Directing osteogenesis of stem cells with hydroxyapatite precipitated electrospun eri-tasar silk fibroin nanofibrous scaffold.

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Directing osteogenesis of stem cells with hydroxyapatite precipitated electrospun eri–tasar silk fibroin nanofibrous scaffold a

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N. Panda , A. Bissoyi , K. Pramanik & A. Biswas a

Department of Biotechnology and Medical Engineering, Center of Excellence for Tissue Engineering, National Institute of Technology, Rourkela, Rourkela 769008, Odisha, India Published online: 04 Aug 2014.

To cite this article: N. Panda, A. Bissoyi, K. Pramanik & A. Biswas (2014) Directing osteogenesis of stem cells with hydroxyapatite precipitated electrospun eri–tasar silk fibroin nanofibrous scaffold, Journal of Biomaterials Science, Polymer Edition, 25:13, 1440-1457, DOI: 10.1080/09205063.2014.943548 To link to this article: http://dx.doi.org/10.1080/09205063.2014.943548

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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, No. 13, 1440–1457, http://dx.doi.org/10.1080/09205063.2014.943548

Directing osteogenesis of stem cells with hydroxyapatite precipitated electrospun eri–tasar silk fibroin nanofibrous scaffold N. Panda, A. Bissoyi, K. Pramanik* and A. Biswas Department of Biotechnology and Medical Engineering, Center of Excellence for Tissue Engineering, National Institute of Technology, Rourkela, Rourkela 769008, Odisha, India


(Received 28 March 2014; accepted 8 July 2014) Stimulating stem cell differentiation without growth factor supplement offers a potent and cost-effective scaffold for tissue regeneration. We hypothesise that surface precipitation of nano-hydroxyapatite (nHAp) over blends of non-mulberry silk fibroin with better hydrophilicity and RGD amino acid sequences can direct the stem cell towards osteogenesis. This report focuses on the fabrication of a blended eri–tasar silk fibroin nanofibrous scaffold (ET) followed by nHAp deposition by a surface precipitation (alternate soaking in calcium and phosphate solution) method. Morphology, hydrophilicity, composition, and the thermal and mechanical properties of ET/nHAp were examined by field emission scanning electron microscopy, TEM, FT-IR, X-ray diffraction, TGA and contact angle measurement and compared with ET. The composite scaffold demonstrated improved thermal stability and surface hydrophilicity with an increase in stiffness and elastic modulus (778 ± 2.4 N/m and 13.1 ± 0.36 MPa) as compared to ET (160.6 ± 1.34 N/m and 8.3 ± 0.4 MPa). Mineralisation studies revealed an enhanced and more uniform surface deposition of HAp-like crystals, while significant differences in cellular viability and attachment were observed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and confocal microscopy study. The cell viability and expression of adhesion molecules (CD 44 and CD 29) are found to be optimum for subsequent stages of growth proliferation and differentiation. The rates of proliferation have been observed to decrease owing to the transition of MSC from a state of proliferation to a state of differentiation. The confirmation of improved osteogenic differentiation was finally verified through the alkaline phosphatase assay, pattern of gene expression related to osteogenic differentiation and morphological observations of differentiated cord blood human mesenchymal stem cells under fluorescence microscope. The results obtained showed the improved physicochemical and biological properties of the ET/nHAp scaffold for osteogenic differentiation without the addition of any growth factors. Keywords: eri–tasar silk fibroin; hydroxyapatite; bone tissue engineering; cell adhesion; proliferation; differentiation

1. Introduction Bone defects arise due to several incidents such as tumour, trauma and accidental injury as well as diseases of old age like osteoporosis. Also, they can occur because of a failure in the repair mechanism of bone, either by infection or by an increased magnitude of defects requiring surgical intervention with autologous bone graft or donor allografts. *Corresponding author. Email: [email protected] © 2014 Taylor & Francis


