Osteoinduction of bone grafting materials for bone repair and regeneration Elena Garc´ıa-Gareta, Melanie J. Coathup, Gordon W. Blunn PII: DOI: Reference:

S8756-3282(15)00279-3 doi: 10.1016/j.bone.2015.07.007 BON 10797

To appear in:

Bone

Received date: Revised date: Accepted date:

28 July 2014 3 July 2015 6 July 2015

Please cite this article as: Garc´ıa-Gareta Elena, Coathup Melanie J., Blunn Gordon W., Osteoinduction of bone grafting materials for bone repair and regeneration, Bone (2015), doi: 10.1016/j.bone.2015.07.007

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ACCEPTED MANUSCRIPT Osteoinduction of bone grafting materials for bone repair and regeneration

RAFT Institute of Plastic Surgery, Mount Vernon Hospital, Northwood HA6 2RN, UK

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Elena García-Garetaa,*, Melanie J Coathupb, Gordon W Blunnb

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John Scales Centre for Biomedical Engineering, Institute of Orthopaedics and

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Musculoskeletal Science, Division of Surgery and Interventional Science, University College

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London, Royal National Orthopaedic Hospital, Stanmore HA7 4LP, UK

Dr Elena García-Gareta

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RAFT

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*Corresponding author:

Leopold Muller Building

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Mount Vernon Hospital Northwood HA6 2RN United Kingdom

Tel: +44 1923 844555 Fax: +44 1923 844031 [email protected]

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ACCEPTED MANUSCRIPT Abstract Regeneration of bone defects caused by trauma, infection, tumours or inherent genetic

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disorders is a clinical challenge that usually necessitates bone grafting materials. Autologous

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bone or autograft is still considered the clinical “gold standard” and the most effective

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method for bone regeneration. However, limited bone supply and donor site morbidity are the most important disadvantages of autografting. Improved biomaterials are needed to match the performance of autograft as this is still superior to that of synthetic bone grafts.

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Osteoinductive materials would be the perfect candidates for achieving this task.

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The aim of this article is to review the different groups of bone substitutes in terms of their most recently reported osteoinductive properties. The different factors influencing

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osteoinductivity by biomaterials as well as the mechanisms behind this phenomenon are also

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presented, showing that it is very limited compared to osteoinductivity shown by bone morphogenetic proteins (BMPs). Therefore, a new term to describe osteoinductivity by

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biomaterials is proposed. Different strategies for adding osteoinductivity (BMPs, stem cells) to bone substitutes are also discussed. The overall objective of this paper is to gather the

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current knowledge on osteoinductivity of bone grafting materials for the effective development of new graft substitutes that enhance bone regeneration.

Keywords Osteoinductivity Bone biomaterials Ectopic bone formation Bone regeneration Review

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ACCEPTED MANUSCRIPT 1. Introduction Regeneration of bone defects caused by trauma, infection, tumours or inherent genetic

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disorders leading to abnormal skeletal development is a clinical challenge. Regeneration of

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bone is also required in spinal fusions and in defects caused by osteolysis adjacent to

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implants. Repair of such bone defects usually necessitates bone grafting materials. Autologous bone or autograft is still considered the clinical “gold standard” and the most effective method for bone regeneration as it promotes bone formation over its surface by

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direct bone bonding (osteoconduction) and induces local stem cells to differentiate into bone

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cells (osteoinduction) without any associated immune response [1]. In autologous bone grafting fresh cortical or trabecular bone or a combination of both, are transplanted from one

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site in the body, such as the iliac crest, to another within the same patient. However,

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autografting presents several disadvantages; with limited bone supply and donor site morbidity being the major drawbacks [1-3]. These disadvantages can be overcome by using

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allograft, transplanted cortical/trabecular bone or demineralised bone matrix from a donor tissue into the patient. Usually harvested from sections of the pelvis from cadavers or from

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removed femoral heads at primary total hip replacements, allogenic bone graft possesses osteoconductivity and when used as fresh frozen or in a demineralised form also presents limited osteoinductivity [3,4]. Disadvantages associated with allogenic bone grafts are disease transmission or bacterial infection. Differences in graft preparation techniques lead to inconsistency, immune response, fracture and non-union due to differences in the bone quality between the donor and the patient [3,4]. The use of bone substitutes or synthetic grafts aims at overcoming the disadvantages of using autologous and allogenic bone grafts. Natural bone serves as the model for the development of bone substitutes, which try to mimic the mineral composition of bone tissue or the structure of interconnected struts of trabecular bone [5,6]. Table 1 summarises the different

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ACCEPTED MANUSCRIPT groups of bone substitutes: natural and synthetic biodegradable polymers, ceramics including bioglasses, metals and composites. All of these materials have advantages as well as

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disadvantages (Table 1). For example, calcium-phosphate ceramics, the most popular bone

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substitutes due to their chemical similarity with bone mineral generally have good

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biocompatibility, osteoconductivity, low cost and are readily available. However these materials are stiff when compared to bone, brittle, present unpredictable dissolution rates in vivo and have limited osteoinductivity [6-8].

