter of 398.7 ± 99.86 nm with
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loading capacity of 4.53 ± 0.08 %. For pore formation, different concentrations of sodium
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chloride (NaCl; 50, 100, 150, and 200 w/v) were applied. it was observed that using NaCl with
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50, 100 and 150 % (w/v) concentrations had no effect on the structure materials during the desalination process; while, 200% NaCl easily damaged and degraded into particles or powder.
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Moreover, rhBMP-2 showed a dual-phase release profile, with both fast and slow rates, from the
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blended composites during 28 days. In the first three days, a burst and the fast release of protein (about 47.3 ± 3.1%) were observed. Afterwards, the cumulative release was about 72.2 ± 4.6%
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detected between days 3 to 14, and about 90.4 ± 5.3 between days 14 and 28 (Chen, Liang et al.,
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2014). The addition of organic materials into inorganic phases exhibits many advantages, such as increasing degradability, promoting migration and recruitment of osteoblast cells on the surface
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of the materials and increasing rates of mineralization.
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The efficacy of BCP/rhBMP-2/SF in a sheep lumbar fusion model was also evaluated (Chen,
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Liang et al., 2015). For this purpose, BCP and rhBMP-2/SF microspheres were blended to form BCP/rhBMP-2/SF composites that were implanted into the disc spaces of 30 sheep at the levels of L1/2, L3/4 and L5/6, randomly. The growth factor exhibited an initial burst release about 39.1 ± 2.8% during the first 4 days, and then had sustained release over 28 days (cumulative release rate ~ 81.3 ± 4.9 %). After implantation, the BCP/rhBMP-2/SF showed significantly better histological aspects such as in semi-quantitative CT scores, fusion rates, histologic scores and fusion stiffness in bending in all directions than the BCP or BCP/rhBMP-2 study groups. The
41
ACCEPTED MANUSCRIPT results recommended that using low doses of rhBMP-2 in the structure of SF microspheres improved bone fusion in sheep via BCP constructs (Chen, Liang et al., 2015). Despite the beneficial characteristics of controlled release systems in biomedical fields, the important parameters for mimicking the natural process of bone repair remain challenging to
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emulate. In many experiments, a single growth factor with controlled release kinetics is used; however, developing a system with the ability to control the release of multiple growth factors is
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more useful for bone regeneration (Kolambkar et al., 2011). Therefore, attempts have been
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pursued to develop delivery systems containing multiple growth factors for the synergistic effect on bone healing (Chen et al., 2010; Farokhi et al., 2016b; Shah et al., 2011).
(Bhattacharjee et al., 2016b). The 4-methacryloyloxyethyl trimellitate
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rhBMP-2 and TGF-β
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The electrospinning method was used to produce nanofibrous NSF/PCL for dual delivery of
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anhydride (4-META) was used to modify the NSF-PCL matrix and the nHAp were deposited on them by electrodeposition at two separate potentials (3V and 5V). The strong ionic interaction
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between nHAp and 4-META promoted the deposition of nHAp on the NSF-PCL matrix. The rhBMP-2 and TGF-β with a 1:1 ratio were loaded on the surfaces of the scaffold. After 4 weeks,
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rhBMP-2 and TGF-β exhibited about 20.02% and 20.93% release kinetics from NSF-PCL
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scaffolds, respectively. It seemed that the covalent interaction between the growth factors and HAp nuclei reduced the release kinetics. This structure had many favorable properties for bone tissue engineering because it contained SF with RGD motifs with the ability to induce cellular attachment
and
proliferation,
calcium phosphate
with high potential in promoting the
proliferation of osteoblasts, and the ability for dual growth factor delivery. The results also revealed that the prepared structures increased ALP production, intensified mineralization and improved the expression of bone-related genes. Random actin-stress fibers with dense cell 42
ACCEPTED MANUSCRIPT colony deposits were observed across the nanofibers loaded with dual growth factors via confocal laser microscopy. However, these outcomes were lower in the single growth factorloaded scaffolds, which confirmed the potential utility of dual growth factor delivery in bone tissue engineering applications (Bhattacharjee et al., 2016b).
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Similarly, chemical deposition method was used for preparing nHAp particles. Electrospun PCL nanofibers were prepared with different contents of nHAp (0%, 25% and 50%) (Bhattacharjee et
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al., 2016c). NSF was then grafted on the nHAp-PCL composites. It was observed that initial
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addition of nHAp had a positive effect in increasing the mechanical strength of NSF grafted PCL nanofibers. However, the higher tensile strength, toughness, and ductility was detected in those
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matrices containing 25% nHAp. Although the samples containing 50% nHAp showed higher mechanical properties and toughness than NSF-PCL nanaofibers without nHAp, but it was
M
weaker than samples containing 25% nHAp. In addition, rhBMP-2 and TGF-β were coupled to
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the scaffolds by using carbodiimide coupling. The release rate of rhBMP-2 and TGF-β was about
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21.34% and 22.6% after 4 weeks, respectively. These low release rates may be related to the strong interactions between the functional groups of growth factors with active binding sites of
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SF. In the first 3 days, a burst release profile of both growth factors was observed, which may be
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due to the presence of non-conjugated or physically conjugated growth factors (about 22%). After 3 days, the remaining growth factors that were covalently bonded to the scaffolds were slowly released during 4 weeks. The fabricated scaffolds containing both growth factors provided optimal microenvironments for cellular migration, proliferation, differentiation, and enhanced expression of bone specific markers (Bhattacharjee et al., 2016c). The cross-talk between signaling of these growth factors and different signaling cascades such as Wnt, Hedgehog, Notch, MAP, and FGF play important roles in triggering the differentiation of
43
ACCEPTED MANUSCRIPT osteoblasts and consequently, bone formation (Chen et al., 2012). As mentioned earlier, multiple biomolecules can help to improve the bone healing process. In addition to growth factor delivery, other biomolecules such as some drugs would be useful in bone tissue engineering applications. Recently, silk–HAp films were prepared using casting method for incorporation of antidrugs
like
clodronate
(non-nitrogenous
bisphosphonate)
and
alendronate
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osteoporotic
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(nitrogenous bisphosphonate) (Hayden et al., 2014). Biological aspects of THP-1 human acute
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monocytic leukemia cell line-derived osteoclasts and human mesenchymal stem cell-derived osteoblasts such as calcium deposition, metabolic activity and ALP production on the prepared during 12 weeks. Clodronate-complexed scaffolds showed higher
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films were evaluated
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metabolic activity and roughness after culturing osteoblasts and co-cultures of osteoblasts with osteoclasts. Although the low dose of alendronate enhanced the metabolic activity and calcium
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deposition of osteoblasts (Figure 7), some toxicity was observed using the high doses against
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osteoclasts, osteoblasts and co-cultures. These data may result from different mechanisms of action of nitrogenous and non-nitrogenous bisphosphonates. The toxicity may be related to drug
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class. Nitrogenous bisphosphonates cause apoptosis in various cell types at concentrations
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similar to those that cause osteoclast apoptosis, and by the same mechanisms (Idris et al., 2008). Moreover, the inhibitory effect of nitrogenous bisphosphonates on mineralization is independent
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of the mechanism, which leads to cell apoptosis (Idris et al., 2008). Additionally, the ALP production was not changed using clodronate; however, alendronate decreased the ALP activity in a dose-dependent manner (Hayden et al., 2014). Generally, drugs can be loaded in CaPs such as HAp by tailoring the porosity with large surface area during synthesis (Arcos and Vallet-Regí, 2013; Vallet-Regí et al., 2007). Although high porosity may decrease mechanical strength, pores with an appropriate size can act as a host for
44
ACCEPTED MANUSCRIPT drug molecules (Arcos and Vallet-Regí, 2013). The release of drug can be controlled by altering the amount of porosity of CaP structures (Kim and Tabata, 2015). Furthermore, a homogeneous distribution of drug can be achieved in well-ordered pores (Vallet-Regí et al., 2008). In addition to the beneficial features of CaPs as drug carriers, nHAp retains an advantage as a major 3
ions that are
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constituent of natural bones. The degradation of CaP produces Ca2+ and PO4
found in high concentrations in the bloodstream. Moreover, CaPs, particularly HAp, are
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relatively insoluble at pH 7.4, thus there is no immunogenic response, and no cytotoxic
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degradation products (Bose and Tarafder, 2012).
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4.5. Silk fibroin/Hydroxyapatite for stem cell differentiation
Several tissues are important source of therapeutically relevant differentiated cells but the
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inevitable difficulties are in harvesting sufficient cells for implantation. Lineages such as neurons
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and cardiac cells, being terminally differentiated and non-regenerative, impose the biggest challenge. Among different stem cell lineages, pluripotent cells have been focus in biomedical
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applications due to high self-renewal capacity, prolonged undifferentiated options, and the ability
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to differentiate into multiple cell types under suitable stimuli (Zhao et al., 2013). Adult stem cells are other potential cells useful for tissue engineering because they can fully regenerate damaged
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tissue and improve the process of tissue repair. Recently, autologous adult stem cells, such as cardiac stem cells (CSCs), were found favorable when compared to induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) for treating myocardial infarction (MI) and chronic heart failure (CHF) (Shafiq et al., 2016). Moreover, adult stem cells do not face the challenges of using ESCs such as ethical, religious and immune rejection or the high costs of isolating and culturing iPSCs. However, iPSCs and ESCs are still good candidates for biomedical and tissue engineering applications due to their beneficial characteristics (Nori et al., 45
ACCEPTED MANUSCRIPT 2011; Song et al., 2010; Takahashi et al., 2007). In recent years, using different stem cell sources for different kinds of tissue engineering purposes has been introduced. Besides, it is more costeffective to promote the differentiation of stem cells using appropriate scaffolds without growth factors for optimal tissue repair. For example, silk/HAp composies have been introduced as a
showed
better
biological
and
physiochemical
properties
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scaffold
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potential scaffold for promoting the stem cell differentiation. Accordingly, the NSF/nHAp for
stimulating
the
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differentiation of bone marrow-derived stem cells (CBhMSC) into an osteogenic lineage in comparison to NSF electrospun scaffolds because the existence of well-dispersed nHAp over
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NSF matrix.After 24 h, the cells were attached and aligned along the axis of fibers that form
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oriented morphology. The ability of the scaffold to promote the osteogenic differentiation of CBhMSC cells were confirmed by the slight decrease in the proliferation rate and the increase in
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the expression level of ALP and osteogenic genes (Panda et al., 2014). Recently, NSF
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composites containing silk fibers and HAp was fabricated as bone substitutes (Gupta et al., 2016). The HAp was uniformly dispersed in the structure of highly porous and interconnected
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composite scaffold that formed an appropriate substrate for inducing the proliferation and
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migration of cells by preparing the suitable environment for transferring oxygen and nutrients. Moreover, the great mechanical behavior of the composite provided a supportive matrix for
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growth and differentiation of MG63 and hBMSCs cells due to high surface area/roughness and osteoconductivity. The osteogenic potential of the scaffolds were further confirmed by assessment the expression level of osteogenic genes like Col I, osteopontin, osteocalcin, and sialoprotein, ALP production, and ECM deposition (Gupta et al., 2016). Another study showed that adding NSF (10 wt%) into PLGA/graphene oxide (GO) nanofibers and
then
mineralization
in
simulated
body fluid
46
(SBF)
can significantly enhance the
ACCEPTED MANUSCRIPT hemocompatibility, protein absorption, surface hydrophilicity, and the mechanical properties. Besides, the conductivity of the electrospinning solution was increased from 0.39 to 0.78 mS/cm after adding 1 wt % GO into PLGA/NSF nanofibers that significantly decreased the fiber diameter from 321 nm to 89 nm. Moreover, biological tests confirmed that scaffolds not only
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support hMSC adhesion and proliferation, but also promote osteogenesis and ALP production
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(Shao et al., 2016c).
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Similarly, GO-HAp/SF composite was prepared by biomineralizing carboxylated GO sheets,
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blending with SF, and freeze-drying for evaluating the differentiation of MSCs. The GO-HAp (1:4)/SF composites showed significantly higher mechanical strength in comparison to HAp/SF
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scaffolds that had better compressive strength (85 MPa) and compressive modulus (938 MPa).
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The protein absorption of GO-HAp (1:4)/SF were also 1.8-fold higher than HAp/SF scaffolds. Furthermore, the GO-HAp/SF composite was able to potentially improve the adhesion and
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proliferation of MSCs, increase the expression level of osteocalcin, and differentiation of
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osteoblast cells (Wang et al., 2017).
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Aqueous-derived porous SF scaffolds were fabricated and considered as templates to deposit apatite on the pore surfaces via mixing with polyaspartic acid (PA) during processing, followed
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by mineralization with CaCl2 and Na2 HPO 4 (Kim et al., 2008). The salt-leaching method using granular NaCl with particle size of 850∼1000 μm was used to prepare the scaffolds. Moreover, the ratios of SF-PA (w/w) blends were as 100/0 (group I), 95/5 (group II), 90/10 (group III) and 80/20 (group IV). The scaffolds were then used for seeding hMSCs under osteogenic condition in the presence or absence of BMP-2 for 6 weeks. After 24h, the aqueous solution of SF/PA converted to hydrogel and formed a porous and water-stable structure. The apatite crystals were deposited using salt deposition because PA produced nucleation sites at the interfaces of the 47
ACCEPTED MANUSCRIPT hydrophobic silk structures. It was found that the increased polyaspartic content increased HAp deposition on the scaffolds. Accordingly, it is well-established that proteins containing aspartic acid-rich motifs are able to interact with calcium salts in a specific fashion (Addadi and Weiner, 1985; Fujisawa et al., 1996). It was also showed that the calcium deposition and ALPase activity
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were almost the same in mineralized silk scaffolds in the absence of BMP-2. Moreover, the
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osteogenic media, whether using BMP-2 or not, also stimulated similar ALPase activity of MSCs
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after 6 weeks in groups I, II, and III. The group IV showed higher ALPase activity in the presence of BMP-2. All the mineralized scaffolds revealed more calcium deposition with BMP-
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2, while no calcium deposition was detected in those scaffolds without mineralization (control
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groups) after 6 weeks. Based on van Kossa staining, more calcium deposition led to higher osteoconductivity of the scaffolds. Immunocytochemistry demonstrated Col I was expressed in
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all groups. Additionally, the expression of Col I was increased in groups II and III in the
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presence of BMP-2 (Figure 8). It was concluded that higher amounts of apatite in the structure of porous silk constructs along with the existence of BMP-2 increased the osteoconductivity of the
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scaffolds (Kim et al., 2008).
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Despite the effectiveness of using silk/HAp as bone constructs, the injectable form of bone
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materials is less investigated. The main drawback of bone substitutes is the lack of suitable structure for filling the irregular bone defects. To provide an injectable biomaterials with osteogenic properties, the silk-HAp composite with silk nanofibers in hydrogels was prepared. For this, an injectable system was fabricated by blending thixotropic silk nanofiber hydrogels with water dispersible silk-HAp nanoparticles to form injectable nanoscale systems. The composites had about 60 % w/w HAp content for optimally mimic the bone environment. The physical strength of the system was also improved to 21 kPa by adding of HAp that was suitable
48
ACCEPTED MANUSCRIPT for stimulating the osteo-differentiation. The osteogenesis was more enhanced by using composite hydrogels in comparison to silk nanofiber hydrogel without HAp. The silk-HAp composite hydrogel was able to treat irregular bone defects after injection (Ding et al., 2017). The main purpose of tissue engineering is to develop functional materials with suitable
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biocompatibility that highly mimics the characteristics of the target tissue. For this, using engineered biomaterials is of interest due to rational design, adequate biocompatibility and tunable
structural properties
and
functionality.
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biodegradability,
Recombinant silk
fusion
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proteins are an example of engineered materials that have these features. Mineral-binding sequences are rich in acidic residues (aspartate, glutamate, phosphoserines) that can increase the
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net negative charge of engineered materials. These sequences can interact with positively
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charged HAp nanocrystals (Addison et al., 2010). Recently, an organic-inorganic hybrid system was developed based on genetically engineered spider silks. The organic phase of spider silk
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inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT)15 maintained the
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stability of the scaffold and facilitated different modes of processing; while, the inorganic domain of SF VTKHLNQISQSY (VTK) for HAp binding was involved in regulating the
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osteogenesis and biomineralization. The VTK peptide localized on recombinant fusion protein
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was able to provoke the calcification but it did not any effect in improving the physical strength of the recombinant structure. It was also observed that adding VTK peptide on both c-terminal and N-terminal sides of recombinant silk could significantly enhance the formation of crystalline HAp. All the prepared constructs was able to provoke the attachment, proliferation, and differentiation of hMSCs (Dinjaski et al., 2017).
Salt-leached SF scaffolds were also prepared
followed by functionalization and mineralization in CaCl2 ethanol solution/K 2 HPO 4 aqueous solution. The scaffolds were then mineralized by immersing in the simulated body fluid (SBF)
49
ACCEPTED MANUSCRIPT for 4 days. The authors claimed that the duration of soaking in SBF would be decreased by using this method that consequently protect the SF-nHAp scaffold from degradation during the mineralization process. The microspores of nHAp with a size range of 100 to 250 nm were formed on the surface wall of SF. Incorporating of nHAp particles with the SF scaffolds
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significantly improved the compressive strength and stiffness of SF (Liu, H. et al., 2015).
