Induction of callus formation by implants of bone morphogenetic protein and associated bone matrix noncollagenous proteins

Callus formation in the periosteal bone interface in response to bone morphogenetic protein (BMP) and associated bone matrix noncollagenous proteins (BMP/NCP) was investigated in mature adult rabbits. For controls byproducts of BMP/NCP purification, bone marrow, eight nonskeletal tissues, purified matrix gamma-carboxyglutamic acid-rich protein (MGP), and a composite of BMP/NCP and polylactic-polyglycolic acid polymer (PLA/PGA) were also implanted in the periosteal bone interface. Quantitative microcomputer image analysis and histologic studies were performed three weeks after the implantation. BMP/NCP and bone marrow or BMP/NCP implanted over a single drill hole into the marrow cavity produced three times more new bone than the bone marrow alone. BMP/NCP alone produced twice as much new bone as bone marrow alone. Control implants of bovine serum albumin or purified MGP produced no new bone. Autogeneic minced muscle and ten nonskeletal tissue controls produced little or no bone formation. Even at one-fifth of the dose of BMP/NCP, a composite of PLA/PGA incorporating BMP/NCP showed almost the same amount of new bone as BMP alone. Histologically, the response to BMP/NCP consisted of an external callus of calcifying cartilage and woven bone. The response to subperiosteal implants of BMP/NCP or BMP/NCP with bone marrow or with minced muscle occurred with the same sequence of developmental events as seen either in embryonic skeletogenesis or in fracture callus.     

Nature of bone morphogenetic protein (BMP) from decalcified rabbit bone matrix

Rabbit bone morphogenetic protein (BMP) from demineralized and defatted rabbit bone matrix was partially purified. BMP activity was examined by the implantation of fractionated materials into the thigh muscle pouch of the mouse. Rabbit BMP was solubilized by both 4M guanidine hydrochloride (GuHCl) and 6M urea solutions. Crude BMP had isoelectric point precipitation at pH 3 in 6M urea and showed bone morphogenesis. Fractions eluted with 0 and 0.2 N NaCl in DEAE CL-6B ion exchange chromatography showed bone morphogenesis in each individual pH of pH 4 to pH 7 but the fraction eluted with 1.0 N NaCl did not show any activity. Sephadex G-75 filtration separated the crude material into three peaks and the peak of about 23,000 showed bone morphogenesis. In sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and isoelectric focusing, rabbit BMP was thought to be an acidic protein having a molecular weight of 24,000 with an isoelectric point around 4.85.     

调控骨形态发生蛋白诱导成骨的相关转录因子

骨形态发生蛋白（Bone morphogenetic protein,BMPs）是一种确切具有诱导成骨活性的生长因子,其主要生物学作用是诱导未分化的间充质细胞分化形成软骨和骨。迄今为止,至少发现了15种BMP,除BMP1外,其他BMPs均属于转化生长因子β（transforming growth factor-beta,TGF-β）超家族成员,它们可通过旁分泌和自分泌的形式诱导骨、软骨及与骨有关的结缔组织的形成,还能诱导骨源和非骨源性细胞的成骨分化。转录因子是一种核蛋白,是一类可以通过与下游靶基因DNA结合,从而调控下游基因转录的分子。随着对BMPs诱导成骨研究的深入,一些与BMPs相关的转录因子日益受到关注,对它们的研究也成为热点。根据目前的研究结果发现,调控BMPs诱导成骨的相关转录因子可分为两类,包括正调控因子（如Cbfαl、Osterix、Dlx5、Hoxc8和Msx2等）和负调控因子（如CIZ、AJ18、Tob和c-Ski等）,本综述对它们的研究进展分别作以介绍。     

Solubilization and purification of bone morphogenetic protein (BMP) from Dunn osteosarcoma

The 4 M GuHCl soluble proteins of Dunn osteosarcoma were fractionated in two steps by means of CsCl density gradient centrifugation. Further purification of bone morphogenetic protein (BMP) was accomplished by molecular sieve techniques utilizing thin-channel diafiltration (Amicon), Sepharose CL-6B and Sephacryl S-200 gel filtration. Partially purified BMP fractions were analyzed by electrophoresis on 7.5% SDS polyacrylamide gel. The molecular weight of BMP active fractions ranges from 30,000 to a few thousand daltons. BMP may be one of the two proteins of MW of 12,500 and 16,000 daltons.     

