Effect of strontium substituted ß-TCP associated to mesenchymal stem cells from bone marrow and adipose tissue on spinal fusion in healthy and ovariectomized rat

Despite alternatives to autogenous bone graft for spinal fusion have been investigated, it has been shown that osteoconductive materials alone do not give a rate of fusion comparable with autogenous bone. This study analyzed a strontium substituted ß-tricalcium phosphate (Sr-ßTCP) associated with syngeneic, unexpanded, and undifferentiated mesenchymal stem cells from bone marrow (BMSC) or adipose tissue (ADSC) as a new tissue engineering approach for spinal fusion procedures. A posterolateral fusion was performed in 15 ovariectomized (OVX) and 15 sham-operated (SHAM) Inbred rats. Both SHAM and OVX animals were divided into three groups: Sr-ßTCP, Sr-ßTCP + BMCSs, and Sr-ßTCP + ADSCs. Animals were euthanized 8 weeks after surgery and the spines evaluated by manual palpation, micro-CT, and histology. For both SHAM and OVX animals, the fusion tissue in the Sr-ßTCP + BMSCs group was more solid. This effect was significantly higher in OVX animals by comparing the Sr-ßTCP + BMCSs group with Sr-ßTCP + ADSCs. Radiographical score, based on micro-CT 2D image, highlighted that the Sr-ßTCP + BMCSs group presented a similar fusion to Sr-ßTCP and higher than Sr-ßTCP + ADSCs in both SHAM and OVX animals. Micro-CT 3D parameters did not show significant differences among groups. Histological score showed significantly higher fusion in Sr-ßTCP + BMSCs group than Sr-ßTCP and Sr-ßTCP + ADSCs, for both SHAM and OVX animals. In conclusion, our results suggest that addition of BMSCs to a Sr-ßTCP improve bone formation and fusion, both in osteoporotic and nonosteoporotic animal, whereas spinal fusion is not enhanced in rats treated with Sr-ßTCP + ADSCs. Thus, for conducting cells therapy in spinal surgery BMSCs still seems to be a better choice compared with ADSCs.     

Utility of tricalcium phosphate and osteogenic matrix cell sheet constructs for bone defect reconstruction

AIM: To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate (TCP) constructs on bone formation in bone defects.METHODS: Osteogenic matrix cell sheets were prepared from bone marrow stromal cells (BMSCs), and a porous TCP ceramic was used as a scaffold. Three experimental groups were prepared, comprised of TCP scaffolds (1) seeded with BMSCs; (2) wrapped with osteogenic matrix cell sheets; or (3) both. Constructs were implanted into a femoral defect model in rats and bone growth was evaluated by radiography, histology, biochemistry, and mechanical testing after 8 wk.RESULTS: In bone defects, constructs implanted with cell sheets showed callus formation with segmental or continuous bone formation at 8 wk, in contrast to TCP seeded with BMSCs, which resulted in bone non-union. Wrapping TCP constructs with osteogenic matrix cell sheets increased their osteogenic potential and resulting bone formation, compared with conventional bone tissue engineering TCP scaffolds seeded with BMSCs. The compressive stiffness (mean ± SD) values were 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6 MPa for BMSC/TCP/Sheet constructs with continuous bone formation, BMSC/TCP/Sheet constructs with segmental bone formation, and BMSC/TCP constructs, respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation was significantly higher than those with segmental bone formation and BMSC/TCP constructs.CONCLUSION: This technique is an improvement over current methods, such as TCP substitution, and is useful for hard tissue reconstruction and inducing earlier bone union in defects.     

