Rational synthesis of a heparan sulfate saccharide that promotes the activity of BMP2.

Heparan sulfate (HS) is a glycosaminoglycan (GAG) found throughout nature and is involved in a wide range of functions including modulation of cell signalling via sequestration of growth factors. Current consensus is that the specificity of HS motifs for protein binding are individual for each protein. Given the structural complexity of HS the synthesis of libraries of these compounds to probe this is not trivial. Herein we present the synthesis of an HS decamer, the design of which was undertaken rationally from previously published data for HS binding to the growth factor BMP-2. The biological activity of this HS decamer was assessed in vitro, showing that it had the ability to both bind BMP-2 and increase its thermal stability as well as enhancing the bioactivity of BMP-2 in vitro in C2C12 cells. At the same time no undesired anticoagulant effect was observed. This decamer was then analysed in vivo in a rabbit model where higher bone formation, bone mineral density (BMD) and trabecular thickness were observed over an empty defect or collagen implant alone. This indicated that the HS decamer was effective in promoting bone regeneration in vivo.

Affinity-selected heparan sulfate collagen device promotes periodontal regeneration in an intrabony defect model in Macaca fascicularis.

It is challenging to regenerate periodontal tissues fully. We have previously reported a heparan sulfate variant with enhanced affinity for bone morphogenetic protein-2, termed HS3, that enhanced periodontal tissue regeneration in a rodent model. Here we seek to transition this work closer to the clinic and investigate the efficacy of the combination HS3 collagen device in a non-human primate (NHP) periodontitis model. Wire-induced periodontitis was generated in ten Macaca fascicularis, and defects were treated with Emdogain or collagen (CollaPlug) loaded with (1) distilled water, (2) HS low (36 µg of HS3), or (3) HS high (180 µg of HS3) for 3 months. At the endpoint, microscopic assessment showed significantly less epithelial down-growth, greater alveolar bone filling, and enhanced cementum and periodontal ligament regeneration following treatment with the HS-collagen combination devices. When evaluated using a periodontal regeneration assessment score (PRAS) on a scale of 0-16, collagen scored 6.78 (± 2.64), Emdogain scored 10.50 (± 1.73) and HS low scored 10.40 (± 1.82). Notably, treatment with HS high scored 12.27 (± 2.20), while healthy control scored 14.80 (± 1.15). This study highlights the efficacy of an HS-collagen device for periodontal regeneration in a clinically relevant NHP periodontitis model and warrants its application in clinical trials.

A Heparan Sulfate Device for the Regeneration of Osteochondral Defects.

IMPACT STATEMENT: Repairing damaged joint cartilage remains a significant challenge. Treatment involving microfracture, tissue grafting, or cell therapy provides some benefit, but seldom regenerates lost articular cartilage. Providing a point-of-care solution that is cell and tissue free has the potential to transform orthopedic treatment for such cases. Glycosaminoglycans such as heparan sulfate (HS) are well suited for this purpose because they provide a matrix that enhances the prochondrogenic activities of growth factors normally found at sites of articular damage. In this study, we show the potential of a novel HS device, which is free of exogenous cells or growth factors, in regenerating osteochondral defects.

Biphasic calcium phosphate with submicron surface topography in an Ovine model of instrumented posterolateral spinal fusion.

As spinal fusions require large volumes of bone graft, different bone graft substitutes are being investigated as alternatives. A subclass of calcium phosphate materials with submicron surface topography has been shown to be a highly effective bone graft substitute. In this work, a commercially available biphasic calcium phosphate (BCP) with submicron surface topography (MagnetOs; Kuros Biosciences BV) was evaluated in an Ovine model of instrumented posterolateral fusion. The material was implanted stand-alone, either as granules (BCPgranules) or as granules embedded within a fast-resorbing polymeric carrier (BCPputty) and compared to autograft bone (AG). Twenty-five adult, female Merino sheep underwent posterolateral fusion at L2-3 and L4-5 levels with instrumentation. After 6, 12, and 26 weeks, outcomes were evaluated by manual palpation, range of motion (ROM) testing, micro-computed tomography, histology and histomorphometry. Fusion assessment by manual palpation 12 weeks after implantation revealed 100% fusion rates in all treatment groups. The three treatment groups showed a significant decrease in lateral bending at the fusion levels at 12 weeks (P < 0.05) and 26 weeks (P < 0.001) compared to the 6 week time-point. Flexion-extension and axial rotation were also reduced over time, but statistical significance was only reached in flexion-extension for AG and BCPputty between the 6 and 26 week time-points (P < 0.05). No significant differences in ROM were observed between the treatment groups at any of the time-points investigated. Histological assessment at 12 weeks showed fusion rates of 75%, 92%, and 83% for AG, BCPgranules and BCPputty, respectively. The fusion rates were further increased 26 weeks postimplantation. Similar trends of bone growth were observed by histomorphometry. The fusion mass consisted of at least 55% bone for all treatment groups 26 weeks after implantation. These results suggest that this BCP with submicron surface topography, in granules or putty form, is a promising alternative to autograft for spinal fusion.

