Bone Morphogenetic Protein-2 cDNA Research Articles

Efficient autocrine and paracrine signaling explain the osteogenic superiority of transgenic BMP-2 over rhBMP-2

Bone morphogenetic protein-2 (BMP-2) is an osteogenic protein used clinically to enhance bone healing. However, it must be applied in very high doses, causing adverse side effects and increasing costs while providing only incremental benefit. Preclinical models of bone healing using gene transfer to deliver BMP-2 suggest that transgenic BMP-2 is much more osteogenic than rhBMP-2. Using a reporter mesenchymal cell line, we found transgenic human BMP-2 cDNA to be at least 100-fold more effective than rhBMP-2 in signaling. Moreover, a substantial portion of the BMP-2 produced by the transduced cells remained cell associated. Signaling by transgenic BMP-2 occurred via binding to the type I receptor, activating the associated kinase and generating phospho-smads. Signaling was partially resistant to noggin, an important extracellular inhibitor of BMP-2, possibly because nascent BMP-2 binds to its cell surface receptor during secretion and thus signals in a protected peri-cellular environment. Although the amounts of BMP-2 secreted by the transduced cells were too low to affect distant cells, transduced cells were able to induce signaling in a paracrine fashion that required close proximity of the cells, possibly cell-to-cell contact. The greater osteogenic potency of transgenic BMP-2 was confirmed with human bone marrow stromal cells.

Construction and characterization of an adenoviral vector encoding human bone morphogenetic protein-2.

ObjectivesConstruction of adenoviral vectors can be complicated and time-consuming. The aim of this study was to construct an adenoviral vector expressing human bone morphogenetic protein 2 (BMP-2).MethodsAn adenoviral vector expressing human BMP-2 was constructed using the Gateway™ technique based on site-specific recombination. Briefly, BMP-2 cDNA was obtained by polymerase chain reaction, inserted into the pMD19-T cloning vector, and subcloned into a shuttle vector. The DNA sequence encoding BMP-2 was then subcloned from pEC3.1-BMP2 into the pAd/BLOCK-iT™-DEST adenoviral vector genome, which was then linearized and used to transfect 293 cells for adenovirus packaging. Adenovirus particles were generated with a titer of 1 × 1010 infectious units/mL.ResultsPotent expression of BMP-2 mRNA and protein was detected in adenovirus-infected 293 cells, confirming that the adenoviral vector encoding BMP-2 was successfully constructed.ConclusionsThis recombinant adenoviral vector encoding BMP-2 can be applied in future studies to explore the roles of BMP-2 in various cell types and tissues.

Bone morphogenetic protein 2 stimulates chondrogenesis of equine synovial membrane-derived progenitor cells.

Bone morphogenetic protein 2 (BMP-2) is critical for skeletal and cartilage development, homeostasis and repair. This study was conducted to clone and characterize equine BMP-2, develop expression constructs for equine BMP-2, and to determine whether BMP-2 can stimulate chondrogenesis of equine synovial membrane-derived progenitor cells (SMPC). Equine BMP-2 cDNA was amplified from chondrocyte RNA, and then transferred into an expression plasmid and adenoviral vector. Effective expression of equine BMP-2 was confirmed using a BMP reporter cell line. SMPC were isolated from synovium, expanded through two passages and transferred to chondrogenic cultures, with recombinant human (rh) transforming growth factor beta 1 (TGF-β1) or rhBMP-2. Chondrogenesis was assessed by up-regulation of collagen types II and X, and aggrecan mRNA, secretion of collagen type II protein and sulfated glycosaminoglycans (sGAG), and by alkaline phosphatase induction. Chondrogenic stimulation of SMPC by the equine BMP-2 adenovirus was assessed by sGAG secretion and histology. The mature equine BMP-2 peptide is identical to human and murine peptides. Recombinant human BMP-2 and TGF-β1 stimulated equivalent amounts of collagen type II protein in SMPC pellets, but sGAG secretion was doubled by BMP-2. Neither factor stimulated hypertrophic marker expression. The equine BMP-2 adenoviral vector induced chondrogenesis comparably to rhBMP-2 protein, with no indication of hypertrophy. Bone morphogenetic protein 2 is a potent inducer of SMPC non-hypertrophic chondrogenesis, supporting the use of this combination for articular cartilage repair applications.

