复合羧甲基纤维素重组合异种骨骨泥的制备

[目的]在制备重组合异种骨的基础上,加入塑型剂,探索其合适的成分比例,研究小牛脱钙骨泥的制备方法。[方法]分别制备小牛松质骨的完全脱钙颗粒和生化方法提取纯化牛骨形态发生蛋白(BMP),通过真空冻干机将松质骨和BMP充分复合后冻干,制成重组合异种骨(RBX)骨粉,按一定比例加入羧甲基纤维素(CMC)作为塑形剂无菌分装,使用时加适量生理盐水或自体血液即制备成骨泥。电镜下观察其孔径、BMP复合情况,大体观察其塑形性。[结果]制备的小牛脱钙骨泥极易塑形,塑形后不易松散,并且具有一定的弯曲强度,一侧应力后材料不会出现裂隙。骨泥复合材料经电镜观察,载体孔隙较多,孔径多在100μm以上,BMP与载体复合良好。[结论]复合BMP小牛脱钙骨泥物理性状稳定,可塑性强,使用方便。骨泥材料经电镜扫描有良好的孔隙结构,BMP与载体复合良好。     

异种脱蛋白骨管移植修复大段骨缺损的力学评估

目的研究异种脱蛋白骨管移植修复大段骨缺损的生物力学变化。方法建立山羊双侧胫骨大段骨缺损模型,36只山羊(72只后肢)随机分为2组,实验组:异种脱蛋白骨管;对照组:异种脱蛋白骨粒;两组均在移植骨中添加了骨形态发生蛋白(BMP),并采用骨板、钢板双重固定修复骨缺损。术后5、10、15周对羊胫骨行影像学观察和生物力学的变化。结果影像学显示实验组在骨缺损修复及成骨方面较对照组高。生物力学测试结果表明,术后5、10、15周时实验组力学强度较对照组明显增高,差异有统计学意义(P<0.05);术后15周,实验组的生物力学强度与正常胫骨已无差异。结论应用异种脱蛋白骨管支撑复合BMP修复大段骨缺损,能够有效加强移植骨修复负重骨缺损区的力学结构。     

抗感染重组合异种骨对犬污染性桡骨缺损的一期植骨修复作用

目的探讨抗感染重组合异种骨(anti-infective reconstituted bone xenograft,ARBX)对犬污染性桡骨缺损的一期植骨修复的效果. 方法在重组合异种骨(reconstituted bone xenograft,RBX)基础上,结合抗生素局部缓释技术,制备具有较强抗感染能力和较高成骨作用的ARBX.取成年杂种犬8只,于双侧桡骨中上段制成15 mm节段性骨缺损,在缺损处注入5×106 CFU/ml金黄色葡萄球菌1 ml,静置15分钟后,于双侧缺损区分别植入ARBX、RBX,并用钢板固定.术后6个月对存活的6只犬进行取材,通过解剖学、X线片、组织学及细菌学检查,比较ARBX和RBX一期植骨修复犬污染性桡骨缺损的效果. 结果术后6个月,ARBX侧有5只完全修复,1只中央部3 mm缺损未修复,均无骨髓炎表现,标本细菌培养均为阴性.RBX侧有1只部分修复,5只未能修复,残留8～13 mm缺损,均有明显骨髓炎表现,标本细菌培养均为阳性. 结论 ARBX具有较高成骨活性和较强的抗菌能力,能够一期植骨修复细菌污染性骨缺损.     

重组合异种骨移植成骨活性及量效关系的实验研究

观察重组合异种骨的成骨活性及其量效关系。方法采用ＲＢＸ移植建立ＢＡＬＢ／Ｃ小鼠股后肌袋模型,术后定期对移植组织进行放射学,病理学及碱性磷酸酶活性检查。结果（１）含不同比例的牛骨形态发生蛋白的ＲＢＸ组,成活性与ｂＢＭＰ含量呈正相关,存在着剂量依赖关系;（２）ＲＢＸＩ组成骨良好,同样含量的纯ｂＭＰ组也出现可见的成骨效应,但最终未能成骨;（３）ＡＬＰ活性以术后７天最高,４２例时仍较明显。结论ＲＢＸ是高效     