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Furthermore, the cDNA microarray demonstrated changes in 23 calcium ion binding genes and
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17 cell adhesion genes of BMSCs seeded on nHAp-SF scaffolds compared with bulk SF constructs. Among these genes, about 15 genes corresponded to cellular adhesion and 20 genes
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were attributed to calcium ion binding were decreased in expression level. Based on these
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observations, the cultured BMSCs on nHAp-SF scaffolds had round shapes due to less surface adhesion. The nHAp-SF scaffolds showed higher osteoinductivity in vitro, significantly higher
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ALP activity on day 14, and more osteogenesis related to signaling pathway genes on day 7
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compared with the SF scaffolds. Moreover, the nHAp-SF scaffolds demonstrated higher capability to induce bone regeneration in cranial bone defects than bulk SF scaffolds. It was also
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shown that soluble IL-1a factor and biomolecules on the surface of BMSCs were played an
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important role in provoking osteogenesis. Upon interaction between BMSCs and nHAp, BMSCs regulated the activity of IL-1a by IL IR2. This study helped to understand better the possible
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mechanism of osteoinductivity by nHAp (Liu, H. et al., 2015). Electrospun SF nanofibers containing nHAp and BMP-2 was prepared as bone construct with the ability to induce the differentiation of BMSCs (Niu et al., 2017). The nHAp particles were successfully dispersed in SF fibers. The increase in the content of HAp resulted in more aggregations of this particles in the SF matrix. The mechanical properties of the composite containing 20% HAp such as tensile strength, yield strength and Young’s modulus was almost
50
ACCEPTED MANUSCRIPT two-fold more than pure SF scaffolds. BMSCs showed significantly more ALP activity on SF/HAp/BMP-2 scaffolds compared with SF scaffolds. Thus, SF/HAp/BMP-2 scaffolds were considered as a potential structure in inducing osteogenesis (Niu et al., 2017). It is assumed that synergistic effect of HAp and BMP-2 improve the osteogenic capacity of the composite scaffold.
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This issue was also confirmed by Lu (Lu et al., 2015) or Liu (Liu, Z. et al., 2015) claimed that
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degradation of SF can increase the exposure of HAp to the cells during time.
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Similarly, the bioactivity and the sustained release rate of BMP-2 was preserved in the nHAp-
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embedded SF scaffold by adjusting the ratio of loaded BMP-2 on SF and nHAp. The suitable BMP-2 release behavior inside the structure of SF-nHAp scaffold increase the expression level
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of Col I, ALP and Runx2 protein that resulted in higher osteo-conductivity. Based on the results
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from in vitro and in vivo, the release of BMP-2 had positive effect in inducing osteogenesis of
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the scaffold (Ding, Zhaozhao et al., 2016).
Shen et al. fabricated porous SF/nHAp composites containing BMP-2 and SDF-1 loaded SF
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microspheres to synthesis the cell-free scaffold. Laminar jet break-up technology was used for
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SF microsphere preparation which provide optimal condition for high encapsulation efficiency (Shen et al., 2016). It was reported by Wenk et al. that preparing SF microspheres using this
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method can preserved the bioactivity of IGF-I and controlling the release rate of growth factor in a sustained manner (Wenk et al., 2008). SDF-1 showed a rapid release rate during the first few days, while it presented a sustained profile in the following three weeks. The scaffold significantly provoked the osteogenic differentiation and recruitment of BMSCs (Shen et al., 2016). In another study, electrospun SF nanofibers containing HAp were fabricated for periosteum regeneration (Ding, X. et al., 2016). Periosteum is the main source of osteo-progenitor cells 51
ACCEPTED MANUSCRIPT around bone, therefore, this tissue plays a crucial role in the initial phase of bone formation and regeneration (Tiyapatanaputi et al., 2004; Zhang, X. et al., 2005). The HAp particles were seen on the surfaces of SF nanofibers containing 10%, 20%, and 30% HAp. Moreover, more HAp were observed on the scaffolds with higher content of HAp. Some aggregations of HAp particles
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were observed on the surface of interconnected pores of SF/HAp nanofibers which may be
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responsible for increasing the diameter of 10%, 20%, and 30% SF/HAp nanofibers. The Young's
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modulus of the scaffolds was also increased with the increase in the content of HAp. Moreover, the results demonstrated that SF scaffolds containing 30% HAp induced the metabolic activity of
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rBMSCs more than those scaffolds containing 0% HAp after 4 days. SF scaffolds containing
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30% HAp promoted the proliferation of rBMSCs and upregulated mRNA levels of ALP and Runx2 more than the other groups. Moreover, rBMSCs showed higher levels of ALP expression
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when cells were cultured on SF scaffolds containing 30% HAp in comparison to 0% HAp-SF
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scaffolds. Higher calcium deposition was found by Alizarian red assay when 30% HAp-SF scaffolds were used versus 0% HAp-SF scaffolds (Ding, X. et al., 2016). Therefore, it was
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suggested that SF scaffolds containing 30% HAp were potent structures to promote bone
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formation due to their ability to induce mineral deposition and consequently, cell differentiation.
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Co-cultures of MSCs between ligament and bone cells on suitable sections of hybrid silk scaffolds induced cellular differentiation into fibrocartilage lineage (He, P. et al., 2012). The section of the scaffold for seeding osteoblast cells was pre-coated with HAp. Real-Time PCR revealed that both osteoblast and fibroblast cells maintained their normal phenotype during coculture. However, co-cultured BMSCs differentiated toward the fibrocartilage lineage. The cocultured BMSCs on the hybrid SF scaffolds exhibited up-regulation of Col II, Sox9, aggrecan and fibrocartilage gene markers in comparison to mono-cultured BMSCs. The data were
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by
histological
analysis
and
immunohistochemistry.
The
immunohistochemistry
staining revealed that aggrecan was the most prominent marker in the enthesis (the connective tissue between tendon or ligament and bone). In addition, the trilineage co-culture was supplemented with 10 ng/mL TGF-β3 and the fibrocartilaginous differentiation of BMSCs by the
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TGF-β3-treated group was similar to those groups without TGF-β3. No calcium deposition was
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process of mineralization in chondrocytes (He, P. et al., 2012).
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observed in the TGF-β3-treated group and it was suggested that TGF-β3 could postpone the
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In another study, leaching paraffin microsphere with the modified temperature gradient-guided thermal-induced phase separation (TIPS) methods were applied to fabricate SF/HAp scaffolds
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with the potential to mimic the integrated trilayered osteochondral substrates (Ding et al., 2014).
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Based on the Micro-CT analysis, the consecutive tri-layer structure consisting a chondral (top) layer, intermediate layer, and bony (bottom) layer was observed in the integrated scaffold. Using
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TIPS method resulted in the formation of oriented microtubularlike porous structure in the
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chondral layer and the paraffin-sphere leaching led to formation of an interconnected macropore structure in the bony layer. The chondral and bony layers had porosities of 85.30% ± 1.80% and
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90.25% ± 2.05%, respectively. Adipose-derived stem cells (ADSCs) were used under
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chondrogenic (with TGF-β1 and IGF) and osteogenic (with dexamethasone) conditions in chondral and bony layers of the scaffold in order to evaluate the potential of these cells for differentiation to cartilage and bone lineages. In the chondral layer which was previously seeded with ADSCs, chondrocyte-like cells were observed, which were enclosed by ECM components, especially collagen type II and glycosaminoglycan (GAG). The amounts of ECM components were also increased during 21 days in the chondral layer. Moreover, chondrogenic-related genes were also up-regulated during culture. Generally, it was proposed that ADSCs seeded chondral
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ACCEPTED MANUSCRIPT layer
provided
suitable
environments
for
inducing
chondrogenesis
under
chondrogenic
conditions. The ADSCs also showed higher expression of osteogenic related genes and bone ECM components such as Col I and calcium. Therefore, osteogenic differentiation also could be promoted under osteogenic conditions. Consequently, the pores were filled with more cartilage-
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related and bone-related matrices as the culture time increased, resulting in an increase in the
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compressive elastic modulus (Ding et al., 2014). The results showed promise for the use of novel
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3D integrated SF scaffolds in osteochondral tissue engineering in vitro. However, whether ADSCs can differentiate into cartilage and bone in the corresponding chondral and bony layers
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of integrated scaffolds needs to be further investigated through in vivo implantation. The
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isolating role of the intermediate layer needs to be confirmed in vivo. Other important studies
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using SF/HAp for stem cell differentiation are listed in Table 5. Totally, primary osteoblast cells are the mostly used cells for bone regeneration; however, their
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limited number and dedifferentiation properties might limit applications. Using potential stem
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cells can overcome these drawbacks (Wang et al., 2006). Stem cells have been largely investigated for creating functional tissues and organs but it is still at infant stage. Bone marrow
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is the favored source of MSCs for both experimental and clinical application. The biological
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features of stem cell such as self-renewal and differentiation capacity are regulated by ECM components. ECM also serves as a supporting structure for various growth factors and biomolecules which can act as structural support with the ability to control signal transduction (Kundu and Kundu, 2010). The suitable morphological aspects of SF/HAp composites make it an appropriate structure for stimulating the differentiation of stem cell into desired lineage and thus promoting the regeneration of defected tissue.
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ACCEPTED MANUSCRIPT Table 5. Simultaneous use of SF/HAp composites and stem cell for bone tissue engineering. Material
Processing method
Structure and properties of the scaffold
Cell
Key findings
Ref.
SF1 /HAp2
Direct-write assembly
Formation of 3D scaffold with an interpenetrating gradient network of pore channels, suitable mechanical integrity
hMSCs3 /
Formation of complex network of hMSCs matrix within the 3D scaffold by hMSCs and hMMECs based on histological analysis, observation of vascular-like structures only in hMSCs and hMMECs coculture groups
Sun et al., ( )2012
Freeze-drying/ co-precipitation
Formation of dense flakelike HAp crystals on the surface and the pore walls of the 3D silk structure
BMSCs5
Increasing the surface roughness and proliferation of BMSCs by incorporating HAp in SF matrix, improved in vitro differentiation of BMSCs toward osteoblast cells
Jiang et al., ( )2013
High and uniform coupling of nHAp on the surface of nano-HAp/SF sheet
MMCs6
Good cellular proliferation and attachment, higher ALP 7 and bonespecific osteocalcin production under osteogenic condition after 14 days of cell cultured on nanoHAp/SF sheets compared with controls
Tanaka et al., ( )2007
Graft polymerization
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SF/nHAp
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SF/HAp
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hMMECs4
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ACCEPTED MANUSCRIPT Altered surface chemistry, increased surface roughness and stiffness after incorporating HAp in the structure of 3D scaffold
SF/Gelatin/HAp
Salt-leaching/
Formation of 3D scaffold with
hMSCs
Higher surface osteoconductivity of SF scaffold by using HAp and suggesting HAp microparticles as nucleation sites for directing mineralization, enhancing the formation of trabecular structure, increasing the mechanical behavior and conductivity of tissue grafts
Bhumiratana ( )et al., 2011
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Salt-leaching
BMSCs/
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co-precipitation
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SF/HAp
MC3T3E1
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approximately 52 ± 5.5% porosity and pore size of 52.3 ± 5.5
Ti/SF/nHAp
Coprecipitation / electrostatic spray
Induction of HAp nanocrystals aggregation in 56
MG-63
Higher Sinlapabodin ( )et al., 2016 proliferation rate of mouse MC3T3E1 cell line seeded on the scaffolds with perfusion flow rate of 1 ml/min than the static culture, increased differentiation of BMSCs into osteoblast using perfusion flow rate of 3 ml/min compared to 1 and 5 ml/min flow rates, statistically higher Ca/P ratio, ALP activity, and calcium contents in flow rate of 3 ml/min compared to other flow rates Better cellular attachment and osteogenic differentiation
Lin et al., ( )2015
ACCEPTED MANUSCRIPT the presence of SF, intact crystalline structure of HAp in the structure of SF/nHAp composites
based on ALP activity, BGP 8 contents, and Col I9 of cells in SF/nHAp compared with nHAp groups
SF/HAp
Freeze-drying
hDPCs10 / Formation of 3D structure hPDLCs11 with interconnected pores, varied pore size between 20 and 80 μm with the diameter of 65 μm
SF/PHBV12 /HAp
Electrospinning
Reduction in the tensile Young’s modulus of the composite fibers with the increase in the content of the nHAp and SF phase from 2% to 5%
-
Park et al., ( )2015
MC3T3E1
Increased cellular proliferation and attachment on composites compared with PHBV control, higher expansion and elongation of cells on the surface of fiber composites after 1 day of culturing, incomplete elongation of cells seeded on PHBV scaffolds
Paşcu et al., ( )2016
PMSCs13
Secretion of calcium crystals by exposing PMSCs into osteogenic medium, secreting extracellular matrix when cell
Jin et al., ( )2014
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Remaining mostly SF scaffold after 4 weeks at the defected site, supporting cellular attachment on SF scaffold and producing extracellular matrix, inducing new bone formation in the edges of defects by using SF fibers
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deposition
SF/HAp
Salt-leaching
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cultured on SF/HAp scaffolds, increasing tissue regeneration by using PMSCs seeded SF/HAp scaffolds in radius segmental bone of rabbit Silk fibroin, 2 Hydroxyapatite,3 Human bone marrow derived mesenchymal stem cells, 4 Human mammary microvascular endothelial cells, 5 Bone marrow derived mesenchymal stromal cells, 6 Marrow mesenchymal cells,7 Alkaline phosphatase, 8 Bone gla protein, 9 Collagen type I, 10 Human dental pulp cells, 11 Human periodontal ligament cells, 12 Polyhydroxybutyrate–polyhydroxyvalerate, 13 Placenta-derived mesenchymal stem cells
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5. Conclusions and future perspectives
Silk scaffolds are biocompatible structures with tunable properties in terms of morphology,
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degradability, and conformation that make them useful candidates for tissue engineering applications. Recently, investigations have been performed to better understand the structure and
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processing of silk-based structures that broadened the applications of different types of silk in
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regenerative medicine. The unique characteristics of silk such as exceptional mechanical and
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physical behavior, and versatile processability to form different structures make it potentially useful for ligament, bone and musculoskeletal tissue engineering. Silk has many suitable
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properties for various applications; however, it has some limitations. For example, the properties
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of silk protein, as a natural polymer, are different based on species source. Moreover, inconsistence degumming processes and different fabrication methods may produce silk structures with various properties if the process is not tightly controlled. It is well established that genetically modified silk proteins may overcome the mentioned limitations pending scale up and cost efficiencies. In order to enhance the potential of silk-based scaffolds for bone tissue engineering applications, this polymer can be blended with other materials such as HAp to enhance osteogenic potential of the scaffolds. HAp has a similar chemical structure to
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ACCEPTED MANUSCRIPT mineralized bone of human tissue. This ceramic material demonstrates affinity to hard tissues. More investigations are to be carried out in order to understand better the application of nanoscale constructs that can be degraded over time and replaced by endogenous hard tissues. It is also necessary to focus on modifying the surfaces of biomaterials with different biomolecules There are many
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with the aim of improving their biological behavior and interfacial functions.
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studies that describe the benefits of using nHAp-SF scaffolds for bone tissue engineering
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applications in vitro and in vivo. However, there is no comprehensive study that shows the adverse effects of using these structures. It is essential to develop effective constructs for clinical
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use despite advantageous properties of SF/HAp in this area. It is noteworthy that many
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investigations have confirmed the potential of SF scaffolds to induce osteogenesis in vivo. Most of these studies are performed on small animal models, thus the results may not fully be
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extended for human use. More investigations are needed to be performed in the future to identify
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the requirements of using silk-based scaffolds in clinical trials and constructing suitable
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Acknowledgments
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commercialized structures for bone tissue regeneration.
This work is supported by Pasteur Institute of Iran
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Disclosure statement
No potential conflict of interest was reported by the authors
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ACCEPTED MANUSCRIPT Figure legends Figure 1. Applications of (SF) as a biomaterial based on Scopus database. (A) SF-related articles (B) SF used for tissue engineering (C) SF used for bone tissue engineering (D) Silk/
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hydroxyapatite (SF-HAp) composites. (E) to (H) remarkable studies related to parts (A) to (D).
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Figure 2. Silk/ hydroxyapatite (SF-HAp) composites for bone tissue engineering.
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Figure 3. Different types and applications of bioceramics.
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Figure 4. Ultrastructure of bone tissue. The extracellular matrix of bone is highly specialized. The organic part of bone is composed of collagen, noncollagenous proteins (NCPs) and lipids.
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The inorganic phase of bone mainly consists of hydroxyapatite (HAp) nanocrystals. This mineral phase is crystallized along the long axis of collagen fibrils and integrates together in a tight
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hierarchical organization. These crystals associate with the collagen fibres, making bone hard
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Figure 5. Signaling pathway of osteoblast cells seeded on hydroxyapatite (HAp) scaffold. Adhesion of osteoblast cells on HAp substrates can trigger some signaling pathways like
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extracellular regulated kinases (ERK). Due to interaction of integrin receptors with such
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extracellular matrix of bone e.g. fibronectin, focal adhesion kinase (FAK) as an intracellular signaling cascades can be activated. This causes activation of ERK pathway and then expression of some osteoblast specific genes e.g. osteocalcin and collagen type I. Figure 6. The role of Notch signaling pathway in osteoblast differentiation of stem cells. a). Notch signaling blocks RUNX2 expression and consequently inhibition of osteocalcin and osteopontin. b). Blocking of Notch signaling cascade after seeding of stem cells on silk fibroin based scaffold. 73
ACCEPTED MANUSCRIPT EGF: epidermal growth factor, LNR: Lin-12/Notch repeats, HD domain: heterodimerization domain, TMD: transmembrane domain, RAM: RBPjκ association module, ANK: ankyrin, NLS: nuclear localization sequences, TAD: transactivation domain, PEST: proline (P), glutamic acid (E), serine (S),
threonine (T) rich motif, NICD: Notch intracellular domain, ADAM: a
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HEY: HES-related with YRPW motif.
