BMP stimulation of alkaline phosphatase activity in pluripotent mouse C2C12 cells is inhibited by dermatopontin, one of the most abundant low molecular weight proteins in demineralized bone matrix

Demineralized bone matrix (DBM) is a complex mixture of osteoinductive bone morphogenetic proteins (BMPs), as well as BMP-binding proteins that regulate BMP bioactivity and localization. Our aim was to use modern proteomic methods to identify additional BMP-binding proteins in DBM, with initial emphasis on the most abundant. Relatively large, water-soluble noncollagenous proteins (NCPs) were preferentially extracted from DBM with alkalinized urea. The insoluble residue, which contained the BMP activity, was extracted with GuHCl/CaCl2, dialyzed versus citrate, defatted, resuspended in GuHCl, dialyzed sequentially against Triton X-100 and water, pelleted, and lyophilized. The proteins in this pellet were fractionated by hydroxyapatite affinity chromatography. Proteins that copurified with BMP bioactivity were separated by SDS-PAGE. Distinct bands were excised, and the proteins in them were reduced and alkylated, digested with trypsin, eluted, and subjected to MALDI/ToF MS (matrix-assisted laser-desorption ionization time-of-flight mass spectrometry). Computer-assisted peptide fingerprint analysis of the MS profiles was used to identify C-terminal lysine-6-oxidase; dermatopontin (DPT); histones H2A2, H2A3, and H2B; and trace amounts of gamma-actin. DPT is a 22-kDa, tyrosine-rich acidic matrix protein not previously recognized to be among the most abundant small proteins to copurify with BMP bioactivity in DBM. We tested the effects of DPT on BMP-2 stimulation of alkaline phosphatase (ALP) activity in C2C12 cells. BMP-2 stimulated ALP activity in C2C12 cells by 6.2-fold above basal levels. DPT alone had no effect on ALP activity in C2C12 cells. When added with BMP-2, DPT blocked 40% of the stimulatory effect of BMP-2 on ALP activity in C2C12 cells. DPT is an abundant protein in DBM, and it can inhibit the stimulatory effects of BMP-2 on ALP activity in C2C12 cells.     

Heparin microparticle effects on presentation and bioactivity of bone morphogenetic protein-2

Biomaterials capable of providing localized and sustained presentation of bioactive proteins are critical for effective therapeutic growth factor delivery. However, current biomaterial delivery vehicles commonly suffer from limitations that can result in low retention of growth factors at the site of interest or adversely affect growth factor bioactivity. Heparin, a highly sulfated glycosaminoglycan, is an attractive growth factor delivery vehicle due to its ability to reversibly bind positively charged proteins, provide sustained delivery, and maintain protein bioactivity. This study describes the fabrication and characterization of heparin methacrylamide (HMAm) microparticles for recombinant growth factor delivery. HMAm microparticles were shown to efficiently bind several heparin-binding growth factors (e.g. bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (FGF-2)), including a wide range of BMP-2 concentrations that exceeds the maximum binding capacity of other common growth factor delivery vehicles, such as gelatin. BMP-2 bioactivity was assessed on the basis of alkaline phosphatase (ALP) activity induced in skeletal myoblasts (C2C12). Microparticles loaded with BMP-2 stimulated comparable C2C12 ALP activity to soluble BMP-2 treatment, indicating that BMP-2-loaded microparticles retain bioactivity and potently elicit a functional cell response. In summary, our results suggest that heparin microparticles stably retain large amounts of bioactive BMP-2 for prolonged periods of time, and that presentation of BMP-2 via heparin microparticles can elicit cell responses comparable to soluble BMP-2 treatment. Consequently, heparin microparticles present an effective method of delivering and spatially retaining growth factors that could be used in a variety of systems to enable directed induction of cell fates and tissue regeneration.     

DRAGON, a GPI-anchored membrane protein, inhibits BMP signaling in C2C12 myoblasts

Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation of myoblasts via binding to cell surface receptors. Repulsive guidance molecules (RGMs) have been identified as BMP co-receptors. We report here that DRAGON/RGMb, a member of the RGM family, suppressed BMP signaling in C2C12 myoblasts via a novel mechanism. All RGMs were expressed in C2C12 cells that were differentiated into myocytes and osteoblastic cells, but RGMc was not detected in immature cells. In C2C12 cells, only DRAGON suppressed ALP and Id1 promoter activities induced by BMP-4 or by constitutively activated BMP type I receptors. This inhibition by DRAGON was dependent on the secretory form of the von Willbrand factor type D domain. DRAGON even suppressed BMP signaling induced by constitutively activated Smad1. Over-expression of neogenin did not alter the inhibitory capacity of DRAGON. Taken together, these findings indicate that DRAGON may be an inhibitor of BMP signaling in C2C12 myoblasts. We also suggest that a novel molecule(s) expressed on the cell membrane may mediate the signal transduction of DRAGON in order to suppress BMP signaling in C2C12 myoblasts.     