复合基因重组人骨形成蛋白-2异种骨的研制及成骨活性研究

目的　研制新型具有诱导成骨活性的异种骨植骨材料。方法　在重组合异种骨 (RBX)基础上 ,以基因重组人骨形成蛋白 - 2 (rh BMP- 2 )取代从牛皮质骨中提取的牛 BMP,与去抗原牛松质骨载体 (BCB)复合 ,制成复合 rh BMP- 2的异种骨 (rh BMP- 2 / BCB) ;将 4周龄雄性 BAL B/ C小鼠 6 0只 ,随机分为实验组和对照组 ,于实验组小鼠左股部肌袋植入 rh BMP- 2 / BCB骨粒 ,对照组小鼠左股部肌袋植入 BCB骨粒 ,术后 7、14及 2 1天取材 ,通过组织学、骨计量学方法检测 rh BMP- 2 / BCB的诱导成骨活性。结果　 1实验组术后 7天在小鼠肌袋可诱导软骨生成 ,14天形成编织骨 ,2 1天改建成板层骨并形成大量骨髓 ;对照组于术后各时间点均未见有软骨及骨形成。 2实验组的碱性磷酸酶活性和钙含量均高于对照组 ,具有统计学意义 (P<0 .0 1)。结论　 rh BMP- 2 / BCB具有较好的骨诱导能力 ,是一种较理想的植骨材料     

Anti-infective reconstituted bone xenograft used for primary bone grafting to repair contaminated defect in the radius in dogs

Objective: To investigate the effect of anti-infective reconstituted bone xenograft as a primary graft to repair a segmental with severe contamination. Methods: A canine model of contaminated defect of 1.5 cm in size in the radius was used, in which antiinfective reconstituted bone xenograft or reconstituted bone xenograft was implanted as a primary graft followed by internal fixation. The effectiveness of the two grafting materials in repairing a contaminated segmental defect was compared. Resuits: The animals which had received implant of anti-infective reconstituted bone xenograft should largely healed defects 6 months after operation while the defects implanted wit]h reconstituted bone xenograft remained unrepaired witla bone infection. Conclusions: Besides its strong osteoinductive and osteoconductive activity, anti-infective reconstituted bone xenograft is highly antibacterial and can be used as a primary graft to repair the severely contaminated segmental defect.     

Immunologic analyses of bovine bone treated with a novel tissue sterilization process

Abstract: Background: This study evaluates the efficacy of removal of xeno‐antigens from bovine bone using a patented BioCleanse^® process for decellularization of allograft tissues for clinical implantation. BioCleanse^® deploys a combination of chemicals and several high pressure rinses to achieve standardized sterility assurance levels. This method produces sterile grafts without reducing allograft bone biomechanical properties and effectively removes cells, lipids, and other sources of antigenic material from human allografts for clinical use. Methods: In this investigation, BioCleanse^® is evaluated for its potential in removing xenograft antigens from bovine bone grafts followed by immunologic evaluation in the subcutaneous pouch of immunocompetent rats. The alpha‐galactosyl (α‐gal) epitope with the structure Galα1‐3Galβ1‐4GlcNAc‐R constitutes a critical component of xenoantigens and its removal using BioCleanse^® from bovine bone was compared with tissue levels of unprocessed bone. The relative degree of antigen removal was also determined through measuring the pro‐inflammatory cytokine, tumor necrosis factor (TNF)‐α, and through the use of histologic grading of cellular infiltrates into bone. Results: Compact cortical bone inhibited immune cell migration but cancellous bone demonstrated cellular increase and bone resorption in the untreated control group. The α‐gal xenoantigen level was significantly lower in both cortical (P < 0.001) and cancellous bone (P < 0.001) compared with controls. TNF‐α levels were significantly (P < 0.001) reduced compared with untreated controls when human acute monocytic leukemia cells were exposed to cortical or cancellous bone. Conclusions: BioCleanse^® effectively removed xenoantigens and inflammatory markers justifying a follow up study in primates to determine these benefits in a model that is primed with preformed xeno‐antibodies responsible for hyperacute rejection in hard tissues.     

庆大霉素重组合异种骨复合体防治开放性骨折骨缺损感染的实验研究

目的 研制庆大霉素重组合异种骨复合体（ｇｅｎｔａｍｉｃｉｎ－ｒｅｃｏｎｓｔｉｕｔｅｄ ｂｏｎｅ ｘｅｎｏｇｒａｆｔ－ｃｏｍｐｏｓｉｔｅ,Ｇ－ＲＢＸ－Ｃ）,为临床防治开放性骨折骨缺损感染提供理论依据。方法 采用具有高效骨诱导及骨肟导能力的重组合民种骨作为载体,复合庆大霉素制备庆大霉素后组合异种骨复合体,并对其体内药物释放特性、异位诱导成骨能力及骨缺损修复能力进行了研究。结果 体内药物释放试验显示：Ｇ     