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Figure 7. Effects of bisphosphonates on calcium deposition. Calcium content of films was measured after 4, 8 and 12 weeks of culture and expressed as mean ± SD. Top left: osteoblasts
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Bottom left: co-cultures cultured on clodronate-loaded films. Bottom right: co-cultures cultured on alendronate-loaded films. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Copyright © 2014 Elsevier
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B.V. Reprinted with permission from (Hayden et al., 2014).
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Figure 8. Immunohistochemical staining of collagen type I. Extracellular matrices (*) stained
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positive for Col I within the scaffold pores. The Col I matrix covered the entire mineralized aqueous-based silk lattice (#) after 6 weeks of culture with BMP-2 (100 ng/ml; b, d, f, h) or
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without BMP-2 (a, c, e, g). Copyright © 2008 Elsevier B.V. Reprinted with permission from
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composites
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Mehdi Farokhi, Fatemeh Mottaghitalab, Saeed Samani, Mohammad Ali Shokrgozar, Subhas C. Kundu, Rui L. Reis, Yousef Fattahi, David L. Kaplan PII: DOI: Reference:
S0734-9750(17)30121-0 doi:10.1016/j.biotechadv.2017.10.001 JBA 7159
To appear in:
Biotechnology Advances
Received date: Revised date: Accepted date:
12 June 2017 12 September 2017 4 October 2017
Please cite this article as: Mehdi Farokhi, Fatemeh Mottaghitalab, Saeed Samani, Mohammad Ali Shokrgozar, Subhas C. Kundu, Rui L. Reis, Yousef Fattahi, David L. Kaplan , Silk fibroin/hydroxyapatite composites for bone tissue engineering. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jba(2017), doi:10.1016/j.biotechadv.2017.10.001
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ACCEPTED MANUSCRIPT Silk fibroin/hydroxyapatite composites for bone tissue engineering Mehdi Farokhia,* , Fatemeh Mottaghitalab b, Saeed Samanic, Mohammad Ali Shokrgozara, Subhas C Kundud, Rui L. Reisd, Yousef Fattahie, David L. Kapland National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
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a
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Sciences, Tehran, Iran
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Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical
Department of Tissue Engineering & Applied Cell Sciences, School of Advanced Technologies
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in Medicine, Tehran University of Medical Sciences, Tehran, Iran d
3Bs Research Group, Headquarters of the European Institute of Excellence on Tissue
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Engineering and Regenerative Medicine, University of Minho, AvePark - 4805-017 Barco, Guimaraes, Portugal Department of pharmaceutical nanotechnology, Faculty of pharmacy, Tehran university of
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medical sciences
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USA
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Department of Biomedical Engineering, Tufts University, 4 Colby St, Medford, MA 02155,
Corresponding author:
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Mehdi Farokhi, E-mail address: [email protected]
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ACCEPTED MANUSCRIPT Abstract Silk fibroin (SF) is a natural fibrous polymer with strong potential for many biomedical applications. SF has attracted interest in the field of bone tissue engineering due to its extraordinary
characteristics
in
terms
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elasticity,
flexibility,
biocompatibility
and
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biodegradability. However, low osteogenic capacity has limited applications for SF in the orthopedic arena unless suitably functionalized. Hydroxyapatite (HAp) is a well-established
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bioceramic with biocompatibility and appropriate for constructing orthopedic and dental
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substitutes. However, HAp ceramics tend to be brittle which can restrict applications in the repair of load-bearing tissues such as bones. Therefore, blending SF and HAp combines the useful
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properties of both materials as bone constructs for tissue engineering, the subject of this review.
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Keywords: Silk fibroin, Hydroxyapatite, Scaffold, Bone tissue engineering
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Contents 1. Introduction 2- Bioceramics for bone tissue engineering applications
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2.2. Signaling responses of osteoblast cells to hydroxyapatite
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2.1. Hydroxyapatite
3. Structure and characteristics of silk fibroin
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3.1. Osteogenic signaling responses of silk fibroin
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3.2. Silk fibroin scaffolds for bone tissue engineering
4. Silk fibroin/hydroxyapatite for bone tissue engineering
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4.1. Silk fibroin/hydroxyapatite composites for bone tissue engineering
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4.2. Silk fibroin/nano-hydroxyapatite composites for bone tissue engineering 4.3. Silk nanofibers containing hydroxyapatite for bone tissue engineering application
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4.4. Silk fibroin/hydroxyapatite for drug delivery in bone tissue engineering
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4.5. Silk fibroin/hydroxyapatite for stem cell differentiation 5. Conclusion and future perspective
References
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Acknowledgment
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ACCEPTED MANUSCRIPT 1. Introduction Bone has an intrinsic capacity for regeneration; however, strategies to improve the repair capacity of bone are still needed in many circumstances. Bone regeneration depends on the type of defect. For example, about 10% of fractures are non-union that do not heal spontaneously and
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lead to inappropriate bone tissue regeneration and delayed unions. The probability of normal bone healing in these fractures is influenced by age, metabolic condition, and severity of the
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trauma. Autologous bone or autografts are still considered the clinical “gold standard” and the
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most effective method for bone regeneration. This approach promotes bone formation by direct bone bonding (osteoconduction) and induces local stem cells to differentiate into bone cells
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(osteoinduction) without any immune responses (Zhang et al., 2014). About 2.2 million bone
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grafts are performed annually (Giannoudis et al., 2005) and are met some limitations due to the limited supply, morbidity of the donor site and are associated with more than 50% failure in
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specific regions (Bajaj et al., 2003; Clavero and Lundgren, 2003). Therefore, many attempts have
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been made to develop suitable bone constructs consisting of natural or synthetic biomaterials in order to increase bone regeneration capacity and avoid the above limitations. Some polymers are
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usually good candidates for this purpose due to their biocompatibility, biodegradability, tunable
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structural properties and ease of fabrication. Degradable polymers are important for these types of repairs to avoid barriers to full regeneration of bone, and are categorized to natural polymers such as alginate, chitosan, collagen, silk and synthetic polymers including polydioxanone (PDS), polycaprolactone (PCL), polyglycolic acid (PGA), polylactic acid (PLA), and poly (lactic-coglycolic acid) (PLGA) (Lee and Yuk, 2007; Rezwan et al., 2006). Recently, more attention has been paid to natural polymers in comparison to synthetic polymers for bone tissue engineering applications due to their unique biocompatibility, versatility and biodegradability. These types of
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ACCEPTED MANUSCRIPT polymers provide well-patterned structures consisting of ligands that can bind to cell surface receptors or provide accessible enzymatic degradation sites (Swetha et al., 2010). Silk fibroin (SF) as a natural protein polymer has potent characteristics for tissue engineering applications, as a biocompatible, biodegradable, and low immunogenic polymer (Farokhi et al., 2014a; Farokhi
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et al., 2016a; Mottaghitalab et al., 2013; Omenetto and Kaplan, 2010; Sugihara et al., 2000).
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Moreover, SF possesses extraordinary properties for stimulating bone repair; for example, the
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fibrous structure of SF is mostly similar to collagen type I (Col I). The amorphous links between the β-sheets in the structure of SF act as sites for deposition of HAp nanocrystals because it can
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mimic the anionic structure of noncollagenous proteins (NCPs) (Marelli et al., 2012). However,
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there are also some reports believed that β-sheets crystal can alone act as a nucleation site for deposition of HAp nanaocrystals (Vetsch et al., 2015). Furthermore, this polymer is introduced
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as a potential bone construct due to appropriate mechanical behaviors and structural integrity
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(Mandal et al., 2012; Sofia et al., 2001). The ultimate tensile strength of SF extracted from Bombyx mori (B. mori) is 300–740 MPa (Cunniff et al., 1994; Shao and Vollrath, 2002) and it
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also has great breaking strain and high toughness more than synthetic fibers such as Kevlar (Du
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et al., 2011; Gosline et al., 1999). Upon searching the Scopus database for studies related to tissue engineering and bone tissue engineering applications with SF from 2005 to Sep 2017, the
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number of peer-reviewed original articles per year and the first ten scientific study fields that attract the most attention and the highest numbers of published articles were assembled (Figure 1). Various studies have also recently focused on the applications of natural polymers as well as SF in bone repair. Although some of these polymers have intrinsic capacities for the bone regeneration, this is insufficient to repair large bone defects. For this reason, incorporating
5
ACCEPTED MANUSCRIPT particles into the polymeric matrix can be advantageous to improve the mechanical behavior and tune the topographical features of the scaffold in order to mimic the structure of natural bone (Abadi et al., 2010; Aboudzadeh et al., 2010; Shokrgozar et al., 2010; Swetha et al., 2010). HAp with similar properties to bone tissue and suitable biocompatibility has been introduced as an
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appropriate bioceramic phase which can be incorporated in different polymeric phases (Ginebra
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et al., 2006a; Ginebra et al., 2006b). This is osteoconductive (Cai et al., 2009), non-toxic (He, X.
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et al., 2012), low in immunogenicity with the ability to directly bind to hard tissues (Azadi et al., 2016; Chen, Li et al., 2015; Li et al., 2011). HAp is the most stable calcium phosphates (CaPs)
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under neutral or basic pHs in the biological fluids and humid conditions (Aoki, 1991). Thus, this
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ceramic is usually formed in the fluids containing phosphates in the bodies of vertebrates that has the pH between 7.2 to 7.4. The chemical structure of HAp is mostly similar to inorganic
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compounds existed in the bone matrix. High-affinity of HAp to bone tissue makes it a good bone
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replacement in comparison to allografts and metallic implants (Bauer et al., 1991). There are two common structures of HAp including monoclinic and hexagonal constructs. In humans and
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animal bones, HAp is mainly in the hexagonal structure and thus synthetic HAp is the
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predominant form of CaP used in biomedical fields (Figueiredo et al., 2010). However, HAp is weakly dispersed in aqueous media which leads to aggregation. Thus, it is necessary to
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functionalize the surface of HAp (Kim, H.H. et al., 2014). In addition, poor fracture toughness of HAp (Azadi et al., 2016; Li et al., 2011; Wang et al., 2004b), migration of nanoparticles from implanted sites (Azadi et al., 2016; Wang et al., 2005) and challenging processability (Azadi et al., 2016) restrict the clinical applications of this biomaterial. In order to overcome these limitations, many efforts have been directed to develop hybrid structures containing HAp and biopolymers or organic molecules (Li et al., 2008; Li et al., 2011; Wang et al., 2004b; Wang et
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ACCEPTED MANUSCRIPT al., 2005). Several natural polymers, including collagen, gelatin, chitosan, silk, and chondroitin sulfate have been used in combination with HAp to enhance bone regeneration (He, J. et al., 2012). Many structures based on SF/HAp composites alone or in combination with bioactive molecules and stem cells for bone tissue regeneration are schematically represented in Figure 2.
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In this review, we focused on recent studies that used SF/HAp as bone constituents. This review starts with a brief description about bioceramics and CaPs, peruses structural properties of HAp
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and SF, and continues with descriptions of the signaling responses. Finally, the combination of
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SF/HAp for orthopedic applications will be discussed in detail.
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2- Bioceramics for bone tissue engineering applications
Ceramics are solid materials consisting of non-metallic, inorganic substances in two crystalline
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and non-crystalline (amorphous) forms (Hench and Jones, 2005). Ceramic and glasses have
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different applications in various fields of human health and in industry, such as diagnostic instruments, thermometers, eyeglasses, tissue culture flasks and fiber optics for endoscopy
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(Dorozhkin, 2010). During past decades, bioceramics have been used for repairing bone defects
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(Eshtiagh-Hosseini et al., 2007) and hard tissues (Hench and Jones, 2005). Bioceramics are included in the ceramic materials category that usually have non-crystalline structures (Baino
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and Vitale-Brovarone, 2014). The main classes of bioceramics and their subset groups and some applications are presented in Figure 3. Nontoxic and biologically inactive ceramics such as Al2 O 3 and ZrO 2 as bioinert bioceramics (Park, 2009) are usually used for joint prostheses because of their mechanical properties. Bioactive glasses and glass-ceramics directly bond to hard tissues and support new bone formation (Baino and Vitale-Brovarone, 2014). In the biomaterial industry, CaPs are used
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ACCEPTED MANUSCRIPT predominantly as bone replacements due to their biocompatibility, low density, chemical stability (Fathi et al., 2008), chemical and crystallographic similarity to bone mineral (Ding, ZZ et al., 2016), and integration into living tissue similar to the healthy bone (Dorozhkin, 2010). These inorganic materials include diverse types from bioresorbable tricalcium phosphate (TCP)
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to bioactive and biostable HAp (Fathi et al., 2008). Most CaP bioceramics are based on HAp,
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beta-tricalcium phosphate (β-TCP), alpha-tricalcium phosphate (α-TCP) or biphasic calcium
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phosphate (BCP) containing a mixture of α-TCP+HAp or β-TCP+HAp (Dorozhkin, 2010). The major physical and structural properties of CaPs and inorganic components of bone and tooth are
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listed in Table 1.
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ACCEPTED MANUSCRIPT Table 1. Major properties of calcium phosphates and inorganic phases of adult human calcified tissues (Dorozhkin, 2012; Ducheyne et al., 2015; Park, 2009; Ratner et al., 2004; Wnek et al., 2008).
Ca/P mola r ratio
Ca(H2 PO4 ) 2 .H2 O
0.5
Densit y (g/cm 3 )
Z1
Lattice structure
a= 5.6261(5), b= 11.889(2), c= 6.4731(8) Å,
2.23
2
T riclinic
a= 7.5577(5), b= 8.2531(6), c= 5.5504(3) Å,
~18 (1.14)
0.0–2.0
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0.5
pH stability range in aqueous solution s at 25 °C
2
T riclinic
~100
~17 (1.14)
2
2.32
4
Monoclinic
~80
~0.088 (6.59)
2.0–6.0
2.89
4
T riclinic
~100
~0.048 (6.90)
1
2.58
Dicalcium phosphate anhydrous (DCPA or DCP), mineral mone tite
1.0
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CaHPO4 .2H2 O
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Dicalcium phosphate dihydrate (DCPD), mine ral brushite
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Ca(H2 PO4 ) 2
~100
Solubilit y at 25°C, g/L (logKa )
transforms into MCPA
α= 98.633(6)°, β= 118.262(6)° ,γ= 83.344(6)° Monocalcium phosphate anhydrous (MCPA or MCP)
Transformatio n temperature (°C)
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Formula
Lattice parameter s
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Monocalcium phosphate monohydrate (MCPM)
Composition
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Che mical name
CaHPO4
α= 109.87(1)°, β= 93.68(1)°, γ = 109.15(1)° a= 5.812(2), b = 15.180(3), c= 6.239(2) Å, β= 116.42(3)°
1.0
a= 6.910(1), b = 6.627(2), c= 6.998(2) Å,
Sinter ~300
α =96.34(2)°, β= 103.82(2)°, γ= 88.33(2)°
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ACCEPTED MANUSCRIPT O ctacalcium phosphate (O CP)
Ca8 (HPO4) 2(PO4)4 .5H2 O
1.33
a= 19.692(4), b= 9.523(2), c = 6.835(2) Å,
2.61
1
T riclinic
2.86
24
Monoclinic
An unstable transient intermediate
~0.0081 (96.6)
5.5–7.0
~0.0025 (25.5)
3
a= 12.887(2), b= 27.280(4), c= 15.219(2) Å, β= 126.20(1)°
β-Tricalcium phosphate (βTCP)
β-Ca3 (PO4 ) 2
Amorphous calcium phosphate s (ACP)
Cax Hy (PO4 ) z.nH2 O, n = 3–4.5, 15–20% H2O
1.22.2
Calciumde ficie nt hydroxyapatit e (CDHA or Ca-de f HA) 7
Ca10-x (HPO4 ) x (PO4 ) 6x (OH) 2-x (0 < x < 1)
1.51.67
Hydroxyapatit e (HA, HAp or O HAp)
Ca10 (PO4 ) 6 (OH) 2
a=b= 10.4183(5), c= 37.3464(23 ) Å, γ = 120°
3.08
21 4
Rhombohedr al
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1.5
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1.5
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α-Ca3 (PO4 ) 2
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α-Tricalcium phosphate (αTCP)
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α= 90.15(2)°, β = 92.54(2)°, γ = 108.65(1)°
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a= 9.84214(8), b = 2a, c = 6.8814(7) Å, γ= 120° (monoclinic )
~1125
~0.0005 (28.9)
3
transforms into α-T CP
-
-
-
-
-
-
-
~700
5
~5–12 6
~0.0094 (385)
6.5–9.5
~250, monoclinic to hexagonal Sinter ~1000
~0.0003 (116.8)
9.5–12
-
~0.0002
7–12
transforms into β-T CP
3.16
4
Monoclinic
2
Hexagonal
a=b= 9.4302(5), c =6.8911(2) Å, γ = 120° (hexagonal) Fluorapatite
Ca10 (PO4 ) 6 F2
1.67
a=b=
3.20
10
2
Hexagonal
ACCEPTED MANUSCRIPT (FA or FAp)
(120)
9.367, c = 6.884 Å,
O xyapatite (O A, O Ap or O XA) 8
Ca10 (PO4 ) 6 O
1.67
a =b = 9.432, c = 6.881 Å, α = 90.3°, β = 90.0°,γ = 119.9°
~3.2
1
Hexagonal
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Te tracalcium phosphate (TTCP or Te tCP), mine ral hilgenstockite
Ca4 (PO4 ) 2 O
2.0
a= 7.023(1), b = 11.986(4), c= 9.473(2) Å, β= 90.90(1)°
3.05
4
Monoclinic
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γ = 120°
Ename l
-
1.63
a=9.441, c= 6.880 Å
-
De ntine
-
1.61
a= 9.421, c= 6.887 Å
-
Bone
-
1.71
a= 9.41, c= 6.89 Å
-
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~0.0007 (38-44)
3
-
-
-
-
-
-
-
-
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3
-
-
Number of formula units per unit cell, 2Stable at temperatures above 100°C, 3Cannot be precipitated from aqueous solutions, Per the hexagonal unit cell, 5Cannot be measured precisely. However, the following values were found: 25.7± 0.1 (pH 7.40), 29.9 ± 0.1 (pH 6.00), 32.7± 0.1 (pH 5.28). The comparative extent of dissolution in acidic buffer is: ACP >> α-TCP >> β-TCP > CDHA >> HA > FA, 6Always metastable, 7Occasionally referred to as “precipitated HA” (PHA), 8The existence of OA remains questionable
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1
-
~0.087 (369)
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ACCEPTED MANUSCRIPT 2.1. Hydroxyapatite The term ‘apatite’, given by Wener in 1788, refers to inorganic compounds with similar structure but different compositions (Kokubo, 2008). These minerals have lattice parameters a=0.9432 nm and c=0.6881 nm and crystallize into hexagonal rhombic prisms (Park, 2009; Park and Lakes, or
pure
HAp
is
introduced
by
Ca10 PO4 6 OH 2 or
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Synthetic
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2007).