由BMP-4基因转入C2C12细胞引起的成肌细胞到成骨细胞的表型转变

目的:研究BMP-4基因对C2C12细胞分化的影响。方法:BMP-4基因在pCI-neo质粒中构建后转入C2C12细胞。转入pCI-neo-BMP-4重组质粒的细胞、转入pCI-neo质粒的细胞及没有转染的细胞在相同的条件下培养。结果:转入pCI-neo质粒的细胞及没有转染的细胞在增殖期和分化期均不表达BMP-4,在未分化期表现为典型的星形形态,并相互融合成细长而多核的肌小管。而转入pCI-neo-BMP-4重组质粒的细胞在增殖期和分化期均大量表达BMP-4,形态上不形成肌小管,而是形成类似成骨细胞的形态,并高水平表达碱性磷酸酶、在培养基中分泌骨钙素。结论:表明将BMP-4基因转入C2C12细胞可改变其肌源的分化途径,而分化为成骨细胞系,且这种分化转向是基因水平上的。     

Enhanced bioactivity of bone morphogenetic protein-2 with low dose of 2-N, 6-O-sulfated chitosan in vitro and in vivo

Bone morphogenetic protein-2 (BMP-2) has been widely used as an effective growth factor in bone tissue engineering. However, large amounts of BMP-2 are required to induce new bone and the resulting side effects limit its clinical application. Sulfated polysaccharides, such as native heparin, and heparan sulfate have been found to modulate BMP-2 bioactivity and play pivotal roles in bone metabolism. Whereas the direct role of chitosan modified with sulfate group in BMP-2 signaling has not been reported till now. In the present study, several sulfated chitosans with different positions were synthesized by regioselective reactions firstly. Using C2C12 myoblast cells as in vitro models, the enhanced bioactivity of BMP-2 was attributed primarily to the stimulation from 6-O-sulfated chitosan (6SCS), while 2-N-sulfate was subsidiary group with less activation. Low dose of 2-N, 6-O-sulfated chitosan (26SCS) showed significant enhancement on the alkaline phosphatase (ALP) activity and the mineralization formation induced by BMP-2, as well as the expression of ALP and osteocalcin mRNA. Moreover, increased chain-length and further sulfation on 26SCS also resulted in a higher ALP activity. Dose-dependent effects on BMP-2 bioactivity were observed in both sulfated chitosan and heparin. Compared with native heparin, 26SCS showed much stronger simultaneous effects on the BMP-2 bioactivity at low dose. Stimulated secreted Noggin protein failed to block the function of BMP-2 in the presence of 26SCS. The BMP-2 ligand bound to its receptor was enhanced by low dose of 26SCS, whereas weakened by the increasing amounts of 26SCS. Furthermore, simultaneous administration of BMP-2 and 26SCS in vivo dose-dependently induced larger amounts of ectopic bone formation compared with BMP-2 alone. These findings clearly indicate that 26SCS is a more potent enhancer for BMP-2 bioactivity to induce osteoblastic differentiation in vitro and in vivo by promoting BMP-2 signaling pathway, suggesting that 26SCS could be used as the synergistic factor of BMP-2 for bone regeneration.     

RGD-grafted thermoreversible polymers to facilitate attachment of BMP-2 responsive C2C12 cells

The purpose of this study was to design thermoreversible biomaterials for enhanced adhesion of bone morphogenetic protein-2 (BMP-2)-responsive cells. Peptides containing the arginine-glycine-aspartic acid (RGD) sequence were conjugated to N-isopropylacrylamide (NiPAM) polymers via amine-reactive N-acryloxysuccinimide (NASI) groups. In monolayer cultures, the adhesion of BMP-2-responsive C2C12 cells to RGD-grafted NiPAM/NASI surfaces was significantly higher than adhesion on ungrafted NiPAM/NASI surfaces. Although the morphology of cells adhered to RGD-grafted NiPAM/NASI surfaces was comparable to cells adhered on tissue culture polystyrene (TCPS), long-term cell growth was limited on the NiPAM/NASI surfaces, even for RGD-grafted surfaces. Treatment of C2C12 cells with recombinant BMP-2 induced dose-dependent osteoblastic differentiation as assessed by alkaline phosphatase (ALP) activity. In the absence of BMP-2, cells cultured on NiPAM/NASI polymers (either grafted with RGD peptide or not) expressed significantly higher levels of ALP activity than the cells cultured on TCPS, indicating that the polymer surfaces induced some osteoblastic activity in C2C12 cells without the need for BMP-2. We conclude that NiPAM-based thermoreversible biomaterials, despite their limited ability to support cell growth, allowed an enhanced expression of the chosen osteogenic marker (ALP) by C2C12 cells in vitro.     