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Autologous grafts are associated with limited availability and donor site morbidity, while allografts are associated with immune response and pathogen transfer. Therefore, the development of tissue-engineered constructs with immunocompatibility brings forth hope for the healing of bone. Scaffold, an integral part of the tissue-engineered constructs, serves as a temporary template for cellular interaction and formation of the extracellular matrix (ECM). The major challenge in the design and fabrication of a scaffold is to mimic the natural ECM of bone, which constitute nanofibres in the size range of 50–250 nm, in addition to proper cellular attachment, growth, proliferation and desired mechanical strength in order to support the bone remodelling process.[1] Electrospinning is a simple technique that produces polymeric fibres in a nanoscale dimension and multiscale pore size distribution from nano to micrometres, providing a large surface area for cell attachment, making the scaffold an excellent material for mimicking the natural ECM in comparison with microporous and microfibre scaffolds.[1] The synthetic polymeric fibres such as poly(caprolactones) and poly(lactic acid) lack cell recognition sites, whereas the natural biopolymers such as chitosan, collagen and fibrins, although they possess cell recognition sites, furnish poor mechanical strength. In this context, the search for new material explores silk fibroin, which has good biocompatibility, impressive mechanical strength and a slow rate of degradation.[2] Earlier attempts have prepared silk fibroin and its composite with hydroxyapatite as filler and poly(lactic acid) to prepare porous scaffold and film, respectively, for tissue regeneration.[3,4] Extensive studies have been undertaken for silk fibroin protein derived from Bombyx mori, as well as its composite with calcium phosphate, for bone tissue engineering applications. Nevertheless, this silk fibroin lacks RGD epitope and an adequate amount of arginine amino acid content which are responsible for cell attachment.[5] Several reports have suggested that non-mulberry silk fibroins from Antheraea mylitta and Philosomia ricini lack cytotoxicity and inflammatory properties and possess an RGD (GenBank: AY136274.1) epitope within their amino acid backbones in addition to a high content of arginine amino acids.[6,7] Additionally, the high content of hydrophilic amino acids is supposed to provide cell adhesive properties to the material.[2,5,8] They have also inherited antibacterial properties.[9] In contrast, the silk fibroin scaffold lacks osteoinductive properties, which can be overcome by the incorporation of hydroxyapatite, the natural mineral constituent of bone (70% of total mineral phase of bone with a Ca:P ratio of 1.67), which possesses bioactivity and osteoinductive properties.[10] Moreover, the incorporation of eri silk fibroin in the osteogenic differentiation of cord blood human mesenchymal stem cells (CBhMSCs) needs to be verified, being not been reported yet. Furthermore, the use of HAp (Ca10(PO4)6(OH)2) promotes cell–material interaction by activating HAp-dependent cellular processes at various levels.[11,12] In addition to osteoblast proliferation, differentiation and ECM mineralisation, calcium stimulates Ca-sensing receptors in bone cells and consequently enhances the expression of growth factors such as IGF-I and IGF-II.[13] Inorganic phosphate is helpful in stimulating the expression of matrix gla protein, an important regulator of bone formation.[14] Notably, its poor fracture strength, resilience and brittleness limit its direct use as an implant.[15] Hence, it is assumed that integrating the silk fibroin protein templates with hydroxyapatite will function as an organisational component, employing complementary interacting and bone tissue induction properties. It is assumed that the application of a direct mineralisation method for HAp precipitation over the polymer fibre, as reported earlier,[16,17] will allow proper electrospinning without the agglomeration of hydroxyapatite.

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Cord blood is a medical waste and a cheap source of stem cells that are capable of multilineage differentiation with similar immunophenotypic and osteogenic differentiation properties to bone marrow-derived stem cells (CBhMSC).[18,19] The high availability of the source and the low toxicity have prompted its use as an alternative source of CBhMSC. In the present study, research work focuses on the development of a CBhMSC-seeded SF/nano-hydroxyapatite (nHAp) construct and verifying its ability towards cell adhesion, proliferation and differentiation. 2. Materials and methods Fibronectin-coated 96-well plate, Dulbecco’s phosphate-buffered saline (DPBS) solution (Cat# D5652), 4′,6-diamidino-2-phenylindole (DAPI), phalloidin, fluorescein diacetate, Alamar Blue, osteogenic primers (Glyceraldehydes phosphate dehydrogenase [GAPDH], osteocalcin (OCN), osteopontin (OPN), osteonectin (ONN) and Runx 2) penicillin, streptomycin, fungizone, gentamicin sulphate, dexamethasone, ascorbic acid, and β-sodium glycerophosphate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Glutaraldehyde solution (25%), calcium chloride, sodium carbonate and ethanol, were obtained from Merck India Ltd (Mumbai, India). Blocking buffer, Falcon tissue culture plate and Slide-A-Lyser dialysis cassette (MW 3500 Da) were obtained from Pierce Thermo Scientific. FITC-conjugated CD 44 (Catalog Number 563029) and CD 29 (Catalog Number 555443) were purchased from BD Bioscience. Dulbecco’s phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM; 4.5 g/l glucose with L-glutamine and without sodium pyruvate) and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay (MTT) were purchased from Life Technologies (Grand Island, NY). Foetal bovine serum was purchased from Atlas Biologicals (Fort Collins, CO). TRIzol for isolation of cellular RNA and trypsin were purchased from Invitrogen (Carlsbad, CA). Alkaline phosphatase (ALP) assay kits were purchased from Bioassay Systems (Hayward, CA). Blocking buffer, Falcon tissue culture plate and Slide-A-Lyser dialysis cassette (MW 3500 Da) were obtained from Pierce Thermo Scientific. FITC-conjugated CD 44 (Catalog Number 563029) and CD 29 (Catalog Number 555443) were purchased from BD Bioscience. CBhMSCs were harvested from human cord blood and cultured in our stem cell culture laboratory. The cells were tested for purity by high expression of CD105, CD 90 and CD 73 and low expression of CD14, CD34 and HLA-DR markers using standard protocol. All other chemicals were procured locally. 2.1. Preparation of SF/HAp nanofibrous scaffold by electrospinning The details of electrospun eri and tasar silk fibroin nanofibrous mat formation can be found in our patent (713/KOL/2012-Applied patent). In brief, the chopped cocoons of eri and tasar were boiled in 0.02 M aq. sodium carbonate (Na2CO3) solution to remove sericin and were dried followed by dissolution in a ternary mixture of CaCl2/H2O/EtOH (1/8/2 M ratio). The solution was dialysed in a Slide-A-Lyser dialysis cassette against distilled water and lyophilised to obtain regenerated silk fibroin powder. The spinnable solution was prepared by dissolving eri and tasar in a 70:30 w/w ratio in a mixture of formic acid and chloroform (60:40 v/v). The nanofibres were prepared using an electrospinning machine (ESPINO-NANO, India) with an applied voltage of 22 kV, a tip-collector distance of 15 cm and a flow rate of 0.5 ml/h. The fibres were collected on a grounded parallel plate collector consisting of an aluminium sheet mounted over a


