Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage.

The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP-2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)-positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.

Recombinant human bone morphogenetic protein-2 stimulates osteoblastic maturation and inhibits myogenic differentiation in vitro.

The in vitro effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on osteogenic and myogenic differentiation was examined in two clonal cell lines of rat osteoblast-like cells at different differentiation stages, ROB-C26 (C26) and ROB-C20 (C20). The C26 is a potential osteoblast precursor cell line that is also capable of differentiating into muscle cells and adipocytes; the C20 is a more differentiated osteoblastic cell line. Proliferation was stimulated by rhBMP-2 in C26 cells, but inhibited in C20 cells. rhBMP-2 greatly increased alkaline phosphate (ALP) activity in C26 cells, but not in C20 cells. The steady-state level of ALP mRNA was also increased by rhBMP-2 in C26 cells, but not in C20 cells. Production of 3',5'-cAMP in response to parathyroid hormone (PTH) was dose-dependently enhanced by adding rhBMP-2 in both C26 and C20 cells, though the stimulatory effect was much greater in the former. There was neither basal expression of osteocalcin mRNA nor its protein synthesis in C26 cells, but they were strikingly induced by rhBMP-2 in the presence of 1 alpha,25-dihydroxyvitamin D3. rhBMP-2 induced no appreciable changes in procollagen mRNA levels of type I and type III in the two cell lines. Differentiation of C26 cells into myotubes was greatly inhibited by adding rhBMP-2. The inhibitory effect of rhBMP-2 on myogenic differentiation was also observed in clonal rat skeletal myoblasts (L6). Like BMP-2, TGF-beta 1 inhibited myogenic differentiation. However, unlike BMP-2, TGF-beta 1 decreased ALP activity in both C26 and C20 cells. TGF-beta 1 induced neither PTH responsiveness nor osteocalcin production in C26 cells, but it increased PTH responsiveness in C20 cells. These results clearly indicate that rhBMP-2 is involved, at least in vitro, not only in inducing differentiation of osteoblast precursor cells into more mature osteoblast-like cells, but also in inhibiting myogenic differentiation.

Novel regulators of bone formation: molecular clones and activities.

Protein extracts derived from bone can initiate the process that begins with cartilage formation and ends in de novo bone formation. The critical components of this extract, termed bone morphogenetic protein (BMP), that direct cartilage and bone formation as well as the constitutive elements supplied by the animal during this process have long remained unclear. Amino acid sequence has been derived from a highly purified preparation of BMP from bovine bone. Now, human complementary DNA clones corresponding to three polypeptides present in this BMP preparation have been isolated, and expression of the recombinant human proteins have been obtained. Each of the three (BMP-1, BMP-2A, and BMP-3) appears to be independently capable of inducing the formation of cartilage in vivo. Two of the encoded proteins (BMP-2A and BMP-3) are new members of the TGF-beta supergene family, while the third, BMP-1, appears to be a novel regulatory molecule.

Ectopic induction of tendon and ligament in rats by growth and differentiation factors 5, 6, and 7, members of the TGF-beta gene family.

Little is known about the regulatory signals involved in tendon and ligament formation, and this lack of understanding has hindered attempts to develop biologically based therapies for tendon and ligament repair. Here we report that growth and differentiation factors (GDFs) 5, 6, and 7, members of the TGF-beta gene superfamily that are most related to the bone morphogenetic proteins, induce neotendon/ligament formation when implanted at ectopic sites in vivo. Analysis of tissue induced by GDF-5, 6, or 7, containing implants by currently available morphological and molecular criteria used to characterize tendon and ligament, adds further evidence to the idea that these GDFs act as signaling molecules during embryonic tendon/ligament formation. In addition, comparative in situ localizations of the GDF-5, 6, and 7 mRNAs suggest that these molecules are important regulatory components of synovial joint morphogenesis.

Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12.

When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor beta1 (TGF-beta1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-beta1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2alphaA/Cbfa1, a global regulator of osteogenesis, as a major TGF-beta1-responsive element binding protein induced by TGF-beta1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-beta1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-beta1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-beta1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-beta and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells.

