不同应力环境对兔骨髓间充质干细胞修复关节软骨缺损的影响

目的探讨不同应力环境对骨髓间充质干细胞(MSCs)修复关节软骨缺损的影响. 方法将日本大耳白兔15只制成髌骨外侧脱位动物模型,平均分成3组,每组5只:即单纯载体脱位组(对照组)、移植物正常应力组及移植物脱位组.对兔MSCs进行分离、培养,以兔MSCs为种子细胞构建自体组织工程移植物修复关节软骨缺损.6周后处死动物,观察修复组织的成分和结构. 结果术后6周,移植物正常应力组修复组织浅层为软骨组织,甲苯胺蓝染色接近正常关节软骨;深层为软骨下骨,与正常关节软骨结构相似.移植物脱位组为骨组织所修复,缺损周围的正常关节软骨变薄,软骨下血管侵入正常关节软骨内,遗留在股骨髁滑车槽内的移植物在滑车槽正常关节软骨表面形成新生类透明软骨组织.单纯载体脱位组为纤维组织修复. 结论 MSCs修复关节软骨缺损,只有在正常应力状态下修复效果最佳;提示维持负重关节正常的应力刺激,对组织工程软骨修复组织的形成和维持必不可少.     

牛透明软骨细胞与组织引导再生胶原膜体外培养后植入体内的研究

目的解决修复重建组织器官缺损所需移植材料的问题,利用组织工程技术,进行组织工程牛透明软骨的实验研究。方法用分离获得的牛软骨细胞,与医用组织引导再生胶原膜体外联合培养1周后,以软骨细胞可降解材料复合物的形式植入裸鼠体内,8周后对所获得的组织工程软骨样组织进行形态学研究。结果在实验获得的组织工程软骨样组织中有软骨细胞的存在,并且可以分泌硫酸软骨素。证实为组织工程牛透明软骨。结论实验结果表明应用组织工程技术,在体外进行组织工程软骨的制作,可能成为解决整形外科领域修复缺损用移植材料不足的方法之一。     

牛透明软骨细胞与组织引导再生胶原膜体外培养后植入 …

目的 解决修复重建组织器官缺移植材料的问题,利用组织工程技术,进行组织工程牛透明软骨的实验研究。方法 用分离获得的牛软骨细胞,与医用组织引导再生胶原膜体外联合培养１周后,以软骨细胞可降解材料复合物的形式植入裸鼠体内,８周后对所获得的组织工程软骨样组织进行形态学研究。结果 在实验 组织工程软骨要组织中和软骨细胞的存在,并且可以分泌硫酸软骨素。证实为组织工程年 骨。结论实验结果表明在体外进行组织工程软     

温敏性高黏度几丁聚糖水凝胶复合脱钙骨基质修复兔膝全层软骨缺损的初步研究

目的将高黏度几丁聚糖水凝胶(high viscous chitosan/glycerol phosphate,HV-C/GP)与异体脱钙骨基质(demineralized bone matrix,DBM)颗粒复合,评估修复兔膝全层软骨缺损的效果。方法将HV-C/GP与DBM按2∶1(W/W)复合,制备HV-C/GP-DBM复合物。取成年健康34周龄新西兰大白兔24只,体重3.5～4.5kg,于两侧股骨髁间窝制备直径为3.5mm、深度为3mm的软骨全层缺损区。右侧缺损区植入HV-C/GP-DBM复合物作为实验组;左侧不作处理,作为对照组。术后4、8、16周,分别处死动物取材,行大体及组织学观察。根据国际软骨修复协会组织学评分(International Cartilage Repair Society Histological Scoring,ICRS)标准对第16周标本评分,评估软骨修复效果。结果大体及组织学观察:术后4、8周,实验组缺损区大部分被透明软骨样组织修复,DBM颗粒部分吸收;对照组缺损区可见少量纤维组织及软骨细胞;术后16周,实验组6例关节表面平滑,缺损区已基本被透明软骨样软骨组织修复,缺损区与周围软骨整合较好,新骨形成活跃,部分DBM颗粒未被爬行替代;对照组仍存在凹陷,修复组织为纤维组织。术后16周ICRS评分结果示,实验组的软骨细胞生成以及与周围软骨整合等6个方面均优于对照组,差异有统计学意义(P<0.05)。结论HV-C/GP-DBM复合物组织相容性好,可降解,可注射,是一种较好的软骨修复材料。     

Repair of osteochondral defects in the knee by resorbable bioimplants in a rabbit model

Background?The intrinsic healing capacity of articular cartilage remains poor, despite various attempts that have been undertaken to treat cartilage defects. This study describes the experimental use of a double-layer bioimplant consisting of a bone-substitute layer and a cartilage-substitute layer.