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glass plate. Electrospun non-woven mats were immersed in a solution of methanol: water (90:10 v/v) for 10 min to induce the β-sheet conformational transition of silk fibroin and were then washed with water for 24 h at room temperature to remove the electrospun fibrous mat from the aluminium sheet. The nanofibrous scaffold was immersed in 0.5 M of calcium chloride solution in tris buffer for 12 h at pH 10.4 and repeatedly washed with distilled water. The scaffolds were then immersed in 0.5 M of aq. Na2HPO4 solution in tris buffer for 12 h at pH 10.4 followed by washing with distilled water. The alternate soaking and rinsing process were repeated for 3/5 cycles which resulted in the precipitation of HAp over the nanofibres. The prepared HApcoated SF scaffold (SF/nHAp) was stored in a dessicator for further use.[20]


2.2. Characterisation of nanofibrous scaffold The nanofibrous mat was sputter-coated with gold (JEOL JFC fine coater) and analysed by field emission scanning electron microscopy (JEM2010, JEOL) to measure the fibre size. The transmission electron microscope analysis was performed to analyse its shape and the structural features of deposited hydroxyapatite. FT-IR spectroscopy (Automatic Infrared microscope, AIM-800, Shimadzu) was used to analyse the sample in scanning range, resolution and scan numbers of 4000–400, 8 and 60 cm−1, respectively. X-ray diffraction (XRD) analyses of SF and SF/nHAp fibres were performed for the scanning region of 2θ = 10°–80°. The TGA analyses of samples were performed between 30 and 600 °C with a scanning rate of 2 °C/min to identify the degradation behaviour. The ultimate tensile strength (UTS) of the nanofibrous silk scaffolds (50 mm × 12 mm × 0.3 mm) were tested in a universal testing machine (Instron 3369, Bioplus) with a 1 kN load cell under standard atmospheric conditions at 65% relative humidity, with a cross head speed of 10 mm/min. The swelling ratio and water uptake % of the nanofibrous scaffolds were determined in PBS at pH 7.4 and 37 °C after 1, 2, 3, 4, 5, 6, 7, 8, 24, 48 and 96 h using Equations (1) and (2). The dry and wet weights of the scaffold were denoted as WD and WT, respectively.[21] Swelling ratio ¼ Water uptake% ¼

WT WD WD

WT WD 100 WT

(1)

(2)

The hydrophilicity of scaffolds was determined by contact angle goniometer (DSA-10, Kruss188, Germany) using axisymmetric drop shape analysis (ADSA-NA) methodology.[22] The mean value of the contact angles was calculated from five individual measurements taken at different locations on the substrates at 25 °C and 65% humidity. The bioactivity of scaffolds was tested by immersing in 35 ml of simulated body fluid (SBF) for a period of 14 days at 37 °C. SBF was prepared according to the study by Zhu et al. [23]. After removal of the scaffold, it was washed and dried at room temperature and analysed by SEM. 2.3. Cell culture Non-woven nanofibrous scaffolds with a diameter of 0.6 and 0.3 cm thickness were autoclaved followed by washing with sterile PBS. Passage 4 CBhMSCs [18] at a concentration of 5 × 104 cells/cm2 were seeded using a static method and incubated at

1444 37 °C with 5% CO2; these growth factor following medium was replaced at 5 × 105 cells/ml in medium

N. Panda et al. were cultured with 2 ml of cell culture medium without any the previously described procedure.[24] The culture a regular interval of 3 days. Cell suspensions containing without scaffold were used as controls (n = 3).


