Insulin‐like growth factor 2 (IGF‐2) potentiates BMP‐9‐induced osteogenic differentiation and bone formation

Efficient osteogenic differentiation and bone formation from mesenchymal stem cells (MSCs) should have clinical applications in treating nonunion fracture healing. MSCs are adherent bone marrow stromal cells that can self‐renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We have identified bone morphogenetic protein 9 (BMP‐9) as one of the most osteogenic BMPs. Here we investigate the effect of insulin‐like growth factor 2 (IGF‐2) on BMP‐9‐induced bone formation. We have found that endogenous IGF‐2 expression is low in MSCs. Expression of IGF‐2 can potentiate BMP‐9‐induced early osteogenic marker alkaline phosphatase (ALP) activity and the expression of later markers. IGF‐2 has been shown to augment BMP‐9‐induced ectopic bone formation in the stem cell implantation assay. In perinatal limb explant culture assay, IGF‐2 enhances BMP‐9‐induced endochondral ossification, whereas IGF‐2 itself can promote the expansion of the hypertropic chondrocyte zone of the cultured limb explants. Expression of the IGF antagonists IGFBP3 and IGFBP4 leads to inhibition of the IGF‐2 effect on BMP‐9‐induced ALP activity and matrix mineralization. Mechanistically, IGF‐2 is further shown to enhance the BMP‐9‐induced BMPR‐Smad reporter activity and Smad1/5/8 nuclear translocation. PI3‐kinase (PI3K) inhibitor LY294002 abolishes the IGF‐2 potentiation effect on BMP‐9‐mediated osteogenic signaling and can directly inhibit BMP‐9 activity. These results demonstrate that BMP‐9 crosstalks with IGF‐2 through PI3K/AKT signaling pathway during osteogenic differentiation of MSCs. Taken together, our findings suggest that a combination of BMP‐9 and IGF‐2 may be explored as an effective bone‐regeneration agent to treat large segmental bony defects, nonunion fracture, and/or osteoporotic fracture. © 2010 American Society for Bone and Mineral Research.     

Additive effects of hyperbaric oxygen and platelet‐derived growth factor‐BB in chondrocyte transplantation via up‐regulation expression of platelet‐derived growth factor‐β receptor

The present study investigated the effects of hyperbaric oxygen (HBO) and platelet‐derived growth factor‐BB (PDGF‐BB) in chondrocyte transplantation. In vitro, chondrocytes were treated with HBO, PDGF‐BB, and HBO combined with PDGF‐BB (H+P). Cell growth was analyzed using cell counting, MTT assay, and FACS analysis. mRNA expression of the PDGF‐α receptor (PDGFR‐α) and β receptor (PDGFR‐β) was detected by RT‐PCR. Protein expression of PDGFR‐β was detected by Western blotting. In vivo, chondrocytes and PDGF‐BB were suspended in alginate as a transplantation system. Cartilage defects were grafted with this system and with or without HBO treatment. Released PDGF‐BB concentration was quantified by ELISA. After 8 weeks, animals were sacrificed and the repaired tissues were examined. In vitro data suggested that each treatment increased cell growth via the up‐regulated mRNA expression of PDGFR‐α and increased cell accumulation in the S‐phase. The H+P treatment was more additive in cell growth and in mRNA and protein expression of PDGFR‐β than HBO or PDGF‐BB. In vivo results suggested that PDGF‐BB delivery lasted for more than 5 weeks. Scoring results showed that each treatment significantly increased the cartilage repair. Safranin‐O and type II collagen staining confirmed the hyaline‐like cartilage regeneration in the repaired tissues. In situ up‐regulation of PDGFR‐β expression partially explains the additive effect of H+P treatment in cartilage repair. Accordingly, H+P offers a potential treatment method for cartilage repair. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1439–1446, 2009     

Native multipotential stromal cell colonization and graft expander potential of a bovine natural bone scaffold

