Evaluation of Partially Demineralized Osteoporotic Cancellous Bone Matrix Combined with Human Bone Marrow Stromal Cells for Tissue Engineering: An In Vitro and In Vivo Study

Allogenous demineralized bone matrix (DBM) represents a potential scaffold for bone tissue engineering due to its close relation in structure and function with autologous bone, but its supply is often restricted by donor availability. Thus, an expanded source of human bone is needed. The aim of this study was to evaluate the capacity of partially DBM scaffolds derived from allogenous cancellous bone of osteoporotic femurs to support osteogenesis of human bone marrow stromal cells (BMSCs) in vitro and in vivo in order to assess their potential use in bone tissue-engineering strategies. Human BMSCs of passage 2 were seeded either on osteoporotic bone–derived DBM scaffolds or on normal bone–derived scaffolds and cultured in osteogenic medium for 14 days. To assess the in vitro proliferation potential and osteogenic differentiation of BMSCs on scaffolds, scanning electronic microscopy observation, DNA content assays, and measurements of alkaline phosphatase activity and osteocalcin content were applied; the results displayed no significant differences between the osteoporotic DBM group and the normal DBM group. After 2 weeks of subculture in vitro, the BMSC/DBM composites were subcutaneously implanted into athymic mice for 8 weeks to evaluate their in vivo bone-forming ability. Histological examination showed tissue-engineered bone formation in the DBM pores in both groups, and no significant differences were observed in either the extent or frequency of new bone formation between these two groups. Based on these results, it can be concluded that osteoporotic bone–derived DBM may serve as a promising scaffold for bone tissue engineering.     

Impaired marrow osteogenesis is associated with reduced endocortical bone formation but does not impair periosteal bone formation in long bones of SAMP6 mice.

We used the SAMP6 osteoporotic mouse to examine the link between marrow osteogenic potential and in vivo cortical bone formation. SAMP6 marrow supported less in vitro osteogenesis than marrow from SAMR1 controls; SAMP6 mice had a corresponding deficit in endocortical mineralizing surface. This marrow/endocortical defect did not affect the periosteum, where SAMP6 mice had normal to enhanced bone formation. INTRODUCTION: With aging, there may be a reduction in the number or proliferative capacity of bone marrow osteoprogenitors that may contribute to age-related decreases in bone formation. To examine the link between the ability of the marrow to support osteogenesis and age-related changes in bone formation, we measured in vitro and in vivo indices of osteogenesis in a model of osteoporosis, the senescence-accelerated mouse SAMP6. MATERIALS AND METHODS: Femora and tibias from SAMP6 and SAMR1 (control) mice were harvested at 2, 4, 6, and 12 months of age (168 bones total). Bone marrow cells were cultured under osteogenic conditions and stained for alkaline phosphatase (ALP) and alizarin red. Dynamic indices of bone formation were assessed histologically from calcein labels. RESULTS: ALP+ and alizarin red-positive areas were significantly less in cultures from SAMP6 bones versus SAMR1 (p < 0.05), indicating less osteogenic potential. For example, SAMP6 tibial cultures had 21% less ALP+ area and 36% less alizarin red-positive area than SAMR1. Marrow from tibias had 2-fold greater osteogenesis than femoral marrow (p < 0.001). SAMP6 mice had a deficit in endocortical mineralizing surface across all age groups (p < 0.05), but no deficit in mineral apposition rate. Last, despite the marrow and endocortical deficits, SAMP6 mice had normal or slightly increased periosteal bone formation, consistent with their larger bone size. CONCLUSION: SAMP6 bone marrow supports less in vitro osteogenesis than SAMR1, consistent with a lower concentration of marrow osteoprogenitors in SAMP6. SAMP6 mice have less endocortical mineralizing surface than SAMR1 at all ages but no detectable deficit in mineral apposition rate, which suggests a reduction in osteoblast number but normal function. Periosteal bone formation is unimpaired in SAMP6 mice, indicating that the marrow/endocortical defect does not affect the periosteal surface.     

