新型脱细胞软骨基质三维多孔支架的制备

目的 探讨脱细胞软骨基质三维多孔支架的制备方法以及将其应用于关节软骨组织工程的可行性. 方法 取天然人软骨粉碎后,采用梯度离心法取100 nm～5μm软骨微丝,脱细胞处理后制备为质量体积比为3%的悬液,采用冷冻冻干法制备脱细胞软骨基质三维多孔支架.254nm紫外线和碳化二亚胺/N-羟基琥珀酰亚胺对支架进行交联.冷冻冻干后,对支架材料进行组织学及扫描电镜观察,测定支架孔径和孔隙率、吸水率,并采用MTT法分析支架浸提液毒性.分离培养犬BMSCs,用TGF-β1成软骨诱导后种植至支架,倒置显微镜、电镜观察细胞在支架上的生长、分化情况. 结果 组织学观察显示,三维多孔支架中无软骨细胞碎片残留,甲苯胺蓝染色、番红O染色、Ⅱ型胶原免疫组织化学染色均呈阳性.扫描电镜显示支架内孔洞相互连通,孔径为(155±34)μm,孔隙率为91.3%±2.0%,吸水率为2 451%±155%.MTT法显示不同浓度支架浸提液与对照DMEM培养液吸光度值比较,差异无统计学意义(P＞0.05),支架无细胞毒性.倒置显微镜观察,细胞在支架上黏附良好;扫描电镜下细胞在支架上均匀分布,细胞呈圆形或椭圆形,并有基质分泌. 结论 制备的脱细胞软骨基质三维多孔支架去细胞彻底,保留了软骨ECM主要成分,无毒,具备合适的孔径和孔隙率,生物相容性良好,是软骨组织工程良好的支架载体.     

可塑形脱细胞软骨基质材料的制备及性状研究

目的 利用牛膝关节透明软骨进行脱细胞处理,制备新型可塑形生物材料,探讨脱细胞软骨基质作为组织工程载体材料的可行性. 方法 采集新鲜牛膝关节,切取关节表面透明软骨,冻干后低温粉碎为软骨微粒,胰酶、Triton X-100及低张Tris-HC1溶液联合作用进行脱细胞处理,冷冻干燥塑形,紫外线交联后制备脱细胞软骨基质材料.采用组织学、免疫组织化学、扫描电镜、孔隙率测定及生物力学检测等对材料的理化性状进行观察分析.取4只成年新西兰白兔骨髓制备BMSCs,传至第3代进行实验.观察浓度分别为100%、10%及1%的材料浸提液培养BMSCs 0、24、48及72 h后相对乳酸脱氢酶(lactate dehydrogenase,LDH)释放率,以含5?S的DMEM培养基作为阴性对照,观察细胞毒性作用;并将浓度为1×107 个/mL的BMSCs单细胞悬液与材料复合培养,观察细胞黏附情况. 结果 制备的脱细胞软骨基质材料呈白色多孔状结构.HE染色示材料由纤维状的软骨微粒形成网状结构,基质内无细胞成分残留;阿尔新蓝染色示微颗粒呈蓝色.材料Ⅱ型胶原免疫组织化学染色呈阳性.扫描电镜材料为多孔状海绵结构,孔径30～150μm.压汞法测定材料平均孔隙率为89.37%,平均孔径为90.8μm.力学分析示脱细胞软骨基质材料的压缩模量为(17.91±0.98)MPa,未经脱细胞处理的软骨微粒材料压缩模量为(15.12 ±0.77)MPa,差异无统计学意义(P>0.05);与正常牛关节软骨的(26.30±1.98)MPa比较差异均有统计学意义(P<0.05).细胞毒性实验显示,培养0、24、48及72 h,100%、10%、1%浓度材料浸提液条件培养基和阴性对照DMEM培养基相对LDH释放率差异无统计学意义(P>0.05).细胞黏附实验显示细胞可黏附于材料上,生长状态良好. 结论 牛关节软骨经脱细胞处理后,制成的多孔状脱细胞软骨基质材料,既保持了软骨基质中的主要成分,又具有良好的理化性质和生物相容性,可作为组织工程研究的一种新型载体材料.     

