胰岛素样生长因子-Ⅰ对软骨细胞移植修复关节软骨缺损的作用

目的：观察胰岛素样生长因子－Ⅰ（insulin-like growth factor-Ⅰ,IGF-Ⅰ)对关节软骨缺损修复的作用。方法：采用组织工程方法制备骨基质明胶（BMG）－软骨细胞移植物。40只4－5个月龄的新西兰兔均分为软骨细胞实验组及软骨细胞对照组,BMG对照组,空白对照组。实验组兔膝关节注射IGF－Ⅰ,各对照组均注射等渗盐水。术后行大体,组织学观察及免疫组化检测。结果：实验组软骨细胞生长较快,术后24周修复的软骨组织Safranin-O染色,Ⅱ型胶原染色与正常关节软骨一致,软骨细胞呈柱状排列,软骨细胞对照组术后24周修复软骨组织Safranin-O染色较淡,部分软骨细胞呈柱状排列,BMG,空白对照组未修复。结论：IGF－Ⅰ能促进关节软骨缺损修复。     

不同细胞移植修复兔全层关节软骨缺损的比较研究

目的 :比较软骨细胞、骨髓基质细胞及成纤维细胞对全层关节软骨缺损的修复作用。材料和方法 :取幼兔的软骨细胞、骨髓基质细胞及成纤维细胞 ,共 3种有生成软骨潜力的细胞进行体外分离培养 ;以聚乳酸 (PLA)为载体 ,将培养的原代细胞植入PLA支架上 ,形成细胞 -PLA复合物。于 2 8只成年新西兰大白兔的股骨滑车关节面上造成直径 4 5mm、深 3 0mm的全层关节软骨缺损 ,将 3种细胞 -PLA复合物分别植入关节软骨缺损处。植入细胞 -PLA复合物为实验组 ,单纯植入PLA支架为对照组。术后 6周、12周观察缺损修复情况及新生组织类型。结果 :软骨细胞移植组为软骨样组织修复 ,分界明显 ,甲苯胺兰及Ⅱ型胶原染色阳性 ;软骨下骨部分重建 ;细胞排列紊乱。骨髓基质细胞移植组为软骨样组织修复 ,分界不明显 ,甲苯胺兰及Ⅱ型胶原染色阳性 ;软骨下骨重建良好 ,软骨下潮线恢复 ;细胞排列趋于正常。成纤维细胞移植组为纤维组织修复 ,甲苯胺兰及Ⅱ型胶原染色阴性 ;软骨下潮线消失。对照组为纤维组织修复。结论 :软骨细胞、骨髓基质细胞移植修复软骨缺损明显优于成纤维细胞及对照组。骨髓基质细胞与软骨细胞移植组的修复结果无统计学差异 ,但骨髓基质细胞修复组织的细胞排列有序 ,软骨下骨重建良好 ,与周围组织融合密切 ,更接近正?     

成肌干细胞在透明质酸凝胶载体中诱导分化为软骨细胞的研究

目的 探讨以透明质酸钠凝胶作为组织工程软骨载体、以成肌干细胞作为种子细胞诱导分化形成软骨细胞的可行性. 方法体外分离培养兔成肌干细胞,采用结蛋白(Desmin)细胞化学染色进行鉴定,并观察兔成肌干细胞在不同浓度透明质酸钠凝胶中的生长状态.在5%透明质酸钠凝胶中以骨形态发生蛋白2(BMP-2)诱导兔成肌干细胞,倒置相差显微镜观察细胞生长情况,并通过甲苯胺蓝染色、免疫细胞化学及RT-PCR技术对诱导后的细胞进行鉴定. 结果原代培养兔成肌干细胞Desmin细胞化学染色呈阳性表达,且在不同浓度透明质酸钠凝胶中均生长良好.BMP-2诱导培养1d后,成肌干细胞即开始于透明质酸钠凝胶中伸展生长,诱导培养10d后,细胞形态由梭形向多边多角形转化,甲苯胺蓝染色阳性,Ⅱ型胶原免疫荧光检测阳性,RT-PCR检测Ⅱ型胶原和聚焦蛋白聚糖mRNA呈阳性表达. 结论兔成肌干细胞可在适当浓度BMP-2诱导下分化为软骨样细胞,透明质酸钠凝胶可作为软骨组织工程的良好载体.     