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Ca10z HPO4 z PO4 6z OH 2z [0 < z < 1] as a general formula. The chemical behavior of HAp depends on stoichiometry status, which is basic for stoichiometric apatite (z = 1) and acidic
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for the others (Faria et al., 2008).
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Inorganic materials coexist with organic molecules in a well-ordered manner in some tissues such as bone, cartilage and skin (Wang et al., 2004a). Natural bone is a nanocomposite
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(Andiappan et al., 2013; Bhattacharjee et al., 2016b) composed of organic components (~90%
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type I collagen, ~5% NCPs,~2% lipids by weight), water and inorganic CaP (Boskey, 2013;
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Šupová, 2015; Wu et al., 2014; Zhao et al., 2009). Recently, NCPs have attracted attention due to their effect on bone tissue behavior, including cell proliferation and attachment, HAp and Ca2+
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binding and degree of mineralization (Young et al., 1992). Osteocalcin, sialoprotein, alkaline
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phosphatase (ALP) and matrix Gla protein are the most abundant NCPs that are a main focus of research; however, it is not well-understood that which of these proteins may have a crucial role in bone behavior (Figure 4). It is reported that the combination of these proteins may influence bone functions synergistically more than each of them alone. It should be noted that some features such as gender, age, health status and ethnicity affect the proportion of these proteins in bone structure of an individual patient. Therefore, the quantity and quality of NCPs can influence the characteristics of bone tissue in terms of structure and function (Boskey, 1989, 2013).
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ACCEPTED MANUSCRIPT The inorganic phase of bone consists of HAp nanocrystals (about 70 wt% (Andiappan et al., 2013; Wang et al., 2004a)) dispersed in Col I (Andiappan et al., 2013; Wang et al., 2004a). This mineral phase is crystallized along the long axis of collagen fibrils (Hu et al., 2015) and integrates together in a tight hierarchical organization (Figure 4) [28]. The X-ray diffraction
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(XRD) spectrum of sintered bone and mineral apatite showed that HAp and fluorapatite are the
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main minerals in bone and teeth. For many years, it was assumed that the inorganic parts of
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calcified tissues were HAp. However, studies focused on synthetic carbonated apatite and biological apatite showed that the bone mineral does not consist of pure apatite but contains
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carbonated apatite (Kokubo, 2008) and some ions such as K +, Mg2+, Na+, F- and CO 3 2- (Li et al.,
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2008; Webster et al., 2004). Moreover, bioactivity of bone mineral is higher than synthetic HAp (Fathi et al., 2008). Therefore, non-stoichiometric HAp substituted with suitable ions during
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synthesis would be useful to mimic the function of bony apatite and osteoinductive activity when
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compared to pure HAp (Ren et al., 2009).
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Different methodologies have been used to prepare HAp-based products in micro- or nanoscale quantities. Co-precipitation (Cao et al., 2013; Ming et al., 2014a), chemical precipitation (He, X.
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et al., 2012; Kweon et al., 2011; Wang et al., 2002), solid-state reaction (Ming et al., 2014a;
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Wang et al., 2002), hydrothermal methods (Cao et al., 2013; Kweon et al., 2011; Ming et al., 2014a; Wang et al., 2002), sol-gel synthesis (Cao et al., 2013; Kweon et al., 2011; Ming et al., 2014a) and wet mechanochemical routes (Cao et al., 2013; Nemoto et al., 2001; Wang et al., 2002) are common methods used for HAp synthesis. Microemulsions, spray drying and plasma melting are also applied for synthesizing spherical HAp powders (Liu, J. et al., 2013). Structural features of the synthesized powders are mainly related to the corresponding preparation method (Kweon et al., 2011). Control of size distribution and morphology of nano-sized HAp powders
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ACCEPTED MANUSCRIPT (nHAps) is critical for tissue engineering and drug delivery systems, which are often ignored in some methods (He, X. et al., 2012; Huang et al., 2014). The different methods suitable for
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synthesis of nHAps are summarized in Table 2.
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ACCEPTED MANUSCRIPT Table 2. Different methods for nHAps fabrication and main properties based on the materials used (El Briak-BenAbdeslam et al., 2008; Gedanken, 2004; Sadat-Shojai et al., 2013; Sasikumar and Vijayaraghavan, 2008, 2010; Tas, 2000). Raw materials
Advantage s
Disadvant ages
Synthetic conditions
Shape categori es 1
Solid-state reaction
Few Ca and PO4 containing chemicals
Relatively simple
Weak precise control over product features
900~1300° C, usually with water vapor flowing
1, 3
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Diverse morpholog y
Usually low purity Variable Ca/P
Irregular shape
e.g.: CaHPO4 .2H2 O + CaO
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(Mechanosynt hesis)
Few Ca and PO4 containing chemicals
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Mechanochem ical
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Low cost
Heterogene ous compositio n of powder
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Using organic templates to control morpholog y
Product features
Very high crystallinity
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Mass production
Usually micronWide
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Presynthesized CaP salts
Size-Size distribut ion
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Synthesis method
CaCO3 +NH4 H2 PO4 Ca3 (PO4 )2 +Ca( OH)2
Simplicity
Cannot be exploited for biomimetic synthesis -
Reproducib ility
Reaction activated
Production of nanocrystal line alloys and ceramics
Ca(OH)2 +P2 O5
Welldefined structure of powder
CaO+Ca(OH)2 +P2 O5
Mass production
Wet or dry medium
by mechanical milling Effective factors: reagents, milling medium, atmosphere , rotational speed, diameter of milling
Low cost
15
1, 2, 3
NanoUsually wide
Diverse morpholog y Very high crystallinity Low purity Nonstoichiomet ric Ca/P
ACCEPTED MANUSCRIPT ball
Using organic templates and molecules to control morpholog y Low cost
Variable chemical reagents
Molecular level mixing of reactants
High cost of some raw materials
Improving chemical homogeneit y
Possible impurities (CaO, Ca2 P2 O7 , Ca3 (PO4 )2 , CaCO3 )
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Sol-gel
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Ca(OEt)2 , Ca(NO3 )2 , P(OEt)3 , P2 O5 , (NH4 )2 HPO4
Low temperature for various steps Stoichiome tric structure
Hydrothermal
Variable reagents Wet
Using organic modifiers to control
Long aging to complete reaction Thermal treatment to get pure HAp Expensive equipment for high temperatur
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Diverse morpholog y Frequently low crystallinity
Aqueous medium
Variable purity
pH 7-12
Usually high cost
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Method to modify a prepared HAp
Usually nanoVariable
Atmospher ic pressure
pH 6-7 for acidic phases (DCPD, DCPA)
1, 3, 5, 7
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Other CaP phase (DCPDc, DCPA TCP)
Distinct method to convert a CaP into HAp
1, 2, 3, 4, 5, 7, 8, 11
VariableVariable
Conversion depends on pH, T and other ions
Aqueous or organic media
Stoichiomet ric Ca/P 1, 2, 3
NanoNarrow
Diverse morpholog y Variable crystallinity Variable purity
Drying at ~100150°C
Stoichiomet ric Ca/P Higher bioresorbab ility than chemical precipitatio n
Heat treatment at ~300900°C
High
Diverse morpholog y
Usually high purity
Aging at room temperatur e
Aqueous solution
Usually nonstoichiomet ric Ca/P
Variable crystallinity
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Few chemical substances
Usually at room temperatur e
Phase impurities (DCPA a, OCPb )
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Hydrolysis
Precise control over conditions necessary to minimize defects
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The simplest route
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Frequently few Ca and PO4 containing reagents
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Chemical precipitation
1, 2, 3, 4, 5, 6, 8, 9
Nano and micronWide
Frequently needle-like Usually
ACCEPTED MANUSCRIPT
growth, and aging Simplicity
-
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Low temperatur e
Usually high purity Stoichiomet ric Ca/P
1, 2, 3, 5
NanoNarrow
Minimal agglomerati on
Frequently low crystallinity
Mild conditions
Variable purity
Aqueous environme nt
Heterogene ous
Using organic modifiers to control or change process
reactions between liquid and solid reactants
Usually low cost
17
Frequently needle-like
Different surfactants
Chemical reactions activated by powerful ultrasound radiation
More uniform, smaller and purer crystals
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e.g.: Ca(OH)2 + Ca(H2 PO4 )2
Increasing rate of crystal growth
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Few Ca ad PO4 containing chemicals
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Agglomerat e-free
Sonochemical
100~200°C (1~2 MPa), 300~600°C (1~2 kbar)
High cost
More effective control over morpholog y and microstruct ure
Very high crystallinity
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Many raw materials
irregular
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Emulsion
Poor control over morpholog y and size distribution
temperatur e and pressure
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Regulating more predictable reactions as crystal nucleation,
e and pressure
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Nonstoichiomet ric Ca/P
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chemically prepared HAp, other calcium phosphates, seeding large crystals
1, 2, 3
NanoUsually narrow
Diverse morpholog y (usually needle-like) Variable crystallinity Usually high purity Variable Ca/P
ACCEPTED MANUSCRIPT
Inexpensiv e raw materials Relatively simple Well chemical homogeneit y of powder
front
used
e.g.: Ca(NO3 )2 , (NH4 )2 HPO4
combustio n of a fuel and oxidiser at elevated
Diverse morpholog y (usually irregular) Variable crystallinity Usually high purity
HAp decomposit ion into αTCP at high temperatur e of flame
Heat sources: flame or furnace
1, 2, 3, 5
Secondary aggregation s leading to decrease in specific surface area
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Usually low cost
1
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Easily scaled-up for continuous production
Usually nanoWide
Variable Ca/P
temperatur es
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Variable chemical materials
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Pyrolysis
1, 2, 3, 5
solution via spontaneou s
Particle morpholog y dependent on fuel
Fairly low temperature Usually low cost
Particles formed from aqueous
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Organic fuels (glycine, urea, sucrose, citric acid, succinic acid)
Single step operation
Mixed phases due to uncontrolla ble high temperatur e reaction
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Oxidants (Ca(NO3 )2 , (NH4 )2 HPO4 and HNO3 )
Quick production of powder
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Few raw materials
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Selfpropagating combustion synthesis
Nano particles embedde d in micron aggregate sVariable
Diverse morpholog y High crystallinity Variable purity Usually stoichiomet ric Ca/P
1-Irregular sphere, 2-(micro and nano)Sphere or ball-shaped, 3-rod- or needle-shaped or strip-like, 4-plate or sheet, 5-self-assembled or bundled nanorod, 6-dendelion or rosette, 7-bundled sheets or leaves, 8-Flower, 9-porous microsphere or mesoporous sphere, 10-Bowknot, 11-Dumbbell a
Anhydrous dicalcium phosphate, b Octacalcium phosphate, cDicalcium phosphate dihydrate
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ACCEPTED MANUSCRIPT 2.2. Signaling responses of osteoblast cells to hydroxyapatite Dynamic interactions between osteoblasts and their extracellular matrix (ECM) regulate their behavior and differentiation potential. Understanding the principal ECM cues promoting osteogenic differentiation can be pivotal for both bone tissue engineering and regenerative
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medicine. HAp can trigger signaling cascades; however, the mechanism of action is not well understood. The bioactivity of any material, here defined as the ability of osteoblasts to adhere
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and spread upon it, depends on these signaling mechanisms (Kokubo, 1991; Zambuzzi et al.,
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2011a). The signaling pathway involved in osteoblasts adhering on HAp scaffolds was recently evaluated (Gemini‐ Piperni et al., 2014). Adhesion of osteoblast cells on HAp substrates actives
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such signaling pathway that leads to activation of protein kinase C (PKC), protein kinase A
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(PKA), Adducin 1 (ADD1) and vascular endothelial growth factor (VEGF).
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Moreover, kinase activation leads to increased phosphorylation of Ser-421 in histone deacetylase 1 (HDAC1), a cyclin-dependent kinase 5 (CDK5) substrate. The surface chemistry of HAp is
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crucial for promoting the differentiation of osteoblasts via phosphorylation (Gemini‐ Piperni et
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al., 2014). In response to HAp, other signaling molecules such as extracellular regulated kinases (ERK) and SOX9 are also activated (Song et al., 2008b). The integrin receptors mediate the
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interaction between osteoblast cells cultured on HAp substrates with some domains of ECM proteins that can activate the cytoskeletal and intracellular signaling cascades like focal adhesion kinase (FAK). FAK is associated with Shc protein activating Ras that stimulates the ERK signaling cascade (Miyamoto et al., 1996). Activated ERK affects the expression level of different osteoblast genes such as osteocalcin and Col I (Figure 5) (Mimori et al., 2007; Xiao et al., 2002). Therefore, ERK signaling is essential for cytoskeletal rearrangement and intracellular signaling (Zambuzzi et al., 2011b). Various signaling cascades are also triggered upon the 19
ACCEPTED MANUSCRIPT interaction between HAp and osteoblast integrins such as BMP/Smad (Liu, H. et al., 2013), Wnt (Thorfve et al., 2014), TGF-β, MAPK, and Notch signaling pathways (Lü et al., 2014). 3. Structure and characteristic of silk fibroin
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Silk protein is found in the gland of silk producing arthropods such as spiders, silkworms, mites,
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scorpions, and bees. During metamorphosis, silk is spun to fibers. Cocoon silk, as a Food and
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Drug Administration (FDA) approved biomaterial for some medical applications, is produced by silkworm B. mori (Kaplan et al., 1994). B. mori silk is composed of two major types of proteins,
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silk fibroin (SF) and sericin. SF is located in the core of B. mori silk fibers and consists of light (~26 kDa) and heavy (~390kDa) chains; while, sericin coats the SF chains. Silk fibers contain
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about 70-75% SF filaments and 25-30% sericin. The primary sequence of sericin consists of
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some repeat motifs (Sutherland et al., 2010). The structure of the SF heavy chain is similar to
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amphiphilic block copolymers because it contains several hydrophobic and hydrophilic domains or blocks. The hydrophobic blocks of SF are comprised of highly conserved (GAGAGS) and less
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conserved (GAGAGX) repeats (where X is either valine or tyrosine) that are responsible for the
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crystalline structure and β-sheet conformation of SF. The hydrophilic part of SF core consists of short and non-repetitive segments in comparison with hydrophobic parts (Bini et al., 2004;
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Kaplan and McGrath, 2012). Silk fibers have useful mechanical properties, including a tensile strength of 0.5 GPa, breaking elongation of 15% and 62,104 J kg−1 toughness (Li et al., 2012). The Young’s modulus of silk obtained from forced silking of silkworm in the laboratory is about 12.4–17.9 GPa, with an ultimate tensile strength of 360–530 MPa and 18-21% breaking elongation (Pérez‐ Rigueiro et al., 2001). Silk fibers have been used for many years as sutures and for textile engineering due to their high toughness (Nova et al., 2010). Some studies have reported comparable mechanical behavior for silk filaments with nylons (Lucas, 1964). These 20
ACCEPTED MANUSCRIPT unique mechanical characteristics make SF a useful polymer candidate for load-bearing applications, such as for bone tissue engineering in composite systems with HAp. 3.1. Osteogenic signaling responses to silk fibroin
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While silk scaffolds do not inherently contain any cell-specific signaling epitopes in the primary
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sequence, silk-based scaffolds provide suitable substrates for supporting the proliferation and
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adhesion of mesenchymal stem cells (MSCs) (Mandal and Kundu, 2009; Mauney et al., 2007). The ability of hydrolyzed silk protein to improve the ALP activity of osteoblast cells and their
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osteogenic differentiation has also been reported (Seo et al., 2011). Moreover, B. mori silk induces mineral deposition from rat bone marrow stromal cells (BMSCs) during 12 days of
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culture in vitro (Nikbakht Dastjerdi, 2006). This study demonstrated that by using silk, no
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dexamethasone additives are required for pre-osteogenic factor, although the compound can
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accelerate the outcomes. Dexamethasone can trigger Wnt/β-catenin signaling dependent Runx2 expression (Langenbach and Handschel, 2013). Furthermore, other signaling factors like Wnt,
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Notch, fibroblast growth factor (FGF), bone morphogenetic protein/Transforming growth factor
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beta (BMP/TGFβ), insulin-like growth factor (IGF), and platelet derived growth factor (PDGF) can regulate the osteogenic differentiation of stem cells on silk-based substrates (Midha et al.,
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2016). Based on pharmacologic and molecular studies, Notch signaling was responsible for differentiation of mesenchymal progenitor cells into osteoblast cells. Silk protein provoked the upregulation of some osteoblast differentiation markers (e.g. ALP, osteorix and Runx2) by inhibiting Notch signaling (Jung et al., 2013). Additionally, the mass of trabecular bone in the skeletogenic mesenchyme of mice limbs was significantly increased by inhibiting the Notch signaling pathway in adolescent mice. Severe osteopenia was observed in mice during aging period when they were without mesenchymal progenitors in their bone marrow. The inhibition of 21
ACCEPTED MANUSCRIPT osteogenic differentiation by Notch signaling is triggered by Hes and Hey proteins, which can suppress Runx2 activity through physical interaction (Figure 6a) (Hilton et al., 2008). Thus, for optimal osteogenic induction of silk scaffolds, suppression of the Notch signaling pathway is advantageous (Figure 6b).
properties
of
SF
such
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outstanding
as
biocompatibility,
biodegradability,
low
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The
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3.2. Silk fibroin-based scaffolds for bone tissue engineering
immunogenicity, and suitable processability make it a useful scaffold system for various tissue
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engineering applications, including bone constructs. Another advantage of SF as a bone substitute is its high mechanical strength (Kundu et al., 2013; Mottaghitalab et al., 2015b;
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Nourmohammadi et al., 2017); it can bear the force produced in vivo (Nazarov et al., 2004).