Variation of the bone forming ability with the physicochemical properties of calcium phosphate bone substitutes.

Because of their bioactive properties and chemical similarity to the inorganic component of bone, calcium phosphate (CaP) materials are widely used for bone regeneration. Six commercially available CaP bone substitutes (Bio-Oss, Actifuse, Bi-Ostetic, MBCP, Vitoss and chronOs) as well as two tricalcium phosphate (TCP) ceramics with either a micron-scale (TCP-B) or submicron-scale (TCP-S) surface structure are characterized and their bone forming potential is evaluated in a canine ectopic implantation model. After 12 weeks of implantation in the paraspinal muscle of four beagles, sporadic bone (0.1 ± 0.1%) is observed in two Actifuse implants (2/4), limited bone (2.1 ± 1.4%) in four MBCP implants (4/4) and abundant bone (21.6 ± 4.5%) is formed in all TCP-S implants (4/4). Bone is not observed in any of the Bio-Oss, Bi-Ostetic, Vitoss, chronOs and TCP-B implants (0/4). When correlating the bone forming potential with the physicochemical properties of each material, we observe that the physical characteristics (e.g. grain size and micropore size at the submicron scale) might be the dominant trigger of material directed bone formation via specific mechanotransduction, instead of protein adsorption, surface mineralization and calcium ion release.

Topography of calcium phosphate ceramics regulates primary cilia length and TGF receptor recruitment associated with osteogenesis.

The surface topography of synthetic biomaterials is known to play a role in material-driven osteogenesis. Recent studies show that TGFß signalling also initiates osteogenic differentiation. TGFß signalling requires the recruitment of TGFß receptors (TGFßR) to the primary cilia. In this study, we hypothesize that the surface topography of calcium phosphate ceramics regulates stem cell morphology, primary cilia structure and TGFßR recruitment to the cilium associated with osteogenic differentiation. We developed a 2D system using two types of tricalcium phosphate (TCP) ceramic discs with identical chemistry. One sample had a surface topography at micron-scale (TCP-B, with a bigger surface structure dimension) whilst the other had a surface topography at submicron scale (TCP-S, with a smaller surface structure dimension). In the absence of osteogenic differentiation factors, human bone marrow stromal cells (hBMSCs) were more spread on TCP-S than on TCP-B with alterations in actin organization and increased primary cilia prevalence and length. The cilia elongation on TCP-S was similar to that observed on glass in the presence of osteogenic media and was followed by recruitment of transforming growth factor-ß RII (p-TGFß RII) to the cilia axoneme. This was associated with enhanced osteogenic differentiation of hBMSCs on TCP-S, as shown by alkaline phosphatase activity and gene expression for key osteogenic markers in the absence of additional osteogenic growth factors. Similarly, in vivo after a 12-week intramuscular implantation in dogs, TCP-S induced bone formation while TCP-B did not. It is most likely that the surface topography of calcium phosphate ceramics regulates primary cilia length and ciliary recruitment of p-TGFß RII associated with osteogenesis and bone formation. This bioengineering control of osteogenesis via primary cilia modulation may represent a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery applications. STATEMENT OF SIGNIFICANCE: The surface topography of synthetic biomaterials plays important roles in material-driven osteogenesis. The data presented herein have shown that the surface topography of calcium phosphate ceramics regulates mesenchymal stromal cells (e.g., human bone marrow mesenchymal stromal cells, hBMSCs) with respect to morphology, primary cilia structure and TGFßR recruitment to the cilium associated with osteogenic differentiation in vitro. Together with bone formation in vivo, our results suggested a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery by the bioengineering control of osteogenesis via primary cilia modulation.

