胫骨牵引成骨过程中骨形态形成蛋白4及其受体的表达

背景：骨形态形成蛋白4及其受体在骨再生和修复过程中起着重要的作用,但具体机制尚不清楚。目的：建立小鼠胫骨牵引成骨模型,分析骨形态形成蛋白4及其受体在牵引成骨过程中的表达,探讨机械牵张力转化为生物信号从而调节骨再生过程的机制。方法：健康雄性8周龄CD-1小鼠36只,按照手术时间随机分成术后第5,9,13,17,24和31天组,每组6只。所有小鼠均接受左胫骨中上段低能截骨,安置体外延长固定架,截骨后5d为静止期;截骨后第6天起开始每天进行胫骨延长,速率为0.2mm,2次/d,共12d,为牵引期;自第18天停止牵引,为固塑期。于术后第5,9,13,17,24和31天分别处死动物,采集胫骨标本,作组织学检查、RT-PCR和原位杂交实验分析骨形态形成蛋白4及其受体激活素样激酶3以及骨钙素的表达。结果与结论：组织学检查显示静止期修复过程基本与骨折愈合过程相似。小鼠骨折断端在持续牵引下有明显的骨痂形成,骨形态形成蛋白4及其受体激活素样激酶3以及骨钙素的mRNA的表达明显增强。结果表明,牵引成骨是一种持续的骨再生过程,机械张力可通过刺激骨形态形成蛋白4及其受体以及骨钙素的持续高表达维持骨痂的不断形成和再塑,以充填连续延长的骨折间隙。     

牵引成骨小鼠早期骨折愈合

背景:牵引成骨技术治疗四肢骨缺损和发育不良等有一定的优势,但是有关骨延长区新骨的成骨方式,目前尚存在争议.目的:观察小鼠胫骨骨折愈合过程中与成骨方式有关的骨基质蛋白组织学与基因水平的表达,论证在外界牵张力的作用下骨折愈合方式.设计、时间及地点:组织学和蛋白基因检测,于2007-08/12在中南大学第三附属医院中心实验室完成.材料:8周龄雄性CD-1小鼠36只,体质量25～30 g,由中南大学湘雅医学院实验动物中心提供.方法:接受左胫骨中上段骨干横行截骨,安置特制延长外固定架,胫骨牵引过程包括5 d静止期,12 d牵引期和70 d固塑期,牵引期的牵引速率为0.1 mm/次,共0.2 mm/d.术后于5,9,13,17,24,31 d采集左胫骨标本,分别作组织学检查和骨基质蛋白mRNA检测.主要观察指标:①小鼠胫骨牵引成骨模型骨折端组织学变化.②小鼠胫骨牵引成骨骨痂内各种基质蛋白mRNA在不同时间点的表达.结果:组织学检查显示:静止期其修复过程基本与骨折愈合相似;牵引期,被牵引骨痂显示3个典型的生物力学功能区:纤维间区,初始骨基质前沿和微骨柱形成区;固塑早期,牵引骨痂骨性愈合.mRNA检测显示:Ihh在牵引后的第4天可检测到表达,静止期及牵引后期无明显表达.Ⅱ型胶原从截骨后第5天即可检测,直到固塑期1周,但其mRNA表达逐渐减弱,到固塑期第2周即停止表达.X型胶原的表达在牵引期第4天达到高峰,然后逐步下降.骨钙素在截骨第5天尚不能被检测,其mRNA的表达在牵引后期和固塑期早期达高峰.结论:胫骨牵引静止期软骨内成骨和膜内成骨同时存在,但在机械牵张力作用下转为以膜内成骨为主的成骨过程.     