Graft expanders are bone scaffolds used, in combination with autografts, to fill large bone defects in trauma surgery. This study investigates the graft expander potential of a natural bone substitute Orthoss® by studying its ability to support attachment, growth and osteogenic differentiation of neighboring multipotential stromal cells (MSCs). Material consisting of bone marrow (BM) aspirate and reamer‐irrigator‐aspirator (RIA)‐harvested autograft bone was co‐cultured with commercially available Orthoss® granules. Native MSCs attached to Orthoss® were expanded and phenotypically characterized. MSCs egress from neighboring cancelous bone was assessed in 3D Matrigel co‐cultures. MSC differentiation was evaluated using scanning electron microscopy and measuring alkaline phosphatase (ALP) activity per cell. CD45⁺ hematopoietic lineage cells and highly proliferative CD90⁺CD73⁺CD105⁺ MSCs preferentially colonized Orthoss® granules, over RIA bone chips. MSC colonization was followed by their intrinsic osteogenic differentiation, assessed as mineral deposition and gradual rise in ALP activity, even in the absence of osteogenic stimuli. When in contact with mixed cell populations and RIA chips, Orthoss® granules support the attachment, growth and osteogenic differentiation of neighboring MSCs. Therefore, natural bone substitutes similar to Orthoss® can be used as void fillers and graft expanders for repairing large bone defects in conjunction with autologous BM aspirates and autografts. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1950–1958, 2013     

Noggin resistance contributes to the potent osteogenic capability of BMP9 in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors and can differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bone morphogenetic proteins (BMPs) play important roles in stem cell proliferation and differentiation. We recently demonstrated that BMP9 is a potent but less understood osteogenic factor. We previously found that BMP9‐induced ectopic bone formation is not inhibited by BMP3. Here, we investigate the effect of BMP antagonist noggin on BMP9‐induced osteogenic differentiation. BMP antagonists noggin, chording, gremlin, follistatin, and BMP3 are highly expressed in MSCs, while noggin and follistatin are lowly expressed in more differentiated pre‐osteoblast C2C12 cells. BMP9‐induced osteogenic markers and matrix mineralization are not inhibited by noggin, while noggin blunts BMP2, BMP4, BMP6, and BMP7‐induced osteogenic markers and mineralization. Likewise, ectopic bone formation by MSCs transduced with BMP9, but not the other four BMPs, is resistant to noggin inhibition. BMP9‐induced nuclear translocation of Smad1/5/8 is not affected by noggin, while noggin blocks BMP2‐induced activation of Smad1/5/8 in MSCs. Noggin fails to inhibit BMP9‐induced expression of downstream targets in MSCs. Thus, our results strongly suggest that BMP9 may effectively overcome noggin inhibition, which should at least in part contribute to BMP9's potent osteogenic capability in MSCs. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1796–1803, 2013     

Pulsed electromagnetic fields enhance BMP‐2 dependent osteoblastic differentiation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) express an osteoblastic phenotype when treated with BMP‐2, and BMP‐2 is used clinically to induce bone formation although high doses are required. Pulsed electromagnetic fields (PEMF) also promote osteogenesis in vivo, in part through direct action on osteoblasts. We tested the hypothesis that PEMF enhances osteogenesis of MSCs in the presence of an inductive stimulus like BMP‐2. Confluent cultures of human MSCs were grown on calcium phosphate disks and were treated with osteogenic media (OM), OM containing 40 ng/mL rhBMP‐2, OM + PEMF (8 h/day), or OM + BMP‐2 + PEMF. MSCs demonstrated minor increases in alkaline phosphatase (ALP) during 24 days in culture and no change in osteocalcin. OM increased ALP and osteocalcin by day 6, but PEMF had no additional effect at any time. BMP‐2 was stimulatory over OM, and PEMF + BMP‐2 synergistically increased ALP and osteocalcin. PEMF also enhanced the effects of BMP‐2 on PGE2, latent and active TGF‐β1, and osteoprotegerin. Effects of PEMF on BMP‐2–treated cells were greatest at days 12 to 20. These results demonstrate that PEMF enhances osteogenic effects of BMP‐2 on MSCs cultured on calcium phosphate substrates, suggesting that PEMF will improve MSC response to BMP‐2 in vivo in a bone environment. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1250–1255, 2008     

