组织工程技术修复同种异体兔关节软骨缺损实验研究

目的：探讨关节软骨缺损治疗的新途径。方法：把几丁糖作为软骨细胞培养的支架。将几丁糖与软骨细胞一起体外培养,然后移植修复同种异体兔的膝关节软骨缺损,并对关节软骨的修复过程进行术后16周大体、组织学、电镜观察及修复组织厚度测定。结果：几丁糖无纺网在术后2周开始降解吸收,术后10-12周完全吸收;术后第16周在实验侧关节软骨缺损处可见成熟的透明软骨,软骨缺损得到完全修复。结论：几丁糖泊生物学特性符合组织工程中对细胞培养支架的要求;几个糖负载软骨细胞移植修复同种异体兔膝关节软骨缺损,兔膝关节全层软骨缺损得到成功修复。为临床上关节软骨缺损的治疗提供了可能的途径。     

组织工程化软骨修复兔膝关节软骨缺损的实验研究

目的 评估同种异体组织工程软骨修复兔膝关节全层软骨缺损的有效性。方法分离收集成年新西兰大白兔软骨细胞进行体外培养。建立双侧兔膝关节软骨缺损模型，用去端肽胶原（atelocollagen）凝胶与所培养的异体兔关节软骨细胞共同植入兔膝关节软骨缺损处，并设对照组。分别于手术后4周、8周观察大体标本以及组织学修复结果，并进行Wakitan的评分，评估此方法的有效性。结果大体观察结果表明，与对照组相比，实验组缺损处由软骨组织修复而对照组缺损处由纤维样组织填充。组织学观察可以见到实验组关节软骨缺损处有密集的软骨细胞而对照组关节缺损处只有纤维细胞无软骨细胞。结论通过短期观察表明以同种异体软骨细胞-去端肽胶原复合物修复全层软骨缺损的方法是有效可行的，为其进一步临床应用提供了参考。     

同种异体组织工程化软骨修复关节软骨缺损

目的：探讨应用同种异体组织工程化软骨修复软骨缺损的可行性。方法：取新西兰大白兔双膝关节软骨细胞，经体外培养扩增，与PlruonicF127混合，植入人为造成的异体兔膝关节软骨缺损。结果：空白对照组和材料对照组只见少许纤维组织修复，缺损凹陷。实验组8周后关节软骨缺损区由部分白色透明样软骨组织充填，Masson三色染色见胶原分布较均匀，软骨陷窝多见，未见明显炎症现象，16周后缺损完全修复，缺损表面较光滑，部分颜色呈淡兰色，软骨陷窝清晰，细胞与基质分布均匀，未见炎症和退变现象，结论：同种异体组织工程化软骨可用于修复关节软骨缺损。     

同种异体软骨细胞移植术后关节软骨蛋白多糖的测定

目的 应用Pluronic F-127负载同种异体软骨细胞移植修复兔全厚关节软骨损伤,对于新生的修复组织进行基质蛋白多糖含量测定,以探讨此方法修复全厚关节软骨损伤的可行性.方法 取3个月龄新西兰大白兔关节软骨细胞体外培养扩增,与20%Plurortic F-127凝胶混合.选27只健康同种成年大白兔,人为造成双侧膝关节软骨缺损.实验组软骨缺损处植入培养的软骨细胞/Pluronic F-127混合物,对照组缺损处单纯注入Pluronic F-127凝胶和空白对照.然后,对修复组织进行大体观察及蛋白多糖含量测定.结果 移植的软骨细胞-载体复合物中的软骨细胞能良好地生长,12周时再生组织与周围正常软骨组织外观相似,界限模糊.实验组与对照组各时期蛋白多糖含量均有非常显著性差异,实验组不同时期的蛋白多糖含量之间均有显著性差异,实验组12周时蛋白多糖含量与正常软骨组织无显著性差异.结论 Pluronic F-127负载同种异体软骨细胞移植是治疗关节软骨缺损的有效方法.     