2.3.1. Cellular attachment and viability The CBhMSCs-seeded scaffold was fixed with 3.7% formaldehyde (Sigma) for 20 min and diluted in blocking buffer at room temperature before being permeabilised for 10 min with 0.5% Triton X-100/PBS. Thereafter, cells were incubated in DPBS solution and blocked by blocking buffers (Pierce, Thermo Scientific) for 30 min to prevent non-specific antibody binding. Cytoskeletal F-actin was detected by Alexa488-conjugated phalloidin (1:200) at 37 °C for 40 min in dark condition. The nuclei were stained with 0.1 μg/ml of DAPI in PBS for 1 min. Then, the cell–scaffold constructs were mounted on glass slide and visualised under Leica TCS SP5 X supercontinuum confocal microscope.[25] 2.3.2. Expression of CMFDA dye by fluorescent microscopy Fluorescent dye 5-chloromethylfluorescein diacetate (CMFDA) (Life Technologies, Carlsbad, CA, USA Cat #c2925) produces a bright fluorescence upon cleavage of its acetates by cytosolic esterases, which is proportional to the metabolic activity of the cell. In brief, the culture media from a cell–scaffold construct was removed, and 180 ml of DMEM medium and 20 ml of CMFDA (25 mM) were added to the cells and incubated at 37 °C for 2 h. Thereafter, the dye–medium was replaced by complete medium and incubated overnight. After removing the culture medium, the cells were washed with PBS and observed under an upright fluorescence microscope (Carl Zeiss E600, wavelength = 450 nm) using a green filter (wavelength = 490 nm). 2.3.3. MTT assay Cellular metabolic activities of CBhMSCs seeded on scaffolds were evaluated by MTT after 3, 5, 7 and 10 days of culture following the protocol published in the literature.[26] 2.4. Cell proliferation study 2.4.1. SEM analysis The cell proliferation over the scaffold was visualised on the 7th and 14th days of culture using a JEOL JSM-840A scanning electron microscope. Briefly, the cell-seeded scaffolds were rinsed in DPBS, and fixed with 2.5% glutaraldehyde in DPBS overnight at 4 °C. The constructs were dehydrated through a gradient series of alcohol followed by aseptic critical point drying and coated with platinum before the SEM studies were performed. 2.4.2. Alamar blue assay The proliferation of CBhMSCs on the scaffolds was quantitatively assessed on days 7, 14, 21 and 28 of culture using the Alamar Blue assay. In brief, cell-seeded constructs were placed in fresh wells in phenol red-free serum-free medium and then Alamar Blue was added followed by overnight incubation. After pipetting the media, the OD was measured in a microplate reader at 570 nm.


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2.5. Expression of adhesion molecules (CD 44 and CD 29) through flow cytometry analysis The expression of CD44 and CD29 surface markers were quantified using a flow cytometer (Fortessa, BD Bioscience, USA) following the procedure described elsewhere.[27] Cell-seeded constructs were trypsinised and washed with PBS after 24 h of culture. The detached cell concentration was adjusted to 1 × 106 cells/ml by the addition of ice-cold PBS. Monolayer of cultured MSC grown over tissue culture plates were taken as control. Then, FITC-conjugated CD44 and CD29 surface markers were added to the cell suspension and analysed for CD expression using flow cytometry.


2.6. Cellular differentiation 2.6.1. ALP assay The ALP activities of CBhMSCs in constructs were measured on 7, 14 and 21 days of culture to assess the osteogenic differentiation, as per the procedure described elsewhere.[28] The constructs were repeatedly washed with PBS after the desired days followed by treatment with 1% Triton X-100 (Fisher Scientific) for 60 min and centrifuged at 10,000 rpm at 4 °C for 15 min. Then, 0.5 ml of supernatant was added to dilute the p-nitro phenyl phosphate (100 μl of p-NPP concentrate per 2 ml of 100 mM sodium bicarbonate/carbonate buffer, pH 10) and incubated at 37 °C for 45mins. The reaction was stopped by adding 50 μl of 1 N NaOH, and the absorbance of the mixture was measured at 405 nm.

2.6.2. Reverse transcriptase-PCR (RT-PCR) for quantitative determination of OCN, ONN, Runx2 and OPN Differentiated CBhMSCs were isolated to extract total RNA by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then 1 μg of total RNA was reversibly transcribed to cDNA synthesis using Superscript First-248 strand synthesis system, GibcoBRL, Life Technologies, following the protocol of the manufacturer. RT-PCR was performed to determine the expression of osteogenic differentiation genes such as OCN, ONN, OPN and Runx2 using primers given in Table 1. GAPDH, a house keeping gene, was used as internal control, and the PCR was carried out using BIO-RAD Mycycler and analysed by software Bio-Rad. Detail description of the procedure was described in the published literature.[29] All the electrophoresis images were analysed quantitatively by NIH Image J software and normalised to their respective GAPDH values for drawing the bar plot (sample n = 3). The morphological observations of CBhMSCs over the scaffold were visualised under a fluorescent microscope using FDA dye to observe cell morphology using a standard protocol for cell visualisation.

2.7. Statistical analysis Statistical significances were determined for all of the samples in triplicates, and p values were generated by ANOVA through the Bonferroni test with multiple comparisons to one control (p0.05 > 3 assay). This assay method relies on an assumption of normality and homogeneity of the variation of distribution.