Human CD133-derived bone marrow stromal cells establish ectopic hematopoietic microenvironments in immunodeficient mice

Cultured adherent bone marrow stromal cells (BMSCs) are capable of forming ectopic hematopoietic microenvironments (HMEs) in immunodeficient mice. However, the cell surface phenotype of the native bone marrow stem/progenitor cell that gives rise to BMSCs that support hematopoiesis remains poorly defined. We recently reported the derivation of human BMSC-like cells (CD133BMSCs) by magnetic cell sorting against Prominin-1 (CD133), an epitope expressed by embryonic, fetal, and adult stem cells. Here we demonstrate that CD133BMSCs are capable of forming ectopic HMEs. Cultured adherent CD133BMSCs derived from sorted CD133-positive cells lacked CD133 expression, but were uniformly positive for CD146, an epitope recently described to identify self-renewing osteoprogenitor cells that could transfer the HME. CD133BMSCs were genetically-tagged by lentivirus, expanded, and seeded into HA/TCP/fibrin constructs that were implanted subcutaneously. After 60 days, CD133BMSCs produced human osteocytes, osteoblasts, adipocytes, and reticular cells that supported murine hematopoiesis. CD133BMSCs that were not transduced with lentivirus also formed HMEs. Control constructs seeded with human dermal fibroblasts formed connective tissue, but failed to form HMEs. Our data indicate that CD133 expression identifies a native human bone marrow stem/progenitor cell that gives rise to BMSCs capable of forming the HME.     

In vivo bone formation by human bone marrow stromal cells: effect of carrier particle size and shape

Successful closure of bone defects in patients remains an active area of basic and clinical research. A novel and promising approach is the transplantation of human bone marrow stromal cells (BMSCs), which have been shown to possess a significant osteogenic potential. The extent and quality of bone formation by transplanted human BMSCs strongly depends on the carrier matrix with which cells are transplanted; to date, hydroxyapatite/tricalcium phosphate (HA/TCP) supports far more osteogenesis than any other matrix tested. In order to further improve the technique of BMSC transplantation, we studied whether commercially available HA/TCP particles, clinically approved as an osteoconductive material and commercially available as particles measuring 0.5-1.0 mm diameter, is an optimum matrix for promoting bone development by BMSCs. HA/TCP and HA particles of varying size were sieved into a variety of size ranges, from <0.044 mm to 1.0-2.0 mm. Transplants were formed by mixing 40 mg aliquots of particles with cultured passaged human BMSCs. They were placed in subcutaneous pockets in immunocompromised Bg-Nu-XID mice and harvested 4 or 10 weeks later. The transplants were examined histologically; the presence of bone within each transplant was evaluated using histomorphometry or blindly scored on a semiquantitative scale. Transplant morphology and the amount of new bone varied in a consistent fashion based on particle size and shape. Transplants incorporating HA/TCP particles of 0.1-0.25 mm size demonstrated the greatest bone formation at both 4 and 10 weeks; larger or smaller particles were associated with less extensive bone formation, while a size of 0.044 mm represented a threshold below which no bone formation could be observed. Flat-sided HA particles measuring 0.1-0.25 mm formed no bone. The differences in bone formation were not attributable to the differences in cell attachment among the groups. Instead, the size and spatial and structural organization of the particles within BMSC transplants appear to determine the extent of bone formation. These findings provide necessary information for the successful clinical application of BMSC transplantation techniques.     

表面改性双相陶瓷复合自体骨髓干细胞修复兔骨缺损的实验研究

目的:应用自体骨髓基质干细胞(Bone Marrow Stromal Cells,BMSCs)复合经低晶态羟基磷灰石(Low Crystalline Hydroxyap- atite,LcHA)涂层的双相陶瓷(Biphasic Calcium Phosphate,BCP)构建的组织工程化骨(LcBCP)修复兔桡骨节段性缺损。方法:体外分离培养、诱导扩增兔BMSCs,取第三代细胞复合LcBCP(实验组)后修复15只兔左侧桡骨15mm缺损;右侧桡骨缺损处植入复合BMSCs的BCP(对照组),于植入后4、8和12周处死动物,通过大体形态、组织学、影像学和扫描电镜检测骨缺损修复效果。结果:BMSCs-LcBCP复合物生长良好,随时间延长,X线显示实验组连接处骨痂形成,对照组连接处始终愈合稍差,12周大体观察实验组骨修复良好,髓腔再通;组织学显示板层骨形成,连接处骨性愈合,实验对照组连接处虽然也为骨性愈合,但尚有较多编织骨形成。结论:自体BMSCs复合LcBCP形成的组织工程化骨可修复兔桡骨节段性缺损,低晶态羟基磷灰石涂层能够增强双相陶瓷的早期成骨。     

