3D microenvironment as essential element for osteoinduction by biomaterials

In order to unravel the mechanism of osteoinduction by biomaterials, in this study we investigated the influence of the specific surface area on osteoinductive properties of two types of calcium phosphate ceramics. Different surface areas of the ceramics were obtained by varying their sintering temperatures. Hydroxyapatite (HA) ceramic was sintered at 1150 and 1250 degrees C. Biphasic calcium phosphate (BCP) ceramic, consisting of HA and beta-tricalcium phosphate (beta-TCP), was sintered at 1100, 1150 and 1200 degrees C. Changes in sintering temperature did not influence the chemistry of the ceramics; HA remained pure after sintering at different temperatures and the weight ratio of HA and beta-TCP in the BCP was independent of the temperature as well. Similarly, macroporosity of the ceramics was unaffected by the changes of the sintering temperature. However, microporosity (pore diameter <10 microm) significantly decreased with increasing sintering temperature. In addition to the decrease of the microporosity, the crystal size increased with increasing sintering temperature. These two effects resulted in a significant decrease of the specific surface area of the ceramics with increasing sintering temperatures. Samples of HA1150, HA1250, BCP1100, BCP1150 and BCP1200 were implanted in the back muscles of Dutch milk goats and harvested at 6 and 12 weeks post implantation. After explantation, histomorphometrical analysis was performed on all implants. All implanted materials except HA1250 induced bone. However, large variations in the amounts of induced bone were observed between different materials and between individual animals. Histomorphometrical results showed that the presence of micropores within macropore walls is necessary to make a material osteoinductive. We postulate that introduction of microporosity within macropores, and consequent increase of the specific surface area, affects the interface dynamics of the ceramic in such a way that relevant cells are triggered to differentiate into the osteogenic lineage.     

Adipose-derived stem cells combined with beta-tricalcium phosphate: A novel possible strategy for periodontal defects regeneration

Regeneration of destroyed periodontal tissue is a complicated problem in periodontal therapy. Conventional strategies such as surgical techniques, occlusive barrier membranes, and bone grafts have disadvantages that limit their application. Tissue engineering is a novel approach to regenerate periodontal tissue overcoming limitations of current strategies. In recent studies, researchers reported the potential of adipose-derived stem cells (ADSCs) to use in periodontal tissue regeneration. Unfortunately, in vivo studies indicated that small amount of alveolar bone formed using ADSCs implantation in the periodontal defects, which would be overcome by inducing osteogenic differentiation of ADSCs using osteoinductive biomaterials combined with ADSCs. Beta-tricalcium phosphate (beta-TCP) is an osteoinductive material which reported to induce osteogenic differentiation of adipose-derived stem cells. Based on the performed studies, we hypothesize that ADSCs from Wistar rat are isolated, induced by appropriate signaling molecules, seeded by beta-TCP, and then the complex was implanted into the periodontal defects in the same Wistar rat to observe the regeneration of periodontal tissue in vivo. This study will explore the possibility and application of ADSCs/beta-TCP complex for reconstructing periodontal defects. We believe it is especially useful to future clinical study and application in this field.     

In vivo evaluation of a porous hydroxyapatite/poly-DL-lactide composite for bone tissue engineering

As reported previously, a porous composite of uncalcined hydroxyapatite (u-HA) and poly-DL-lactide (PDLLA) showed excellent osteoconductivity and biodegradability as a bone substitute in rabbit model. In this study, to investigate the usefulness of this composite as a scaffold loaded with cells, we estimated whether this material showed osteogenesis on implantation to extraosseous site. On loading with syngeneic bone marrow cells and implantation into rat dorsal subcutaneous tissue, osteogenesis with enchondral ossification was seen both on and in the material at 3 weeks after implantation. The osteogenesis in the u-HA/PDLLA had progressed, and newly formed bone tissue was found in the material by 6 weeks. To investigate the osteoinductive properties of the material, we implanted this porous composite material into extraosseous canine dorsal muscle. At 8 weeks, osteogenesis was seen in the pores of the material. Newly formed bone could be observed adjacent to the material. In addition, cuboidal osteoblasts adjacent to the newly formed bone were evident. Neither cartilage nor chondrocytes were found. These results might indicate that the material induced osteogenesis by intramembranous ossification. Conversely, similar porous PDLLA did not induce osteogenesis during the observation period. Therefore, porous HA/PDLLA, which has osteoconductive and osteoinductive properties, might be a useful material for use as a bone substitute and cellular scaffold.     