Using poly(lactic-co-glycolic acid) microspheres to encapsulate plasmid of bone morphogenetic protein 2/polyethylenimine nanoparticles to promote bone formation in vitro and in vivo

Repair of large bone defects is a major challenge, requiring sustained stimulation to continually promote bone formation locally. Bone morphogenetic protein 2 (BMP-2) plays an important role in bone development. In an attempt to overcome this difficulty of bone repair, we created a delivery system to slowly release human BMP-2 cDNA plasmid locally, efficiently transfecting local target cells and secreting functional human BMP-2 protein. For transfection, we used polyethylenimine (PEI) to create pBMP-2/PEI nanoparticles, and to ensure slow release we used poly(lactic-co-glycolic acid) (PLGA) to create microsphere encapsulated pBMP-2/PEI nanoparticles, PLGA@pBMP-2/PEI. We demonstrated that pBMP-2/PEI nanoparticles could slowly release from the PLGA@pBMP-2/PEI microspheres for a long period of time. The 3–15 μm diameter of the PLGA@pBMP-2/PEI further supported this slow release ability of the PLGA@pBMP-2/PEI. In vitro transfection assays demonstrated that pBMP-2/PEI released from PLGA@pBMP-2/PEI could efficiently transfect MC3T3-E1 cells, causing MC3T3-E1 cells to secrete human BMP-2 protein, increase calcium deposition and gene expressions of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), SP7 and I type collagen (COLL I), and finally induce MC3T3-E1 cell differentiation. Importantly, in vivo data from micro-computed tomography (micro-CT) and histological staining demonstrated that the human BMP-2 released from PLGA@pBMP-2/PEI had a long-term effect locally and efficiently promoted bone formation in the bone defect area compared to control animals. All our data suggest that our PLGA-nanoparticle delivery system efficiently and functionally delivers the human BMP-2 cDNA and has potential clinical application in the future after further modification.

Evaluation of BMP‐2 gene‐activated muscle grafts for cranial defect repair

Large, osseous, segmental defects heal poorly. Muscle has a propensity to form bone when exposed to an osteogenic stimulus such as that provided by transfer and expression of cDNA encoding bone morphogenetic protein-2 (BMP-2). The present study evaluated the ability of genetically modified, autologous muscle to heal large cranial defects in rats. Autologous grafts (8 mm × 2 mm) were punched from the biceps femoris muscle and transduced intraoperatively with recombinant adenovirus vector containing human BMP-2 or green fluorescent protein cDNA. While the muscle biopsies were incubating with the vector, a central parietal 8 mm defect was surgically created in the calvarium of the same animal. The gene-activated muscle graft was then implanted into the cranial defect. After 8 weeks, crania were examined radiographically, histologically, and by micro-computed tomography and dual energy X-ray absorptiometry. Although none of the defects were completely healed in this time, muscle grafts expressing BMP-2 deposited more than twice as much new bone as controls. Histology confirmed the anatomical integrity of the newly formed bone, which was comparable in thickness and mineral density to the original cranial bone. This study confirms the in vivo osteogenic properties of genetically modified muscle and suggests novel strategies for healing bone.

Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo

Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs) and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP) production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix. We conclude that BMP-2 cDNA incorporated in alginate hydrogel appears to be a promising new strategy for minimal-invasive delivery of growth factors in bone regeneration.

The Repair of Critical-Sized Bone Defects Using Expedited, AutologousBMP-2Gene-Activated Fat Implants

The repair of bone defects can be induced experimentally with bone morphogenetic protein-2 (BMP-2) producing fat-derived stem cells, but this ex vivo tissue engineering method requires the isolation and long-term culture of autologous cells. To develop an expedited bone repair strategy, we transferred BMP-2 cDNA directly to autologous fat tissue fragments that were held in culture for only 24 h before implantation. We evaluated the ability of such gene-activated fat grafts to regenerate large segmental bone defects in rats. Fat tissue was harvested from 2 of 35 male Fischer 344 rats used for this study. The fat tissue fragments were incubated with an adenoviral vector carrying the cDNA encoding either BMP-2 or green florescent protein (GFP), or they remained unmodified. According to their group, the segmental femoral bone defects of 33 rats were filled press fit with either BMP-2-activated fat tissue, GFP-transduced fat tissue, or unmodified fat tissue. Another control group remained untreated. Femora were evaluated by radiographs, microcomputed tomography, biomechanical torsional testing, and histology. Radiographically and histologically, 100% of the femora treated with BMP-2-activated fat grafts were bridged at 6 weeks after surgery. The femora of this group exceeded the bone volume and the biomechanical stability of intact, contralateral femora. Control defects receiving no treatment, unmodified fat tissue, or GFP-transduced fat were filled with fibrous or adipose tissue, as evaluated by histology. The use of BMP-2 gene-activated fat tissue grafts represents an expedited and effective bone repair strategy that does not require the extraction and expansion of stem cells.