牵引成骨与小鼠早期骨折愈合

背景：牵引成骨技术治疗四肢骨缺损和发育不良等有一定的优势,但是有关骨延长区新骨的成骨方式,目前尚存在争议。目的：观察小鼠胫骨骨折愈合过程中与成骨方式有关的骨基质蛋白组织学与基因水平的表达,论证在外界牵张力的作用下骨折愈合方式。设计、时间及地点：组织学和蛋白基因检测,于2007-08/12在中南大学第三附属医院中心实验室完成。材料：8周龄雄性CD-1小鼠36只,体质量25～30g,由中南大学湘雅医学院实验动物中心提供。方法：接受左胫骨中上段骨干横行截骨,安置特制延长外固定架,胫骨牵引过程包括5d静止期,12d牵引期和70d固塑期,牵引期的牵引速率为0.1mm/次,共0.2mm/d。术后于5,9,13,17,24,31d采集左胫骨标本,分别作组织学检查和骨基质蛋白mRNA检测。主要观察指标：①小鼠胫骨牵引成骨模型骨折端组织学变化。②小鼠胫骨牵引成骨骨痂内各种基质蛋白mRNA在不同时间点的表达。结果：组织学检查显示：静止期其修复过程基本与骨折愈合相似;牵引期,被牵引骨痂显示3个典型的生物力学功能区：纤维间区,初始骨基质前沿和微骨柱形成区;固塑早期,牵引骨痂骨性愈合。mRNA检测显示：Ihh在牵引后的第4天可检测到表达,静止期及牵引后期无明显表达。Ⅱ型胶原从截骨后第5天即可检测,直到固塑期1周,但其mRNA表达逐渐减弱,到固塑期第2周即停止表达。X型胶原的表达在牵引期第4天达到高峰,然后逐步下降。骨钙素在截骨第5天尚不能被检测,其mRNA的表达在牵引后期和固塑期早期达高峰。结论：胫骨牵引静止期软骨内成骨和膜内成骨同时存在,但在机械牵张力作用下转为以膜内成骨为主的成骨过程。     

携带LacZ的腺病毒载体在牵引骨痂局部转染成纤维细胞和成骨细胞的表达

目的:利用小鼠胫骨牵引成骨动物模型,在牵引成骨过程中局部给予携带了LacZ的腺病毒载体后观察其表达情况,以探讨基因治疗促进骨折愈合的可行性。方法:实验于2004-10/2006-01在中南大学湘雅三医院完成。①实验分组:取雄性8周龄CD-1小鼠20只,随机数字表法分为实验组和对照组,每组各10只。②实验方法:所有小鼠接受左胫骨中上段骨干横行截骨安置特制延长外固定架,胫骨牵引过程包括5d静止期,10d牵引期,牵引速率为0.1mm/次,2次/d,共0.2mm/d。牵引期第7天实验组牵引骨痂局部注射5μL携带了LacZ的腺病毒载体,对照组局部注入等量未含LacZ腺病毒液。③实验评估:注射后第3天麻醉后杀取动物,采集左胫骨标本,分别作组织学检查和组织化学分析。结果:纳入小鼠20只,均进入结果分析。在牵引第10天,牵引骨痂中纤维间区形成,两端的新生骨由骨折两端中心生长延伸。骨髓腔内外可见大量新生骨形成。X-Gal底物染色显示,在实验组新生骨组织内可见大量细胞呈阳性染色;而对照组未发现阳性染色细胞。结论:在牵引成骨过程中,骨痂局部注射携带LacZ的腺病毒,能成功转染局部的成纤维细胞及成骨细胞,并表达LacZ基因,为临床应用基因治疗促进骨牵引延长或骨折愈合提供可行性。     

糖尿病骨折大鼠骨痂组织中成骨细胞增殖和骨钙素表达与骨形态发生蛋白2干预的关系

背景:有研究表明,糖尿病患者骨折愈合过程中局部骨形态发生蛋白2明显降低,成骨细胞增殖能力下降,骨折延迟愈合,或者不愈合.目的:观察骨形态发生蛋白2治疗糖尿病大鼠骨折愈合过程中骨痂组织成骨细胞增殖和骨钙素的表达,分析骨形成蛋白在糖尿病大鼠骨折愈合过程中的作用.设计、时间及地点:随机对照动物实验,于2008-09/12在昆明医学院动物实验中心完成.材料:SD雄性大鼠30只,20只腹腔内注射链脲佐菌素破坏胰岛细胞诱导为糖尿病大鼠后,随机均分为骨形态发生蛋白2组、模型对照组,另10只为正常对照大鼠,腹腔内注射等容量生理盐水.方法:3组大鼠行右胫骨上段骨折建立大鼠胫骨骨折模型.骨形态发生蛋白2组用10 μ g/kg骨形态发生蛋白2治疗,模型 对照组及正常对照组给予等渗量生理盐水.主要观察指标:伤后第1,2,4,6周X射线片观察骨折愈合情况,光镜下观察骨痂内成骨细胞数量,放射免疫法测定血清骨钙素表达.结果:骨形态发生蛋白2、正常对照组X射线片显示骨痂生成量多,骨折愈合程度明显优于模型对照组(P<0.01)骨形态发生蛋白2组成骨细胞数量明显多于模型对照组,略低于正常对照组.各组血清骨钙素均呈上升趋势,在第2周达到峰值.第4用下降,至第6周进一步下降.模型对照组骨钙素升高幅度明显低于正常对照组(P<0.01),并且低于骨形态发生蛋白2组.骨形态发生蛋白2组、正常对照组的骨钙素差异无显著性意义.结论:糖尿病大鼠骨折愈合过程中较正常大鼠,骨钙索水平降低,成骨细胞增殖下降,采用骨形态发生蛋白2治疗可提高骨钙素水平,促进成骨细胞增殖.促进骨折愈合.     