Tethering of Epidermal Growth Factor (EGF) to Beta Tricalcium Phosphate (βTCP) via Fusion to a High Affinity,Multimeric βTCP-Binding Peptide: Effects on Human Multipotent Stromal Cells/Connective Tissue Progenitors

Transplantation of freshly-aspirated autologous bone marrow, together with a scaffold, is a promising clinical alternative to harvest and transplantation of autologous bone for treatment of large defects. However, survival proliferation, and osteogenic differentiation of the marrow-resident stem and progenitor cells with osteogenic potential can be limited in large defects by the inflammatory microenvironment. Previous studies using EGF tethered to synthetic polymer substrates have demonstrated that surface-tethered EGF can protect human bone marrow-derived osteogenic stem and progenitor cells from pro-death inflammatory cues and enhance their proliferation without detriment to subsequent osteogenic differentiation. The objective of this study was to identify a facile means of tethering EGF to clinically-relevant βTCP scaffolds and to demonstrate the bioactivity of EGF tethered to βTCP using stimulation of the proliferative response of human bone-marrow derived mesenchymal stem cells (hBMSC) as a phenotypic metric. We used a phage display library and panned against βTCP and composites of βTCP with a degradable polyester biomaterial, together with orthogonal blocking schemes, to identify a 12-amino acid consensus binding peptide sequence, LLADTTHHRPWT, with high affinity for βTCP. When a single copy of this βTCP-binding peptide sequence was fused to EGF via a flexible peptide tether domain and expressed recombinantly in E. coli together with a maltose-binding domain to aid purification, the resulting fusion protein exhibited modest affinity for βTCP. However, a fusion protein containing a linear concatamer containing 10 repeats of the binding motif the resulting fusion protein showed high affinity stable binding to βTCP, with only 25% of the protein released after 7 days at 37^oC. The fusion protein was bioactive, as assessed by its abilities to activate kinase signaling pathways downstream of the EGF receptor when presented in soluble form, and to enhance the proliferation of hBMSC when presented in tethered form on commercial βTCP bone regeneration scaffolds.     

Dexamethasone-loaded hydroxyapatite enhances bone regeneration in rat calvarial defects

A combination of bioceramics and osteogenic factors is potentially useful for bone regeneration applications. In the present study, hydroxyapatite particles (HA) were loaded with dexamethasone (Dex) and then characterized using SEM and drug release study. The bone regeneration ability of Dex-loaded HA (Dex/HA) was investigated in a rat critical size bone defect using digital mammography, multislice spiral-computed tomography (MSCT) imaging, and histological analysis. The HA and Dex/HA showed nano and micro-scale morphology with a nearly homogenous distribution of diameter. In addition, about 90 % of the drug was released from Dex/HA over a period of three days. After 8 weeks of implantation in rat calvarial defects, no sign of inflammation or complication was observed at the site of surgery. According to digital mammography and MSCT, Dex/HA showed the highest bone regeneration in rat bone defects compared to those received drug-free HA. Histological studies confirmed these data and showed osteointegration to the surrounding tissue. Taking all together, it was demonstrated that Dex/HA can be used as an appropriate synthetic graft for bone tissue engineering applications. These newly developed bioceramics can be used as new bone graft substitutes in orthopaedic surgery and is capable of enhancing bone regeneration.     

