自体骨膜游离移植的实验研究--不同龄动物大块关节软骨缺损修复的比较

目的研究比较自体骨膜移植软骨再生修复不同龄动物大块关节软骨缺损。方法用52只不同龄家兔自体骨膜游离移植修复大块关节软骨缺损,比较移植骨膜生发层朝向关节腔与松质骨时再生软骨的差别。结果经不同时期肉眼和组织学检查证实,幼年兔和成年兔的骨膜移植都能生成软骨,修复大块关节软骨缺损。在成年兔骨膜再生的软骨与成年兔本身周围正常软骨的厚度、组织结构一样。移植骨膜生发层朝向关节腔与松质骨二者间再生软骨结果无明显差别。结论骨膜具有再生软骨的能力,可用来移植修复关节软骨的缺损。骨膜移植生发层不同朝向对软骨再生无明显影响。成年后骨膜移植修复关节软骨缺损能够生成与自身相适应的软骨。     

活动对自体骨膜游离移植修复关节软骨缺损的影响实验研究

为了探索活动对骨膜游离移植修复关节软骨缺损的影响，选用成年健康家兔６０只，随机分三组：笼外活动组，笼内活动组及制动组。手术造成双后肢骨宾股关节股骨关节面０．５ｃｍ×１ｃｍ全层软骨缺损各一处，将自体胫骨骨膜游离移植于左侧软骨缺损处，右侧作为对照。分别于术后第４，８和１２周处死动物，取关节软骨标本。通过大体组织学、组织化学及电镜观察软骨再生情况，并比较活动量的多少对骨膜化生软骨的影响。结果表明，游离骨膜产生类透明软骨能有效地修复全层关节软骨缺损；统计学分析，笼外活动组与笼内活动组差异显著（Ｐ＜０．０５），笼内活动组与制动组差异显著（Ｐ＜０．０５），笼外活动组与制动组差异高度显著（Ｐ＜０．０１）。证明，活动量的大小对骨膜化生软骨有着重要的影响     

筋膜蒂骨膜移植修复关节软骨缺损的实验研究

目的：应用筋膜蒂骨膜移植修复关节软骨缺损，观察骨膜在关节腔内再生关节软骨的情况，探讨骨膜再生软骨的机制。方法：用成年家兔２２只，在右侧膝关节造成胫骨关节面软骨缺损，行筋膜蒂骨膜移植；在左侧膝关节行游离骨膜移植，作为对照。观察方法包括形态学观察、Ｘ线和组织学检查。结果：实验组再生关节软骨的质与量均优于对照组。结论：筋膜蒂骨膜移植，由于有血液供应，能够再生关节软骨，修复关节软骨缺损。     

兔自体耳软骨膜复合游离移植形成工程化软骨的实验研究

目的：探求用软骨腹复合移植的方法形成自体软骨的可行性。方法：将兔自体耳软骨膜包裹异体耳软骨脱细胞基质材料复合游离移植于兔背部皮下（I组），并在移植物附近皮下游离移植双层耳软骨膜（Ⅱa组）及单纯脱细胞的异体耳软骨基质（Ⅱb组）作为对照。在术后3周、6周分别取标本进行研究。结果：在3周、6周时，I组与Ⅱa组成软骨细胞的增殖变化情况差异有显性（P＜0．05），I组比Ⅱa组成软骨细胞的增殖明显旺盛，有的已经向软骨细胞转化，并有少量细胞外基质分泌，基质内软骨囊出现。Ⅱb组无成软骨细胞及软骨囊出现。结论：自体耳软骨膜与异体耳软骨脱细胞后的基质材料复合移植促进自体软骨生成。提示该基质材料有促进软骨膜细胞分化和生长的作用。     

