自体骨髓基质干细胞辅助Mosaicplasty技术促进骨软骨缺损修复整合

目的探讨以基于骨髓基质干细胞(BMSCs)的组织工程技术与自体骨软骨柱镶嵌移植术(Mosaicplasty)相结合的方法修复骨软骨及促进缺损间隙的整合效果。方法12只中国山羊于术前2周抽取骨髓,体外培养自体BMSCs。术中以自制器械分别制造山羊双后肢股骨内髁负重区直径5 mm、深3 mm的复合骨软骨缺损各一处。在Mosaicplasty技术填充缺损后,即以动物自体BMSCs与透明质酸凝胶相复合,注射填充于左后肢骨软骨柱之间及与周围组织的间隙内,右后肢单纯自体骨软骨柱移植作为对照组。术后第4、8、16周分别取材进行组织学、组织化学及蛋白聚糖含量等检测。比较16周时两组的缺损区惨复软骨组织与正常软骨的蛋白聚糖含量。结果两组自体骨软骨柱移植软骨均以透明软骨存活,与周围正常软骨间无明显差异。实验组骨软骨柱的间隙内可见新生软骨修复,组织学表现与周围正常软骨相同。交界区整合良好,间隙消失;对照组各时间点软骨间的间隙为纤维组织或纤维软骨填充,仍有间隙存留。移植软骨的基质、实验组骨软骨柱间隙内的新生软骨基质及Ⅱ型胶原免疫组化染色均为阳性。蛋白聚糖含量比较显示,对照组骨软骨柱间隙内新生组织的蛋白聚糖含量均低于正常软骨和实验组,差异有显著性意义(P< 0．05)。结论基于BMSCs的组织工程技术结合Mosaicplasty技术,可以有效地促进骨软骨缺损间隙的整合,改善修复效果好,有望成为一种理想的促进骨软骨缺损修复的方法。     

骨髓基质干细胞复合藻酸钙凝胶修复全层半月板无血运区缺损

目的 观察骨髓基质干细胞复合藻酸钙凝胶注射式修复全层半月板无血运区缺损的效果.方法 2008年6月至2009年2月制造成年山羊半月板前角无血运区全层缺损模型.以自体骨髓基质干细胞复合可注射藻酸钙凝胶修复半月板缺损(Ⅰ组),同时设立单纯载体组(Ⅱ组)和空白对照组(Ⅲ组).术后4、8、16周处死动物,行大体观察,组织学观察、电镜观察和MRI检查并比较修复效果.结果 Ⅰ组缺损完全被修复组织填充,结合紧密,与正常半月板组织相似,在4～16周效果逐渐改善,大体观察优于其他各组.光镜见细胞随凝胶纤维排列分布,载体纤维间隙大多为细胞分泌基质所充填,细胞排列密集,基质分布均匀.透射电镜见Ⅰ组细胞呈软骨细胞样形态,细胞突起较多,细胞器丰富,细胞为不同走向排列的纤维包绕.MRI检查发现Ⅰ组修复效果较好.结论 骨髓基质干细胞复合藻酸钙凝胶注射可有效地修复全层半月板无血运区缺损.     