基于鸡蛋清制备的多孔支架用于组织工程软骨构建的实验研究

目的探究鸡蛋清制备多孔支架用于组织工程软骨构建的可行性。方法将鸡蛋清与去离子水以不同体积比(1:0、1:1和1:2)混合,通过冷冻干燥制备成多孔支架,行大体观、孔隙率及力学性能检测。取兔耳软骨细胞接种于不同体积比支架,于体外培养1 d、4 d、7 d后进行细胞染色和细胞增殖实验;体外培养至3周后,行组织学检测,观察其成软骨情况。结果三种不同体积比的蛋清均能制备成多孔结构的支架材料。随着去离子水比例的增加,其孔径大小明显提高,但是力学强度却呈下降趋势。活死细胞染色及细胞增殖实验证明,细胞可以在支架上黏附、增殖。延长体外培养时间至3周,HE染色显示三组蛋清支架上均有稚嫩的新生软骨基质形成,证实蛋清支架体外构建软骨的可行性。值得注意的是,随着去离子水比例的增加,残余的支架材料减少,新生软骨基质成分增多。结论基于鸡蛋清制备的多孔支架适合用于组织工程软骨构建。     

医用聚氨酯/脱细胞软骨支架的研制

我们将医用聚氨酯(PU)材料与脱细胞软骨基质生成一种新的PU/脱细胞软骨基质材料,评价该材料是否适合作为软骨组织工程支架. 一、材料与方法 1:分组:根据PU与脱细胞软骨基质的构成比进行实验分组,单用PU组为对照组,构成比为2:1组为实验1组,构成比为4:1组为实验2组.每组又按3种不同的实验温度(0、-10、-20 ℃)分为3组.各组样本量均为35份.     

微粒软骨脱细胞基质复合纤维蛋白凝胶构建软骨组织工程支架材料的研究

目的：应用异体微粒软骨脱细胞基质与纤维蛋白凝胶结合作为可注射性支架材料，进行体内构建良好可塑性和生物特性的组织工程化软骨。方法：制备普通家猪耳廓软骨微粒脱细胞基质，与体外扩增的小型实验猪第二代软骨细胞结合，以纤维蛋白凝胶为支架材料，利用其可注射性回植于实验猪自体腹壁外侧皮下，8周后取财进行大体及组织学检测，并与不合微粒脱细胞软骨基质实验组相比较。结果：培养出的组织工程化软骨组织细胞生长良好，具有分泌软骨基质功能，含微粒脱细胞软骨基质实验组表现出更佳的生物学性能。结论：将异体微粒软骨脱细胞基质与纤维蛋白凝胶结合，可以作为良好的可注射性复合支架材料应用于组织工程化软骨的构建。     

脱细胞软骨基质三维支架的制备及特性研究

[目的]探索脱细胞软骨基质三维多孔支架的制备及其应用于关节软骨组织工程的可行性。[方法]新鲜牛膝关节软骨粉碎后,梯度离心法获取软骨微粒,采用改进的Courtman改良法处理细胞后,再冷冻干燥,制备脱细胞软骨基质三维多孔支架。然后,采用京尼平对三维支架进行交联,再次冷冻干燥后,对支架材料进行大体、组织学染色及扫描电镜观察,分别测定支架的孔隙率、溶胀率、降解率。最后,分离培养兔骨髓基质细胞(BMSCs),采用MTT法检测BMSCs在支架材料上的生长、增殖情况,以柱形图表示。[结果]大体观察显示支架呈疏松多孔状,京尼平交联后整体呈深蓝色。组织学观察显示支架材料无软骨细胞碎片残留,HE染色、甲苯胺兰染色观察均未见软骨细胞残留。测量示支架孔隙率为90%,溶胀率为(1314±337)%,降解率2周为(13.69±7.3)%,4周为(25.99±8.9)%。MTT法显示细胞在支架上生长良好,与对照组DMEM培养液吸光度值比较,差异无统计学意义(P0.05),提示支架无细胞毒性。扫描电镜显示支架内孔洞较明显,BMSCs通过细胞突起黏附于支架表面,黏附良好,能较好地在其上生长。[结论]经改进的Courtman改良法处理的软骨基质三维多孔支架脱细胞更彻底,保留了软骨的天然细胞外基质成分,天然交联剂京尼平交联后支架的细胞相容性好,抗降解性得到了提高,是一种适用于软骨组织工程的良好载体。     