Dexamethasone modulates BMP‐2 effects on mesenchymal stem cells in vitro

Dexamethasone/ascorbic acid/glycerolphosphate (DAG) and bone morphogenic protein (BMP)‐2 are potent agents in cell proliferation and differentiation pathways. This study investigates the in vitro interactions between dexamethasone and BMP‐2 for an osteoblastic differentiation of mesenchymal stem cells (MSCs). Bone marrow‐derived human MSCs were cultured with DAG (group A), BMP‐2 + DAG (group B), and DAG + BMP‐2 combined with a porous collagen I/III scaffold (group C). RT‐PCR, ELISA, immuncytochemical stainings and flow cytometry analysis served to evaluate the osteogenic‐promoting potency of each of the above conditions in terms of cell morphology/viability, antigen presentation, and gene expression. DAG induced collagen I secretion from MSCs, which was further increased by the combination of DAG + BMP‐2. In comparison, the collagen scaffold and the control samples showed no significant influence on collagen I secretion of MSCs. DAG stimulation of MSCs led also to a steady but not significant increase of BMP‐2 level. A DAG and more, a DAG + BMP‐2, stimulation increased the number of mesenchymal cells (CD105+/CD73+). All samples showed mRNA of ALP, osteopontin, Runx2, Twist 1 and 2, Notch‐1/2, osteonectin, osteocalcin, BSP, and collagen‐A1 after 28 days of in vitro culture. Culture media of all samples showed a decrease in Ca²⁺ and PO concentration, whereas a collagen‐I‐peak only occurred at day 28 in DAG‐ and DAG + BMP‐2‐stimulated bone marrow cells. In conclusion, BMP‐2 enhances DAG‐induced osteogenic differentiation in mesenchymal bone marrow cells. Both agents interact in various ways and can modify osteoblastic bone formation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1440–1448, 2008     

Mechanical loading increased BMP‐2 expression which promoted osteogenic differentiation of tendon‐derived stem cells

This study aimed to investigate the effect of repetitive tensile loading on the expression of BMP‐2 and the effect of BMP‐2 on the osteogenic differentiation of tendon‐derived stem cells (TDSCs) in vitro. Repetitive stretching was applied to TDSCs isolated from rat patellar tendon at 0%, 4%, and 8%, 0.5 Hz. The expression of BMP‐2 was detected by Western blotting and qPCR. To study the osteogenic effects of BMP‐2 on TDSCs, BMP‐2 was added to the TDSC monolayer for the detection of ALP activity and calcium nodule formation in a separate experiment. TDSCs adhered, proliferated, and aligned along the direction of externally applied tensile force while they were randomly oriented in the control group. Western blotting showed increased expression of BMP‐2 in 4% and 8% stretching groups but not in the control group. Up‐regulation of BMP‐2 mRNA was also observed in the 4% stretching group. BMP‐2 increased the osteogenic differentiation of TDSCs as indicated by higher ALP cytochemical staining, ALP activity, and calcium nodule formation. Repetitive tensile loading increased the expression of BMP‐2 and addition of BMP‐2 enhanced osteogenic differentiation of TDSCs. Activation of BMP‐2 expression in TDSCs during tendon overuse might provide a possible explanation of ectopic calcification in calcifying tendinopathy. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:390–396, 2011     

脂联素通过调节BMP信号通路促进骨髓干细胞成骨分化

目的 研究脂联素（adiponectin, ApN）对骨髓间充质干细胞（bone marrow mesenchymal stem cells, BMSCs）成骨分化的作用,并探讨其可能的机制。方法 体外培养BMSCs,构建ApN过表达质粒及干扰质粒,转染至BMSCs中。将BMSCs随机分为5组：对照组、过表达组、过表达空载组、干扰组和干扰空载组。茜素红染色观察各组细胞钙化沉积。碱性磷酸酶（alkaline phosphatase, ALP）染色观察各组细胞成骨分化能力。qRT-PCR检测ApN受体、骨形态发生蛋白（bone morphogenetic protein, BMP）信号通路及成骨相关基因表达情况。结果 与对照组相比,过表达组BMSCs中钙化沉积和ALP阳性表达增多,AdipoR1、AdipoR2、BMP2、RUNX2、Smad1和Smad5 mRNA表达量显著升高（P<0.05）;干扰组BMSCs中钙化沉积和ALP阳性表达减少,AdipoR1、AdipoR2、BMP2、RUNX2、Smad1和Smad5 mRNA表达量显著降低（P<0.05）。结论 ApN可能通过上调BMP信号通路促进BMSCs成骨分化。     

NVP‐BEZ235, a dual pan class I PI3 kinase and mTOR inhibitor,promotes osteogenic differentiation in human mesenchymal stromal cells