Identification of Sparus aurata bone morphogenetic protein 2: Molecular cloning, gene expression and in silico analysis of protein conserved features in vertebrates

Bone morphogenetic protein 2 (BMP-2) is a secreted signaling molecule that acts as an inducer of bone formation and a regulator of embryonic development. The objectives of this work were as follows: (1) to clone the full-length cDNA of BMP-2 in a marine fish model, (2) analyze its gene expression during development, in adult tissues and in cell lines, and (3) identify protein conserved features of vertebrate BMP-2. Using a combination of RT- and 5′-RACE-PCR, a 1653-bp fragment corresponding to Sparus aurata BMP-2 cDNA (SaBMP-2) was amplified. Levels of SaBMP-2 gene expression were estimated using quantitative real-time PCR and shown to be strongly increased (150-fold induction) at gastrulation, thus suggesting a key role for BMP-2 in fish development. Tissue distribution of SaBMP-2 mRNA revealed highest levels in the calcified tissues bone, caudal fin and scales and in liver. BMP-2 was also found to be highly expressed in S. aurata bone-derived cell lines VSa13 and VSa16 and to be up-regulated (more than 10-fold induction) in mineralized VSa13 chondrocyte-like cells. Using bioinformatic tools and all vertebrate protein sequences available, conserved features of BMP-2 were characterized. The mature protein was shown to be highly conserved across 20 species indicating that BMP-2 function has been conserved throughout evolution, a finding that is in agreement with the widely accepted view of the important role played by BMPs in vertebrate development.

In vivo molecular imaging of adenoviral versus lentiviral gene therapy in two bone formation models

Regional gene therapy techniques are promising methods to enhance bone formation in large bone defects that would be difficult to treat with allograft or autograft bone stock. In this study, we compared in vivo temporal expression patterns of adenoviral- and lentiviral-mediated gene therapy in two bone formation models. Primary rat bone marrow cells (RBMC) were transduced with lentiviral or adenoviral vectors containing luciferase (Luc) or BMP-2 cDNA, or cotransduced with vectors containing Luc and bone morphogenetic protein 2 (BMP-2). In vitro protein production was determined with luciferase assay or ELISA (for BMP-2 production) weekly for 12 weeks. Two bone formation models were used -- a hind limb muscle pouch or radial defect -- in SCID mice. A cooled charged-coupled device (CCD) camera was used to image in vivo luciferase expression weekly for 12 weeks. In vitro, adenoviral expression of BMP-2 and luciferase was detected by ELISA or luciferase assay, respectively, for 4 weeks. Lentiviral expression of BMP-2 and luciferase was sustained in culture for 3 months. Using the CCD camera, we found that adenoviral vectors expressed luciferase expression for up to 21 days, but lentiviral vectors expressed target gene expression for 3 months in vivo in both bone formation models. There was no detectable difference in the amount of bone formed between the adenoviral and lentiviral groups. Lentiviral-mediated delivery of BMP-2 can induce long term in vitro and in vivo gene expression, which may be beneficial when developing tissue engineering strategies to heal large bone defects or defects with a compromised biologic environment.

Direct Adenoviral Transfer of Bone Morphogenetic Protein-2 cDNA Enhances Fracture Healing in Osteoporotic Sheep

Osteoporosis, a major public health burden, is associated with increased fracture risk. Fracture healing in osteoporosis is delayed, with reduced callus formation and impaired biomechanical properties of newly formed bone leading to high risk of fixation failure. Adenoviral gene transfer of bone morphogenetic protein-2 (BMP-2) has been shown to enhance fracture healing. This study evaluated the ability of gene transfer to enhance bone healing in osteoporosis. An established sheep model of osteoporosis with well-characterized alterations in fracture healing was used. Osteotomies were created surgically in the tibias of adult female sheep and monitored for 8 weeks, using radiographic, biomechanical, and histological methods. For pilot experiments, primary ovine osteoblasts and mesenchymal stem cells were transduced with a recombinant adenovirus carrying BMP-2 cDNA (Ad.BMP-2). Large increases in alkaline phosphatase production and mineralization confirmed the ability of human BMP-2 to stimulate osteoblastic differentiation in sheep. In vivo bending stiffness measurements during fracture healing as well as ex vivo torsional stiffness measurements demonstrated stiffer callus tissue after treatment with Ad.BMP-2. The differences were found mainly in the early fracture-healing period. Computed tomography demonstrated that animals receiving the BMP-2 cDNA had larger cross-sectional callus area and higher callus density. Histological examination of the tibias confirmed enhanced callus formation. Direct, local adenoviral delivery of an osteogenic gene thus led to enhanced healing of fractures in an ovine model of osteoporosis. These promising data encourage the further development of genetic approaches to enhance bone healing in patients suffering osteoporosis-associated fractures.

