组织工程化软骨修复软骨损伤

背景:目前临床上多采用自体或异体软骨移植、软骨膜或骨膜移植、软骨细胞移植等方法进行软骨损伤修复,但均因取材局限、免疫排斥等导致效果不佳。利用组织工程的方法再造软骨,为软骨缺损的临床修复提供了新的思路与途径。目的:利用SCI数据库文献检索和深度分析功能,对于组织工程软骨的文献资料趋势进行多层次探讨分析,根据检索文献结果分析组织工程软骨的发展趋势。方法:通过计算机检索SCI数据库中2001/2010有关组织工程软骨的文献,检索词为"软骨(cartilage);组织工程(tissueengineering);软骨细胞(chondrocyte/cartilage cell);软骨组织(cartilage tissue);支架材料(scaffold materials);种子细胞(seed cell)",以文字和图表的形式进行统计和结果分析,描述其分布特征。结果与结论:SCI数据库2001-01/2010-12收录组织工程软骨相关文献总计3137篇,中国在该领域的文献产出量逐渐增多,总体呈上升趋势,Biomaterials《生物材料》杂志发表文献量最多。美国在该领域文献产出量多于其他国家,对该领域研究起到重要作用,发表文献较多的机构集中在美国的哈佛大学,莱斯大学,麻省理工学院,哥伦比亚大学,加利福尼亚大学圣地亚哥分校等。     

Advanced glycation end products‐induced chondrocyte apoptosis through mitochondrial dysfunction in cultured rabbit chondrocyte

Advanced glycation end products (AGEs) are an important mediator in osteoarthritis (OA) and cause apoptosis in articular chondrocytes. Mitochondrial function is involved in modulating apoptosis of articular chondrocytes. This study was performed to investigate the mechanism of AGEs‐induced chondrocyte apoptosis. The ratio of apoptotic cell and cell viability was surveyed by TUNEL, MTT,LDH release assay. The reactive oxygen species was determined by the fluorescent probe 2’, 7’‐dichlorofluorescein diacetate. The expression of caspase‐3 and cytochrome c was detected by Western blot. The mitochondrial membrane potential (?Ψm) was evaluated by rhodamine‐123 fluorescence. We found that AGEs induced apoptosis in primary rabbit chondrocytes, upregulation of ROS production, cytochrome c, and caspase‐3 levels. Simultaneously, AGEs decreases the levels of ?Ψm and ATP production; however, the antibody of AGEs (sRAGE) and antioxidant‐N‐acetylcys‐teine (NAC) significantly reversed AGEs‐induced the above damage thus to protect the cells from apoptosis. These observations suggested that the mechanism of AGEs‐induced chondrocyte apoptosis was primarily via ROS production and mitochondria‐mediated caspase‐3 activation.     

IDO-EGFP表达载体的构建及其在人软骨细胞中的表达

目的 克隆人吲哚胺双加氧酶(IDO)基因并构建IDO与增强型绿色荧光蛋白(EGFP)融合蛋白哺乳动物细胞表达载体。方法 利用RT-PCR方法从人白细胞克隆IDO基因并构建IDO-EGFP融合蛋白表达载体。通过细胞核转染仪将表达载体转人人软骨细胞进行瞬时表达，以共聚焦显微镜对表达的绿色荧光进行分析。结果 从活化的人白细胞中克隆到IDO基因编码区全长，并构建了IDO-EFFP融合蛋白表达载体。以该表达载体转染原代人软骨细胞，表达的融合蛋白较均匀地分布于整个细胞。结论 成功构建了IDO-EGFP哺乳动物细胞表达载体，为进一步研究IDO奠定了基础。     

三维立体空间动态培养及骨髓间充质干细胞软骨向分化的研究

目的三维立体空间动态诱导骨髓间充质干细胞软骨向分化，分析培养状态对软骨向分化的影响。方法分离培养骨髓间充质干细胞（MSC），以海藻酸钠凝胶载体诱导软骨向分化，于转壁反应器制造周期应力性三维立体环境，比较凝胶立体结构、细胞聚集状态、细胞构型等因素对软骨向分化的影响，分别以凝胶微球悬浮高密度细胞、凝胶包埋离心细胞团、凝胶覆盖细胞及普通细胞培养等方式．进行空间动态诱导培养：同时各培养方式分别设立相应静止培养方式作为对照。10d后观察大体和组织学形态．测量生物化学指标并筛选。结果三维诱导方式比平面诱导有效，动态诱导环境优于静止诱导环境．结合三维立体空间培养和动态环境可进一步提高软骨诱导效率。其中．凝胶微球悬浮高密度细胞的培养方式分化效果最好．优于凝胶包埋细胞团的培养方式。结论海藻酸钠凝胶微球立体动态培养可有效诱导MSC的软骨向分化．为改进软骨组织工程种子细胞的立体空间动态培养提供新思路。     