Bone morphogenetic proteins-2 and -4 are involved in the retinoic acid-induced differentiation of embryonal carcinoma cells.

Bone morphogenetic proteins-2 and -4 (BMPs-2 and -4) are transforming growth factor beta-related proteins that can induce bone formation in vivo. We observed that the level of endogenous BMP-2 mRNA increased an average of 11-fold on differentiation of F9 embryonal carcinoma cells into parietal endoderm after treatment with retinoic acid (RA) and cAMP, whereas the message for the closely related BMP-4 decreased 12-fold after this treatment. Therefore, the effects of exogenous recombinant BMP-2 protein on the RA-induced differentiation of F9 embryonal carcinoma cells were investigated. BMP-2 addition altered the growth and morphology of RA-treated but not untreated cells. Moreover, the abundance of several messages was affected by exogenous BMP-2 treatment. Notably, the BMP-2 and -4 messages themselves were reduced by the addition of exogenous BMP-2. The observations suggest that RA, which is known to affect bone morphogenesis, may regulate the osteoinductive proteins, BMP-2 and -4. Furthermore, BMP-2 and -4 may be involved in preimplantation embryogenesis.

Effects of dentin proteins, transforming growth factor beta 1 (TGF beta 1) and bone morphogenetic protein 2 (BMP2) on the differentiation of odontoblast in vitro.

We have studied the effects of dentin proteins, of Transforming Growth Factor beta 1 (TGF beta 1) and Bone Morphogenetic Protein (BMP2) on the differentiation of odontoblasts in vitro. The total EDTA-soluble fraction of dentin proteins, prepared from rabbit incisors was further separated by chromatography on DEAE-Cellulose and heparin-agarose columns. While the total EDTA-soluble fraction of dentin had no effect on cultured dental papillae, fractions retained on both columns were able to initiate functional differentiation of preodontoblasts of isolated day-17 first lower mouse molar dental papillae cultured in vitro. TGF beta 1 and BMP2, both stimulated the matrix secretion by dental papillae cells. TGF beta 1 and BMP2, combined with the inactive total EDTA-soluble fraction, stimulated odontoblast differentiation. An active fraction retained on DEAE-Cellulose completely lost the inductive activity after incubation with a neutralizing anti-TGF beta antibody. These results demonstrate that a TGF beta-like molecule present in dentin could interact with some component which acts as a modulator of its activity on the initiation of the cytological and functional differentiation of odontoblasts.

Perturbation of BMP signaling in somitogenesis resulted in vertebral and rib malformations in the axial skeletal formation.

Axial skeletons such as vertebrae, ribs, and scapulae develop from the embryonic somitic mesoderm through interactions with neural tube/notochord and skin ectoderm. Bone morphogenetic proteins (BMPs) seem to play important roles in these tissue interactions; however, the relationship between BMP signaling and the early development of axial skeletons is poorly understood. In this report, we investigated possible roles of BMP signaling in axial skeletal formation. First, we describe the expression patterns of BMP4 and type I receptors for BMP during somitogenesis in chick embryos based on whole mount in situ hybridization. Next, the effects of BMP on axial skeletal morphogenesis were investigated by implantation of BMP proteins into the dorsal mesoderm at the time of somitogenesis. Transcripts for both BMP4 ligand and its receptors are expressed in the dorsal ectoderm and mesoderm. Implantation of BMP4 and BMP2 into the dorsal regions of embryos result in subsequent anomalies of vertebrae, ribs, and scapulae. The effects of BMP implantation on the skeleton are shown to be dependent upon the somitic stage. Vertebral anomalies are restricted to the dorsolateral elements of the vertebrae and specifically observed after BMP implantation into embryonic day 2 (E2) embryos, but not E3 embryos. These results indicate that implantation of BMP into the dorsal part of embryos where endogenous BMP ligand and BMP receptors are expressed perturbs BMP signaling and causes axial skeletal malformations. The findings presented here suggest that BMP signaling may be involved in the early developmental process of the axial skeleton.

Responsiveness of clonal limb bud cell lines to bone morphogenetic protein 2 reveals a sequential relationship between cartilage and bone cell phenotypes.

