The osteoinductive activity of bone morphogenetic protein (BMP) purified by repeated extracts of bovine bone

Native bone morphogenetic proteins (BMPs) extracted from bone have been used clinically to stimulate bone regeneration and repair. However, preparation of purified BMP is a laborious process. This study investigated the yield, activity and cost effectiveness of repeatedly extracting the same bone matrix to produce purified BMP. While repeated extraction was able to increase the yield 62% the activity of the partially purified BMP in later extracts decreased both in vitro and in vivo. This decline in activity appears to be due to an increase in non-BMP contaminants, such as collagen, in the extracts. When the first three extracts were combined and processed together activity was equivalent to that of the first extract. A simple analysis based on the cost of reagents used and the time required for purification indicates that separate processing of the extracts is inefficient while combining the first and second extracts and processing them together would result in a small cost saving. Based on this study we would recommend that the demineralized bone matrix be extracted no more than twice and that the extracts be combined for further processing.     

Expression of bone morphogenetic proteins and cartilage-derived morphogenetic proteins during osteophyte formation in humans

Bone‐ and cartilage‐derived morphogenetic proteins (BMPs and CDMPs), which are TGFβ superfamily members, are growth and differentiation factors that have been recently isolated, cloned and biologically characterized. They are important regulators of key events in the processes of bone formation during embryogenesis, postnatal growth, remodelling and regeneration of the skeleton. In the present study, we used immunohistochemical methods to investigate the distribution of BMP‐2, ‐3, ‐5, ‐6, ‐7 and CDMP‐1, ‐2, ‐3 in human osteophytes (abnormal bony outgrowths) isolated from osteoarthritic hip and knee joints from patients undergoing total joint replacement surgery. All osteophytes consisted of three different areas of active bone formation: (1) endochondral bone formation within cartilage residues; (2) intramembranous bone formation within the fibrous tissue cover and (3) bone formation within bone marrow spaces. The immunohistochemistry of certain BMPs and CDMPs in each of these three different bone formation sites was determined. The results indicate that each BMP has a distinct pattern of distribution. Immunoreactivity for BMP‐2 was observed in fibrous tissue matrix as well as in osteoblasts; BMP‐3 was mainly present in osteoblasts; BMP‐6 was restricted to young osteocytes and bone matrix; BMP‐7 was observed in hypertrophic chondrocytes, osteoblasts and young osteocytes of both endochondral and intramembranous bone formation sites. CDMP‐1, ‐2 and ‐3 were strongly expressed in all cartilage cells. Surprisingly, BMP‐3 and ‐6 were found in osteoclasts at the sites of bone resorption. Since a similar distribution pattern of bone morphogenetic proteins was observed during embryonal bone development, it is suggested that osteophyte formation is regulated by the same molecular mechanism as normal bone during embryogenesis.     

The epididymis and its development in ratite birds (ostrich, emu, rhea)

Summary The epididymis of ratitae is subdivided into a main part and an appendix epididymidis. The appendix epididymidis consists of the ductus aberrans and ductuli aberrantes. The ductus aberrans is the cranial continuation of the ductus epididymidis. The appendix epididymidis is cranially attached to the adrenal gland. In the main part of the epididymis the largest part of the rete testis is found.The rete testis is composed of an intratesticular rete (also named tubuli recti), an intracapsular rete (with a longitudinal cistern and a true rete), and an extratesticular rete (predominantly consisting of approximately 20 longitudinal channels). The rete testis develops most likely embryonally from buds of the glomerular capsules of the mesonephros. The ductuli efferentes proximales also arise from these capsules, while the ductuli efferentes distales develop from the proximal and distal tubules and intermediate-segments of the mesonephros. The ductus epididymidis originates from the Wolffian duct and meanders dorsolaterally through the epididymis.Dedicated to Professor Dr. Seiferle (Professor emeritus at the Department of Veterinary Anatomy of the University of Zurich) on the occasion of his 80th birthday     

