体外重建组织工程关节软骨的实验研究

目的　用胶原蛋白和人血纤维蛋白混合物为载体在体外进行软骨细胞三维立体培养 ,构建人工软骨组织。方法　取 2周龄的新生兔关节软骨 ,经消化 ,将获得的软骨细胞与牛 型胶原、人血冻干纤维蛋白原、凝血酶按一定比例混合 ,制成软骨培养物并在体外培养。培养第 3周时 ,取材进行 HE、甲苯胺蓝染色和透射电镜检查。结果　体外培养 3周 ,培养物内细胞均存活 ,形成软骨陷窝 ,同源性细胞簇出现 ,并分泌软骨基质。透射电镜下可见丰富的粗面内质网和线粒体 ,及少量的高尔基复合体。结论　用胶原蛋白和人血纤维蛋白为载体支架体外培养软骨细胞 ,可构建较大的组织工程软骨     

应用自体软骨细胞移植物修复关节软骨缺损

目的 通过体外扩增关节软骨细胞，构建软骨组织工程移植物，观察移植物对自体关节软骨损伤的修复作用。方法 由8周龄成年兔髌骨外侧缘获取关节软骨，分离软骨细胞，体外扩增后，与牛Ⅰ型胶原、人血冻干纤维蛋白原、凝血酶按一定比例混合，制成软骨培养物，体外培养4d。用于对成兔自体关节软骨损伤的移植修复。结果 成年家兔关节软骨细胞体外单层培养至第2代时开始出现去分化现象，可利用生长因子抑制去分化；该移植物的移植修复效果良好，修复后12周移植物与周边正常组织结合紧密、齐高，细胞趋于柱状排列，已形成透明软骨组织。结论 利用体外扩增的方法能获得足够量表型良好的关节软骨细胞，当应用胶原-纤维蛋白为载体制备出软骨组织工程培养物后，能较好地修复自体关节软骨损伤。     

松质骨骨基质明胶负载软骨细胞移植修复兔膝关节软骨缺损

目的 应用松质骨骨基质明胶(bone matrix gelatin，BMG)负载兔软骨细胞移植修复同种异体关节软骨缺损。方法 BMG载软骨细胞，体外培养12d后植入同种异体新西兰兔膝关节软骨缺损。对照组植入BMG或不作任何植入。移植前用胰蛋白酶处理关节软骨缺损。术后2、4、8、12、24周行解剖显微镜、组织学、电镜观察和免疫组织化学染色。结果 BMG术后8周降解。实验组术后24周关节软骨缺损以软骨组织修复，与周围软骨及软骨下骨愈合良好。Safranin-O、免疫组织化学染色证实其基质有蛋白多糖和Ⅱ型胶原，电镜观察表明修复组织软骨细胞及基质与正常关节软骨一致。BMG对照组以部分软骨组织修复，空白对照组以纤维组织修复。结论 松质骨BMG可作为软骨组织工程的天然细胞支架，其负载软骨细胞移植能修复同种异体兔关节软骨缺损。     

组织工程软骨修复全层大面积关节软骨损伤的实验研究

目的观察由软骨细胞体外培养的人工软骨组织对家兔膝关节软骨全层缺损修复的可行性.方法分离收集兔软骨细胞(4周龄),用培养液悬浮,经离心管体外培养成组织工程软骨,然后植入兔膝关节股骨负重面的缺损(5 mm×4 mm)部位,观察2,4,8,12周软骨缺损的修复情况,并进行大体和组织学观察.结果移植后第8周,移植部位填充物与宿主已完全整合,缺损处表面光滑;组织学显示,缺损部位新生软骨与所移植软骨有较好的连续性,并且缺损底部有软骨下骨及海绵状骨生成;对照组(未移植组),从组织学观察到缺损部位呈纤维样修复,未见有由新生软骨细胞形成软骨组织.结论用体外培养的组织工程软骨做移植物修复家兔关节软骨组织深层缺损具有较好的疗效.     