Cells responding to surface structure of calcium phosphate ceramics for bone regeneration.

Surface structure largely affects the inductive bone-forming potential of calcium phosphate (CaP) ceramics in ectopic sites and bone regeneration in critical-sized bone defects. Surface-dependent osteogenic differentiation of bone marrow stromal cells (BMSCs) partially explained the improved bone-forming ability of submicron surface structured CaP ceramics. In this study, we investigated the possible influence of surface structure on different bone-related cells, which may potentially participate in the process of improved bone formation in CaP ceramics. Besides BMSCs, the response of human brain vascular pericytes (HBVP), C2C12 (osteogenic inducible cells), MC3T3-E1 (osteogenic precursors), SV-HFO (pre-osteoblasts), MG63 (osteoblasts) and SAOS-2 (mature osteoblasts) to the surface structure was evaluated in terms of cell proliferation, osteogenic differentiation and gene expression. The cells were cultured on tricalcium phosphate (TCP) ceramics with either micron-scaled surface structure (TCP-B) or submicron-scaled surface structure (TCP-S) for up to 14 days, followed by DNA, alkaline phosphatase (ALP) and quantitative polymerase chain reaction gene assays. HBVP were not sensitive to surface structure with respect to cell proliferation and osteogenic differentiation, but had downregulated angiogenesis-related gene expression (i.e. vascular endothelial growth factor) on TCP-S. Without additional osteogenic inducing factors, submicron-scaled surface structure enhanced ALP activity and osteocalcin gene expression of human (h)BMSCs and C2C12 cells, favoured the proliferation of MC3T3-E1, MG63 and SAOS-2, and increased ALP activity of MC3T3-E1 and SV-HFO. The results herein indicate that cells with osteogenic potency (either osteogenic inducible cells or osteogenic cells) could be sensitive to surface structure and responded to osteoinductive submicron-structured CaP ceramics in cell proliferation, ALP production or osteogenic gene expression, which favour bone regeneration. Copyright © 2017 John Wiley & Sons, Ltd.

Biocompatibility evaluation of dicalcium phosphate/calcium sulfate/poly (amino acid) composite for orthopedic tissue engineering in vitro and in vivo.

In vitro cytocompatibility of ternary biocomposite of dicalcium phosphate (DCP) and calcium sulfate (CS) containing 40 wt% poly (amino acid) (PAA) was evaluated using L929 fibroblasts and MG-63 osteoblast-like cells. Thereafter, the biocompatibility of biocomposite in vivo was investigated using an implantation in muscle and bone model. In vitro L929 and MG-63 cell culture experiments showed that the composite and PAA polymer were noncytotoxic and allowed cells to adhere and proliferate. The scanning electron microscope (SEM) confirmed that two kinds of cells maintained their phenotype on all of samples surfaces. Moreover, the DCP/CS/PAA composite showed higher cellular viability than that of PAA; meanwhile, the cell proliferation and ALP activity were much higher when DCP/CS had added into PAA. After implanted in muscle of rabbits for 12 weeks, the histological evaluation indicated that the composite exhibited excellent biocompatibility and no inflammatory responses were found. When implanted into bone defects of femoral condyle of rabbits, the composite was combined directly with the host bone tissue without fibrous capsule tissue, which shown good biocompatibility and osteoconductivity. Thus, this novel composite may have potential application in the clinical setting.

Submicron-surface structured tricalcium phosphate ceramic enhances the bone regeneration in canine spine environment.

Calcium phosphate ceramics with submicron-scaled surface structure can trigger bone formation in non-osseous sites and are expected to enhance bone formation in spine environment. In this study, two tricalcium phosphate ceramics having either a submicron-scaled surface structure (TCP-S) or a micron-scaled one (TCP-B) were prepared and characterized regarding their physicochemical properties. Granules (size 1-2 mm) of both materials were implanted on either left or right side of spinous process, between the two lumbar vertebrae (L3-L4), and in paraspinal muscle of eight beagles. After 12 weeks of implantation, ectopic bone was observed in muscle in TCP-S explants (7.7 ± 3.7%), confirming their ability to inductively form bone in non-osseous sites. In contrast, TCP-B implants did not lead to bone formation in muscle. Abundant bone (34.1 ± 6.6%) was formed within TCP-S implants beside the two spinous processes, while limited bone (5.1 ± 4.5%) was seen in TCP-B. Furthermore, the material resorption of TCP-S was more pronounced than that of TCP-B in both the muscle and spine environments. The results herein indicate that the submicron-scaled surface structured tricalcium phosphate ceramic could enhance bone regeneration as compared to the micron-scaled one in spine environment. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1865-1873, 2016.