兔胫骨骨延长模型在低强度脉冲超声刺激条件下的骨再生与成熟

背景:实验证实,低强度脉冲超声有促进骨折愈合的作用.但低强度脉冲超声对骨折愈合早期骨质再生成熟的影响及其相关的作用机制仍不十分清楚.目的;创新性提出低强度脉冲超声能够在兔胫骨骨延长动物模型牵拉成骨早期骨再生成熟中发挥正效效应的理论假设,并期望以超声干预与不干预进行对照比较予以验证. 方法:36只成年健康新西兰兔随机数字表法分为超声治疗组和对照组,每组18只.所有动物均行胫骨中段截骨,以Orthofix M103型迷你外固定架延长器固定,术后7 d以0.5 mm/12 h延长10 d,总延长长度为10 mm,超声治疗组在延长完成后以低强度脉冲超声骨折治疗仪治疗,20 min/d,1次/d,对照组不给予低强度脉冲超声治疗.治疗4,8,12周后,X射线评定胫骨骨折断端愈合程度,采用Image J图像分析软件分析计算骨痂生成率;延长区新骨作苏木精-伊红染色、Masson三色染色、VG染色后光镜下组织学分析,并在显微镜下测新骨占总骨痂的面积.结果与结论:36只兔均进入结果分析.治疗4周后对照组仅见外骨痂连接形成骨桥,骨痂区密度低,还可见部分骨痂缺如:超声治疗组骨早期再生成熟优于对照组,表现在两断端骨痂影密度增高,数量增多,骨痂影由两端向中央生长,被密度高的骨痂取代,外骨痂已开始被吸收,骨痂充满延长区,密度增高增粗,骨折处皮质骨密度接近正常皮质骨,骨痂生成率明显提高(P＜0.05),与8,12周无差异.组织学分析显示,超声治疗组治疗4,8,12周时新骨形成早于对照组,治疗4周新骨占总骨痂面积的百分比大于对照组,治疗8,12周后两组新骨占总骨痂面积的百分比无差别.提示低强度脉冲超声可促进新骨形成,增加骨痂面积,在兔胫骨骨延长动物模型牵拉成骨后的骨再生成熟中有促进作用.     

豪猪蛋白在成年小鼠胫骨骨折愈合中对骨形成的作用

目的:观察骨痂组织中豪猪蛋白IndianHedgehog,Ihh)基因的时空表达(模式,分析豪猪蛋白在骨折愈合过程中可能的作用,探索骨组织石蜡切片的原位杂交技术。方法:实验动物模型选用标准的小鼠非稳定性胫骨骨折模型,采用常规石蜡切片原位杂交技术,观察豪猪蛋白mRNA在骨折愈合过程中不同时期的基因表达情况。结果:骨折第5天起,软骨细胞中出现豪猪蛋白mRNA的低水平表达。骨折后第7天,豪猪蛋白mRNA在骨痂肥大前软骨细胞中明显表达。到骨折后第14天,软骨痂中的肥大软骨细胞豪猪蛋白mRNA表达逐渐消失。骨折后第28天,软骨完全被骨取代,此时骨痂中没有豪猪蛋白mRNA表达。结论:豪猪蛋白在小鼠胫骨骨折愈合中的肥大前软骨细胞中表达,提示调节胚胎骨骼发育过程中软骨细胞分化的豪猪蛋白基因,可能在成年小鼠骨折愈合过程中也参与调节骨形成而影响骨折愈合。     