冷冻保存胎兔颅骨骨膜修复关节软骨缺损的实验研究

为探讨关节软骨缺损的修复方法，采用家兔３０只，平均分成两组。在每个动物的双侧髌股关节股骨关节面作４ｍｍ×７ｍｍ全层软骨缺损。将经二步冷冻法保存的胎兔颅骨骨膜移植于一组动物一侧后肢作为实验组，另一侧用自体骨膜移植作为自体对照组。在另一组动物的一侧后肢用新鲜胎兔颅骨骨膜移植作为新鲜对照组，另一侧关节软骨缺损不修复作为空白对照组。术后肢体不作外固定。１６周后取材，经大体观察、组织学观察及氨基酸成分分析。结果发现，经冷冻保存的胎兔颅骨骨膜移植后能形成透明样软骨，与自体对照组无显著性差异（Ｐ＞０．０５），与新鲜对照组及空白对照组主要指标有显著性差异（Ｐ＜０．０５）。氨基酸分析示实验组新生物接近纤维软骨（Ｐ＞０．０５）。认为，冷冻胎兔颅骨骨膜移植为关节软骨缺损的修复提供了实验依据。     

带血运骨膜管移植和骨充填物修复桡骨长段缺损的研究

目的：探讨联合应用带血运骨膜管移植和骨充填物治疗兔桡骨长段缺损的效果。方法：实验分两部分，分别选用幼兔和成年兔各40只，根据填充物的不同分为4组，将兔双侧桡骨干中段切除3cm制成骨长段缺损模型，保留切骨段骨膜，重新重原缝合后作带血运骨膜管移植模型，左侧分别用自体骨，同种异体脱钙骨，磷酸三钙陶瓷和羟基磷灰石进行填充，右侧不行任何填作为对照。观察3个月。通过X线片，髓强度，骨密度和组织学检查等方法，了解骨缺损的修复效果。结果：幼兔术后6周，所有实验组双侧的骨缺损均得到修复，术后12周，磷酸三钙陶瓷和羟基磷灰石组桡骨抗弯曲强度较差与自体骨组、同种异体脱钙骨组和对照侧比较具有统计学意义（P＜0．05）；骨愈合为膜内成骨和软骨成骨，以膜内成骨为主，成年兔；各组实验侧骨缺损修复率分别为：自体骨组50％；同种异体脱钙骨组40％；磷酸三钙陶瓷和羟基磷灰石组为30％。对照侧骨缺损修复率为42．5％，结论：幼兔单行单血运骨管移植或结合应用骨充填物均可有效修复骨长段缺损，但置换较慢的骨充填物不利于再生骨强度的恢复，成年兔带血运骨膜移植联合应用骨填充物不能有效修复骨长段缺损。     

自体软骨细胞团块植入修复兔陈旧性关节软骨缺损的实验研究

目的 观察自体软骨细胞团块植入对兔关节软骨缺损的修复作用. 方法 24只成年新西兰大白兔48侧膝关节,随机分为三组(n=16)并制备双膝关节股骨滑车软骨缺损模型.空白对照组无特殊处理,骨膜移植组将骨膜覆盖缺损并缝合于缺损两侧的股骨髁上,实验组将自体软骨细胞团块植入缺损中.术后3、6个月分别取材(n=8),进行大体和组织学观察,修复组织行Wakitani评分并进行比较. 结果实验组共成功取材11个缺损关节,9个为透明软骨修复,2个因植入细胞生长状态差未修复;骨膜移植组修复组织为纤维软骨或纤维组织,修复组织薄,基质异染弱;空白对照组仅有少量纤维组织填充缺损底部.修复组织Wakitani评分:实验组3.82分,骨膜移植组6.71分,空白对照组9.23分,差异有统计学意义(F=5.96,P=0.00). 结论自体软骨细胞团块植入能较好修复关节软骨缺损,修复的质量与植入细胞的质量有关.     