Ectopic bone formation in mice associated with a lactic acid/dioxanone/ethylene glycol copolymer-tricalcium phosphate composite with added recombinant human bone morphogenetic protein-2

A new putty-like material with bone-inducing capacity was made by combining a block copolymer of poly d,l-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG) and beta-tricalcium phosphate (beta-TCP) powder with added recombinant human bone morphogenetic protein-2 (rhBMP-2). To optimize the material's efficacy for bone formation, we formulated the optimal composition ratio of the respective constituent that gives the greatest osteoinductive efficacy in a mouse model of ectopic bone formation. In this series of studies, we investigated the size of ectopic bone mass induced 3 and 6 weeks after implantation of the materials composed of 30 mg of PLA-DX-PEG with 2 microg of rhBMP-2 and 0, 15, 30, or 60 mg of beta-TCP powder. An additional experiment was designed to investigate how content ratios of beta-TCP powder in 30 mg-putty implants (0%, 16.7%, 33.3%, 50%, 66.7%, 83.3%, or 100%) for a fixed dose (5 microg) of the rhBMP-2 altered the size of the induced ossicle. The results from the first experiment indicated that the bone yields were linearly dependent on the amount of additional beta-TCP powder. In the second experiment, the largest ossicles induced by 5 microg of rhBMP-2 were obtained when the polymer/beta-TCP ratio was 1/2 in mice. The data provide important insights into the fabrication of implants that provide efficacious delivery of rhBMP-2. The new putty-like material may be valuable for repairing or regenerating bone in a clinical setting.     

骨诱导性磷酸钙陶瓷材料修复牙槽突裂

BACKGROUND: In our previous studies, we had prepared calcium phosphate ceramics with better ectopic osteogenesis. OBJECTIVE: To explore the effect of novel osteoinductive calcium phosphate ceramics in the repairing of alveolar cleft. METHODS: Bilaterl alveolar defects were created in nine immature beagles. Three months later, osteoinductive calcium phosphate ceramics with high modular surface (experimental group) and smooth surface (control group) were randomly implanted in each side of the defect. Meanwhile, the corresponding material was implanted into the thigh muscle. New bone formation in the implanted region, osteogenesis in the implanted region and muscle, and respair results were respectively observed by fluorescence microscope, light microscope and CT at 4, 8 and 12 weeks after implantation. RESULTS AND CONCLUSION: (1) Fluorescence microscope observation: A circular permutation of red, yellow and green fluorescent strip could be observed in both two groups. (2) Light microscope observation: At 12 weeks after implantation, in the experimental group, the bone reconstruction was obvious, the implant material was decomposed gradually, the gap was filled with a large number of mature bone that combined with the rest material closely, and numerous Haversian canals appeared; the control group was similar but slightly inferior to the experimental group in the quality of new bone. The experimental group material successfully induced heterotopic osteogenesis in muscle, while the control did not. (3) CT examination: The two group materials restored the appearance and continuity of the alveolar ridge, and made no effect on the eruption of permanent teeth in both sides of the defect. To conclude, our findings suggest that the novel osteoinductive calcium phosphate ceramic exhibits advantages in alveolar cleft repair with earlier osteogenesis activation, faster osteogenesis rate and more bone formation than those traditional materials.     

The influence of genetic factors on the osteoinductive potential of calcium phosphate ceramics in mice

The efficacy of calcium phosphate (CaP) ceramics in healing large bone defects is, in general, not as high as that of autologous bone grafting. Recently, we reported that CaP ceramics with osteoinductive properties were as efficient in healing an ilium defect of a sheep as autologous bone graft was, which makes this subclass of CaP ceramics a powerful alternative for bone regeneration. Although osteoinduction by CaP ceramics has been shown in several large animal models it is sporadically reported in mice. Because the lack of a robust mouse model has delayed understanding of the mechanism, we screened mice from 11 different inbred mouse strains for their responsiveness to subcutaneous implantation of osteoinductive tricalcium phosphate (TCP). In only two strains (FVB and 129S2) the ceramic induced bone formation, and in particularly, in FVB mice, bone was found in all the tested mice. We also demonstrated that other CaP ceramics induced bone formation at the same magnitude as that observed in other animal models. Furthermore, VEGF did not significantly increase TCP induced bone formation. The mouse model here described can accelerate research of osteoinductive mechanisms triggered by CaP ceramics and potentially the development of therapies for bone regeneration.     