Direct Adenoviral Transfer of Bone Morphogenetic Protein-2 cDNA Enhances Fracture Healing in Osteoporotic Sheep

Direct Adenoviral Transfer of Bone Morphogenetic Protein-2 cDNA Enhances Fracture Healing in Osteoporotic Sheep

De Novo Osteogenesis From Human Ligamentum Flavum by Adenovirus-Mediated Bone Morphogenetic Protein-2 Gene Transfer

In vitro and in vivo experiment using degenerated human ligamentum flavum (LF) and Type 5 adenovirus construct with bone morphogenetic protein-2 (BMP-2) cDNA. To demonstrate in vitro and in vivo osteogenic effect of BMP-2 gene transfer to human LF and to propose genetically modified LF as a substitute for autogenous bone graft in spinal fusion. Spinal fusion is still considered to be an important option for treating various spinal disorders. To induce solid spinal fusion, osteoinductive and/or osteoconductive agents have been widely adopted. Autogenous LF, however, has never been seriously considered as a carrier for ex vivo osteoinductive gene therapy for spinal fusion. In vitro experiment: Degenerated human LF was harvested and cultured. Type 5 adenovirus lacZ (Ad/lacZ) and BMP-2 construct (Ad/BMP-2) were produced. LF cell cultures were then exposed to Ad/BMP-2. Expressions of osteocalcin and BMP-2 mRNA were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis was performed to detect osteocalcin protein. Alkaline phosphatase and von Kossa stains were used to detect osteogenic markers and bone nodule formation, respectively. In vivo experiment: Human LF tissues treated with Ad/lacZ, Ad/BMP-2, and saline were implanted into the subcutaneous tissue of nude mice. After 4 weeks, nude mice were radiographed and killed. Implanted LF tissues were harvested and histologically stained. LF cell cultures with Ad/BMP-2 revealed strong expression of BMP-2 and osteocalcin mRNA in RT-PCR and osteocalcin protein in western blot analysis. LF cell culture with saline showed baseline expression of BMP-2, osteocalcin mRNA, and osteocalcin protein, respectively. Furthermore, LF cell culture with Ad/BMP-2 demonstrated the expression of alkaline phosphatase and bone nodule formation in the aforementioned histochemical stain. LF tissues with Ad/BMP-2 revealed de novo osteogenesis in nude mice, whereas LF with Ad/lacZ or saline showed only remaining LF tissue without sign of bone formation. Human LF cells transduced with Ad/BMP-2 exhibited the expression of osteogenic phenotype and bone nodule formation. Additionally, genetically modified human LF with BMP-2 cDNA clearly demonstrated de novo osteogenesis, which supports the concept that biologically modified LF can be a substitute for autogenous bone graft in spinal fusion surgery.

Transgene-activated mesenchymal cells for articular cartilage repair: a comparison of primary bone marrow-, perichondrium/periosteum- and fat-derived cells

