Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells

The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-β gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.     

Regulation of tenascin expression in bone

Tenascins regulate cell interaction with the surrounding pericellular matrix. Within bone, tenascins C and W influence osteoblast adhesion and differentiation, although little is known about the regulation of tenascin expression. In this study we examined the effect of osteogenic differentiation, bone morphogenetic protein (BMP) and Wnt growth factors, and mechanical loading on tenascin expression in osteogenic cells. Osteogenic differentiation increased tenascin C (TnC), and decreased tenascin W (TnW), expression. Both growth factors and mechanical loading increased both TnC and TnW expression, albeit via distinct signaling mechanisms. Both BMP-2 and Wnt5a induction of tenascin expression were mediated by MAP kinases. These data establish a role for BMP, Wnts, and mechanical loading in the regulation of tenascin expression in osteoblasts.     

Regulation of cartilage and bone differentiation by bone morphogenetic proteins.

Quantum advances have recently been made in the understanding of the regulation of cartilage and bone differentiation through the identification, purification, genetic cloning and expression of recombinant bone morphogenetic proteins. Bone morphogenetic proteins are a family of pleiotropic differentiation factors with actions on chemotaxis, mitosis, initiation and promotion of chondrogenic and osteogenic phenotypes. They bind extracellular matrix components, heparin and type IV collagen and initiate bone repair. The cascade of cartilage and bone differentiation consists of several continuous phases: initiation, promotion, maintenance and termination.     

Perspectives in the biological function, the technical and therapeutic application of bone morphogenetic proteins

The bone morphogenetic proteins (BMPs) belong to the transforming growth factor beta superfamily of growth and differentiation factors and have been characterized by their ability to induce new bone formation in ectopic (non-skeletal) sites. BMPs are secreted molecules and are key regulators in early embryogenesis and organogenesis. One of the many functions of BMPs is to induce cartilage, bone, and connective tissue formation in vertebrates. This osteo-inductive capacity of BMPs has long been considered very promising for applications in bone repair, in the treatment of skeletal diseases, and in oral applications such as dentiogenesis and cementogenesis during regeneration of periodontal wounds. We discuss here biological roles of the BMPs in the organism and their signaling cascades leading to bone and cartilage formation in particular. It is also the aim of this review to evaluate the potential and the problems of BMPs in skeletal tissue engineering for the regeneration of bone damaged by disease or trauma and to serve as therapeutic agents for periodontal defects.     

Symbiosio of biotechnology and biomaterials: Applications in tissue engineering of bone and cartilage

The three ingredients for the successful tissue engineeping of bone and cartilage are ragulatory signals, cells and extracellular matrix. Recent advance in cellular and molecular biology of thde growth and differentiation factors have set the stage for a symbiosis of biotechnology and biomaterials. Recent advances permit one to enunciate the rules of architechure for tissue engineering of bone and cartilage. The purification and cloning of bone morphogenetic proteins (BMPs) and growth factors such as platelet derived growth factors (PDGF), tranforming growth factor-β (TGF-β), and insulin-like growth factors (IGF-I) Will allow the design of an optimal combinatiol of signals to initiate and promote development of skeletal stem cells into cartilage and bone. Successful and optimal bone and motion. BMPs function as inductive signals. Biomaterials (Both natural and synthetic) mimic the extracellular matrix and play a role in conduction of bone and cartiage. Examples of biomaterials include hydroxyapatite, polyanhydrides, polyphosphoesters, polylactic acid, and polyglycolic acid. The prospects for novel biomaterials are immense, and they likely will be a fertile erowth industpy. Cooperative ventures between academia and industry and teahnology transfer from the federal government augur well for an exciting future fop clinical applications.     