Osteoblasts are bone‐forming cells derived from mesenchymal stromal cells (MSCs) that reside within the bone marrow. In response to a variety of factors, MSCs proliferate and differentiate into mature, functional osteoblasts. Several studies have shown previously that suppression of the PI3K and mTOR signaling pathways in these cells strongly promotes osteogenic differentiation, which suggests that inhibitors of these pathways may be useful as anabolic bone agents. In this study we examined the effect of BEZ235, a newly developed dual PI3K and mTOR inhibitor currently in phase I–II clinical trials for advanced solid tumors, on osteogenic differentiation and function using primary MSC cultures. Under osteoinductive conditions, BEZ235 strongly promotes osteogenic differentiation, as evidenced by an increase in mineralized matrix production, an upregulation of genes involved in osteogenesis, including bone morphogenetic proteins (BMP2, ‐4, and ‐6) and transforming growth factor β1 (TGF‐β1) superfamily members (TGFB1, TGFB2, and INHBE), and increased activation of SMAD signaling molecules. In addition, BEZ235 enhances de novo bone formation in calvarial organotypic cultures. Using pharmacologic inhibitors to delineate mechanism, our studies reveal that suppression of mTOR and, to a much lesser extent PI3K p110α, mediates the osteogenic effects of BEZ235. As confirmation, shRNA‐mediated knockdown of mTOR enhances osteogenic differentiation and function in SAOS‐2 osteoblast‐like cells. Taken together, our findings suggest that BEZ235 may be useful in treating PI3K/mTOR‐dependent tumors associated with bone loss, such as the hematologic malignancy multiple myeloma. © 2010 American Society for Bone and Mineral Research.     

体外诱导家猪骨髓间充质干细胞向成骨细胞分化

目的观察家猪骨髓间充质干细胞(marrow mesenchymal stem cells,MSCs)在体外诱导条件下成骨分化的特征及相关基因的表达。方法选取3月龄雌性长白猪6头,无菌条件下于胫骨近端抽取骨髓15ml。采用密度梯度离心法并根据细胞贴壁特性对MSCs进行分离、纯化,倒置相差显微镜观察原代细胞生长情况,于培养第7天计算MSCs百分比含量及群体倍增值。将第1代细胞置于含1×10-8mmol/L地塞米松(dexamethasone,Dex)、10mmol/Lβ-磷酸甘油(β-glycerophosphate,β-GP)和82μg/ml抗坏血酸(ascorbic acid,Asc)的成骨诱导培养液中培养21d,作为实验组;DMEM培养液中培养作为对照组。分别行细胞形态学观察、碱性磷酸酶(alkaline phosphatase,ALP)组织化学染色、钙沉积和细胞增殖测定,采用实时定量PCR分析成骨分化的相关基因表达。结果原代MSCs特征:培养第1天,有核细胞大部分由悬浮的圆形血源性细胞组成;第3天换液弃除非贴壁细胞,MSCs克隆开始形成,细胞呈成纤维细胞样生长;第7天,镜下观察到大小不一的克隆。细胞在培养后12～14d基本长满,原代细胞群体倍增值平均为13。MSCs成骨分化:诱导培养14d实验组细胞形态由成纤维细胞样变成立方体样,而对照组细胞始终保持成纤维细胞样。培养5d,对照组细胞计数为11723±4040,实验组为10276±5513,二者差异无统计学意义(P>0.05)。与对照组相比,实验组诱导培养14d,ALP染色呈强阳性,21d钙沉积明显增加(P<0.01)。实验组MSCs成骨相关基因:核心结合因子α1(corebinding factorα1,Cbfα1)、osterix、ALP、型胶原、骨连接素(osteonectin,ON)、骨钙素(osteocalcin,OC)表达逐渐增强;Cbfα1、ALP、ON在分化早期增加明显;与第7天比较,第21天osterix和OC基因表达明显上调(P<0.05);第14天,型胶原表达也上调(P<0.05)。结论密度梯度离心法分离的猪MSCs,在体外诱导条件下能通过上调分化特异基因表达向成骨细胞分化。     

Peri‐prosthetic tissue cells show osteogenic capacity to differentiate into the osteoblastic lineage