自体软骨细胞移植修复猪膝关节软骨缺损的实验研究

目的评价传统和复层高密度培养（复层培养）的自体软骨细胞移植修复关节软骨缺损的效果。方法在8头猪16膝髌切迹上下共建立32个全层软骨缺损，右膝为空白和自体骨膜移植对照组，左膝为传统的自体骨膜覆盖下细胞注射移植组和自体骨膜覆盖下复层培养细胞移植组（上下缺损随机进入各对照和实验组）。术后5-6个月对缺损部位行大体、组织学、免疫组织化学检查。采用O’Driscoll软骨组织形态学评分评价软骨缺损的修复质量。结果四组的O’Driscoll软骨组织形态学评分分别为（3．14±1．95）分、（10．57±3．60）分、（16．29±2．63）分、（20．43±1．81）分，各组之间差异均有统计学意义（P〈0．01）。空白对照组缺损被少量的纤维组织覆盖；骨膜移植组被纤维组织和少量的纤维软骨修复，且与周围组织整合差。自体软骨细胞移植（注射组和复层培养组）缺损被塑形良好的修复组织覆盖，组织学、基质特殊染色示修复组织为纤维软骨和透明软骨，与周边软骨和软骨下骨整合良好。复层培养组修复组织的细胞形态、基质染色比注射细胞组更接近正常软骨。结论自体软骨细胞移植可成功修复全层关节软骨缺损。扩增后的软骨细胞经短期复层培养更有利于软骨缺损的修复。     

骨髓间充质干细胞复合消旋聚乳酸/明胶修复兔关节软骨缺损的实验研究

目的 研究骨髓间充质干细胞（marrow mesenchymal stem cells，MSCs）复合消旋聚乳酸（poly-L-lacticacid，PLLA）／明胶修复兔膝关节全层软骨缺损的效果。方法 4～6月龄青紫兰兔36只，体重2．5～3．5kg，雌雄不拘。体外分离培养MSCs，取第2代MSCs种植于PLLA／明胶支架体外复合培养。将36只青紫兰兔制备双侧膝关节全层软骨缺损模型，根据修复方法不同随机分为A、B、C3组（n=12）。A组，将MSCs与PLLA／明胶支架材料复合物植入兔双膝缺损处；B组，将单纯PLLA／明胶支架材料植入兔双膝缺损处；C组，缺损处不作任何处理，作为对照。A、B组在植入时均加入25μg／L转化生长因子β（transforming growth factorp1，TGF-β1）0．4ml。分别于术后4、8和12周，取材行大体、组织学及免疫组织化学染色观察，并将12周大体及组织学标本按照O’driscoll等评分标准进行评分。结果大体观察：术后12周，A组修复组织与正常软骨结合处完整，表面光滑，界限模糊；B、C组缺损处修复组织呈纤维组织或无修复，表面不平整或呈虫蚀样改变。组织学观察：A组术后4周，细胞数较多，呈梭形、圆形或椭圆形；8周修复组织与周围软骨大部分结合，细胞呈圆形，以透明软骨样细胞为主，有软骨陷窝，表层有梭形纤维样细胞；12周修复组织细胞为透明软骨样细胞，柱状排列，与周围软骨及软骨下骨整合良好；B组，术后4～8周细胞数较少，细胞层次排列差；12周修复组织菲薄，呈纤维软骨样，基质染色接近正常；C组，术后4～8周缺损组织由薄层纤维组织覆盖；12周缺损区纤维组织进一步增厚，与周围软骨未结合。免疫组织化学染色显示，A组修复组织Ⅱ型胶原染色呈阳性，B组呈弱阳性，C组无表达。术后12周大体观察总评分，A、B及C组分别为2．75±0．89、4．88±1．25和7．38±1．18，A组优于B、C组，B组优于C组，且差异均有统计学意义（P〈0．05）；组织学观察总评分，A、B及C组分别为3．88±1．36、8．38±1．06和13．13±1．96，A组与B、C组以及B组与C组差异均有统计学意义（P〈0．05）。结论 应用软骨组织工程原理，以PLLA／明胶为支架材料复合自体MSCs移植是一种修复软骨缺损行之有效的方法。     