骨软骨组织中Ⅰ、Ⅱ型胶原基因表达的检测

目的：用原位杂交法研究骨和软骨组织中Ⅰ、Ⅱ型胶原基因的表达。方法：以与Ⅰ型、ⅡＡ型和ⅡＢ型胶原ｍＲＮＡ特异性互补的寡核苷酸序列为探钍,用末端转移酶将地高辛标记于３′-末端,在兔胫骨近端组织切片上进行Ｎｏｒｔｈｅｎ原位杂交,检测关节软骨、骺板和新生骨小梁区三种胶原基因的表达情况。结果：关节软骨和骺板区均检测到软骨细胞中有ⅡＢ型胶原基因表达,新生骨小梁区检测到成骨细胞中有Ⅰ型胶原基因表达。结论：本实验方     

运用双重免疫组织化学方法研究骨性关节炎关节软骨细胞的胶原表型

目的 :研究原发性骨性关节炎 (osteoarthritis,OA)关节软骨中软骨细胞的胶原表型。方法 :运用抗Ⅱ型、Ⅲ型及Ⅹ型胶原单克隆抗体 ,对OA关节软骨标本分别进行Ⅹ型和Ⅱ型胶原以及Ⅹ型和Ⅲ型胶原双重免疫组织化学染色。结果 :在原发性OA关节软骨的深层和钙化层软骨细胞中 ,存在Ⅹ型和Ⅱ型胶原以及Ⅹ型和Ⅲ型胶原的共同分布现象。同一个簇聚软骨细胞团中不同细胞的胶原表型也各不相同。结论 :原发性OA关节软骨细胞在疾病环境中发生进一步分化 ,在不同的分化阶段细胞的胶原表型不同 ,其中早期肥大软骨细胞能够同时合成Ⅹ型和Ⅱ型胶原以及Ⅹ型和Ⅲ型胶原。     

组织工程重建兔颞下颌关节盘软骨

目的 应用组织工程学方法重建颞下颌关节盘软骨。方法 分离6只日本大耳白兔髁状突软骨细胞。进行细胞的微载体大规模扩增,将扩增后的软骨细胞接种于组织引导再生胶原膜,体外适当培养后植入4只同种成年兔皮下,植入后12周,对所获组织进行组织形态学观察。结果 髁状[突软骨细胞在胶原膜内生长良好,植入动物体内12周后可形成乳白色类软骨样组织,其表面光滑,有弹性。甲苯胺蓝染色,细胞周围基质呈异染性。结论 应用胶原膜结合软骨细胞共同培养,可形成软骨样组织,该方法将有可能成为软骨缺损及关节盘破损修复的有途径。     

人胰岛素样生长因子1基因转染关节软骨细胞及其稳定表达

目的　研究人胰岛素样生长因子 1 (hIGF 1 )基因转染关节软骨细胞获得稳定表达的可行性。方法　用脂质体转染法将含有hIGF 1cDNA的真核表达载体转染兔关节软骨细胞 ,经G4 1 8筛选 ,形成阳性细胞克隆。继续培养 4周 ,原位杂交检测hIGF 1的表达 ,免疫细胞化学检测Ⅱ型胶原的表达。结果　G4 1 8筛选后所获得的阳性细胞克隆 ,原位杂交检测表明hIGF 1基因得到稳定表达 ,免疫细胞化学检测显示转染后的软骨细胞仍表达Ⅱ型胶原。结论　外源性hIGF 1基因能够在软骨细胞内获得稳定表达 ,而转染后的软骨细胞仍能稳定表达Ⅱ型胶原     