During the process of aseptic loosening of prostheses, particulate wear debris induces a continuous inflammatory‐like response resulting in the formation of a layer of fibrous peri‐prosthetic tissue at the bone‐prosthesis interface. The current treatment for loosening is revision surgery which is associated with a high‐morbidity rate, especially in old patients. Therefore, less invasive alternatives are necessary. One approach could be to re‐establish osseointegration of the prosthesis by inducing osteoblast differentiation in the peri‐prosthetic tissue. Therefore, the aim of this study was to investigate the capacity of peri‐prosthetic tissue cells to differentiate into the osteoblast lineage. Cells isolated from peri‐prosthetic tissue samples (n = 22)−obtained during revision surgeries−were cultured under normal and several osteogenic culture conditions. Osteogenic differentiation was assessed by measurement of Alkaline Phosphatse (ALP), mineralization of the matrix and expression of several osteogenic genes. Cells cultured in osteogenic medium showed a significant increase in ALP staining (p = 0.024), mineralization of the matrix (p < 0.001) and ALP gene expression (p = 0.014) compared to normal culture medium. Addition of bone morphogenetic proteins (BMPs), a specific GSK3β inhibitor (GIN) or a combination of BMP and GIN to osteogenic medium could not increase ALP staining, mineralization, and ALP gene expression. In one donor, addition of GIN was required to induce mineralization of the matrix. Overall, we observed a high‐inter‐donor variability in response to osteogenic stimuli. In conclusion, peri‐prosthetic tissue cells, cultured under osteogenic conditions, can produce alkaline phosphatase and mineralized matrix, and therefore show characteristics of differentiation into the osteoblastic lineage. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1732–1742, 2017.

     

Expression of osterix inhibits bone morphogenetic protein-induced chondrogenic differentiation of mesenchymal progenitor cells

Osteoblasts and chondrocytes arise from common bipotential mesenchymal progenitor cells. Although the differentiation of these two cell lineages can be induced by treatment with bone morphogenetic proteins (BMPs), the responses of mesenchymal progenitors to BMP differ from cell line to cell line. Here we demonstrate that C3H/10T1/2 cells preferred chondrogenic differentiation, primary bone marrow stroma cells (MSCs) tended to convert to osteoblasts, and ST-2 cells differentiated into both the osteoblastic and chondrocytic lineages simultaneously, suggesting that a molecular switch functions to select cell fate. Osterix, the secondary master regulator of osteoblastogenesis, was induced by BMP at high and low levels in MSCs and ST-2 cells, respectively; in contrast, C3H/10T1/2 cells demonstrated only faint expression. As osterix has been suggested as a negative regulator of chondrogenesis, we hypothesized that the intense chondrocyte differentiation of C3H/10T1/2 cells may have resulted from an absence of osterix. We therefore restored osterix gene expression in C3H/10T1/2 cells using an adenovirus vector. Following BMP treatment, infection with an osterix-encoding virus dramatically inhibited the chondrocytic differentiation of C3H/10T1/2 cells, resulting instead in prominent osteoblast differentiation. These results indicate the chondrogenic potential of C3H/10T1/2 cells was abrogated by osterix expression. Chondrocyte differentiation of MSCs, however, was not enhanced by silencing the osterix gene using lentivirus-mediated shRNA, despite successful suppression of osteoblast differentiation. These results suggest that the low levels of osterix expression remaining after knockdown are sufficient to block chondrogenesis, whereas higher expression may be required to promote osteoblastic differentiation.     

Neurotrophin‐3 Induces BMP‐2 and VEGF Activities and Promotes the Bony Repair of Injured Growth Plate Cartilage and Bone in Rats