应用人胚软骨细胞修复关节软骨缺损的研究

目的了解应用人胚胎关节软骨细胞修复家兔膝关节软骨大面积深层缺损的效果,为临床上进一步应用人胚胎关节软骨细胞进行同种异体细胞移植的可行性提供佐证.方法将分离的人胚胎关节软骨细胞以天然胶原作为细胞外基质网架,进行体外三维培养1周后,植入家兔关节软骨大面积深层(5 mm×5 mm×4 mm)缺损处,对修复组织进行大体观察及组织学评估.结果移植术后12周,缺损部位平坦、光滑,已为类似关节软骨的半透明组织填充,修复组织与宿主关节软骨组织整合较好,界限模糊;移植术后24周,缺损表面为透明软骨填充,缺损深部的软骨下骨组织已经重建.结论人胚胎关节软骨细胞可以作为软骨组织工程的种子细胞用于修复关节软骨组织的缺损.     

生长因子与软骨细胞-胶原海绵复合体联合应用修复软骨缺损的研究

目的研究在体条件下碱性成纤维生长因子（bFGF）与软骨细胞-胶原海绵复合体联合应用修复软骨缺损的疗效. 方法取3日龄异体幼兔膝关节软骨细胞,体外培养后接种在胶原海绵支架上,再做移植.同一大白兔双侧膝关节内外髁分为4组：空白对照组、软骨细胞-胶原海绵复合体移植组（复合体移植组）、bFGF应用组和软骨细胞-胶原海绵复合体与bFGF联合应用组.移植术后不同时间分批处死,进行大体、光镜和电镜观察. 结果移植术后12周联合应用组软骨缺损修复以完全修复为主,而复合体移植组以不完全修复为主,差异有统计学意义;20周时2组均得到修复,差异无统计学意义,组织学分析均为软骨组织;在不同时间点空白对照组和单纯bFGF应用组软骨缺损修复差异无统计学意义,但组织学上修复的组织,前者为纤维组织,而后者同时有软骨组织存在. 结论 bFGF在软骨组织工程中有促进软骨缺损早期修复的作用.     

应用转染BMP7基因组织工程软骨修复兔膝关节软骨缺损观察

目的 构建转染骨形态发生蛋白-7(BMP7)基因组织工程软骨,观察对家兔膝关节软骨缺损的修复作用.方法 将转染BMP7基因的软骨细胞(5×109个/L)接种于胶原-纤维蛋白凝胶的支架中,培养14 d后移植于家兔膝关节软骨表面,直径为5.0 mm,深达软骨下骨板的全层软骨缺损部位.移植后4、8、12周处死家兔,通过创面形态观察、组织形态的光镜观察、BMP7基因在移植部位的表达,评价转染BMP7基因组织工程软骨对兔膝关节软骨缺损的修复作用.结果 转染BMP7和未转染BMP7基因组织工程软骨培养物的体积分别为9 mm × 9 mm×2 mm和8 mm×8 mm×2 mm.转染BMP7基因组移植于家兔膝关节软骨后4、8、12周,均有软骨缺损修复作用,且移植部位有红褐色BMP7 mRNA和棕红色BMP7蛋白表达.移植后12周修复效果佳.优于未转染BMP7基因组.结论 转染BMP7和未转染BMP7基因组织工程软骨对兔膝关节软骨缺损均有修复作用,但转染BMP7基因组织工程软骨修复作用更强.     

组织工程软骨修复兔膝关节软骨大面积深层缺损的实验研究

目的　将培养的软骨细胞埋在胶原凝胶中,观察其对家兔漆关节软骨大面积深层缺损的修复作用。方法体外培养软骨细胞,然后与胶原以一定比例混合培养,植入免膝关节股骨负重面的缺损（５ｍｍ ×４ｍｍ）部位,观察 ２、４、８．１２周软骨缺损的修复情况,并进行大体和组织学观察。结果移植后第 ８周,所修复组织的边缘变得模糊,与周围宿主整合得很好,缺损边缘不清晰。组织学观察,缺损处表面光滑,边缘与宿主完全整合,缺损处呈典型的关节软骨样组织,并且底部有新形成的软骨及骨小梁生成。对照组（未移植组）组织学观察缺损部位主要为纤维组织所填充,未见有由新生软骨细胞形成的软骨组织。结论埋在胶原凝胶中软骨细胞对家兔膝关节软骨深层缺损的修复作用。     