Directly auto‐transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model

Bone tissue engineering approaches increasingly focus on the use of mesenchymal stem cells (MSC). In most animal transplantation models MSC are isolated and expanded before auto cell transplantation which might be critical for clinical application in the future. Hence this study compares the potential of directly auto‐transplanted versus in vitro expanded MSC with or without bone morphogenetic protein‐2 (BMP‐2) to induce bone formation in a large volume ceramic bone substitute in the sheep model. MSC were isolated from bone marrow aspirates and directly auto‐transplanted or expanded in vitro and characterized using fluorescence activated cell sorting (FACS) and RT‐PCR analysis before subcutaneous implantation in combination with BMP‐2 and β‐tricalcium phosphate/hydroxyapatite (β‐TCP/HA) granules. Constructs were explanted after 1 to 12 weeks followed by histological and RT‐PCR evaluation. Sheep MSC were CD29⁺, CD44⁺ and CD166⁺ after selection by Ficoll gradient centrifugation, while directly auto‐transplanted MSC‐populations expressed CD29 and CD166 at lower levels. Both, directly auto‐transplanted and expanded MSC, were constantly proliferating and had a decreasing apoptosis over time in vivo. Directly auto‐transplanted MSC led to de novo bone formation in a heterotopic sheep model using a β‐TCP/HA matrix comparable to the application of 60 μg/ml BMP‐2 only or implantation of expanded MSC. Bone matrix proteins were up‐regulated in constructs following direct auto‐transplantation and in expanded MSC as well as in BMP‐2 constructs. Up‐regulation was detected using immunohistology methods and RT‐PCR. Dense vascularization was demonstrated by CD31 immunohistology staining in all three groups. Ectopic bone could be generated using directly auto‐transplanted or expanded MSC with β‐TCP/HA granules alone. Hence BMP‐2 stimulation might become dispensable in the future, thus providing an attractive, clinically feasible approach to bone tissue engineering.     

BrdU体内示踪骨髓基质干细胞生物学状态的效果分析

目的:探讨5-溴脱氧尿嘧啶核苷(Brd U)体内示踪骨髓基质干细胞(BMSCs)生物学状态的效果。方法:抽取健康成年比格狗骨髓,在传代培养中进行Brd U标记并鉴定,体外实验中测定细胞周期、凋亡率和细胞活力;在体内实验中将标记Brd U的骨髓基质干细胞植入自体股骨头缺损处,另一侧单纯植入自体骨作为对照,记录成骨量与分子标记物的表达情况。结果:骨髓基质干细胞的Brd U体外标记率为85.2%。Brd U组的细胞凋亡率为3.62±1.33%,未标记组为3.52±1.08%;Brd U组与未标记组的细胞成活率分别为96.31±1.39%和95.20±2.10%,两组对比差异均无统计学意义(P0.05)。移植侧Brd U标记的骨髓基质干细胞免疫组化观察可见Brd U免疫组化染色阳性,阳性率为81.6%。骨髓基质干细胞移植侧缺损区的骨钙素、Ⅰ型胶原阳性细胞表达数量与强度明显高于对照侧缺损区;骨髓基质干细胞移植侧成骨量为17.46±2.12%,对照侧为9.06±1.24%,两两对比差异有统计学意义(P0.05)。结论:Brd U在体外示踪骨髓基质干细胞能有效反映细胞的生物学状态,体内示踪显示移植的骨髓基质干细胞能成活,能促进骨组织形成和坏死骨修复。     

Alkbh1‐mediated DNA N6‐methyladenine modification regulates bone marrow mesenchymal stem cell fate during skeletal aging

ObjectivesDNA N6‐methyladenine (N6‐mA) demethylase Alkbh1 participates in regulating osteogenic differentiation of mesenchymal stem cell (MSCs) and vascular calcification. However, the role of Alkbh1 in bone metabolism remains unclear.Materials and MethodsBone marrow mesenchymal stem cells (BMSCs)‐specific Alkbh1 knockout mice were used to investigate the role of Alkbh1 in bone metabolism. Western blot, qRT‐PCR, and immunofluorescent staining were used to evaluate the expression of Alkbh1 or optineurin (optn). Micro‐CT, histomorphometric analysis, and calcein double‐labeling assay were used to evaluate bone phenotypes. Cell staining and qRT‐PCR were used to evaluate the osteogenic or adipogenic differentiation of BMSCs. Dot blotting was used to detect the level of N6‐mA in genomic DNA. Chromatin immunoprecipitation (Chip) assays were used to identify critical targets of Alkbh1. Alkbh1 adeno‐associated virus was used to overexpress Alkbh1 in aged mice.ResultsAlkbh1 expression in BMSCs declined during aging. Knockout of Alkbh1 promoted adipogenic differentiation of BMSCs while inhibited osteogenic differentiation. BMSC‐specific Alkbh1 knockout mice exhibited reduced bone mass and increased marrow adiposity. Mechanistically, we identified optn as the downstream target through which Alkbh1‐mediated DNA m6A modification regulated BMSCs fate. Overexpression of Alkbh1 attenuated bone loss and marrow fat accumulation in aged mice.ConclusionsOur findings demonstrated that Alkbh1 regulated BMSCs fate and bone‐fat balance during skeletal aging and provided a potential target for the treatment of osteoporosis.     