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Performance of autologous bone graft is still the gold standard and preferentially used and

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therefore, improved biomaterials are needed to match the performance of autograft. Osteoinductive synthetic materials that induce differentiation down the osteogenic lineage of

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local stem cells would be appropriate materials for replacing autograft [9].

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The historical background and definitions of osteoinduction were thoroughly reviewed by Barradas and co-authors in 2011 [10]. It is generally accepted and well established that an

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osteoinductive material should induce bone formation upon implantation in non-osseous sites, also known as heterotopic or ectopic sites [6,9,10]. The osteoinductive potential of new

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materials can be assessed in vitro by studying the osteogenic differentiation of undifferentiated mesenchymal stem cells or osteoprogenitor cells in contact with candidate bone graft substitutes [11-14]. We believe that while such in vitro tests give an indication of the potential osteoinductivity of a given material, confirmation of in vivo bone formation in ectopic sites is necessary before a material is classified as osteoinductive [15]. The aim of this paper is to review the different groups of bone substitutes in terms of their most recently reported osteoinductive properties. Different strategies for adding osteoinductivity to bone substitutes, e.g. by adding bone morphogenetic proteins (BMPs) or stem cells, are also discussed. The overall objective of this paper is to gather the current knowledge on osteoinductivity of bone grafting materials for the effective development of

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ACCEPTED MANUSCRIPT new osteoinductive bone substitutes that match the clinical performance of autologous bone

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graft, considered the “gold standard” for bone repair and regeneration.

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2. Polymers

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2.1 Natural polymers

Natural polymers such as collagen, fibrin, gelatin, starch, hyaluronic acid or chitosan offer the advantage of good biocompatibility and biodegradability as they compose the structural

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native extracellular matrix of tissues. Natural polymers are bioactive as they have the

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potential to interact with the host’s tissue. Some of them such as starch or chitosan offer the advantage of an almost unlimited source [7,8,16-19]. Electrospinning of natural polymers to

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biomimetic biomaterials [20].

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create extracellular matrix analogues is a promising research area towards the development of

Collagen is one of the most useful biomaterials with many biomedical applications such as

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drug delivery systems, nanoparticles for gene delivery and basic matrices for cell culture systems [12,21-23]. Osteoconductivity of collagen scaffolds was shown: anionic collagen

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matrices were able to heal bone defects in rats therefore demonstrating bone formation [24]. However, osteoinductivity of collagen or other natural polymers has not yet been reported. The great disadvantages of natural polymers are insufficient mechanical strength and high rates of degradation. Thus, they are often used in composites or are chemically modified to improve mechanical properties and degradation rates [7,8,16,25].

2.2 Synthetic polymers Synthetic polymers offer great versatility as they can have different porosities, pore sizes, degradation rates, mechanical properties and forms [7,8,16,17,25-27]. The most commonly used are poly-(α-hydroxyacids), such as poly-glycolic acid (PGA) and poly-lactic acid (PLA),

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ACCEPTED MANUSCRIPT and poly-(ε-caprolactone). The degradation products of these polymers are glycolic acid and lactic acid, which are naturally found in the human body and therefore are removed by

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natural metabolic pathways. The use of poly-(lactide-co-glycolide) (PLGA), which is a

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copolymer formed by PLA and PGA, for bone regeneration has been extensively studied and

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approved for human clinical use by the American Food and Drug Administration [28-30]. The main disadvantage of synthetic polymers is their poor mechanical properties, even when they are in the form of rods or solid screws, and have therefore been applied in low

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mechanical stress applications [7]. Another potential disadvantage is high local

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concentrations of acidic degradation products that can affect cell differentiation on the scaffolds in vitro and could induce an inflammatory response in vivo. Moreover, dissolution

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of the polymer is often accompanied by breakup into smaller particles which then dissolve

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inducing an inflammatory reaction [31-32]. The only synthetic polymer to date to have shown osteoinductivity is poly-

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hydroxyethylmethacrylate (poly-HEMA): subcutaneous bone formation upon implantation of poly-HEMA sponge in pigs was reported by Winter and Simpson in 1969 [33]. A year later

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Winter described the same osteoinductive effect by poly-HEMA sponge subcutaneously implanted in rats [34]. Interestingly, in both cases the poly-HEMA sponge calcified after implantation but before any cell differentiation into bone could be identified, suggesting for the first time that calcification and therefore calcium-phosphates may be important for osteoinduction [9,10,35].