骨软骨镶嵌成形术修复骨软骨复合缺损的比较研究

目的观察采用骨软骨镶嵌成形术（Mosaicplasty）修复膝关节中等和大面积骨软骨复合缺损的效果,为临床应用提供理论依据。方法24只成年山羊随机分成3组（n=8）。中等面积缺损组在股骨内髁制造直径6mm缺损,植入直径2mm骨软骨柱修复;大面积缺损组于股骨内髁制造9mm直径缺损,以直径3mm骨软骨柱修复;对照组于股骨内髁制造直径6mm缺损后不修复。自股骨髁间窝和滑车沟两侧非负重区用自制Mosaicplasty器械钻取骨软骨柱,推出器嵌入缺损处镶嵌填满。术后4、8、16及24周处死动物,取修复骨软骨组织行大体观察、HE及甲苯胺蓝染色。术后24周,取大面积缺损组和对照组膝关节摄X线片,观察骨软骨缺损修复情况,并分别取修复组织及正常软骨组织行蛋白聚糖（glycosaminogly cans,GAG）含量测定。结果中等面积缺损组术后4周,移植的骨软骨柱与基底部骨床结合牢固;8-24周软骨层之间以及与正常软骨间界限仍清晰。大面积缺损组术后4周,移植的骨软骨柱与基底骨床结合牢固,部分骨软骨柱被压入骨床内;8-24周压陷程度加重,与股骨髁相对关节面的部分软骨被磨损。对照组24周缺损仍无明显修复迹象,与股骨髁相对关节面的软骨磨损剥脱。组织学观察结果类似大体观察,术后24周中等及大面积缺损组软骨柱间均有缝隙存在,大面积缺损组毗邻软骨细胞稀疏肥大。术后24周,X线片可见大面积缺损组软骨下骨愈合良好,而对照组仍可见骨质缺损,与股骨髁相对关节面的软骨局部骨质硬化;软骨GAG含量测定显示正常软骨和大面积缺损组修复组织间差异无统计学意义（P〉0.05）;前两者与对照组修复组织比较,差异均有统计学差异（P〈0.05）。结论Mosaicplasty可修复中等面积骨软骨复合缺损,但无法有效修复大面积缺损,效果有待改进。     

可注射性藻酸钙凝胶修复整合兔膝关节骨软骨缺损实验研究

目的: 使用骨髓基质干细胞 (BMSCs)、藻酸钙及两种生长因子, 修复兔股骨内髁负重区骨软骨缺损, 寻找合适的关节骨软骨缺损修复方法。方法: 将藻酸钙、体外培养扩增的自体BMSCs及IGF I TGF β复合材料注射于兔膝关节股骨内髁负重区上的骨软骨缺损。结果: BMSCs在藻酸钙中能分化为软骨细胞和成骨细胞, 材料无毒副作用; 关节骨软骨缺损后注入BMSCs 藻酸钙 IGF I TGF β复合体, 各期修复较对照组均有统计学意义 (P<0. 05)。结论: 藻酸钙能为BMSCs提供良好三维生长环境并能使之保持正常形态; BMSCs 藻酸钙 IGF I TGF β复合体能用于关节骨软骨急性缺损修复, 形成正常透明软骨样结构, 软骨下骨修复良好。     

hBMP-2转染自体兔骨髓基质细胞附和异体兔脱钙骨基质的成骨实验研究

目的 探讨人骨发生蛋白2(hBMP-2)转染兔自体骨髓基质细胞(rMSCs)的骨生成诱导作用以及附和异体兔脱钙骨基质(DBM)后的成骨效能。方法 取兔自体骨髓基质细胞培养扩增，用脂质体介导的方法转染人骨发生蛋白2基因，将转染和未转染人骨发生蛋白2基因的自体骨髓基质细胞附和于异体兔脱钙骨基质形成新的生物植骨材料，连同空白及单纯脱钙骨基质对照组植入兔前肢肌袋和桡骨缺损。2、4、6、8周分别取材进行大体、放射线和病理观察。结果 肌袋试验6周后，转染人骨发生蛋白2基因的自体骨髓基质细胞附和异体兔脱钙骨基质材料中出现骨组织细胞，未转染组和单纯异体兔脱钙骨基质组为纤维组织。骨缺损试验中，三组均能产生骨的形成和缺损的修复，但仍然是转染复合材料组的骨修复再生能力最大。结论 局部应用hBMP-2基因转染的rMSCs—DBM材料可以诱导骨形成促进骨修复。     

经碱性成纤维细胞生长因子修饰骨髓基质干细胞注射式修复全层软骨缺损

目的 探讨经碱性成纤维细胞生长因子(bFGF)修饰的骨髓基质干细胞(BMSCs)注射式修复全层软骨缺损的可行性.方法 体外培养家兔自体骨髓基质干细胞,并以bFGF处理修饰细胞,免疫组织化学Ⅱ型胶原蛋白表达、逆转录-聚合酶链反应(RT-PCR)检测蛋白聚糖表达.将其与藻酸钙凝胶支架复合注射式植入兔股骨髁全层软骨缺损处,同时设立凝胶支架对照组和空白对照组.术后8周取材观察修复效果,并行苏木素-伊红(HE)、甲苯胺蓝、Ⅱ型胶原免疫组织化学染色检测;透射电镜观察修复组织的微观结构.结果 BMSCs的细胞群体倍增时间(PDT)为33.8 h,经bFGF处理的BMSCs可检测到Ⅱ型胶原和蛋白聚糖的表达.至术后8周可见质硬的类白色修复组织完全充填软骨缺损处,组织学检查可见大量软骨样细胞分布于深染的细胞外基质中,检测到Ⅱ型胶原的表达.透射电镜可见丰富的细胞器和胞外基质.凝胶支架对照组和空白对照组仅有部分质软组织充填缺损处,未检测到Ⅱ型胶原的表达.结论 经hFGF修饰的自体骨髓基质干细胞藻酸钙凝胶复合物可用于修复全层软骨缺损.     