块型抗感染重组合异种骨Ⅰ期植骨修复犬桡骨污染性节段性骨缺损

目的：研究和开发能够Ⅰ期植骨修复细菌污染的节段性骨缺损的植骨材料。方法：在块型重组织异种骨（MRBX）基础上，结合抗生素局部缓释技术。研制出兼具高效成骨作用和强力抗感染能力的块型抗感染重组合异种骨（MARBX）；并采用细菌污染的犬桡骨，1.5cm节段性骨缺损模型，通过解剖学、放射学、组织学及细菌学检查，对比研究MARBX和MRBXⅠ期植骨内固定修复效果。结果：术后6个月，MARBXⅠ期植骨，可有效预防感染，并基本完成骨缺损的修复；而MRBXⅠ期植骨，则导致骨髓炎，不能修复骨缺损。结论：MARBX由于既具有高效成骨活性，又有非常强的抗菌能力，能Ⅰ期植骨、有效修复污染性节段性骨缺损。     

干细胞移植和BMP2基因治疗修复骨损伤和坏死的实验研究

目的:根据老年骨缺损和股骨头坏死实验模型的研究结果来评价干细胞移植和BMP2基因治疗的方法是否可用于一些特殊损伤和疾病的治疗.方法:从不同年龄段大鼠、羊骨髓中分离培养骨髓间充质干细胞,利用含BMP-2基因或βal基因的腺病毒载体感染干细胞,通过酶联免疫测定方法检测基因转染细胞培养上清中BMP-2蛋白的含量.利用基因转染细胞和多孔三磷酸钙复合,回植后修复24月龄老年大鼠股骨干6毫米节段性缺损和实验性羊股骨头坏死.通过组织学观察和生物力学测定来评价比较BMP-2治疗组和βgal对照组的新骨形成情况和修复组织的强度.结果:基因转染细胞培养上清中BMP-2蛋白的含量随时间延长而逐渐上升,不同年龄大鼠干细胞BMP-2转染后蛋白质的分泌水平没有明显差异.组织学观察表明BMP-2基因转染细胞和多孔三磷酸钙复合物已成功修复24月龄老年大鼠股骨干6毫米节段性缺损和实验性羊股骨头坏死,BMP-2治疗组的新骨形成明显多于βgal对照组(P＜0.05).治疗后第16周,BMP-2治疗组股骨头修复组织的最大压缩强度和弹性模量也明显高于Bgal对照组(P＜0.05).结论:BMP-2基因转染的自体骨髓间充质干细胞和多孔三磷酸钙复合后回植可有效修复老年大鼠骨缺损并重建羊坏死股骨头的功能.     

Enhanced healing of goat femur‐defect using BMP7 gene‐modified BMSCs and load‐bearing tissue‐engineered bone

Segmental defect regeneration is still a clinical challenge. In this study, we investigated the feasibility of bone marrow stromal cells (BMSCs) infected with adenoviral vector containing the bone morphogenetic protein 7 gene (AdBMP7) and load‐bearing to enhance bone regeneration in a critically sized femoral defect in the goat model. The defects were implanted with AdBMP7‐infected BMSCs/coral (BMP7 group) or noninfected BMSCs/coral (control group), respectively, stabilized with an internal fixation rod and interlocking nails. Bridging of the segmental defects was evaluated by radiographs monthly, and confirmed by biomechanical tests. Much callus was found in the BMP7 group, and nails were taken off after 3 months of implantation, indicating that regenerated bone in the defect can be remodeled by load‐bearing, whereas after 6 months in control group. After load‐bearing, it is about 5 months; the mechanical property of newly formed bone in the BMP7 group was restored, but 8 months in control group. Our data suggested that the BMP7 gene‐modified BMSCs and load‐bearing can promote bone regeneration in segmental defects. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:412–418, 2010     

重组PcDNA3.1-hBMP-2转染骨髓基质干细胞及复合异种骨支架体外构建组织工程骨

目的：构建人骨形态发生蛋白-2(human bone morphogenetic protein,，hBMP-2)真核表达载体PcDNA3．1-hBMP-2，转染兔骨髓基质干细胞(bane marrow stromal cells，BMSCs)，种植去抗原牛松质骨(bovine cancellous bone，BCB)支架体外构建组织工程骨。方法：蛋白印迹法检测转染后细胞BMP-2的表达，碱性磷酸酶(ALPase)活性检测分析基因转染对细胞分化的影响。然后将转染后细胞接种到BCB支架上，扫描电镜观察细胞贴附、生长状况。结果：转染后，BMSCs表达BMP-2，ALP活性明显增高。扫描电镜见转染细胞分布均匀，伸展良好。结论：在脂质体介导下，BMP-2基因可导入细胞且稳定表达基因产物促进自身增殖分化，转染后细胞在支架材料上贴附生长良好，为进一步应用携带BMP-2基因的人工骨修复骨缺损奠定了实验基础。     