Long-term follow-up evaluation of autologous chondrocyte implantation for symptomatic cartilage lesions of the knee: A single-centre prospective study

IntroductionAutologous Chondrocyte Implantation (ACI) has been the first technique in reconstruction of a valid articular surface. The aim of this study was to evaluate clinical results of this technique at an average follow up of 162 ± 27 months (range 88–208) in a group of patients who underwent ACI.Materials and methods32 patients were operated between 1997 and 2007 for chondral lesions or osteochondritis dissecans of the knee. Mean size of the defect was 5.48 cm² ± 1.53 (range 2–9). Nine patients were treated with I generation technique and 23 with II generation. All patients were evaluated with Subjective IKDC and Tegner Activity Scales for clinical outcomes and with EQ-VAS for a quantitative measure of health after intervention, starting from pre-operative period and at regular follow up (minimum 88 months-maximum 208 months).ResultsA significant increment of all scores was noticed comparing preoperative and postoperative results. In particular medium IKDC score increased from 40.3 ± 9.6 in preoperative evaluation to 74.2 ± 11.6 at one year (p < 0.00001) and to 83.9 ± 10.4 at 5 years follow up (p < 0.001). Mean IKDC values at the last follow-up were 80.3 ± 14.2, showing no statistical differences with those obtained at five-year follow-up. Tegner Activity Scale values increased from 2.8 ± 1.1 preoperatively to 4.1 ± 1.1 (p < 0.0001) after one year and to 6 ± 1.1 at five years (p < 0.0001). Mean Tegner Activity Scale values decreased to 4.8 ± 1.4 at the last follow-up. EQ-VAS evaluation showed superposable results comparing the 5 years evaluation with the ones at a medium follow up of 162 ± 27 months.DiscussionThe most important finding is the reliability at long-term of ACI technique, which in our series gave excellent clinical results. No statistical differences were observed between first- and second-generation. Clinical outcomes were significantly better for defects in the femoral condyles, influenced by age (worse results over 30 years old).ConclusionsACI represents a valid technique for chondral and osteochondral lesions of the knee in a population heterogeneous for age, sex and activity level with good results even at a long term follow up.     

兔关节软骨细胞体外培养 形成基质能力的观察

目的：观察兔关节软骨细胞体外培养形成基质能力。方法：通过Ⅱ型胶原酶消化法获得大量高活性兔关节软骨细胞,倒置显微镜及透射电镜下观察细胞形态及超微结构,用含１５％胎牛血清的ＤＭＥＭ培养液连续培养,观察软骨基质的形成情况。结果：体外培养的软骨细胞为多角形,透射电镜下见胞浆内有丰富的粗面内质网系统及线粒体,胞膜下及胞浆中有较多分泌泡;连续培养１０天时,培养瓶底部出现肉眼可见的白色结节,蕃红－Ｏ染色显示白色结节含大量氨基葡聚糖阳性物质,２０天时形成火山口样结构,也被蕃红－Ｏ染成深红色。结论：软骨细胞在体外也具有很强的基质形成能力并能形成软骨样组织,因而可以用于进行软骨体外再造。     

Development of a biocomposite to fill out articular cartilage lesions. Light, scanning and transmission electron microscopy of sheep chondrocytes cultured on a collagen I/III sponge

The regenerative capacity of hyaline articular cartilage is limited. Thus, lesions of this tissue are a proarthrotic factor, and up to now the conservative treatment of cartilage lesions and arthrosis does not yield satisfying results. Therefore, autologous transplantation of articular chondrocytes is being investigated in a variety of different assays. The aim of our study was to create a mechanically stable cell-matrix implant with viable and active chondrocytes which could serve to fill out articular lesions created in the knees of sheep. For this purpose, articular cartilage was collected from knee lesions, chondrocytes were liberated enzymatically and seeded in culture flasks and cultured till confluency. Cells were then trypsinized and grown on a type I/III collagen matrix (Chondro-Gide™, Geistlich Biomaterials, Wolhusen, Switzerland) for 3, 6 and 10 days before being fixed and embedded for electron microscopy by routine methods. Scanning electron microscopy was performed after dehydration in acetone, critical point drying and sputter-coating with gold-paladium.

Light microscopically, clusters of chondrocytes can be seen on the surface of the matrix with a few cells growing into the matrix. Transmission electron microscopic photographs yield a rather differentiated chondrocyte-like appearance, which is evidence of a matrix-induced redifferentiation after dedifferentiation during the growth period in the culture flasks. Scanning electron microscopic results show large, flattened chondrocytes without signs of differentiation on plastic, whereas chondrocytes grown on the Chondro-Gide™ sponge show a more roundish aspect wrapping firmly around the collagen fibrils, exhibiting numerous contacts with the matrix. This cell-matrix biocomposite can now serve to fill out articular cartilage lesions created in the knees of sheep.     