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These properties of SF have attracted many researchers to use this biomaterial in bone tissue
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engineering applications. Recent investigations on the SF-based scaffold as a bone construct are summarized in Table 3.
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Table 3. SF based scaffolds indicating types of material used for blending, methods for
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applications.
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preparation of matrices, cell types used and their main findings in bone tissue engineering
Material
Processing method
Scaffold structure
Cell
Key findings
Ref.
TSF1 / PLA2
Electrospinning
Nanofibers
mMSCs 3
Inducing the proliferation and attachment of MSC, increasing the rate of ALP4 production, osteogenesis, and bone mineralization
(Shao et al., 2016a)
Electrospinning
Nanofibers
-
Stimulating the early bone
(Bhattacharjee
(90:10)
NSF5 /PCL6
22
ACCEPTED MANUSCRIPT et al., 2016a)
Increased proliferation, adhesion and differentiation of hMSCs confirmed by ALP production, transcription of RUNX2, and expressions of osteocalcin and type1 collagen
(Singh et al., 2016)
MG-63
Capability of the modified scaffold for alveolar bone repair due to outstanding biofunctionality
(Sangkert et al., 2016)
3D scaffold
MC3T3-E1
Increased wettability, suitable porosity, high mechanical strength, and good biocompatibility by incorporating diopside nanopowders into the SF matrix
(Ghorbanian et al., 2013)
3D scaffold
HGF12
Formation of a porous scaffold with appropriate pore size for cell infiltration, improved compressive strength and water uptake, and decreased porosity
(Teimouri et al., 2015)
Electrospinning
Nanofibers
SF/Decellularized pulp/Collagen/Fi bronectin
Freeze-drying
3D scaffold
SF/Diopside
Freeze-drying
hMSCs 9
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SF7 /CMC8
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formation, infiltration of implants and formation of strong bonds at the interface of boneimplant, activating the initial immune reaction which reaching to normal after 4 weeks postimplantation
SF/CS 10 /Nano ZrO2 11
Freeze-drying
23
ACCEPTED MANUSCRIPT after adding zirconia Electrospinning/ Freeze-drying
3D scaffold
SF
Casting
Film
hOBs 15
Providing an optimal microenvironment for the biological behavior of osteoblast cells due to acceptable porosity, chemical and physical properties with ability to sustain the release rate of VEGF16 within 28 days, stimulating the new bone formation at the site of injury 10 weeks after implantation
(Farokhi et al., 2014b)
BMSC17
More efficient delivery of BMP218 after immobilizing this biomolecule on the surface of biomaterial in comparison to soluble delivery, retained in vitro biological activity of BMP-2 based on its potential to induce the osteogenic markers of BMSCs
(Karageorgiou et al., 2004)
OEC19 /hOBs
Extracellular matrix deposition by hOBs during 4 weeks, enhancing expression of osteogenic markers, retained functionality of both cell types in provoking the formation of capillary like structures and
(Fuchs et al., 2009)
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SF/CaP13 /PLGA14
SF
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Mesh
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ACCEPTED MANUSCRIPT differentiation of hOBs Salt leaching
3D scaffold
hMSC
Increased bone formation in calvarial critical size defects in mice during 5 weeks by using tissueengineered bone implants, detecting less bone formation by using stem cell loaded SF scaffolds and scaffolds alone
(Meinel et al., 2005a)
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SF
Tussah silk fibroin, 2 Polylactic acid, 3 Mouse mesenchymal stem cells, 4 Alkaline phosphatase, 5 Nonmulberry silk fibroin, 6 Silk fibroin, 7 Poly(ε-caprolactone), 8 Carboxymethyl cellulose, 9 Human mesenchymal stem cells, 10 Chitosan, 11 Zirconia, 12 Human gingival fibroblast, 13 Calcium phosphate, 14 Poly (lactic-co-glycolic acid), 15 Human osteoblast cell, 16 Vascular endothelial growth factor, 17 Bone marrow mesenchymal stem cells, 18 Bone morphogenetic protein-2, 19 Outgrowth endothelial cells
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4. Silk fibroin/hydroxyapatite for bone tissue engineering
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4.1. Silk fibroin/hydroxyapatite composites for bone tissue engineering Bone tissue engineering offers suitable constructs for restoring and repairing bone defects (Ma et
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al., 2005). Mimicking the structure and composition of natural tissues by engineering biomaterials, such as developing inorganic-organic composites, has grown in interest and focus
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over the past decades. Some typical ceramics such as bioactive glass, HAp, β-TCP, and calcium
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citrate are suitable candidates for bone tissue engineering applications (Chouzouri and Xanthos, 2007; Maquet et al., 2004). The most common organic material used as bone constructs is collagen. This protein polymer can be obtained from some animals (e.g. skin of pig and cow) or generated through transgenic techniques. However, collagen is not cost-effective, provoke immune responses, and retains a risk for contamination (Du et al., 2009). For these limitations, some researchers have tried to use other natural biomaterials such as silk for bone tissue engineering. 25
ACCEPTED MANUSCRIPT Blending silk with HAp enhanced crystal formation of HAp along the c-axis and the coordinative effect on the structure and properties between SF and HAp was found during the composite film fabrication. The nucleation of HAp could enhance the molecular orientation and crystallinity of SF (Du et al., 2009). Recently, Jo et al. evaluated alginate/HAp/SF composites as a bone
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replacement (Jo et al., 2017). Four weeks after implantation, the rate of bone formation in the
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defected site was significantly higher using alginate/HAp/SF scaffold than alginate and control
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(unfilled defects) groups. The suitable biocompatibility of alginate/HAp/SF scaffold led to less immunogenic response or formation of giant cells around the degraded grafts. Significantly
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lower expression level of tumor necrosis factor-alpha (TNF-α) was also observed in
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alginate/HAp/SF group compared with alginate and alginate/HAp groups; while, the osteogenic markers such as Runx2, osteoprotegerin, and FGF-23 showed higher expression rate. The
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alginate/HAp/SF scaffold was introduced as a potential bone construct for repairing calvarial
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defects in rat model that was stably biodegraded without provoking the immunogenic responses
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(Jo et al., 2017).
The homogenous dispersion and interfacial bonding between polymeric matrix and HAp is still
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challenging in fabricating composites. The interfacial bonding between HAp and the organic
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phase is dependent to the density of contact numbers and the chemical interactions. In this context, pretreatment of SF fibrils with an alkali increased the exposed active sites on the surface that further improved the interactions between the organic and mineral phases. The alkali pretreatment effected the crystallographic characteristics of HAp in comparison to pretreatment with proteinase K, a protease that digests the SF. Applying both pretreatments enhanced the exposed active sites to interact with the mineral phases. It was suggested that SF surface fibrils were mostly disentangled by alkali, while the enzymatic pretreatment was effective in
26
ACCEPTED MANUSCRIPT disentanglement of the SF blocks into smaller fragments (Wang et al., 2007). An increase in number of contact sites enhanced the interactions between organic and inorganic phases to improve microhardness (>50%) (Wang et al., 2007; Wang et al., 2004b; Wang et al., 2005). However, the remaining sodium ions from the alkali reagent may have unexpected side effects
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on crystallographic properties of HAp due to replacement of calcium with sodium ions. During the past decade, many efforts have been attempted to develop hybrid biomaterials based
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on organic and inorganic materials to mimic the structure and function of natural biomaterials.
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The existence of organic materials like polymers and proteins in the structure of hybrid materials regulate the nucleation of inorganic crystals, and enhance the physicochemical properties of the
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microstructure. As mentioned above, adding HAp to SF improve the biological and mechanical
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properties of SF. HAp/SF composites are so similar to the natural bone structure that mimic the inorganic and organic phases of native bone tissue. Thus, combination of inorganic and organic
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materials in a unique hybrid composite can improve the flexibility, mechanical strength, and the
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toughness of the bone tissue scaffolds. Some applications of SF/HAp composites for bone tissue
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engineering are listed in Table 4.
Table 4. The composites based on SF/HAp, methods for preparation. Types of cells, properties
Materials SF1 /HAp2
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of the matrices and findings are reported for bone tissue engineering. Processing method
Cell
Structural properties
In vitro/in vivo key findings
Ref.
MAPLE3
SaOs2
Exhibition of 1540 cm−1 amide II, 1654 cm−1 amide I, 1243 cm−1 amide III peaks in the FTIR spectrum of SF, and observing of
Improved proliferation of cells with normal morphology after 72h culture on SF and HAp/SF films
(Miroiu et al., 2010)
27
ACCEPTED MANUSCRIPT 1027 cm−1 only for HAp and HAp/SF composite groups SF/HAp
Layer solvent casting/Freeze drying
BMSCs 4
Decreasing pore average size with increasing SF content from 5% (350 ± 67 μm) to 10% (112 ± 89 μm), more uniform pores in the scaffolds containing 5% HAp
SF/HAp
Casting
-
Exhibition of silk II conformation in the secondary structure of SF films, no impact of HAp content in the solution on the structure of SF films, Significant effect of HAp content on rheological behavior of SF solution
-
(Ming et al., 2014b)
NSF5 /HAp
Electrospinning
Osteoblast
Higher crystallinity and thermal stability of electrospun NSF/HAp scaffold compared with pure NSF and NSF/HAp formed by soaking method
Higher cell viability on electrospun NSF/HAp scaffold than pure NSF and NSF/HAp formed by soaking method
(Sekar et al., 2016)
MG-63
Scaffolds exhibited 90.5– 96.1% porosity with 100–300 µm pore size, better mechanical
Suitable for increasing the proliferation and adhesion of cells, higher efficacy of CS/SF/nHA composite porous
(Qi et al., 2014)
(Gholipourmalekabadi et al., 2015)
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No variation in the proliferation rate of BMSCs on HAp/SF and no toxicity, good in vivo biocompatibility of SF containing 5% HAp, no significant increase in the average number of lymphocytes in long and short periods
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and soaking
CS 6 /SF/nHAp
Salt fractionation
28
ACCEPTED MANUSCRIPT behavior of tricomponent scaffold compared with bi-component scaffolds
scaffold than other groups
-
Coprecipitation
-
Characteristics of primary HAp crystals: needlelike shape, 20-50 nm long, and ~10nm wide, Chemical interactions between amino group of chitosan or amid bands of SF with Ca2+ ions
SF/COL7 /HAp
Particulate leaching
MG-63
Displaying porous structure with adjustable macropores with high interconnectivity, uniform dispersion of SF and collagen within the scaffold
Significantly higher biocompatibility at days 7 and 14 compared with day 3, suitable features of scaffold in inducing the cellular proliferation, and potential of the scaffold to stimulate the cellular infiltration and migration
(Mou et al., 2013)
PVA8 /SF/HAp
Freezing and thawing
No agglomeration and uniform distribution of silk in the composite hydrogel, high impact of silk on elastic modulus of PVA-HApSilk composite hydrogel
-
(Zhang et al., 2012)
5
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-
(Wang and Li, 2007)
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CS/SF/HAp
Silk fibroin, 2 Hydroxyapatite, 3 Matrix Assisted Pulsed Laser Evaporation, 4 Bone marrow stromal cells, Non-mulberry silk fibroin, 6 Chitosan, 7 Collagen, 8 Polyvinyl alcohol
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ACCEPTED MANUSCRIPT 4.2. Silk fibroin/nano-hydroxyapatite composites for bone tissue engineering Recently, nanoparticles have attracted attention in biomedical fields due to their exceptional properties such as high surface to volume ratio, tunable structural properties in terms of surface chemistry, size and shape (Brannon-Peppas and Blanchette, 2012). Today, different applications
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are considered for nanoparticles including theranostics therapy, molecular imaging, drug delivery and cell labeling (Hassani Besheli et al., 2017; Kim, E.-S. et al., 2014; Mottaghitalab et al.,
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2015a; Mottaghitalab et al., 2017). It is also observed that the alterations in nanoscale features
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can affect cellular responses. For example, nano-patterned structures were potent in directing stem cell differentiation without the using of exogenous biochemical agents (Kim et al., 2012a;
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Kim et al., 2012b). Controlling the cellular behavior by using nano-patterned structures is a key
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application for nanotechnology in biomedical fields. In the orthopedic field, HAp nanocrystals were sintered and improved densification because of the high surface area. The high surface area
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can also increase fracture toughness and mechanical strength of HAp nanocrystals. However,
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agglomeration is the most challenging issue in using nano-powders (LeGeros, 1993). nHAp showed better bioactivity in comparison to coarser crystals that broaden its applications in tissue-
have
high
bioresorption
properties
and
similar
crystallographic
and
chemical
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nHAp
CE
engineered implants when compared to other constructs (Stupp and Ciegler, 1992). Moreover,
characteristics to the natural apatite in bone (Murugan and Ramakrishna, 2005). Composites containing nHAp induced both osteogenesis and angiogenesis by stimulating the proliferation, adhesion, and differentiation of osteoblasts; therefore, these composites are potent for bone tissue engineering applications (Kilian et al., 2008; Nageeb et al., 2012; Wei and Ma, 2004). Besides, in many studies SF protein extracted from B.mori alone or in blended with CaP has been used for bone tissue engineering. However, there is no RGD motifs or sufficient arginine 30
ACCEPTED MANUSCRIPT amino acids in the structure of SF derived B.mori to support cellular adhesion (Sen and Babu, 2004). Some reports suggesting the presence of RGD epitopes and high arginine content in the structure of non-mulberry silk fibroins (NSF) that reduced the cytotoxicity and inflammatory responses (Datta et al., 2001; Mandal and Kundu, 2008). Moreover, presence of hydrophilic
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amino acids in the structure of NSF promotes cellular attachment to the surface of the materials
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(Meinel et al., 2005b; Sen and Babu, 2004; Zhang, Y.-Q. et al., 2005). The in situ reinforcement
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of NSF-nHAp scaffold and the NSF scaffold with external nHAp deposition showed higher mechanical properties and biocompatibility in comparison to NSF-nHAp reinforced fibroin
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scaffolds. Both composite scaffolds also showed less immunogenicity by using osteoblast-
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macrophage co-culture model (Behera et al., 2017). However, no significant anti-inflammatory differences were observed through the modified scaffold indicating the native anti-inflammatory
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properties of fibroin structures. It was also observed that by culturing the macrophages on the
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modified scaffolds, the level of interleukin 1 beta (IL1-β) increased that might be related to the role of HAp (Loi et al., 2016). Similarly, nHAp/SF powders were synthesized using a co-
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precipitation method, added to SF solution in order to fabricate nHAp/SF composite material
CE
(Liu et al., 2011). These composites showed improved compressive properties. The good compressive properties were due to the uniform dispersion of nHAp/SF in SF solution, the high
AC
surface area of needle-like nHAp/SF powders, and good molecular interaction between SF molecules in the structure of nHAp/SF powder and SF matrix. The good interfacial interactions between the nHAp/SF powders and the SF matrix promoted cellular proliferation, attachment and differentiation (Liu et al., 2011). The superior biological behavior of nHAp powders when compared to micro-scaled versions provides options for many biomedical applications. Recently, nHAp particles 30-100 nm were homogeneously dispersed in a collagen/silk fibroin (Col-SF)
31
ACCEPTED MANUSCRIPT matrix and the electrostatic interaction between Ca2+ of the inorganic phase and carboxyl or amino groups of organic phase generated nanocomposites (Col-SF/HAp) with suitable structural properties and
biocompatibility.
