Ectopic osteochondral formation of biomimetic porous PVA-n-HA/PA6 bilayered scaffold and BMSCs construct in rabbit

In this work, the novel poly vinyl alcohol/gelatin-nano-hydroxyapatite/polyamide6 (PVA-n-HA/PA6) bilayered scaffold with biomimetic properties for articular cartilage and subchondral bone is developed. Furthermore, when these osteochondral scaffolds were seeded with induced bone mesenchymal stem cells (BMSCs) and implanted at ectopic sites, showed the potential for an engineered cartilage tissue and the corresponding subchondral bone. BMSCs were expanded in vitro and induced to chondrogenic or osteogenic potential by culturing in suitable media for 14 days. Subsequently, these induced cells were seeded into PVA-n-HA/PA6 separately, and the constructs were implanted into the rabbit muscle pouch for upto 12 weeks. Ectopic neocartilage formation in the PVA layer and reconstitution of the subchondral bone which remained confined within the n-HA/PA6 layer with the alteration of the cellular phenotype were identified with Masson's trichrome stain. Simultaneously, the RT-PCR results confirmed the expression of specific extracellular matrix (ECM) markers for cartilaginous tissue, such as collagen type II (Col-II), or alternatively, markers for osteoid tissue, such as collagen type I (Col-I) at the corresponding layers. During ectopic implantation, the underlying subchondral bone layer was completely integrated with the cartilage layer. The result from the ectopic osteochondral scaffolds implantation suggests that PVA-n-HA/PA6 with induced BMSCs is a possible substitute with potential in cartilage repair strategies.     

Biocompatibility and osteogenesis of biomimetic nano-hydroxyapatite/polyamide composite scaffolds for bone tissue engineering

In this study, we prepared nano-hydroxyapatite/polyamide (n-HA/PA) composite scaffolds utilizing thermally induced phase inversion processing technique. The macrostructure and morphology as well as mechanical strength of the scaffolds were characterized. Mesenchymal stem cells (MSCs) derived from bone marrow of neonatal rabbits were cultured, expanded and seeded on n-HA/PA scaffolds. The MSC/scaffold constructs were cultured for up to 7 days and the adhesion, proliferation and differentiation of MSCs into osteoblastic phenotype were determined using MTT assay, alkaline phosphatase (ALP) activity and collagen type I (COL I) immunohistochemical staining and scanning electronic microscopy (SEM). The results confirm that n-HA/PA scaffolds are biocompatible and have no negative effects on the MSCs in vitro. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both pure n-HA/PA scaffolds and MSC/scaffold constructs were implanted in rabbit mandibles and studied histologically and microradiographically. The results show that n-HA/PA composite scaffolds exhibit good biocompatibility and extensive osteoconductivity with host bone. Moreover, the introduction of MSCs to the scaffolds dramatically enhanced the efficiency of new bone formation, especially at the initial stage after implantation. In long term (more than 12 weeks implantation), however, the pure scaffolds show as good biocompatibility and osteogenesis as the hybrid ones. All these results indicate that the scaffolds fulfill the basic requirements of bone tissue engineering scaffold, and have the potential to be applied in orthopedic, reconstructive and maxillofacial surgery.     

新型人工生物活性接骨板治疗犬股骨骨折*◆

背景：研究表明纳米羟基磷灰石/聚酰胺66是一种具有良好的生物相容性、生物安全性、生物活性和骨传导能力的高强度高韧性复合材料。 目的：分析转染LIM矿化蛋白1基因的骨髓间充质干细胞复合纳米羟基磷灰石/聚酰胺66接骨板治疗犬股骨骨折的可行性和效果。 方法：收集犬骨髓间充质干细胞,经LIM矿化蛋白1转染后与纳米羟基磷灰石/聚酰胺66接骨板复合制备新型生物活性接骨板。48只杂交犬建立右侧股骨中段横形骨折模型,分别用转染/未转染LIM矿化蛋白1基因的骨髓间充质干细胞复合纳米羟基磷灰石/聚酰胺66接骨板、单纯纳米羟基磷灰石/聚酰胺66接骨板和钢板螺钉进行修复。 结果与结论：修复后8周及修复后12周转染组内固定失败率均低于未转染组和单纯接骨板组(P < 0.05),与钢板螺钉修复比较差异无显著性意义(P > 0.05)。转染组骨折愈合时间明显短于其他组。修复后12周转染组接骨板与犬股骨外侧皮质完全融合,骨干间有明显新骨生成。未转染组和单纯接骨板组未见或少量骨组织生成。说明新型生物活性接骨板能促进骨折愈合并与自体骨融合,不需要二次手术取出,但该新型接骨板的强度有一定的局限性,用于犬股骨骨折的治疗时必须辅加外固定。      

Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold

To investigate the potential application of bone marrow stromal cells (BMSCs) and an injectable sodium alginate/gelatin scaffold for bone tissue engineering (BTE). The phenotype of osteogenic BMSCs was examined by mineralized nodules formation and type I collagen expression. Cell proliferation was evaluated by MTT assay. The biocompatibility of scaffold and osteogenic cells were examined by hematoxylin and eosin (H&E) staining. Ectopic bone formation as well as closure of rabbit calvarial critical-sized defects following scaffold-cell implantation were analyzed by histological examination and computed tomography (CT) scanning. Spindle-shaped osteogenic cells of high purity were derived from BMSCs. The osteogenic cells and sodium alginate/gelatin (2:3) scaffold presented fine biocompatibility following cross-linking with 0.6% of CaCl(2). After implantation, the scaffold-cell construct promoted both ectopic bone formation and bone healing in the rabbit calvarial critical-sized defect model. Our data demonstrated that the sodium alginate/gelatin scaffold could be a suitable biomaterial for bone engineering, and the scaffold-osteogenic cells construct is a promising alternative approach for the bone healing process.     

人骨形成蛋白7修饰骨髓间充质干细胞复合仿生型支架材料的体内成骨研究

目的 应用携带人骨形成蛋白7(hBMP7)基因的兔骨髓间充质干细胞(BMSC)与仿生型生物玻璃-胶原-透明质酸-磷脂酰丝氨酸(BG-COL-HYA-PS)支架材料复合培养,植入兔桡骨缺损模型中观察其在体内成骨的能力.方法 携带hBMP7基因或增强型绿色荧光蛋白(EGFP)基因的2型重组腺相关病毒(rAAV2)载体在体外分别转染兔BMSC,再将转染后和未转染的兔BMSC分别与BG-COL-HYA-PS支架材料复合培养7 d后植入3组兔桡骨缺损模型,每组6只兔.各组在分别术后8周、12周通过大体标本观察、影像学、组织学等方法观察骨缺损的修复情况.以正常兔桡骨为对照组(n=3),术后12周比较各组骨缺损修复组织生物力学差异.结果 rAAV2-hBMP7转染的兔BMSC与BG-COL-HYA-PS复合支架材料有良好的生物相容性,植入兔桡骨缺损模型内表现出明确的成骨能力和骨修复能力,而形成的新骨最大压力载荷值低于正常桡骨组织[(188.46±12.24)N比(203.25±19.29)N,P＜0.05].结论 用hBMP7修饰BMSC复合仿生型BG-COL-HYA-PS支架材料构建的组织工程骨具有较强的骨修复能力,但形成的新生骨组织与正常骨组织比较仍然有早期生物力学方面的不足.     

Electroporation-mediated transfer of Runx2 and Osterix genes to enhance osteogenesis of adipose stem cells