硅橡胶植入对关节软骨Ⅰ、Ⅱ、Ⅲ型胶原合成的影响

目的观察植入硅橡胶对兔关节软骨细胞Ⅰ、Ⅱ、Ⅲ型胶原合成的影响.方法18只成年新西兰大白兔,每只动物右膝为实验组,在膝关节滑车处植入硅橡胶;左膝为对照组,膝关节滑车处造成关节软骨缺损后不植入硅橡胶.分别于术后4周、24周和48周取材,采用原位杂交的方法观察关节软骨细胞Ⅰ、Ⅱ、Ⅲ型前胶原mRNA的表达,同时运用免疫组织化学的方法观察关节软骨细胞Ⅰ、Ⅱ、Ⅲ型胶原蛋白的合成.结果实验组中关节软骨可见Ⅱ型前胶原mRNA和胶原蛋白的稳定表达.Ⅰ型前胶原mRNA从术后4周起即有表达,且随时间延长而表达增加.但Ⅰ型胶原染色仅在24周时方开始呈现阳性.Ⅲ型前胶原的mRNA则在各期均未探测到,但从24周起Ⅲ型胶原染色呈阳性.对照组关节软骨在术后4周时即可见到Ⅰ、Ⅱ、Ⅲ型前胶原mRNA和胶原蛋白同时表达,且对照组中Ⅰ、Ⅱ、Ⅲ型前胶原mRNA和胶原蛋白表达均呈减少趋势.结论(1)前胶原mRNA的表达早于胶原分泌.Ⅰ型前胶原mRNA表达在实验组中比对照组晚,而且实验组中Ⅰ型前胶原mRNA的表达时间较长,容易观察到阳性的表达信号,所以Ⅰ型前胶原mRNA可以成为早期骨关节炎(OA)的分子标志物.(2)Ⅲ型前胶原mRNA由于表达时间可能较短,所以即使在实验组有Ⅲ型胶原蛋白分泌,但不易查到有Ⅲ型前胶原mRNA的表达,因此Ⅲ型前胶原mRNA不是一个合适的早期OA的分子标志物.(3)硅橡胶植入对关节软骨的胶原合成有一定影响,但对关节软骨的影响小于对照组.硅橡胶植入修补关节软骨缺损可延缓骨关节炎的发病过程.     

人半月板纤维软骨细胞培养及生物学特性研究

为探索分离培养人半月板纤维软骨细胞的简便实用方法，采用胰蛋白酶和胶原酶联合消化的方法，简便快速地获得大量成活率高的人半月板纤维软骨细胞，在F－12培养液中进行原代和传代培养，并对传代培养纤维软骨细胞进行了免疫组化鉴定，光镜及超微结构的观察，结果显示，光镜下，原代培养细胞呈线形，单层排列，电镜可见细胞内有丰富的粗面内质网及线粒体，细胞呈多极性，表面有突起，纤维软骨细胞免疫组化Ⅱ型胶原染色阳性，提示本实验建立的人纤维软骨细胞分离培养方法是一种可行的方法。，     

转胰岛素样生长因子-Ⅰ基因软骨细胞体内构建组织工程软骨

目的探讨转胰岛素样生长因子-Ⅰ(insu lin like growth factorⅠ,IGF-Ⅰ)基因软骨细胞与交联透明质酸复合后在裸鼠体内形成组织工程软骨的能力。方法分离培养人关节软骨细胞,将IGF-Ⅰ基因转染人关节软骨细胞,转基因细胞(转染组)、未转基因的软骨细胞(对照组)分别与交联透明质酸材料在体外孵育2 d后,移植裸鼠皮下。移植后6周,取出裸鼠皮下新生组织进行大体形态学观察(HE染色、阿尔新蓝染色),逆转录-聚合酶链反应(RT-PCR)法检测新生组织中Ⅰ型、Ⅱ型前胶原和IGF-Ⅰ基因mRNA的表达,判断裸鼠体内新生组织的结构和功能。结果转染组裸鼠体内新生组织6周仍能表达IGF-ⅠmRNA,而对照组不能;转染组和对照组在6周后均能形成软骨组织陷窝,但对照组的陷窝数量明显低于转染组;转染组的Ⅱ型前胶原mRNA的相对表达量为1.204±0.139,高于对照组(P<0.05);Ⅰ型前胶原mRNA的相对表达量为0.069±0.019,低于对照组(P<0.01)。结论转IGF-Ⅰ基因软骨细胞与交联透明质酸在体内能构建出软骨样组织,其结构更接近于自然软骨。     

