Autogenous regulation of a network of bone morphogenetic proteins (BMPs) mediates the osteogenic differentiation in murine marrow stromal cells

The expression patterns of (bone morphogenetic proteins) BMPs during fracture repair and pre-natal bone development suggest that these processes are regulated through the coordinated actions of multiple BMPs. Murine bone marrow stromal cells (MSCs) in culture provide a well recognized ex vivo system of mesenchymal stem cell differentiation in which the effects of BMPs can be examined. Studies were performed to determine if MSC differentiation is dependent on the endogenous expression of multiple BMPs and to characterize their interactions. MSCs were harvested from the bone marrow of tibiae and femora of 8 to 10-week-old male C57/B6 mice and prepared by standard methods. Osteogenic differentiation was assessed by histological assays, alkaline phosphatase enzyme activity and assays for the expression of multiple mRNAs for BMPs and osteogenic development. The role of autogenously expressed BMPs in controlling the osteogenic differentiation of marrow stromal cells in vitro was assessed in both gain-of-function and loss-of-function experiments. Gain of function experiments were carried out in the presence of exogenously added BMP-2 or -7 and loss-of-function experiments were carried out by BMP antagonism with noggin and BMP-2 antibody blockade. Osteogenic differentiation was concurrent with and proportional to increases in the expression of BMPs-2, -3, -4, -5, -6 and -8A. BMP antagonism with either noggin or BMP-2 antibody blockade inhibited osteogenic differentiation by 50% to 80%, respectively, and reduced the expression of endogenous levels of BMPs-2, -3, -5 and -8A. In contrast, antagonism induced the expression of BMP-4 and -6. The addition of rhBMP-2 or -7 enhanced osteogenic differentiation and produced a reciprocal expression profile in the endogenous BMPs expression as compared to BMP antagonism. BMP antagonism could be rescued through the competitive addition of rhBMP-2. These studies demonstrated that osteogenic differentiation was regulated by a complex network of multiple BMPs that showed selective increased and decreased expression during differentiation. They further demonstrated that BMP-2 was a central regulator in this network.     

Osteogenic activity of the fourteen types of human bone morphogenetic proteins (BMPs)

BACKGROUND: Bone morphogenic proteins (BMPs) are known to promote osteogenesis, and clinical trials are currently underway to evaluate the ability of certain BMPs to promote fracture-healing and spinal fusion. The optimal BMPs to be used in different clinical applications have not been elucidated, and a comprehensive evaluation of the relative osteogenic activity of different BMPs is lacking. METHODS: To identify the BMPs that may possess the most osteoinductive activity, we analyzed the osteogenic activity of BMPs in mesenchymal progenitor and osteoblastic cells. Recombinant adenoviruses expressing fourteen human BMPs (BMP-2 to BMP-15) were constructed to infect pluripotent mesenchymal progenitor C3H10T1/2 cells, preosteoblastic C2C12 cells, and osteoblastic TE-85 cells. Osteogenic activity was determined by measuring the induction of alkaline phosphatase, osteocalcin, and matrix mineralization upon BMP stimulation. RESULTS: BMP-2, 6, and 9 significantly induced alkaline phosphatase activity in pluripotential C3H10T1/2 cells, while BMP-2, 4, 6, 7, and 9 significantly induced alkaline phosphatase activity in preosteoblastic C2C12 cells. In TE-85 osteoblastic cells, most BMPs (except BMP-3 and 12) were able to induce alkaline phosphatase activity. The results of alkaline phosphatase histochemical staining assays were consistent with those of alkaline phosphatase colorimetric assays. Furthermore, BMP-2, 6, and 9 (as well as BMP-4 and, to a lesser extent, BMP-7) significantly induced osteocalcin expression in C3H10T1/2 cells. In C2C12 cells, osteocalcin expression was strongly induced by BMP-2, 4, 6, 7, and 9. Mineralized nodules were readily detected in C3H10T1/2 cells infected with BMP-2, 6, and 9 (and, to a lesser extent, those infected with BMP-4 and 7). CONCLUSIONS: A comprehensive analysis of the osteogenic activity of fourteen types of BMPs in osteoblastic progenitor cells was conducted. Our results suggest an osteogenic hierarchical model in which BMP-2, 6, and 9 may play an important role in inducing osteoblast differentiation of mesenchymal stem cells. In contrast, most BMPs are able to stimulate osteogenesis in mature osteoblasts.     