Animals and methods?In group A, 12 implants were placed into osteochondral defects of the load-bearing area of rabbit femoral condyles. In group B, 12 implantations were done in the same manner, with a separating membrane consisting of cement between both layers to investigate ingrowth of mesenchymal stem cells from the subchondral marrow space. Group C, with 12 similar defects but without treatment, served as control. Investigations by microscopy and immunohistochemistry were done after 12 weeks.

Results?All bioimplants showed coverage of the defect with a regeneration tissue that contained cartilage-like regions. Implants with a cement layer showed less cartilage and more fibrous tissue. The bioimplant group showed more cartilage-like regeneration tissue than the control groups, which only showed incomplete fibrous filling of the defects. Results from the second group supported the hypothesis that the subchondral space must be opened for adequate regeneration. Additional examinations were done using an established semiquantitative score. The bioimplant group showed a significant improvement in results compared to the group with the separating layer and the control group.

Interpretation?Our findings indicate that cartilage repair by resorbable bioimplants seems to be a promising new approach, especially if mesenchymal stem cells are present and can differentiate under mechanical load.     

组织工程软骨移植修复兔膝关节软骨缺损

研究用组织工程的方法进行差了软骨缺损修复的基本原理。方法取新生兔关节－干骺复后体软骨,胶原酶消化,将所获软骨细胞种入９６孔板内几丁质纤维无纺布上。结果培养２１天时形成“膜状软骨”,至１１０天时形成直径为４．４ｍｍ的“圆盘状软骨”。经Ｓａｆｒａｎ“Ｏ”ｉｖｓ ｑｃ ｙｇｈ ｐｕ 其基质富含蛋白多糖,原位杂交法 产其软骨细胞表达Ⅱ型胶原ｍＲＮＡ。将培养２１天的“膜状软骨”移植于成年兔膝关节圆形全厚缺损     

Repair of osteochondral defects in the knee by resorbable bioimplants in a rabbit model

BACKGROUND: The intrinsic healing capacity of articular cartilage remains poor, despite various attempts that have been undertaken to treat cartilage defects. This study describes the experimental use of a double-layer bioimplant consisting of a bone-substitute layer and a cartilage-substitute layer. ANIMALS AND METHODS: In group A, 12 implants were placed into osteochondral defects of the load-bearing area of rabbit femoral condyles. In group B, 12 implantations were done in the same manner, with a separating membrane consisting of cement between both layers to investigate ingrowth of mesenchymal stem cells from the subchondral marrow space. Group C, with 12 similar defects but without treatment, served as control. Investigations by microscopy and immunohistochemistry were done after 12 weeks. RESULTS: All bioimplants showed coverage of the defect with a regeneration tissue that contained cartilage-like regions. Implants with a cement layer showed less cartilage and more fibrous tissue. The bioimplant group showed more cartilage-like regeneration tissue than the control groups, which only showed incomplete fibrous filling of the defects. Results from the second group supported the hypothesis that the subchondral space must be opened for adequate regeneration. Additional examinations were done using an established semiquantitative score. The bioimplant group showed a significant improvement in results compared to the group with the separating layer and the control group. INTERPRETATION: Our findings indicate that cartilage repair by resorbable bioimplants seems to be a promising new approach, especially if mesenchymal stem cells are present and can differentiate under mechanical load.     

The role of mandibular condylar cartilage in articular cartilage repair.

The articular hyaline cartilage of synovial joints has a very limited capacity for repair after injury. In contrast, the mandibular condylar cartilage of the temporomandibular joint possesses as intrinsic potential for regeneration. This study aimed to test the hypothesis that cultured allografts of mandibular condylar cartilage could be used to promote biological repair of injured orthotopic joint surfaces. Using a primate animal model, cultures of mandibular condylar cartilage cells were grafted into surgically created defects in a recipient hyaline cartilage joint surface. Articular wound healing was assessed macroscopically and histologically over a postoperative period of 52 weeks. Mandibular condylar cartilage cells scheduled for allogenous transplantation were initially characterised in vitro. Expansion of primary colonies in organ culture provided the allogenic cellular material for in vivo grafting. Grafting of osteochondral articular wounds with 5-week cultures of mandibular cartilage cells led to wound regeneration with complete reconstitution of articular surface continuity by 52 weeks. There was novel synthesis of cartilage collagens and sulphated glycosaminoglycans within the repair tissue and no evidence of immunological rejection. Healing of grafted defects was thought to occur by a combination of donor cell proliferation and ingress of host mesenchymal cells. In contrast, grafted control wounds underwent largely fibrous repair with incomplete articular regeneration. In conclusion, transplanted allografts of cultured mandibular condylar cartilage appeared to have the ability, in this primate model, to promote cartilaginous repair and regeneration of orthotopic articular wounds.     