Analysis of bone morphogenetic protein (BMP) derived from human and bovine bone matrix

Recently, Bone Morphogenetic Protein (BMP) has attracted the attention of a number of investigators, but its elucidation remains incomplete. At present, the determination of its amino acid sequence, which is necessary for its synthesis, and screening for a carrier that allows BMP to be effective in small amounts are unsolved problems. Bone morphogenetic protein is studied here to clarify its clinical applications. BMP was extracted from human and bovine bone matrix with 4 M guanidine-HCl and purified by liquid chromatography. Acrylamide electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) showed that the purified BMP was homogeneous. We used type I collagen as the carrier in the bioassay. This BMP induced new bone in situ three weeks after implantation in muscle pouches in Wistar rats. The molecular weights of human and bovine bone matrix-derived BMP are 17.0 and 18.0 kDa by SDS-PAGE, and pI values for both are 4.9 by IEF. Human and bovine bone matrix-derived BMP are peptides containing 165 and 163 amino acids, respectively, according to amino acid analysis. The NH2-terminal sequence of bovine bone matrix-derived BMP was obtained from the bovine band, electroblotted onto polyvinylidene difluoride membrane, that corresponded to the final purified fraction. The sequence differs from previously designated BMPs25 and other proteins reported to have similar activity, but the physicochemical characteristics are comparable to the native preparations.     

Solubilized bone morphogenetic protein (BMP) from mouse osteosarcoma and rat demineralized bone matrix

A selection of proteins including bone morphogenetic protein (BMP) was extracted in a disaggregated form from Dunn osteosarcoma or rat demineralized bone matrix by 4M guanidine hydrochloride (GuHCl) solution without losing its biological activity. The GuHCl extracts of Dunn osteosarcoma were divied into 4 different fractions by cesium chloride (CsCl) density gradients. Under a dissociative condition, the highest new bone yield was obtained in the low dense top one-third fraction, and BMP acitivity declined with increase in the density of each fraction. No BMP potential was observed in the surface-gel fraction under dissociative conditions. Under an associative condition (low GuHCl concentrations), BMP activity appears in the surface-gel fraction, while under a dissociative condition (high concentrations of GuHCl) BMP appears in the fraction below the surface gel. These facts suggest that under associative conditions, BMP aggregates with other low dense proteins in the surface-gel fraction and that this may be the state of aggregation of BMP in cells and matrix in nature. Present observations support the assumption that BMP is a relatively low density protein and excludes the idea of BMP activity in the collagen molecule, per se. A specific protein, with an apparent molecular weight of 63,000 daltons, is present in all fractions that exhibit BMP activity, and absent in fractions that do not exhibit this activity. BMP is not species-specific; rat BMP induces bone formation in mice. CsCl density-gradient centrifugation is an efficient tool for further purification and isolation of BMP.     

A possible role of bone morphogenetic protein (BMP) in the regulation of bone remodeling

Bone morphogenetic protein (BMP) from an osteosarcoma powder was found to be solubilized in weak acid (pH 2.6–3.0), but not in acidic solutions of a lower or higher pH. This indicates that the solubility of BMP strictly depends on the hydrogen ion concentration of the solution. Ectopic osteogenesis induced by the osteosarcoma powder was greatly enhanced by treating the powder with acid solutions below pH 3.0. The acid-treated preparations were neutralized before bioassay. Acid treatment resulted in almost a five-fold increase in the amount of new bone formation. This finding suggests that a high concentration of hydrogen ion is important in regulating the activity of BMP. Lyophilized cultured cells of the osteosarcoma preserved the osteogenic activity even after incubation in acidic and neutral buffers at 37°C for six days. This observation suggests that BMP is stable to endogenous enzymes such as lysosomal or proteolytic enzymes. Based on these results, the role possibly played by BMP in the regulation of bone remodeling is discussed.     