Additionally,
the existence of SF in the structure of
nanocomposites enhanced elastic modulus in comparison to Col/nHAp (Chen, Li et al., 2014). In
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another study, it was observed that SF affected the mineralization of HAp nanocrystals. SF was
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able to significantly increase the growth of HAp nanocrystals at pH 8 in room temperature. The
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strong chemical interaction between HAp and the SF protein shifted the amide II peak from 1517-1539 cm-1 in FTIR spectra, which indicated that HAp crystals were replaced by carbonated
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HAp (Kong et al., 2004). SF not only influenced the morphological aspects of HAp crystals but
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also affected the water dispersibility of HAp. Moreover, SF surrounded HAp nanocrystals to form a layer with a negative charge to prevent aggregation of nHAp particles in aqueous solution
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(Huang et al., 2014). Previous studies also confirmed the regulatory activity of SF on
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biomineralization of calcium salts (Takeuchi et al., 2005; Wang et al., 2012). However, the distance between SF and HAp nanocrystals played an important role in the intensity of their
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interactions. At longer distances, the interactions became weaker (Zhang et al., 2013). Thus, SF
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and nHAp particle ratios are important factors in controlling the growth of nanocrystals and crystal morphology. Accordingly, regular SF nanostructures were implemented in order to
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evaluate their effect on the formation and morphology of HAp nanocrystals. SF nanospheres induced the formation of rice-like nHAp, while SF nanofibers stimulated the formation of HAp nanofibers (He, X. et al., 2012). Therefore, it was suggested that different nanostructures of SF can be considered as templates for the formation of different morphologies of HAp. Silk fibroin/sodium alginate (SF/SA) hydrogels induced the formation of HAp nanorods with rectangular column morphologies at room temperature. The strong interaction between SF/SA
32
ACCEPTED MANUSCRIPT nanofibers and Ca2+ ions in HAp had significant effect on the morphology of nanocrystals (Ming et al., 2014a). Generally, the phosphorylated motifs on the surface of SF play an important role in triggering the nucleation and formation of apatite crystals. Each of apatite crystals grew in size to form a thick mineralized layer during mineralization. Adding foreign molecules may trigger
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the initial formation of HAp crystals; however, mineral proliferation may be delayed due to
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inhibition of the growth sites (Boskey, 1998; LeGeros, 1990). Furthermore, one of the main
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challenges of using biomaterials to induce the process of bone regeneration is the bacterial resistance before complete bone repair. Thus, the biomaterials should have the ability to
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stimulate the regeneration before the adhesion of bacterial species. For this purpose, SF/nHAp
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hydrogen was modified with gold nanoparticles (AuNPs) and AgNPs in order to induce the antimicrobial effect (Ribeiro et al., 2017). The obtained results revealed that the hydrogel
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containing high concentration of AgNPs had strong capability to reduce the number of different
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gram positive and gram negative bacterial strains such as S. aureus (MSSA), S. aureus (MRSA), S. epidermidis, Escherichia coli (E. coli), and P. aeruginosa. However, the hydrogen comprising
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AuNPs did not show a linear antimicrobial effect against S. epidermidis. The hydrogels
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containing AuNPs≥0.5% had suitable antibacterial activity toward MRSA and P. aeruginosa; while, those containing AuNPs≥0.1% reduced the number of MSSA and E. coli. It seems that the
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higher surface activity of AgNPs in comparison to AuNPs might be responsible for this observation (Ribeiro et al., 2017). Taken together, the biological HAp existed in the natural bone has nanometric thickness and length with nanoscopic plate-like or rod-like shape. Thus, applying nHAp would be a good bone substitute with high regeneration capacity that highly mimic the mineral phase of
natural bone
tissue (Vallet-Regi and González-Calbet, 2004). nHAp possess great characteristics compared
33
ACCEPTED MANUSCRIPT with HAp such as high sinterability and densification as a result of high surface area that lead to improved mechanical strength and toughness (LeGeros, 1993). Additionally, the appropriate biocompatibility, osteoconductivity, the ability to promote the angiogenesis, and inducing the attachment, migration, and differentiation of osteoblast make it useful for bone regeneration
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(Hassan et al., 2016). However, nHAp is fragile, less flexible with intrinsic hardness that make it
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difficult to shape into the desired form which limits its applications in repairing load-bearing
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bone defects. In order to hamper the aforementioned drawbacks, nHAp in blended with other polymers such as SF to form nanocomposites with high osteoconductivity suitable for orthopedic
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surgery (Sun et al., 2011).
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4.3. Silk nanofibers containing hydroxyapatite for bone tissue engineering application
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The high surface area of nanofiber scaffolds makes them useful for protein absorption and
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binding to cell membrane receptors. The absorbed proteins can change the structural conformation of the nanoscale scaffolds along with generating more binding sites that make
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them more suitable for tissue engineering. Electrospinning is one of the most useful techniques
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for fabricating nanofibrous scaffolds. Electrospun mats are 3D structures with appropriate porosity and high surface area to volume ratio that mimic to some extent the structure of the
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ECM. Therefore, electrospun nanofibers are considered as potential scaffolds for tissue engineering applications due to their ability to support cellular proliferation, adhesion, and differentiation (Agarwal et al., 2008; Liang et al., 2007). However, using electrospun SF nanofibers is still limited for bone tissue applications because of their low mechanical properties. In order to overcome this limitation, inorganic ceramics can be incorporated into the polymeric matrix. However, the aggregation of a ceramic particle in the SF matrix can interrupt the electrospinning process. Therefore, further investigations are needed to fabricate appropriate 34
ACCEPTED MANUSCRIPT SF/HAp nanofibers for bone tissue engineering purposes. In several studies, the aggregation of HAp particles in SF/HAp nanocomposites was reported (Kim et al., 2008; Li et al., 2006; Zhang et al., 2010). The aggregation can reduce the mechanical strength of the SF/HAp composites (Kim, 2007; Song et al., 2008a) and can result in non-uniform physical and chemical structures
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in these scaffolds. A useful strategy for enhancing the uniform dispersion of HAp particles in the SF matrix is the
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surface modification of the SF polymer by γ-glycidoxypropyltrimethoxysilane (GPTMS). The
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epoxide functional groups of GPTMS are able to attach to amino groups in the side chains of SF. Concomitantly, another side group of GPTMS can interact with the hydroxyl groups on the HAp
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surface (Arafat et al., 2011; Kim, H. et al., 2014). Electrospun SF nanofibers containing 20%) disrupted the polymeric chains
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that affect the mechanical behavior of the scaffolds (Kim, H. et al., 2014). Similarly, using more
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(Yang et al., 2016).
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than 20% HAp content showed decreased bending modulus due to local aggregation of nHAp
Electrospinning of SF/HAp blends faces another limitation such as immediate precipitation of
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nHAp before ejection. A three-way stopcock connector was developed for rapid mixing of various polymeric solutions and then rapidly injecting under the electric field to form blended nanofibers (Sheikh et al., 2013). Based on FTIR analysis, the chemical interaction between SF and nHAp changed the conformation of SF from random coil to β-sheet, which improved the mechanical strength of SF nanofibers (Sheikh et al., 2013). It was also showed that combining electrospun SF nanofibers with high pure HAp and silver nanoparticles (AgNPs) produced a suitable scaffold with antimicrobial properties for tissue engineering (Sheikh et al., 2014). 35
ACCEPTED MANUSCRIPT AgNPs can enhance the antimicrobial activity of nanofibrous structures. SF nanofibers containing 0.5% silver nitrate showed no toxicity toward NIH 3T3 fibroblast cells; however, using higher concentrations (1.0 or 1.5%) of silver nitrate revealed typical toxicity (Sheikh et al., 2014). New biocomposites based on electrospun SF nanofibers containing mesoporous bioactive
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glass/hydroxyapatite nanocomposite (MGHA) were reported (Liu et al., 2014). Recently,
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mesoporous bioactive glass (MBGs) has been introduced as potential candidates for drug
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delivery and bone tissue engineering applications. The ternary SiO2 –CaO–P2 O5 structure of MBG possess useful properties such as high surface area, degradability and large pore volume in
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comparison to non-mesoporous bioactive glasses that make them suitable for inducing bone
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formation (Wu et al., 2013; Xia and Chang, 2006). Moreover, adding MGHA particles into SF solution increased fiber diameter related to conductivity affecting fiber diameter and particle
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dispersion. The decrease in the conductivity of the blend solution was controlled by using higher
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MGHA content and less SF solution in the matrix. Using MGHA as a reinforcing component in the structure of SF-based nanocomposites improved swelling, surface hydrophilicity, and pore
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size, while tensile strength decreased. SF/MGHA scaffolds induced the formation of larger bones
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than pure SF after 4 and 8 weeks post-implantation. Additionally, in both implants, new bones containing osteocytes were directly formed near the scaffolds. The capability of SF/MGHA
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composites in bone regeneration may be related to the chemical interactions between the implant and host tissue, along with their surface bioactivity (Liu et al., 2014). In a recent study, coaxial electrospinning was applied in order to prepare a core-shell structure consisting of HAp and tussah silk fibroin (TSF) (Shao et al., 2016b). The crystalline region of TSF has a higher amount of alanine (Ala) in comparison to B. mori silk; therefore, Ala repeats are the main sequences in the structure of TSF, while domesticated silk is mostly comprised of
36
ACCEPTED MANUSCRIPT Gly-Ala-Gly-Ser repeats. In addition, the existence of an Arg-Gly-Asp motif in TSF structure induced specific cellular attachment, useful for biomedical purposes (He et al., 2013; Zhang et al., 2009). An increased content of nanoparticles in the core of prepared fibers increased the tensile strength. In other words, the content of core nanoparticles can be used to gradually
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change the mechanical properties of the composite from soft to rigid. A similar trend was also
tissue engineering applications
due to
potential in inducing cellular proliferation,
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bone
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seen in the Young's modulus of the composites. This scaffold showed promising properties for
attachment, ALP production, and biomineralization (Shao et al., 2016b). It was also found that
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the robust interaction between nHAp and SF nanofibrills induced the formation of flower-like
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structure that potentially facilitated the mineralization of HAp in situ. Moreover, the good mechanical properties and thixotropy with storage modulus (G’) make SF/HAp hydrogel useful
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to recover to over 85% in 50 seconds after applying a large shearing strain (5000%) (Mi et al.,
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2016).
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Generally, it is necessary to use nanofibrous structure for bone tissue engineering applications due to the nanometric nature of ECM components existed in the natural bone. The bone is a
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natural nanocomposite comprising a nanoscale HAp with about 20-80 nm in length that are
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dispersed in an organic collagenous matrix (Rho et al., 1998). The individual collagen chains have the length about 10 nm which reach to 500 nm after collagen fiber formation. Thus, using nanoscale bone constructs not only mimic the structure of natural bone but also can provoke the cellular activities in terms of adhesion, growth, differentiation, and expression of various proteins (Jiang et al., 2015). Despite the potential of electrospun mats in resembling the structure of collagenous fibers, the weak mechanical behavior compared with porous materials might be challenging for bone tissue engineering applications. Therefore, it is crucial to find a way for
37
ACCEPTED MANUSCRIPT increasing the mechanical strength of fibrous SF nanofibers to reach to adequate tensional or compressional stresses similar to skeletal bone (Fu et al., 2011; Wei and Ma, 2004). It seems that addition of ceramic phases in SF fibers are good strategies to improve the mechanical properties of fibrous scaffolds (Ito et al., 2005).
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4.4. Silk fibroin/Hydroxyapatite for drug delivery in bone tissue engineering
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To date, many attempts have been made to develop an appropriate carrier based on novel materials for drug delivery purposes. These systems control the release rate of drugs in a more
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sustained manner that can be helpful for decreasing side effects. Different structures such as membranes, micro- and nanoparticles, fibers and three-dimensional scaffolds have been
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introduced as carriers for different biomolecules (Balmayor, 2015; Zarrabi et al., 2014). Most of
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these structures are mainly comprised of polymers; however, using inorganic materials can be
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also useful.
Bioceramics are attractive inorganic materials to incorporate bioactive molecules or even cells,
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without denaturation or loss of bioactivity during preparation or implantation (Ginebra et al.,
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2012). Large surface area, narrow pore size distribution, and well-ordered pore systems make CaPs useful candidates for drug delivery (Bose and Tarafder, 2012; Vallet-Regí et al., 2007).
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Moreover, CaP is useful for controlling the local release rate of antibiotics (Arcos and ValletRegí, 2013; Verron et al., 2010), anticancer, anti-inflammatory, and analgesic drugs (Ginebra et al., 2006a) and growth factors (Bose and Tarafder, 2012; Ginebra et al., 2006b). Oral administration of CaPs is impossible because of rapid degradation at low pH of the gastric system. Therefore, drug delivery systems for the intestinal system (pH 8-9) with a controlled and targeted release profile are useful. However, current investigations have been focused more on drug delivery to locally or directly to bone tissue or as coatings (Kolmas et al., 2016). 38
ACCEPTED MANUSCRIPT CaPs also absorb many chemical molecules on their surface, which is useful for applications such as chromatography, and different biomolecules isolation and purification (Ginebra et al., 2006a; Tiselius et al., 1956). For instance, this criterion has been used for purification of bone growth factor through HAp chromatography (Urist et al., 1984). The high affinity of HAp to
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different biomolecules can provide options for developing novel matrixes for drug delivery
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systems. However, applying HAp-based carriers faces challenges due to their low capacity for
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loading drugs and the inability to control the release kinetics of such biomolecules. In contrast, silk is a useful biomaterial with high drug loading capacity and capability in controlling the
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release rate of drugs. Different drugs and biological macromolecules also can be loaded on silk-
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based structures without changing their bioactivity (Kundu et al., 2010; Shi et al., 2013a). For this reason, considerable efforts have been devoted to developing combinations of the intrinsic
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bone regeneration potential of HAp blended with SF, with the ability to incorporate drugs or
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other active molecules relevant for different therapeutic needs.
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Electrospun SF fibers containing nHAp/bone morphogenetic protein 2 (BMP-2) were used to induce bone formation in vitro using human bone marrow-derived mesenchymal stem cells
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(hMSCs) (Li et al., 2006). The authors found that it was impossible to electrspin the SF solution
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with the concentration more than 18% because the insufficient viscosity and surface tension of this solution. Higher concentrations of SF solution induced the gel formation during the electrospinning process. Furthermore, nHAp had a tendency to form aggregations after suspending in aqueous media and to overcome to this issue the SF and polyethylene glycol (PEG) solutions were prepared in 0.001M phosphate buffer (pH 6.8) that reduced the aggregation. It was observed that the nHAp were effectively embedded in the SF/ PEO fiber scaffold. The nanoparticles showed different fashions in dispersing in the polymeric matrix in
39
ACCEPTED MANUSCRIPT which they were well-oriented along the fiber axis in some regions while agglomerated in the other parts. It seems that high viscosity of SF/PEO blend solution led to non-homogenous dispersion of individual particles. Moreover, this study revealed that co-processed BMP-2 supported more calcium deposition and more upregulation of bone-specific markers (BMP-2,
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Col I and bone sialoprotein-II) in comparison to control groups. This observation suggested that
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silk nanofibers were useful for BMP-2 delivery, which broadened the application of this
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biomaterial in bone tissue engineering applications. Based on XRD spectra, the crystallinity of apatite on SF/BMP-2 scaffolds was higher than bulk SF scaffolds. Additionally, the
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incorporation of nHAp with SF/BMP-2 during electrospinning induced bone formation (Li et al.,
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2006). In another study, silk was used as a surface stabilizer and template in order to enhance surface properties of nHAp (Ding, ZZ et al., 2016). The core-shell structure of these composite
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materials facilitated the interaction between silk on the outer surface of nanoparticles with BMP-
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2 resulting increase the loading capacity of nHAp. This study showed that removing silk from nHAp had no effect on the morphological aspects of nHAp; however, aggregation particles was
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observed because of the elimination of repulsive forces from SF. Several studies confirmed that
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the high loading capacity of silk was related to electrostatic interactions with BMP-2 (Shi et al., 2013a; Shi et al., 2013b). Moreover, HAp particles alone showed 90% release of BMP-2 during
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2h, it was about 50% and 70% for silk alone and silk-nHAp composite groups after 21 days, respectively. Despite the faster release kinetics of BMP-2 from silk-nHAp compared to silk particles, the trend was linear. Moreover, BMP-2 loaded silk-nHAp showed more potential for inducing osteogenesis from bone mesenchymal stem cells (BMSCs) in comparison to BMP-2 loaded HAp particles or silk scaffolds (Ding, ZZ et al., 2016).
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ACCEPTED MANUSCRIPT In a study, a BCP consisting of HAp/β-TCP (60/40% ratio) and rhBMP-2/SF microsphere was developed which were then integrated into a BCP/rhBMP-2/SF composite (Chen, Liang et al., 2014). Based on SEM, HAp particles showed a needle-like structure, while β-TCP had hexahedral-like morphology. The rhBMP-2/SF had the mean diameter of 398.7 ± 99.86 nm with
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loading capacity of 4.53 ± 0.08 %. For pore formation, different concentrations of sodium
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chloride (NaCl; 50, 100, 150, and 200 w/v) were applied. it was observed that using NaCl with
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50, 100 and 150 % (w/v) concentrations had no effect on the structure materials during the desalination process; while, 200% NaCl easily damaged and degraded into particles or powder.