Influence of β-tricalcium phosphate granule size and morphology on tissue reaction in vivo

In this study the tissue reaction to five different β-tricalcium phosphate (β-TCP)-based bone substitute materials differing only in size, shape and porosity was analyzed over 60 days, at 3, 10, 15, 30 and 60 days after implantation. Using the subcutaneous implantation model in Wistar rats both the inflammatory response within the implantation bed and the resulting vascularization of the biomaterials were qualitatively and quantitatively assessed by means of standard and special histological staining methods. The data from this study showed that all investigated β-TCP bone substitutes induced the formation of multinucleated giant cells. Changes in size, shape and porosity influenced the integration of the biomaterials within the implantation bed and the formation of tartrate-resistant acid phosphatase (TRAP)-positive and TRAP-negative multinucleated giant cells, as well as the rate of vascularization. While a high porosity generally allowed cell and fiber in-growth within the center of the bone substitute, a lower porosity resulted in a mosaic-like integration of the materials, with the granules serving as place holders. The number of multinucleated giant cells located in the implantation bed positively correlated with the vascularization rate. These data emphasize that all biomaterials investigated were capable of inducing the formation of TRAP-positive multinucleated giant cells as a sign of biomaterial stability. Furthermore, these cells directly influenced vascularization by secretion of vascular endothelial growth factor (VEGF), as well as other chemokines. Based on these findings, the role of multinucleated giant cells in the foreign body reaction to biomaterials might need to be reconsidered. This study demonstrates that variations in the physical properties of a bone substitute material clearly influence the (extent of the) inflammatory reaction and its consequences.     

The osteoinductive potential of printable, cell-laden hydrogel-ceramic composites

Hydrogels used as injectables or in organ printing often lack the appropriate stimuli to direct osteogenic differentiation of embedded multipotent stromal cells (MSCs), resulting in limited bone formation in these matrices. Addition of calcium phosphate (CaP) particles to the printing mixture is hypothesized to overcome this drawback. In this study we have investigated the effect of CaP particles on the osteoinductive potential of cell-laden hydrogel-CaP composite matrices. To this end, apatitic nanoparticles have been included in Matrigel constructs where after the viability of embedded progenitor cells was assessed in vitro. In addition, the osteoinductive potential of cell-laden Matrigel containing apatitic nanoparticles was investigated in vivo and compared with composites containing osteoinductive biphasic calcium phosphate (BCP) microparticles after subcutaneous implantation in immunodeficient mice. Histological and immunohistochemical analysis of the tissue response as well as in vivo bone formation revealed that apatitic nanoparticles were osteoinductive and induced osteoclast activation, but without bone formation. The BCP particles were more effective in inducing elaborate bone formation at the ectopic location.     

缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导骨形成

背景：重组人骨形态发生蛋白2在体内半衰期短、易降解代谢,达不到理想的骨再生效果。 目的：制备缓释型重组人骨形态发生蛋白2/壳聚糖生物骨修复材料,并观察其缓释性能、骨诱导活性。 方法：将重组人骨形态发生蛋白2与壳聚糖混合制备壳聚糖膜,涂覆于生物骨修复材料表面,ELISA方法检测其体外释药性能。茜素红染色检测缓释型人骨形态发生蛋白2/壳聚糖生物骨材料、重组人骨形态发生蛋白2生物骨材料、单纯骨填充材料诱导C2C12细胞骨钙蛋白的形成,观察其诱导成骨细胞能力。同时将3种骨修复材料植入清洁级KM小鼠股部肌袋内,2周后检测新生骨Ca2+离子含量,评价其异位骨诱导能力。 结果与结论：材料表面的壳聚糖膜分布均匀,负载的重组人骨形态发生蛋白2呈团簇状。重组人骨形态发生蛋白2/壳聚糖生物骨修复材料体外释药存在突释,前4 d释放量达总药量的50%,持续至12 d,释药量达到90%,第18天时释放完全。与单纯骨填充材料、重组人骨形态发生蛋白2生物骨材料相比,缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料诱导C2C12细胞向成骨晚期分化能力与异位骨形成能力显著增强(P < 0.05)。结果提示缓释型人骨形态发生蛋白2/壳聚糖生物骨修复材料缓释性能好,促进骨形成能力强。     