1446 Table 1.

N. Panda et al. Primer sequences used for semi-quantitative PCR.

Gene primer sequences OCN OPN ONN Runx2


GAPDH

Product Forward: CCC AGG CGC TAC CTG TAT CAA Reverse: GGT CAG CCA ACT CGT CAC AGT C Forward: TCAGCATTTTGGGAA TGGCC Reverse: GAGGTTGTTGTCTTC GAGGT Forward: AGTAGGGCCTGGATC TTC TT Reverse: CTGCTTCTCAGTCAG AAGGT Forward: CAC TGG CGC TGC AAC AAG A Reverse: CAT TCC GGA GCT CAG CAG AAT Forward: GCA CCG TCA AGG CTG AGA AC Reverse: ATG GTG GTG AAG ACG CCA GT

Size (bp) Accession 112

NM_199173

666

J05213

575 127

BC072457 BC004974 NM_004348

142

NM_002046

3. Result and discussion 3.1. Characterisation of the nanofibrous scaffold Nanofibrous scaffolds closely mimic the natural ECM for tissue regeneration. The SEM image (Figure 1(a)) shows the precipitation of randomly agglomerated nHAp particles (>50 nm) not only over the ET nanofibrous scaffold but also from inside and below the fibre surface. The sample images are taken after several washes, hence this verified the strong bonding between nHAp crystals and the nanofibre surface, which is corroborated by TEM analysis (Figure 1(b) and (c)), showing semi-crystallinity and non-uniformity

Figure 1. (a) Scanning electron micrograph showing deposition of apatite particles over eri–tasar nanofiber (b) and (c) TEM micrographs showing growth of n-HAp crystals over the nanofibers. The particles show strong attachment with fibers (d) EDX analysis evaluates the ratio of Ca and P ions over the particles being 1.6.



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in shape. The elemental analysis (Figure 1(d)) showed that Ca and P were at a ratio of 1.6 confirming the apatite deposition. The FT-IR spectra of ET/nHAp (Figure 2(a)) shows the characteristic absorption peaks of 697, 612 and 563 cm−1 for o–p–o bending vibration at 958 cm−1 for p–o stretching vibration, indicating the presence of the PO3 4 groups, which are absent in the spectra of the ET nanofibrous scaffold. The C=O stretch vibration peak of amide I at 1658 cm−1 and N–H bending of the amide II band at 1524–1530 cm−1 can be observed for both scaffolds. It has been shown that the intensity of two amide peaks present in the ET scaffold is severely repressed in the ET/nHAp scaffold. The repression may be due to interactions between Ca2+ ions of HAp and C=O bonds in amino acids of the proteins.[30] The XRD analysis of the ET and ET/nHAp scaffolds (Figure 2(b)) showed diffraction peaks at 21° (2θ) and 29.54° (2θ), which correspond to α-helix structure and β-pleated sheet, respectively. A peak at 39.26° corresponds to the characteristic peak of deposited semi-crystalline hydroxyapatite.[31–33] The thermal stability of the ET and ET/nHAp composite scaffolds are shown in Figure 2(c). The weight loss for ET/nHAp scaffold was 6–7% compared to 10–12% for the ET scaffold at 100 °C, implying reduction in free water loss. The ET scaffold

Figure 2. (a) FT-IR, (b) XRD and (c) TGA analysis of SF blend and SF/nHAp nanofibrous scaffold showing difference in structural and thermal behavior. FT-IR study shows the presence of PO3 4 functional group and decrease in absorbance of C=O stretching vibration due to interaction with Ca2+. The XRD analysis shows semi crystalline nature of deposited HAp particles rather than fully crystalline one. (c) The TGA analysis found increase in stability of HAp coated ET nanofibrous scaffold compared to uncoated one.


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started to decompose around 250–300 °C, with residual weight loss of about 20–25%; at 350 °C, weight loss of about 45–50% indicated severe decomposition because of peptide bond breakage in amino acids. In contrast, the ET/nHAp scaffold showed decomposition above 350 °C with 15% residual weight loss, and about 30–35% weight loss was noticed at 375 °C. Due to the presence of inorganic salt in HAp over ET nanofibres, their thermal stability was improved, as reported previously.[31] The stress–strain graph of the mineralised nanofibrous scaffold (SF/nHAp with n = 4) (Figure 3(a)) shows the UTS and % extension 0.392 ± 0.14 MPa and 2.895 ± 0.31%, respectively, which are lower than the results for the ET scaffold (1.83 ± 0.21 MPa and 22.78 ± 1.75%, respectively; Applied patent: 713/KOL/2012), as reported in our previous work. Further calculation of stiffness and elastic modulus has been found to be 778 ± 2.4 N/m and 13.1 ± 0.36 MPa which is significantly higher than those from pure ET scaffold (160.6 ± 1.34 N/m and 8.3 ± 0.4 MPa). The decrease in extension % is significantly higher than the decrease in UTS which enhances the stiffness and elastic modulus of nanofibrous scaffold. An increase in elastic modulus and stiffness has shown to enhance the ability of scaffold to absorb impact effectively. The decrease in UTS may be attributed to the restricted free moments of nanofibres owing to the deposition of nHAp, making the nanofibrous matrix stiffer and less plastic in nature. The hard inorganic phase and high charge density of HAp also decrease the ductile nature of the ET/nHAp scaffold. However, the elastic modulus is still sufficient to direct the osteogenic differentiation of CBhMSCs. Similar findings are reported for PCL/HAp and PLA/HAp-electrospun scaffolds.[33–35] The deposition of nHAp has enhanced the rigidity and brittleness of the scaffold, which is clinically useful for retaining the shape during implantation and ideally bearing mechanical loads after implantation in bone.[36] Figure 3(b) demonstrates the increase in % of water uptake capacity and swelling ratio of the ET/nHAp nanofibrous scaffold, that is 80% and 3.6, respectively, after 96 h of treatment in PBS, which is higher than the results of the pure ET scaffold (38% and 1.9, respectively), thus indicating better hydrophilicity. This was further supported by the water contact angle measurement (53.4° ± 2.7°), which was less than that of the ET (57.4°) scaffold. Therefore, this composite scaffold has shown better surface properties

Figure 3. (a) Stress strain curve of HAp precipitated ET nanofibrous scaffold. (b) Water uptake capacity of ET and ET/nHAp scaffold during a period of 96 h of treatment in SBF. The HAp deposition show an improvement in water uptake capacity of nanofibrous scaffold.