Induced regeneration of calvaria by bone morphogenetic protein (BMP) in dogs

In the adult dog, a 14-mm skull trephine defect regenerates only incompletely in the lifetime of the individual. Only about half of the defect is repaired; the regenerated part develops by extension of growth from the bony rim. Correlated roentgenographic and histomorphometric methods demonstrate that new bone develops by proliferation of preexisting osteoprogenitor cells lining the diplo? and perivascular cells of the bone marrow stroma. An autogeneic bone graft, including bone marrow, provides a supplementary supply of cells in a homostructural framework and generally completes the repair process. Transplants of bone marrow alone fail to repair the defect. Implants of bovine bone morphogenetic protein (bBMP) and a carrier consisting of matrix gamma-carboxyglutamic acid rich protein (without any additional bone or bone marrow) induce repair almost as completely as an autograft. BMP-induced bone regeneration is incomplete in the thin lateral temporal marrow-deficient part of the cranium. Implants of BMP plus bone marrow induce complete repair, suggesting that calvarial bone regeneration is bone marrow stroma-dependent for a supply of target cells. The target for BMP in cranial bone regeneration is the perivascular connective tissue cells (pericyte) of the host bed marrow stroma and endosteum. The molecular mechanism of differentiation of pericytes into osteoprogenitor cells is not known, but the process is irreversible, heritable, and presents a solvable problem.     

Noggin regulation of bone morphogenetic protein (BMP) 2/7 heterodimer activity in vitro

Bone morphogenic proteins (BMPs) are growth factors important for skeletal development and bone growth. Noggin, one of the soluble BMP antagonists, regulates the action of BMPs on mesenchymal precursor cells, partially through a feedback type of inhibition. In this study, we constructed a novel BMP2/7 'fusion gene' that encodes both BMP2 and BMP7 genes in tandem by a linker. Polymerase chain reaction (PCR) and Western blotting showed that the BMP2/7 fusion gene construct led to the production of BMP2/7 heterodimers in A549 'producer' cells. When applied to C2C12 myoblastic cells, BMP2/7 heterodimers increased alkaline phosphatase (ALP) activity and osteocalcin (OCN) expression (markers of osteoblastic differentiation) more effectively than either BMP2 or BMP7 homodimers. Moreover, this heterodimer induced significantly lower levels of Noggin expression in C2C12 cells than respective homodimers at similar doses. The addition of Noggin did not affect the heterodimer's activities in increasing osteoblastic differentiation in C2C12 cells. In contrast, BMP2 and BMP7 homodimers were largely inhibited by Noggin. Our finding suggests that the 'fusion gene' construct led to the production of bioactive BMP2/7 heterodimers, which were not antagonized by Noggin as effectively as it to BMP homodimers. The weaker Noggin antagonism on BMP heterodimers compared to homodimers may contribute to increased osteogenic potency of heterodimers in vitro and in vivo.     

骨形态发生蛋白5(BMP5)基因在骨折愈合中的定位研究

目的：应用骨形态发生蛋白5（BMP5）cDNA探针检测骨折愈合过程中外骨痂内BMP5基因表达的定位与分布,探讨BMP5基因表达在闭合性骨折愈合外骨痂形成中的作用。方法：以64只健康SD大鼠,制备闭合性胫骨骨折动物模型。骨折后分别于12小时,1,3,5,7,9,14及28天取材,取材后行恒冷切片,用地高辛素标记的BMP5 cDNA探针进行原位杂交,设立对照。结果：骨折后12小时及1天,骨折周围血肿内细胞及肌肉中新出现的间充质细胞内BMP5检测为阳性信号。结论：本实验表明创伤激活BMP5 cDNA的表达,并呈区域性参与骨折的修复,也说明骨折血肿及周围软组织在骨折愈合过程中具有非常重要的地位。     

Cartilaginous transdifferentiation of rat tenosynovial cells under the influence of bone morphogenetic protein in tissue culture

In an in vitro system cartilage tissue was formed from the patellar ligament of young adult rats in the crevices of bone matrix gelatin (BMG) under the influence of bone morphogenetic protein (BMP). The rate of cartilage induction was 75% (six of eight) for the explants using patellar ligaments of rat knee joints. Almost the same rate was observed, 67% (four of six), for explants of ligament with fatty tissue. Tenosynovial cells and synovial cells from the surface of ligaments are potent responders to BMP, and this property may be applied to accelerate the biologic attachment of excised tendons and ligaments in clinical practice.     