Spatial patterning of BMP-2 and BMP-7 on biopolymeric films and the guidance of muscle cell fate

In the cellular microenvironment, growth factor gradients are crucial in dictating cell fate. Towards developing materials that capture the native microenvironment we engineered biomimetic films that present gradients of matrix-bound bone morphogenetic proteins (BMP-2 and BMP-7). To this end layer-by-layer films composed of poly(l-lysine) and hyaluronan were combined in a simple microfluidic device enabling spatially controlled growth factor diffusion along the film. Linear long-range gradients of both BMPs induced the trans-differentiation of C2C12 myoblasts towards the osteogenic lineage in a dose dependent manner with a different signature for each BMP. The osteogenic marker alkaline phosphatase (ALP) increased in a linear manner for BMP-7 and non-linearly for BMP-2. Moreover, an increased expression of the myogenic marker troponin T was observed with decreasing matrix-bound BMP concentration, providing a substrate that it is both osteo- and myo-inductive. Lastly, dual parallel matrix-bound gradients of BMP-2 and -7 revealed a complete saturation of the ALP signal. This suggested an additive or synergistic effect of the two BMPs. This simple technology allows for determining quickly and efficiently the optimal concentration of matrix-bound growth factors, as well as for investigating the presentation of multiple growth factors in their solid-phase and in a spatially controlled manner.     

IRAK-4对成骨细胞分化BMP-Smad通道的影响

目的　探讨白细胞介素-l受体相关激酶-4对成骨细胞分化过程BMP-Smad通道的影响。 方法　 C2C12细胞是肌卫星细胞,不同培养条件下有不同分化潜能。它可分化为成骨细胞,是研究体外成骨细胞分化的理想模型。C2C12细胞培养于培养板中,随机分为3组,每组加入不同培养物,模拟干细胞向成骨细胞分化过程中受到的不同刺激。检测ALP活性、Smad1 mRNA、P-Smad1蛋白表达,观察不同刺激对成骨细胞分化的影响。 结果　与正常对照组比较,BMP-2组ALP活性明显增加,与BMP-2组比,BMP2+IRAK-4siRNA转染组ALP活性增加,BMP2+IRAK-4siRNA转染组和BMP-2组比Smadl 　mRNA的表达只是轻微增加,P-Smad1蛋白表达明显增加。 结论　BMP-2可增强C2C12细胞成骨化,IRAK-4可抑制C2C12细胞被BMP-2诱导的成骨化,其机制可能是通过减弱BMP-Smad通道中Smad1磷酸化水平实现的。     

Osteoinductivity of partially purified bovine, ostrich and emu bone morphogenetic proteins in vitro

The aim of this study was to observe the osteogenic activity of native bone morphogenetic proteins (BMPs) obtained from different species including bovine, ostrich and emu sources in order to compare mammalian and avian BMPs. Rat mesenchymal progenitor marrow stromal cells and pre-osteoblastic C2C12 cell cultures, were exposed to the native BMPs and alkaline phosphatase (ALP) and creatine kinase (CK) levels were determined by assay. The results showed that the ALP activity in C2C12 cultures was elevated by bovine BMP by 2- to 10-fold (p < 0.05-0.001) from day 3 during 14 days. There were no significant differences in avian BMP related elevations of ALP activity except with ostrich BMPs at day 14 (p < 0.05). However, exposure of MSCs cultures to BMPs derived from bovine, ostrich or emu sources resulted in elevated ALP from day 3 (p < 0.05). Bovine BMP resulted in more ALP elevation than with either of the avian BMPs. All of BMPs elevated Creatine kinase (CK) activity from day 1 and climbed until peaking at day 7. Compared with control cultures, CK was elevated more with exposure to emu BMP and was more elevated with greater statistical significance than with bovine and ostrich BMP before day 5. These higher levels remained until day 14 (p < 0.05). The results of this study suggest that both bovine and avian BMPs are able to stimulate osteogenesis in mature osteoblasts in vitro. The strongest synergistic effect on osteogenesis was detected in cells stimulated with bovine BMP. Avian BMPs had lower effects on ALP and CK activity, emu BMP being more effective than ostrich BMP.     