3. Ceramics Ceramics have been widely used in the biomedical engineering field and for clinical applications for many years. As biodegradable polymers they can be from a natural or a synthetic origin and can be synthesized to different forms, porosities, pore sizes or

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ACCEPTED MANUSCRIPT topographies. An example of natural ceramics is coralline hydroxyapatite (HA) while synthetic HA or β-tricalcium phosphate (β-TCP) are among the synthetic ceramics more

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commonly used [7,16,36-38].

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3.1 Calcium-phosphate ceramics

Calcium-phosphate (CaP) ceramics resemble the bio-minerals that are naturally found in the body as part of bone or teeth [5]. The main properties that these CaP materials offer are

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excellent biocompatibility, biodegradability and osteoconductivity [6,7,16,36,37,39]. CaP

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materials have been shown to be able to form a bioactive apatite layer on their surfaces thus enhancing osteointegration [6,36,40]. Another reason for the good osteointegration shown by

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these materials in vivo is that natural cytokines and adhesive proteins such as fibronectin are

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able to bind to CaP materials. The proteins and cytokines adsorbed to a scaffold surface and provide a matrix for cell attachment [41,42].

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Generally, CaP materials are regarded as not osteoinductive as they are not able to form bone de novo [6]. However, Zhang et al. demonstrated that more bone formation in non-osseous as

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well as osseous sites was obtained using HA with a 75-550µm pore size and 60% porosity [43]. Similarly, Yuan and colleagues reported bone formation in CaP materials with a microporous structure when implanted into the muscles of dogs [44]. These studies, among many others [45-56], suggested that CaP materials can show osteoinductive properties when they exhibit specific chemical and structural characteristics. Indeed, the influence of macrostructure (i.e. macro-porosity, concavities) has been demonstrated in several studies [44-51] where bone formation always occurred in the inner pores and on the concave surfaces and never on the convex surface. Interestingly, it has been shown in vitro that osteoblasts grown on grooved surfaces on biocompatible materials mineralise preferentially within the groove rather than on the flat surface [57]. The influence

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ACCEPTED MANUSCRIPT of surface structure (i.e. micro-porosity, strut porosity, particle size) on in vivo osteoinductivity has also been demonstrated in a number of publications [52-56,58]. As an

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example, the effect of strut porosity, micro-pores within the struts of the material, has been

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studied by Chan and colleagues and Coathup and colleagues [54,56], that have shown that

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bone grafting materials with greater strut porosity (30% versus 20% in the study by Coathup et al. and 46% compared to 22.5% in Chan et al.) are more osteoinductive. In both studies, materials were studied in an ectopic ovine model, inserted into the paraspinalis muscle.

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Chemical composition also affects the osteoinductivity of calcium-phosphate ceramics.

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Coathup and co-workers showed that silicate substitution may increase osteoinduction [53,54] while Wang and colleagues very recently reported that phase composition of calcium-

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phosphate ceramics may regulate the amount of osteoinductive factors in the local

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microenvironment of the implant [59]. Strontium, a trace element in the human body of special interest in the treatment of osteoporosis, shows a role in both the stimulation of bone

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formation and the reduction in bone resorption and has recently received attention for the fabrication of strontium substituted calcium-phosphate ceramics. These materials have shown

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higher bone formation in osseous sites than the non-substituted calcium-phosphate ceramic controls [60-62]. Whether strontium substitution affects osteoinductivity or not has not been reported so far.

Thus, it can be concluded that when CaP materials present certain chemical compositions, specific surface structures, geometries or pore sizes they are osteoinductive [6,63]. Table 2 offers a summary of the different factors that influence osteoinductivity by biomaterials and the proposed explanations for the observed influence.

3.2 Bioglasses and glass-ceramics

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ACCEPTED MANUSCRIPT In spite of their brittleness, bioglasses and glass-ceramics present unique properties. They are biodegradable and their degradation rate can be controlled. As they degrade they release ions

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that can promote osteogenesis and angiogenesis. More importantly, they convert to a

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biologically active carbonated HA material that firmly binds to both hard and soft tissues

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[64,65]. Bioglasses exhibit an amorphous structure while glass-ceramics are crystallised glasses. There is a relationship between mechanical strength and bioactivity: while the crystalline glass-ceramics are mechanically stronger they possess greatly reduced bioactivity

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compared to the amorphous bioglasses. Thus, amorphous bioglasses are also referred to as

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bioactive glasses [64-67]. Different types of bioactive glasses include silicate glasses such as 45S5 or 13-93, which have been shown to support proliferation and differentiation of osteoblast precursor cell lines and bone marrow stromal cells in vitro [68.69], or

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borate/borosilicate glasses such as 13-93B2, 13-93B3 or Pyrex®. Cell proliferation and

infiltration [72].