There is growing evidence to suggest that BMPs are among the signals necessary to create the embryonic skeleton, but how these regulatory molecules enter the pathways of embryonic bone formation remains to be defined. The earliest steps of endochondral bone formation, consisting of mesenchymal condensation and chondrogenesis, have been shown to result directly from BMP-2 action. To determine whether the transition from chondrogenesis to osteogenesis occurring later in endochondral bone formation is also the result of BMP activity, we tested the effects of BMP-2 on immortalized endochondral skeletal progenitor cells derived from mouse limb bud. The cell lines established by this process were found to fall into three general categories: undifferentiated skeletal progenitor cells, which in the presence of BMP-2 first express cartilage matrix proteins and then switch to production of bone matrix proteins; prechondroblast-like cells that constitutively express a subset of markers associated with chondrogenesis and, in the presence of BMP-2, shut off synthesis of these molecules and are induced to produce bone matrix molecules; and osteoblast-like cells that are not significantly affected by BMP-2 treatment. These data suggest that BMP-2 initiates the differentiation of limb bud cells into cells of both the cartilage and bone lineages in a sequential manner, making BMP-2 a potent regulator of skeletal cell differentiation.

Recombinant human bone morphogenetic protein-2 enhances osteotomy healing in glucocorticoid-treated rabbits.

The objectives of this study were to evaluate the effect of chronic prednisolone treatment on osteotomy healing in rabbits and to determine whether recombinant human bone morphogenetic protein-2 (rhBMP-2) would enhance healing in the presence of chronic glucocorticoid therapy. Forty-nine skeletally mature, male rabbits were injected with either prednisolone (n = 26; 0.35 mg/kg per day, three times a week) or saline (n = 23). After a 6-week pretreatment period, bilateral ulnar osteotomies were created surgically. One osteotomy was treated with rhBMP-2 (0.2 mg/ml of rhBMP-2, 40 microg of rhBMP-2 total) delivered on an absorbable collage sponge (ACS), whereas the contralateral osteotomy remained untreated. Prednisolone or saline treatment was continued until the rabbits were killed either 6 weeks or 8 weeks after creation of the osteotomy. Osteotomy healing was evaluated by radiography, peripheral quantitative computed tomography (pQCT), torsional biomechanics, and undecalcified histology. Because we observed similar responses to both prednisolone and rhBMP-2/ACS treatment in the 6-week and 8-week cohorts, the results from these time points were combined. Serum osteocalcin and vertebral trabecular bone density were lower in the prednisolone-treated rabbits. Prednisolone treatment dramatically inhibited osteotomy healing. In the untreated ulnas, callus area and torsional strength were 25% and 55% less, respectively, in the prednisolone-treated rabbits than in the saline group (p < 0.001 for both). rhBMP-2/ACS enhanced healing in both the prednisolone- and the saline-treated groups, although the effect was larger in the prednisolone-treated rabbits. In the prednisolone-treated rabbits, callus area and torsional strength were 40% and 165% greater (p < 0.001 for both), respectively, in osteotomies treated with rhBMP-2/ACS compared with the contralateral, untreated osteotomies. Histological evaluation confirmed that osteotomy healing was inhibited by prednisolone and accelerated by rhBMP-2/ACS. In summary, a single application of rhBMP-2/ACS counteracted the inhibition of osteotomy healing caused by prednisolone exposure. These results suggest that rhBMP-2/ACS may be a useful treatment for enhancing fracture healing in patients who are undergoing chronic glucocorticoid therapy.

Effect of recombinant human bone morphogenetic protein-2 on fracture healing in a goat tibial fracture model.