Activation of demineralized bone matrix by genetically engineered human bone morphogenetic protein-2 with a collagen binding domain derived from von Willebrand factor propolypeptide

There is a large demand for new bone regeneration to restore the function during bone injuries. Bone filling materials are important in bone tissue restoration. In this study, the demineralized bone matrix (DBM) was activated with the engineering human bone morphogenetic protein-2 (BMP-2). To enhance the binding of BMP-2 to the DBM scaffolds, a collagen-binding peptide was fused to the N-terminal of BMP-2. The in vitro results showed that the engineered collagen-targeted BMP-2 (rhBMP2-v) bound to DBM scaffolds specifically and the rhBMP2-v had increased alkaline phosphatase activity in C2C12 cells. In vivo, the DBM scaffolds impregnated with rhBMP2-v showed greater effect on ectopic bone formation. Our data suggested that the collagen-based BMP-2 targeting bone repair system had greater bone inducing ability than DBM loaded with regular BMP-2.     

白藜芦醇对维生素D_3和尼古丁诱导的大鼠血管钙化的抑制作用

目的:观察白藜芦醇(RES)对维生素D3和尼古丁诱导的大鼠血管钙化的抑制作用和可能机制。方法:32只雄性Sprague Dawley大鼠随机分为4组,每组各8只:对照组(CON组);血管钙化组(VDN组);RES低剂量处理组[VDN+RES(L)组];RES高剂量处理组[VDN+RES(H)组]。实验第6周末进行各组大鼠主动脉Von Kossa染色,比色法测定碱性磷酸酶(ALP)活性及钙含量,Western Blot检测成骨细胞标志性蛋白骨桥蛋白(OPN)和骨形态发生蛋白-2(BMP-2)表达,并比较各组间差异。结果:与CON组比较,VDN组大鼠主动脉Von Kossa染色显示主动脉中膜弹力纤维排列紊乱,其间广泛分布黑色银染颗粒,主动脉ALP活性、钙含量、OPN和BMP-2水平均显著升高(P0.01);与VDN组比较,两种剂量RES干预组大鼠主动脉中膜弹力纤维紊乱程度下降、黑色银染颗粒减少,主动脉ALP活性、钙含量、OPN和BMP-2水平均明显降低(P0.05或P0.01),高剂量RES干预组较低剂量RES干预组降低更明显(均P0.05)。结论:RES能够有效抑制维生素D3和尼古丁诱导的大鼠血管钙化,其机制可能与其能降低主动脉ALP活性及抑制OPN和BMP-2表达有关。     

Homogeneous osteogenesis and bone regeneration by demineralized bone matrix loading with collagen-targeting bone morphogenetic protein-2

Considerable research has been focused on the development of bone morphogenetic protein-2 (BMP-2) delivery system for homologous and efficient bone regeneration. The aim of the present study was to develop a collagen-based targeting bone repair system. A collagen-binding domain (CBD) was added to the N-terminal of native BMP-2 to allow it bind to collagen specifically. We showed that the collagen-binding bone morphogenetic protein-2 (named bone morphogenetic protein2-h, BMP2-h) had maintained the full biological activity as compared to rhBMP2 lacking the CBD. In vitro functional study also demonstrated that collagen matrix could maintain higher bioactivity of BMP2-h than native BMP-2. When demineralized bone matrix (DBM) impregnated with BMP2-h was implanted subcutaneously in rats, homogeneous bone formation was observed. Moreover, in a rabbit mandible defect model, surgical implantation of collagen matrix loaded with BMP2-h exhibited remarkable osteoinductive properties and excellent homogeneous bone formation. Our studies suggested that this novel collagen-based BMP-2 targeting bone repair system induced better bone formation not only in quantity but also in quality. Similar approaches may also be used for the repair of other tissue injuries.     

Immobilized recombinant human bone morphogenetic protein-2 enhances the phosphorylation of receptor-activated Smads

Bone morphogenetic protein (BMP)-2 plays an important role in bone growth and regeneration; however, BMP-2 is easily lost by diffusion through body fluid and has some inhibitory pathways. To address this problem, we previously immobilized recombinant human BMP-2 (rhBMP-2) on succinylated type I atelocollagen. Here, we examined the effect of immobilized rhBMP-2 in vitro and vivo. In ST2, MC3T3-E1, and C2C12 cells, alkaline phosphatase activity, which is a marker of osteoblast differentiation, was enhanced more by immobilized than nonimmobilized rhBMP-2. In addition, the phosphorylation of receptor-activated Smads, part of the signaling pathway activated by BMP-2, was prolonged by immobilized rhBMP-2 in these cells. Furthermore, implantation of immobilized rhBMP-2 into the backs of rats promoted the formation of mature bone-like structure. These results demonstrate that immobilized rhBMP-2 has higher bioactivity than nonimmobilized rhBMP-2, and, therefore, immobilization of rhBMP-2 can prolong BMP signaling.     