豪猪蛋白在成年小鼠胫骨骨折愈合中对骨形成的作用

目的：观察骨痂组织中豪猪蛋白(Indian Hedgehog，IHh)基因的时空表达模式，分析豪猪蛋白在骨折愈合过程中可能的作用，探索骨组织石蜡切片的原位杂交技术。方法：实验动物模型选用标准的小鼠非稳定性胫骨骨折模型，采用常规石蜡切片原位杂交技术，观察豪猪蛋白mRNA在骨折愈合过程中不同时期的基因表达情况。结果：骨折第5天起，软骨细胞中出现豪猪蛋白mRNA的低水平表达。骨折后第7天，豪猪蛋白mRNA在骨痂肥大前软骨细胞中明显表达。到骨折后第14天，软骨痂中的肥大软骨细胞豪猪蛋白mRNA表达逐渐消失。骨折后第28天，软骨完全被骨取代，此时骨痂中没有豪猪蛋白mRNA表达。结论：豪猪蛋白在小鼠胫骨骨折愈合中的肥大前软骨细胞中表达，提示调节胚胎骨骼发育过程中软骨细胞分化的豪猪蛋白基因，可能在成年小鼠骨折愈合过程中也参与调节骨形成而影响骨折愈合。     

骨形态发生蛋白2在骨质疏松症大鼠骨折愈合过程中的表达

目的:骨折愈合过程中,间充质细胞向骨折部位迁徙并分化为软骨细胞及成骨细胞是其中的关键性环节,骨形态发生蛋白2在其中具有重要作用.观察骨形态发生蛋白2在绝经后骨质疏松症大鼠胫骨骨折早期愈合过程中表达的变化,探讨骨质疏松症骨折延迟愈合的原因.方法:实验于2005-08/2006-10在教育部口腔生物医学工程重点实验室完成.①实验材料及分组:64只6个月龄雌性SD大鼠随机分为去势组和对照组,每组32只.②实验过程:去势组行双侧卵巢切除建立Ⅰ型骨质疏松动物模型,对照组切除少量脂肪组织.3个月后造成胫骨骨折.③实验评估:两组于术后7,14,21,28 d麻醉状态下各取大鼠8只,取胫骨周围少量软组织和外骨痂的骨折断端、骨痂、骨皮质及骨髓腔的全层标本,分别进行组织学、免疫组织化学,半定量反转录-聚合酶链反应方法检测.结果:①组织学观察结果:显示骨折处膜内成骨和软骨成骨两种方式,术后第21天及28天时去势组骨痂中仍可见大量软骨细胞.②免疫组织化学结果:术后第7天及14天对照组骨形态发生蛋白2平均吸光度值大(阳性细胞计数高)于去势组,而在术后第21天低于去势组.③反转录-聚合酶链反应结果:术后第7天对照组骨形态发生蛋白2条带强度高于去势组,而在第14天弱于去势组.结论:骨形态发生蛋白2在骨折早期骨痂形成中具有重要作用,骨质疏松症骨折愈合早期骨形态发生蛋白2的表达量减少且延迟,可能是其愈合延迟的重要影响因素.     

失神经状态下胫骨骨折断端骨痂成骨的组织学变化

背景:有研究表明神经系统在骨折的修复重建中发挥重要的作用,但其具体机制尚未阐明,中枢神经及神经生长因子对骨痂中成骨的影响至今少有报道.目的:观察失神经状态下胫骨骨折断端骨痂成骨的组织学变化.方法:用SD大鼠建立左胫骨闭合骨折髓内固定大鼠模型,建模成功后随机为3组,左胫骨骨折组;T10脊髓横断组:于左胫骨骨折后,横断切除T10段脊髓约0.3 cm,制成T10脊髓完全性损伤大鼠胫骨骨折模型;神经生长因子治疗组:建立T10脊髓完全性损伤大鼠胫骨骨折模型后,肌注神经生长因子.术后各组均用苏木精伊红染色法观察骨组织形态变化,骨钙素免疫组织化学法观察骨痂中成骨情况,透射电镜观察成骨细胞内微结构变化.结果与结论:T10脊髓横断组大鼠在失神经状态下,骨痂中成骨细胞的数量减少,骨钙素表达减少,成骨细胞内细胞器功能低下.神经生长因子治疗组的以上各组织学观察指标均较T10脊髓横断组均有不同程度的改善.结果显示,神经生长因子可部分改善脊髓横断失神经状态下的骨组织再生情况.     