游离骨膜与复合rhBMP2的异种骨联合移植修复骨缺损的实验研究

目的 探讨复合基因重组人ＢＭＰ２（ｒｈＢＭＰ２）的异种骨与游离骨膜（ＦＰ）联合移植修复节段性骨缺损的效果。方法 将ｒｈＢＭＰ２与去抗原牛松质骨载体（ＢＣＢ）复合,制成ｒｈＢＭＰ２／ＢＣＢ;选用新西兰大耳白兔２８只,制成烧骨干１５ｍｍ缺损动物模型,分别行ＦＰ和ｒｈＢＭＰ２／ＢＣＢ复合移植（ｎ＝６）、ｒｈＢＭ２／ＢＣＢ移植（ｎ＝６）和单纯ＢＣＢ移植（ｎ＝２）;术后４、８、１２、１６周取材,通过Ｘ线、生     

Bovine bone implant with bovine bone morphogenetic protein in healing a canine ulnar defect

Xenograft is considered an alternative material for bone transplantation, but its bone healing capacity is inferior compared to that of autografts and allografts. Here, we tested whether bone morphogenetic protein (BMP) addition enhances the suitability of demineralized xenogeneic bovine bone for bone grafting in dogs, and whether xenogeneic bone is a suitable carrier material for BMPs. The capacity of demineralized bovine bone implants, with and without native partially purified bovine BMP, to heal a 2-cm ulnar defect was determined in six dogs over a follow-up time of 20 weeks. No instances of bone union were seen, but there was slightly more bone formation in the xenografts with BMP, though the difference was not statistically significant. The ulnas treated with an implant with BMP were also mechanically stronger, but the difference was not significant. Computed tomography scans showed no differences in the implant area in bone density, bone mineral content, or bone cross-sectional area. It is concluded that native, partially purified BMP does not sufficiently improve the suitability of bovine demineralized xenografts as a bone substitute material for dog. Demineralized xenogeneic bone does not seem to be a feasible carrier material for BMP.     

血管化骨-重组异种骨移植修复骨缺损

目的 探讨血管化骨-重组异种骨(RBX)移植修复骨缺损的意义。方法 27例骨缺损随机分成3组，A组为血管化骨-RBX移植修复组(9例)；B组为血管化骨移植修复组(10例)；C组为RBX移植修复组(8例)。临床评价以术后3个月、6个月、12个月随访的X线片来判断骨缺损是否修复、骨愈合时间长短以及是否再吸收等。结果 术后3个月A组8例、B组6例、C组3例骨愈合，6个月A组1例、B组3例、C组3例骨愈合，12个月B组1例、C组2例发生骨移植区部分再吸收。结论 血管化骨一重组异种骨移植的骨折愈合率明显优于单纯重组异种骨移植。     

治疗节段性骨缺损的一种新方法—新型重组合异种骨与带血循骨膜联合移植

目的探讨治疗节段性骨缺损新方法一新型重组合异种骨(NRBX)与带血循骨膜联合移植方法的效果。方法将基因重组人BMP2(rhBMP     

Autogeneic cancellous bone grafts in extensive segmental ulnar defects in dogs. Effects of xenogeneic bovine bone morphogenetic protein without and with interposition of soft tissues and interruption of blood supply

Xenogeneic (bovine) bone morphogenetic protein (bBMP) and associated insoluble noncollagenous proteins (NCP) were implanted in inbred adult beagle dogs with 3-4 cm diaphyseal defects in the ulna. Defects were stabilized with internal plate fixation, and the control defects were not stabilized. The defects were implanted with either autogeneic cancellous bone grafts (ACG), bBMP/NCP, or a composite of ACG and bBMP/NCP. Of the plated ulnae, 18 of 19 ACG controls restored bone continuity; six of seven defects healed under the influence of bBMP/NCP plus ACG. Two of four defects with bBMP/NCP plus ACG healed and two were filled with osseous tissue, but fibrous tissue developed at one or both bone ends. Eight of nine defects implanted with bBMP/NCP capsules alone were repaired with fibrous tissue only. Of the nonplated defects, four were implanted with bBMP/NCP plus ACG and only one regenerated; three of four showed hypertrophic bone growth around a pseudarthrosis. Of six nonplated defects implanted with bBMP/NCP without ACG, all developed atrophic bone ends and fibrous tissue repair. Thus, to restore continuity of large segmental defects three times greater than the critical size for spontaneous regeneration, xenogeneic bBMP/NCP failed to induce bone regeneration in dogs. To exclude cell-mediated immune reactions and soft-tissue ingrowth, one defect was bridged with a polytetrafluoroethylene semipermeable tube (pore size 0.45 micron) containing implants of bBMP/NCP. In response to bBMP/NCP, cells from the host bone ends produced ossicles of induced woven bone formation. The observation that bBMP/NCP induced bone formation across the defect inside of semipermeable cylindrical chambers suggests that the experiments on bone defects larger than the critical size for spontaneous repair should be repeated with: (1) allogeneic dog BMP/NCP; (2) semipermeable cylinders to protect against muscle interposition; (3) compartment angiograms to evaluate blood supply; (4) treatment of the recipient with immunosuppressants and immunostaining to observe the concentration gradient of BMP; and (5) histologic observations on the first three days after implantation to evaluate cell-mediated immune barriers to the response of BMP.     