藻酸钙复合自体软骨细胞修复羊膝关节负重区软骨缺损的实验研究

[目的]以中国山羊为动物模型,观察藻酸钙复合自体软骨细胞修复膝关节负重区软骨缺损的可行性。[方法]取羊肩关节软骨,分离、培养软骨细胞,蕃红"O"、 Giemsa及Ⅱ型胶原免疫组织化学染色对其进行鉴定。将自体软骨细胞与藻酸钙凝胶复合,修复山羊股骨髁负重区全层软骨缺损(直径6 mm),实验分为四组:(1)缺损旷置组:缺损内未植入任何组织;(2)骨膜覆盖组:自体骨膜覆盖缺损区;(3)藻酸钙+骨膜组:凝胶植入软骨缺损区,并用自体骨膜覆盖;(4)藻酸钙+细胞+骨膜组:藻酸钙复合自体软骨细胞植入软骨缺损区,自体骨膜覆盖;分别于手术后3、6个月取材,通过大体观察及组织学评分检测修复效果。[结果]软骨细胞复合物蕃红"O"、 Giemsa染色及Ⅱ型胶原免疫组化染色结果均为阳性,将藻酸钙凝胶-软骨细胞复合物用于羊负重区关节面软骨缺损修复,从大体观察和组织学评分进行比较,发现各组均有不同程度的组织修复,藻酸钙+细胞+骨膜组效果最好,与其他组差异有统计学意义(P<0.05)。[结论]藻酸钙凝胶-软骨细胞复合物结合自体骨膜覆盖,可较好修复山羊膝关节负重区软骨缺损。     

含自体富集骨髓干细胞松质骨移植与自体松质骨镶嵌移植修复关节软骨缺损的对比研究

目的：评价含富集骨髓干细胞松质骨镶嵌移植与自体松质骨镶嵌移植两种方法修复兔膝关节骨软骨缺损的修复效果,为临床应用提供实验依据。方法：选健康3月龄新西兰兔16只,随机分成A、B两组,每组8只16膝。A组采用含富集自体骨髓干细胞的自体松质骨移植,B组采用自体松质骨移植。术后第12周处死动物取材,分别进行大体观察、光镜观察,并对观察指标采用Wakitani组织学评分法进行评分和秩和检验。结果：大体观察及光镜观察显示含富集骨髓干细胞松质骨镶嵌移植组大体观察关节面大多呈乳白色,表面比较平整,与周围软骨融合较好,未发现裂隙;光镜检查见关节软骨的厚度为正常软骨的2/3左右,表面较光滑,与周围组织接合较好,大多为透明软骨细胞;组织学评分为（4.44±1.41）分。自体松质骨镶嵌移植组大体观察见关节面呈灰白色,表面稍有凹陷,欠光整;光镜检查见关节软骨的厚度较薄,为正常软骨的1/3～1/2,移植物中央区域厚度更薄,见成纤维细胞和极少量透明软骨细胞;组织学评分为（8.93±1.18）分。两组组织学评分比较差异有统计学意义（P〈0.01）。结论：含富集骨髓干细胞松质骨镶嵌移植能以类透明软骨组织修复全层关节软骨缺损,可用于较大面积软骨缺损的修复。自体松质骨移植修复软骨缺损的能力较差。     