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differentiation in vitro is supported by borate glasses [70,71] as well as in vivo tissue

Osteoinductivity of porous 45S5 bioactive glass was reported by Yuan and colleagues who

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showed that in vivo bone formation in a non-osseous site was preceded by calcification [73], as with poly-HEMA sponge implants [33,34]. In vitro studies suggest that the osteogenic properties of 45S5 bioactive glass are due to their dissolution products which stimulate osteoprogenitor cells at the genetic level [67,69]. Interestingly, although 13-93B borate glasses have not been shown to be osteoinductive, greater bone formation after 12 weeks in critical-sized calvarial defects has been reported for 13-93B3 glass than for 45S5 silicate glass. However, blood vessel area was significantly higher for 45S5. Both 45S5 and 13-93B3 glasses completely converted to HA in vivo [74]. The only other bioglass that shows osteoinductivity is Pyrex®, which as early as in 1960, was shown to form bone 60 days after subcutaneous implantation in rats of 30mm diameter by

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ACCEPTED MANUSCRIPT 20mm length Pyrex® glass tubes [75]. Histological analysis also revealed the presence of

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cartilage and haematopoietic tissue along with bone inside the Pyrex® glass tubes.

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3.3 Other ceramics

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Yuan and colleagues reported osteoinduction of porous alumina ceramics [76], which are able to form an apatite layer in vitro when immersed in a supersaturated calcium-phosphate

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solution [77].

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4. Metals

Metals possess excellent mechanical properties that make them ideal candidates for load-

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bearing orthopaedic applications [7]. Investigation into porous metals is very active as they

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provide bone ingrowth potential that leads to early fixation of the implant. These metals are morphologically and mechanically similar to trabecular bone [78] and disadvantages include

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lack of tissue adherence, which may result in implant loosening with a necessary second surgery to remove it [79], and a risk of toxicity due to accumulation of metal ions from

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corrosion which is increased due to the high surface area associated with porosity [80-82].

4.1 Titanium and its alloys Because of their biocompatibility, strength, lightness and high resistance to corrosion titanium and its alloys are the metal materials more commonly used for biomedical applications [83,84]. The biocompatibility of titanium materials is based on a thin titanium dioxide (TiO2) layer formed on the surface of the bulk material. Titanium is a very reactive element even at room temperature. Therefore a newly polished titanium surface will have a thin layer of TiO2. Coating of titanium implants with a TiO2 improves cell adhesion and osseointegration[85-86]. Porous titanium materials have been developed to achieve material

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ACCEPTED MANUSCRIPT properties compared to bone [83,87,88] and new families of titanium alloys are constantly under research [89]. Osteoinductivity of titanium was demonstrated by Fujibayashi and

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colleagues when they implanted 4 different types of titanium implants in the dorsal muscles

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of mature beagle dogs: porous blocks and fibre mesh cylinders that were chemically treated

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(immersion in aqueous 5M NaOH solution at 60°C for 24h), followed by a thermal treatment (heating to 600°C at a rate of 5°C/min, maintained at 600°C for 1h and then allowed to cool down to the natural rate of the furnace) to create a bioactive surface composed of micro-

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pores. The non-treated implants displayed smooth surfaces. Bone formation was only

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demonstrated on treated porous blocks after 12 months of implantation and was observed to appear within the pores and extend throughout the porous network [90]. Only the treated

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implants had shown formation of an apatite layer in vitro. The authors concluded that the

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complex interconnective macro-porous structure of the porous blocks, the 3D surface microporous structure and the ionic concentration of calcium and phosphorus played an important

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role in the displayed osteoinductivity of this material. This study highlighted the importance of calcium-phosphate and structural characteristics on the osteoinductive potential of

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materials, already seen for calcium-phosphate ceramics.

4.2 Porous tantalum or trabecular metal Trabecular metal, a highly porous (80%) biomaterial made of porous tantalum with structural and mechanical resemblance with trabecular bone, was approved by the Foods and Drugs Administration for use in acetabular cups in 1997 [78,91]. The ingrowth potential of this material was demonstrated [92] and has been used in primary and in revision total hip arthroplasty components with excellent early clinical results [93-96]. Although osteoinductivity of tantalum materials has not been reported yet, Wang and colleagues showed that by modifying the tantalum surface with a coating of tantalum oxide (Ta2O5)

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ACCEPTED MANUSCRIPT nano-tube films improved the anticorrosion, biocompatibility and osteoinductive potential in vitro of pure tantalum [97], suggesting that by modifying pure tantalum surfaces with Ta2O5

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nano-coatings this metal could become osteoinductive in vivo.