血管内皮细胞生长因子/骨形态发生蛋白2联合修饰骨髓间充质干细胞修复股骨头坏死

背景:将血管内皮细胞生长因子及骨形态发生蛋白二者联合修复坏死骨,有可能在保持种子细胞成骨表型的同时,又能持续高效地分泌血管内皮细胞生长因子及骨形态发生蛋白2,从而有效促进血管再生,促进组织工程化骨组织的形成和再血管化。目的:观察血管内皮细胞生长因子165/骨形态发生蛋白2体外修饰骨髓间充质干细胞移植治疗兔股骨头缺血性坏死效果。方法:建立兔右侧股骨头缺血性坏死模型,并以抽签法随机分为3组:单纯髓芯减压组、髓芯减压+骨髓间充质干细胞组、髓芯减压+血管内皮细胞生长因子165/骨形态发生蛋白2转染骨髓间充质干细胞组。关节镜监视下将坏死骨清除,组织学检查成骨及血管生成情况。结果与结论:通过关节镜观察显示各组坏死骨清除干净,有新鲜血流出。组织形态学检测发现,髓芯减压+血管内皮细胞生长因子165/骨形态发生蛋白2转染骨髓间充质干细胞组移植后不同时间点血管数量及修复区新骨面积比显著高于单纯髓芯减压组、髓芯减压+骨髓间充质干细胞组(P<0.05)。提示血管内皮细胞生长因子165/骨形态发生蛋白2基因转染加强了骨髓间充质干细胞成骨作用,提高了新生骨的数量与质量,加快了股骨头缺血性坏死的修复。     

Polyethylenimine-alginate nanocomposites based bone morphogenetic protein 2 gene-activated matrix for alveolar bone regeneration

The repair and treatment of lost or damaged alveolar bone is of great significance in dentistry. Gene-activated matrix (GAM) technology provides a new way for bone regeneration. It is a local gene delivery system, which can not only recruit cells, but also influence their fate. For this purpose, we fabricated a bone morphogenetic protein 2 (BMP-2) gene-loaded absorbable gelatin sponge (AGS) and studied its effect on promoting alveolar bone formation and preventing resorption following tooth extraction in rats. In order to obtain better transfection efficiency, polyethylenimine-alginate (PEI-al) nanocomposites were synthesized and used as gene vectors to deliver BMP-2 cDNA plasmids (PEI-al/pBMP-2). The transfection efficiency, BMP-2 protein expression and osteogenic differentiation of the cells were investigated in vitro. In vivo, we established an alveolar bone regeneration model by extracting the rats'' left mandibular incisors. The rats were randomly assigned into 3 groups: control group, unfilled sockets; AGS group, sockets filled with PEI-al solution-loaded gelatin sponges; AGS/BMP group, sockets filled with PEI-al/pBMP-2 solution-loaded gelatin sponge. Radiological and histological assays were performed at 4 and 8 weeks later. In vitro transfection assays indicated that PEI-al/pBMP-2 complexes could effectively transfect MC3T3-E1 cells, promoting the secretion of BMP-2 protein for at least 14 days, as well as increasing the expression of osteogenesis-related gene, ALP activity and calcium deposition. In vivo, western blot analysis showed BMP-2 protein was expressed in bone tissues of AGS/BMP group. The relative height of the residual alveolar ridge and bone mineral density (BMD) of the AGS/BMP group were significantly greater than those in the AGS and control groups at 4 and 8 weeks, respectively. Histological examination showed that, at 4 weeks, osteoblasts had grown in a cubic shape around the new bone in the AGS/BMP group, suggesting new bone formation. In conclusion, the combination of PEI-al/pBMP-2 complexes and gelatin sponge could promote alveolar bone regeneration, which may provide an easy and valuable method for alveolar ridge preservation and augmentation.

Polyethylenimine-alginate nanocomposites based bone morphogenetic protein 2 gene-activated matrix may provide an easy and valuable method for alveolar ridge regeneration.     

An analysis of bone regeneration at a segmental bone defect by controlled release of bone morphogenetic protein 2 from a biodegradable sponge composed of gelatin and β-tricalcium phosphate