Bone induction by AdBMP-2/collagen implants

BACKGROUND: Demineralized bone matrix and recombinant human bone morphogenetic protein-2 or 7 (BMP-2 or BMP-7)-containing collagenous matrix have been shown to induce new bone formation in orthotopic and heterotopic sites. We examined the ability of subcutaneous implants of collagen combined with adenoviral vector containing the BMP-2 gene (AdBMP-2) to induce bone formation in rats. We also evaluated whether targeting the AdBMP-2 vector through an alternative receptor pathway, fibroblast growth factor (FGF), would increase the vector's potency. METHODS: In a time-course study, rat subcutaneous sites were implanted with (1) AdBMP-2 in rat-bone-derived collagen or (2) rat-bone-derived collagen alone. Samples were collected three, seven, fourteen, or thirty-five days after treatment. In a dose-response study, bone induction by AdBMP-2 in collagen (AdBMP-2/collagen) or by AdBMP-2 and FGF2 Fab' anti-adenovirus knob protein antibody in collagen (FGF2-AdBMP-2/collagen) was tested at fourteen days. Viral vector doses of 1 x 10(9) PN (viral particle number), 3 x 10(9) PN, 1 x10(10) PN, 3 x 10(10) PN, or 1 x 10(11) PN per implant were used. Equal amounts of collagen (25 mg) were used to formulate all implants. Explanted tissues were evaluated histologically to determine bone formation, specific activity of alkaline phosphatase, and calcium content. RESULTS: AdBMP-2/collagen implants induced robust bone formation. New bone was formed by the fourteenth day after implantation. In contrast, little or no bone was induced by the implant containing collagen alone. FGF2-AdBMP-2/collagen implants stimulated significantly more bone formation (p < 0.05) than did AdBMP-2/collagen implants, regardless of the dose of viral particles. CONCLUSIONS: Local delivery of AdBMP-2 in a collagen matrix rapidly induces bone formation, and targeting the virus through FGF receptors enhances the osteogenic potential of AdBMP-2.     

Chondrocytes provide morphogenic signals that selectively induce osteogenic differentiation of mesenchymal stem cells.

During endochondral bone development cartilage formation always precedes that of bone, leading to the hypothesis that chondrocytes provide inductive signals for osteogenesis. To test this hypothesis, C3H10T1/2 mesenchymal stem cells were cocultured in membrane separated trans-well culture chambers with nonhypertrophic chondrocytes, hypertrophic chondrocytes, calvaria osteoblasts, or tendon fibroblasts derived from embryonic chickens to assess if individual cell types would selectively promote osteogenic differentiation. Then, differentiation of C3H10T1/2 mesenchymal stem cells in coculture were compared with that induced by bone morphogenetic protein 7 or osteogenic protein-1 (BMP-7; OP-1) treatment. Osteogenesis, as determined by the expression of Cbfa1 and osteocalcin (OC) messenger RNAs (mRNAs), was induced strongly in C3H10T1/2 cells cocultured with both chondrocyte cell populations but was not induced by coculture with either osteoblasts or skin fibroblasts. Interestingly, treatment of C3H10T1/2 cells with BMP-7 induced both chondrogenesis and osteogenesis, and only osteogenic differentiation was observed in the C3H10T1/2 cells cocultured with chondrocytes. No alterations in the expression of mRNAs for BMP-1 to -8 were observed in the C3H10T1/2 cells under any of the coculture conditions. This shows that the induction of endogenous BMPs by coculture does not regulate osteogenesis in an autocrine manner. These results show that chondrocytes express soluble morphogenetic factors that selectively promote osteogenesis, and this selective effect is not mimicked by an exogenously added BMP.     

Isolation and characterization of a mesenchymal cell line that differentiates into osteoblasts in response to BMP-2 from calvariae of GFP transgenic mice

We established the clonal mesenchymal cell line, GFP-C3 (C3), which differentiates into osteoblasts in response to BMP-2 from calvariae of newborn green fluorescence protein (GFP) transgenic mice. This cell line cultured with control medium expressed low levels of alkaline phosphatase (ALP) activity and osterix mRNA and undetectable ALP and osteocalcin mRNA. Incubation of these cells with rhBMP-2 increased ALP activity dose-dependently and induced substantial levels of ALP, osteocalcin and osterix mRNA expression. C3 cells infected with adenovirus vector encoding BMP-2 (AdBMP-2) or Runx2 (AdRunx2) showed greatly increased ALP mRNA expression in a time-dependent fashion. Transduction with AdRunx2-induced expression of ALP and osteocalcin mRNA, but not osterix mRNA by day 3. Transduction with AdBMP-2 induced apparent expression of ALP and osterix mRNA by day 1 after transduction, but induced only weak expression of osteocalcin mRNA day 3 after transduction. Transplantation of C3 cells transduced with AdBMP-2 into back subfascia in wild-type mice with a complex of poly-d,l-lactic-co-glycolic acid/gelatin sponge (PGS) generated ectopic bone formation involving GFP-positive osteoblasts and osteocytes 2 weeks after transplantation. C3 cells transduced with AdRunx2 or AdLacZ failed to induce ectopic bone formation. Transplantation of C3 cells transduced with AdBMP-2 into craniotomy defects in wild-type mice using PGS as a carrier induced bone formation 2 weeks after transplantation, and replaced defects 4 weeks after transplantation. C3 cells transduced with AdRunx2 failed to induce bone repair after transplantation into craniotomy defects. These results indicate that C3 cells retain differentiation potential into osteoblasts in response to BMP-2. They are useful tools for analyzing the process of osteoblast differentiation in vivo after transplantation.     