A mathematical framework to study the effects of growth factor influences on fracture healing

During fracture healing, multipotential stem cells differentiate into specialized cells responsible for producing the different tissues involved in the bone regeneration process. This cell differentiation has been shown to be regulated by locally expressed growth factors. The details of their regulatory mechanisms need to be understood. In this work, we present a two-dimensional mathematical model of the bone healing process for moderate fracture gap sizes and fracture stability. The inflammatory and tissue regeneration stages of healing are simulated by modeling mesenchymal cell migration; mesenchymal cell, chondrocyte and osteoblast proliferation and differentiation, and extracellular matrix synthesis and degradation over time. The effects of two generic growth factors on cell differentiation are based on the experimentally studied chondrogenic and osteogenic effects of bone morphogenetic proteins-2 and 4 and transforming growth factor-beta-1, respectively. The model successfully simulates the progression of healing and predicts that the rate of osteogenic growth factor production by osteoblasts and the duration of the initial release of growth factors upon injury are particularly important parameters for complete ossification and successful healing. This temporo-spatial model of fracture healing is the first model to consider the effects of growth factors. It will help us understand the regulatory mechanisms involved in bone regeneration and provides a mathematical framework with which to design experiments and understand pathological conditions.     

Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor beta and bone morphogenetic protein

Transforming growth factors beta (TGF-betas) inhibit growth of epithelial cells and induce differentiation changes, such as epithelial-mesenchymal transition (EMT). On the other hand, bone morphogenetic proteins (BMPs) weakly affect epithelial cell growth and do not induce EMT. Smad4 transmits signals from both TGF-beta and BMP pathways. Stimulation of Smad4-deficient epithelial cells with TGF-beta 1 or BMP-7 in the absence or presence of exogenous Smad4, followed by cDNA microarray analysis, revealed 173 mostly Smad4-dependent, TGF-beta-, or BMP-responsive genes. Among 25 genes coregulated by both factors, inhibitors of differentiation Id2 and Id3 showed long-term repression by TGF-beta and sustained induction by BMP. The opposing regulation of Id genes is critical for proliferative and differentiation responses. Hence, ectopic Id2 or Id3 expression renders epithelial cells refractory to growth inhibition and EMT induced by TGF-beta, phenocopying the BMP response. Knockdown of endogenous Id2 or Id3 sensitizes epithelial cells to BMP, leading to robust growth inhibition and induction of transdifferentiation. Thus, Id genes sense Smad signals and create a permissive or refractory nuclear environment that defines decisions of cell fate and proliferation.     

Comparison of bone morphogenetic protein-2 and osteoactivin for mesenchymal cell differentiation: effects of bolus and continuous administration

Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morphogenetic proteins (BMPs), which is costly, and may not replicate normal bone healing. The limited in vivo biologic activity of BMPs requires the investigation of growth factors that may enhance this activity. In this study, we utilized the C3H10T1/2 murine mesenchymal stem cell line to test the hypotheses that osteoactivin (OA) has comparable osteoinductive effects to bone morphogenetic protein-2 (BMP-2), and that sustained administration of either growth factor would result in increased osteoblastic differentiation as compared to bolus administration. Sustained release biodegradable hydrogels were designed, and C3H10T1/2 cells were grown on hydrogels loaded with BMP-2 or OA. Controls were grown on unloaded hydrogels, and positive controls were exposed to bolus growth factor administration. Cells were harvested at several time points to assess osteoblastic differentiation. Alkaline phosphatase (ALP) staining and activity, and gene expression of ALP and osteocalcin were assessed. Treatment with OA or BMP-2 resulted in comparable effects on osteoblastic marker expression. However, cells grown on hydrogels demonstrated osteoblastic differentiation that was not as robust as cells treated with bolus administration. This study shows that OA has comparable effects to BMP-2 on osteoblastic differentiation using both bolus administration and continuous release, and that bolus administration of OA has a more profound effect than administration using hydrogels for sustained release. This study will lead to a better understanding of appropriate delivery methods of osteogenic growth factors like OA for repair of fractures and segmental bone defects.     

Application of bone morphogenetic proteins in spinal fusion

We are now entering an exciting new era in spinal surgery where the inherent osteoinductive capacity of the body has been harnessed for bone formation for therapeutic purposes. Recombinant bone morphogenetic proteins have been extensively studied in both the pre-clinical and clinical arena for spinal fusion with considerable success. The challenges facing spine surgeons now is the development of site-specific carriers and optimal doses for these growth factors. This review highlights the recent advances in this regard.     