"双相"组织工程软骨修复兔关节骨软骨缺损

目的探讨“双相”异体骨基质明胶（bonematrixgelatin,BMG）作为组织工程软骨载体,与同体骨髓间充质干细胞（marrowmesenchymalstemcells,MSCs）结合,构建组织工程软骨修复兔关节骨软骨缺损的效果。方法4月龄新西兰兔32只,雌雄不限,体重2～3kg。①体外实验：取5只新西兰兔,处死后取髂骨和四肢骨,制备一侧松质骨,一侧皮质骨的“双相”异体BMG载体,扫描电镜观察。另取新西兰兔18只,抽取骨髓,分离MSCs并诱导成软骨分化;将诱导而来的软骨前体细胞与“双相”BMG载体复合构建组织工程软骨,分别于1、3和5周取材行Masson、PAS染色和扫描电镜观察。②体内实验：将抽取骨髓的18只及余下的9只新西兰兔制成双侧股骨内髁骨软骨缺损模型,将前期制备的组织工程软骨同体植入18只兔的右股骨内髁骨软骨缺损（A组）,左侧缺损移植异体BMG（B组）,其余9只双侧软骨缺损未予处理作为空白对照（C组）,分别于术后1、3和6个月取材,行大体、组织学和Ⅱ型胶原mRNA原位杂交观察,改良Wakitani法评分,比较各组修复效果差异。结果①体外实验：“双相”BMG松质骨面孔隙大小100-800μm,细胞于其中增生,形成富含细胞的软骨层;皮质骨面孔隙大小10～40pm,细胞层状覆盖于其表面,可作为起支撑作用的软骨下骨。②体内实验：A组术后1个月即可重建关节骨软骨缺损;修复软骨在观察期内逐渐变薄,但在6个月内始终保持关节面及软骨下骨结构完整。B、C组未能修复缺损,缺损周边软骨磨损加剧。改良Wakitani评分显示A组在3个时间点的各项评分结果,除6个月软骨厚度外,其它指标均优于B、C组,且差异有统计学意义（P〈0．01）。Ⅱ型胶原mRNA原位杂交显示,A组缺损区修复组织中细胞阳性染色率明显高于B、C组,且差异有统计学意义（P〈0．01）。结论“双相”异体BMG可作为组织工程软骨载体材料,其结合自体MSCs诱导的软骨前体细胞制备的组织工程软骨,可修复兔关节软骨和软骨下骨。     

Reconstruction of Articular Cartilage Defects with Free Periosteal Grafts: An Experimental Study

The chondrogenic potential of free autogenous periosteal grafts was studied histologically in 6-month-old rabbits. The grafts were taken from the tibia and transplanted to 7 × 14 mm large artificial defects of the femoral articular cartilage. The results revealed that the defects were repaired and filled after 4 weeks with a hyaline-like cartilage which was histologically similar to the cartilage adjacent to the transplant. The tissue maintained this morphology after 1 year of observation. In control animals where no periosteum was transplanted to the defect, no real cartilage was found. The tissue which partially filled the defect was a variable mixture of fibrous tissue and fibrocartilage.     

Comparative Study of the Reconstruction of Articular Cartilage Defects with Free Costal Perichondrial Grafts and Free Tibial Periosteal Grafts: An Experimental Study on Rabbits

The purpose of this study was to compare the chondrogenic potential of free perichondrial with free periosteal grafts in the resurfacing of full-thickness defects of patellar articular cartilage in rabbits. We used adolescent New Zealand rabbits weighing between 2.4 and 3.6 kg. A 6-mm wide and 3-mm thick defect was created on the patellar articular surface. A total of 30 rabbits were randomly divided into a control group and two test groups. One test group received free perichondrial grafts (PC); the other received free periosteal grafts (PO). All the animals were killed 8 weeks after surgery. All the histological samples were scored from 0 to 17 according to a standard scoring system. Differences in the quality of the regenerated tissue were only found between the control and the test groups. There were no statistically significant histological differences between the grafted defects of the PC and the PO groups that there are not on any of the variables. The results of this study support that there are not significant differences in the quality of the repair tissue when using these two types of biological grafts. Received: 7 July 1998 / Accepted: 8 January 1999     