同种异体软骨细胞移植术后关节软骨蛋白多糖的测定

目的应用Pluronic F-127负载同种异体软骨细胞移植修复兔全厚关节软骨损伤，对于新生的修复组织进行基质蛋白多糖含量测定，以探讨此方法修复全厚关节软骨损伤的可行性。方法取3个月龄新西兰大白兔关节软骨细胞体外培养扩增，与20％Pluronic F-127凝胶混合。选27只健康同种成年大白兔，人为造成双侧膝关节软骨缺损。实验组软骨缺损处植入培养的软骨细胞／Pluronic F-127混合物，对照组缺损处单纯注入Pluronic F-127凝胶和空白对照。然后，对修复组织进行大体观察及蛋白多糖含量测定。结果移植的软骨细胞-载体复合物中的软骨细胞能良好地生长，12周时再生组织与周围正常软骨组织外观相似，界限模糊。实验组与对照组各时期蛋白多糖含量均有非常显著性差异，实验组不同时期的蛋白多糖含量之间均有显著性差异，实验组12周时蛋白多糖含量与正常软骨组织无显著性差异。结论Pluronic F-127负载同种异体软骨细胞移植是治疗关节软骨缺损的有效方法。     

异体软骨细胞复合Pluronic修复关节软骨缺损

目的 探讨运用同种异体软骨细胞复合Pluronic修复关节软骨缺损的可行性，并应用^3H—TdR放射自显影方法鉴别软骨缺损修复的细胞来源。方法 取同种异体软骨细胞体外培养至第2代，用^3H—TdR标记后复合Pluronic植入兔关节软骨缺损区作为实验组，并采用单纯Pluronic植入作为材料对照组，不作任何处理组为空白对照组，分别于4、8及16周取材，观察其修复效果，并应用放射自显影方法鉴别修复组织的细胞来源。结果 实验组术后8周，缺损表面可见新生软骨形成，术后16周缺损完全修复，表面光滑，与周围界限模糊，放射自显影证实所修复组织的细胞来源于植入细胞。材料对照组及空白对照组缺损均未见明显修复。结论 ①同种异体软骨细胞复合Pluronic修复关节软骨缺损是可行的；②^3H—TdR标记细胞可作为鉴别细胞来源的一种简便可行的方法。     

同种异体软骨移植修复关节软骨缺损实验研究

目的 采用兔膝关节软骨标本经打孔梯度降温冻存后行同种异体移植,研究打孔梯度降温冻存对兔关节软骨的影响及其修复关节软骨缺损的效果.方法 自16只2月龄新西兰白兔膝关节股骨髌面取分别取3块骨软骨移植物,随机分为3组.Ⅰ组为实验组,在软骨上以3 mm×3 mm矩阵打孔,Ⅱ、Ⅲ组为对照组,不打孔.分别将软骨标本置于二甲基亚砜冷冻保护溶液中,并经梯度降温至-80℃(Ⅰ、Ⅱ组)或直接置于-80℃(Ⅲ组)保存1周,复温后移植到成年兔相应膝关节部位.术后分批处死动物,通过对移植物的大体形态学、组织学、免疫组化染色光镜观察,研究各组移植物保存效果的差异.结果 Ⅰ、Ⅱ组光镜观察结果明显优于Ⅲ组;Ⅰ组与Ⅱ组结果差异不明显,但Ⅰ组对中间层软骨组织的保护明显加强.结论 关节软骨的梯度降温冷冻保存效果明显优于快速降温冷冻保存,且关节软骨打孔冷冻保存对深层软骨细胞有一定的保护作用,可提高软骨细胞存活率,延缓移植软骨组织的退变过程.     

同种幼年异体软骨微粒植入关节软骨缺损的早期组织反应

[目的]研究同种幼年异体软骨微粒植入猪软骨缺损的早期组织反应,为进一步的研究创造条件。[方法]取12头成年贵州小香猪制备膝关节股骨软骨缺损模型,随机分为异体组、自体组,每组6头。另取2头幼年贵州小香猪制取异体软骨微粒,植入异体组软骨缺损处;自体组植入在制备软骨缺损时产生的自体软骨微粒。两组于术后1个月取材分析,标本来自关节滑膜、软骨缺损区域组织,检测指标包括大体观察、HE染色和IL-6免疫组化染色。[结果] 12头贵州小香猪手术顺利,两组动物术后无感染和切口愈合障碍。术后1个月取材时,两组术侧膝关节未见局部明显红肿及大量关节腔积液,术区软骨缺损处可见纤维软骨样白色组织填充。组织学观察(HE染色)示,两组标本的滑膜和软骨缺损组织区域可见轻度炎症反应,组间淋巴细胞和中性粒细胞计数差异无统计学意义;免疫组化染色显示,两组标本的IL-6表达量差异无统计学意义(P0.05)。[结论]同种幼年异体软骨微粒植入猪膝关节软骨缺损区域的早期,局部产生轻度炎症反应,但与自体微粒植入相比,组织反应差异不明显。     