Injured growth plate is often repaired by bony tissue causing bone growth defects, for which the mechanisms remain unclear. Because neurotrophins have been implicated in bone fracture repair, here we investigated their potential roles in growth plate bony repair in rats. After a drill‐hole injury was made in the tibial growth plate and bone, increased injury site mRNA expression was observed for neurotrophins NGF, BDNF, NT‐3, and NT‐4 and their Trk receptors. NT‐3 and its receptor TrkC showed the highest induction. NT‐3 was localized to repairing cells, whereas TrkC was observed in stromal cells, osteoblasts, and blood vessel cells at the injury site. Moreover, systemic NT‐3 immunoneutralization reduced bone volume at injury sites and also reduced vascularization at the injured growth plate, whereas recombinant NT‐3 treatment promoted bony repair with elevated levels of mRNA for osteogenic markers and bone morphogenetic protein (BMP‐2) and increased vascularization and mRNA for vascular endothelial growth factor (VEGF) and endothelial cell marker CD31 at the injured growth plate. When examined in vitro, NT‐3 promoted osteogenesis in rat bone marrow stromal cells, induced Erk1/2 and Akt phosphorylation, and enhanced expression of BMPs (particularly BMP‐2) and VEGF in the mineralizing cells. It also induced CD31 and VEGF mRNA in rat primary endothelial cell culture. BMP activity appears critical for NT‐3 osteogenic effect in vitro because it can be almost completely abrogated by co‐addition of the BMP inhibitor noggin. Consistent with its angiogenic effect in vivo, NT‐3 promoted angiogenesis in metatarsal bone explants, an effect abolished by co‐treatment with anti‐VEGF. This study suggests that NT‐3 may be an osteogenic and angiogenic factor upstream of BMP‐2 and VEGF in bony repair, and further studies are required to investigate whether NT‐3 may be a potential target for preventing growth plate faulty bony repair or for promoting bone fracture healing. © 2016 American Society for Bone and Mineral Research.     

Immunomodulatory and osteogenic differentiation effects of mesenchymal stem cells by adenovirus‐mediated coexpression of CTLA4Ig and BMP2

Many reports have previously utilized a human bone morphogenetic protein 2 (BMP2)‐expressing recombinant adenoviral vector (AdBMP2) and mesenchymal stem cells (MSCs) for osteoinductive gene therapy. However, immunosuppression is essential for osteoinduction by AdBMP2, and this is one of the major impediments to its clinical use. In the present study, in vitro propagated MSCs were transduced using an adenoviral (Ad) vector to express the gene encoding cytotoxic T lymphocyte antigen 4‐immunoglobulin (CTLA4Ig). Lymphocyte response was induced by allogeneic‐irradiated MSCs as stimulators. We also examined the effects of cotransfection with a combination of the CTLA4Ig and the BMP2 gene on osteoblastic cell differentiation. The results showed that BMP2 gene transfected MSC elicited significant stimulatory responses, and one‐way MLR reactions were significantly blunted by CTLA4Ig. Further study demonstrates that cotransfection of MSCs with the combination of the CTLA4Ig and the BMP2 gene stimulates osteoblastic cell differentiation in vitro. The findings suggest that genetic engineering of MSCs to express an immunosuppressive molecule in combination with an osteogenic protein gene may have potential application in the treatment of several genetic diseases and in bone reconstruction. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:314–321, 2008     

Expression of preosteoblast markers and Cbfa-1 and Osterix gene transcripts in stromal tumour cells of giant cell tumour of bone

In giant cell tumour of bone (GCT), mononuclear stromal cells, which represent the neoplastic component of this lesion, regulate the formation of multinucleated osteoclast-like giant cells which are the characteristic hallmark of this tumour. However, the origin of stromal tumour cells has not yet been clearly defined. In this study, we evaluated several osteoblast markers including collagen type I, bone sialoprotein (BSP), osteonectin and osteocalcin in GCT using immunohistochemical techniques. Amongst the 13 GCT specimens and 7 GCT stromal cell (GCTSC) cultures studied, majority of the GCTSC synthesized type I collagen, BSP and osteonectin proteins but did not produce the differentiated osteoblast marker, osteocalcin. We further examined the regulation of several important osteogenic genes such as Cbfa-1, osterix and osteocalcin, and regulation of ALP activity in GCTSC in culture by bone morphogenetic protein 2 (BMP-2). Real-time PCR analysis indicated that Cbfa-1, osterix and osteocalcin mRNA were present in primary cultures of GCTSC. The addition of BMP-2 upregulated Cbfa-1 and osterix gene expression within 12 h and the enhancement was still observed at 24 h. ALP activity was minimal in untreated GCTSC in cultures. The number of ALP-positive GCTSC was significantly increased following treatment with BMP-2 or combinations with beta-glycerophosphate and ascorbic acid. In contrast, BMP enhancement of osterix mRNA level and ALP activity was also seen in SaOS2 osteoblast-like cells, but not in the primary culture of normal human skin fibroblasts. In summary, our data suggest that GCT stromal tumour cells may have an osteoblastic lineage and retain the ability to differentiate into osteoblasts.