Telopeptide-depleted bovine skin collagen as a carrier for bone morphogenetic protein

The telopeptide of type I collagen is thought to be responsible for causing immunogenic response when introduced into xenogenic hosts. To eliminate this problem, solubilized bovine skin collagen was filtered through a millipore membrane, digested repeatedly with pepsin to remove telopeptides, and used as a carrier for a water-soluble, partially-purified fraction of bone morphogenetic protein (BMP) in mice. Composite implants of this telopeptide-depleted collagen and the partially-purified BMP fraction consistently elicited ectopic bone formation in mice 3 weeks postimplantation. When implanted alone, collagen or BMP failed to show this response. Collagens, prepared by use of conventional methods (acid-solubilized collagen, or collagen-digested once with pepsin) were also assessed as carriers for BMP, but were found to be inferior in terms of consistency of bone formation and amount of induced bone mass. The results suggest that telopeptide-depleted collagen permitted a gradual release of purified BMP for induction of bone, with minimal immunogenic interference. Consequently, this collagen carrier represents an important development for future clinical application of BMP.     

Osteoinductivity assay of the variability of repeated extractions of bone morphogenetic proteins from bovine bone at different times

BOrthobiologicsLaboratory ,DepartmentofOralMaxillofacialSurgery,FacultyofDentistry ,UniversityofToronto ,Canada (HuZH ,SeanA .F .PeelandCameronM .L .Clokie)DepartmentofOrthopaedicSurgery,TheAffiliatedSecondHospital,KunmingMedicalCollege ,1Mayuan ,Kunming ,Yunnan6 5 0 10 1,China (HuZH) Correspondingauthor:Tel:86 871 5 35 12 81 2 5 95 ,E mail:zminghu @hotmail.comonemorphogeneticproteins (BMPs)aremembersoftransforminggrowthfactor (TGF )superfamilyencodingpolypeptidesthatshareco…     

同种异体微小颗粒骨复合BMP胶原修复骨缺损的实验研究

目的探讨以同种异体微小颗粒骨复合BMP胶原修复节段性骨缺损的效果.方法取34只新西兰白兔,于两侧桡骨干制成长1.5 cm骨膜骨缺损,左侧(A组)植入由200mg同种异体微小颗粒骨、10 mg BMP与0.2 ml胶原的复合物;右侧(B组)植入复合0.2 ml胶原的200mg同种异体微小颗粒骨.分别于术后2、4、8、12周进行影像学,组织学检查;术后8、12周行骨密度检测;术后12周行生物力学检查.另取8只仅加入0.2 ml胶原作为空白对照组(即C组).结果放射学以及组织学检查结果显示A,B两组骨缺损区均得到了比较彻底的修复,但A组在成骨速度、骨再生量、再生髓腔结构等方面均优于B组.8、12周骨密度测试结果显示A组的骨密度值高于B组.12周的生物力学实验结果显示三点弯曲实验结果表明A组极限强度值明显高于B组,而且A组的弯曲刚度也好于B组;轴向压缩实验结果说明A组的抗压刚度优于B组;扭转实验结果指出A组的抗扭转刚度和最大扭矩分别高于B组,差异均有显著性意义(P<0.05).骨密度测定和生物力学实验结果证实了两组成骨情况的差异,这与新生骨的成熟程度不同有关.复合了BMP的A组因髓腔再通时间早,骨改建塑形更完善,新生板层骨的成熟度亦较高,耐受应力情况也更好.C组则不能产生骨性愈合.结论同种异体微小颗粒骨修复节段性骨缺损的成骨作用令人满意,复合BMP后效果更佳.     