Bone morphogenetic protein-2 and -7 mediate the anabolic function of nucleus pulposus cells with discrete mechanisms

Bone morphogenetic proteins (BMPs) play roles in promoting cell anabolism, especially in extracellular matrix production. The difference between BMP members in their capacity to modulate intervertebral disc cell activity is yet to be defined. BMP-7/OP-1 has been shown to retard disc degeneration. We compared the activity of BMP-7 with that of BMP-2 on nucleus pulposus (NP) cell phenotype and function, and investigated how they differentially affect the gene expression profiles of signaling cascade components in human NP cells under degenerative states. We found that while both BMP-2 and BMP-7 enhanced matrix production of bovine NP cells, BMP-7 is more potent than BMP-2 at various dosages (50–800 ng/ml). BMP-7 exerted a relatively stronger stimulation on sulfated glycosaminoglycan production and proliferation in human NP cells. Degenerated NP cells showed an overall weaker response to the BMPs than non-degenerated cells, and were more sensitive to BMP-7 than BMP-2 stimulation. Compared to BMP-2, BMP-7 not only induced the gene expression of canonical BMP components, but also evoked changes in MAPKs as well as CREB1 and EP300 gene expression in degenerated NP cells, suggesting potential activation of the cAMP dependent protein kinase related pathways. In contrast to BMP-2, BMP-7 concomitantly inhibited the expression of profibrotic genes. We propose that BMP-2 and BMP-7, and likely other BMPs, may operate multifaceted but discrete molecular machineries that give rise to their different capacity in regulating NP cell phenotype. Further investigations into such differential capacity may possibly derive alternative cues important for IVD repair or engineering.     

A comparison of osteocyte bioactivity in fine particulate bone powder grafts vs larger bone grafts in a rat bone repair model

The osteogenic potential for bone grafts is based on numbers and activities of cells that survive transplantation. In this study, we compared the bioactivity of osteocytes in 300–500 μm fine particulate bone powder grafts to 2 mm larger bone grafts in a rat radial defect model. Expression levels of bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta 1 (TGF-β1), alkaline phosphatase (ALP), and collagen I were semi-quantified by both immunohistochemistry and RT-PCR at days 1 and 4, as well as weeks 1, 2, 4, 6 and 10 post-transplantation. Within two weeks post-transplantation, more cells stained positively for BMP-2, TGF-β1, ALP, and collagen I within the bone grafts and in the surrounding tissues in the group transplanted with the fine particulate bone powder grafts than in those with larger bone grafts (P < 0.05). The mRNA levels of all four markers in the group transplanted with fine particulate bone powder graft peaked earlier and were expressed more highly than in the larger bone graft group, suggesting that fine particulate bone powder grafts provide more viable and active osteocytes to accelerate bone defect healing than larger bone grafts.     

Heterodimeric Bone Morphogenetic Proteins Show Enhanced Activity In Vitro and In Vivo

Abstract

The bone morphogenetic proteins (BMPs), a subgroup of the TGF-β gene super-family, are dimeric molecules involved in the growth, differentiation and repair of a wide variety of tissues. Based on the observation that several of the BMPs co-purify when isolated from bovine bone and that a pattern of co-localization exists during mouse embryogenesis, we co-expressed various combinations of BMPs in Chinese hamster ovary cells to test for possible heterodimer formation and activity. Transient co-expression of BMP-2 with either BMP-5, BMP-6 or BMP-7, or BMP-4 transiently co-expressed with BMP-7, resulted in more BMP activity than expression of any single BMP. Stable cell lines were then made in order to purify and characterize co-expressed BMPs in more detail. Co-expression of BMP-2 with BMP-7 yielded heterodimeric BMP-2/7 with a specific activity about 20-fold higher than BMP homodimers in an in vitro alkaline phosphatase induction assay. These heterodimers were also 5- to 10-fold more potent than BMP-2 in inducing cartilage and bone in an in vivo assay. Similar results were obtained with BMP-2/6 heterodimer. These experiments demonstrate the increased potency of several BMP heterodimers relative to BMP homodimers and support the hypothesis that such heterodimeric forms are likely to have natural biological functions.