Adult primary mesenchymal cells of different origin which can be obtained with minor donor site morbidity are considered for articular cartilage repair. This study aims at a comparison of their chondrogenic potential. Mesenchymal cells were isolated from perichondrium/periosteum, bone marrow or fat of adult rats and found to be positive for the stem-cell-related antigens Sca-1, c-Kit, CD10, CD13 and CD90 by reverse transcription polymerase chain reaction (RT-PCR). Chondrogenic differentiation was induced by applying recombinant bone morphogenetic protein-2 (BMP-2) or adenoviral vectors carrying BMP-2 cDNA, followed by micromass culture. The stimulated cells were characterized by RT-PCR, cell proliferation and apoptosis assays. Expression of aggrecan, collagen type I, II, IX and X and alkaline phosphatase genes was analyzed by RT-PCR, immunofluorescence and immunohistochemistry in comparison with unstimulated control cells. Adenovirally stimulated cells were transplanted into mechanically generated partial-thickness cartilage lesions in the patellar groove of the rat femur. Quality and integration of the repair tissues were assessed by histochemical and immunohistochemical methods. Stimulation with BMP-2 or AdBMP-2 led to an up-regulation of cartilage-specific gene expression in all three cell populations studied, most rapidly and prominently in the perichondrial/periosteal cells, which showed a 3200-fold increase of type II collagen mRNA and reached the highest absolute levels of type II and IX collagen transcripts after stimulation. Similar results were obtained for the bone marrow stromal cells (BMSC), while the respective transcript levels in fat stromal cells declined after an initial more than 30-fold elevation. Following transplantation in vivo, AdBMP-2-infected perichondrial/periosteal cells produced a proteoglycan-rich, type II collagen-positive matrix with only faint staining for type I collagen. The repair tissue originating from AdBMP-2-infected BMSC showed less intense type II collagen staining, but a relatively proteoglycan-rich matrix, weakly positive for type I collagen. Transgene-activated fat stromal cells formed rather fibrous tissue mainly composed of type I collagen. Unstimulated cells of the three different populations gave only rise to fibrous tissue. Perichondrium/periosteum-derived cells and BMSC seem superior to cells isolated from fat with respect to forming hyaline cartilaginous tissue. A chondrogenic stimulus, e.g. by transfer of BMP-2 cDNA, appears to be required for initiation and support of chondrogenic differentiation.

Biologic modification of ligamentum flavum cells by marker gene transfer and recombinant human bone morphogenetic protein-2.

The study involves an in vitro experiment using human ligamentum flavum (LF), adenovirus lacZ construct (Ad/lacZ), and recombinant human bone morphogenetic protein-2 (BMP-2). To demonstrate the feasibility of marker gene transfer to human LF cells and the effect of BMP-2 on the osteogenic differentiation of human LF cells. BMP-2 is a widely known pivotal osteoinductive agent. Clinically and experimentally, BMP-2 has proven to be an effective in spinal fusion. Degenerated LF has only been implicated to be of pathophysiological significance in spinal stenosis. However, biologic modifications of LF to enhance osteogenesis have not been attempted previously. Human LF and cancellous bone from the ilium were harvested from patients with lumbar spinal stenosis. LF cells and osteoblasts were isolated and cultured, and adenovirus lacZ construct (Ad/ lacZ), luciferase construct (Ad/luciferase), and BMP-2 were designed and produced. LF cell cultures were then exposed to various concentrations of Ad/lacZ (25, 50, 75, 100, 150 multiplicity of infection) and BMP-2 (50, 100, 500, 1,000, and 1,500 ng/mL). Osteoblast cultures were used as a positive control for LF culture. LF cell cultures with Ad/luciferase served as viral controls for culture with Ad/ lacZ. The transgene expression of lacZ was assessed by X-gal stain and beta-galactosidase assay. Alkaline phosphatase, Von Kossa, and Alizarin red-S stains were used to confirm osteogenic differentiation and bone nodule formation. Immunocytochemical staining was also performed to detect osteocalcin expression. LF cell cultures transduced with Ad/lacZ showed extensive X-gal expression and increased beta-galactosidase activity compared to viral (Ad/luciferase) and saline controls. In LF cultures treated with BMP-2, robust alkaline phosphatase expression, and bone nodule formations were observed as evidenced by positive Von Kossa and Alizarin red-S staining, and the strong expression of osteocalcin. The osteogenic response of LF cells to BMP-2 was dose dependent. Human LF cells were found to be susceptible to adenovirus-mediated marker gene transfer, which offers the possibility of a new range of possible genetic modifications. In human LF cells, BMP-2 was found to markedly up-regulate the expression of osteogenic phenotypes and to induce bone nodule formation. The results of this study support the notion that biologically modified LF cells, i.e., LF cells treated with BMP-2, or with adenovirus-mediated BMP-2 cDNA gene transfer, may facilitate spinal fusion.

Bone morphogenetic protein-2 facilitates expression of chondrogenic, not osteogenic, phenotype of human intervertebral disc cells.