软骨脱细胞基质复合脂肪源性干细胞构建软骨组织的实验研究

[目的] 探讨软骨脱细胞基质支架材料的制备,及其体外复合脂肪源性干细胞构建软骨组织的技术方法.[方法] 成年新西兰大白兔脂肪源性干细胞获得,培养,扩增.成年新西兰大白兔新鲜软骨,低温冻干12 h,后经曲拉通、DNA、RNA酶等处理制备成为软骨脱细胞基质支架材料,终浓度为2×107/L的脂肪干细胞种植于软骨脱细胞基质中于软骨细胞方向诱导培养基中培养2周,构建软骨组织.新鲜制备的软骨脱细胞基质及构建的软骨组织分别行组织学、免疫组织化学及透射电镜检测.[结果] 实验制备的软骨脱细胞基质支架材料内无细胞结构存在,仅残留空白软骨陷窝.具有合适的孔隙率和孔径大小;复合脂肪源性干细胞后细胞向材料内部迁移,粘附,生长良好.部分载体内细胞Ⅱ型胶原免疫组化染色阳性.[结论] 软骨脱细胞基质可作为支架材料应用于软骨组织工程,复合脂肪源性干细胞培养可成功构建软骨组织.     

软骨细胞在异体脱细胞软骨基质上的体外培养实验

目的探讨脱细胞软骨基质对软骨细胞体外生长的影响。方法以脱细胞异体兔耳软骨基质为支架材料,以脱细胞异体兔耳软骨膜为对照,体外种植兔耳软骨细胞进行常规培养,观察软骨细胞的生长状况和增殖情况。结果软骨细胞进入脱细胞异体软骨基质构架裸露的空穴,生长旺盛。而在脱细胞异体软骨膜上,软骨细胞不易生长。结论支架材料的表面性状对细胞生长有显著影响,异体脱细胞软骨基质可提供适宜于软骨细胞生长的良好环境,有可能发展成为软骨组织工程的一种天然支架材料。     

软骨细胞在异体脱细胞软骨基质上的体外培养实验

目的探讨脱细胞软骨基质对软骨细胞体外生长的影响。方法以脱细胞异体兔耳软骨基质为支架材料,以脱细胞异体兔耳软骨膜为对照,体外种植兔耳软骨细胞进行常规培养,观察软骨细胞的生长状况和增殖情况。结果软骨细胞进入脱细胞异体软骨基质构架裸露的空穴,生长旺盛。而在脱细胞异体软骨膜上,软骨细胞不易生长。结论支架材料的表面性状对细胞生长有显著影响,异体脱细胞软骨基质可提供适宜于软骨细胞生长的良好环境,有可能发展成为软骨组织工程的一种天然支架材料。     

纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞修复兔膝关节软骨缺损的实验研究

目的：探讨纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞作为软骨组织工程支架的可行性及有效性,并为后续研究可注射性材料做基础。方法：体外分离培养软骨细胞后接种到纤维蛋白凝胶和脱钙骨基质支架材料体外培养4周,然后植入兔膝关节软骨缺损区继续培养4、8、12周后取材,分别行大体、组织学、Ⅱ型胶原免疫组织化学观察。并进行Wakitani评分,观察其体内修复关节缺损效果。结果：大体观察4周后,实验组软骨缺损区可有乳白色组织修复,12周可修复完全,并无明显凹凸感。光镜下8周可见大量软骨细胞修复,并在TB染色下见Ⅱ型胶原比4周时明显增多。12周时软骨陷窝结构形成,细胞形态排列及Ⅱ型胶原与正常软骨组织相近。结论：纤维蛋白凝胶和脱钙骨基质支架材料复合软骨细胞可以作为软骨组织工程支架材料,能够用于再生修复软骨的缺损。并为构建可注射性修复材料提供途径。     

Analysis of collagens solubilized from cartilage of normal and spontaneously osteoarthritic rhesus monkeys

Osteoarthritis (OA) is a disorder which results in the destruction of the articular cartilage and the remodeling of the subchondral bone in synovial joints. We have analyzed the cartilage collagen from normal and osteoarthritic free-ranging rhesus monkeys from the Cayo Santiago colony. The cartilage samples were assigned a severity score based on histological staging system and were divided into four groups (normals, mild OA, moderate OA and severe OA). After a 4.0 M guanidinium chloride (GuCl) extraction, the remainder of the cartilage was digested with pepsin and the collagen was salt precipitated at 2.5 M and 4.3 M NaCl. The GuCl solubility of the osteoarthritic cartilage increased compared to normals. Collagen extractability by GuCl also increased with the severity of disease. Pepsin digestion followed by salt precipitation shows that collagen from rhesus osteoarthritis cartilage is more easily extracted than from normal cartilage. With an anti-type I collagen antibody we have detected the presence of type I collagen in the severe OA cartilage samples but not in the milder OA groups or in normal cartilage. Total collagen content decreases with severity of OA, which is not due to changes in propyl hydroxylation because examination of collagen hydroxylation, based on hydroxyproline analysis, shows no difference between OA and normal cartilage.     