Regeneration of an enchondroma defect under the influence of an implant of human bone morphogenetic protein

A 29-year-old woman with an enchondroma that was expanding and eroding the palmar cortex of the middle phalanx was successfully treated by curettage and implantation of bone morphogenetic protein. The metaphysis and cortex were repaired by lamellar bone within 2 months. The medulla was completely filled with trabecular bone by 9 months. The full range of motion and normal functions of finger and hand joints were restored, and there was no recurrence or abnormalities at follow-up visits 2 1/2 years after the operation.     

Direct stimulation of osteoclastic bone resorption by bone morphogenetic protein (BMP)-2 and expression of BMP receptors in mature osteoclasts

Bone morphogenetic proteins (BMPs) play an important role in various kinds of pattern formation and organogenesis during vertebrate development. In the skeleton, BMPs induce the differentiation of cells of chondrocytic and osteoblastic cell lineage and enhance their function. However, the action of BMPs on osteoclastic bone resorption, a process essential for pathophysiological bone development and regeneration, is still controversial. In this study, we examine the direct effect of BMPs on osteoclastic bone-resorbing activity in a culture of highly purified rabbit mature osteoclasts. BMP-2 caused a dose- and time-dependent increase in bone resorption pits excavated by the isolated osteoclasts. BMP-4 also stimulated osteoclastic bone resorption. The increase in osteoclastic bone resorption induced by BMP-2 was abolished by the simultaneous addition of follistatin, a BMP/activin binding protein that negates their biological activity. Just as it increased bone resorption, BMP-2 also elevated the messenger RNA expressions of cathepsin K and carbonic anhydrase II, which are key enzymes for the degradation of organic and inorganic bone matrices, respectively. Type IA and II BMP receptors (BMPRs), and their downstream signal transduction molecules, Smad1 and Smad5, were expressed in isolated osteoclasts as well as in osteoblastic cells, whereas type IB BMPR was undetectable. BMPs directly stimulate mature osteoclast function probably mediated by BMPR-IA and BMPR-II and their downstream molecules expressed in osteoclasts. The results presented here expand our understanding of the multifunctional roles of BMPs in bone development.     

人骨形态发生蛋白对体外培养胎儿关节软骨块的影响

为了解人骨形态发生蛋白（ｈＢＭＰ）对软骨细胞的影响,作者观察了ｈＢＭＰ对体外培养关节软骨块中软骨细胞表达骱钙素（ＢＧＰ）。ｈＢＭＰ的影响及对软骨基质中钙沉积的影响。结果显示：ｈＢＭＰ可诱导软骨块中的关节软骨的细胞表达ＢＧＰ、ｈＢＭＰ并可促进软骨基质中的钝沉积。作者认为,ｈＢＭＰ有诱导软骨块中软骨细胞进一步分化为成骨细胞样细胞的作用,并推测这可能是软骨内化骨过程中,成骨细胞的另一来源的。     

Matrix vesicles are carriers of bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF), and noncollagenous matrix proteins

Matrix vesicles (MVs) are well positioned in the growth plate to serve as a carrier of morphogenetic information to nearby chondrocytes and osteoblasts. Bone morphogenetic proteins (BMPs) carried in MVs could promote differentiation of these skeletal cells. Vascular endothelial growth factor (VEGF) in MVs could stimulate angiogenesis. Therefore, a study was undertaken to confirm the presence of bone morphogenetic protein (BMP)-1 through-7, VEGF, and the noncollagenous matrix proteins, bone sialoprotein (BSP), osteopontin (OPN), osteocalcin (OC), and osteonectin (ON) in isolated rat growth plate MVs. MVs were isolated from collagenase-digested rachitic rat tibial and femoral growth plates. The presence of BMP-1 through BMP-7, VEGF, BSP, ON, OPN, and OC was evaluated by Western blot, plus ELISA analyses for BMP-2 and-4 content. The alkaline phosphatase-raising ability of MV extracts on cultured rat growth plate chondrocytes was measured as a reflection of MV ability to promote chondroosseous differentiation. BMP-1 through-7, VEGF, BSP, ON, OPN, and OC were all detected by Western blot analyses. Chondrocytes treated with MV extracts showed a two-to threefold increase in alkaline phosphatase activity over control, indicating increased differentiation. Significant amounts of BMP-2 and BMP-4 were detected in MVs by ELISA. Combined, these data suggest that MVs could play an important morphogenetic role in growth plate and endochondral bone formation.     