In the present study, we tested the hypothesis that electroporation-mediated transfer of Runx2, Osterix, or both genes enhances the in vitro and in vivo osteogenesis from adipose stem cells (ASCs). ASCs were transfected with Runx2, Osterix, or both genes using electroporation, and further cultured in monolayer or in PLGA scaffold under osteogenic medium for 14 days, then analyzed for in vitro osteogenic differentiation. Transfected ASC-PLGA scaffold hybrids were also implanted on nude mice to test for in vivo ectopic bone formation. Runx2 and Osterix genes were strongly expressed in ASCs transfected with each gene on day 7, decreasing rapidly on day 14. Runx2 protein was strongly expressed in ASCs transfected with the Runx2 gene, while Osterix protein was strongly expressed in ASCs transfected with either or both Runx2 and Osterix genes. Overexpression of Runx2 and Osterix significantly increased the gene expression of osteogenic differentiation markers (alkaline phosphatase [ALP], osteocalcin [OCN], type I collagen [COL1A1], and bone sialoprotein [BSP]) in ASCs. Transfection of Runx2 and Osterix genes enhanced the protein expression of OCN, type I collagen, and BSP, as demonstrated by Western blot analysis, and ALP activity as well as enhancing mineralization in the monolayer culture and ASC-PLGA scaffold hybrids. Runx2- or Osterix-transfected ASC-PLGA scaffold hybrids promoted bone formation in nude mice after 6 weeks of in vivo implantation.     

辛伐他汀复合Bio-Oss修复兔下颌骨缺损

背景：研究发现辛伐他汀具有促进新骨形成的作用,但其成骨机制及成骨效果目前仍然存在争议。 目的：对比观察Bio-Oss/辛伐他汀复合材料与单纯Bio-Oss修复材料修复兔下颌骨骨缺损区的成骨效果。 方法：12只新西兰大白兔下颌骨双侧制备缺损,随机将一侧采用辛伐他汀复合Bio-Oss修复缺损区;另一侧采用单纯Bio-Oss修复缺损区。两组均覆盖Bio-Gide胶原膜。植骨后4,8,12周分别处死各组兔子4只,通过大体观察,X射线及口腔锥形束CT影像学观察,组织学切片观察,定性定量对比分析植骨区牙槽骨形成情况。 结果与结论：植骨后4,8,12周新骨形成逐渐增多,随着高阻射的Bio-Oss骨粉逐渐降解,在各时间点密度值测量结果辛伐他汀复合Bio-Oss组均显著低于单纯Bio-Oss组(P < 0.05)。新生骨百分比测量结果辛伐他汀复合Bio-Oss均显著高于单纯Bio-Oss组(P < 0.05)。提示辛伐他汀具有促进Bio-Oss骨粉吸收的效果,在骨缺损修复中具有促进新骨生成的作用。     

In vitro and in vivo evaluation of differentially demineralized cancellous bone scaffolds combined with human bone marrow stromal cells for tissue engineering

Mineralized and partially or fully demineralized biomaterials derived from bovine bone matrix were evaluated for their ability to support human bone marrow stromal cell (BMSC) osteogenic differentiation in vitro and bone-forming capacity in vivo in order to assess their potential use in clinical tissue-engineering strategies. BMSCs were either seeded on bone-derived scaffolds and cocultured in direct cell-to-scaffold contact, allowing for the exposure of soluble and insoluble matrix-incorporated factors, or cocultured with the scaffold preparations in a transwell system, exposing them to soluble matrix-incorporated factors alone. Osteoblast-related markers, alkaline phosphatase (ALP) activity and bone sialoprotein (BSP) and osteopontin (OP) mRNA expression were evaluated in BMSCs following 14 days of cocultivation in both systems. The data demonstrate that BMSCs from some donors express significantly higher levels of all osteoblast-related markers following cocultivation in direct cell-to-scaffold contact with mineralized scaffolds in comparison to fully demineralized preparations, while BMSCs from other donors display no significant differences in response to various scaffold preparations. In contrast, BMSCs cocultured independently with soluble matrix-incorporated factors derived from each scaffold preparation displayed significantly lower levels of ALP activity and BSP mRNA expression in comparison to untreated controls, while no significant differences were observed in marker levels between cells cocultured similarly with different biomaterial preparations. In addition, BMSCs were seeded directly on mineralized and partially or fully demineralized biomaterials and implanted in subcutaneous sites of athymic mice for 8 weeks to evaluate their in vivo bone-forming capacity. The ex vivo incorporation of BMSCs into all bone-derived scaffold preparations substantially increased the mean extent and frequency of samples containing de novo bone formation over similar nonseeded controls, as determined by histological and histomorphometrical analysis. No statistically significant differences were observed in the extent or frequency of bone formation between various scaffold preparations seeded with BMSCs from different donors. These results demonstrate that the in vivo osteoinductivity of bone-derived scaffolds can be modulated by ex vivo incorporated BMSCs and the extent of scaffold demineralization plays a significant role in influencing in vitro osteogenic differentiation of BMSCs depending on the coculture system and BMSC donor.     