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva‐derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK‐8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT‐qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT‐qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90‐positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.     

Sema3A and HIF1α co-overexpressed iPSC-MSCs/HA scaffold facilitates the repair of calvarial defect in a mouse model

Mesenchymal stem/stromal cells (MSCs) play an important role in bone tissue engineering because MSCs possess multilineage potential of differentiation to mesenchymal tissues. Semaphorin 3A (Sema3A) and hypoxia-inducible factor-1α (HIF1α) are proved as important regulatory factors for osteogenesis and angiogenesis. The aim of this study was to investigate the effects of Sema3A and HIF1α co-overexpression on the osteogenesis and angiogenesis in induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs). Importantly, we assessed the potential osteogenic effectiveness of Sema3A and HIF1α co-overexpressed iPSC-MSCs seeded on hydroxyapatite (HA) scaffold in a mouse calvarial defect model. The overexpression for Sema3A, HIF1α, or Sema3A-HIF1α fusion in iPSC-MSCs was performed by separately infecting with conducted lentiviral vector. We determined the cell proliferation, the expressions of osteogenic, and endothelial markers of iPSC-MSCs cultured in osteogenic or endothelial induction medium in vitro. A mouse model calvarial defect was created and implanted with the Empty implant, HA scaffold alone, HA scaffold combined with iPSC-MSCs that infected with negative control or Sema3A-HIF1α fusion for 8 weeks in vivo. The results showed that Sema3A and HIF1α co-overexpression reversed the reduced cell proliferation that reduced by Sema3A overexpression alone. Importantly, the co-overexpression significantly increased the expressions of osteogenic and angiogenic related-genes compared with negative control after induction. Moreover, the Sema3A-HIF1α co-overexpressed iPSC-MSCs seeded on HA scaffold boosted the new bone and collagen fiber formation and facilitated repair of calvarial defect in a mouse model, which might have the potential application for bone defect reconstruction.     

Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats

OBJECTIVE: The aim of this study was to develop a feasible approach to promote bone healing in osteoporotic rats using autogenous bone tissue-engineering and gene transfection of human bone morphogenetic protein 2 (hBMP-2). METHODS: Bone marrow stromal cells (BMSCs) from the left tibia of osteoporotic rats were transfected with the hBMP-2 gene in vitro which was confirmed by immunohistochemistry, in situ hybridization and Western blotting. Autogenous transfected or untransfected BMSCs were seeded on macroporous coral hydroxyapatite (CHA) scaffolds. Each cell-scaffold construct was implanted into a defect site which was created in the ramus of the mandible of osteoporotic rats. Four or eight weeks after implantation in situ hybridization was performed in BMSCs transfected with hBMP-2, X-ray examinations, histological and histomorphological analyses were used to evaluate the effect of tissue-engineered bone on osseous defect repair. RESULTS: Newly formed bone was observed at the margin of the defect 4 weeks after implantation with BMSCs transfected with BMP-2. Mature bone was observed 8 weeks after treatment. In the control group there was considerably less new bone and some adipose tissue was observed at the defect margins 8 weeks after implantation. CONCLUSIONS: Autogenous cells transfected with hBMP-2 promote bone formation in osteoporotic rats. BMSC-mediated BMP-2 gene therapy used in conjunction with bone tissue engineering may be used to successfully treat bone defects in osteoporotic rats. This method provides a powerful tool for bone regeneration and other tissue engineering.     