关于关节软骨愈合和再生研究的现代进展(四)

3 .2 .3　同种异体软骨细胞移植 (ｔｒａｎｓｐｌａｎｔａｔｉｏｎｏｆａｌｌｏｇｅｎｉｃｃｈｏｎｄｒｏｃｙｔｅｓ)　同种异体软骨细胞移植治疗关节软骨缺损 ,在一些实验研究 (特别是兔 )中 ,获得成功[1] 。但是 ,那些成功的动物实验 ,并不包括马的实验在内[2 ,3] 。Ｆｒｅｅｄ等[1] 在观察研究同种异体软骨细胞移植修复关节软骨缺损时发现 ,将取自一个兔膝的关节软骨细胞经术后多羟基乙酸 (ｐｏｌｙｇｌｙｃｏｌｌｉｃａｃｉｄ)培养 1个月 ,植于另一兔膝关节的关节软骨缺损上时 ,仅产生很轻微的免疫反应。实验研究者们指出培养可使由细胞…     

同种异体组织工程化软骨修复关节软骨缺损

目的　探讨应用同种异体组织工程化软骨修复软骨缺损的可行性。方法　取新西兰大白兔双膝关节软骨细胞 ,经体外培养扩增 ,与PlruonicF12 7混合 ,植入人为造成的异体兔膝关节软骨缺损。结果　空白对照组和材料对照组只见少许纤维组织修复 ,缺损凹陷 ;实验组 8周后关节软骨缺损区由部分白色透明样软骨组织充填 ,Masson三色染色见胶原分布较均匀 ,软骨陷窝多见 ,未见明显炎症现象。 16周后缺损完全修复 ,缺损表面较光滑 ,部分颜色呈淡兰色 ,软骨陷窝清晰 ,细胞与基质分布均匀 ,未见炎症和退变现象。结论　同种异体组织工程化软骨可用于修复关节软骨缺损。     

Rabbit articular cartilage defects treated by allogenic chondrocyte transplantation

Articular cartilage defects have a poor capacity for repair. Most of the current treatment options result in the formation of fibro-cartilage, which is functionally inferior to normal hyaline articular cartilage. We studied the effectiveness of allogenic chondrocyte transplantation for focal articular cartilage defects in rabbits. Chondrocytes were cultured in vitro from cartilage harvested from the knee joints of a New Zealand White rabbit. A 3 mm defect was created in the articular cartilage of both knees in other rabbits. The cultured allogenic chondrocytes were transplanted into the defect in the right knees and closed with a periosteal flap, while the defects in the left knees served as controls and were closed with a periosteal flap alone, without chondrocytes. Healing of the defects was assessed at 12 weeks by histological studies. Allogenic chondrocyte transplantation significantly increased the amount of newly formed repair tissue (P=0.04) compared with that found in the control knees. The histological quality score of the repair tissue was significantly better (P=0.05), with more hyaline characteristics in the knees treated with allogenic chondrocytes than in the control knees. Articular cartilage defects treated with allogenic chondrocyte transplantation result in better repair tissue formation with hyaline characteristics than those in control knees.     

Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel

In an attempt to repair articular cartilage, allograft articular chondrocytes embedded in collagen gel, were transplanted into full-thickness defects in rabbit articular cartilage. Twenty-four weeks after the transplantation, the defects were filled with hyaline cartilage, specifically synthesising Type II collagen. These chondrocytes were autoradiographically proven to have originated from the transplanted grafts. Assessed histologically the success rate was about 80%, a marked improvement over the results reported in previous studies on chondrocyte transplantation without collagen gel. By contrast, the defects without chondrocyte transplantation healed with fibrocartilage. Immunological enhancement induced by transplanted allogenic chondrocytes or collagen was not significant at eight weeks after treatment, so far as shown by both direct and indirect blastformation reactions. Thus, allogenic transplantation of isolated chondrocytes embedded in collagen gel appears to be one of the most promising methods for the restoration of articular cartilage.     