骨形成蛋白-2转染兔骨髓基质细胞修复软骨缺损的研究

目的 观察骨形成蛋白-2(BMP-2)基因转染兔骨髓基质细胞(MSCs)复合藻酸钙修复兔膝关节软骨缺损的效果.方法 取4周龄的新西兰兔骨髓细胞,体外培养得到MSCs.选用24只成年新西兰兔,在两侧股骨髁非负重区造成全层软骨缺损模型,A组(实验组)植入BMP-2转染的MSCs+藻酸钙,B组(对照组Ⅰ)植入空质粒转染的MSCs+藻酸钙,C组(对照组Ⅱ)植入不含MSCs的藻酸钙,D组(对照组Ⅲ)旷置.12周后,观察软骨缺损修复情况及组织学评价.结果 实验组修复组织基本光滑,有大量Ⅱ型胶原蛋白表达,3个对照组修复组织Ⅱ型胶原蛋白表达量少,仍有明显缺损.实验组和3个对照组组织学评分差异有统计学意义(P＜0.05).结论 BMP-2转染兔MSCs修复软骨缺损效果优于其他对照组.     

体内构建骨软骨复合体修复骨软骨缺损的实验研究

[目的]探讨软骨组织工程方法修复骨软骨缺损理想的种子细胞和支架材料。[方法]体外诱导骨髓基质干细胞向软骨细胞定向分化，分别与PLGA支架复合培养，并模仿马赛克骨软骨移植术在犬关节骨软骨缺损深层置入MSC-支架复合体，浅层置入诱导培养后的MSC-支架复合体，紧密压配，体内构建骨软骨复合体，观察其修复的情况。[结果]16周实验组缺损区完全由透明软骨修复，与周围正常软骨完全融合，组织观察显示以软骨细胞修复为主，软骨下骨形成，潮线基本恢复；阴性对照组缺损区主要由纤维组织修复，部分由透明软骨修复，组织观察显示以纤维细胞修复为主，混有部分软骨细胞；空白对照组完全由纤维组织修复。[结论]MSCs经向软骨细胞定向分化后复合PLGA支架材料，通过紧密压配方式与MSCs-PLGA支架在体内构建骨软骨复合体，可以有效修复骨软骨缺损。     

Mosaicplasty associated with gene enhanced tissue engineering for the treatment of acute osteochondral defects in a goat model

Objective  To compare single mosaicplasty, mosaicplasty associated with gene enhanced tissue engineering and mosaicplasty associated with the gels of non-gene transduced bone mesenchymal stem cells (BMSCs) in alginate for the treatment of acute osteochondral defects in a goat model. Methods  The principle and methods of tissue engineering were used. BMSCs were separated and amplified in vitro, and human transforming growth factor-β1 (hTGF-β1) gene was transduced to the cells. Then, the cells were suspended in the alginate. At the same time using mosaicplasty to repair the defects on the medial femoral condyle, the dead space between the cylindrical grafts were filled with the gels of hTGF-β1 gene transduced BMSCs in alginate. Single mosaicplasty and mosaicplasty associated with the gels of non-gene transduced BMSCs in alginate were compared by the different time observation. Results  All of the three treatments could repair the acute osteochondral defects. Mosaicplasty associated with gene enhanced tissue engineering had a better integration than single mosaicplasty, and mosaicplasty associated with the gels of non-gene transduced BMSCs in alginate. Conclusion  Mosaicplasty associated with tissue engineering could solve the problem of the poor concrescence of the remnant defect and the integration of single mosaicplasty.     

胶原/羟基磷灰石支架负载软骨细胞修复兔膝关节骨软骨缺损

目的 探讨胶原复合梯度羟基磷灰石(Col/HA)双相支架负载软骨细胞修复兔膝关节骨软骨缺损的可行性及疗效.方法 构建Col/HA双相支架,将软骨细胞种植于支架培养1周,再将软骨细胞-支架复合体移植修复兔膝关节股骨髁的骨软骨缺损,并对骨软骨缺损的修复进行检测.结果 光镜及扫描电镜观察显示软骨细胞在Col/HA支架中贴附良好,表型维持稳定,分泌胞外基质.大体观察和组织学检测显示,植入体内16周后实验组软骨层呈透明软骨样修复,软骨下骨缺损有新骨构建;对照组骨软骨缺损修复不良,组织学检测以纤维性组织或纤维软骨组织形成.Wakitani评分显示实验组修复组织优于对照组,差异有统计学意义(P＜0.05).结论 双相Col/HA复合支架可作为骨软骨组织工程支架,负载软骨细胞可修复兔膝关节骨软骨缺损,重建关节软骨的结构和功能.     