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4.3 Other metals

Recently, magnesium and its alloys have been receiving great interest as biomaterials for orthopaedic applications. These materials possess elastic moduli (41-45GPa) closer to those

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of cortical bone (12-18GPa) than titanium materials (110-117GPa) and show good

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biocompatibility and biodegradability [98,99]. As magnesium is one of the reported substituting ions (Mg2+) in bone mineral [5], the release of magnesium ions from these

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materials could benefit cell attachment, proliferation and migration of bone cells [100].

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Several in vivo studies have demonstrated that magnesium based materials promote bone formation when implanted in osseous sites [101-103]. However, bone formation of

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5. Composites

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magnesium-based materials in non-osseous sites has not yet been shown.

Each individual material discussed so far in this review has its advantages and drawbacks as bone grafting materials for bone regeneration and repair, which could be overcome by combining different materials. For instance, Kasuga et al. fabricated a composite consisting of the synthetic polymer PLA and calcium carbonate. The resulting composite showed no brittleness and an improved modulus of elasticity compared to that of PLA alone. Moreover, the composite was able to form a bone-like apatite layer on its surface when soaked in simulated body fluid, thus showing bioactive and osteoconductive potential [104]. PLGA/HA composites have been shown to be osteoconductive [105] and fibrin based scaffolds with incorporated nano-crystalline HA supported bone formation when used in a mouse calvarial

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ACCEPTED MANUSCRIPT defect model [106]. Recently a combination of three natural polymers (gelatin, hyaluronic acid and alginate) into a 3D porous composite scaffold, synthesized by freeze-drying

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followed by ionic crosslinking with CaCl2, showed high load bearing capacity without

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fractural deformation [107].

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We have seen that few of the materials show osteoinductivity, mostly CaP ceramics, when they present certain chemical compositions, macrostructure and surface characteristics (Table 2). Several strategies have been developed to make composite materials with potential

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osteoinductivity. Ceramic based coatings on metals are a popular strategy for such means as

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these coatings increase the biocompatibility and osteointegration of metals [108-116] and in some cases are osteoinductive [117-118]. Combination of ceramics with natural [119-122],

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synthetic [15,123-126] or both types of polymers [127,128] is another promising strategy:

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PLA/HA composites have been shown to be osteoinductive by different groups [15,123,124]. In two of these studies the HA was nano-sized [15,124] while in another the composite had an

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average interconnective pore diameter of 62µm [123]. Finally, Fricain and colleagues recently reported osteoinduction of a nano-HA-pullulan/dextran polysaccharide composite

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with macro-porosity and 37% porosity after 30 and 60 days of subcutaneous implantation in mice and after 1 and 6 months of intramuscular implantation in goat[129]. In both models, no ectopic bone formation was seen for the polysaccharide matrices without nano-HA.

6. Adding osteoinductivity to bone grafting materials So far we have reviewed the different groups of materials used for bone repair in terms of their osteoinductive properties. We can draw the conclusion that osteoinductivity by biomaterials is an isolated phenomenon heavily influenced by chemistry and structure. In terms of how the ectopic bone is formed with calcium phosphate materials the mechanism for osteoinduction is usually intramembranous and seen inside pores. The majority of the studies

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ACCEPTED MANUSCRIPT describing bone formation by biomaterials in ectopic sites cited so far in this review use large animal models where the ectopic bone formation is slow, often requiring months (Table 3).

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For example, in the mentioned study by Chan et al [56], osteoinduction was only identified

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after 8 weeks of implantation and the maximal amount of bone formation within the silicate

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bone graft substitute was 30.00% after 24 weeks. A way of reducing the time for osteointegration and increasing the overall amount of bone formation would be extremely clinically relevant. Moreover, after implantation of hydroxyapatite specimens in the rectus

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abdominus of adult rabbits, dogs and baboons Ripamonti reported minimal amounts of new

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bone formation in rabbits and dogs while substantial bone formation occurred in baboons [47]. Combination of the different materials into composites also shows limited potential for

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osteoinductivity. Therefore, other strategies are necessary to add osteoinductivity to bone

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grafting materials. Scientists have been focusing on two strategies for this purpose: the use of

6.1 BMPs

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BMPs and seeding of scaffolds with stem cells to create bone tissue engineered constructs.

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BMPs are grouped into the TGF-β superfamily due to their similarities in protein structure and sequence with TGF-β. Back in 1965, Urist discovered that demineralised bone matrix (DBM) could induce bone formation when implanted ectopically in subcutaneous tissue in rats [130]. This capability was later attributed to a protein called bone morphogenetic protein, which was purified in 1984 based on its potential to induce bone formation [131,132]. Unlike calcium phosphate materials, bone formation with BMPs usually occurs by endochondral ossification. Growth factors, cytokines secreted by many cell types that function as signalling molecules, promote and/or prevent cell adhesion, proliferation, migration and differentiation by up-regulating or down-regulating the synthesis of proteins, growth factors and receptors. They are essential for tissue formation and play an important role in tissue repair [133-136].