Differentiation of cartilage from calvarial bone under the influence of bone matrix gelatin in vitro.

Fetal rat calvarium in an optimum (CMRL-1066) culture medium (with respect to amino acids. Tween 80, DNA precursors and hydrogen acceptors used in oxidative metabolism) produces new lamellar bone. The explanted osteoprogenitor cells survive, proliferate on living membrane bone surfaces and remodel membrane bone into lamellar bone. In the same culture medium, calvarial connective tissue and osteoprogenitor cells grow out of the membrane bone interstices onto a substratum consisting of bone matrix gelatin (BMG) and differentiate not into bone but into hyaline cartilage. Outgrowths from either bone or muscle onto a substratum of BMG prepared from bone autodigested in neutral buffer solutions, produce only fibrous connective tissue. In BGJ, a culture medium containing suboptimal ingredients for cell proliferation, calvarial bone produces neither lamellar bone nor new cartilage but is gradually resorbed and replaced by fibroblasts. In CMRL culture medium, outgrowths of mesenchymal-like cells from muscle onto a substratum of living membrane bone produce an epiphyseal plate-like columnar deposit of new hyaline cartilage. These observations suggest that the function of BMG is to evoke mesenchymal cell differentiation into prechondroblasts during the latent or migratory morphogenetic phase while the effect of the culture medium is to provide the bionutritional requirements for synthesis of hyaline cartilage matrix by chondrocytes during the patent phase of development.     

Microfracture and bone morphogenetic protein 7 (BMP-7) synergistically stimulate articular cartilage repair

OBJECTIVE: Microfracture is used to treat articular cartilage injuries, but leads to the formation of fibrocartilage rather than native hyaline articular cartilage. Since bone morphogenetic protein 7 (BMP-7) induces cartilage differentiation, we hypothesized that the addition of the morphogen would improve the repair tissue generated by microfracture. We determined the effects of these two treatments alone and in combination on the quality and quantity of repair tissue formed in a model of full-thickness articular cartilage injury in adolescent rabbits. DESIGN: Full-thickness defects were made in the articular cartilage of the patellar grooves of forty, 15-week-old rabbits. Eight animals were then assigned to (1) no further treatment (control), (2) microfracture, (3) BMP-7, (4) microfracture with BMP-7 in a collagen sponge (combination treatment), and (5) microfracture with a collagen sponge. Animals were sacrificed after 24 weeks at 39 weeks of age. The extent of healing was quantitated by determining the thickness and the surface area of the repair tissue. The quality of the repair tissue was determined by grading specimens using the International Cartilage Repair Society Visual Histological Assessment Scale. RESULTS: Compared to controls, BMP-7 alone increased the amount of repair tissue without affecting the quality of repair tissue. Microfracture improved both the quantity and surface smoothness of repair tissue. Compared to either single treatment, the combination of microfracture and BMP-7 increased both the quality and quantity of repair tissue. CONCLUSIONS: Microfracture and BMP-7 act synergistically to stimulate cartilage repair, leading to larger amounts of repair tissue that more closely resembles native hyaline articular cartilage.     

The reaction of the dura to bone morphogenetic protein (BMP) in repair of skull defects.

Trephine defects in the adult rat skull 0.8 cm in diameter, which do not spontaneously heal, were filled with a bovine bone morphogenetic protein (BMP) fraction. The defects healed not only by bony ingrowth from the trephine rim, but also by proliferation of pervascular mesenchymal-type cells (pericytes) of the dura mater. Under the influence of BMP, dural pericytes differentiated into chondroid and woven bone. Between three and four weeks postimplantation, sinusoids formed and the woven bone remodelled into lamellar bone. Concurrently, blood-borne bone marrow cells colonized the bone deposits, and the diploe were restored. Demonstrating that it is soluble in interstitial fluid, and diffusible across a nucleopore membrane (which isolated the bony margins of the skull), BMP induced new bone formation in the underlying dura and complete repair of the defect. The response of the dura to the BMP fraction produced more new bone than the response to allogeneic bone matrix. The BMP-induced repair was dose dependent; the quantity of new bone was proportional to the dose of the implanted BMP.