Adenovirus-mediated direct gene therapy with bone morphogenetic protein-2 produces bone

The need to improve bone healing permeates the discipline of orthopedic surgery. Bone morphogenetic proteins (BMPs) are capable of inducing ectopic and orthotopic bone formation. However, the ideal approach with which to deliver BMPs remains unknown. Gene therapy to deliver BMPs offers several theoretical advantages over implantation of a recombinant BMP protein, including persistent BMP delivery and eliminating the need for a foreign body carrier. A replication defective adenoviral vector was constructed to carry the rhBMP-2 gene (AdBMP-2). The direct in vivo gene therapy approach was applied in both immunodeficient and immunocompetent animals to produce intramuscular bone as early as 2 weeks following injection. Radiographic and histologic analysis revealed radiodense bone containing mature bone marrow elements. Adenovirus-mediated delivery of a marker gene (β-galactosidase) into control animals produced no bone but indicated the cells transduced with the AdBMP-2 vector. Furthermore, comparisons between immunodeficient and immunocompetent animals illustrated the magnitude and significance of the immune response. Gene therapy to deliver BMP-2 has innumerable potential clinical applications from bone defect healing to joint replacement prosthesis stabilization. This study is the first to establish the feasibility of in vivo gene therapy to deliver active BMP-2 and produce bone.     

Nell-1 induced bone formation within the distracted intermaxillary suture

Maxillary bone deficiencies, such as cleft palate and underdeveloped maxilla that require bone graft or regeneration after orthopedic or surgical expansion, pose a significant biomedical burden. Nell-1 is a secreted molecule that possesses chordin-like domains and induces cranial suture bone growth and osteoblast differentiation. To accelerate bone formation in acutely distracted palatal sutures, rat organ cultures were stimulated with Nell-1 or BMP-7 for 8 days in vitro. We hypothesized that Nell-1 stimulation to the distracted palatal suture would accelerate bone formation. Distracted palates of 4-week-old male rats were maintained in an organ culture system, and tissue was either unstimulated or stimulated with Nell-1 or BMP-7 for 8 days. MicroCT was conducted to quantitate bone formation, while alcian blue staining was conducted for cartilage localization. Immunohistochemistry of Sox9 for chondrocyte proliferation, type X collagen for hypertrophic cartilage in endochondral bone formation, and bone sialoprotein for bone formation was conducted to characterize the cellular mechanism of newly developed tissues. Distracted palates cultured in the presence of Nell-1 or BMP-7 produced statistically significantly (P < 0.05) more bone and cartilage within the intermaxillary suture, relative to unstimulated control samples. While both BMP-7 and Nell-1 induced similar bone formation in the distracted suture, BMP-7 induced both chondrocyte proliferation and differentiation, while Nell-1 accelerated chondrocyte hypertrophy and endochondral bone formation. While both Nell-1 and BMP-7 are effective in forming bone in the distracted palatal suture, they are suggested to have distinctively different mechanisms. The ability of Nell-1 to accelerate bone formation within the palate suture demonstrates the versatility of Nell-1 within the craniofacial complex as well as an exciting advance in palate suture defect healing.     