Spatial arrangement of physeal cartilage chondrocytes and the structure of the primary spongiosa

Using laser confocal microscopy and 5-chloromethyl-fluoresceindiacetate (CMFDA) loading of chondrocytes we have investigated the structure of the ovine physis during late fetal development and its relationship to the structure observed in the primary spongiosa. Chondrocytes within the ovine growth plate form nests that together span the growth plate. We propose that all growth plates may be composed of nests of cells, but that the length of the individual nests changes between growth plates and with gestational age. The continuous column of cells seen within some growth plates is a nest of cells that is in the process of being absorbed by the invading metaphyseal front. Scanning electron microscopy of the mineralized portion of the primary spongiosa revealed structures that were consistent with the hypothesis that the cartilage surrounding the nest structure gives rise to the structure in the primary spongiosa. Although mineralization does not occur between cells within a nest, bands of mineral form between nests in the lower hypertrophic region and around the end of the nest as it reaches the hypertrophic region. This pattern of mineralization around and between nest termini yields the complex three-dimensional network of mineralized trabeculae observed in the primary spongosia. Received for publication on Aug. 11, 1999; accepted on Oct. 22, 1999     

5种黄酮有效部位的不同组方的细胞抗炎、免疫与骨细胞修复活性的比较

目的:以腰痛宁中生物碱加黄酮有效部位拆方为基础比较不同中药黄酮有效部位对细胞抗炎、免疫及骨细胞修复的影响,为风湿骨病处方候选药物筛选有效部位提供依据。方法:在腰痛宁马钱子、麻黄生物碱基础上,分别加入50%的甘草黄酮、淫羊藿黄酮、红花黄酮、骨碎补黄酮、桑寄生黄酮以及5种黄酮的等比例混合物,得到6个有效部位组方。采用半数有效浓度(EC50)或半数抑制浓度(IC50)评价各样品对巨噬Ana-1细胞分泌白细胞介素1β(IL-1β),白细胞介素-6(IL-6),肿瘤坏死因子-α(TNF-α)炎症因子的促进作用;对脂多糖(LPS)诱导的Ana-1细胞释放前列腺素E2(PGE2)的抑制作用;对IL-1β诱导的软骨细胞增殖的影响。同时采用最小二乘优化方法,计算各样品的EC50或IC50叠加值,根据EC50或IC50叠加值与实验值之间的差异分析各有效部位间的相互作用关系。结果:骨碎补黄酮为主的组方促进IL-1β分泌的活性最强;红花黄酮为主的组方促进IL-6分泌的活性最强且其他活性仅次于最佳的组方;由马钱子、麻黄生物碱+5种黄酮混合物构成的组方抑制PGE2分泌及促进TNF-α分泌、促进软骨细胞增殖的活性最强,且各模型下该组方中有效部位间有极强的协同作用。促软骨细胞增殖模型下,单一黄酮有效部位与生物碱构成的组方中的有效部位之间均表现出较强的拮抗作用。结论:不同药材黄酮之间有出色的协同增效作用,红花黄酮、骨碎补黄酮及各药材黄酮混合物具有良好的抗炎、免疫及骨细胞修复等综合药理活性,提示红花黄酮、骨碎补黄酮及不同药材黄酮混合物可作为风湿骨病药物的优选有效部位。     

苏红透骨散对骨关节炎模兔去分化软骨细胞影响的实验研究

目的：探讨苏红透骨散对膝骨性关节炎模兔软骨细胞增殖、凋亡的影响。方法：60只新西兰大白兔用HuIth法造骨关节炎动物模型,随机分成空白对照组、实验组;实验组用苏红透骨散灌胃,空白对照组用生理盐水。治疗第4、8、12、24周后每组各取6只动物,用亚硝酸盐还原酶法测定关节滑液NO水平。关节软片组织HE染色,光镜观察形态学变化、电镜观察软骨细胞内结构变化及原位末端标记（TLNEL）法观察软骨细胞凋亡病理改变,进行组织炳理学评估。结果：空白对照组NO活性在不同时期均高于实验组（8周,P〈0.05;12、24周,P〈0.01）,两组间比较有显著性差异（P〈0.05,0.01）：实验组的关节软骨病理改变总积分、软骨细胞病理改变积分和软片表层病理改变积分均明显低于空白对照组（P〈0.05）;实验组的软骨细胞凋亡指数与对照组的软骨细胞凋亡有显著性差异（P〈0.05）,与关节液中NO含量显著相关。结论：苏红透骨散对兔膝关节实验性骨关节炎软骨退变具有保护作用,通过NO诱导途径减少软骨细胞的凋亡,可能是其作用机制之一。