关节软骨细胞体外培养时生物学性状的变化

<正> 在运动医学领域中,由于运动员的关节软骨伤病发生率远高于普通人,同时又缺乏有效的治疗方法,影响训练和运动成绩的提高,因此对关节软骨伤病的研究一直是重点科研项目之一。对关节软骨进行研究的手段近20年来有了巨大进步,其标志是关节软骨细胞在体外分离培养取得成功。它作为探索软骨细胞性状的先进手段正越来越广泛地被应用到关节软骨损伤和疾患的研究工作中。我所1982年在体外分离培养关节软骨细胞获得成功。近年来,对关节软骨细胞在体外培养过程中所     

Autologous chondrocyte implantation: cells phenotype and proliferation analysis

The phenotype and proliferation of human chondrocytes in culture were analyzed before they were implanted as autologous graft in cartilage lesions. During ten autologous chondrocyte implantations in articular cartilage lesions of the knee in ten patients, small amounts of cells to be implanted were collected and analyzed by morphology, cytochemistry (alcian blue, safranin-O), and immunocytochemistry (antibodies anti-S100 protein, anti-collagen types I and II, anti-chondroitin-S). In four cases the cells were also cultured for 3 weeks. At 1, 10, and 20 days of culture cells were incubated with 1 microCi/ml [3H]thymidine for proliferation analysis. In all cases the cells showed the morphological appearance of mature chondrocytes, stained positively for alcian blue and safranin-O, and revealed a strong immunoreaction for S-100 protein, type II collagen, and chondroitin-S but not for type I collagen. Radioisotope assay of chondrocyte proliferation at 1, 10, and 20 days of culture revealed a progressive increase in [3H]thymidine incorporation. These findings indicate that the cells before autologous implantation maintain their differentiated phenotype of mature chondrocytes and proliferate greatly. This analysis is an essential step preceding wider use of this treatment in humans. However, other biological aspects of the autologous chondrocyte graft remain to be elucidated.     

In vitro bonding of pre-seeded chondrocytes

Abstract This study investigated the capacity of seeded chondrocytes to join separate cartilage disc matrices in an in vitro model. Articular cartilage discs were harvested from pigs and devitalized by multiple freeze/thaw cycles. The devitalized cartilage discs were incubated in the presence (experimental group) or absence (control group) of chondrocytes for 10 days in order to allow chondrocytes to adhere to the matrix. After culturing, pairs of cartilage discs were held in apposition in a 48-multiwell plate and cultured for two and eight weeks. Twelve experimental composites (with cells) and twelve controls (without cells) were prepared per each time point. Samples were retrieved from culture and grossly inspected for adherence and processed for histological evaluation. Histological sections demonstrated the presence of new cartilage matrix formed by seeded chondrocytes bonding the two matrix discs together and producing glycosaminoglycans (GAG) able to diffuse within the devitalized tissue. Generally, gross adherence between the discs was demonstrated in the experimental samples, while the controls did not show any bonding. We conclude that isolated and seeded chondrocytes produce a new cartilaginous matrix, capable to join devitalized cartilage discs in vitro.     

A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen type I/III scaffold: in vitro and in vivo studies

Purpose

To develop a nano-structured porous polycaprolactone (NSP-PCL) scaffold and compare the articular cartilage repair potential with that of a commercially available collagen type I/III (Chondro-Gide^®) scaffold.

Methods

By combining rapid prototyping and thermally induced phase separation, the NSP-PCL scaffold was produced for matrix-assisted autologous chondrocyte implantation. Lyophilizing a water–dioxane–PCL solution created micro and nano-pores. In vitro: The scaffolds were seeded with rabbit chondrocytes and cultured in hypoxia for 6 days. qRT–PCR was performed using primers for sox9, aggrecan, collagen type 1 and 2. In vivo: 15 New Zealand White Rabbits received bilateral osteochondral defects in the femoral intercondylar grooves. Autologous chondrocytes were harvested 4 weeks prior to surgery. There were 3 treatment groups: (1) NSP-PCL scaffold without cells. (2) The Chondro-Gide^® scaffold with autologous chondrocytes and (3) NSP-PCL scaffold with autologous chondrocytes. Observation period was 13 weeks. Histological evaluation was made using the O’Driscoll score.