长期动态机械拉伸力诱导类骨细胞的分化及成熟

目的探讨长期动态机械拉伸力对类骨细胞分化及成熟的影响。方法利用机械形变发生装置拉伸类骨细胞,分析类骨细胞是否因拉伸力而使分化更趋成熟。应用Flexercell Strain Unit机械力拉伸装置对MG63及C2C12细胞株施加0～10%的拉伸形变72h。通过碱性磷酸酶(ALP)染色、RT-PCR反应及骨形态发生蛋白-2(BMP-2)添加测试来检测长期动态机械拉伸力对类骨细胞分化的影响。结果拉伸力使MG63细胞的ALP活性约增加2倍,RT-PCR检测显示骨标志物[骨钙素(OCN)及ALP]表达均因拉伸力作用而增加。而单独施加拉伸力并不能增加C2C12肌原细胞内ALP的活性。添加BMP-2培养能促进C2C12细胞向骨细胞分化,并增加细胞ALP的活性。在BMP-2刺激下拉伸力可使C2C12细胞内ALP的活性增加2倍以上。结论本研究确立10%拉伸力能促进类骨细胞向骨细胞分化。对于未分化的肌原细胞,拉伸力刺激并不能使其直接向骨分化,但却能使分化中的骨细胞加速分化或成熟。     

Uniaxial cyclic stretch induces osteogenic differentiation and synthesis of bone morphogenetic proteins of spinal ligament cells derived from patients with ossification of the posterior longitudinal ligaments

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To elucidate this mechanism, we investigated the effects of in vitro sinusoidal cyclic stretch (120% peak to peak, at 1 Hz) on cultured spinal ligament cells derived from OPLL and non-OPLL patients. The mRNA expressions of alkaline phosphatase (ALP), osteopontin, bone morphogenetic protein (BMP)-2, BMP-4, and BMP receptors as well as ALP activity in cell layers and production of BMPs into the conditioned medium were significantly increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. A stretch-activated Ca(2+) channel blocker, Gd(3+), the voltage-dependent L-type Ca(2+) channel blockers diltiazem and nifedipine, and Ca(2+)-free medium suppressed stretch-induced ALP activity, which suggests a role of Ca(2+) influx in the signal transduction of mechanical stress to the osteogenic response of OPLL cells. Our study provides first evidences that mechanical stress plays a key role in the progression of OPLL through the induction of osteogenic differentiation in spinal ligament cells and the promotion of the autocrine/paracrine mechanism of BMPs in this lesion.     

Transplantation of skin fibroblasts expressing BMP-2 promotes bone repair more effectively than those expressing Runx2

We investigated the osteogenic potential of skin fibroblasts that overexpressed BMP-2 or Runx2 by using adenoviral vectors. In in vitro experiments, skin fibroblasts infected with adenovirus vector encoding BMP-2 (AdBMP-2) released substantial levels of BMP-2 proteins into culture media, and those infected with adenovirus vector encoding Runx2 (AdRunx2) produced its protein. Transduction of BMP-2 or Runx2, respectively, increased alkaline phosphatase (ALP) activity and induced expression of mRNAs of ALP, osteocalcin, and osterix in skin fibroblasts. In in vivo experiments, we investigated the bone induction activity by transplantation of a complex composed of carrier [poly-D,L-lactic-co-glycolic acid/gelatin sponge (PGS)] and skin fibroblasts (PGS/SF complex). Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2-induced ectopic bone formation when transplanted into the subfascia of back muscle, unlike those infected with AdRunx2. Transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdBMP-2 into craniotomy defects induced bone formation from 2 weeks after transplantation, and almost all PGS was replaced by newly synthesized bone at 6 weeks. To investigate the fate of the transplanted cells, we transplanted skin fibroblasts isolated from green fluorescence protein transgenic mice into craniotomy defects. Transplantation of these skin fibroblasts transfected with AdBMP-2 generated green fluorescence protein-positive osteoblasts and osteocytes, indicating that the transplanted skin fibroblasts differentiated into osteoblastic lineage cells during bone repair. In contrast, transplantation of PGS/SF complexes composed of skin fibroblasts transduced with AdRunx2 induced a few ALP-positive cells at 1 week after transplantation, but their number decreased depending on time after transplantation. In addition, transplantation of these complexes was insufficient to induce bone repair. Taken together, our results suggest that skin fibroblasts expressing BMP-2 are more suitable for cell-mediated therapy of bone repair than those expressing Runx2.     

Enhanced expression of the inorganic phosphate transporter Pit-1 is involved in BMP-2-induced matrix mineralization in osteoblast-like cells.