Bone morphogenetic proteins (BMPs) are considered to have important regulatory roles in skeletal embryogenesis and bone healing. Recombinant human BMPs (rhBMPs) have been shown to heal critical size defects and promote spinal fusion. We studied the effects of rhBMP-2 in an absorbable collagen sponge (ACS) on bone healing in a large animal tibial fracture model. Bilateral closed tibial fractures were created in 16 skeletally mature goats and reduced and stabilized using external fixation. In each animal, one tibia received the study device (0.86 mg of rhBMP-2/ACS or buffer/ACS), and the contralateral fracture served as control. The device was implanted as a folded onlay or wrapped circumferentially around the fracture. Six weeks following fracture, the animals were sacrificed and the tibiae harvested for torsional testing and histomorphologic evaluation. Radiographs indicated increased callus at 3 weeks in the rhBMP-2/ACS treated tibiae. At 6 weeks, the rhBMP-2/ACS wrapped fractures had superior radiographic healing scores compared with buffer groups and controls. The rhBMP-2/ACS produced a significant increase in torsional toughness (p = 0.02), and trends of increased torsional strength and stiffness (p = 0.09) compared with fracture controls. The device placed in a wrapped fashion around the fracture produced significantly tougher callus (p = 0.02) compared with the onlay application. Total callus new bone volume was significantly increased (p = 0.02) in the rhBMP-2/ACS fractures compared with buffer groups and controls regardless of the method of device application. The rhBMP-2/ACS did not alter the timing of onset of periosteal/endosteal callus formation compared with controls. Neither the mineral apposition rates nor bone formation rates were affected by rhBMP-2/ACS treatment. The increased callus volume associated with rhBMP-2 treatment produced only moderate increases in strength and stiffness.

Delivering on the promise of bone morphogenetic proteins.

The advent of bone growth factors has been widely anticipated since their successful production using recombinant DNA technology. Bone morphogenetic proteins (BMPs) are an important class of bone growth factors and will be the focus of this article. In the near future these therapeutics might revolutionize how clinicians treat such diverse orthopedic applications as the healing of broken bones, increasing bone density lost through aging, and strengthening the spine. These potent proteins require application directly at the site of repair via a delivery system. The choice of delivery system has a profound effect on the clinical outcome. In the past decade, researchers have focused on developing efficient delivery systems and advancing these factors from the bench to the clinic.

Recombinant human bone morphogenetic protein-2 induces osteoblastic differentiation in W-20-17 stromal cells.

To better understand the in vivo bone-inductive properties of recombinant human (rh) BMP-2, we examined the ability of the protein to alter the phenotype of a bone marrow stromal cell line. W-20-17. rhBMP-2 increased alkaline phosphatase activity in W-20-17 cells in a dose-responsive manner in the absence of an effect on proliferation. The induction of alkaline phosphatase activity was not apparent until 12 h after rhBMP-2 treatment had begun and was effectively eliminated by cotreatment with cycloheximide, suggesting a requirement for protein synthesis. Continued treatment of W-20-17 cells with rhBMP-2 for 8 days resulted in a significant increase, compared to control cultures, in the production of cellular cAMP in response to a PTH challenge. In addition, 4-day treatment with rhBMP-2 induced osteocalcin levels in W-20-17 cells. These results indicate that rhBMP-2 induces the expression of several markers associated with the osteoblast phenotype in W-20-17 cells and raises the possibility that BMP-2 may be involved in the differentiation of osteoblasts from progenitor cells resident in bone marrow.

Glucocorticoid-induced differentiation of fetal rat calvarial osteoblasts is mediated by bone morphogenetic protein-6.

Glucocorticoids (GCs) at physiological concentrations promote osteoblast differentiation from fetal calvarial cells, calvarial organ cultures, and bone marrow stromal cells; however, the cellular pathways involved are not known. Bone morphogenetic proteins (BMPs) are recognized as important mediators of osteoblast differentiation. Specific roles for individual BMPs during postembryonic membranous bone formation have yet to be determined. We recently reported that GC potentiated the osteoblast differentiation effects of BMP-2 and BMP-4, but not of BMP-6, which, by itself, was the most potent of the three. In the present study, we used fetal rat secondary calvarial cultures to study the role of BMP-6 during early osteoblast differentiation. Treatment with the GC triamcinolone (10(-9) M) resulted in a 5- to 8-fold increase in BMP-6 steady-state messenger RNA levels, peaking at 12 h. In contrast, BMPs -2, -4, -5, -7, and transforming growth factor (TGF)-beta1 messenger RNA levels increased by less than 2-fold, after GC treatment, compared with untreated control cultures at 24 h. BMP-6 protein secretion increased 6- to 7-fold by 12 h and 12-fold (from 7.5 to 90 ng/ml) by 24 h, as measured by quantitative Western analysis. Treatment of cells with oligodeoxynucleotides antisense to BMP-6 diminished secretion of BMP-6 protein and significantly inhibited the GC-induced differentiation, as determined by a 10-fold decrease in the number of mineralized bone nodules, compared with controls that were treated with sense oligonucleotides or no oligonucleotides (ANOVA, P < 0.05). The antisense oligonucleotide inhibition of differentiation was rescued by treatment with exogenous recombinant human BMP-6. We conclude that GC-induced differentiation of osteoblasts from the pluripotent precursors is mediated, in part, by BMP-6. These results suggest that BMP-6 has an important and unique role during early osteoblast differentiation.