转碱性成纤维细胞生长因子基因组织工程骨修复兔骨缺损

背景：生物活性玻璃是一类具有良好生物活性及骨修复特性的生物医用材料,将其与血、脱钙骨基质或与骨形态发生蛋白等混合来促进成骨形成,增加强度,骨愈合更接近于天然骨结构。 目的：观察生物活性玻璃结合转碱性成纤维细胞生长因子基因骨髓间充质干细胞构建组织工程骨修复兔骨缺损的效果。 方法：以生物活性玻璃作为转碱性成纤维细胞生长因子基因骨髓间充质干细胞的可吸附载体,体外构建组织工程骨,将其植入兔桡骨中段10 mm骨缺损处,以自体骨移植组、生物活性玻璃/骨髓间充质干细胞组和空白组作为对照,术后2,4,8,12周进行影像学、病理组织切片、生物力学测试,观察各组骨缺损修复效果。 结果与结论：以生物活性玻璃作为转碱性成纤维细胞生长因子基因骨髓间充质干细胞的可吸附载体,体外构建的组织工程骨植入兔桡骨骨缺损的成骨效应、骨愈合后的抗扭转强度明显优于其他各组(P < 0.01)。说明生物活性玻璃结合转碱性成纤维细胞生长因子基因骨髓间充质干细胞构建的组织工程骨可用于骨缺损修复,优于自体骨移植。     

The performance of poly-epsilon-caprolactone scaffolds in a rabbit femur model with and without autologous stromal cells and BMP4

The ability of a cellular construct to guide and promote tissue repair strongly relies on three components, namely, cell, scaffold and growth factors. We aimed to investigate the osteopromotive properties of cellular constructs composed of poly-epsilon-caprolactone (PCL) and rabbit bone marrow stromal cells (BMSCs), or BMSCs engineered to express bone morphogenetic protein 4 (BMP4). Highly porous biodegradable PCL scaffolds were obtained via phase inversion/salt leaching technique. BMSCs and transfected BMSCs were seeded within the scaffolds by using an alternate flow perfusion system and implanted into non-critical size defects in New Zealand rabbit femurs. In vivo biocompatibility, osteogenic and angiogenic effects induced by the presence of scaffolds were assessed by histology and histomorphometry of the femurs, retrieved 4 and 8 weeks after surgery. PCL without cells showed scarce bone formation at the scaffold-bone interface (29% bone/implant contact and 62% fibrous tissue/implant contact) and scarce PCL resorption (16%). Conversely, PCL seeded with autologous BMSCs stimulated new tissue formation into the macropores of the implant (20%) and neo-tissue vascularization. Finally, the BMP4-expressing BMSCs strongly favoured osteoinductivity of cellular constructs, as demonstrated by a more extensive bone/scaffold contact.     

Fate of bone marrow stromal cells in a syngenic model of bone formation

Bone marrow stromal cells (BMSCs) have been demonstrated to induce bone formation when associated to osteoconductive biomaterials and implanted in vivo. Nevertheless, their role in bone reconstruction is not fully understood and rare studies have been conducted to follow their destiny after implantation in syngenic models. The aim of the present work was to use sensitive and quantitative methods to track donor and recipient cells after implantation of BMSCs in a syngenic model of ectopic bone formation. Using polymerase chain reaction (PCR) amplification of the Sex determining Region Y (Sry) gene and in situ hybridization of the Y chromosome in parallel to histological analysis, we have quantified within the implants the survival of the donor cells and the colonization by the recipient cells. The putative migration of the BMSCs in peripheral organs was also analyzed. We show here that grafted cells do not survive more than 3 weeks after implantation and might migrate in peripheral lymphoid organs. These cells are responsible for the attraction of host cells within the implants, leading to the centripetal colonization of the biomaterial by new bone.     