自体骨软骨移植治疗股骨髁关节软骨缺损

目的探讨关节镜下自体骨软骨移植治疗关节软骨缺损的可行性。方法16例膝关节软骨缺损患者,关节镜下在其非负重区的软骨面上用专用器械凿取圆柱状骨软骨,移植至软骨缺损部位以修复缺损。术后行系统功能锻炼和MRI检查。结果随访7～20个月,患者关节症状消失,关节活动度正常,MRI显示原关节软骨缺损区表面平整,移植骨软骨位置良好。Brittberg-Peterson评分：13例0分,2例2分,1例1分。结论关节镜下自体镶嵌式骨软骨移植术创伤小,操作简单,能保持关节面曲度,可用于修复关节软骨缺损。     

Fate of transplanted bone-marrow-derived mesenchymal cells during osteochondral repair using transgenic rats to simulate autologous transplantation

OBJECTIVE: The fate of transplanted cells used in tissue engineering strategies should be followed. With this aim in view, the survival of transplanted bone-marrow-derived mesenchymal cells within osteochondral defects was determined using transgenic rats to simulate autologous transplantation. DESIGN: An autologous transplantation model was simulated using transgenic rats - whose transgenes produce no foreign proteins - as donors, and wild-type rats as recipients. Dense masses of mesenchymal cells were prepared from the transgenic rats using the hanging-drop culture technique. These cell masses were then transplanted into osteochondral defects created within the medial femoral condyle of wild-type rats, wherein they are affixed with fibrin glue. The course of repair was assessed histologically. The survival of the transplanted cells was ascertained by in situ hybridization of the transgenes. RESULTS: Twenty-four weeks after transplantation, the defects were repaired with hyaline-like cartilage, which was thicker than normal, and with subchondral bone. Using the in situ hybridization technique, cells derived from the transplanted ones were detected within both the cartilaginous and the subchondral bone layers. CONCLUSION: Using this simulated autologous transplantation model, the survival of transplanted mesenchymal cells was monitored in vivo. The findings indicate that the transplanted mesenchymal cells contributed to the repair of the osteochondral defects.     

不同比例骨软骨镶嵌成形术联合基因增强组织工程方法治疗骨软骨缺损

目的：研究骨软骨镶嵌成形术与联合基因增强组织工程方法治疗急性骨软骨缺损,并观察在不同比例下的组织修复情况,以期发现两者间的最佳组合。方法：对携带hTGF-β1的重组腺病毒转染BMSCs（hTGF-β1转染组）,与采用Adv-βgal转染BMSCs（Adv-βgal转染组）及未转染BMSCs（未转染空白对照组）进行WesternBlot检测hTGF-β1、Ⅱ型胶原及Aggrecan表达。雄性6月龄崇明山羊18只,体重22～25kg,取自体骨髓进行BMSCs分离及培养,传至第3代。每只动物双膝股骨内髁进行实验,分为AR、AL、BR、BL、CR、CL6组。AR为骨软骨移植覆盖面积为44.44%单纯组,AL为44.44%联合组,BR为33.33%单纯组,BL为33.33%联合组,CR为22.22%单纯组,CL为22.22%联合组。于双膝股骨内髁负重区采用骨钻制备直径为9mm,深为3mm的骨软骨缺损后,单纯组采用骨软骨镶嵌成形术的自体骨软骨柱修复,联合组同时将转染hTGF-β1的BMSCs藻酸钠混合液注入空隙,加入氯化钙产生凝胶。术后24周取材,行大体及组织学观察,并参照O＇Driscoll,KeeleyandSalter组织形态学评分标准进行评分,行免疫组织化学及透射电镜观察。结果：hTGF-β1转染组细胞hTGF-β1、Ⅱ型胶原及Aggrecan表达均强于Adv-βgal转染组及未转染空白对照组。大体观察,组织学染色以及透射电镜检查显示各组的缺损有不同程度的修复。33.33%联合组、44.44%单纯组、44.44%联合组的整体修复效果差别无统计学意义,33.33%单纯组、22.22%单纯组、22.22%联合组的整体修复效果差于前3组。结论：骨软骨镶嵌成形术联合基因增强组织工程的方法可以有效修复骨软骨缺损。随着自体骨软骨移植覆盖缺损面积的减少,骨软骨镶嵌成形术联合基因增强组织工程方法的修复优越性可以得到更好的体现。     