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ACCEPTED MANUSCRIPT Bone tissue possesses a plethora of growth factors, including BMPs, fibroblast growth factors (FGFs), insulin growth factor I and II (IGF I/II) and platelet derived growth factor (PDGF).

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The most heavily studied cytokines are BMPs [8,135]. In 1988, Wozney and colleagues

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cloned these molecules and since then over 30 different BMPs have been identified with

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promising efficacy as therapeutic molecules for bone formation [134,137,138]. It is well established that bone formation by BMPs is easily induced in small animals, as early as 2 to 3 weeks after ectopic implantation in rodents, and that the new induced bone is

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the carrier (Table 3) [9,10,46,139-142].

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observed on the periphery of the carrier and even in the soft tissue distant from the surface of

However, multiple questions regarding a suitable carrier for BMPs, dosage, repeat exposure,

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carcinogenesis and long-term results have hampered the potential benefits these molecules

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could offer for bone formation [143]. Moreover, there has been concern over the use of BMPs for anterior cervical spinal fusion where inflammation has induced pressure on the trachea

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leading to death of patients in a number of instances [144]. This may be associated with the use of excessive amounts of BMPs which lead to many fold increase in local concentrations

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compared with natural levels when commercial preparations are used. Therefore, extensive research is being carried out on incorporating BMPs into biomaterials as scaffolds and delivery systems [145-154]. Another approach is gene therapy to engineer populations of progenitor cells over-expressing the production of BMP, which offers the advantage of continuous delivery of cytokines during a prolonged period [134,135,138,155-157]. Several studies have demonstrated that osteoinductivity can be added to materials used for bone repair by immobilising BMPs throughout their structure [147,149,151,158,159]. Lu and colleagues spatially immobilised BMP-4 in a collagen/PLGA composite using a fusion BMP4 that was composed of an additional collagen-binding domain derived from fibronectin. The

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ACCEPTED MANUSCRIPT BMP-4-collagen/PLGA material showed strong osteoinductivity in vivo [147]. Earlier, Kim and co-workers demonstrated ectopic and orthotopic bone formation of BMP-2 protein

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immobilised in an absorbable collagen sponge [149]. Recently Stenfelt et al. fabricated

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chemically cross-linked hyaluronan based hydrogels loaded with HA and BMP-2 that after

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implantation for 5 weeks in an ectopic site showed cancellous bone formation 151]. Studies such as the ones discussed here show the potential for fabricating hybrid materials using

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BMPs for added osteoinductivity.

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6.2 Stem cells

Stem cells are undifferentiated cells, capable of self-renewal and production of a large

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number of undifferentiated progeny. They have a high proliferation capability and multi-

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lineage differentiation potential; therefore they are involved in the regeneration of tissues [133,160]. Embryonic stem (ES) cells are pluripotent as they can differentiate into a wide

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range of cell types, a plasticity that is essential in the early development of the embryo [133,161,162], while adult stem cells are found in the fully differentiated tissues and are

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responsible for the regeneration of damaged tissue and the maintenance of tissue homeostasis. Adult stem cells have been found in the bone marrow, periosteum, muscle, fat, umbilical cord blood, placenta, brain or skin [133,160,163]. In 2006 Takahashi and Yamanaka reported the seminal discovery that differentiated cells can be reprogrammed to a pluripotent state by introducing transcription factors that maintain the pluripotency in both early embryos and ES cells [164]. Such reprogrammed cells are named induced pluripotent stem cells (iPSCs) and offer exciting features for regenerative medicine such as autogenicity and ease of ethical issues. All of the above types of stem cells have been used in combination with biomaterials to create tissue engineered constructs with great potential for bone regeneration [165-169].

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ACCEPTED MANUSCRIPT Korda and colleagues showed in 2010 that mesenchymal stem cells from bone marrow used in combinations of allograft and HA and subjected to forces used during impaction

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allografting enhanced ectopic bone formation in a ovine model [168]. Recently, a study by He

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et al. reported that osteogenically induced bone marrow mesenchymal stem cells in

7. Discussion and concluding remarks

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bone formation and scaffold biodegradation [167].

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combination with a chitosan scaffold and implanted into SD rat thigh muscles showed ectopic

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As stated in the introduction the objective of this review article is to gather the current knowledge on osteoinductivity so effective bone substitutes that mimic the performance of

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autograft can be developed.