骨形成蛋白信号转导及调控*

背景：骨形成蛋白属于转化生长因子β超家族成员,具有很强的诱导成骨能力。 目的：对骨形成蛋白功能、信号转导及调控机制及其信号通路异常与人类疾病的关系进行综述。 方法：由第一作者用计算机检索中国期刊全文数据库(CNKI：2010)Medline(1999/2010)数据库,检索词分别为“骨形成蛋白、骨形成蛋白受体、Smad 蛋白、信号转导、信号调控”和“Bone morphogenetic proteins,Bone morphogenetic proteins receptors, Smads,Signal transduction,Signaling regulation”。从骨形成蛋白的结构与功能,骨形成蛋白信号传导通路及调控,骨形成蛋白信号通路异常与人类疾病3方面进行总结。共检索到110篇文章,按纳入和排除标准对文献进行筛选,共纳入47篇文章。 结果与结论：骨形成蛋白主要通过 Smad 依赖性和Smad 非依赖性2条信号转导途径发挥作用,此过程受到细胞内外许多蛋白的调控,此外骨形成蛋白信号通路异常与人类某些疾病密切相关。     

骨形态发生蛋白研究进展

骨形态发生蛋白BMPs具有异位和原位的骨诱导活性。BMPs研究经历了自然BMPs研究阶段、重组BMPs阶段和骨组织工程阶段。随着基因工程学、骨组织工程学以及基因治疗的发展 ,BMPs在骨折及骨缺损的治疗中将发挥重要作用     

骨形态发生蛋白2基因转染犬牙髓细胞

背景：研究发现骨形态发生蛋白2具有诱导间充质细胞向成骨细胞表型分化以及诱导新骨及修复性牙本质形成的能力。 目的：通过对细胞超微结构的分析,探讨骨形态发生蛋白2基因转染的犬牙髓细胞,向成牙本质细胞转化的过程。 方法：将培养的性状稳定的第4代犬牙髓细胞分为3组：基因转染组转染pEGFP-N1-BMP-2基因,空白载体转染组转染EGFP-N1空白荧光载体,未转染组不转染。 结果与结论：成功构建pEGFP-N1-BMP-2真核表达质粒,并成功将其转染犬牙髓细胞, pEGFP-N1-BMP-2质粒转染促进犬牙髓细胞分泌骨形态发生蛋白2。pEGFP-N1-BMP-2质粒转染有促进牙髓细胞碱性磷酸酶活性的作用。透射电镜观察结果显示：转染后的犬牙髓细胞具有成牙本质细胞的形态特点。说明pEGFP-N1-BMP-2 真核表达质粒转染后的牙髓细胞,具有成牙本质细胞的特征。     

Activation of bovine neutrophils by partially purified Pasteurella haemolytica leukotoxin.

In this study we developed a new method for the partial purification of Pasteurella haemolytica leukotoxin by size-exclusion high-performance liquid chromatography. The partially purified leukotoxin had a molecular weight of 104,000, as estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reacted on an immunoblot with an antileukotoxin monoclonal antibody. As expected, high concentrations of the leukotoxin were inhibitory or lethal to bovine neutrophils. Incubation of bovine neutrophils with diluted leukotoxin, however, resulted in significant neutrophil activation that was comparable in magnitude to that obtained with standard activating agents such as opsonized zymosan or zymosan-activated serum. Dilute leukotoxin (1:128 to 1:8,192 dilutions) stimulated an oxidative burst (luminol-dependent chemiluminescence) by bovine neutrophils that was comparable in magnitude to that obtained with opsonized zymosan. Preincubation with leukotoxin did not significantly prime the neutrophils for an enhanced oxidative burst when they were then exposed to opsonized zymosan as a second stimulus. Dilute leukotoxin (1:100 to 1:1,000 dilutions) also stimulated cytoskeletal alterations in bovine neutrophils, as measured by a significant shape change response. Preferential release of secondary granule constituents (lactoferrin) occurred when neutrophils were incubated with 1:100 to 1:500 dilutions of leukotoxin. Significant release of primary granules, as measured by beta-glucosaminidase activity, was not observed except at low dilutions (1:20) of leukotoxin that resulted in significant release of cytosolic constituents (i.e., lactate dehydrogenase activity). The neutrophil-activating activity of the leukotoxin was heat labile, unaffected by polymyxin B, and abrogated by a leukotoxin-neutralizing monoclonal antibody. These data indicate that P. haemolytica leukotoxin, like the closely related Escherichia coli hemolysin, is a potent neutrophil-activating agent. Leukotoxin-stimulated release of neutrophil oxygen intermediates and granule constituents may contribute to the intense inflammation that characterizes bovine pulmonary pasteurellosis.     