新鲜同种异体骨软骨移植修复软骨缺损

目的联合应用新鲜同种异体骨软骨移植,和局部注射碱性成纤维细胞生长因子(basic fibroblast growthfactor,bFGF),探讨能否促进关节软骨缺损区新生软骨的形成,提高软骨缺损修复的成功率。方法48只青紫兰兔,96个实验关节,随机分为A、B、C、D组。无菌条件下制作骨软骨缺损模型。在A组缺损区单纯植入新鲜的同种异体骨软骨,B组单纯局部注射重组人bFGF,C组局部注射bFGF后同时植入新鲜的同种异体骨软骨,D组用作空白对照。术后第4、8、12周作大体观察、X线摄片、组织学检查及免疫组化检查。结果移植加注射bFGF组促进软骨缺损修复的效果均好于其他组,图像分析仪进行软骨细胞记数有显著差异(P<0.05),有统计学意义。修复软骨型胶原免疫组化染色强阳性。结论采用新鲜的同种异体骨软骨移植及联合应用碱性成纤维细胞生长因子,二者能起交互作用,促进了新生软骨的形成。     

Transplantation of free tibial periosteal grafts for the repair of articular cartilage defect: An experimental study

Background:

Articular chondrocytes have got a long lifespan but rarely divides after maturity. Thus, an articular cartilage has a limited capacity for repair. Periosteal grafts have chondrogenic potential and have been used to repair defects in the articular cartilage. The purpose of the present study is to investigate the differentiation of free periosteal grafts in the patellofemoral joint where the cambium layer faces the subchondral bone and to investigate the applicability of periosteal grafts in the reconstruction of articular surfaces.

Materials and Methods:

The study was carried out over a period of 1 year on 25 adult, male Indian rabbits after obtaining permission from the institutional animal ethical committee. A full-thickness osteochondral defect was created by shaving off the whole articular cartilage of the patella of the left knee. The defect thus created was grafted with free periosteal graft. The patella of the right knee was taken as a control where no grafting was done after shaving off the articular cartilage. The first animal was used to study the normal histology of the patellar articular cartilage and periosteum obtained from the medial surface of tibial condyle. Rest 24 animals were subjected to patellectomy, 4 each at serial intervals of 2, 4, 8, 16, 32 and 48 weeks and the patellar articular surfaces were examined macroscopically and histologically.

Results:

The grafts got adherent to the underlying patellar articular surface at the end of 4 weeks. Microscopically, graft incorporation could be appreciated at 4 weeks. Mesenchymal cells of the cambium layer were seen differentiating into chondrocytes by the end of 4 weeks in four grafts (100%) and they were arranged in a haphazard manner. Till the end of 8 weeks, the cellular arrangement was mostly wooly. At 16 weeks, one graft (25%) had wooly arrangement of chondrocytes and three grafts (75%) had columnar formation of cells. Same percentage was maintained at 32 weeks. Four grafts (100%) at 48 weeks showed columnar orientation. The control side showed no changes over the shaved off articular surface in all the rabbits. One rabbit at 4 weeks had a dislocation of the patella on the control side. None of the rabbits developed any infection or wound dehiscence.

Conclusion:

Autologous periosteal graft transplantation can be a promising substitute for articular cartilaginous defects.     

CS/PVA凝胶负载Ad-hTGF-β1转染的BMSCs移植修复兔关节软骨缺损

目的 探讨温敏型CS/PVA凝胶负载Ad-hTGF-β1转染的骨髓间充质干细胞(BMSCs)移植修复兔关节软骨缺损的实验效果.方法 体外分离培养兔BMSCs,在Ad-hTGF-β1转染1周后,用细胞免疫化学方法检测hTGF-β1在细胞内的表达.用24只成年新西兰大白兔制造关节软骨缺损模型,双侧后肢均用于实验,动物模型随机分为4组,各组动物6只.A组:凝胶复合转染BMSCs修复组;B组:凝胶复合未转染BMSCs修复组;C组:凝胶修复组;D组:空白对照组.在术后16周时处死动物取材,通过大体标本和组织学染色观察评价各组修复效果,按照改良Pinoda法评分,对各组修复效果进行统计学分析.结果 免疫组化证实体外培养的BMSCs在Ad-hTGF-β1转染后表达hTGF-β1蛋白,阳性率为85.4%.术后16周取材见凝胶复合转染细胞组关节软骨缺损部位为软骨样组织填充,组织学观察见再生的软骨组织细胞排列及细胞密度与正常软骨相似,Ⅱ型胶原免疫组化阳性,Pineda评分同其它各组相比差异有统计学意义(P＜0.05).结论 CS/PVA凝胶作为一种温敏型可注射支架材料,其负载hTGF-β1转染的BMSCs移植可用于兔关节软骨缺损修复.