Reversibility of joint changes produced by immobilization in rabbits.

Rabbits knee joints which had been immobilized for only 2 weeks regained motion during the first 2 months of remobilization. Histologically, joint tissues were normal. Only 9 of the 14 rabbits immobilized for 6 weeks regained normal or partial knee motion. Animals that regained knee motion generally did so during the first 4 months of remobilization. There are 2 types of repair of the damaged cartilage; replacement by fibrocartilage and proliferation of chondrocytes. Normal chondrocyte alignment and normal collagen fibril orientation was not restored. Repaired cartilage did not withstand normal joint stress.     

Chondrocyte ultrastructure in exercise and experimental osteoarthrosis. A stereologic morphometric study of articular cartilage of young rabbits using transmission electron microscopy

The effects of physical exercise (running) and immobilization (splinting) on chondrocyte ultrastructure were studied in the knee joint articular cartilage of 24 young rabbits. Synthetic activity of the chondrocytes was quantified by measuring the amount of rough endoplasmic reticulum (RER) from electron micrographs using a sterologic point-counting method. Extra loading of the joint by running, or by increased weight-bearing after splinting of the contralateral limb, caused a 20% and 30% increment of RER in the middle and deep zones of the cartilage, respectively, while immobilization decreased the amount of RER by 30% in the superficial zone. Some attempts to repair and regenerate were observed, especially in the deep zone of articular cartilage. Hypertrophy of cells and organelles, and cell replication were considered as signs of reparative processes. The accumulation of fine intracytoplasmic filaments (FIF) in chondrocytes, regarded as a sign of cell degeneration, was reduced in the exercise group. FIF also decreased in the deep zone chondrocytes of the immobilized group, which could be indicative of improved or retained viability of the chondrocytes.     

冷冻保存对软骨细胞存活率及代谢活性的影响

目的了解各种冷冻保存方法对软骨细胞的存活率和代谢活性的影响，寻找满意的软骨组织冻存方法。方法采用梯度慢速降温法和连续慢速降温法对兔关节软骨进行低温冷冻保存处理，通过荧光染色及^35SO4摄入率了解冻存后软骨细胞的存活率和代谢活性。结果采用梯度降温法的软骨细胞存活率为61％，显著高于连续降温法；冷冻保存对软骨细胞的代谢活性有一定的影响．但与对照组的差异并无统计学意义。结论梯度降温法较传统保存方法能显著提高冷冻保存后软骨细胞的存活率，并能维持软骨细胞的代谢活性，是理想的关节软骨冷冻保存方法。     

同种异体软骨细胞与自体骨髓基质干细胞混合共培养构建软骨皮下移植的可行性研究

背景：软骨组织工程的种子细胞问题是目前研究的热点和难点,如何找到一种既能够避免对自体软骨进行取材又能够达到稳定软骨构建目的的方法呢？本研究尝试利用少量同种异体羊软骨细胞作为软骨诱导微环境提供者,与扩增后的羊自体BMSC混合共培养并植入皮下环境,探讨利用同种异体软骨细胞共培养构建软骨皮下移植的可行性。方法：本实验对山羊软骨细胞和BMSC分别进行取材和分离培养扩增,并将以上细胞分为以下四组进行混合并接种在PGA支架材料上：A组：100%自体软骨细胞;B组：30%自体软骨细胞＋70%自体BMSCs;C组：30%同种异体软骨细胞＋70%自体BMSCs;D组：100%同种异体软骨细胞。经过体外构建6周后植入羊皮下进行体内构建12周,对所形成的组织块进行大体观察和组织学染色等评价。结果：自体软骨细胞组和自体软骨细胞混合自体BMSC组皮下移植后可见成熟软骨组织形成,但同种异体软骨细胞参与的两组（包括同种异体软骨细胞混合自体BMSC的实验组和单纯异体软骨细胞组）在皮下环境中都因为较强的免疫反应未能形成软骨组织。结论：同种异体软骨细胞以及PGA支架材料的存在对于组织工程软骨在羊皮下环境的构建有负面影响。     