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Figure 4. Bioactivity study. Scanning electron micrograph of (a) ET and (b) ET/nHAp scaffold after 14 days treatment in SBF. The ET scaffold show irregular deposition of nHAp, while ET/ nHAp shows a thick deposition of HAp.

for cell growth and proliferation as a result of its capacity to store and assimilate nutrients, growth factors and waste products.[23] The deposition of hydroxyapatite over a scaffold surface determines its osteogenic properties. Figure 4 shows the SEM images following apatite deposition over ET (a) and mineralised ET (b) scaffolds after 14 days of treatment in SBF. The irregular and thin depositions of HAp are observed on the ET scaffold surface compared to the deposition of a substantially thicker layer of nHAp, demonstrating the enhanced osteogenic property of the composite scaffold. This may be a result of previously deposited HAp, acting as a nucleus for growth of HAp crystals. 3.2. In vitro cell culture study 3.2.1. Cellular attachment and metabolic activity of CBhMSCs This study investigated the possibility of using non-mulberry silk fibroin (eri and tasar) and a hydroxyapatite nanocomposite as an osteogenic scaffold and determined its innate ability to home and differentiate CBhMSCs. The initial interaction between CBhMSCs and scaffolds are more decisive for the subsequent growth, proliferation and differentiation,[37] all of which depend on the physico-chemical and surface properties of the scaffold. Apatite crystals are reported to reduce cell attachment.[38] Furthermore, Kundu and colleagues showed that tasar silk favours the formation of chondral tissue. In this context, the tricomposite scaffold (tasar, eri and HAp) was investigated for cell attachment, proliferation and in the line of intimation to osseous tissue formation for the first time. Moreover, investigations were performed to observe the expression of early and late osteogenic differentiation markers.[39] The ability of the ET/nHAp scaffold to support the attachment of CBhMSCs was observed after 12 h of culture through immunofluorescence studies. Elongated and well-spread cells were observed to be deposited in layers and elongated in the direction of nanofibres (Figure 5(d)), suggesting a favourable substrate for growth and proliferation. Intimate cell–cell and cell–scaffold adhesions with pseudopods were clearly observed in the figure. The successful cell retention in the scaffold ensured that cells were not migrating away from the scaffold. Figure 5(a) and (b) demonstrates the relative number and metabolic activity of CBhMSCs grown over ET and ET/nHAp stained with CMFDA dye under a fluorescent microscope after 5 days of culture. The measured fluorescence intensity per cell was


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Figure 5. Fluorescence microscopic images (CMFDA dye image) showing cell viability and proliferation after 5 days on (a) ET and (b) ET/nHAp composite scaffold (50× magnification). Confocal laser scanning microscope images of (c) SF/nHAp scaffold (d) MSCs attachment over ET/nHAp nanofibrous scaffold. Arrow mark indicates cell-cell interaction and growth in layers. MSC cultures were stained for β-actin (Phalloidin cytoskeleton-green) and DAPI (nuclei, blue) scale bar 25 μm. (Please see the online article for the colour version of this figure: http://dx.doi. org/10.1080/09205063.2014.943548.)

comparatively higher in the case of the ET scaffold, indicating that the CBhMSCs were metabolically more active. Additionally, the cell number per area of observation in the ET scaffold was higher than that of the ET/nHAp scaffold. Further quantifications of cell viability were performed on days 3, 5, 7 and 10 days of culture using the MTT assay, which showed an enhanced growth rate over time (Figure 8(a)). Additionally, metabolically more active cells were found over ET scaffold as compared to ET/nHAp on day 5, 7 and 10. The cells were naturally metabolically more active in the G1 phase and directed cell division,[40] indicating the supply of an adequate amount of nutrients and cell mass to support the division of CBhMSCs. 3.2.2. Cellular proliferation The proliferation of CBhMSCs on ET and ET/nHAp scaffolds was quantitatively estimated for a period of 28 days by Alamar Blue assay (Figure 8(b)) and morphologically visualised on days 7 and 14 under SEM to investigate the effect of deposited nHAp. As indicated in Figure 6, substantially higher aggregation and overcrowding of spindleshaped elongated cells were observed on the 14th day of culture (c) and (d) when compared to day 7 (a) and (b) in both types of scaffold. Because of the bare visibility of scaffold surface and the deposition of salt-like white particles, we failed to assess the deposition of ECM over the scaffold. The Alamar Blue assay demonstrated that the