Influence of defective bone marrow osteogenesis on fracture repair in an experimental model of senile osteoporosis

Bone marrow osteogenesis in senile osteoporotic bone is impaired and, as such, may have significant implications on the successful outcome of fracture repair. Here we utilize a well‐established murine model of senile osteoporosis, the P6 strain of senescence‐accelerated mice (SAMP6), to investigate fracture healing in aged osteoporotic bone. A femoral osteotomy was created in SAMP6 and in non‐osteoporotic age‐matched control R1 senescence‐resistant mice (SAMR1). The course of fracture healing was evaluated over a period of 42 days using quantitative µCT and histological analysis. The differentiation capabilities of bone mesenchymal progenitor cells derived from SAMP6 and SAMR1 mice was examined, and their osteogenic potential determined. Although preliminary in vitro analysis confirmed that bone marrow‐derived stem cells (BMSC) isolated from SAMP6 mice had a reduced osteogenic capacity, no significant deficit in fracture repair as determined by quantitative µCT could be detected. This was supported by histology assessment, where complete bridging of the fracture gap was evident by day 28 and was fully healed day 42 in both SAMP6 and SAMR1 mice. Further in vitro studies revealed that periosteal‐derived progenitor cells (PDPC) isolated from SAMP6 mice had an osteogenic potential comparable to that observed in SAMR1 mice. In conclusion, fracture healing in SAMP6 mice is not detrimentally affected by impairment of BMSC osteogenesis, suggesting that bone marrow‐mediated repair processes are dispensable for normal bone healing in this senile osteoporotic fracture model. Furthermore, the influence of PDPC in the repair process may partly explain the absence of any detectable deficits in fracture repair in SAMP6 mice. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:798–804, 2010     

Bone morphogenetic protein-2b stimulation of growth and osteogenic phenotypes in rat osteoblast-like cells: comparison with TGF-beta 1.

The biologic effects of recombinant human bone morphogenetic protein-2b (BMP-2b = BMP-4) were studied and compared with transforming growth factor-beta 1 (TGF-beta 1) in fetal rat osteoblast-like (ROB) cells. Similar to the effects of TGF-beta 1, BMP-2b stimulated DNA and collagen synthesis as well as protein accumulation. Unlike TGF-beta 1, which inhibited alkaline phosphatase activity, BMP-2b enhanced enzyme activity eight-to ninefold over the control level. The present study demonstrates direct actions of BMP-2b on bone-associated cells to stimulate osteogenic phenotypes in vitro and provides a cellular mechanism for the induction of bone formation by BMP-2b in vivo.     

A micro-architectural evaluation of osteoporotic human femoral heads to guide implant placement in proximal femoral fractures

Background and purpose The micro-architecture of bone has been increasingly recognized as an important determinant of bone strength. Successful operative stabilization of fractures depends on bone strength. We evaluated the osseous micro-architecture and strength of the osteoporotic human femoral head.

Material and methods 6 femoral heads, obtained during arthroplasty surgery for femoral neck fracture, underwent micro-computed tomography (microCT) scanning at 30 μm, and bone volume ratio (BV/TV), trabecular thickness, structural model index, connection density, and degree of anisotropy for volumes of interest throughout the head were derived. A further 15 femoral heads underwent mechanical testing of compressive failure stress of cubes of trabecular bone from different regions of the head.

Results The greatest density and trabecular thickness was found in the central core that extended from the medial calcar to the physeal scar. This region also correlated with the greatest degree of anisotropy and proportion of plate-like trabeculae. In the epiphyseal region, the trabeculae were organized radially from the physeal scar. The weakest area was found at the apex and peripheral areas of the head. The strongest region was at the center of the head.

Interpretation The center of the femoral head contained the strongest trabecular bone, with the thickest, most dense trabeculae. The apical region was weaker. From an anatomical and mechanical point of view, implants that achieve fixation in or below this central core may achieve the most stable fixation during fracture healing.     