In vitro experiment using bone morphogenetic protein-2 (BMP-2) and human intervertebral disc (IVD) cells. To demonstrate the effect of BMP-2 on mRNAs expression (collagen type I, collagen type II, aggrecan, and osteocalcin), proteoglycan synthesis, expression of alkaline phosphatase, bone nodule formation in human IVD cells. BMP-2 was widely known as a powerful agent for osteoinduction and a crucial growth factor for early chondrogenesis and maintenance of cartilaginous phenotype. BMP-2 proved to be effective in stimulating proteoglycan synthesis in articular chondrocytes and IVD cells. Nevertheless, the effect of BMP-2 on IVD cells, whether chondrogenic or osteogenic, was not thoroughly elucidated in transcriptional level and histochemical stains. Human IVDs were harvested and enzymatically digested. Then IVD cells were cultured three-dimensionally in alginate beads. Osteoblasts were cultured from cancellous bone of ilium for histochemical stains. Recombinant human BMP-2 (rhBMP-2) was produced by Chinese hamster ovary cells after transduction of BMP-2 cDNA, then concentrated and purified. Then IVD cell cultures were exposed to various concentrations of rhBMP-2. Reverse transcription-polymerase chain reaction for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin was performed. Newly synthesized proteoglycan was measured by 35S-sulfate incorporation on Sephadex G-25 M in PD 10 columns. As a histochemical examination, alkaline phosphatase and Alizarin red-S stains were used to detect osteogenic marker and bone nodule formation, respectively. In the rhBMP-2 treated cultures, there was increased newly synthesized proteoglycan (67% in 300 ng/mL and 200% in 1,500 ng/mL of rhBMP-2) and up-regulated expression of aggrecan, collagen type I, and collagen type II mRNA over untreated control. However, rhBMP-2 did not up-regulate expression of osteocalcin mRNA in the given dose and culture period. IVD cell cultures with rhBMP-2 showed no evidence of bone formation in histochemical stains, i.e., alkaline phosphatase and Alizarin red-S, while osteoblast culture exhibited strong positive stains. The rhBMP-2 clearly up-regulated mRNA expression of chondrogenic components and also stimulated proteoglycan synthesis without expression of osteogenic phenotype. Taken together, this study raise the possibility of rhBMP-2 can be anabolic agent for regenerating matrix of intervertebral disc.

Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene transfer of BMP-2

The multipotential murine embryonic C3H10T1/2 mesenchymal cell line is able to undergo chondrogenesis in vitro, in a high density micromass environment, following treatment with soluble human bone morphogenetic protein-2 (BMP-2). To enhance this process, the human BMP-2 cDNA was cloned into a retroviral expression vector and a high titer, infectious retrovirus (replication defective) was generated. Infection of C3H10T1/2 cells with this retroviral construct resulted in an infection efficiency of 90 – 95 % and was highly effective in converting cells in micromass culture to a chondrocyte phenotype, as assessed by positive Alcian blue staining for extracellular matrix proteoglycans, increased sulfate incorporation, increased expression of the cartilage marker genes collagen type II and aggrecan, and decreased expression of collagen type I. Interestingly, BMP-2 expression in the micromass cultures also induced the expression of the cell cycle inhibitory protein/differentiation factor p21/WAF1, suggesting its functional involvement in chondrogenesis. The chondrogenic effect of retrovirally expressed BMP-2 in these high-density cultures was limited to the infected cells, since uninfected cells did not chondrify when co-cultured as a nonoverlapping micromass adjacent to BMP-2 expressing cells. These data indicate that retrovirally expressed BMP-2 is highly effective at inducing a chondrocyte phenotype in a multipotential mesenchymal cell line in vitro, and its action is restricted to the infected cell population. These findings should provide a framework for the optimization of chondrogenesis in culture using mesenchymal stem cells and retroviral gene transfer.

Bone Morphogenetic Protein-2 Causes Commitment and Differentiation in C3Hl0T1/2 and 3T3 Cells

C3H10T1/2 cells are an established mesenchymal stem cell line which can differentiate into muscle, fat and cartilage cells when treated with azacytidine. Bone morphogenetic protein-2 (BMP-2) caused a dose dependent differentiation of these cells into fat, cartilage and bone cells-low concentrations favoring adipocytes and high concentrations chondrocytes and osteoblasts. The differentiated phenotypes were stable in the absence of BMP-2. Furthermore, the addition of other growth factors during the differentiation process altered the frequency of the differentiated colony formation. Transfection of the C3H10T1/2 cells with a BMP-2 cDNA also induced a phenotypic change from the parental fibroblast to adipocytes and osteoblasts. Our results in this model system indicate that a single protein factor can cause differentiation of a stem cell line to multiple phenotypes, that phenotypes induced can be regulated by factor concentration, and that other factors can also influence BMP-2 induced differentiation.