关节软骨组织体外保存力学环境构建

<正>目前多种治疗方法运用于关节软骨缺损的修复,但大多数疗效不理想,修复组织也以纤维软骨为多,缺乏正常透明软骨的生物力学性能及耐用性。同种异体骨软骨移植术是将供体关节的活性骨软骨块移植到受体关节软骨及软骨下骨缺损区,以修复关节软骨损伤〔1,2〕。患者软骨缺损区完全被透明软骨修复;关节软骨细胞完全包埋在周围软骨基质中,为"免疫豁免"组织。关节软骨是一种特殊的结缔组织,表面光滑位于骨两端关节面位置,在骨与骨间的关节处为其活动提供低磨损和摩擦     

β-TCP—HA—AC—BMSCs复合体修复兔膝关节骨、软骨缺损的实验研究

目的观察磷酸三钙人工骨（β-TCP）－透明质酸（HA）－脱细胞耳软骨（AC）-骨髓间充质干细胞（BM-SCs）复合体修复兔膝关节骨、软骨缺损的效果。方法获取新西兰大白兔BMSCs,体外诱导成软骨细胞并培养,取异体兔耳软骨并进行脱细胞处理,并与β-TCP—AC结合形成复合物接种软骨细胞。取6月龄新西兰大白兔12只,手术制备膝关节缺损模型,并随机分为A、B两组。A组（8只）缺损区植入β-TCP—AC—BMSCs,并于关节腔内注入HA;B组（4只）缺损空置作为空白对照。结果BMSCs在体外生长稳定,增殖能力强,可被诱导为软骨细胞。第16周,A组缺损区内充填白色半透明新生软骨组织,色泽与正常软骨相似,质韧,表面平整,与正常软骨界限消失,表面细胞平行于关节面,深层细胞排列紊乱,细胞呈团状,基质异染广泛,与周围正常软骨连接良好。B组缺损区未修复,底部为白色纤维组织。结论 β-TCP—HA-AC—BMSCs复合体修复兔膝关节骨、软骨缺损效果良好。     

Transglutaminase activity in aging articular chondrocytes and articular cartilage vesicles

Objective. Transglutaminases (TGases) (E.G. 2.3.2.13) catalyze a posttranslational modification of proteins and are associated with biomineralization in growth plate cartilage. Type II TGase participates in the activation of latent transforming growth factor β (TGFß), a crucial factor for both normal cartilage mineralization and the pathologic mineralization that results in calcium pyrophosphate dihydrate (CPPD) crystal formation in aging articular cartilage. To explore a possible association between TGase levels and CPPD crystal formation in mature articular cartilage, TGase activity in articular chondrocytes from old and young pigs and in the articular cartilage vesicle (ACV) fraction of porcine articular cartilage was examined. In addition, the effects of TGase inhibitors on the production of inorganic pyrophosphate (PPi), a process necessary for CPPD crystallogenesis, were determined. Methods. TGase activity was measured with a radiometric assay in cultured articular chondrocytes from the knee joints of old (3–5 years old) and young (2–6 weeks old) pigs and in the ACVs. PPi levels were measured in chondrocyte-conditioned media in the presence of TGase inhibitors or control compounds. Results. Levels of TGase activity in the cytosolic fraction of old chondrocytes were 7-fold higher than those in identically cultured young chondrocytes. The mean ± SD activity level in the membrane fraction of lysed chondrocytes was 6.0 ± 0.6 units/mg protein in old articular chondrocytes and was undetectable in young chondrocytes. In ACVs, the mean ± SD TGase activity level was 1.23 ± 0.1 units/mg protein. Type II TGase protein was present in chondrocyte cytosol and in ACVs. TGase activity was increased by TGFß to 120% of control values (P < 0.01), and decreased by insulin-like growth factor 1 to 80% of control values (P < 0.01). TGase inhibitors blocked media accumulation of PPi, an essential precursor of CPPD crystal formation, and a sensitive marker of TGFß effect. Conclusion. These data suggest a potential link between TGase activity and processes of pathologic biomineralization that result in CPPD crystal formation in aging articular cartilage.     