Results

In vitro: The expressions of sox9 and aggrecan were higher in the NSP-PCL scaffold, while expression of collagen 1 was lower compared to the Chondro-Gide^® scaffold. In vivo: Both NSP-PCL scaffolds with and without cells scored significantly higher than the Chondro-Gide^® scaffold when looking at the structural integrity and the surface regularity of the repair tissue. No differences were found between the NSP-PCL scaffold with and without cells.

Conclusion

The NSP-PCL scaffold demonstrated higher in vitro expression of chondrogenic markers and had higher in vivo histological scores compared to the Chondro-Gide^® scaffold. The improved chondrocytic differentiation can potentially produce more hyaline cartilage during clinical cartilage repair. It appears to be a suitable cell-free implant for hyaline cartilage repair and could provide a less costly and more effective treatment option than the Chondro-Gide^® scaffold with cells.     

聚乙醇酸负载同种异体软骨细胞移植修复兔关节软骨缺损

目的：应用聚乙醇酸（ＰＧＡ）负载的兔软骨细胞培养移植修复同种异体关节软骨缺损．方法：应用在生物体内可降解吸收、纤维状多孔态的ＰＧＡ作为支架行兔软骨细胞培养．培养１４天后，软骨细胞在ＰＧＡ提供的三维空间中大量分裂、增殖并合成大量软骨基质，形成ＰＧＡ－软骨细胞复合体，然后利用该复合体移植修复同种异体兔膝关节全层软骨缺损，对侧膝关节作对照．术后行大体、组织学、电镜动态观察及修复组织厚度测定．结果：ＰＧＡ在术后８周完全降解吸收，实验侧与对照侧修复组织的厚度有显著性差异（Ｐ＜０．０１）；术后１６周在实验侧可见典型的软骨组织，电镜下为成熟的软骨细胞，而对照侧为纤维组织修复．结论：应用ＰＧＡ－软骨细胞复合体移植，可修复同种异体的兔关节软骨缺损，为临床治疗关节软骨缺损奠定了基础．     

骨形态发生蛋白对培养胎儿关节软骨细胞代谢的影响及意义

为了解骨形态发生蛋白（ＢＭＰ）在关节软骨损伤与修复及异位化骨中的作用，本研究观察了部分纯化的牛骨形态发生蛋白（ｂＢＭＰ）和高度纯化的人骨形态发生蛋白（ｈＢＭＰ）对体外贴壁培养原代、反分化人胎儿关节软骨细胞及鼠３Ｔ３成纤维细胞ＤＮＡ、胶原和蛋白多糖合成的影响。结果发现：部分纯化的ｂＢＭＰ和高度纯化的ｈＢＭＰ均不促进原代人关节软骨细胞合成ＤＮＡ和胶原，并显著抑制其蛋白多糖的合成。部分纯化的ｂＢＭＰ对反分化关节软骨细胞和鼠３Ｔ３成纤维细胞的ＤＮＡ合成、胶原合成和蛋白多糖合成却有明显促进作用。因此，本研究认为，ＢＭＰ诱导下的关节软骨细胞不会发生反分化，而有可能进一步向肥大软骨细胞样细胞或成骨细胞样细胞方向分化，并可能使反分化的软骨细胞重新表达软骨细胞表型。     

Membrane-seeded autologous chondrocytes: cell viability and characterization at surgery

The implantation of chondrocytes, seeded on matrices such as hyaluronic acid or collagen membranes, is a method that is being widely used for the treatment of chondral defects. The aim of the present study was to evaluate the distribution, viability and phenotype expression of the cells seeded on a collagen membrane just at the time of the implantation. Twelve patients who were suffering from articular cartilage lesions were treated by the MACI^® procedure. The residual part of each membrane was tested by colorimetric assay (MTT) and histochemical and ultrastructural analyses were carried out. In all of the samples a large number of viable cells, quite homogenously distributed, was detected. The cells expressed the markers of the differentiated hyaline chondrocytes. These data reassure in that the MACI procedure provides a suitable engineered tissue for cartilage repair, in line with the clinical evidences emerging in the literature.     

关节软骨表层组织的免疫防护作用

文中的实验采用免疫荧光组织学方法观察到正常的关节软骨表层组织可阻止抗Ⅱ型胶原抗体侵入软骨深部组织,防止抗体与软骨细胞结合,具有免疫防护作用。