Zum Verhalten künstlich erzeugter Epiphysenfugendefekte

Drillholes of 8 mm diameter were made through the epiphyseal cartilage of the distal femora in 20 6-week-old Göttingen minipigs. The defects were filled with autologous or with homologous cartilage. This was intended to prevent epi-metaphyseal osseous bridge formation. If of appropriate size and location, the latter whould lead to subsequent misgrowth. In group A (autologous costal cartilage), at all drillholes the transplantated costal cartilage can prevent ossification of the defect. An epiphyseal boundary lamella was formed over the transplant which precluded penetration of vessels into the defect. In group B (homologous costal cartilage), the transplanted costal cartilage showed a tendency to mineralization in all preparations at the 20 drillholes available. The cartilage was integrated into the primary cancellous bone developing within the defect. An epiphyseal boundary lamella had not formed in the period of the experiment (in contrast to autogenous transplantation). These investigations show that autologous costal cartilage is superior to homologous costal cartilage in the potential clinical application of costal cartilage transplants in the treatment of Brodie abscesses or post-traumatic epiphyseodeses.     

Poor outcome after a surgically treated chondral injury on the medial femoral condyle: early evaluation with dGEMRIC and 17-year radiographic and clinical follow-up in 16 knees

Background and purpose — The optimal treatment for traumatic cartilage injuries remains unknown. Contrast-enhanced MRI of cartilage (dGEMRIC) evaluates cartilage quality and a low dGEMRIC index may predict radiographic osteoarthritis (OA). The purpose of this study was (a) to explore the results 17 years after surgical treatment of an isolated cartilage knee injury and (b) to evaluate the predictive value of dGEMRIC.

Patients and methods — 16 knees with an isolated traumatic cartilage injury of the medial femoral condyle had cartilage repair surgery either by microfracture or autologous cartilage implantation. dGEMRIC of the injured knee was performed 2 years after surgery and radiographic examinations were performed 17 years after the operation.

Results — Radiographic OA was present in 12 of 16 knees. Irrespective of surgical method, the dGEMRIC index was lower in repair tissue compared with adjacent cartilage in the medial compartment, 237?ms vs. 312?ms (p < 0.001), which in turn had lower value than in the non-injured lateral cartilage, 312?ms vs. 354?ms (p < 0.008). The dGEMRIC index in the cartilage adjacent to the repair tissue correlated negatively with radiographic osteophyte score, r = –0.75 (p = 0.03).

Interpretation — A traumatic cartilage injury is associated with a high prevalence of OA after 17 years. The low dGEMRIC index in the repair tissue 2 years postoperatively indicates fibrocartilage of low quality. The negative correlation between the dGEMRIC index in the adjacent cartilage and future OA suggests that the quality of the surrounding cartilage influences outcome after cartilage repair surgery.     

Microscopic localization of active gelatinases in equine osteochondritis dissecans (OCD) cartilage

OBJECTIVE: To investigate the relationship between matrix metalloproteinase (MMP) activity and osteochondritis dissecans (OCD) in the equine joint. METHODS: Equine articular cartilage was obtained from normal (N = 8) and osteochondrotic (OCD) (N = 6) femoropatellar joints from horses at necropsy. The activity of gelatinase MMPs was determined in sections of cartilage by in situ gelatin zymography. RESULTS: Gelatinase activity was markedly increased in articular cartilage obtained from OCD samples and was particularly prominent in the deep cartilage zone. Activity was only seen in the pericellular area of chondrocytes. In addition, in OCD cartilage there were vertical lines of activity, starting from the deep zone and radiating towards the articular surface. In contrast, normal cartilage showed only a very small amount of gelatinolytic activity, which was not restricted to specific cartilage zones. Gelatin zymography of culture supernatants from isolated chondrocytes demonstrated increased production of MMP-2 and MMP-9 from OCD chondrocytes. CONCLUSIONS: Sections of articular cartilage from OCD lesions revealed MMP activity, especially in the deep zone adjacent to the calcified subchondral bone. This MMP activity could account for the loss of cartilage integrity in the deep cartilage zone and the vertical lines of activity could represent areas of mechanical weakness, likely to result in fissures and the release of cartilage fragments into the joint space.     