Immortalized murine osteoblasts derived from BMP 2-T-antigen expressing transgenic mice.

Osteoblast cell lines capable of undergoing bone formation in vitro would provide useful models for understanding gene expression during bone cell differentiation. To that end, transgenic mice were produced using a 2.9-kilobase bone morphogenetic protein 2 (BMP-2) promoter fragment, driving simian virus 40 T antigen as the transgene. The expression of simian virus 40 T antigen driven by the BMP-2 promoter immortalizes the cells. From the calvaria of the transgenic mouse, several osteoblastic cell lines were isolated and cloned. One clonal osteoblast cell line, called 2T3, has been characterized and shown to produce mineralized bone nodules. Recombinant human BMP-2 (rhBMP-2) accelerates the formation of these mineralized bone nodules. 2T3 cells express alkaline phosphatase, collagen type I, osteocalcin, and endogenous BMP-2 messenger RNA (mRNA) in a similar chronological order as normal freshly isolated fetal rat calvarial cells during early nodule formation and subsequent mineralization. The 2T3 cells also exhibit extensive growth and multilayering during differentiation, as demonstrated by growth curves and transmission electron microscopy. As with freshly isolated fetal rat calvarial cells, 1,25-dihydroxyvitamin D3 inhibited alkaline phosphatase activity and alkaline phosphatase mRNA expression, but stimulated osteocalcin mRNA expression, but stimulated osteocalcin mRNA expression. rhBMP-2 also accelerated the expression of alkaline phosphatase activity and mRNA, osteocalcin mRNA, and BMP-2 mRNA in 2T3 cells along with the formation of larger and more mineralized bone nodules. The 2T3 cell exhibits autoregulation at the mRNA and transcriptional levels. The 2T3 osteoblast cell line offers a system for examining autoregulation of the BMP-2 gene and downstream gene expression during osteoblast differentiation. 2T3 cells are reclonable and maintain their differentiation capabilities.

Differential effects and glucocorticoid potentiation of bone morphogenetic protein action during rat osteoblast differentiation in vitro.

Bone morphogenetic proteins (BMPs) induce cartilage and bone differentiation in vivo and promote osteoblast differentiation from calvarial and marrow stromal cell preparations. Functional differences between BMP-2, -4, and -6 are not well understood. Recent investigations find that these three closely related osteoinductive proteins may exert different effects in primary rat calvarial cell cultures, suggesting the possibility of unique functions in vivo. In this study, we use a fetal rat secondary calvarial cell culture system to examine the differential effects of BMP-2, -4, and -6 on early osteoblast differentiation. These cells do not spontaneously differentiate into osteoblasts, as do cells in primary calvarial cultures, but rather require exposure to a differentiation initiator such as glucocorticoid or BMP. We determined that BMP-6 is a 2- to 2.5-fold more potent inducer of osteoblast differentiation than BMP-2 or -4. BMP-6 induced the formation of more and larger bone nodules as well as increased osteocalcin secretion. The effects of all three of these BMPs were potentiated up to 10-fold by cotreatment or pretreatment with the glucocorticoid triamcinolone (Trm). The Trm effects were synergistic with those of BMP-2 or -4, suggesting that this glucocorticoid may increase the cell responsiveness to these BMPs. Finally, BMP-6 did not require either cotreatment or pretreatment with Trm to achieve greater amounts of osteoblast differentiation than seen with BMP-2 or BMP-4 treatment, suggesting that BMP-6 may act at an earlier stage of cell differentiation.