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Moreover, rhBMP-2 showed a dual-phase release profile, with both fast and slow rates, from the
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blended composites during 28 days. In the first three days, a burst and the fast release of protein (about 47.3 ± 3.1%) were observed. Afterwards, the cumulative release was about 72.2 ± 4.6%
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detected between days 3 to 14, and about 90.4 ± 5.3 between days 14 and 28 (Chen, Liang et al.,
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2014). The addition of organic materials into inorganic phases exhibits many advantages, such as increasing degradability, promoting migration and recruitment of osteoblast cells on the surface
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of the materials and increasing rates of mineralization.
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The efficacy of BCP/rhBMP-2/SF in a sheep lumbar fusion model was also evaluated (Chen,
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Liang et al., 2015). For this purpose, BCP and rhBMP-2/SF microspheres were blended to form BCP/rhBMP-2/SF composites that were implanted into the disc spaces of 30 sheep at the levels of L1/2, L3/4 and L5/6, randomly. The growth factor exhibited an initial burst release about 39.1 ± 2.8% during the first 4 days, and then had sustained release over 28 days (cumulative release rate ~ 81.3 ± 4.9 %). After implantation, the BCP/rhBMP-2/SF showed significantly better histological aspects such as in semi-quantitative CT scores, fusion rates, histologic scores and fusion stiffness in bending in all directions than the BCP or BCP/rhBMP-2 study groups. The
41
ACCEPTED MANUSCRIPT results recommended that using low doses of rhBMP-2 in the structure of SF microspheres improved bone fusion in sheep via BCP constructs (Chen, Liang et al., 2015). Despite the beneficial characteristics of controlled release systems in biomedical fields, the important parameters for mimicking the natural process of bone repair remain challenging to
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emulate. In many experiments, a single growth factor with controlled release kinetics is used; however, developing a system with the ability to control the release of multiple growth factors is
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more useful for bone regeneration (Kolambkar et al., 2011). Therefore, attempts have been
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pursued to develop delivery systems containing multiple growth factors for the synergistic effect on bone healing (Chen et al., 2010; Farokhi et al., 2016b; Shah et al., 2011).
(Bhattacharjee et al., 2016b). The 4-methacryloyloxyethyl trimellitate
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rhBMP-2 and TGF-β
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The electrospinning method was used to produce nanofibrous NSF/PCL for dual delivery of
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anhydride (4-META) was used to modify the NSF-PCL matrix and the nHAp were deposited on them by electrodeposition at two separate potentials (3V and 5V). The strong ionic interaction
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between nHAp and 4-META promoted the deposition of nHAp on the NSF-PCL matrix. The rhBMP-2 and TGF-β with a 1:1 ratio were loaded on the surfaces of the scaffold. After 4 weeks,
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rhBMP-2 and TGF-β exhibited about 20.02% and 20.93% release kinetics from NSF-PCL
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scaffolds, respectively. It seemed that the covalent interaction between the growth factors and HAp nuclei reduced the release kinetics. This structure had many favorable properties for bone tissue engineering because it contained SF with RGD motifs with the ability to induce cellular attachment
and
proliferation,
calcium phosphate
with high potential in promoting the
proliferation of osteoblasts, and the ability for dual growth factor delivery. The results also revealed that the prepared structures increased ALP production, intensified mineralization and improved the expression of bone-related genes. Random actin-stress fibers with dense cell 42
ACCEPTED MANUSCRIPT colony deposits were observed across the nanofibers loaded with dual growth factors via confocal laser microscopy. However, these outcomes were lower in the single growth factorloaded scaffolds, which confirmed the potential utility of dual growth factor delivery in bone tissue engineering applications (Bhattacharjee et al., 2016b).
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Similarly, chemical deposition method was used for preparing nHAp particles. Electrospun PCL nanofibers were prepared with different contents of nHAp (0%, 25% and 50%) (Bhattacharjee et
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al., 2016c). NSF was then grafted on the nHAp-PCL composites. It was observed that initial
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addition of nHAp had a positive effect in increasing the mechanical strength of NSF grafted PCL nanofibers. However, the higher tensile strength, toughness, and ductility was detected in those
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matrices containing 25% nHAp. Although the samples containing 50% nHAp showed higher mechanical properties and toughness than NSF-PCL nanaofibers without nHAp, but it was
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weaker than samples containing 25% nHAp. In addition, rhBMP-2 and TGF-β were coupled to
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the scaffolds by using carbodiimide coupling. The release rate of rhBMP-2 and TGF-β was about
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21.34% and 22.6% after 4 weeks, respectively. These low release rates may be related to the strong interactions between the functional groups of growth factors with active binding sites of
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SF. In the first 3 days, a burst release profile of both growth factors was observed, which may be
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due to the presence of non-conjugated or physically conjugated growth factors (about 22%). After 3 days, the remaining growth factors that were covalently bonded to the scaffolds were slowly released during 4 weeks. The fabricated scaffolds containing both growth factors provided optimal microenvironments for cellular migration, proliferation, differentiation, and enhanced expression of bone specific markers (Bhattacharjee et al., 2016c). The cross-talk between signaling of these growth factors and different signaling cascades such as Wnt, Hedgehog, Notch, MAP, and FGF play important roles in triggering the differentiation of
43
ACCEPTED MANUSCRIPT osteoblasts and consequently, bone formation (Chen et al., 2012). As mentioned earlier, multiple biomolecules can help to improve the bone healing process. In addition to growth factor delivery, other biomolecules such as some drugs would be useful in bone tissue engineering applications. Recently, silk–HAp films were prepared using casting method for incorporation of antidrugs
like
clodronate
(non-nitrogenous
bisphosphonate)
and
alendronate
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osteoporotic
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(nitrogenous bisphosphonate) (Hayden et al., 2014). Biological aspects of THP-1 human acute
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monocytic leukemia cell line-derived osteoclasts and human mesenchymal stem cell-derived osteoblasts such as calcium deposition, metabolic activity and ALP production on the prepared during 12 weeks. Clodronate-complexed scaffolds showed higher
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films were evaluated
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metabolic activity and roughness after culturing osteoblasts and co-cultures of osteoblasts with osteoclasts. Although the low dose of alendronate enhanced the metabolic activity and calcium
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deposition of osteoblasts (Figure 7), some toxicity was observed using the high doses against
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osteoclasts, osteoblasts and co-cultures. These data may result from different mechanisms of action of nitrogenous and non-nitrogenous bisphosphonates. The toxicity may be related to drug
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class. Nitrogenous bisphosphonates cause apoptosis in various cell types at concentrations
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similar to those that cause osteoclast apoptosis, and by the same mechanisms (Idris et al., 2008). Moreover, the inhibitory effect of nitrogenous bisphosphonates on mineralization is independent
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of the mechanism, which leads to cell apoptosis (Idris et al., 2008). Additionally, the ALP production was not changed using clodronate; however, alendronate decreased the ALP activity in a dose-dependent manner (Hayden et al., 2014). Generally, drugs can be loaded in CaPs such as HAp by tailoring the porosity with large surface area during synthesis (Arcos and Vallet-Regí, 2013; Vallet-Regí et al., 2007). Although high porosity may decrease mechanical strength, pores with an appropriate size can act as a host for
44
ACCEPTED MANUSCRIPT drug molecules (Arcos and Vallet-Regí, 2013). The release of drug can be controlled by altering the amount of porosity of CaP structures (Kim and Tabata, 2015). Furthermore, a homogeneous distribution of drug can be achieved in well-ordered pores (Vallet-Regí et al., 2008). In addition to the beneficial features of CaPs as drug carriers, nHAp retains an advantage as a major 3
ions that are
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constituent of natural bones. The degradation of CaP produces Ca2+ and PO4
found in high concentrations in the bloodstream. Moreover, CaPs, particularly HAp, are
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relatively insoluble at pH 7.4, thus there is no immunogenic response, and no cytotoxic
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degradation products (Bose and Tarafder, 2012).
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4.5. Silk fibroin/Hydroxyapatite for stem cell differentiation
Several tissues are important source of therapeutically relevant differentiated cells but the
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inevitable difficulties are in harvesting sufficient cells for implantation. Lineages such as neurons
ED
and cardiac cells, being terminally differentiated and non-regenerative, impose the biggest challenge. Among different stem cell lineages, pluripotent cells have been focus in biomedical
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applications due to high self-renewal capacity, prolonged undifferentiated options, and the ability
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to differentiate into multiple cell types under suitable stimuli (Zhao et al., 2013). Adult stem cells are other potential cells useful for tissue engineering because they can fully regenerate damaged
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tissue and improve the process of tissue repair. Recently, autologous adult stem cells, such as cardiac stem cells (CSCs), were found favorable when compared to induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) for treating myocardial infarction (MI) and chronic heart failure (CHF) (Shafiq et al., 2016). Moreover, adult stem cells do not face the challenges of using ESCs such as ethical, religious and immune rejection or the high costs of isolating and culturing iPSCs. However, iPSCs and ESCs are still good candidates for biomedical and tissue engineering applications due to their beneficial characteristics (Nori et al., 45
ACCEPTED MANUSCRIPT 2011; Song et al., 2010; Takahashi et al., 2007). In recent years, using different stem cell sources for different kinds of tissue engineering purposes has been introduced. Besides, it is more costeffective to promote the differentiation of stem cells using appropriate scaffolds without growth factors for optimal tissue repair. For example, silk/HAp composies have been introduced as a
showed
better
biological
and
physiochemical
properties
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scaffold
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potential scaffold for promoting the stem cell differentiation. Accordingly, the NSF/nHAp for
stimulating
the
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differentiation of bone marrow-derived stem cells (CBhMSC) into an osteogenic lineage in comparison to NSF electrospun scaffolds because the existence of well-dispersed nHAp over
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NSF matrix.After 24 h, the cells were attached and aligned along the axis of fibers that form
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oriented morphology. The ability of the scaffold to promote the osteogenic differentiation of CBhMSC cells were confirmed by the slight decrease in the proliferation rate and the increase in
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the expression level of ALP and osteogenic genes (Panda et al., 2014). Recently, NSF
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composites containing silk fibers and HAp was fabricated as bone substitutes (Gupta et al., 2016). The HAp was uniformly dispersed in the structure of highly porous and interconnected
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composite scaffold that formed an appropriate substrate for inducing the proliferation and
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migration of cells by preparing the suitable environment for transferring oxygen and nutrients. Moreover, the great mechanical behavior of the composite provided a supportive matrix for
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growth and differentiation of MG63 and hBMSCs cells due to high surface area/roughness and osteoconductivity. The osteogenic potential of the scaffolds were further confirmed by assessment the expression level of osteogenic genes like Col I, osteopontin, osteocalcin, and sialoprotein, ALP production, and ECM deposition (Gupta et al., 2016). Another study showed that adding NSF (10 wt%) into PLGA/graphene oxide (GO) nanofibers and
then
mineralization
in
simulated
body fluid
46
(SBF)
can significantly enhance the
ACCEPTED MANUSCRIPT hemocompatibility, protein absorption, surface hydrophilicity, and the mechanical properties. Besides, the conductivity of the electrospinning solution was increased from 0.39 to 0.78 mS/cm after adding 1 wt % GO into PLGA/NSF nanofibers that significantly decreased the fiber diameter from 321 nm to 89 nm. Moreover, biological tests confirmed that scaffolds not only
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support hMSC adhesion and proliferation, but also promote osteogenesis and ALP production
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(Shao et al., 2016c).
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Similarly, GO-HAp/SF composite was prepared by biomineralizing carboxylated GO sheets,
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blending with SF, and freeze-drying for evaluating the differentiation of MSCs. The GO-HAp (1:4)/SF composites showed significantly higher mechanical strength in comparison to HAp/SF
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scaffolds that had better compressive strength (85 MPa) and compressive modulus (938 MPa).
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The protein absorption of GO-HAp (1:4)/SF were also 1.8-fold higher than HAp/SF scaffolds. Furthermore, the GO-HAp/SF composite was able to potentially improve the adhesion and
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proliferation of MSCs, increase the expression level of osteocalcin, and differentiation of
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osteoblast cells (Wang et al., 2017).
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Aqueous-derived porous SF scaffolds were fabricated and considered as templates to deposit apatite on the pore surfaces via mixing with polyaspartic acid (PA) during processing, followed
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by mineralization with CaCl2 and Na2 HPO 4 (Kim et al., 2008). The salt-leaching method using granular NaCl with particle size of 850∼1000 μm was used to prepare the scaffolds. Moreover, the ratios of SF-PA (w/w) blends were as 100/0 (group I), 95/5 (group II), 90/10 (group III) and 80/20 (group IV). The scaffolds were then used for seeding hMSCs under osteogenic condition in the presence or absence of BMP-2 for 6 weeks. After 24h, the aqueous solution of SF/PA converted to hydrogel and formed a porous and water-stable structure. The apatite crystals were deposited using salt deposition because PA produced nucleation sites at the interfaces of the 47
ACCEPTED MANUSCRIPT hydrophobic silk structures. It was found that the increased polyaspartic content increased HAp deposition on the scaffolds. Accordingly, it is well-established that proteins containing aspartic acid-rich motifs are able to interact with calcium salts in a specific fashion (Addadi and Weiner, 1985; Fujisawa et al., 1996). It was also showed that the calcium deposition and ALPase activity
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were almost the same in mineralized silk scaffolds in the absence of BMP-2. Moreover, the
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osteogenic media, whether using BMP-2 or not, also stimulated similar ALPase activity of MSCs
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after 6 weeks in groups I, II, and III. The group IV showed higher ALPase activity in the presence of BMP-2. All the mineralized scaffolds revealed more calcium deposition with BMP-
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2, while no calcium deposition was detected in those scaffolds without mineralization (control
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groups) after 6 weeks. Based on van Kossa staining, more calcium deposition led to higher osteoconductivity of the scaffolds. Immunocytochemistry demonstrated Col I was expressed in
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all groups. Additionally, the expression of Col I was increased in groups II and III in the
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presence of BMP-2 (Figure 8). It was concluded that higher amounts of apatite in the structure of porous silk constructs along with the existence of BMP-2 increased the osteoconductivity of the
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scaffolds (Kim et al., 2008).
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Despite the effectiveness of using silk/HAp as bone constructs, the injectable form of bone
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materials is less investigated. The main drawback of bone substitutes is the lack of suitable structure for filling the irregular bone defects. To provide an injectable biomaterials with osteogenic properties, the silk-HAp composite with silk nanofibers in hydrogels was prepared. For this, an injectable system was fabricated by blending thixotropic silk nanofiber hydrogels with water dispersible silk-HAp nanoparticles to form injectable nanoscale systems. The composites had about 60 % w/w HAp content for optimally mimic the bone environment. The physical strength of the system was also improved to 21 kPa by adding of HAp that was suitable
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ACCEPTED MANUSCRIPT for stimulating the osteo-differentiation. The osteogenesis was more enhanced by using composite hydrogels in comparison to silk nanofiber hydrogel without HAp. The silk-HAp composite hydrogel was able to treat irregular bone defects after injection (Ding et al., 2017). The main purpose of tissue engineering is to develop functional materials with suitable
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biocompatibility that highly mimics the characteristics of the target tissue. For this, using engineered biomaterials is of interest due to rational design, adequate biocompatibility and tunable
structural properties
and
functionality.
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biodegradability,
Recombinant silk
fusion
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proteins are an example of engineered materials that have these features. Mineral-binding sequences are rich in acidic residues (aspartate, glutamate, phosphoserines) that can increase the
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net negative charge of engineered materials. These sequences can interact with positively
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charged HAp nanocrystals (Addison et al., 2010). Recently, an organic-inorganic hybrid system was developed based on genetically engineered spider silks. The organic phase of spider silk
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inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT)15 maintained the
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stability of the scaffold and facilitated different modes of processing; while, the inorganic domain of SF VTKHLNQISQSY (VTK) for HAp binding was involved in regulating the
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osteogenesis and biomineralization. The VTK peptide localized on recombinant fusion protein
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was able to provoke the calcification but it did not any effect in improving the physical strength of the recombinant structure. It was also observed that adding VTK peptide on both c-terminal and N-terminal sides of recombinant silk could significantly enhance the formation of crystalline HAp. All the prepared constructs was able to provoke the attachment, proliferation, and differentiation of hMSCs (Dinjaski et al., 2017).
Salt-leached SF scaffolds were also prepared
followed by functionalization and mineralization in CaCl2 ethanol solution/K 2 HPO 4 aqueous solution. The scaffolds were then mineralized by immersing in the simulated body fluid (SBF)
49
ACCEPTED MANUSCRIPT for 4 days. The authors claimed that the duration of soaking in SBF would be decreased by using this method that consequently protect the SF-nHAp scaffold from degradation during the mineralization process. The microspores of nHAp with a size range of 100 to 250 nm were formed on the surface wall of SF. Incorporating of nHAp particles with the SF scaffolds
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significantly improved the compressive strength and stiffness of SF (Liu, H. et al., 2015).