骨形成蛋白7在糖尿病大鼠肾组织中的表达及意义

目的 观察糖尿病大鼠肾组织中骨形成蛋白7(BMP-7)的表达及探讨其在糖尿病肾病中的可能作用。方法 将大鼠分为糖尿病组及正常对照组，分别在第30、60、90、120、150及180 d测定各组的血糖、Scr、24 h尿白蛋白、尿肌酐(Ucr)水平。以HE、PASM染色及Col Ⅳ蛋白表达衡量肾脏病理改变。用免疫组化及RT-PCR方法检测肾组织中BMP-7、TGF-β1的蛋白及mRNA表达水平。 结果 糖尿病组各时间点血糖、尿白蛋白量(24 h)均明显高于对照组(P < 0.01)。糖尿病组伴随病理改变的加重和Col Ⅳ表达的逐渐增强，BMP-7蛋白及mRNA水平(A%，30 d 95.87±1.19；180 d 26.43±1.26)逐渐降低，与对照组(A%，30 d 98.64±0.80；180 d 98.80±0.49)相比，差异有统计学意义(P < 0.01或P < 0.05)；相反，肾组织中TGF-β1的mRNA(A%，30 d 40.26±0.98； 180 d 102.12±6.45)及蛋白水平较对照组显著增加(P < 0.01)。BMP-7与TGF-β1的蛋白及mRNA表达水平均呈显著负相关(P < 0.01)。结论 BMP-7可能是维持肾脏功能稳定的一个重要因子。BMP-7在糖尿病肾病中具有与TGF-β1相反的作用，其水平下降可能参与了糖尿病肾病早期小管间质的损伤。     

骨髓间充质干细胞在丝素蛋白/骨形态发生蛋白中的生物学行为观察

目的 观察丝素蛋白与牛骨形态发生蛋白的载体系统与骨髓间充质干细胞的体外相容性.方法 将多孔丝素蛋白修剪成20×10×5 mm长方体状物,置入6孔板中;将粉状bBMP用PBS液溶解制成2mg/ml混悬液,0.5ml无菌移液管滴定于多孔丝素蛋白上(0.5毫升/个).骨髓间充质干细胞以107/ml接种到载体系统上,复合4小时后加入生长液继续体外培养,每1、4、8天时进行碱性磷酸酶、矿化结节染色观察以及扫描电镜和激光共聚焦显微镜观察;另外同时设立单纯丝素蛋白与细胞复合培养对照组进行观察.结果 倒置显微镜、扫描电镜和共聚焦显微镜下观察到细胞在两种材料上生长形态、活性正常,体外培养24小时时细胞已贴附材料上,呈匍匐状伸展,以后逐渐融合生长,8天时大量细胞成片状攀附在材料表面,并分泌有大量的网状细胞外基质,实验组可见结节状基质生成;碱性磷酸酶、矿化结节染色观察发现实验组细胞碱性磷酸酶和矿化结节染色阳性反应,对照组阴性.结论 丝素蛋白是一种良好细胞外基质材料,也是一种良好生长因子的缓释载体.     

柠檬酸钙载体复合骨形态发生蛋白-2成骨的实验研究

目的 研究柠檬酸钙与骨形态发生蛋白-2(BMP-2)复合后成骨的作用.方法 取2月龄雄性昆明小鼠15只,通过体外实验获取柠檬酸钙与BMP-2最佳比例,在体外将10 mg柠檬酸钙与2mg BMP-2复合后,植入小鼠股部肌肉上中,行肌袋实验.以柠檬酸钙与BMP-2复合物为实验组,单独BMP-2为对照组,术后2、4、6周取材进行大体标本观察与组织学观察,计算新骨形成面积比及新生骨密度比.结果 大体标本观察与组织学观察示实验组成骨明显高于对照组,成骨面积较对照组明显增多.2、4、6周时比较差异均有统计学意义(P＜0.05);2、4、6周时骨密度测量实验组较对照组高,差异均有统计学意义(P＜0.05).实验组骨质2周时出现骨小梁结构,成熟度高,成骨细胞数量多,分化明显,成骨密度高;4周时骨的成熟度增高,更多粉红色骨质形成,骨质基本成熟,骨小梁增粗增大,出现大面积成骨区域;6周时血管化及骨髓腔形成充分,骨质密度高.结论 柠檬酸钙与BMP-2复合后具有良好的诱导成骨作用,可促进骨形成,是一种较理想的载体材料,可作为一种新型复合人工骨修复骨缺损.     