成骨诱导脂肪基质细胞在骨组织工程体内成骨中的作用

背景：以往研究认为,经过成骨诱导后的脂肪基质细胞通过转化为成骨细胞分泌骨基质进而修复骨缺损,然而并没有明确结论证实。 目的：将经过体外成骨诱导的脂肪基质细胞复合支架材料分别植入骨缺损区和非骨区,根据是否成骨,验证经过成骨诱导后的脂肪基质细胞是否转化为成骨细胞。 方法：取12月龄犬背部皮下脂肪,经胶原酶消化法获得单个核细胞,将培养的第3代细胞与双相磷酸钙陶瓷形成复合物。在犬下颌骨两侧制备长20 mm、高10 mm的箱状缺损,拔除术区牙齿,将细胞支架复合物植入一侧术区,空白侧留作对照;另外在犬背部皮下肌肉区植入细胞支架复合物及骨诱导性磷酸钙陶瓷材料,术后6周及12周经组织学检测骨缺损修复情况。 结果与结论：脂肪基质细胞复合双相磷酸钙陶瓷在骨缺损区成骨,在肌肉区未形成新骨;骨诱导性磷酸钙陶瓷在肌肉区形成新骨。提示成骨诱导并不能将脂肪基质细胞转化为成骨细胞,其确切机制有待进一步研究。     

Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants inserted at an ectopic site in rabbits

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is known for its osteoinductive potential in bone tissue engineering. Calcium phosphate (Ca-P) cements are injectable, osteoconductive ceramic materials in which a macroporous structure can be induced during the setting reaction. In this study, the osteoinductive capability of rhBMP-2 loaded porous Ca-P cement was evaluated. Porous Ca-P cement discs were made and loaded with rhBMP-2 in vitro and implanted subcutaneously in the back of New Zealand white rabbits. The implantation period was either 2 or 10 weeks. Histological analysis of retrieved specimens revealed evident bone formation in the rhBMP-2 loaded Ca-P cement discs (pore fill: 18+/-6%) after 10 weeks of implantation. Bone formation occurred only in rhBMP-2 loaded porous Ca-P cement discs. Degradation of the Ca-P cement could not be confirmed after 10 weeks of implantation. The scaffold maintained its shape and stability during this time period. We conclude that porous Ca-P cement is a suitable carrier material for ectopic bone engineering.     

Mathematical modelling of the distribution of newly formed bone in bone tissue engineering

New bone formation in bone substitutes is usually investigated by histomorphometric global analysis. This study provides a novel mathematical modelling approach of new bone formation in the use of osteoinductive and functionalized biomaterials for bone tissue engineering. We discuss here the repartition and the probability to get new bone formation inside Biphasic Calcium Phosphate (BCP) loaded with autologous osteogenic cells, functionalized with a cyclo RGD peptide, after implantation in rabbits for 2 and 4 weeks. This local analysis allowed us to complement classical global findings and to demonstrate that after 2 weeks of implantation, the probability of new bone formation was significantly higher in RGD-grafted BCP and that new formed bone was largely distributed from the edge to the centre of the implant. While no significant differences were obtained after 4 weeks of implantation between RGD-grafted and non-grafted materials, distribution of new bone formation inside RGD-grafted materials was significantly more homogeneous as demonstrated by our mathematical modelling approach. In conclusion, local analysis of new bone formation inside macroporous substitutes coupled with mathematical modelling constitutes a potential quantitative approach for the evaluation of the osteoconductive and osteoinductive characteristics of such biomaterials.     

Calcium phosphate ceramics in bone tissue engineering: A review of properties and their influence on cell behavior

Calcium phosphate ceramics (CPCs) have been widely used as biomaterials for the regeneration of bone tissue because of their ability to induce osteoblastic differentiation in progenitor cells. Despite the progress made towards fabricating CPCs possessing a range of surface features and chemistries, the influence of material properties in orchestrating cellular events such as adhesion and differentiation is still poorly understood. Specifically, questions such as why certain CPCs may be more osteoinductive than others, and how material properties contribute to osteoinductivity/osteoconductivity remain unanswered. Therefore, this review article systematically discusses the effects of the physical (e.g. surface roughness) and chemical properties (e.g. solubility) of CPCs on protein adsorption, cell adhesion and osteoblastic differentiation in vitro. The review also provides a summary of possible signaling pathways involved in osteoblastic differentiation in the presence of CPCs. In summary, these insights on the contribution of material properties towards osteoinductivity and the role of signaling molecules involved in osteoblastic differentiation can potentially aid the design of CPC-based biomaterials that support bone regeneration without the need for additional biochemical supplements.     