CKIP-1过表达对大鼠体外破骨细胞增殖影响的研究

目的分离培养大鼠破骨细胞,构建CKIP-1过表达慢病毒,观察被CKIP-1过表达慢病毒感染的体外大鼠破骨细胞的细胞增殖情况,探讨CKIP-1基因对体外大鼠破骨细胞增殖的影响。方法分离、培养SD大鼠原代破骨细胞,用抗酒石酸酸性磷酸酶染色法鉴定。构建CKIP-1过表达载体,完成慢病毒包装。将破骨细胞分为A组(破骨细胞空白对照组)、B组(破骨细胞+空载病毒组)、C组(破骨细胞+CKIP-1过表达病毒组),分别进行对应处理,qRT-PCR检测CKIP-1基因表达效果,CCK-8试剂盒检验破骨细胞的相对数量。结果成功鉴定大鼠原代破骨细胞分离培养。qRT-PCR检测破骨细胞中CKIP-1的mRNA水平结果显示:相比A组和C组,在感染CKIP-1病毒的C组,CKIP-1的基因水平有较高表达(P0.01),CKIP-1过表达载体过表达效果明显,载体构建成功。CCK-8试剂盒检测各组细胞增殖的结果显示:相比A组和C组,感染CKIP-1病毒的C组的细胞增殖比例显著升高(P0.01)。结论成功分离培养出大鼠破骨细胞。成功构建CKIP-1过表达慢病毒,并感染体外大鼠破骨细胞。CKIP-1的过表达具有促进体外大鼠破骨细胞增殖的功能。     

Different Behavior of Human Osteoblast-Like Cells Isolated from Normal and Heterotopic Bone In Vitro

In this study, a characterization of human bone-forming cells responsible for heterotopic ossification was carried out in vitro. The biological and biochemical cell characteristics of the heterotopic osteoblast-like (HOB) cells were compared with those of orthotopic osteoblast-like (OB) cells from normal bone and stromal bone marrow cells believed to contain a subpopulation of osteogenic precursor cells. We found that HOB's from the spongiosa of heterotopic ossification required less time until the beginning of migration and the achievement of confluence in vitro compared with OBs from femoral shaft spongiosa. The fraction of mitotically active cells assessed by a clonogenic assay was higher as well in HOB cells. The in vitro studies of mitogenesis and the efficiency of colony formation of osteogenic cells indicate that with increasing differentiation and relative age they become more dependent on growth factors in the medium, otherwise the morphology of osteoblast-like cells changes and they pass irreversibly into the postmitotic stage of the cell cycle. The activity of the alkaline phosphatase is distinctly higher in the HOB than in the OB cells, HOB cells exhibit a lower level of osteocalcin expression compared with OB cells. No significant difference was found between OB and HOB cells in the amount of procollagen of type I sequestered by the cells. After 30 days, HOB and OB cells formed a mineralized matrix on exposure to 2 mM β-glycerophosphate. Since HOBs were isolated from heterotopic bone that had developed within 3–6 months after hip surgery, the differences in cellular behavior compared with OBs may be attributed to the relatively young age of HOB cells. Received: 29 March 1996 / Accepted: 21 May 1997     

Cortical Bone Porosity: What Is It,Why Is It Important,and How Can We Detect It?

There is growing recognition of the role of micro-architecture in osteoporotic bone loss and fragility. This trend has been driven by advances in imaging technology, which have enabled a transition from measures of mass to micro-architecture. Imaging trabecular bone has been a key research focus, but advances in resolution have also enabled the detection of cortical bone micro-architecture, particularly the network of vascular canals, commonly referred to as ‘cortical porosity.’ This review aims to provide an overview of what this level of porosity is, why it is important, and how it can be characterized by imaging. Moving beyond a ‘trabeculocentric’ view of bone loss holds the potential to improve diagnosis and monitoring of interventions. Furthermore, cortical porosity is intimately linked to the remodeling process, which underpins bone loss, and thus a larger potential exists to improve our fundamental understanding of bone health through imaging of both humans and animal models.     

The anti-diabetic drug metformin does not affect bone mass in vivo or fracture healing

Summary

The present study shows no adverse effects of the anti-diabetic drug metformin on bone mass and fracture healing in rodents but demonstrates that metformin is not osteogenic in vivo, as previously proposed.

Introduction

In view of the increased incidence of fractures in patients with type 2 diabetes mellitus (T2DM), we investigated the effects of metformin, a widely used T2DM therapy, on bone mass and fracture healing in vivo using two different rodent models and modes of metformin administration.

Methods

We first subjected 12-week-old female C57BL/6 mice to ovariectomy (OVX). Four weeks after OVX, mice received either saline or metformin administered by gavage (100 mg/kg/daily). After 4 weeks of treatment, bone micro-architecture and cellular activity were determined in tibia by micro-CT and bone histomorphometry. In another experiment, female Wistar rats aged 3 months were given only water or metformin for 8 weeks via the drinking water (2 mg/ml). After 4 weeks of treatment, a mid-diaphyseal osteotomy was performed in the left femur. Rats were sacrificed 4 weeks after osteotomy and bone architecture analysed by micro-CT in the right tibia while fracture healing and callus volume were determined in the left femur by X-ray analysis and micro-CT, respectively.