Morphometric and histological analysis of low-power laser influence on bone morphogenetic protein in bone defects repair

Bone morphogenetic proteins (BMPs) are secreted signaling molecules belonging to the transforming growth factor-β (TGF-β) superfamily. The objective of this study was to determine how gallium–aluminum–arsenium (GaAlAs) 650 nm laser influenced the action of BMPs on bone defects created in rat femurs. The sample consisted of 24 male albino Wistar rats. Group 1 was composed of rats with bone defects filled with bone-inducing substance, with the application of low-power laser. Group 2 contained rats with bone defects filled with a bone-inducing substance, without the application of low-power laser. Group 3 rats had bone defects not filled with a bone-inducing substance, with the application of low-power laser. Group 4 rats had bone defects and no treatment (control group). A bone defect was produced with drills. In groups 1 and 2 the defects were filled with a bone-inducing substance. The animals were treated with GaAlAs (50 mW) laser, energy density 4J/cm², for 80 ss on a 1 cm² area. Groups 2 and 4 were used as control. Bone samples were removed for histological procedures and morphometric analysis on the 7th, 14th and 21st days after surgery. Results obtained were subjected to statistical analysis. Rejection level for the null hypothesis was 0.05. Statistical differences were found in the comparison between group 1 (G1), G2, G3 and G4 [analysis of variance (ANOVA); P < 0.0134]. There was a statistically significant correlation between groups 1 and 4 (P < 0.01). The results of other correlations by Tukey’s post-hoc test were: group 1 vs group 3 (P = 0.341), group 1 vs group 2 (P = 0.862), group 2 vs group 4 (P = 0.061), group 2 vs group 3 (P = 0.744), and group 3 vs group 4 (P = 0.249). We concluded that the association of low-power laser with a bone-inducing substance produced better results than when low-power laser or BMPs were used alone.     

双膦酸盐对骨形成蛋白诱导骨骨吸收抑制作用的研究

目的研究双膦酸盐(bisphosphonate YM 175)对骨形成蛋白(bone morphogenetic protein．简称BMP)诱导骨骨吸收的抑制作用。方法 42只大白鼠背部植入BMP,诱导出异位骨后,将大白鼠分成2组,即投药组和对照组。投药组在BMP植入后第3w至第7w,双膦酸盐(YM 175)每周投药3次,剂量1(μg／kg．d)。对照组按同样的方式给予等量的生理盐水。在BMP植入第3w、 4w、7w和10w,将BMP诱导骨取出,采用TRAP和cathepsin K染色方法来观察双膦酸盐对破骨细胞的作用。结果 BMP诱导骨3w时(即未投双膦酸盐药前),在BMP植入体周边形成编织骨 (woven bone),大量破骨细胞出现在新生骨组织表面。在4 w时,双膦酸盐投药组和对照组,新生骨组织均向BMP植入体内生长,但双膦酸盐投药组的破骨细胞较对照组有减少。在10 w时,双膦酸盐投药组和对照组均可观察到骨细胞变小并且有规律地排列的板层状骨(lamellar bone)的特征。而双膦酸盐投药组,破骨细胞死亡,与对照组比较,破骨细胞数目明显减少。结论双膦酸盐对破骨细胞性骨吸收有明显的抑制作用。     

聚乳酸作为骨形态发生蛋白载体修复骨缺损的实验研究

目的 探讨聚乳酸（ｐｏｌｙｌｄａｃ ａｃｉｄ,ＰＬＡ）作为骨形态发生蛋白（ｂｏｎ ｅ ｍｏｒｐｈｏｇｅｎｅｔｉｃ ｐｒｏ－ｔｅｉｎ,ＢＭＰ）载体的可行性及观察其诱导成骨能力。方法 手术造成日本大耳白兔左尺骨中上段１２ｍｍ骨缺损实验模型。随机分为实验。对照及空白组,实验组植入以ＰＬＡ为载体的ＢＭＰ１０ｍｇ、对照组植入以牛松质骨基质为载体的ＢＭＰ１０ｍｇ、空白组不做任何处理,术后摄Ｘ线片观察各组不同时相骨缺损修复情况,并于术后第４、８、１２周观察各组缺损内组织学变化。图像分析骨小梁的生成量。结果 实验修复情况优于对照组,无论是骨连接发生时间还是骨成熟时间,实验组均较对照组提前２周左右,同期骨生成量也明显多于对照组,而空白组缺损内主要形成纤维组织。结论 ＰＬＡ可以作为ＢＭＰ的载体修复骨缺损,它比异种松质骨基质载体的成骨效     

Experimental study of carriers of bone morphogenetic protein used for spinal fusion