Effect of low loading and joint immobilization for spontaneous repair of osteochondral defect in the knees of weightless (tail suspension) rats

Background Mechanical stimulation has a great influence on articular cartilage regeneration. The objective of this study was to clarify the temporal sequences of spontaneous repair of weightless or immobilized joints. Methods An osteochondral defect was created in the femoral patellar groove of F344 rats. A tail-suspension procedure was performed to control the mechanical environment of the hindlimbs. The experimental knee joints were classified into three conditions: CONT, normal gait; LLB, low load-bearing; LLI, low load and immobilized. The repair processes up to 4 weeks were evaluated histologically. Results The knee defects in the CONT and LLB conditions were repaired to a smooth surface with fibrous tissue and highly developed subchondral bone. The knees in the LLI condition had the lowest reformation rate of subchondral bone, although partial regeneration of hyaline cartilage-like tissue was seen at 4 weeks after the operation. Bulges of fibrous tissue from the defects were observed in the LLI condition. Conclusions These results, combined with our previous report, suggest that dynamic compressive strain stimulates regeneration of the joint surface structures. They also suggest that the contact condition of the defect with surface cartilage may play a role in the hyaline cartilage repair.     

关节－干骺端软骨细胞移植修复兔桡骨缺损

目的研究组织工程软骨移植于成年兔桡骨缺损后的生长、分化与转归特点,以及引导性骨再生和骨缺损的修复机制。方法取自一日龄新生兔关节－干骺端复合物的软骨细胞在几丁质纤维网中增殖21d后装入硅胶管内,套接在成年兔桡骨干1cm的缺损处（实验组12只）;对照组10只在缺损处套接空硅胶管,2只仅填入裸几丁质纤维。术后4周两组各处死3只动物取材,其余在术后16周取材。结果实验组术后4周3只动物的工程软骨组织在骨缺损内形成软骨样组织,术后16周9只动物中有2只动物的缺损愈合。对照组术后4周已开始骨愈合,术后16周9只动物的骨缺损全部愈合。结论新生兔关节－干骺端复合物的软骨细胞在成年兔桡骨缺损区（套管内）未肥大钙化,未再现软骨内化骨过程。缺损内的工程软骨可能因占据空间、阻碍成骨成分进入而中断了骨缺损修复过程。引导性骨再生的机制可能是人工膜管加强了骨膜的天然引导作用而促进了骨愈合。     

培养软骨移植修复关节软骨缺损的实验研究

目的：为探讨一种新的关节软骨缺损修复方法。方法：将体外培养２周形成软骨样组织,移植修复兔关节软骨全层缺损。于移植术后２、４、８周分别行功能评价、大体形态及组织学检查。结果：全部实验兔于术后２周内恢复正常活动。２周时移植修复组织由非成熟透明软骨组成。４周时部分移植组出现成熟透明软骨。８周时移植组关节软骨缺损全部由成熟透明软骨充填修复,修复组织与邻近关节软骨融合。培养软骨移植修复关节软骨全层缺损明显优于自身修复（Ｐ＜００１）。结论：本实验提示使用具有高有丝分裂率的软骨细胞,经离心管培养形成骺软骨样组织,植入关节软骨全层缺损后,软骨细胞生长良好,逐渐成熟和转化,能发挥良好的修复作用。     

应用犬真皮成纤维细胞构建组织工程半月板修复犬半月板缺损的实验研究

目的 探讨体外构建组织工程半月板修复半月板缺损的可行性.方法 将体外扩增至第4代的犬真皮成纤维细胞(Dermal fibroblasts,DFs)接种于PGA/PLA支架材料上,应用软骨形态发生蛋白1(Cartilage-derived morphogenetic protein-1,CDMP1)细胞诱导液体外诱导培养2周后,回植于犬自体半月板缺损模型体内,移植后6个月取材观察半月板的再生情况.结果 诱导组模型内形成较大的新生半月板组织,组织学染色显示诱导组新生组织更接近于半月板组织的生理特点,尤其是内侧部分大部分细胞表现出软骨细胞特有的陷窝结构;非诱导组及空白对照组新生的组织较小,且组织学表现更类似纤维结缔组织.新生半月板Ⅰ型胶原偏振光检测显示,诱导组胶原纤维排列较非诱导组致密,出现Ⅰ型胶原的横纹特征.扫描电镜检测显示,诱导组细胞外基质与正常半月板相比较疏松,非诱导组细胞外基质则更加疏松.膝关节胫骨平台关节软骨的HE染色显示,诱导组关节面的外侧有新生半月板组织覆盖的部位关节软骨尚有部分存余,而诱导组已基本消失.结论 组织工程化细胞材料复合物能在犬自体半月板缺损模型上实现半月板再生.     