Candidacidal activities of proteins partially purified from rat epidermis.

Proteins with approximate molecular weights of greater than 300,000 (EP greater than 300K) and 49,000 (EP 49K) were partially purified from terminally differentiated cells of 2-day-old rat epidermis. They were extracted in 0.34 M sucrose containing 0.01 M citric acid and purified by Sephacryl S-300 chromatography followed by reverse-phase column chromatography. The major constituents of EP greater than 300K and EP 49K were focused around pH 10 to 11 by sucrose gradient isoelectric focusing. Both proteins were effective at inhibiting colony formation of Candida albicans and C. tropicalis, but neither inhibited the growth of C. parapsilosis. The effect was maximum below pH 5.0 and reduced considerably above pH 5.0. The activity of EP greater than 300K on C. albicans TIMM 1623 (group A) was much stronger than that of EP 49K, whereas both proteins similarly inhibited C. albicans TIMM 1604 (group B). Their effects against C. albicans TIMM 1623 were dose dependent and were activated after a longer preincubation time, and NaCl concentration influenced their potency. At a low salt concentration and a 60-min preincubation at pH 4.5, the 50% effective dose for EP greater than 300K was calculated to be 1.7 x 10(-9) M, whereas that for EP 49K was 1.8 x 10(-7) M.     

骨形态发生蛋白2，7治疗骨不连的效果评价

文题释义：骨形态发生蛋白：具有成骨活性,可通过不同的信号通路促进骨髓间充质干细胞的成骨分化。 骨不连：骨损伤和骨折后超过9个月,连续超过3个月观察未见进一步愈合的倾向。 背景：自体骨移植辅以坚强固定被认为是治疗骨不连的金标准,目前临床上使用骨形态发生蛋白2,7对骨不连进行治疗的案例较多。 目的：从基因水平描述骨形态发生蛋白的成骨通路,对临床上骨形态发生蛋白治疗骨不连的案例进行分析,同时对比骨形态发生蛋白2,7对骨不连的治疗效果,并对二者效果进行评价及分析。方法：由第一作者用计算机检索中国期刊全文数据库、万方数据库和PubMed数据库,中文检索词为“骨形态发生蛋白、骨不连、信号通路、外固定架、内固定、植骨、感染性骨不连、骨缺损、成骨细胞、骨分化”,英文检索词为“BMP,nonunion,pathway,External fixator,ORIF,bone graft,infected nonunion,bone defect,osteoblast,osteoporosis”,最终纳入文献59篇。 结果与结论：①通过对文献的分析,认为骨形态发生蛋白的基因水平通路可以为其临床应用提供治疗思路;②在对于骨不连的治疗上,骨形态发生蛋白2,7是有效的,但目前缺乏使用骨形态发生蛋白治疗骨不连的规范及标准,例如使用量及适应证;③从总体治疗效果及对感染性骨不连的治疗效果两方面,对比骨形态发生蛋白2,7,认为骨形态发生蛋白2的效果优于骨形态发生蛋白7,尤其是在感染性骨不连的治疗中。 ORCID: 0000-0002-6610-7252(从凯) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Neutral lipids facilitate transfer of bone morphogenetic proteins and other noncollagenous proteins