To treat large bone defects is a clinically challenging problem and utilizing tissue engineering technology is an attractive approach for overcoming such a problem. Previously, a biodegradable sponge incorporating bone morphogenic protein-2 (BMP-2), which can control the release of BMP-2 for a prolonged time in an in vivo environment, was reported. In addition, a biodegradable sponge composed of gelatin and β-tricalcium phosphate (βTCP), gelatin-βTCP sponge to develop a more ideal scaffold for enhancing bone regeneration was also created and previously reported. The purpose of this study was to investigate the effectiveness of the gelatin-βTCP sponge for the promotion of bone regeneration in a critical-sized bone defect site in vivo. Apparent bone regeneration was induced by the gelatin sponge incorporating BMP-2 and the gelatin-βTCP sponge with BMP-2 incorporation. In contrast, no apparent bone formation was induced by either the gelatin sponge only or the gelatin-βTCP sponge without BMP-2. To investigate the quality of the regenerated bone, we conducted a biomechanical evaluation with a three-point bending test. We found no significant difference between the gelatin sponge incorporating BMP-2 and the gelatin-βTCP sponge incorporating BMP-2 groups. Incorporation of βTCP into the gelatin sponge was expected to enhance biomechanical strength during the initial bone regeneration. However, our observations showed that the gelatin-βTCP sponge did not significantly improve the quality of regenerated bone from the viewpoint of biomechanical assessment, even though it did not impair the effectiveness of the promotion of bone regeneration by BMP-2 in the bone defect.     

Characterization of the distinct orthotopic bone-forming activity of 14 BMPs using recombinant adenovirus-mediated gene delivery

Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.     

血管内皮细胞生长因子/骨形态发生蛋白2联合修饰骨髓间充质干细胞修复股骨头坏死

背景：将血管内皮细胞生长因子及骨形态发生蛋白二者联合修复坏死骨,有可能在保持种子细胞成骨表型的同时,又能持续高效地分泌血管内皮细胞生长因子及骨形态发生蛋白2,从而有效促进血管再生,促进组织工程化骨组织的形成和再血管化。目的：观察血管内皮细胞生长因子165/骨形态发生蛋白2体外修饰骨髓间充质干细胞移植治疗兔股骨头缺血性坏死效果。方法：建立兔右侧股骨头缺血性坏死模型,并以抽签法随机分为3组：单纯髓芯减压组、髓芯减压＋骨髓间充质干细胞组、髓芯减压＋血管内皮细胞生长因子165/骨形态发生蛋白2转染骨髓间充质干细胞组。关节镜监视下将坏死骨清除,组织学检查成骨及血管生成情况。结果与结论：通过关节镜观察显示各组坏死骨清除干净,有新鲜血流出。组织形态学检测发现,髓芯减压＋血管内皮细胞生长因子165/骨形态发生蛋白2转染骨髓间充质干细胞组移植后不同时间点血管数量及修复区新骨面积比显著高于单纯髓芯减压组、髓芯减压＋骨髓间充质干细胞组（P〈0.05）。提示血管内皮细胞生长因子165/骨形态发生蛋白2基因转染加强了骨髓间充质干细胞成骨作用,提高了新生骨的数量与质量,加快了股骨头缺血性坏死的修复。     

深低温冷冻骨的形态学及免疫组织化学检测

背景:深低温冷冻技术能够降低同种异体骨的免疫原性,对修复骨缺损具有重要意义。目的:观察深低温冷冻对骨材料中骨形态发生蛋白2表达的影响。方法:18只新西兰兔随机分为3组,深低温冷冻3个月组,深低温冷冻6个月组,新鲜骨对照组,取右侧胫骨中上1/3,分别于-80℃保存保存3个月,6个月及不保存,进行苏木精-伊红染色和骨形态发生蛋白2免疫组织化学染色。结果与结论:深低温冷冻后骨材料形态学,与新鲜骨差异不大;深低温冷冻3个月及6个月骨组织中骨形态发生蛋白2均有表达,但较新鲜骨降低。     