Biological performance of uncoated and octacalcium phosphate-coated Ti6Al4V

The in vivo behavior of a porous Ti6Al4V material that was produced by a positive replica technique, with and without an octacalcium phosphate (OCP) coating, has been studied both in the back muscle and femur of goats. Macro- and microporous biphasic calcium phosphate (BCP) ceramic, known to be both osteoconductive and able to induce ectopic bone formation, was used for comparison purpose. The three groups of materials (Ti6Al4V, OCP Ti6Al4V and BCP) were implanted transcortically and intramuscularly for 6 and 12 weeks in 10 adult Dutch milk goats in order to study their osteointegration and osteoinductive potential. In femoral defects, both OCP Ti6Al4V and BCP were performing better than the uncoated Ti6Al4V, at both time points. BCP showed a higher bone amount than OCP Ti6Al4V after 6 weeks of implantation, while after 12 weeks, this difference was no longer significant. Ectopic bone formation was found in both OCP Ti6Al4V and BCP implants after 6 and 12 weeks. The quantity of ectopically formed bone was limited as was the amount of animals in which the bone was observed. Ectopic bone formation was not found in uncoated titanium alloy implants, suggesting that the presence of calcium phosphate (CaP) is important for bone induction. This study showed that CaPs in the form of coating on metal implants or in the form of bulk ceramic have a significantly positive effect on the bone healing process.     

Osteoinduction of hydroxyapatite/β-tricalcium phosphate bioceramics in mice with a fractured fibula

Many studies have shown that calcium phosphate ceramics can induce bone formation in non-osseous sites without the application of any osteoinductive biomolecules, but the mechanisms of this phenomenon (intrinsic osteoinduction of bioceramics) remain unclear. In this study, we compared the intrinsic osteoinduction of porous hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) implanted in mice at different sites. In 30 mice the left fibula was fractured and the right fibula was kept intact. A porous HA/β-TCP cylinder was implanted into both the left (group 1) and right (group 2) leg muscles of each animal. In addition, two HA/β-TCP cylinders were bilaterally implanted into leg subcutaneous pockets (group 3) in each of the remaining 15 mice. New bone formation was studied in the three groups by histology, histomorphometry and immunostaining. In group 1 new bone was observed at week 6 and bone marrow appeared at week 12. In group 2 new bone was observed at week 8 and bone marrow appeared at week 12. The new bone area percentage in group 1 was significantly higher than in group 2 at both weeks 8 and 12. In contrast, group 3 did not show any new bone within the period studied. These differences were explained based on the location of the implants and thus their proximity to the osteogenic environment of fracture healing. The results support the hypothesis that intrinsic osteoinduction by calcium phosphate ceramics is the result of adsorption of osteoinductive substances on the surface.     

Synchrotron X-ray microtomography (on a micron scale) provides three-dimensional imaging representation of bone ingrowth in calcium phosphate biomaterials

This study used synchrotron X-ray microtomography on a micron scale to compare three-dimensional (3D) bone ingrowth after implantation of various calcium phosphate bone substitutes in a rabbit model. The advantage of using this new method for the study of biomaterials was then compared with histomorphometry for analysis of interconnection and bone ingrowth. The study focused on the newly formed bone-biomaterial interface. Macroporous Biphasic Calcium Phosphate (MBCP) ceramic blocks and two different injectable calcium phosphate biomaterials [an injectable bone substitute (IBS) consisting of a biphasic calcium phosphate granule suspension in hydrosoluble polymer and a calcium phosphate cement material (CPC)] were studied after in vivo implantation.Absorption or phase-contrast microtomography was performed with the dedicated set-up at beamline ID22. Experimental spatial resolution was between 1 and 1.4 microm, depending on experimental radiation. All calcium phosphates tested showed osteoconduction. IBS observations after 3D reconstruction showed interconnected bioactive biomaterial with total open macroporosity and complete bone ingrowth as early as 3 weeks after implantation. This experimentation was consistent with two-dimensional histomorphometric analysis, which confirmed its suitability for biomaterials. This 3D study relates the different types of bone substitution to biomaterial architecture. As porosity and interconnection increase, bone ingrowth becomes greater at the expense of the bone substitute: IBS>MBCP>CPC.     