Mechanics, degradability, bioactivity, in vitro, and in vivo biocompatibility evaluation of poly(amino acid)/hydroxyapatite/calcium sulfate composite for potential load-bearing bone repair.

A ternary composite of poly(amino acid), hydroxyapatite, and calcium sulfate (PAA/HA/CS) was prepared using in situ melting polycondensation method and evaluated in terms of mechanical strengths, in vitro degradability, bioactivity, as well as in vitro and in vivo biocompatibility. The results showed that the ternary composite exhibited a compressive strength of 147 MPa, a bending strength of 121 MPa, a tensile strength of 122 MPa, and a tensile modulus of 4.6 GPa. After immersion in simulated body fluid, the compressive strength of the composite decreased from 147 to 98 MPa for six weeks and the bending strength decreased from 121 to 75 MPa for eight weeks, and both of them kept stable in the following soaking period. The composite could be slowly degraded with 7.27 wt% loss of initial weight after soaking in phosphate buffered solution for three weeks when started to keep stable weight in the following days. The composite was soaked in simulated body fluid solution and the hydroxyapatite layer, as flower-like granules, formed on the surface of the composite samples, showing good bioactivity. Moreover, it was found that the composite could promote proliferation of MG-63 cells, and the cells with normal phenotype extended and spread well on the composite surface. The implantation of the composite into the ulna of sheep confirmed that the composite was biocompatible and osteoconductive in vivo, and offered the PAA/HA/CS composite promising material for load-bearing bone substitutes for clinical application.

Strontium-containing apatite/polylactide composites enhance bone formation in osteopenic rabbits.

Strontium (Sr) has been shown to favor bone formation and is used clinically to treat osteoporosis. We have previously reported that Sr addition in apatite/polylactide composites could enhance the BMP-induced bone formation around implants at ectopic site in healthy animals. In this study we aimed to investigate the effectiveness of Sr addition on the local bone formation in osteoporosis. Apatite/polylactide composite granules with different Sr content were loaded with equal amount of rhBMP-2 and implanted intramuscularly in healthy rabbits (Con) and rabbits that received bilateral ovariectomy and daily injection of glucocorticoid (OP) for 12 weeks. The potential effect of Sr on the final volume of BMP-induced bone in both groups was investigated histologically and histomorphometrically. The de novo bone formed in OP implants was significantly less than in Con group when the implants contained no Sr, indicating that the BMP-induced osteogenesis was impaired in OP animals. Sr substitution as low as 0.5 mol% in apatite increased the bone volume in OP implants to levels comparable to that in the Con group, indicating a positive effect of Sr addition on the local bone formation in OP animals. In addition, more adipose tissue formed in parallel with the appearance of cartilage tissue in OP implants, suggesting that the differentiation potential of stem cell in OP animals may have shifted towards adipogenesis and chondrogenesis. From these results, we conclude that the use of Sr addition to enhance the bone growth surrounding implants in osteoporosis merits further study. STATEMENT OF SIGNIFICANCE: The impaired bone healing capacity of osteoporotic patients might result in poor osteointegration and surgical failure in case implants are placed. In this study we aimed to enhance the bone formation around implants under such scenario by adding strontium as the stimulus. Different from other studies, the samples were loaded with rhBMP-2 and implanted at an ectopic site (spinal muscles of New Zealand rabbits) to exclude the influence of conductive bone repair. The results showed that the addition of strontium could enhance the BMP-2-induced bone formation on implants in osteopenic rabbits to levels comparable to that in healthy rabbits. Secondarily, we observed more adipose tissue and cartilage tissue in osteopenic implants, suggesting the role of adipogenesis and chondrogenesis in osteopenia/osteoporosis.

Degradability and cytocompatibility of tricalcium phosphate/poly(amino acid) composite as bone tissue implants in orthopaedic surgery.