Basic fibroblast growth factor enhances the osteogenic differentiation induced by bone morphogenetic protein-6 in vitro and in vivo

Some members of the bone morphogenetic protein subfamily (BMP-2 and -7) are currently used in orthopedic surgery for several applications. Although their use is considered safe at short term, the high doses of growth factors needed make these treatments expensive and their safety uncertain at long term. BMP-6 has been much less studied than BMP-2 and -7, but some authors suggest that this BMP might have a stronger osteogenic activity than the previously mentioned. Having in mind that angiogenesis plays a well-known role during bone formation, the aim of this work was to study the effect of combining BMP-6 with bFGF on both the growth and differentiation of MC3T3-E1 mouse preosteoblasts and rat bone marrow-derived mesenchymal stem cells (MSCs), as well as on in vivo osteogenesis. We demonstrate that a low dose of bFGF enhances the osteogenic differentiation of MSCs induced by BMP-6 in vitro. Furthermore, we also demonstrate that bone formation in vivo induced by BMP-6 can be accelerated and enhanced by adding a low dose of bFGF, what might suggest a synergic effect between these growth factors on in vivo osteogenesis.     

Characterization and evaluation of mesenchymal stem cells derived from human embryonic stem cells and bone marrow

Embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) have been studied for years as primary cell sources for regenerative biology and medicine. MSCs have been derived from cell and tissue sources, such as bone marrow (BM), and more recently from ESCs. This study investigated MSCs derived from BM, H1- and H9-ESC lines in terms of morphology, surface marker and growth factor receptor expression, proliferative capability, modulation of immune cell growth and multipotency, in order to evaluate ESC-MSCs as a cell source for potential regenerative applications. The results showed that ESC-MSCs exhibited spindle-shaped morphology similar to BM-MSCs but of various sizes, and flow cytometric immunophenotyping revealed expression of characteristic MSC surface markers on all tested cell lines except H9-derived MSCs. Differences in growth factor receptor expression were also shown between cell lines. In addition, ESC-MSCs showed greater capabilities for cell proliferation, and suppression of leukocyte growth compared to BM-MSCs. Using standard protocols, induction of ESC-MSC differentiation along the adipogenic, osteogenic, or chondrogenic lineages was less effective compared to that of BM-MSCs. By adding bone morphogenetic protein 7 (BMP7) into transforming growth factor beta 1 (TGFβ1)-supplemented induction medium, chondrogenesis of ESC-MSCs was significantly enhanced. Our findings suggest that ESC-MSCs and BM-MSCs show differences in their surface marker profiles and the capacities of proliferation, immunomodulation, and most importantly multi-lineage differentiation. Using modified chondrogenic medium with BMP7 and TGFβ1, H1-MSCs can be effectively induced as BM-MSCs for chondrogenesis.     

Protein-based tissue engineering in bone and cartilage repair

Bioactive proteins signal host or transplanted cells to form the desired tissue type. Matrix systems are utilized to locally deliver the proteins and to maintain effective protein concentrations. For some indications, a matrix is required to define the physical form of the regenerated tissue. Substantial progress has been made in bone tissue engineering in recent years, based on the results of controlled clinical studies using bone morphogenetic proteins. Ongoing research in this area centers on the design of additional delivery matrices to expand the clinical indications, using synthetic delivery systems that mimic biological qualities of the natural materials currently in use. Although a similar rationale exists for the regeneration of articular cartilage with bioactive factors, advancement in this area has not been as substantial.     