Pi handling by osteogenic cells is important for bone mineralization. The role of Pi transport in BMP-2-induced matrix calcification was studied. BMP-2 enhances Pit-1 Pi transporters in osteogenic cells. Experimental analysis suggest that this response is required for bone matrix calcification. INTRODUCTION: Bone morphogenetic proteins (BMPs) are produced by osteogenic cells and play an important role in bone formation. Inorganic phosphate (Pi) is a fundamental constituent of hydroxyapatite, and its transport by osteogenic cells is an important function for primary calcification of the bone matrix. In this study, we investigated the role of Pi transport in BMP-2-induced matrix mineralization. MATERIALS AND METHODS: Confluent MC3T3-E1 osteoblast-like cells were exposed to BMP-2 for various time periods. Pi and alanine transport was determined using radiolabeled substrate, Pit-1 and Pit-2 expression by Northern blot analysis, cell differentiation by alkaline phosphatase activity, matrix mineralization by alizarin red staining, and the characteristics of mineral deposited in the matrix by transmission electron microscopy, electron diffraction analysis, and Fourier transformed infrared resolution (FTIR). RESULTS: BMP-2 time- and dose-dependently stimulated Na-dependent Pi transport in MC3T3-E1 cells by increasing the V(max) of the transport system. This effect was preceded by an increase in mRNA encoding Pit-1 but not Pit-2. BMP-2 also dose-dependently enhanced extracellular matrix mineralization, an effect blunted by either phosphonoformic acid or expression of antisense Pit-1. Enhanced Pi transport and matrix mineralization induced by BMP-2 were blunted by a specific inhibitor of the c-Jun-N-terminal kinase (JNK) pathway. CONCLUSIONS: Results presented in this study indicate that, in addition to its well-known effect on several markers of the differentiation of osteoblastic cells, BMP-2 also stimulates Pi transport activity through a selective increase in expression of type III Pi transporters Pit-1. In MC3T3-E1 cells, this effect is mediated by the JNK pathway and plays an essential role in bone matrix calcification induced by BMP-2.     

Enhancement of human peripheral blood mononuclear cell transplantation-mediated bone formation

Recent studies have demonstrated the existence of osteoblast progenitor cells in circulating blood. Here we show that local delivery of bone morphogenetic protein-2 (BMP-2) to cell transplantation sites induces in situ osteogenic differentiation of transplanted human peripheral blood mononuclear cells (PBMNCs) and enhances in vivo bone formation mediated by PBMNC transplantation. Human PBMNCs were seeded on scaffolds with or without BMP-2 and implanted subcutaneously into athymic mice. Nonseeded scaffolds with BMP-2 were also implanted. Eight weeks later, radiographic and histological analyses showed that the PBMNC + BMP-2 group had undergone much more extensive bone formation than either the PBMNC group or BMP-2 group. Only the PBMNC + BMP-2 group expressed human Cbfa1, osteonectin, and osteocalcin, suggesting in situ osteogenic differentiation of and bone formation by transplanted human PBMNCs, while the other groups did not express these genes. This study provides a method to enhance human PBMNC transplantation-mediated bone formation.     

Hippo信号通路成骨、成肌分化功能研究进展

Hippo信号通路在器官发育和组织修复等多种生物学行为中起着重要作用。Hippo通路下游效应因子YAP和TAZ与TEAD转录因子共同构成Hippo信号转导网络的连接点来调节基因表达。越来越多的证据表明，Hippo通路既可通过调节骨髓间充质干细胞（bone mesenchymal stem cells，BMSCs）和骨形态发生蛋白（bone morphogenetic protein，BMPs）来影响成骨分化功能，又可通过调节骨骼肌卫星细胞和成肌细胞来影响成肌分化功能。本文对Hippo信号通路的结构、以及成骨、成肌的研究进展作一综述，以期更深入理解Hippo通路成骨、成肌功能，并为肌少-骨质疏松症的临床治疗提供新的研究方向。     

Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells

Bone morphogenetic protein (BMP)-2, a member of the transforming growth factor-beta (TGF-beta) superfamily, is able to induce osteoblastic differentiation of C2C12 cells. Both Smad and mitogen-activated protein kinase (MAPK) pathways are essential components of the TGF-beta superfamily signaling machinery. Although Smads have been demonstrated to participate in the BMP-2-induced osteoblastic differentiation of C2C12 cells, the role of MAPK has not been addressed. This report shows that BMP-2 activates ERK and p38, but not JNK, in C2C12 cells. Pretreatment of cells with the p38 inhibitor, SB203580, dramatically reduced BMP-2-induced expression of the osteoblast markers alkaline phosphatase (ALP) and osteocalcin (OC). Nevertheless, overexpression of MKK3, a protein kinase that phosphorylates and activates p38, failed to induce ALP or OC expression in the absence of BMP-2, indicating that p38 activation is necessary but not sufficient for the acquisition of the osteoblast phenotype by these cells. Although ALP induction was increased slightly in the presence of PD-98059, a selective inhibitor of the ERK cascade, this compound significantly inhibited both steady-state and BMP-2-induced OC RNA levels. Our results indicate that p38 and ERK cascades play a crucial role in the osteoblast differentiation of C2C12 cells mediated by BMP-2.