自体外周血及自体红骨髓干细胞与脱钙骨复合移植治疗骨缺损的比较研究

目的 对比观察自体外周血干细胞（APBSC）／脱钙骨（DB）复合移植与自体外红骨髓（ARBM）／DB复合移植治疗骨缺损的疗效。方法 36只家兔双侧桡骨造成1cm骨缺损,随机分为DB组、ARBM／DM级和APBSC／DB组,每组12只,分别进行X线片、生物力学和组织学检查,然后作对比分析。结果 术后第2、4、8、14周,APBSC／DB组和ARBM／DB组X线片、改进的GaryX线评分和光镜观察结果,以及术后第14周整骨破坏载荷和骨缺损修复形态学评分均明显优于DB组;但APBSC／DB组与ARBM／DB组间差异无显著性。结论 APBSC与ARBM都能在DB的骨形态蛋白诱导下促进成骨细胞的形成,复合移植疗效明显优于单纯DB移植。     

An injectable composite material containing bone morphogenetic protein‐2 shortens the period of distraction osteogenesis in vivo

To investigate new methods that can decrease the duration of bone transport (BT) distraction osteogenesis, we injected composite materials containing recombinant human bone morphogenetic protein‐2 (BMP‐2) and induced the generation of a callus bridge by rapid segmental transport (4 mm/day) in a rabbit bone defect model. The composite materials consisted of BMP‐2 (0, 30, or 100 µg), β‐tricalcium phosphate powder (βTCP, 100 mg/animal; particle size, <100 µm), and polyethylene glycol (PEG; 40 mg/animal). A paste of equivalent composition was percutaneously injected at the lengthening and the docking sites after surgery and after BT, respectively. The radiographic, mechanical, and histological examinations 12 weeks post‐operative revealed that the generation of bridging callus in the presence and in the absence of BMP‐2 was significantly different. The callus mass in the bone defect region was adequately and consistently developed in the presence of 100 µg of BMP (administered for 6 weeks), and the bones were consolidated in 12 weeks. Such an adequate callus formation was not observed in the control animals without BMP‐2 treatment. The result of this experimental study suggests the potential application of BMP‐2 in accelerating callus formation and in enabling rapid bone transporting, thereby shortening the treatment period for the repair of diaphyseal bone defects by distraction osteogenesis. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:452–456, 2011     

硅胶膜管联合BMP/HA修复大块骨缺损机制初探

目的 探讨硅胶膜（SGM）管与骨形态发生蛋白（BMP）／羟基磷灰石（HA）复合物联合修复兔长骨缺损的机制。方法 制备兔桡骨中段1.2cm缺损,实验组缺损区外围包绕SGM，其内分别填充BMP／HA，HA材料，对照组仅填充HA；空白组骨缺损区未填充。通过X线摄片，光学显微镜和扫描电镜观察骨缺损区的影像学，组织学和超微结构变化。结果 术后1个月，SGM＋BMP／HA组骨缺损区有大量的类骨质形成。术后2个月，SGM＋BMP／HA组大量骨膜来源的分化细胞沿SGM管的内，外面及HA间隙向骨缺损区内生长，植入区可见大片的膜内成骨征象。术后3个月，GM＋BMP／HA组骨缺损区愈合。此时，SGM＋HA组与对照组骨缺损区有少量新骨形成，空白组缺损区为纤维组织充填。结论 SGM管与BMP／HA联合修复骨缺损的机制为SGM的屏障作用使骨缺损区受到引导成骨和诱导成骨的双重作用，其中诱导成骨方式为膜内骨发生。