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.     

骨诱导性磷酸钙陶瓷为支架的组织工程骨修复狗下颌骨箱状缺损*☆◆

背景：应用不外加生长因子或细胞而具有骨诱导性的生物材料,在非骨部位构建骨移植物,即体内组织工程骨,其在修复箱状及节段性骨缺损方面,具有更可行的前景。 目的：采用骨诱导性钙磷陶瓷材料构建体内组织工程化类骨移植物,探索其应用于修复实验动物下颌骨箱状骨缺损的可行性。 方法：以骨诱导性磷酸钙陶瓷材料为支架植入狗肌肉内构建体内组织工程骨,同期在狗自体下颌骨左右两侧各拔除牙弓中段牙2颗,形成约20 mm无牙区。8周后在无牙区形成箱状缺损,同期取出支架即刻移植入一侧自体下颌骨缺损区,对侧骨缺损区直接移植入未经体内构建的磷酸钙陶瓷作为对照。 结果与结论：经肌肉内构建的体内组织工程骨移植物的力学性能较单纯磷酸钙陶瓷有明显提高。颌骨缺损区的核吸收强度明显强于对照区,其移植物内长入的骨组织较多,两者的成骨面积差异有非常显著性意义(P < 0.01)。说明在修复颌骨大范围缺损中,体内组织工程骨移植物较单纯骨磷酸钙陶瓷替代材料表现出明显的力学和生物学优势,修复效果显著,有良好的应用前景。关键词：骨诱导性磷酸钙陶瓷;体内组织工程骨;骨缺损;支架;组织工程 缩略语注释：BCP：biphasic calcium phosphate,双相磷酸钙 doi:10.3969/j.issn.1673-8225.2012.16.001     

Brief review of models of ectopic bone formation

Ectopic bone formation is a unique biologic entity--distinct from other areas of skeletal biology. Animal research models of ectopic bone formation most often employ rodent models and have unique advantages over orthotopic (bone) environments, including a relative lack of bone cytokine stimulation and cell-to-cell interaction with endogenous (host) bone-forming cells. This allows for relatively controlled in vivo experimental bone formation. A wide variety of ectopic locations have been used for experimentation, including subcutaneous, intramuscular, and kidney capsule transplantation. The method, benefits and detractions of each method are summarized in the following review. Briefly, subcutaneous implantation is the simplest method. However, the most pertinent concern is the relative paucity of bone formation in comparison to other models. Intramuscular implantation is also widely used and relatively simple, however intramuscular implants are exposed to skeletal muscle satellite progenitor cells. Thus, distinguishing host from donor osteogenesis becomes challenging without cell-tracking studies. The kidney capsule (perirenal or renal capsule) method is less widely used and more technically challenging. It allows for supraphysiologic blood and nutrient resource, promoting robust bone growth. In summary, ectopic bone models are extremely useful in the evaluation of bone-forming stem cells, new osteoinductive biomaterials, and growth factors; an appropriate choice of model, however, will greatly increase experimental success.     

Ectopic bone formation in rats: the importance of vascularity of the acceptor site

Bone graft substitutes (BGS) can be fabricated by the combination of three key ingredients: (1) competent bone-forming cells, (2) a suitable framework or scaffold, and (3) the presence of biological stimulants. Although much research has been done to develop the ideal BGS, still the results are not very consistent. In view of this, the cellularity and vascularity of the recipient site are supposed to be important for the osteoinductive capacity of BGS. Therefore, we hypothesized that a muscle recipient site could favor bone formation in a cell-based BGS compared to a subcutaneous recipient site due to the higher vascularity of muscle tissue. To prove this hypothesis, 48 titanium fiber mesh implants were seeded with rat bone marrow stromal cells (RBM) and implanted subcutaneously and intramuscularly in the adductor thigh muscle of rats. The amount of bone formation after 1, 3 and 6 weeks was evaluated by histology and histomorphometry as well as by calcium content. Analysis revealed that the bone formation increased during implantation. However, bone formation did not exceed 12% of the implant surface, both for the intramuscular and subcutaneous recipient site. Also, no significant differences in bone amount between these two sites existed. Consequently, our hypothesis could not be confirmed.