In this study, a tricalcium phosphate (TCP) and poly (amino acid) copolymer (PAA) biocomposite were fabricated for bone repair and characterized. The results show that the compressive strength of the TCP/PAA composites increased with an increase in the TCP content at TCP contents less than 40 w%. The weight loss of the composite after soaking in phosphate buffered saline for 12 weeks significantly increased with an increase in the TCP content, revealing its good degradability. In addition, the composite maintained adequate mechanical strength during the degradation period because it underwent a surface erosion process. In vitro MG63 cell culture experiments showed that the composite is non-cytotoxic and thus allows cells to adhere, proliferate and differentiate. Osteoid formation was evidenced on the composite surfaces 12 weeks after its implantation into the femoral bone of dogs. Furthermore, the composite combined directly with the host bone tissue without fibrous capsule tissue, and no inflammatory responses were found, showing the good biocompatibility of the composite. It is expected that the composite may be used for the development of bone implants for orthopaedic surgery.

Osteoclast resorption of beta-tricalcium phosphate controlled by surface architecture.

A resorbable bone graft substitute should mimic native bone in its capacity to support bone formation and be remodeled by osteoclasts (OCl) or other multinucleated cells such as foreign body giant cells (FBGC). We hypothesize that by changing the scale of surface architecture of beta-tricalcium phosphate (TCP), cellular resorption can be influenced. CD14(+) monocyte precursors were isolated from human peripheral blood (n = 4 independent donors) and differentiated into OCl or FBGC on the surface of TCP discs comprising either submicron- or micron-scale surface topographical features (TCPs and TCPb, respectively). On submicrostructured TCPs, OCl survived, fused, differentiated, and extensively resorbed the substrate; however, on microstructured TCPb, OCl survival, TRAP activation, and fusion were attenuated. Importantly, no resorption was observed on microstructured TCPb. By confocal microscopy, OCl formed on TCPs contained numerous actin rings allowing for resorption, but not on TCPb. In comparison, FBGC could not resorb either TCP material, suggesting that osteoclast-specific machinery is necessary to resorb TCP. By tuning surface architecture, it appears possible to control osteoclast resorption of calcium phosphate. This approach presents a useful strategy in the design of resorbable bone graft substitutes.

The size of surface microstructures as an osteogenic factor in calcium phosphate ceramics.

The microporosity of calcium phosphate (CaP) ceramics has been shown to have an essential role in osteoinduction by CaP ceramics after ectopic implantation. Here we show that it is not the microporosity but the size of surface microstructural features that is the most likely osteogenic factor. Two tricalcium phosphate (TCP) ceramics, namely TCP-S and TCP-B, were fabricated with equivalent chemistry and similar microporosity but different sizes of surface microstructural features. TCP-S has a grain size of 0.99 ± 0.20 µm and a micropore size of 0.65 ± 0.25 µm, while TCP-B displays a grain size of 3.08 ± 0.52 µm and a micropore size of 1.58 ± 0.65 µm. In vitro, both cell proliferation and osteogenic differentiation were significantly enhanced when human bone marrow stromal cells were cultured on TCP-S without any osteogenic growth factors, compared to TCP-B ceramic granules. The possible involvement of direct contact between cells and the TCP ceramic surface in osteogenic differentiation is also shown with a trans-well culture model. When the ceramic granules were implanted in paraspinal muscle of dogs for 12 weeks, abundant bone was formed in TCP-S (21 ± 10% bone in the available space), whereas no bone was formed in any of the TCP-B implants. The current in vitro and in vivo data reveal that the readily controllable cue, i.e. the size of the surface microstructure, could be sufficient to induce osteogenic differentiation of mesenchymal stem cells, ultimately leading to ectopic bone formation in calcium phosphate ceramics.

Zinc in calcium phosphate mediates bone induction: in vitro and in vivo model.

Zinc-containing tricalcium phosphate (Zn-TCP) was synthesized to investigate the role of zinc in osteoblastogenesis, osteoclastogenesis and in vivo bone induction in an ectopic implantation model. Zinc ions were readily released in the culture medium. Zn-TCP with the highest zinc content enhanced the alkaline phosphatase activity of human bone marrow stromal cells and tartrate-resistant acid phosphatase activity, as well as multinuclear giant cell formation of RAW264.7 monocyte/macrophages. RAW264.7 cultured with different dosages of zinc supplements in medium with or without zinc-free TCP showed that zinc could influence both the activity and the formation of multinuclear giant cells. After a 12-week implantation in the paraspinal muscle of canines, de novo bone formation and bone incidence increased with increasing zinc content in Zn-TCP - up to 52% bone in the free space. However, TCP without zinc induced no bone formation. Although the observed bone induction cannot be attributed to zinc release alone, these results indicate that zinc incorporated in TCP can modulate bone metabolism and render TCP osteoinductive, indicating to a novel way to enhance the functionality of this synthetic bone graft material.