Many materials have been used experimentally as carriers of osteoinductive growth factors. However, there is some doubt about whether the biomechanical strength of the materials affects spinal fusion from early stages of recovery. The aim of this study was to clarify which carrier was biomechanically more effective for bone morphogenetic proteins in spinal fusion. Three biomaterials, each having a different structure and biomechanical strength, were selected as carriers of recombinant human bone morphogenetic protein-2: (1) -tricalcium phosphate cement, which has sufficient biomechanical strength; (2) sintered bovine bone (True Bone Ceramics) coated by type I collagen, which is similar to artificial hydroxyapatite; and (3) type I collagen sheet. Bilateral lumbar intertransverse process arthrodeses were designed in a rabbit model. Spinal fusions were evaluated by radiographic analysis, manual palpation, biomechanics (uniaxial tensile test), and histologic analysis (hematoxylin and eosin, and Villanueva-Goldners trichrome stains) 3 and 6 weeks after surgery; they were then compared for the three carriers. For achieving the earliest solid spinal fusion, -tricalcium phosphate cement (which has good inherent strength) and True Bone Ceramics (which has good porosity to allow bone penetration) did better than plain collagen (the commonly used carrier).     

异种脱蛋白BMP复合骨修复骨缺损实验研究

异种骨移植常由于强烈的免疫排斥反应失败。本文报告将小牛骨经脱蛋白处理（即脱去主要的抗原物质），后再复合进去骨形成蛋白（ｂｏｖｉｎｅＢｏｎｅＭｏｒｐｈｏｇｅｎｅｔｉｃｐｒｏｔｅｉｎ，简称ｂ－ＢＭＰ）使其成为既无抗原性，又利于骨形成的异种脱蛋白ＢＭＰ复合骨。将其植入新西兰白兔尺骨缺损（２ｃｍ），观察愈合结果。免疫学、放射学和组织学检查表明，植入异种脱蛋白ＢＭＰ复合骨，各组实验动物术后无任何免疫反应。４周均显示植入骨与骨床界线模糊，８周植入骨内可见大量成片新生骨细胞及新生血管长入。实验结果表明经处理后的大块异种骨移植，不但无任何免疫排斥反应，且可达到预期修复骨缺损的目的。     

The effect of different mineral frames on ectopic bone formation in mouse hind leg muscles induced by native reindeer bone morphogenetic protein

Introduction Bone morphogenetic proteins (BMPs) require carrier material for slow release and framing material for osteoconduction.Materials and methods The effect of a frame on early bone formation induced by partially purified native reindeer BMP in composite implants containing 3 mg of BMP, type IV collagen and tricalcium phosphate (TCP/Col/BMP) or hydroxyapatite (HA/Col/BMP) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA/Col/BMP) or biocoral (NC/Col/BMP) was evaluated using a mouse hind leg muscle pouch model. Collagen with native reindeer BMP (Col/BMP) and corresponding implants without native reindeer BMP served as controls. Evaluation was done by incorporation of ⁴⁵Ca, radiographically and histologically 3 weeks after the implantation.Results None of the implants without native reindeer BMP were able to induce new bone visible on radiographs. The area of new bone formation in the Col/BMP (p=0.026) and TCP/HA/Col/BMP (p=0.012) groups was significantly greater than in the TCP/Col/BMP group. The optical density of the new bone area was significantly greater in the TCP/HA/Col/BMP group than in the TCP/Col/BMP (p=0.036) or Col/BMP (p=0.02) groups. ⁴⁵Ca incorporation was many times greater in all the groups containing native reindeer BMP than in the corresponding groups without BMP. In the Col/BMP (p=0.046) and TCP/HA/Col/BMP (p=0.046) groups, ⁴⁵Ca incorporation was significantly greater than in the TCP/Col/BMP group. No significant differences were found in any parameters between HA/Col/BMP and NC/Col/BMP groups and the other BMP-containing groups.Conclusions Hydroxyapatite, biocoral and biphasic tricalciumphosphate-hydroxyapatite are equally good as framing material for native reindeer BMP, while tricalciumphosphate is somewhat worse. Osteoinduction of native reindeer BMP works well with collagen alone.     