纳米左旋聚乳酸/聚己内酯复合骨髓基质源性软骨细胞修复犬膝关节软骨缺损

目的探讨纳米左旋聚乳酸/聚己内酯(纳米PLLA-b-PCL)复合骨髓基质源性软骨细胞修复犬膝关节软骨缺损的可行性。方法将纳米PLLA-b-PCL支架/骨髓基质源性软骨细胞复合物植入犬膝关节软骨缺损,其为实验组;另一组膝关节造成缺损后旷置,作为空白对照。术后12周,24周取材,行大体及组织学观察,组织学评分。结果术后12周、24周大体形态学和组织学观察均表明实验组得到较好修复,而对照组缺损未修复。结论骨髓基质细胞源性软骨细胞是修复关节软骨缺损较理想的种子细胞;纳米PLLA-b-PCL在新生软骨形成的同时,逐渐降解吸收,是组织工程修复关节软骨缺损的适宜支架材料,其具有良好的应用前景。     

Restoration of arthritic cartilage defects using autologous chondrocytes transplantation is superior to cartilage-paste graft in rabbits

This study compared the articular cartilage repair potential of cultured chondrocytes transplantation with bone-cartilage paste-graft in the resurfacing of full-thickness defects without breaching of the subchondral bone plate in rabbit knees. A 5 x 5-mm articular cartilage defect was created in the patellar groove of the femur. Three months following creation, the defect was filled with cultured autologous chondrocytes (group 1) or bone-cartilage paste (group 2). A control group of untreated defects was followed for 1 year. The reparative tissue was analyzed macroscopically, histologically, and by immunohistochemistry 3-12 months post-transplantation. The surfaces of the reparative tissue in group 1 were smooth, and the defects were filled with reparative tissue that resembled hyaline cartilage. The composition of the repair tissue more closely resembled cartilage, as demonstrated by cartilage-specific stains. In contrast, the reparative tissue in group 2 was fibrous and exhibited markers of mesenchymal stem cells and bone formation. Transplantation of cultured chondrocytes into a full-thickness defect in the rabbit generates a biologic substitute tissue that resembles native articular cartilage with living cells capable of synthesizing the surrounding cartilage matrix. In contrast, analysis of the healing response to the paste-graft technique failed to show cartilage-like characteristics. This information may be clinically applicable to direct the use of these treatments in chondral injuries.     

Repair of articular cartilage defects with cultured chondrocytes on polysulphonic membrane: experimental studies in rabbits

INTRODUCTION: Autologous osteochondral transplantation is one method that can be used to create hyaline or hyaline-like repair in a defect area. The purpose of the present study was to repair full-thickness articular cartilage defects in 9 rabbit knee joints with autologous cultured chondrocytes. METHODS: An articular cartilage defect was created on the patellar groove of the femur. The defect was filled with chondrocytes cultured in vitro and placed into the knee on a polysulphonic membrane. At 8 weeks after the operation, the reparative tissue was analyzed macroscopically and histologically. RESULTS: At 8 weeks after the operation, the surfaces of the reparative tissue were smooth, and the defects were filled with mature hyaline cartilage in 5 cases. In 2 cases, the reparative hyaline cartilage was immature and there was worse integration of grafted tissue into the adjacent normal cartilage. In 2 cases, the surface of the grafted area was irregular, and the reparative tissue was disintegrated and incompletely differentiated. CONCLUSION: The results suggest that transplantation of autologous chondrocytes cultured in vitro and placed into the knee on polysulphonic membrane is effective in repairing an articular cartilage defect.     