Tracing transduced cells in osteochondral defects

In this study the authors explored the feasibility of using transduced cells for gene therapy to induce healing of osteochondral defects. Both a mouse mesenchymal cell line and mixed rabbit adherent stromal cells were transduced with either liposomal transfection or retroviral transduction using a traceable gene. Transduction efficiency was more than 95% with the retroviral construct and expression was maintained for over 6 months of passage. The liposomal transfection led to a transient expression with an efficiency of 50%. The expression of osteochondral genes was diminished but preserved after transduction in vitro. Transduced rabbit cells were transplanted into osteochondral defects in rabbit femoral condyles. Cells transplanted in vivo could be detected for 4 weeks in the repair tissue. The authors' data demonstrate that mesenchymal cells from bone marrow, stably transduced with a traceable gene product, retain the bone and cartilage phenotype and can be followed in vivo after transplantation into cartilage defects.     

Value of osteo-chondral paste autologous transplantation in experimental cartilage defects reconstruction. Part III--Microscopic analysis of reconstructed cartilage thickness and surface regularity]

INTRODUCTION: A limited ability of the cartilage to heal after trauma was the reason to start research on new methods concerning better cartilage reconstruction. The aim of the study was evaluation of repair tissue thickness and surface regularity after osteochondral paste transplantation. MATERIAL AND METHODS: Full thickness defect (IV(o)--ICRS scale) on distal rabbit femur joint surface was made. Three groups were specified: A--defect with paste graft (cartilage and contiguous bone collected from joint surface, crushed into homogenous paste; B--defect with the paste graft covered with periosteum; C--defect left unfilled. The follow-up periods were established at 4, 8, 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale. RESULTS: Newly formed tissue was well integrated with surrounding cartilage in group A (paste graft). That trait of repair tissue in group A was much better than in other groups, especially in late observations. Structural integrity of tissue filling the defect was similar to integrity of normal cartilage in groups A and C, but tissue formed in group C didn't represent a hyaline-like cartilage character. In all the examined groups reconstruction of subchondral bone exhibited similar rate. 12 weeks from the procedure, around 80% of subchondral bone was rebuilt. The obtained results indicate, that osteochondral paste autologous transplantation in cartilage defects treatment effects with forming well integrated (structurally and with surrounding cartilage) cartilage tissue, of almost complete subchondral bone rebuilding.     

Integration of periosteum covered autogenous bone grafts with and without autologous chondrocytes. An animal experiment using the Göttinger minipig

Autologous osteochondral transplantation has the major disadvantage of significant damage to a healthy joint surface at the donor site.The purpose of this study was to examine the effect of autogenous chondrocytes injected into the periosteum of autologous bone grafts in order to provide an alternative method for cartilagerepair. A total of 22 Göttinger minipigs were operated twice on both knees.The first operation served for cartilage biopsy for the chondrocyte culture.During the second operation an osteochondral defect was created in the medial facet of the trochlear groove.The defect was treated differently with an autologous cortico-cancellous bone cylinder,harvested from the proximal tibia.Group A: untreated defect (control);B: bone-graft;C: bone-graft covered with periosteum; D: bone-graft with periosteum and injected autologous chondrocytes.The animals were killed after 6,12,26 and 52weeks.The regenerated areas were evaluated macroscopically, tested biomechanically (long-term specimens; indentation-test) and a histological, blind evaluation was carried out according to a semi-quantitative scoring system. The periosteum covered bone cylinders in Groups C and D showed good repair of the bone and cartilage defect.The repaired tissue consisted predominantly of fibrocartilage with the partial formation of hyalin like tissue.The regenerated areas were integrated with the adjacent cartilage and were biomechanically superior when compared with the other groups. The additional injection of chondrocytes did not produce significantly better results. Our findings suggest that the transplantation of periosteum-covered bone cylinders may provide an alternative method for treating chondral and osteochondral defects and can be recommended for filling large donor site defects in joint surgery.The additional transplantation of chondrocytes does not seem to be justified.