Brain-derived neurotrophic factor (BDNF) mediates bone morphogenetic protein-2 (BMP-2) effects on cultured striatal neurones

Bone morphogenetic proteins are members of the transforming growth factor-beta superfamily that have multiple functions in the developing nervous system. One of them, bone morphogenetic protein-2 (BMP-2), promotes the differentiation of cultured striatal neurones, enhancing dendrite growth and calbindin-positive phenotype. Bone morphogenetic proteins have been implicated in cooperative interactions with other neurotrophic factors. Here we examined whether the effects of BMP-2 on cultured striatal neurones are mediated or enhanced by other neurotrophic factors. BMP-2 had a cooperative effect with low doses of brain-derived neurotrophic factor or neurotrophin-3 (but not with other neurotrophic factors such as glial cell line-derived neurotrophic factor, neurturin or transforming growth factor-beta 2) on the number of calbindin-positive striatal neurones. Moreover, BMP-2 induced phosphorylated Trk immunoreactivity in cultured striatal neurones, suggesting that neurotrophins are involved in BMP-2 neurotrophic effects. The addition of TrkB-IgG or antibodies against brain-derived neurotrophic factor abolished the effects of BMP-2 on the number and degree of differentiation of calbindin-positive striatal neurones. Indeed, BMP-2 treatment increased brain-derived neurotrophic factor protein levels in treated cultures media and BDNF immunocytochemistry revealed that this neurotrophin was produced by neuronal cells. Taken together, these results indicate that brain-derived neurotrophic factor mediates the effects of BMP-2 on striatal neurones.     

Synthetic osteogenic growth peptide promotes differentiation of human bone marrow mesenchymal stem cells to osteoblasts via RhoA/ROCK pathway

The osteogenic growth peptide (OGP) is a naturally occurring tetradecapeptide that has attracted considerable clinical interest as a bone anabolic agent and hematopoietic stimulator. In vitro studies have demonstrated that OGP directly regulates the bone marrow mesenchymal stem cells' (BMSCs) differentiation into osteoblasts. However, the exact mechanism of this process remains unknown. In the present study, we investigated the role of RhoA/ROCK signaling in differentiation along this lineage using human BMSCs. OGP treatment increased the mRNA level of bone morphogenetic protein-2 and alkaline phosphatase activity after osteogenic induction. Analysis of BMSCs induced in the presence of OGP revealed an increase in RhoA activity, and phosphorylation of FAK and cofilin. The ROCK-specific inhibitors, Y27632, blocked the OGP-induced regulation of BMSC differentiation. Taken together, these data suggest that OGP not only acts on BMSCs to stimulate osteogenic differentiation, but also in a dose-dependent manner, and this effect is mediated via the activation of RhoA/ROCK pathway.     

Suppression of tumor necrosis factor-mediated apoptosis by nuclear factor kappaB-independent bone morphogenetic protein/Smad signaling

The activation of nuclear factor kappaB (NF-kappa B) plays a pivotal role in the regulation of tumor necrosis factor (TNF)-mediated apoptosis. However, little is known about the regulation of TNF-mediated apoptosis by other signaling pathways or growth factors. Here, unexpectedly, we found that bone morphogenetic protein (BMP)-2 and BMP-4 inhibited TNF-mediated apoptosis by inhibition of caspase-8 activation in C2C12 cells, a pluripotent mesenchymal cell line that has the potential to differentiate into osteoblasts depending on BMP stimulation. Utilizing both a trans-dominant IkappaBalpha inhibitor of NF-kappaB expressed in C2C12 cells and IkappaB kinase beta-deficient embryonic mouse fibroblast, we show that BMP-mediated survival was independent of NF-kappaB activation. Rather, the antiapoptotic activity of BMPs functioned through the Smad signaling pathway. Thus, these findings provide the first report of a BMP/Smad signaling pathway that can inhibit TNF-mediated apoptosis, independent of the prosurvival activity of NF-kappaB. Our results suggest that BMPs not only stimulate osteoblast differentiation but can also promote cell survival during the induction of bone formation, offering new insight into the biological functions of BMPs.     

Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily that has been used for bone grafting. We were interested in exploring the functions of BMP-2 in other disease areas and focused on expressing and purifying active BMP-2 proteins. We have developed a new approach which involves using FoldIt refolding buffer to refold BMP-2 followed by a heparin affinity column to separate correctly folded dimer from monomer. A high yield of 29.4 mg BMP-2 dimer per gram cell wet weight was achieved. The purified BMP-2 dimer was shown to possess the same level of activity as BMP-2 from CHO cells as tested by the induction of alkaline phosphatase activity in C2C12 cells. This approach has potential application in refolding and purifying other homodimeric proteins.