应用骨形态发生蛋白(BMP)修复关节软骨缺损的实验研究

目的探讨关节软骨全层缺损应用骨形态发生蛋白修复的效果。方法于2004年5月至2005年12月,30只新西兰种成年兔随机分为A,B,C三组,每只兔子左膝股骨髁间凹做一大小为4mm×5mm×2.5mm的全层关节软骨缺损。A,B组缺损内分别填充骨形态发生蛋白/纤维蛋白胶(BMP/FG)及FG,C组为空白。术后28周对缺损修复情况行大体形态、组织学和电镜观察。结果BMP/FG组,缺损组织以透明软骨修复,接近正常组织,而FG组和空白组则以纤维组织修复为主。结论BMP/FG能较好的完成关节骨软骨全层缺损的修复,并随着时间的延长修复的软骨越接近正常软骨,但修复软骨缺损的组织与邻近正常软骨组织连接性仍不是十分理想。     

骨形态发生蛋白对骨骼肌卫星细胞粘附特性的影响及其机制

目的：探讨骨形态发生蛋白（BMP）对骨骼肌卫星细胞粘附特性的影响。方法：体外获取与培养骨骼肌卫星细胞，分别用0、50、100、500、1000μg/L的BMP诱导培养基诱导培养48h。通过荧光法测定接种后1h的粘附细胞率，利用放射免疫法测定细胞层粘连蛋白的含量。结果：BMP可促进骨骼肌卫星细胞的粘附，在500μg/L的浓度时粘附率最高，当BMP浓度进一步增加时，细胞粘附率不再增加；BMP诱导后可促进骨骼肌卫星细胞层粘连蛋白的表达，当BMP浓度为500μg/L时这种作用最强。结论：BMP可增强骨骼肌卫星细胞的粘附特性，其机制与层粘连蛋白的表达增高有关。     

神经切除对骨形态发生蛋白异位诱导成骨的影响

[目的]通过rhBMP-2异位诱导成骨模型，观察坐骨神经和股神经切除对骨再生的影响，探讨神经支配在骨再生中的作用以及失神经所致骨折骨痂增大的部分机制。[方法]1CR小鼠36只随机分为实验组和对照组。行右侧股后部肌袋模型，实验组行右侧坐骨神经和股神经切除后植入含0．125mgrhBMP-2胶原复合物。对照组仅进行神经暴露后植入等量rhBMP-2胶原复合物。于术后7、14、21d取材，行湿重测量、放射学、生化检测、组织学观察和形态计量分析以及破骨细胞TRAP染色。[结果]湿重检测显示实验组组织块湿重明显大于对照组。X线检测实验组成骨范围较对照组明显增大，成骨组织密度不及对照组。生化检测结果显示术后第7d实验组AKP含量明显高于对照组，术后第14d，实验组钙含量高于对照组，术后21d，实验组钙、磷含量均低于对照组。组织学观察显示实验组成骨范围大于对照组，成骨后期破骨细胞活跃，骨小梁稀疏。组织形态计量分析显示实验组术后21d破骨细胞相对数增多，骨小梁体积密度、平均宽度均低于对照组。TRAP染色显示实验组破骨细胞明显较对照组活跃。[结论]在外源性BMP-2异位诱导过程中，神经切除引起骨诱导早期成骨活动的增加，在成骨中后期失神经导致破骨细胞活动增强引起骨小梁的稀疏和骨密度的降低，提示神经支配可能通过直接或间接的方式影响骨再生活动。     

重组人骨形态蛋白复合肌瓣诱导自体异体气管软骨再生

目的在肌瓣包裹气管移植段促进血液循环重建的基础上复合重组人骨形态蛋白(rhBMP)-2,观察rhBMP-2对自体及异体移植段气管软骨的诱导作用。方法16条犬随机等分为4组,取颈部5环气管为移植段。A1组:自体复合BMP/I型胶原组;A2组:自体移植组;B1组:异体复合BMP/I型胶原组;B2组:异体移植组。术后4周取材,观察比较大体及组织学改变。结果A1组软骨再生最明显(P<0.05);A2、B1组均有不同程度的软骨再生;B2组软骨再生最少。结论在肌瓣包裹移植段气管的基础上,BMP可明显诱导移植段软骨的再生,且自体移植软骨再生明显。     

Recombinant bone morphogenetic protein-2 enhances bone healing,guided by osteopromotive e-PTFE membranes: An experimental study in rats