Abstract：

Objective To investigate the experiment effects of rabbit joint articular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cells. Methods Bone marrow mesenchymal stem cells were isolated and cultured in vitro. The positive rate of transfection was defected by cell immunohistochemistry methods after Ad-hTGF-β1 transfected for 1 week. Twenty-four adult New Zealand white rabbits with full articular cartilage defects were randomly divided into 4 groups, each group had 6 animals, both hind limbs were used in the experiment. Group A: hydrogel combined with transfected cells; Group B: hydrogel combined with untransfected cells; Group C: hydrogel group; Group D: blank control group. Specimens and histological observation were used to evaluate the repair effect after 16 weeks according to Pineda's score. Results The positive rate of hTGF-β1 expression in BMSCs was about 85.4% after transfection. After 16 weeks the defects of group A were repaired by cartilage-like tissue, the cell arrangement and densities of regenerated cartilage were similar to normal cartilage, type Ⅱ collagen immunohistochemistry were positive. There was a significant difference in Pineda's score compaired with other groups (P ＜ 0.05). Conclusion Rabbit articiular cartilage defects could be repaired by CS/PVA hydrogel engineered hTGF-β1-transfected bone marrow mesenchymal stem cells.     

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。     

Effects of hyaluronan on periosteal grafts for large full-thickness defects in rabbit articular cartilage

We studied the effects of hyaluronan (HA) on chondrogenesis in periosteal grafts in rabbit knees to elucidate the effects of this agent in the repair of articular cartilage. Large full-thickness defects of the articular cartilage were created in the anteromedial part of the femoral articular surface of bilateral knee joints. Periosteal grafts were then harvested and sutured onto the defects. HA was injected in the right knee immediately after the operation and then once a week for 4 weeks (HA group). The same volume of saline was injected in the left knee in the control group. The animals were killed 2, 5, 8, and 12 weeks after the operation. Macroscopic and histological findings of the regenerated tissue were evaluated with a semiquantitative histological grading system. The total histological scores of the HA group were better than those in the control group at each time examination point. At 12 weeks, in particular, the scores for surface regularity and integration to adjacent articular cartilage were significantly better in the HA group than in the control group (P < 0.05). No significant differences were observed between the two groups in regard to the area healed (%). HA may have beneficial effects on the repair of large full-thickness defects of the articular cartilage with autologous periosteal grafts. Received for publication on Feb. 18, 1998; accepted on Oct. 20, 1998     

骨髓基质细胞与几丁质复合移植修复关节软骨缺损的实验研究

目的 :探讨骨髓基质细胞 (MSCs)与几丁质复合移植对关节软骨缺损的修复效果。方法 :分离兔骨髓基质细胞并体外培养增殖后 ,与几丁质无纺网复合培养 ;制作兔膝关节软骨全层缺损模型 ,分别用MSCs 几丁质复合物移植、单纯几丁质移植及空白对照组 ,术后第 4、 8、 12、 16周处死动物 ,大体观察并做组织形态学观察。结果 :几丁质 MSCs组术后 16周关节软骨缺损其修复组织表面与正常软骨完全相同 ,软骨及软骨下骨修复 ;单纯几丁质移植组为透明软骨修复 ,表面不平整 ,细胞排列不规则 ,软骨下骨基本修复 ;空白对照组术后各期均为纤维组织修复。结论 :MSCs与几丁质复合移植对关节软骨缺损有较好的修复效果