Results

In both models, our results show no significant differences in cortical and trabecular bone architecture in metformin-treated rodents compared to saline. Metformin had no effect on bone resorption but reduced bone formation rate in trabecular bone. Mean X-ray scores assessed on control and metformin fractures showed no significant differences of healing between the groups. Fracture callus volume and mineral content after 4 weeks were similar in both groups.

Conclusions

Our results indicate that metformin has no effect on bone mass in vivo or fracture healing in rodents.     

A novel strategy of spine defect repair with a degradable bioactive scaffold preloaded with adipose-derived stromal cells

Background contextAlthough the use of mesenchymal stem cells (MSC) with scaffolds for bone repair has been considered an effective method, the interactions between implanted materials and bone tissues have not been fully elucidated. At some specific sites, such as the vertebral body (VB) of the spine, the process of bone repair with implanted biomaterials is rarely reported. Recently, adipose tissue was found to be an alternative source of MSC besides bone marrow. However, the strategy of using adipose-derived stromal (ADS) cells with bioactive scaffold for the repair of spinal bone defects has seldom been studied.PurposeTo use a sintered poly(lactide-co-glycolide) acid (PLGA) microspheres scaffold seeded with induced rat ADS cells to repair a bone defect of the VB in a rat model.Study designBasic science and laboratory study.MethodsA sintered porous microspheres scaffold was manufactured by PLGA. ADS cells were isolated from Fischer 344 rats and then induced by osteogenic medium with growth and differentiation factor 5 (GDF5) in vitro. Before implantation, cells were cultured with inductive media for 2 weeks as a monolayer situation and 1 more week on a PLGA scaffold as a three-dimensional structure. These assembled bioactive scaffolds then were implanted in lumbar VB bone defects in Fischer 344 rats. The ex vivo differentiation of the cells was confirmed by von Kossa staining and real-time polymerase chain reaction. The performance of cells on the scaffold was detected by scanning electron microscopy and (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. In vivo bone formation was quantitatively measured by computed tomography study. And the effect of tissue repair was also evaluated by histological studies.ResultsProliferation and differentiation of cells were confirmed before in vivo implantation. Quantification of bone formation in vivo through serial three-dimensional computed tomography images revealed that the VB implanted with GDF5-induced cells demonstrated more bone formation than the control groups. Besides the bone formation period that occurred between 2 and 4 weeks in all groups, a second bone formation period was found to occur only in the groups that received cells with previous induction in vitro. This second period of significant bone formation happened simultaneously with collapsing of the scaffolds. It was then demonstrated histologically that vascularization early in the process and cooperation between host bone and implanted cells accompanied by collapse of the scaffold may be the factors that influence bone formation. This study not only provides a therapeutic strategy of using biomaterial for bone repair in the spine, but also may lead to a technological method for studying the relationship between implanted stem cells and host tissue.ConclusionsAdipose-derived stromal cells maintained in culture on a scaffold and treated with osteogenic induction with growth factor ex vivo could be used to enhance bone repair in vivo.     

成骨细胞株与强韧化纳米人工骨支架联合培养实验研究

目的 :观察成骨细胞株 ( 3T3 E1)在新型纳米氧化锆强韧化高孔隙率人工骨支架 (下文简称人工骨支架 )材料上生长情况。方法 :成骨细胞株 ( 3T3 -E1)与人工骨支架联合培养 ,通过光镜观察和细胞计数的方法了解成骨细胞与支架结合能力 ,扫描电镜观察细胞生长的情况。结果 :新型强韧化纳米人工骨材料与建株成骨细胞有良好的结合能力 ,成骨细胞株 ( 3T3 E1)在人工骨材料上生长良好。结论 :纳米骨支架是较理想的骨组织工程支架材料 ,成骨细胞复合支架用于骨缺损的修复 ,具有广阔的临床应用前景。     

CKIP-1与骨质疏松的最新研究进展

骨质疏松是严重威胁中老年人健康的骨科常见病。酪蛋白激酶2相互作用蛋白1(casein kinase 2 interaction protein 1,CKIP-1)是近年来发现的一种重要分子,它通过与其他分子的相互作用在许多细胞行为中都发挥着重要的作用。CKIP-1是参与调节骨形成的最重要的蛋白因子,与骨质疏松有密切联系,并已成为药物设计的新靶点。因此,对其的深入研究将为骨质疏松、病理机制阐明及骨疾病防治带来积极影响。本文就CKIP-1与骨质疏松症的关系进行综述。     