In the process of separation of bone morphogenetic proteins from bone matrix, lipids were found in unexpected amounts closely associated with noncollagenous proteins soluble in guanidine hydrochloride. Lipids representing 33.7–49.9% by weight were recovered with various solvents. Composites of noncollagenous proteins and lipids soluble in either chloroform- methanol or acetone implanted in the hindquarter muscles of mice induced the formation of large deposits of heterotopic bone. The protein-lipid aggregates formed microspherules which were stained by Sudan Black B. Implants of bone morphogenetic proteins and noncollagenous proteins-lipid microspherules stained with Sudan Black B induced bone development in the same manner as unstained delipidized bone morphogenetic proteins associated with noncollagenous proteins. Lipid-free osteocalcein, osteonectin, albumin and other bone matrix proteins did not induce bone formation or bind Sudan Black B. The more highly purified the noncollagenous proteins, with or without activity of bone morphogenetic proteins, the lower the level of binding with Sudan Black B. Acetone-soluble bone matrix lipids consisted chiefly of triglycerides, cholesterol and saturated short chain fatty acids, and included little or no phospholipids or monounsaturated fatty acids. Composites of recombinant bone morphogenetic proteins-2 and acetone-soluble lipids induced larger deposits of bone than implants of recombinant bone morphogenetic proteins-2 without acetone-soluble lipids. The hypothesis that an association of bone lipids with protein facilitates the local transport of bone morphogenetic proteins warrants further investigation.     

骨形态形成蛋白在下颌骨快速牵张成骨中的应用*

背景：牵张成骨已经成为治疗不同类型颅面畸形和骨缺损的有效的方法,但是牵张成骨的主要缺点是牵张期和稳定期比较长,可能导致牵张过程中严重的并发症。 目的：总结骨形态形成蛋白在快速牵张成骨过程中作用的研究现状。 方法：电子检索计算机Pubmed数据库(1989/2011)收录的骨形态形成蛋白和牵张成骨相关综述和论文报告。 结果与结论：共纳入骨形态形成蛋白在快速牵张成骨中的作用相关文献32篇。骨形态形成蛋白具有很强的成骨活性,能促进骨再生和骨改建。目前的研究显示应用骨形态形成蛋白能加快牵张成骨过程中新骨形成和缩短治疗的疗程。但是骨形态形成蛋白应用于临床还需要进一步的研究。     

Alkali-urea extraction of demineralized bone matrix removes noggin, an inhibitor of bone morphogenetic proteins

Demineralized bone matrix (DBM) and native bone morphogenetic protein (nBMP) are complex mixtures of non-collagenous bone proteins. These mixtures contain many of the BMPs that are available as recombinant molecules. Information regarding the presence in these materials of molecules that may affect the availability and activity of the BMPs is very limited. We have devised a simple chemical extraction of DBM using alkali-urea that produces a water soluble extractate that inhibits the osteogenic activity of DBM. We have demonstrated the presence of noggin, an extracellular BMP ligand antagonist, in this material. We conclude that differential chemical extraction may be a useful means of removing inhibitory molecules from DBM and nBMP.     

骨形态发生蛋白4和骨形态发生蛋白4/7基因转染对骨髓基质干细胞成骨活性差异的比较

背景：有文献报道骨形态发生蛋白(bone morphogenetic proteins,BMPs)异源二聚体比同源二聚体的活性高,目前国内外尚无BMP-4/7异源二聚体与BMP-4同源二聚体间诱导成骨活性比较的报道。 目的：观察不同基因BMP-4、BMP-4/7对骨髓基质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨活性的差异,探讨融合基因BMP-4/7的生物学活性。 方法：经脂质体转染分离培养的兔BMSCs,G418筛选出稳定表达株,利用RT-PCR法证明目的基因在BMSCs中的表达情况;以BMP-4和BMP4/7融合基因修饰的AAV载体,转染兔BMSCs,MTT法检测不同基因转染细胞的增殖能力;以BMP-4单基因和BMP-4/7融合基因修饰的AAV载体转染兔BMSCs 7 d后,测定两组细胞内碱性磷酸酶及骨钙素水平,观察成骨活性的变化。 结果与结论：实验成功构建高滴度的分别携带BMP-4单基因、BMP-4/7融合基因的重组腺相关病毒载体AAV-BMP-4、AAV-BMP-4/7;RT-PCR结果证明目的基因能够在BMSCs中得到稳定表达。AAV-BMP-4及AAV-BMP-4/7载体转染效率分别为68.20%、72.18%;转染BMSCs后,细胞增殖能力均明显提高,BMP-4/7融合基因的细胞增殖能力活性高于BMP-4单基因。以BMP-4和BMP-4/7融合基因修饰的AAV转染细胞7 d后,细胞内碱性磷酸酶活性明显上调,骨钙素水平升高,成骨活性均增强;两种基因比较,BMP-4/7融合基因成骨活性强于BMP-4单基因(P < 0.01)。提示BMP-4、BMP-4/7修饰的AAV载体转染效率高,对BMSCs均有成骨活性,其中BMP-4/7融合基因成骨活性强于BMP-4单基因。     