Effects of recombinant human bone morphogenetic protein-2 on differentiation of cells isolated from human bone, muscle, and skin.

We investigated the effects of recombinant human BMP-2 (rhBMP-2) on differentiation of cells isolated from human bone, muscle, and skin. Cells isolated from bones of six patients (HBM-1 to HBM-6), muscle from five patients (HM-1 to HM-5), and skin from three patients (HF-1 to HF-3) were used. rhBMP-2 had no effects on proliferation of two HBM cells, but had a stimulatory effect on three HM cell samples. rhBMP-2 stimulated both alkaline phosphatase (ALP) activity in all HBM cells and parathyroid hormone (PTH)-dependent cAMP production in three of the four HBM cell samples, although the magnitudes of these stimulatory effects differed among the cells tested. Although none of the HBM cells examined produced detectable amounts of osteocalcin in the absence of 1,25-(OH)2vitamin D3, they synthesized measurable amounts of osteocalcin in its presence. rhBMP-2 inhibited 1,25-(OH)2vitamin D3-dependent osteocalcin production in all of the HBM cell samples. Transplantation of HBM-6 cells with rhBMP-2 using diffusion chambers into the peritoneal cavity of athymic mice induced formation of cartilage and bone in the diffusion chambers, but neither cartilage nor bone was formed in chambers transplanted without rhBMP-2. rhBMP-2 also stimulated ALP activity in all of the HM and HF cell samples examined and PTH-dependent cAMP production in three of four HM cell samples. rhBMP-2 induced no osteocalcin production in any of the HM or HF cells in the presence of 1,25-(OH)2vitamin D3. rhBMP-2 markedly inhibited myotube formation by all of the HM cell samples. Transplantation of HM-4 cells with rhBMP-2, using diffusion chambers, into athymic mice induced ALP-positive cells in the chambers, but neither cartilage nor bone was observed. These results suggest that rhBMP-2 is a potent stimulator of osteoblast differentiation and bone formation in human cells.

Locally delivered rhBMP-2 enhances bone ingrowth and gap healing in a canine model.

The purpose of the present study was to determine if recombinant human bone morphogenetic protein-2 (rhBMP-2) enhances bone ingrowth into porous-coated implants and gap healing around the implants. In the presence of a 3-mm gap between the implant and host bone, porous-coated implants were placed bilaterally for four weeks in the proximal humeri of skeletally mature, adult male dogs. In three treatment groups, the test implant was treated with HA/TCP and rhBMP-2 in buffer at a dose of 100 microg/implant (n=5), 400 microg/implant (n=6), or 800 microg/implant (n=5) and placed in the left humerus. In these same animals, an internal control implant was treated only with HA/TCP and buffer and placed in the right humerus. These groups were compared with a previously reported external control group of seven animals in which no growth factor was delivered [J. Orthop. Res. 19 (2001) 85]. The BMP treated implants in the two lower dose groups had significantly more bone ingrowth than the external controls with the greatest effect in the 100 g/implant group (a 3.5-fold increase over the external control, p=0.008). All three dose groups had significantly more bone formation in the 3-mm gap surrounding the BMP treated implants than the external controls with the greatest effect in the 800 microg group (2.9-fold increase, p<0.001). Thus, application of rhBMP-2 to a porous-coated implant stimulated local bone ingrowth and gap healing. The enhancement of bone formation within the implant (bone ingrowth) was inversely related to dose.

Recombinant bone morphogenetic protein (BMP)-2 regulates costochondral growth plate chondrocytes and induces expression of BMP-2 and BMP-4 in a cell maturation-dependent manner.