Ectopic bone formation in collagen sponge self-assembled peptide-amphiphile nanofibers hybrid scaffold in a perfusion culture bioreactor

The objective of this study was to enhance ectopic bone formation in a three-dimensional (3-D) hybrid scaffold in combination with bioreactor perfusion culture system. The hybrid scaffold consists of two biomaterials, a hydrogel formed through self-assembly of peptide-amphiphile (PA) with cell suspensions in media, and a collagen sponge reinforced with poly(glycolic acid) (PGA) fiber incorporation. PA was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. A 3-D network of nanofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. Osteogenic differentiation of mesenchymal stem cells (MSC) in the hybrid scaffold was greatly influenced by the perfusion culture method compared with static culture method. When the osteoinduction activity of hybrid scaffold was studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds when perfusion culture was used compared with static culture method. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture method. We conclude that combination of MSC-seeded hybrid scaffold and the perfusion method was promising to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.     

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。     

Evaluation of the osteoconductive potential of poly(propylene carbonate)/nano-hydroxyapatite composites mimicking the osteogenic niche for bone augmentation

Nano-hydroxyapatite (n-HA) reinforced poly(propylene carbonate) (PPC) composites were prepared for bone repair and reconstruction. The effects of reinforcement on the morphology, mechanical properties and biological performance of n-HA/PPC composites were investigated. The surface morphology and mechanical properties of the composites were characterized by scanning electron microscopy (SEM) and universal material testing machine. The analytical data showed that good incorporation and dispersion of n-HA crystals could be obtained in the PPC matrix at a 30:70 weight ratio. With the increase of n-HA content, the tensile strength increased and the fracture elongation rate decreased. In vitro cell culture revealed that the composite was favorable template for cell attachment and growth. In vivo implantation in femoral condyle defects of rabbits confirmed that the n-HA/PPC composite had good biocompatibility and gradual biodegradability, exhibiting good performance in guided bone regeneration. The results demonstrates that the incorporation of n-HA crystals into PPC matrix provides a practical way to produce biodegradable and cost-competitive composites mimicking the osteogenic niche for bone augmentation.     

异种脱钙骨基质联合骨髓间充质干细胞植入豚鼠鼓泡腔增强成骨作用*

背景：目前在矫形外科和颅面重建中已有脱钙骨基质和骨髓间充质干细胞的相关应用,但用于乳突腔填塞尚缺乏研究。 目的：探寻异种脱钙骨基质和骨髓间充质干细胞联合植入乳突腔的成骨效能。 方法：全骨髓贴壁培养法分离近交系雌性豚鼠骨髓间充质干细胞,牛股骨头松质骨制备牛髂骨脱钙骨基质。20只豚鼠随机数字表法分为脱钙骨基质与骨髓间充质干细胞联合植入鼓泡腔的实验组和取自体髂骨植入鼓泡腔的对照组。 结果与结论：植入1个月后两组的成骨量差异无显著性意义;植入后2个月实验组成骨量多于对照组,差异有显著性意义    (P < 0.05)。说明异种脱钙骨基质与骨髓间充质干细胞联合植入乳突腔能有效促进乳突填塞后骨的形成。     