Expression of bone morphogenetic proteins in colon carcinoma with heterotopic ossification

Here we report the case of a 50-year-old woman with adenocarcinoma of the colon, showing heterotopic ossification. The patient was referred to our hospital for investigation of anemia secondary to occult gastrointestinal blood loss. By colonoscopy, an irregular polypoid mass was found in the ascending colon. A biopsy of the lesion revealed moderately to poorly differentiated adenocarcinoma with heterotopic ossification. A right hemicolectomy was done and revealed areas of heterotopic bone within the tumor, but no ossification was evident in the metastatic lesions within the mesenteric lymph nodes. The formation of heterotopic bone in gastrointestinal tumors is rare and its exact mechanism is unknown. Immunohistochemical localization of bone morphogenetic proteins (BMP), known to be primary inducers of new bone formation, was determined. BMP-5 and -6 were prominent in the cytoplasm of tumor cells, and they stained weakly in osteoblast-like cells adjacent to newly formed bone. Cytoplasmic staining for BMP-2 and -4 was weak in tumor cells, osteoblast-like cells, and stromal fibroblast cells. BMP may play an important role in heterotopic ossification in colon adenocarcinoma.     

The in vitro bioactivity of two novel hydrophilic, partially degradable bone cements

Composite bone cements were prepared with bioactive glasses (MgO-SiO(2)-3CaO.P(2)O(5)) of different reactivities. The matrix of these so-called hydrophilic, partially degradable and bioactive cements was composed of a starch/cellulose acetate blend and poly(2-hydroxyethyl methacrylate). The addition of 30 wt.% of glasses to this system made them bioactive in acellular medium: a dense apatite layer formed on the surface after 7 days of immersion in simulated body fluid. This was demonstrated both by microscopic and infrared spectroscopic techniques. The composition of the glass and, consequently, its structure was found to have important effects on the rate of the apatite formation. The combination of reactivity obtained by one formulation with the hydrophilic and degradable character of these cements makes them a very promising alternative to conventional acrylic bone cements, by allowing a better stabilization of the implant and a stronger adhesion to the bone.     

骨形成蛋白信号分子在小鼠椎间盘发育中的表达

目的:探索促进成骨相关的骨形成蛋白(BMP)-2、-4、-6、-7、-9以及抑制成骨作用的BMP-3在小鼠不同年龄段椎间盘中表达情况,为椎间盘发育过程中BMP信号途径的研究奠定重要基础.方法:解剖获取出生后1d、3d、1周、1个月、3个月、6个月龄小鼠的椎间盘及胸腰椎脊柱.RT-PCR检测BMP-2、-3、-4、-6、-7、-9在生后不同年龄段小鼠椎间盘的表达情况,免疫印迹及免疫组织化学验证在RT-PCR中有明显表达差异的BMP-3、-6.结果:RT-PCR结果显示BMP-2、-7、-9的表达在小鼠椎间盘组织中有一定的基础表达,但随着年龄增大没有明显的变化趋势,而BMP-3和BMP-6的表达整体呈现出下降的趋势,BMP-4在椎间盘中无表达,免疫印迹及免疫组织化学结果也证实BMP-3和BMP-6随着小鼠椎间盘的发育表达降低.结论:正常小鼠脊柱,随着年龄的增加,椎间盘中BMP-2、-7、-9的表达无明显变化趋势,各年龄段都维持着一定量的表达,BMP-3和BMP-6的表达整体呈现出下降的趋势,具有明显成骨作用的BMP-4则没有表达.