不同剂量辛伐他汀载药体系对兔骨质疏松模型骨修复的实验研究

目的研发一种新型可注射、可降解及具有抗骨质疏松作用的载药缓释体系,观察其理化性能及对兔骨质疏松模型的骨修复效果。方法载药缓释体系以磷酸钙为基体,将载有不同剂量辛伐他汀(Simvastatin,SIM)的聚左乳酸(Poly-L-Lactic Acid,PLLA)微球与磷酸钙物理共混获得一种可注射、可降解及抗骨质疏松的载药体系,实验分为对照组、低剂量SIM-PLLA缓释组及高剂量SIM-PLLA缓释组,分别测定载药缓释体系的可注射性、凝固时间、力学强度、药物缓释规律及骨质疏松兔模型体内成骨活性。结果与对照组比较,含SIM-PLLA缓释体系凝固时间有所延长(P0.05)、可注射性提高(P0.05)且具有合适的抗压强度(P0.05),植入骨质疏松兔体内4周及12周后,三组材料随着材料的降解均有新生骨长入,但SIM-PLLA缓释组材料的降解率及新骨生成率均显著优于对照组(P0.05),其中以高剂量SIM-PLLA缓释组最高。结论 SIM-PLLA缓释体系具有良好的注射性能及合适的力学强度,对兔骨质疏松模型有明显的成骨活性及抗骨质疏松效果,有望成为治疗骨质疏松性椎体压缩骨折及不规则骨缺损的新型生物医学材料。     

Exogenous heparin binds and inhibits bone morphogenetic protein 6 biological activity

Purpose

The purpose of this study was to explore the effect of heparin on bone morphogenetic protein 6 (BMP6) osteogenic activity.

Methods

Western blot analysis was used to confirm the binding of BMP6 to heparin and to observe its effect on BMP6 signaling in C2C12-BRE-Luc myoblasts. Real-time RT-PCR was performed for the expression analysis of alkaline phosphatase (ALP) and osteocalcin (OC) in C2C12 myoblasts treated with BMP6 and heparin for 72 hours. Rat ectopic bone formation assay was performed to explore the effect of heparin on BMP6 osteogenic activity. Two weeks following implantation the implants were analysed morphologically and histologically. A mouse osteoporotic model was used to test the ability of BMP6 to improve the bone quality in vivo in the presence of heparin, followed by DEXA and μCT analyses. Blood coagulation was tested in rats previously treated with BMP6.

Results

BMP6 specifically bound to heparin and induced Smad1/5/8 phosphorylation which was inhibited by heparin. After 48 and 72 hours of treatment, heparin inhibited BMP6-induced ALP and OC expression in C2C12 cells. Heparin dose dependently inhibited BMP6-induced new bone and cartilage formation in the rat ectopic bone formation assay, while in osteoporotic mice heparin inhibited the BMP6 potential to improve the bone quality as evidenced by decreased bone mineral density and trabecular bone parameters. Interestingly, BMP6 prevented the effect of heparin on the blood coagulation parameters.

Conclusion

The interaction of BMP6 with heparin might contribute to the heparin-induced osteoporosis and blood coagulation.     

Comparison of the osteogenic potential of bone dust and iliac bone chip

Background contextThere is no comparative study of the in vitro and in vivo osteogenic potential of iliac bone chips (autogenous iliac cancellous bone chips) compared with bone dusts generated during the decortication process with a high-speed burr in spine fracture or fusion surgery.PurposeTo compare the osteogenic potential of three sizes of bone dusts with iliac bone chips and to determine whether bone dusts can be used as a bone graft substitute.Study designIn vitro and in vivo study.MethodsBone chips were harvested from the posterior superior iliac spine and bone dusts from the vertebrae of 15 patients who underwent spinal fracture surgery. Bone dust was divided into three groups: small (3 mm), middle (4 mm), and large (5 mm) according to the size of the burr tip. A comparison was made using a cell proliferation assay, alkaline phosphatase (ALP) activity, the degree of mineralization in an in vitro model, and radiographic and histologic studies (the change of absorbable area and tissue density) after implantation of the various materials into back muscles of nude mice.ResultsAlthough all three bone dust groups were less active with regard to cell proliferation, ALP activity, and the degree of mineralization, than were bone chips, they still exhibited osteogenic potential. Furthermore, there was no significant difference among the three bone dust groups. The three bone dust groups did show greater absorbable area and change of the tissue density than did the iliac bone chip group. Again, there was no significant difference among the three bone dust groups in this regard. Histologically, specimens from the bone dust groups had a higher osteoclast cell number than specimens from the iliac bone chip group.ConclusionsThe osteogenic potential of bone dusts is lower than that of iliac bone chips, and the absorption speed of bone dusts in vivo is faster than that of iliac bone chips. The increased resorption speed appeared to result from an increase in osteoclast cell number. Therefore, caution needs to be used when surgeons employ bone dust as a bone graft substitute.     