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Osteoinductivity by biomaterials appears to be a local phenomenon influenced by chemical composition and structure [6,15,51-56,63-74,90,123,124]. The level of osteoinductivity

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achieved with biomaterials appears to be very limited when compared with BMPs [9,10,56]. Therefore, hybrid materials incorporating BMPs for added osteoinductivity seems the

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obvious strategy to follow. Yet, questions over dosage and long terms results remain unsolved for BMPs [143,144]. Alternatively stem cells have also shown the potential of adding osteoinductivity to a priori non-osteoinductive biomaterials [167,168]. However, the development of cell constructs is laborious and, in the case of autologous cells, may need a preliminary additional procedure to obtain the cells. Unless allogenic cells can be used, a biomaterial without added stem cells would be ideal in terms of “off the shelf” availability. Understanding the mechanism of osteoinduction by biomaterials is key to develop such bone substitutes. Unfortunately, the number of available studies showing ectopic bone formation by biomaterials is limited and understanding the mechanism of bone induction in these materials is unknown. Chemistry, porosity and shape of the graft influences osteoinduction.

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ACCEPTED MANUSCRIPT Studies to understand these mechanisms require in vivo work in large animals which take months to complete and therefore progression in this area is slow. The development of a

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standard in vitro model to test osteoinductivity as applicable to clinical use would be greatly

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advantageous.

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Several groups have attempted to develop an in vitro model of osteoinductivity [10,170-172]. Important factors to take into account are cell type (stem cells, osteoprogenitor cells, fully differentiated cells, co-culture of different cell types), culture conditions (media with or

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without osteogenic or angiogenic factors, calcium/phosphate enriched media, static or

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dynamic culture, mechanical stimulation, electrical stimulation, timeframe) and which output parameters should be measured at the end or throughout the culture period (expression of osteogenic markers such as ALP, Runx-2 or osteocalcin, calcium and phosphate ions release,

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mineralisation, expression of inflammatory markers such as TNF-α or IL-6, BMPs binding/release, deposition of a bone-like extracellular matrix with collagen type-I) (Fig. 1).

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Unfortunately, more work in this area is needed to develop a reliable and consistent in vitro model of osteoinductivity.

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Finally, we have seen that the mechanisms behind osteoinductivity by biomaterials or by BMPs are very different. It is important to realise that bone formation induced by BMPs is much faster, and it is more reproducible than that seen on the surface of CaP materials. The clinical reliability associated with osteoinductivity for BMPs is therefore much greater than other materials. Osteoinductivity by bone grafts is limited, late and until now not proven to provide meaningful clinical osteoinduction. Osteoinductivity for CaP bone grafts substitutes does occur although it is a bio-product associated with using these materials as an osteoconductive graft. Even DBM does not show as great osteoinductivity as BMPs and just as with CaP materials the attractiveness of using DBM is also that structurally acts as a scaffold. Even autograft, which contains osteoprogenitor cells, stem cells and osteoblasts

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ACCEPTED MANUSCRIPT together with bone extracellular matrix that may contain low levels of BMPs is not as osteoinductive as BMPs. The downside with BMPs is the selection of an appropriate dose

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together with the use of a scaffold that provides maximum osteoinduction.

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The question therefore arises as to whether ectopic bone formation by biomaterials should be

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called osteoinduction at all or a new term should be coined to name this phenomenon. Since the end result is the same in both processes, ectopic bone formation, we believe that a more descriptive expression such as “limited osteoinduction” or “delayed osteoinduction” should

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be used when describing osteoinductivity by biomaterials.

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The current knowledge on osteoinductivity by biomaterials should be a starting point from which the continuous investigation of this clinically important area should progress. Recent

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advances in fabrication techniques and additive manufacturing (not reviewed here as they are

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not within the scope of this paper; see references 173-178 for comprehensive reviews on

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substitute.

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these fields) will be key for tuning the structural properties of the “perfect” bone graft

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ACCEPTED MANUSCRIPT Acknowledgements This work was supported by the Restoration of Appearance and Function Trust (UK,

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registered charity number 299811) charitable funds.

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The authors have no conflicts of interest to declare.

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Disclosure

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ACCEPTED MANUSCRIPT Tables and figures legends

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Table 1. Bone grafting materials used for bone repair and regeneration: examples, advantages

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and disadvantages.

Table 2. Factors that influence osteoinduction by biomaterials: proposed explanations and

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examples.

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Table 3. Mechanism of osteoinduction by biomaterials and bone morphogenetic proteins

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(BMPs).

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Figure 1. In vitro model of osteoinductivity and factors to take into account: cell type,

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culture conditions and measurable output parameters.