聚乳酸/聚己内酯生物可降解人工骨复合骨形态发生蛋白2基因转染骨髓基质干细胞修复骨缺损的血管化过程

学术背景:组织工程骨植入体内后,再血管化是关系其能否充分发挥成骨作用,有效修复骨缺损的关键环节.目的:评价经骨形成蛋白2基因修饰的骨髓间充质干细胞复合聚乳酸/聚己内酯人工骨修复兔桡骨缺损的血管化过程,观察骨形成蛋白2基因对促进移植骨血管化的影响.设计:随机对照动物实验.单位:实验于2005-01/12在中国医科大学中心实验室完成.材料:聚乳酸/聚己内酯生物可降解材料块由中国科学院长春应用化学研究所提供,孔隙直径150～250 μm,孔隙率90%以上;实验动物为3月龄新西兰大耳白兔60只.方法:60只新西兰大耳白兔双侧桡骨中段制成1.5cm骨缺损模型,随机分为4组,每组15只(30侧),植入经不同处理的人工骨.①AD-BMP-2组:转染骨形成蛋白2基因的细胞 聚乳酸/聚己内酯.②对照基因组:转染β-半乳糖酐酶基因的细胞 聚乳酸/聚己内酯.③未转染组:未转染细胞 聚乳酸/聚己内酯.④单纯聚乳酸/聚己内酯组:植入单纯聚乳酸/聚己内酯支架.主要观察指标:于术后4,8和12周行X射线片观察新骨形成情况,立体显微镜观察微血管分布,苏木精-伊红染色观察微血管与骨形成关系,透射电镜观察成骨细胞和血管内皮细胞间的联系,并行血管内皮生长因子表达检测及微血管计数.结果:①AD-BMP-2组术后4周时见移植骨内片状成骨影,有较多新生血管长入,支架孔隙内充满软骨痂,功能活跃的成骨细胞围绕微血管生长,血管内皮生长因子表达及微血管数均明显高于其他各组;术后8周时移植骨内成骨逐渐增多,微血管迂曲扩张并相互连接,软骨痂转变为小梁骨;术后12周时皮质骨连续,髓腔再通,微血管呈规则地纵向排列.②对照基因组和未转染组成骨能力较弱,血管再生缓慢,12周时骨缺损得到初步修复,微血管沿新生骨小梁孔隙分布.③单纯聚乳酸/聚己内酯组各时间点新生血管少见,术后12周时骨端硬化,缺损区被纤维组织填充.结论:骨形成蛋白2基因转染可通过上调血管内皮生长因子表达,间接诱导移植骨血管化,促进种子细胞的成活,加速新骨形成.     

Ex vivo gene therapy in autologous bone marrow stromal stem cells for tissue-engineered maxillofacial bone regeneration

This study examines the clinical relevance of tissue engineering integrating gene therapy and polymer science to bone regeneration. Bilateral maxillary defects (3 x 1.2 cm(2)) in 20 miniature swine were bridged with a bioresorbable internal splint. Constructs were created using ex vivo adenovirus bone morphogenetic protein (BMP)-2-mediated gene transfer to the expanded bone marrow mesenchymal stem cells (MSCs) 7 days before implantation. Controls were performed using adenovirus beta-galactosidase. The BMP-2 cell/construct displayed white solid bone formation after 3 months. Meanwhile, the hematoxylin and eosin and Von Kossa stains demonstrated exhibited mature woven bone with good mineralization. Additionally, three-dimensional computer tomography imaging revealed a nearly complete infraorbital rim repair. Quantitative analysis demonstrated a significant difference (P<0.001) in bone formation. Finally, biomechanical testing revealed no statistically significant difference in the maximal compressive strength of new bone formed by BMP-2 cell constructs and the normal maxilla. The data evidenced de novo bone formation capable of sustaining axial compressive loads. The measurement results showed that ex vivo replication defective adenovirus-mediated human BMP-2 gene transfer to MSCs enhances autologous bone formation in the repair of maxillary defects.     

Targeted Gene-and-host Progenitor Cell Therapy for Nonunion Bone Fracture Repair

Nonunion fractures present a challenge to orthopedics with no optimal solution. In-vivo DNA electroporation is a gene-delivery technique that can potentially accelerate regenerative processes. We hypothesized that in vivo electroporation of an osteogenic gene in a nonunion radius bone defect site would induce fracture repair. Nonunion fracture was created in the radii of C3H/HeN mice, into which a collagen sponge was placed. To allow for recruitment of host progenitor cells (HPCs) into the implanted sponge, the mice were housed for 10 days before electroporation. Mice were electroporated with either bone morphogenetic protein 9 (BMP-9) plasmid, Luciferase plasmid or injected with BMP-9 plasmid but not electroporated. In vivo bioluminescent imaging indicated that gene expression was localized to the defect site. Microcomputed tomography (µCT) and histological analysis of murine radii electroporated with BMP-9 demonstrated bone formation bridging the bone gap, whereas in the control groups the defect remained unbridged. Population of the implanted collagen sponge by HPCs transfected with the injected plasmid following electroporation was noted. Our data indicate that regeneration of nonunion bone defect can be attained by performing in vivo electroporation with an osteogenic gene combined with recruitment of HPCs. This gene therapy approach may pave the way for regeneration of other skeletal tissues.