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Figure 6. SEM images show cellular proliferation of ET and ET/nHAp scaffold after 7 ((a) and (b)) and 14 days ((c) and (d)) of culture respectively. The cellular proliferation and aggregation are comparatively higher after 14 days as compared to 7 days of proliferation.

proliferation rate of CBhMSCs over ET/nHAp scaffold was comparable to ET scaffold. The rate of proliferation in both cases followed a decline beyond 14 days of growth. The slowdown of cellular metabolism and proliferation has also been reported previously.[41] The decrease in the rate of proliferation beyond 14 days may be a result of the transition from the proliferative phase to the differentiation phase. 3.3. Cell adhesion molecules: CD 44 and CD 29 (beta 1 integrin receptors) The expression of CD 44 and CD 29 is dependent on matrix environment which has a compelling role in cell retention and directing the CBhMSC towards a particular lineage.[42] Figure 7 depicts expression of these markers over CBhMSCs on ET and ET/ nHAp nanofibrous scaffolds by flow cytometry analysis after 24 h of culture. The expression level of these markers with and without FITC-conjugated CD 44 and CD 29 antibody were treated as positive and negative controls, respectively. The positive control showed 98 and 97.6% ((b) and (f)) for CD 44 and CD 29, respectively. The negative control showed 0 and 1.2% for CD 44 and CD 29 ((a) and (e)), respectively. The expression of CD 44 and CD 29 over CBhMSCs grown over ET are 89.3 and 94.1%, respectively. However, their expression reduces significantly to 67.7 and 71.3% in ET/ nHAp scaffold. The reduction in expression may be explained by the reduced availability of RGD ligands due to nHAp surface deposition. However, the increase in roughness, surface wettability and hydrophilicity because of HAp deposition may compensate the scaffold property for optimum expression these adhesion molecules.[43] 3.4. Osteogenic differentiation of CBhMSCs The matrix elasticity plays an important role in the osteogenic differentiation of CBhMSCs, that is an elastic modulus greater than 100 kPa promotes osteogenic lineages.[44] In contrast, a high density of cells (caused by proliferation) may affect the


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Figure 7. Expression of cell adhesion molecules CD 44 and CD 29 (beta 1 integrin) on CBhMSCs grown over fibronectin coated 96 well plate ((a) and (e) used as negative control, (b) and (f) used as positive control), ET (c) and (g) and ET/nHAp (d) and (h) nanofibrous scaffold. Though higher expression of both markers are observed for MSCs grown over ET, the MSCs grown over ET/nHAp express adequate amount of markers to facilitate cell adhesion.

differentiation process, even if the elastic modulus of the matrix is favourable for osteogenic differentiation.[45] ET/nHAp with a low proliferation rate may be better for osteogenic differentiation, than ET scaffold. 3.4.1. ALP assay CBhMSCs undergoing differentiation usually show high levels of ALP activity implying an early stage of differentiation. Figure 8 demonstrates the ALP activity of CBhMSCs over gelatine, ET and ET/nHAp composite scaffolds after 7, 14 and 21 days of culture. The absorption intensity, which is a measure of ALP activity, showed an increase in activity of 65 and 78% for ET and ET/nHAp during the period of 7–14 days, respectively. Notably, during the culture period from 14 to 21 days, it remained nearly constant. It may be argued, therefore, that the initial events of differentiation are consequently switched over to late stage differentiation and mineralisation of the scaffold. This triggering of osteogenic differentiation of CBhMSC followed by mineralisation provides a osteoinductive environment because of HAp.[46] The quantitative expression of genes related to osteogenic differentiation such as OCN (a late and specific marker of bone formation),[47] Runx2 (a critical transcriptional factor that regulates skeletogenesis),[48] ONN (a protein synthesised by cells of the osteoblastic lineage; binds hydroxyapatite and calcium) [49] and OPN (a highly sulphated, phosphorylated and glycosylated protein that mediates cell attachment through a RGD motif to extracellular matrices) were studied by RT-PCR analysis (Figure 9).[50] Figure 9(b) and (c) indicates the intensity of different genes with respect to GAPDH in a bar plot on 14th and 21th days of culture. The expression of OCN is 22 and 98.6% higher in ET/nHAp than ET after 14 and 21 days of culture. Similarly, the expression level of OPN is 28 and 76% higher in ET/nHAp than ET after 14 and 21 days of culture. The expression of ONN and Runx2 is 83 and 87% higher than their expression in ET after 14 days of culture. In contrast, their expression is 38 and 24% higher than ET after 21 days of culture, respectively. Runx2 is an important transcription factor for the



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Figure 8. (a) MTT assay after 3, 5, 7, 10 days on ET and ET/nHAp scaffold show comparatively more number of viable cells on ET nanofibrous scaffold. (b) Alamar blue assay for hMSC proliferation on ET and ET/nHAp composite scaffolds over a period of 28 days of cell culture shows comparatively similar cellular proliferation rate. They follow initial log phase followed by a stationary phase (c) hMSCs on ET and ET/nHAp composite scaffolds after 7, 14 and 21 days of culture show enhanced ALP activity over ET/nHAp as compared to ET nanofibrous scaffold.