Icariin promotes osteogenic differentiation of bone marrow stromal cells and prevents bone loss in OVX mice via activating autophagy

Osteoporosis (OP) results from the impaired function of endogenous bone marrow mesenchymal stem cells (BMSCs). Icariin (ICA) has shown potential osteoprotective effects. However, the molecular mechanism for the anabolic action of ICA remains largely unknown. The objective of the present study is to investigate whether ICA prevents bone loss by acting on BMSCs via affecting the level of autophagy after ovariectomy (OVX). The BMSCs were extracted from BALB/c mice treated with ICA, chloroquine (CQ, an autophagy inhibitor) or ICA + CQ. The OVX mice were injected with ICA, CQ, or ICA + CQ for 1 month. We performed Alizarin Red staining and alkaline phosphatase staining to detect osteogenic differentiation of BMSCs. Micro-CT, hematoxylin and eosin staining, Oil Red O staining, and tartrate-resistant acid phosphatase staining were used to assess the bone mass, lipid droplets and osteoclasts in femurs. Autophagy activity in BMSCs from different groups was evaluated by Western blot analysis. The osteogenic differentiation of BMSCs from OVX-induced OP mice was decreased. Treatment with ICA reduced bone loss and formation of osteoclasts and increased osteogenic differentiation of BMSCs in vitro and vivo. In addition, autophagy was enhanced in BMSCs of OVX mice treated with ICA. Our results indicate that ICA prevents OVX-induced bone loss possibly by strengthening the osteogenic differentiation of BMSCs via increasing autophagic activity.     

Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo

Stimulation of bone formation by osteoinductive materials is of great clinical importance in spinal fusion surgery, repair of bone fractures, and in the treatment of osteoporosis. We previously reported that specific naturally occurring oxysterols including 20(S)-hydroxycholesterol (20S) induce the osteogenic differentiation of pluripotent mesenchymal cells, while inhibiting their adipogenic differentiation. Here we report the characterization of two structural analogues of 20S, Oxy34 and Oxy49, which induce the osteogenic and inhibit the adipogenic differentiation of bone marrow stromal cells (MSC) through activation of Hedgehog (Hh) signaling. Treatment of M2-10B4 MSC with Oxy34 or Oxy49 induced the expression of osteogenic differentiation markers Runx2, Osterix (Osx), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN), as well as ALP enzymatic activity and robust mineralization. Treatment with oxysterols together with PPARγ activator, troglitazone (Tro), inhibited mRNA expression for adipogenic genes PPARγ, LPL, and aP2, and inhibited the formation of adipocytes. Efficacy of Oxy34 and Oxy49 in stimulating bone formation in vivo was assessed using the posterolateral intertransverse process rat spinal fusion model. Rats receiving collagen implants with Oxy 34 or Oxy49 showed comparable osteogenic efficacy to BMP2/collagen implants as measured by radiography, MicroCT, and manual inspection. Histological analysis showed trabecular and cortical bone formation by oxysterols and rhBMP2 within the fusion mass, with robust adipogenesis in BMP2-induced bone and significantly less adipocytes in oxysterol-induced bone. These data suggest that Oxy34 and Oxy49 are effective novel osteoinductive molecules and may be suitable candidates for further development and use in orthopedic indications requiring local bone formation.     

Periostin regulates osteogenesis of mesenchymal stem cells from ovariectomized rats through actions on the ILK/Akt/GSK-3β Axis

Osteoporosis is a condition of the skeleton that mainly results from estrogen deficiency. Periostin is a matricellular component in bone that is involved in osteoblast differentiation. However, how Periostin promotes osteogenesis remains largely unknown. Here, we isolated bone marrow skeletal stem cells (BMSCs) derived from an ovariectomy (OVX)-induced osteoporosis rat model and the effects of periostin on BMSCs derived from OVX rats (OVX-BMSCs) were assessed. Overexpression of periostin enhanced alkaline phosphatase (ALP) and alizarin red staining in OVX-BMSCs as well as the osteogenic genes OCN, BSP and Runx2. ILK is a downstream effector of signals from the extracellular matrix and participates in bone homeostasis. Overexpression of periostin also increased expression of protein levels for ILK, as well as the downstream targets pAkt and pGSK3β. Suppression of ILK or Akt partially suppressed the enhancement of osteogenic ability induced by periostin overexpression in OVX-BMSCs. Thus, periostin may promote the osteogenic ability of OVX-BMSCs through actions on the ILK/Akt/GSK3β axis.