聚左旋乳酸/壳聚糖纳米纤维三维多孔支架复合骨髓间充质干细胞修复兔骨缺损**△

背景：骨髓间充质干细胞具有向多种间质细胞谱系分化的能力,且支架材料的性能对骨缺损的修复有重要影响。 目的：观察聚左旋乳酸/壳聚糖纳米纤维三维多孔支架复合骨髓间充质干细胞治疗骨缺损。 方法：对骨缺损模型兔分别采用空白植入、髂后上棘自体松质骨移植、聚左旋乳酸/壳聚糖纳米纤维多孔支架移植和复合了骨髓间充质干细胞的聚左旋乳酸/壳聚糖纳米纤维多孔支架移植修复缺损部位。 结果与结论：至移植12周,移植复合了骨髓间充质干细胞的聚左旋乳酸/壳聚糖纳米纤维多孔支架的实验兔的缺损处有骨组织生成,支架材料降解,已完成缺损修复,其修复情况接近松质骨组;髂后上棘自体松质骨移植的实验兔的缺损修复完好,新形成的骨组织较规则;只植入聚左旋乳酸/壳聚糖纳米纤维多孔支架的实验兔有少量骨组织形成,材料部分降解;空白植入的实验兔缺损处无新生骨组织生成,主要由纤维结缔组织填充。说明新型的生物支架材料聚左旋乳酸/壳聚糖纳米纤维三维多孔支架与来源于新西兰大白兔的骨髓间充质干细胞复合培养后,植入同种异体兔股骨髁缺损处,使骨缺损的修复速度加快,表现为较好的体内诱导成骨的作用。     

比较纳米羟基磷灰石/聚酰胺66人工椎体与自体髂骨在颈椎前路减压融合中的应用

背景：目前用于颈椎前路重建的材料较多,如自体髂骨、同种异体骨、钛网等,但各种材料均存在一定的不足。纳米羟基磷灰石/聚酰胺66人工椎体具有良好的生物相容性及生物安全性,是一种比较理想的椎体植骨替代材料。 目的：评估纳米羟基磷灰石/聚酰胺66人工椎体应用于颈椎前路减压融合治疗脊髓型颈椎病的临床效果,并与自体髂骨进行对比。 方法：2009-01/2010-03对40例脊髓型颈椎病患者行颈前路椎体次全切减压融合钛板内固定。22例行纳米羟基磷灰石/聚酰胺66人工椎体植骨,18例行自体髂骨块植骨,采用JOA评分法评价神经功能的恢复情况,测量Cobb角评价融合节段曲度以及融合节段椎体前缘、后缘高度。 结果与结论：患者均获得 6~14个月随访,JOA评分较治疗前明显改善。人工椎体组及自体髂骨组融合节段后缘高度和前凸Cobb角治疗后3个月与治疗后即刻差值、治疗后6个月与治疗后3个月差值差异均有显著性意义(P < 0.01)。根据融合标准,治疗后6个月两组融合情况差异无显著性意义(P  > 0.05)。提示纳米羟基磷灰石/聚酰胺66人工椎体作为颈椎前路植骨材料,融合率同自体髂骨相似,可以有效保持颈椎生理曲度及椎间高度,长期效果有待进一步观察。     

In vivo bone formation from human embryonic stem cell-derived osteogenic cells in poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffolds

We have previously reported the efficient osteogenic differentiation of human embryonic stem cells (hESCs) by co-culture with primary human bone-derived cells (hPBDs) without the use of exogenous factors. In the present study, we explored whether osteogenic cells derived from hESCs (OC-hESCs) using the previously reported method would be capable of regenerating bone tissue in vivo. A three-dimensional porous poly(d,l-lactic-co-glycolic acid)/hydroxyapatite composite scaffold was used as a cell delivery vehicle. In vivo implantation of OC-hESC-seeded scaffolds showed significant bone formation in the subcutaneous sites of immunodeficient mice at 4 and 8 weeks after implantation (n=5 for each time point). Meanwhile, implantation of the control no cell-seeded scaffolds or human dermal fibroblast-seeded scaffolds did not show any new bone formation. In addition, the presence of BMP-2 (1 microg/scaffold) enhanced new bone tissue formation in terms of mineralization and the expression of bone-specific genetic markers. According to FISH analysis, implanted OC-hESCs remained in the regeneration sites, which suggested that the implanted cells participated in the formation of new bone. In conclusion, OC-hESCs successfully regenerated bone tissue upon in vivo implantation, and this regeneration can be further enhanced by the administration of BMP-2. These results suggest the clinical feasibility of OC-hESCs as a good alternative source of cells for bone regeneration.