Low-intensity pulsed ultrasound affects RUNX2 immunopositive osteogenic cells in delayed clinical fracture healing

IntroductionOsteogenic cell proliferation and differentiation play an important role in adequate fracture healing, and is target for osteoinductive therapies in delayed fracture healing. The aim of this study was to investigate whether low-intensity pulsed ultrasound enhances fracture healing at the tissue level in patients with a delayed union of the osteotomized fibula through an effect on the presence of RUNX2 immunopositive osteogenic cells. The effect was studied in both atrophic and hypertrophic delayed unions.Materials and methodsBiopsies were obtained from 6 female and 1 male patient (age 43–63) with a delayed union of the osteotomized fibula after a high tibial osteotomy treated for 2–4 months with or without low-intensity pulsed ultrasound in a randomized prospective double-blind placebo-controlled trial. Immunolocalization of RUNX2 protein was performed to identify osteogenic cells. Histomorphometrical analysis was performed to determine the number of cells expressing RUNX2 located within and around the newly formed woven bone at the fracture end (area of new bone formation), and up to 3 mm distant from the fracture end.ResultsCells expressing RUNX2 were present in all histological sections of control and low-intensity pulsed ultrasound-treated bone evaluated. Within the area of new bone formation, RUNX2 immunopositive cells were found in the undifferentiated soft connective tissue, at the bone surface (presumably osteoblasts), and within the newly formed woven bone. Low-intensity pulsed ultrasound treatment of fibula delayed unions significantly reduced the number of RUNX2 immunopositive cells within the soft connective tissue at the fracture ends, whereas the number of RUNX2 immunopositive cells at the bone surface was not affected. The number of RUNX2 immunopositive cells was similar for the atrophic and hypertrophic delayed unions.ConclusionsImmunolocalization of RUNX2 positive cells in delayed unions of the fibula reveals that delayed clinical fracture healing does not result in impairment of osteogenic cell proliferation and/or differentiation at the tissue level, even if delayed unions are clinically regarded as atrophic. Reduced number of osteogenic RUNX2 immunopositive cells within the soft connective tissue, and unchanged number of RUNX2 immunopositive cells at the bone surface, implicate that low-intensity pulsed ultrasound does not increase osteogenic cell presence, but likely affects osteogenic cell differentiation.     

In Vivo Osteogenic Capability of Human Mesenchymal Cells Cultured on Hydroxyapatite and on β-Tricalcium Phosphate

The aim of the current study was to examine in vitro osteogenic capability and in vivo bone formation of mesenchymal stromal cells (MSCs) on two kinds of calcium phosphate ceramics. MSCs derived from human bone marrow were seeded on either hydroxyapatite (HA) ceramic or β-tricalcium phosphate (β-TCP) ceramic and then cultured in a medium supplemented with a donor's serum, vitamin C, β-glycerophosphate, and dexamethasone. The culture revealed the expression of alkaline phosphatase activity, indicating the osteogenic differentiation of the MSCs on the ceramics (fabrication of tissue-engineered construct). The constructs were then implanted subcutaneously into nude rats for 8 weeks. New bone formation was observed in both types of ceramics, and human-specific Alu sequence was detected by in situ hybridization analysis. Quantitative microcomputed tomography showed that the volume of the new bone in the HA ceramic was greater than that in the β-TCP ceramic in six of seven cases. These results suggest that human MSCs cultured on ceramics could retain their osteogenic capability even after ectopic implantation and provide a rationale for the use of tissue-engineered constructs derived from a patient's MSCs and calcium phosphate ceramics in bone tissue regeneration.