It has been shown earlier that it is possible to improve bone healing, to regenerate previously existing bone, and to create new bone by means of an osteopromotive membrane technique. The present study addresses the question of whether it is possible to combine this technique with a locally applied factor, stimulatory to osteogenesis. Circular transosseous critical size defects in mandibles of rats were either implanted with recombinant human bone morphogenetic protein type 2 (rhBMP-2) or were left empty; half the number of implanted and half the number of empty defects were covered with an expanded polytetrafluoroethylene (e-PTFE) membrane (GORE-TEX^®). Results were evaluated after 12 and 24 days of healing by a histomorphological scoring system. Implantation of rhBMP-2 alone resulted in bony bridging of the defect after only 12 days, but also in voluminous amounts of new bone outside the original defect area. When rhBMP-2 was combined with membrane, newly formed woven bone bridged the defect and the bone contour was maintained by the membrane. The combined treatment with membrane and rhBMP-2 demonstrated a significantly better bone healing than with e-PTFE membrane alone at both 12 days and 24 days of healing. It was concluded that rhBMP-2 has a strong osteoinductive potential and, in contrast to what was found earlier with other types of BMP preparations, this potential was retained when combining the rhBMP-2 with the osteopromotive membrane technique, yielding better bone healing than with the membrane alone, and at the same time maintaining the bone contour. This combination may have important therapeutic applications for osseous healing and in reconstructive surgery. The study also shows the importance of an appropriate carrier material when applying stimulatory substances to enhance bone formation in combination with a membrane.     

一种新型生物活性人工骨的制备及成骨活性的研究

目的：研制CPC/BMP复合人工骨,检测其成骨活性。方法：制备CPC/BMP及CPC骨块,扫描电子显微镜观察表面结构。用小鼠肌袋植入实验观察材料的成骨活性。结果：BMP在CPC中呈微球状均匀分布。CPC植入小鼠肌袋内不能诱导,CPC/BMP植入后1周有软骨细胞出现,2周有编织骨,4周以后小梁骨生成,16周出现成熟的板层骨。同时材料出现降解迹象。有机质含量、碱性磷酸酶浓度在CPC/BMP组出现升高,扫描电镜结果同样证实有新骨形成。结论：CPC/BMP生物活性人工骨可异位诱导成骨,可望成为新型的骨缺损修复材料。     

Experimental anterior spine fusion using bovine bone morphogenetic protein: a study in rabbits

We developed an experimental model to study the merit of bovine bone morphogenic protein (bBMP) injection into the intervertebral disc to induce anterior interbody fusion. A total of 24 rabbits, divided into three groups of 8 animals each, were used. One hundred and fifty μg of partially purified bBMP was employed in the first group and 10 μg bBMP in the second group. In the control group, a sham operation was performed. The animals were followed radiographically at weekly intervals and animals were killed 3, 6, and 12 weeks postoperatively. After sacrifice, a mechanical and histologic evaluation of fusion was performed. Results of radiographic and histologic evaluation showed bone formation, which had resulted in the bridging of adjacent endplates, in the 150-μg group. In the 10-μg group, new bone formation was less extensive. In the control group, intradiscal bone formation was seen in only 1 animal. Range of motion measurements on flexion/extension films showed significantly decreased motion in segments that were fused with 150-μg of BMP. This study demonstrated the utility of an experimental model which allowed investigation of how anterior spine fusion may be further studied. Intradiscal injection of BMP could ultimately play a role in the development of minimally invasive techniques for anterior spinal fusion. Received for publication on Jan. 6, 1999; accepted on Aug. 18, 1999     

转染人骨形态发生蛋白在兔骨髓间充质干细胞中的表达

目的采用基因转移技术将人骨形态发生蛋白7(hBMP-7)基因转染兔骨髓间充质干细胞(BMSc),检测外源基因的表达.方法常规分子生物学技术构建hBMP-7逆转录病毒载体,制备含目的基因的重组逆转录病毒液,感染兔骨髓间充质干细胞,使用原位杂交以及免疫组织化学的方法检测hBMP-7在BMSc中的表达.结果原位杂交和免疫组化检测经hBMP-7基因转染的BMSc中出现阳性结果,未转染的BMSc中未见阳性结果出现.结论采用逆转录病毒介导的方法可以将hBMP-7转染至BMSc中,并有外源性基因的表达.