This study examined the effect of recombinant human bone morphogenetic protein-2 on several parameters of growth, differentiation, and matrix synthesis and on the endogenous production of mRNA of bone morphogenetic proteins 2 and 4 by growth plate chondrocytes in culture. Chondrocytes from resting and growth zones were obtained from rat costochondral cartilage and cultured for 24 or 48 hours in medium containing 0.05-100 ng/ml recombinant human bone morphogenetic protein-2 and 10% fetal bovine serum. Incorporation of [3H]thymidine, cell number, alkaline phosphatase specific activity, incorporation of [3H]proline into collagenase-digestible protein and noncollagenase-digestible protein, and incorporation of [35S]sulfate were assayed as indicators of cell proliferation, differentiation, and extracellular matrix synthesis. mRNA levels for bone morphogenetic proteins 2 and 4 were determined by Northern blot analysis. Recombinant human bone morphogenetic protein-2 increased the incorporation of [3H]thymidine by quiescent resting-zone and growth-zone cells in a similar manner, whereas it had a differential effect on nonquiescent cultures. At 24 and 48 hours, 12.5-100 ng/ml recombinant human bone morphogenetic protein-2 caused a dose-dependent increase in cell number and DNA synthesis in resting-zone chondrocytes. No effect was seen in growth-zone cells. Recombinant human bone morphogenetic protein-2 stimulated alkaline phosphatase specific activity in resting-zone chondrocytes in a bimodal manner, causing significant increases between 0.2 and 0.8 ng/ml and again between 25 and 100 ng/ml. In contrast, alkaline phosphatase specific activity in growth-zone chondrocytes was significantly increased only between 12.5 and 100 ng/ml. Recombinant human bone morphogenetic protein-2 increased the production of both collagenase-digestible protein and noncollagenase-digestible protein by resting-zone and growth-zone cells, but incorporation of [35S]sulfate was unaffected. Administration of recombinant human bone morphogenetic protein-2 also increased incorporation of [3H]uridine in both resting-zone and growth-zone chondrocytes; these cells produced mRNA for bone morphogenetic proteins 2 and 4. Bone morphogenetic protein-2 mRNA levels in both resting-zone and growth-zone chondrocytes increased in the presence of recombinant human bone morphogenetic protein-2; however, bone morphogenetic protein-4 mRNA levels in growth-zone cells decreased under its influence, and those in resting-zone cells were upregulated only with a dose of 10 ng/ml. This indicates that recombinant human bone morphogenetic protein-2 regulates chondrocyte proliferation, differentiation, and matrix production, and the effects are dependent on the stage of cell maturation. Resting-zone chondrocytes were more sensitive, suggesting that they are targeted by bone morphogenetic protein-2 and that this growth factor may have autocrine effects on these cells.

rhBMP-2 injected in a calcium phosphate paste (alpha-BSM) accelerates healing in the rabbit ulnar osteotomy model.

This study evaluated the ability of recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered in an injectable calcium phosphate carrier (alpha-BSM) to accelerate healing in a rabbit ulna osteotomy model compared to untreated surgical controls. Healing was assessed by radiography, histology and biomechanics. Bilateral mid-ulnar osteotomies were created in 16 skeletally mature rabbits. One limb in each animal was injected with either 0.1 mg rhBMP-2/alpha-BSM (BMP) (N=8) or buffer/alpha-BSM (BSM) (N=8). Contralateral osteotomies served as untreated surgical controls (SXCT). Gamma scintigraphy showed 75%, 45% and 5% of the initial 125I-rhBMP-2 dose was retained at the osteotomy site at 3 h, 1 week and 3 weeks. The biological activity of rhBMP-2 (alkaline phosphatase activity from bioassay) extracted from alpha-BSM incubated in vitro up to 30 days at 37 degrees C was unchanged. Radiographs demonstrated complete bridging of the BMP limbs at 4 weeks whereas none of the BSM or SXCT limbs were bridged. Post-mortem peripheral quantitative computed tomography determined mineralized callus area was 62% greater in BMP limbs compared to SXCT limbs. Torsional stiffness and strength were 63% and 103% greater in BMP limbs compared to SXCT limbs. There was no difference in torsional properties between BSM and SXCT limbs. Failure occurred outside the osteotomy in four out of seven of the BMP limbs. All BSM and SXCT limbs failed through the osteotomy. Histology showed bony bridging of the osteotomy and no residual carrier in the BMP limbs. BSM and SXCT groups showed less mature calluses composed of primarily fibrocartilaginous tissue and immature bone in the osteotomy gap. These data indicate rhBMP-2 delivered in alpha-BSM accelerated healing in a rabbit ulna osteotomy model compared to BSM and SXCT groups.