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ACCEPTED MANUSCRIPT



POLYMERS

  Synthetic     

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Calciumphosphate

 

CERAMICS

    

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Bioglasses and Glassceramics

  

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Others

METALS



     

COMPOSITES

 

 

Biodegradability Biocompatibility Versatility



 

REFS

Low mechanical strength High rates of degradation High batch to batch variations

7,8,12,16 -25

Low mechanical strength High local concentration of acidic degradation products

7,8,16,17 ,25-34

Brittleness Low fracture strength Degradation rates difficult to predict

6,7,16,36 -56, 5974,76,77

Lack of tissue adherence Corrosion Risk of toxicity due to release of metal ions

7,78-103

Combination of the above

7,104129

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Biodegradability Biocompatibility Bioactivity Unlimited source (some of them)

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Natural

   

DISADVANTAGES

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

Protein: collagen, fibrin, gelatin, silk fibroin Polysaccharides: hyaluronic acid, chondroitin sulphate, cellulose, starch, alginate, agarose, chitosan, pullulan, dextran Poly-glycolic acid (PGA) Poly-lactic acid (PLA) Poly-(ε-caprolactone) (PCL) Poly-(lactide-coglycolide) (PLGA) Polyhydroxyethylmethacr ylate (poly-HEMA) Coralline or synthetic hydroxyapatite (HA) Silicate-substituted HA β-Tricalcium phosphate (β-TCP) Dicalcium phosphate dehydrate (DCPD) Silicate bioactive glasses (45S5, 13-93) Borate/borosilicate bioactive glasses (13-93B2, 13-93B3, Pyrex®) Alumina ceramic (Al2O3)

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

ADVANTAGES

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EXAMPLES

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BONE GRAFTING MATERIALS

Titanium and its alloys Tantalum Stainless Steel Magnesium and its alloys Calcium-phosphate coatings on metals HA/Poly-(D,Llactide) HA/Chitosan-gelatin



 

Biocompatibility Biodegradability Bioactivity Osteoconductivit y Osteoinductivity (subject to structural and chemical properties)

  

Excellent mechanical properties (high strength and wear resistance, ductility) Biocompatibility



Combination of the above



 

Table 1

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REFS

 Dissolution/reprecipitation of relevant ions for bone formation  Formation of a bone-like apatite layer on the surface of the biomaterial  Calcium-phosphate coatings and biomaterials direct mesenchymal stem cells towards the osteogenic lineage

Calcium-phosphate Silicon substitution in calciumphosphate ceramics Strontium substitution in calcium-phosphate ceramics? (only in vivo data on osseous sites) Phase composition of calciumphosphate ceramics Ta2O5 nano-tube film coating on pure tantalum? (only in vitro data) Magnesium materials? (only in vivo data on osseous sites)

15,3335,43-56, 59-62,6474, 98-103, 114-118



Macrostructure

Invasion of blood vessels: supply of nutrients and oxygen and removal of waste products and metabolites Infiltration of cells and tissue Protection against high mechanical forces or body fluid shear stress

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 

Table 2

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 Increased surface area for binding of proteins including BMPs, dissolution/reprecipitation of ions, mineral deposition from body fluids  Nano-topography directs mesenchymal stem cells towards the osteogenic lineage

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Porosity: macro-pores Porosity: interconnectivity

6,9,10, 45-50, 90,123

Geometry: concavities Micro-porosity Strut porosity Grain size Particle size of injectable biomaterials Nano-porosity

15,41-44, 52-56, 90,114,124

Nano-topography

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Surface structure

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EXAMPLES

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Chemical composition

PROPOSED EXPLANATIONS

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FACTORS THAT INFLUENCE OSTEINDUCTION BY BIOMATERIALS

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ACCEPTED MANUSCRIPT By Biomaterials

By BMPs

Intramembranous

YES (Always)

YES

Endochondral

NO

Small animals (rodents)

YES (Rarely)

YES

YES (Mostly)

YES

YES (Always)

NO

Rapid bone formation (2 to 3 weeks)

NO

YES (Always)

Periphery of implant/carrier

NO

YES

YES (Always)

YES

NO

YES

Animal model Large animals (dog, goat, sheep, baboon, rabbit)

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Slow bone formation (months)

Innner pores of implant/carrier

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Location of new ectopic bone

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Timeframe

YES (Mostly)

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In distant soft tissue from implant/carrier

Table 3

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Type of bone formation

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MECHANISM OF OSTEOINDUCTION

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ACCEPTED MANUSCRIPT

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Figure 1

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ACCEPTED MANUSCRIPT Ref No.: BONE-D-14-00678R1. Review title: Osteoinduction of bone grafting materials for bone repair and regeneration.

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Authors: Elena García-Gareta, Melanie J Coathup, Gordon W Blunn.

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Highlights

 Autograft is still the clinical “gold standard” for regeneration of bone defects.  Autograft has major limitations such as limited supply and donor site morbidity.

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 Osteoinductive bone grafting materials could match the performance of autograft.

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 Osteoinductivity shown by biomaterials is very limited compared to BMPs.

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 Current knowledge should be used for effective development of new biomaterials.

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