前交叉韧带断裂和重建对膝关节软骨退变影响的实验研究

目的：研究前交叉韧带（ACL）断裂和不同时期重建对膝关节软骨继发损伤的影响。方法：以新西兰大白兔为实验对象,共14只。共分4组,每组7个膝关节,实验组Ⅰ：右膝前交叉韧带切断后随即重建,左膝的前交叉韧带切断后不予重建作为对照组Ⅰ;组Ⅱ：右膝前交叉韧带切断后3周重建,左膝行单纯关节切开术作为对照组Ⅱ。术后8周通过墨汁染色,常规组织学及扫描电镜方法观察各组膝关节软骨退变的情况。结果：（1）实验组Ⅰ关节软骨退变程度明显轻于对照组Ⅰ（Hc=5.9889,P=0.0144);(2)实验组Ⅱ关节软骨退变程度和对照组Ⅰ相比差异无显著性意义（Hc=0.7143,P=0.785)。结论：（1）ACL断裂后即刻重建可以有效阻止关节软骨继发损伤的发生;（2）ACL断裂后已继发关节软骨退变时再行重建,其对关节软骨退行性变的缓解作用不明显。ACL 裂后应进行早期重建,恢复膝关节稳定性,减少或延缓远期骨性关节炎的发性。     

Continuous passive motion applied to whole joints stimulates chondrocyte biosynthesis of PRG4

Continuous passive motion (CPM) is currently a part of patient rehabilitation regimens after a variety of orthopedic surgical procedures. While CPM can enhance the joint healing process, the direct effects of CPM on cartilage metabolism remain unknown. Recent in vivo and in vitro observations suggest that mechanical stimuli can regulate articular cartilage metabolism of proteoglycan 4 (PRG4), a putative lubricating and chondroprotective molecule found in synovial fluid and at the articular cartilage surface. OBJECTIVES: (1) Determine the topographical variation in intrinsic cartilage PRG4 secretion. (2) Apply a CPM device to whole joints in bioreactors and assess effects of CPM on PRG4 biosynthesis. METHODS: A bioreactor was developed to apply CPM to bovine stifle joints in vitro. Effects of 24h of CPM on PRG4 biosynthesis were determined. RESULTS: PRG4 secretion rate varied markedly over the joint surface. Rehabilitative joint motion applied in the form of CPM regulated PRG4 biosynthesis, in a manner dependent on the duty cycle of cartilage sliding against opposing tissues. Specifically, in certain regions of the femoral condyle that were continuously or intermittently sliding against meniscus and tibial cartilage during CPM, chondrocyte PRG4 synthesis was higher with CPM than without. CONCLUSIONS: Rehabilitative joint motion, applied in the form of CPM, stimulates chondrocyte PRG4 metabolism. The stimulation of PRG4 synthesis is one mechanism by which CPM may benefit cartilage and joint health in post-operative rehabilitation.     

Inhibition of Chondrocyte Terminal Differentiation and Matrix Calcification by Soluble Factors Released by Articular Chondrocytes

Chondrocytes do not undergo terminal differentiation in normal articular cartilage, whereas growth plate chondrocytes synthesize ALPase and induce matrix calcification terminally. Articular chondrocytes in osteoarthritic joints have been reported to express the terminal differentiation phenotypes, suggesting that terminal differentiation of articular chondrocytes is inhibited in normal joints. In the present study, we investigated the underlying inhibitory mechanism of the terminal differentiation in articular cartilage using a culture on type II collagen-coated dishes or a novel culture model on Millipore filters. ALPase activity increased from day 7 to day 8 in growth plate chondrocyte cultures on the collagen-coated dishes, but not in articular chondrocyte cultures. The ALPase expression of growth plate chondrocytes on the collagen-coated dish was completely inhibited when the same number of articular chondrocytes was mixed in the growth plate chondrocyte cultures. When articular chondrocytes or growth plate chondrocytes were maintained on Millipore filters held in 16-mm dishes, they started to synthesize ALPase. The ALPase expression of the chondrocytes on Millipore filters was inhibited by the presence of articular chondrocytes maintained on the bottom collagen-coated substratum in the same dishes. These results indicate that factors that diffused into the medium through the Millipore filters are involved in the inhibition of terminal differentiation. Since the conditioned medium from articular chondrocyte cultures did not affect the ALPase expression, it is considered that the soluble factors, which are continuously released from articular chondrocytes, are responsible for the inhibition of terminal differentiation. Received: 23 April 1998 / Accepted: 12 March 1999