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Furthermore, the cDNA microarray demonstrated changes in 23 calcium ion binding genes and
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17 cell adhesion genes of BMSCs seeded on nHAp-SF scaffolds compared with bulk SF constructs. Among these genes, about 15 genes corresponded to cellular adhesion and 20 genes
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were attributed to calcium ion binding were decreased in expression level. Based on these
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observations, the cultured BMSCs on nHAp-SF scaffolds had round shapes due to less surface adhesion. The nHAp-SF scaffolds showed higher osteoinductivity in vitro, significantly higher
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ALP activity on day 14, and more osteogenesis related to signaling pathway genes on day 7
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compared with the SF scaffolds. Moreover, the nHAp-SF scaffolds demonstrated higher capability to induce bone regeneration in cranial bone defects than bulk SF scaffolds. It was also
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shown that soluble IL-1a factor and biomolecules on the surface of BMSCs were played an
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important role in provoking osteogenesis. Upon interaction between BMSCs and nHAp, BMSCs regulated the activity of IL-1a by IL IR2. This study helped to understand better the possible
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mechanism of osteoinductivity by nHAp (Liu, H. et al., 2015). Electrospun SF nanofibers containing nHAp and BMP-2 was prepared as bone construct with the ability to induce the differentiation of BMSCs (Niu et al., 2017). The nHAp particles were successfully dispersed in SF fibers. The increase in the content of HAp resulted in more aggregations of this particles in the SF matrix. The mechanical properties of the composite containing 20% HAp such as tensile strength, yield strength and Young’s modulus was almost
50
ACCEPTED MANUSCRIPT two-fold more than pure SF scaffolds. BMSCs showed significantly more ALP activity on SF/HAp/BMP-2 scaffolds compared with SF scaffolds. Thus, SF/HAp/BMP-2 scaffolds were considered as a potential structure in inducing osteogenesis (Niu et al., 2017). It is assumed that synergistic effect of HAp and BMP-2 improve the osteogenic capacity of the composite scaffold.
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This issue was also confirmed by Lu (Lu et al., 2015) or Liu (Liu, Z. et al., 2015) claimed that
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degradation of SF can increase the exposure of HAp to the cells during time.
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Similarly, the bioactivity and the sustained release rate of BMP-2 was preserved in the nHAp-
US
embedded SF scaffold by adjusting the ratio of loaded BMP-2 on SF and nHAp. The suitable BMP-2 release behavior inside the structure of SF-nHAp scaffold increase the expression level
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of Col I, ALP and Runx2 protein that resulted in higher osteo-conductivity. Based on the results
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from in vitro and in vivo, the release of BMP-2 had positive effect in inducing osteogenesis of
ED
the scaffold (Ding, Zhaozhao et al., 2016).
Shen et al. fabricated porous SF/nHAp composites containing BMP-2 and SDF-1 loaded SF
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microspheres to synthesis the cell-free scaffold. Laminar jet break-up technology was used for
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SF microsphere preparation which provide optimal condition for high encapsulation efficiency (Shen et al., 2016). It was reported by Wenk et al. that preparing SF microspheres using this
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method can preserved the bioactivity of IGF-I and controlling the release rate of growth factor in a sustained manner (Wenk et al., 2008). SDF-1 showed a rapid release rate during the first few days, while it presented a sustained profile in the following three weeks. The scaffold significantly provoked the osteogenic differentiation and recruitment of BMSCs (Shen et al., 2016). In another study, electrospun SF nanofibers containing HAp were fabricated for periosteum regeneration (Ding, X. et al., 2016). Periosteum is the main source of osteo-progenitor cells 51
ACCEPTED MANUSCRIPT around bone, therefore, this tissue plays a crucial role in the initial phase of bone formation and regeneration (Tiyapatanaputi et al., 2004; Zhang, X. et al., 2005). The HAp particles were seen on the surfaces of SF nanofibers containing 10%, 20%, and 30% HAp. Moreover, more HAp were observed on the scaffolds with higher content of HAp. Some aggregations of HAp particles
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were observed on the surface of interconnected pores of SF/HAp nanofibers which may be
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responsible for increasing the diameter of 10%, 20%, and 30% SF/HAp nanofibers. The Young's
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modulus of the scaffolds was also increased with the increase in the content of HAp. Moreover, the results demonstrated that SF scaffolds containing 30% HAp induced the metabolic activity of
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rBMSCs more than those scaffolds containing 0% HAp after 4 days. SF scaffolds containing
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30% HAp promoted the proliferation of rBMSCs and upregulated mRNA levels of ALP and Runx2 more than the other groups. Moreover, rBMSCs showed higher levels of ALP expression
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when cells were cultured on SF scaffolds containing 30% HAp in comparison to 0% HAp-SF
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scaffolds. Higher calcium deposition was found by Alizarian red assay when 30% HAp-SF scaffolds were used versus 0% HAp-SF scaffolds (Ding, X. et al., 2016). Therefore, it was
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suggested that SF scaffolds containing 30% HAp were potent structures to promote bone
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formation due to their ability to induce mineral deposition and consequently, cell differentiation.
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Co-cultures of MSCs between ligament and bone cells on suitable sections of hybrid silk scaffolds induced cellular differentiation into fibrocartilage lineage (He, P. et al., 2012). The section of the scaffold for seeding osteoblast cells was pre-coated with HAp. Real-Time PCR revealed that both osteoblast and fibroblast cells maintained their normal phenotype during coculture. However, co-cultured BMSCs differentiated toward the fibrocartilage lineage. The cocultured BMSCs on the hybrid SF scaffolds exhibited up-regulation of Col II, Sox9, aggrecan and fibrocartilage gene markers in comparison to mono-cultured BMSCs. The data were
52
ACCEPTED MANUSCRIPT confirmed
by
histological
analysis
and
immunohistochemistry.
The
immunohistochemistry
staining revealed that aggrecan was the most prominent marker in the enthesis (the connective tissue between tendon or ligament and bone). In addition, the trilineage co-culture was supplemented with 10 ng/mL TGF-β3 and the fibrocartilaginous differentiation of BMSCs by the
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TGF-β3-treated group was similar to those groups without TGF-β3. No calcium deposition was
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process of mineralization in chondrocytes (He, P. et al., 2012).
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observed in the TGF-β3-treated group and it was suggested that TGF-β3 could postpone the
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In another study, leaching paraffin microsphere with the modified temperature gradient-guided thermal-induced phase separation (TIPS) methods were applied to fabricate SF/HAp scaffolds
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with the potential to mimic the integrated trilayered osteochondral substrates (Ding et al., 2014).
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Based on the Micro-CT analysis, the consecutive tri-layer structure consisting a chondral (top) layer, intermediate layer, and bony (bottom) layer was observed in the integrated scaffold. Using
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TIPS method resulted in the formation of oriented microtubularlike porous structure in the
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chondral layer and the paraffin-sphere leaching led to formation of an interconnected macropore structure in the bony layer. The chondral and bony layers had porosities of 85.30% ± 1.80% and
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90.25% ± 2.05%, respectively. Adipose-derived stem cells (ADSCs) were used under
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chondrogenic (with TGF-β1 and IGF) and osteogenic (with dexamethasone) conditions in chondral and bony layers of the scaffold in order to evaluate the potential of these cells for differentiation to cartilage and bone lineages. In the chondral layer which was previously seeded with ADSCs, chondrocyte-like cells were observed, which were enclosed by ECM components, especially collagen type II and glycosaminoglycan (GAG). The amounts of ECM components were also increased during 21 days in the chondral layer. Moreover, chondrogenic-related genes were also up-regulated during culture. Generally, it was proposed that ADSCs seeded chondral
53
ACCEPTED MANUSCRIPT layer
provided
suitable
environments
for
inducing
chondrogenesis
under
chondrogenic
conditions. The ADSCs also showed higher expression of osteogenic related genes and bone ECM components such as Col I and calcium. Therefore, osteogenic differentiation also could be promoted under osteogenic conditions. Consequently, the pores were filled with more cartilage-
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related and bone-related matrices as the culture time increased, resulting in an increase in the
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compressive elastic modulus (Ding et al., 2014). The results showed promise for the use of novel
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3D integrated SF scaffolds in osteochondral tissue engineering in vitro. However, whether ADSCs can differentiate into cartilage and bone in the corresponding chondral and bony layers
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of integrated scaffolds needs to be further investigated through in vivo implantation. The
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isolating role of the intermediate layer needs to be confirmed in vivo. Other important studies
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using SF/HAp for stem cell differentiation are listed in Table 5. Totally, primary osteoblast cells are the mostly used cells for bone regeneration; however, their
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limited number and dedifferentiation properties might limit applications. Using potential stem
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cells can overcome these drawbacks (Wang et al., 2006). Stem cells have been largely investigated for creating functional tissues and organs but it is still at infant stage. Bone marrow
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is the favored source of MSCs for both experimental and clinical application. The biological
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features of stem cell such as self-renewal and differentiation capacity are regulated by ECM components. ECM also serves as a supporting structure for various growth factors and biomolecules which can act as structural support with the ability to control signal transduction (Kundu and Kundu, 2010). The suitable morphological aspects of SF/HAp composites make it an appropriate structure for stimulating the differentiation of stem cell into desired lineage and thus promoting the regeneration of defected tissue.
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ACCEPTED MANUSCRIPT Table 5. Simultaneous use of SF/HAp composites and stem cell for bone tissue engineering. Material
Processing method
Structure and properties of the scaffold
Cell
Key findings
Ref.
SF1 /HAp2
Direct-write assembly
Formation of 3D scaffold with an interpenetrating gradient network of pore channels, suitable mechanical integrity
hMSCs3 /
Formation of complex network of hMSCs matrix within the 3D scaffold by hMSCs and hMMECs based on histological analysis, observation of vascular-like structures only in hMSCs and hMMECs coculture groups
Sun et al., ( )2012
Freeze-drying/ co-precipitation
Formation of dense flakelike HAp crystals on the surface and the pore walls of the 3D silk structure
BMSCs5
Increasing the surface roughness and proliferation of BMSCs by incorporating HAp in SF matrix, improved in vitro differentiation of BMSCs toward osteoblast cells
Jiang et al., ( )2013
High and uniform coupling of nHAp on the surface of nano-HAp/SF sheet
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Good cellular proliferation and attachment, higher ALP 7 and bonespecific osteocalcin production under osteogenic condition after 14 days of cell cultured on nanoHAp/SF sheets compared with controls
Tanaka et al., ( )2007
Graft polymerization
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hMMECs4
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Formation of 3D scaffold with
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Higher surface osteoconductivity of SF scaffold by using HAp and suggesting HAp microparticles as nucleation sites for directing mineralization, enhancing the formation of trabecular structure, increasing the mechanical behavior and conductivity of tissue grafts
Bhumiratana ( )et al., 2011
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approximately 52 ± 5.5% porosity and pore size of 52.3 ± 5.5
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Induction of HAp nanocrystals aggregation in 56
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Higher Sinlapabodin ( )et al., 2016 proliferation rate of mouse MC3T3E1 cell line seeded on the scaffolds with perfusion flow rate of 1 ml/min than the static culture, increased differentiation of BMSCs into osteoblast using perfusion flow rate of 3 ml/min compared to 1 and 5 ml/min flow rates, statistically higher Ca/P ratio, ALP activity, and calcium contents in flow rate of 3 ml/min compared to other flow rates Better cellular attachment and osteogenic differentiation
Lin et al., ( )2015
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based on ALP activity, BGP 8 contents, and Col I9 of cells in SF/nHAp compared with nHAp groups
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hDPCs10 / Formation of 3D structure hPDLCs11 with interconnected pores, varied pore size between 20 and 80 μm with the diameter of 65 μm
SF/PHBV12 /HAp
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Reduction in the tensile Young’s modulus of the composite fibers with the increase in the content of the nHAp and SF phase from 2% to 5%
-
Park et al., ( )2015
MC3T3E1
Increased cellular proliferation and attachment on composites compared with PHBV control, higher expansion and elongation of cells on the surface of fiber composites after 1 day of culturing, incomplete elongation of cells seeded on PHBV scaffolds
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deposition
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cultured on SF/HAp scaffolds, increasing tissue regeneration by using PMSCs seeded SF/HAp scaffolds in radius segmental bone of rabbit Silk fibroin, 2 Hydroxyapatite,3 Human bone marrow derived mesenchymal stem cells, 4 Human mammary microvascular endothelial cells, 5 Bone marrow derived mesenchymal stromal cells, 6 Marrow mesenchymal cells,7 Alkaline phosphatase, 8 Bone gla protein, 9 Collagen type I, 10 Human dental pulp cells, 11 Human periodontal ligament cells, 12 Polyhydroxybutyrate–polyhydroxyvalerate, 13 Placenta-derived mesenchymal stem cells
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5. Conclusions and future perspectives
Silk scaffolds are biocompatible structures with tunable properties in terms of morphology,
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degradability, and conformation that make them useful candidates for tissue engineering applications. Recently, investigations have been performed to better understand the structure and
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processing of silk-based structures that broadened the applications of different types of silk in
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regenerative medicine. The unique characteristics of silk such as exceptional mechanical and
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physical behavior, and versatile processability to form different structures make it potentially useful for ligament, bone and musculoskeletal tissue engineering. Silk has many suitable
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properties for various applications; however, it has some limitations. For example, the properties
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of silk protein, as a natural polymer, are different based on species source. Moreover, inconsistence degumming processes and different fabrication methods may produce silk structures with various properties if the process is not tightly controlled. It is well established that genetically modified silk proteins may overcome the mentioned limitations pending scale up and cost efficiencies. In order to enhance the potential of silk-based scaffolds for bone tissue engineering applications, this polymer can be blended with other materials such as HAp to enhance osteogenic potential of the scaffolds. HAp has a similar chemical structure to
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ACCEPTED MANUSCRIPT mineralized bone of human tissue. This ceramic material demonstrates affinity to hard tissues. More investigations are to be carried out in order to understand better the application of nanoscale constructs that can be degraded over time and replaced by endogenous hard tissues. It is also necessary to focus on modifying the surfaces of biomaterials with different biomolecules There are many
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with the aim of improving their biological behavior and interfacial functions.
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studies that describe the benefits of using nHAp-SF scaffolds for bone tissue engineering
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applications in vitro and in vivo. However, there is no comprehensive study that shows the adverse effects of using these structures. It is essential to develop effective constructs for clinical
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use despite advantageous properties of SF/HAp in this area. It is noteworthy that many
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investigations have confirmed the potential of SF scaffolds to induce osteogenesis in vivo. Most of these studies are performed on small animal models, thus the results may not fully be
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the requirements of using silk-based scaffolds in clinical trials and constructing suitable
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Acknowledgments
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commercialized structures for bone tissue regeneration.
This work is supported by Pasteur Institute of Iran
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Disclosure statement
No potential conflict of interest was reported by the authors
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hydroxyapatite (SF-HAp) composites. (E) to (H) remarkable studies related to parts (A) to (D).
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Figure 2. Silk/ hydroxyapatite (SF-HAp) composites for bone tissue engineering.
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Figure 3. Different types and applications of bioceramics.
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Figure 4. Ultrastructure of bone tissue. The extracellular matrix of bone is highly specialized. The organic part of bone is composed of collagen, noncollagenous proteins (NCPs) and lipids.
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The inorganic phase of bone mainly consists of hydroxyapatite (HAp) nanocrystals. This mineral phase is crystallized along the long axis of collagen fibrils and integrates together in a tight
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hierarchical organization. These crystals associate with the collagen fibres, making bone hard
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and strong. This matrix is organized into numerous thin layers, known as lamellae.
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Figure 5. Signaling pathway of osteoblast cells seeded on hydroxyapatite (HAp) scaffold. Adhesion of osteoblast cells on HAp substrates can trigger some signaling pathways like
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extracellular regulated kinases (ERK). Due to interaction of integrin receptors with such
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extracellular matrix of bone e.g. fibronectin, focal adhesion kinase (FAK) as an intracellular signaling cascades can be activated. This causes activation of ERK pathway and then expression of some osteoblast specific genes e.g. osteocalcin and collagen type I. Figure 6. The role of Notch signaling pathway in osteoblast differentiation of stem cells. a). Notch signaling blocks RUNX2 expression and consequently inhibition of osteocalcin and osteopontin. b). Blocking of Notch signaling cascade after seeding of stem cells on silk fibroin based scaffold. 73
ACCEPTED MANUSCRIPT EGF: epidermal growth factor, LNR: Lin-12/Notch repeats, HD domain: heterodimerization domain, TMD: transmembrane domain, RAM: RBPjκ association module, ANK: ankyrin, NLS: nuclear localization sequences, TAD: transactivation domain, PEST: proline (P), glutamic acid (E), serine (S),
threonine (T) rich motif, NICD: Notch intracellular domain, ADAM: a
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disintegrin and metalloproteinase, MAMAL: Mastermind-like, HES: hairy and enhancer of split,
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HEY: HES-related with YRPW motif.
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Figure 7. Effects of bisphosphonates on calcium deposition. Calcium content of films was measured after 4, 8 and 12 weeks of culture and expressed as mean ± SD. Top left: osteoblasts
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cultured on clodronate-loaded films. Top right: osteoblasts cultured on alendronate-loaded films.
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Bottom left: co-cultures cultured on clodronate-loaded films. Bottom right: co-cultures cultured on alendronate-loaded films. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Copyright © 2014 Elsevier
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B.V. Reprinted with permission from (Hayden et al., 2014).
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Figure 8. Immunohistochemical staining of collagen type I. Extracellular matrices (*) stained
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positive for Col I within the scaffold pores. The Col I matrix covered the entire mineralized aqueous-based silk lattice (#) after 6 weeks of culture with BMP-2 (100 ng/ml; b, d, f, h) or
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without BMP-2 (a, c, e, g). Copyright © 2008 Elsevier B.V. Reprinted with permission from
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(Kim et al., 2008).
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