Repair of articular cartilage defects with cultured chondrocytes in Atelocollagen gel

We attempted to repair full-thickness articular cartilage defects in rabbit knee joints with allogeneic cultured chondrocytes embedded in Atelocollagen gel. An articular cartilage defect was created on the patellar groove of the femur. The defect was filled with chondrocytes cultured in the collagen gel and covered with periosteal flap (G group). In three other experimental groups, the same defects were transplanted with chondrocytes in monolayer culture with periosteal flap (M group), periosteal graft only (P group), or left empty (E group). At 4, 12, and 24 weeks after operation, the reparative tissue was analyzed macroscopically and histologically. At 4 weeks after operation, the surfaces of the reparative tissue were smooth, and the defects were filled with reparative tissues that resembled hyaline cartilage in all four groups. However, the reparative tissues degenerated gradually with time in the M, P, and E groups. In contrast, in the G group, the reparative tissue retained its thickness, and there was a steady integration of the grafted tissue into the adjacent normal cartilage at 24 weeks after operation. The results suggest that transplantation of allogeneic chondrocytes cultured in Atelocollagen gel is effective in repairing an articular cartilage defect.     

胶原复合梯度TCP修复关节软骨的形态学观察

目的采用新型双向三维可降解生物活性材料胶原复合梯度TCP（collagen complexTCP，Col／TCP）对兔关节软骨缺损进行修复，并对再生软骨进行组织形态学观察。方法取30只成年大白兔，体重2．0～2．5kg，雌雄不限，于双侧股骨外侧髁制作关节软骨缺损模型。于右侧植入Col／TCP修复缺损，作为实验组，左侧不予处理作为对照组。术后4、6、8、12和24周分别处死6只动物，取股骨外侧髁关节面行大体、组织学、透射电镜及Ⅱ型胶原免疫组织化学染色观察。采用Wakitanifa法软骨组织形态学评分评价修复组织质量。结果大体观察：实验组术后4周，缺损区由白色组织完全充填，表面较光滑，有光泽；12周，修复关节软骨组织与周围正常软骨基本一致，且与关节下骨结合紧密；24周，再生软骨未见明显退变。对照组观察期内均未见软骨组织形成，缺损由纤维组织填充，修复组织表面粗糙，与正常组织界线清楚。实验组术后4、6、8、12和24周组织学评分分别为（7．60&#177;0．98）、（5．69&#177;0．58）、（4．46&#177;0．85）、（4．35&#177;0．12）、（4．41&#177;0．58）分，对照组分别为（10．25&#177;1．05）、（9．04&#177;0．96）、（8．96&#177;0．88）、（8．88&#177;0．68）、（8．66&#177;0．54）分；Ⅱ型胶原含量实验组分别为0．28％&#177;0．01％、0．59％&#177;0．03％、0．68％&#177;0．02％、0．89％&#177;0．02％和0．90％&#177;0．01％，对照组为0．08％&#177;0．02％、0、09％&#177;0．04％、0．11％&#177;0．03％、0．25％&#177;0．03％和0．29％&#177;0．01％；两组各指标比较差异均有统计学意义（P〈0．05）。透射电镜观察实验组可见典型软骨细胞，而对照组为粗大胶原纤维，细胞少见。结论双向三维可降解生物活性材料Col／TCP在动物体内可诱导关节软骨缺损后的软骨修复。     

Value of autologous chondrocyte transplantation in the reconstruction of experimental cartilage defects. Part I. Extent of defect, macroscopic appearance of reconstructed articular surface and microscopic analysis of predominant tissue]

Articular cartilage defect is one of the main reasons of osteoarthritis. Currently, tissue engineering techniques are the methods concerning better cartilage reconstruction. The aim of this part of the study was macroscopic evaluation of degree of defect feeling, macroscopic appearance of repair tissue and microscopic analysis of predominant tissue after autologous chondrocytes transplantation. Repair of partial thickness cartilage defect on distal part of femur was evaluated (25 adolescent rabbits). Procedures were performed in II groups: I--autologous chondrocytes transplantation under periosteal flap, II--periosteal graft. Chondrocytes were isolated from the cartilage specimens by enzymatic digestion and cultured in vitro. The regenerates were inspected 4, 8 and 12 weeks after the operation. Macroscopic analysis in group I, in most cases revealed filling of the defect with tissue resembling surrounding cartilage. In group II the defect was partially filled, and there was many fissures and cracks in all regenerates. In microscopic analysis in group I, after 4 and 8 weeks following the transplantation the tissue similar to juvenile hyaline cartilage predominated. After 12 weeks it resembled mature hyaline cartilage. In group II, in all cases fibrous cartilage was observed after 4, 8, 12 weeks. Obtained results indicate, that macroscopic and microscopic characteristics of repair tissue after autologous chondrocytes transplantation more closely resembled hyaline cartilage, than in periosteal graft group. 12 weeks after autologous chondrocytes transplantation the repair tissue reached maturity, and demonstrated microscopic characteristics of hyaline-like cartilage. The method of autologous chondrocytes transplantation provides potential for clinical application.     

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.