Influence of polymer molecular weight in osteoinductive composites for bone tissue regeneration.

In bone tissue regeneration, certain polymer and calcium-phosphate-based composites have been reported to enhance some biological surface phenomena, facilitating osteoinduction. Although the crucial role of inorganic fillers in heterotopic bone formation by such materials has been shown, no reports have been published on the potential effects the polymer phase may have. The present work starts from the assumption that the polymer molecular weight regulates the fluid uptake, which determines the hydrolysis rate and the occurrence of biological surface processes. Here, two composites were prepared by extruding two different molecular weight L/D,L-lactide copolymers with calcium phosphate apatite. The lower molecular weight copolymer allowed larger fluid uptake in the composite thereof, which was correlated with a higher capacity to adsorb proteins in vitro. Further, the large fluid absorption led to a quicker composite degradation that generated rougher surfaces and enhanced ion release. Following intramuscular implantation in sheep, only the composite with the lower molecular weight polymer could induce heterotopic bone formation. Besides influencing the biological potential of composites, the molecular weight also regulated their viscoelastic behaviour under cyclic stresses. The results lead to the conclusion that designing biomaterials with appropriate physico-chemical characteristics is crucial for bone tissue regeneration in mechanical load-bearing sites.

Controlling dynamic mechanical properties and degradation of composites for bone regeneration by means of filler content.

Bone tissue is a dynamic composite system that adapts itself, in response to the surrounding daily (cyclic) mechanical stimuli, through an equilibrium between growth and resorption processes. When there is need of synthetic bone grafts, the biggest issue is to support bone regeneration without causing mechanically-induced bone resorption. Apart from biological properties, such degradable materials should initially support and later leave room to bone formation. Further, dynamic mechanical properties comparable to those of bone are required. In this study we prepared composites comprising calcium phosphate and L-lactide/D-lactide copolymer in various content ratios using the extrusion method. We evaluated the effect of the inorganic filler amount on the polymer phase (i.e. on the post-extrusion intrinsic viscosity). We then studied their in vitro degradation and dynamic mechanical properties (in dry and humid conditions). By increasing the filler content, we observed significant decrease of the intrinsic viscosity of the polymer phase during the extrusion process. Composites containing higher amounts of apatite had faster degradation, and were also mechanically stiffer. But, due to the lower intrinsic viscosity of their polymer phase, they had larger damping properties. Besides this, higher amounts of apatite also rendered the composites more hydrophilic letting them absorb more water and causing them the largest decrease in stiffness. These results show the importance of filler content in controlling the properties of such composites. Further, in this study we observed that the viscoelastic properties of the composite containing 50wt% apatite were comparable to those of dry human cortical bone.

Bone substitute biomedical material of multi-(amino acid) copolymer: in vitro degradation and biocompatibility.

Degradable polymers with good mechanical strength as bone repair biomaterials have been paid more attention in biomedical application. In this study, a multi-(amino acid) copolymer consisting of 6-aminocaproic acid and five natural amino acids was prepared by a reaction of acid-catalyzed condensation. The results revealed that the copolymer could be slowly degradable in Tris-HCl solution, and lost its initial weight of 31.9 wt% after immersion for 12 weeks, and the changes of pH value of Tris-HCl solution were in range from 6.9 to 7.4 during soaking. The compressive strength of the copolymer decreased from 107 to 68 MPa after immersion for 12 weeks. The proliferation and differentiation of MG-63 cells on the copolymer significantly increased with time, and the cells with normal phenotype extended and spread well on the copolymer surfaces. When the copolymer was implanted in muscle and bone defects of femoral cortex of dogs for 12 weeks, the histological evaluation confirmed that the copolymer exhibited excellent biocompatibility and more effective osteogenesis in vivo. When implanted into cortical bone defects of dogs, the copolymer could be combined directly with the natural bone without fibrous capsule tissue between implants and host bone. The results indicated that the multi-(amino acid) copolymer with sufficient strength, good biocompatibility and osteoconductivity had clinical potential for load-bearing bone repair or substitution.