Lipid peroxides in human articular cartilage

Summary The hypothesis that increased generation of lipid peroxides (LP) causes articular cartilage damage in older patients and in those with osteoarthritis was tested by directly measuring LP tissue levels in various layers of human articular cartilage. The LP content was significantly greater in the superficial than in the deeper portion of the cartilage, but lower in cartilage than in liver, kidney, adrenal glands and synovium. When LP were related to the total lipid content of these tissues, a high peroxide per lipid ratio was obtained for articular cartilage. The relevance of these findings to the mechanism of cartilage fibrillation is discussed.     

Role of tenascin-C in articular cartilage

Tenascin-C (TN-C) is a glycoprotein component of the extracellular matrix (ECM). TN-C consists of four distinct domains, including the tenascin assembly domain, epidermal growth factor-like repeats, fibronectin type III-like repeats, and the fibrinogen-like globe (FBG) domain. This review summarizes the role of TN-C in articular cartilage. Expression of TN-C is associated with the development of articular cartilage but markedly decreases during maturation of chondrocytes and disappears almost completely in adult articular cartilage. Increased expression of TN-C has been found at diseased cartilage and synovial sites in osteoarthritis (OA) and rheumatoid arthritis (RA). TN-C is increased in the synovial fluid in patients with OA and RA. In addition, serum TN-C is elevated in RA patients. TN-C could be a useful biochemical marker for joint disease. The addition of TN-C results in different effects among TN-C domains. TN-C fragments might be endogenous inducers of cartilage matrix degradation; however, full-length TN-C could promote cartilage repair and prevent cartilage degeneration. The deficiency of TN-C enhanced cartilage degeneration in the spontaneous OA in aged joints and surgical OA model. The clinical significance of TN-C effects on cartilage is not straightforward.     

Horizontal splitting in patellar articular cartilage

Horizontal splits at the interface between the uncalcified and the calcified layers of the articular cartilage have been found in 31 of 50 transverse slabs of left patellae from a random series of 50 necropsy subjects aged 18 to 96 years. Evidence is presented that the splits are not due to autolysis nor to technical artefact, and it is here suggested that they result from shearing damage at the uncalcified-calcified cartilage interface during life.     

Immobilization ameliorates chemically-induced articular cartilage damage

We have previously shown that immobilization protects against the development of mechanically-induced osteoarthritis following-anterior cruciate ligament transection in dogs. Herein, we examine the effect of immobilization of the leg on the chemically-induced degeneration of femoral articular cartilage caused by intraarticular injection of iodoacetate in guinea pigs. One week after the injection, cartilage from animals which were not immobilized exhibited a decrease in Safranin O staining and a 10-20% reduction in the number of chondrocytes. Three weeks after injection, cell death and loss of Safranin O staining had progressed, and surface fibrillation and osteophytes had developed. Articular cartilage from the contralateral (uninjected) knees of guinea pigs which received iodoacetate, and from knees of animals which were immobilized for 1 week but did not receive iodoacetate, was histologically and histochemically normal. However, specimens from 2 of 4 untreated knees which were immobilized for 3 weeks showed a reduction in Safranin O staining. Immobilization of the knee did not alter the loss of Safranin O staining seen after intraarticular iodoacetate injection, but did reduce the depletion of chondrocytes (P less than 0.05). Furthermore, neither osteophytes nor fibrillation developed in any of the animals which were constrained after iodoacetate injection. Thus, immobilization was clearly protective in this model of chemically-induced cartilage injury.