commitment of multipotent mesenchymal cells into the osteoblastic lineage by triggering the gene expression of bone matrix, while hampering the transition of osteoblasts to osteocytes.[51] Its expression is upregulated in immature osteoblasts, but downregulated in mature osteoblasts. It regulates the expression of terminal differentiation marker OCN. A significantly higher expression of OCN after 21 days than 14 days clearly demonstrate the transition of osteoblasts to osteocytes. ONN is an important noncollagen calcium binding glycoprotein related to mineralisation at the early stage of bone formation secreted by osteoblasts. Its expression is comparatively higher in ET/ nHAp as compared to ET after 14th day than 21th day. Our data are supported by the previous findings that it is highly expressed at early stage of bone formation. OPN, another mineral-binding protein found in bone ECM, is associated with cell attachment, proliferation, and biomineralisation of ECM into bone and osteogenic lineage commitment.[50] The overall study demonstrates that directing osteogenesis is significantly better on ET/nHAp scaffold those on ET scaffold. The slow rate of proliferation and high differentiation potential of the ET/nHAp scaffold without the presence of soluble factors may be associated with some molecular pathways, which needs to be investigated in details.


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Figure 9. Electrophoretic RT-PCR analysis of OCN, OPN, ONN and Runx2 gene expression with respect to GAPDH from CBhMSCs cultured for 14 and 21 days of culture. Data presented are from three independent experiments (N = 3 total). **p < 0.05 and ***p < 0.01.

The higher osteogenic potential of ET/nHAp scaffold may be explained through the expression of calcium receptors over osteoblast-like cells.[52] These cells reside in the bone matrix which consists of hydroxyapatite crystals that play a significant role in osteogenic differentiation. It is reported that the adsorption of proteins and other biologically active molecules to HAp is different to that seen for ET scaffolds, which mediates the interactions between cells and scaffolds, resulting in the better osteogenic potential of the ET/nHAp scaffold.[53] Finally, Figure 10 shows the fluorescence images of differentiated CBhMSCs cultured over ET ((a) and (c)) and ET/nHAp ((b) and (d)), nanofibrous matrices on 21 and 28 days, which resemble osteocytes. The cell count confirmed that ET/nHAp scaffolds possess a maximum number of osteocytes compared to the ET scaffold, showing the improved osteogenic property of the ET/nHAp scaffold.

Figure 10. Fluorescence microscopy images (stained with FDA) of differentiated CBhMSCs over ET and ET/nHAp (a) and (c) after 21 days of culture and (b) and (d) after 28 days of culture over nanofibrous scaffolds respectively. The differentiated MSCs show osteocyte like morphology with maximum number of differentiated cells on ET/nHAp scaffold after 28 days (40× magnification).


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4. Conclusion The study has evaluated the physicochemical (physical stability and surface property) and biological (bone cell compatibility) properties of CBhMSC-seeded ET/nHAp and ET scaffolds as bone tissue engineering grafts. The nHAp precipitated over ET nanofibres was able to differentiate the seeded CBhMSCs into osteogenic lineages better than ET scaffolds. The significance of nHAp (>50 nm) deposition has been verified through the improvements seen in bioactive properties such as hydrophilicity and mineralisation. It has been observed that the cells firmly adhered and oriented themselves in the direction of fibres after 24 h The cell proliferation was decreased to a small amount, while the expression of ALP and expression of osteogenic genes were found to be enhanced, confirming a novel composite for the osteogenic differentiation of CBhMSC. Overall, the prepared nanocomposite can enhance osteogenesis through material characteristics without the application of growth factors, thereby minimising and reducing costs, which may benefit patients with osteoporotic diseases and bone injuries. Acknowledgements The authors acknowledges Department of Biotechnology, Govt. of India and Bose Institute of Nuclear Physics, Kolkata for providing the Lab facility for performing various characterisation facility.

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silk fibroin nanofibrous scaffolds for bone tissue engineering.

Mechanically-reinforced electrospun composite silk fibroin nanofibers containing hydroxyapatite nanoparticles.

Biocompatibility evaluation of electrospun silk fibroin nanofibrous mats with primarily cultured rat hippocampal neurons.

Characterization of dielectrophoresis-aligned nanofibrous silk fibroin-chitosan scaffold and its interactions with endothelial cells for tissue engineering applications.

poly(lactide-co-ε-caprolactone) nanofibrous scaffolds for bone regeneration.

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hydroxyapatite composites for bone tissue engineering.

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Optimization of nanofibrous silk fibroin scaffold as a delivery system for bone marrow adherent cells: in vitro and in vivo studies.

hydroxyapatite composite scaffold via solid freeform fabrication.

Collagen Nerve Scaffold Seeded with a Co-Culture of Schwann Cells and Adipose-Derived Stem Cells for Sciatic Nerve Regeneration.

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A novel silk fibroin nanofibrous membrane for guided bone regeneration: a study in rat calvarial defects.

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Potential Neurogenesis of Human Adipose-Derived Stem Cells on Electrospun Catalpol-Loaded Composite Nanofibrous Scaffolds.

nanohydroxyapatite nanofibrous membrane scaffolds.

Enhancing surface properties of breast implants by using electrospun silk fibroin.