过氧化物酶Ⅰ在骨关节炎软骨组织中的表达与分布及意义

目的观察过氧化物酶Ⅰ（PrxⅠ）在正常和骨关节炎（OA）膝关节软骨组织中的表达和分布特点,探讨PrxⅠ与活性氧歧化物和凋亡等在OA软骨表层聚集现象之间的关联。方法分别从正常膝关节提取正常软骨（NC,n=21）和接受膝关节表面置换术的OA患者提取OA软骨（OA,n=21）,应用Westernblot技术检测PrxⅠ在正常软骨细胞和OA软骨细胞中表达水平的总体差异,并用免疫组织化学技术观察PrxⅠ蛋白在正常与OA软骨表/中/深各层组织表达和分布的特点。结果 Western blot证实PrxⅠ在OA软骨组织中的表达水平较正常软骨组织显著提高2.89倍（t=18.34,P〈0.01）。免疫组织化学显示PrxⅠ在正常软骨组织的表层、中层和深层呈现较均一的表达,但是,在OA软骨组织中,PrxⅠ的表达水平存在显著层间差异。PrxⅠ在软骨组织深层表达水平显著升高,但在浅层细胞中,PrxI的表达水平反而显著减低甚至缺如。结论虽然总体表达水平升高,但PrxⅠ在OA软骨组织表层的表达缺如,可能与OA软骨组织表层活性氧歧化物蓄积和细胞凋亡聚集现象相关。     

创伤后关节软骨中MMP-3的表达及意义

目的探讨基质金属蛋白酶-3(MMP-3)在创伤后关节软骨退变中的作用。方法手术建立单纯软骨缺损的动物模型,术后4周、8周对软骨退变进行大体评分,用免疫组织化学方法检测MMP-3在关节软骨中的表达。结果术后关节软骨发生退变,MMP-3在关节软骨中的表达与退变有关。结论MMP-3在创伤后软骨退变过程中发挥重要作用。     

Time course evaluation of reparative cartilage with MR imaging after autologous chondrocyte implantation

The aim of this study was to evaluate the qualitative change in reparative cartilage after autologous chondrocyte implantation (ACI). Ten knees of 10 patients were studied. The signal intensities of reparative and normal cartilage were evaluated by fat-suppressed three-dimensional spoiled-gradient recalled (FS 3D-SPGR) MR imaging. The signal intensity (SI) index (signal intensity of reparative cartilage divided by that of normal cartilage) was defined and the change in SI index was investigated. Histological and biochemical evaluation was done at the second look arthroscopy. The SI index was at its lowest level immediately after ACI and increased with time to 9 months thereafter. After 9-12 months, the SI index settled to almost level and was maintained at that value for at least 2-3 years postoperatively. The average of the SI indexes after 12 months to the last examination was 74.2 +/- 4.6 (range 64.2-82.8), which means signal intensity of reparative cartilage was maintained at a value lower than that of normal cartilage. The total ICRS score was 11.6 +/- 2.3 points (mean +/- SD). The GAG concentration was 107.9 +/- 17.0 microg/mg (mean +/- SD) in normal cartilage and 65.9 +/- 9.4 microg/mg in reparative cartilage. The quality of reparative cartilage as hyaline cartilage was inferior to that of normal cartilage. In the present study, the time course change in the SI index indicates that the major maturation process of implanted chondrocytes neared completion in 9-12 months. Minor changes, such as matrix remodeling with reorganization of the collagen fibers in reparative cartilage, may continue, but an almost identical condition seemed to be maintained during the first 2-3 years of follow-up. SI index does not always reflect all properties of reparative cartilage but may be a useful parameter for noninvasive evaluation.     

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。     

Analysis of experimental immune synovitis cartilage using monoclonal antibodies reactive to rabbit proteoglycan

This study details the macromolecular changes in cartilage involving proteoglycan molecules in an animal model of rheumatoid arthritis. In experimental chronic immune synovitis, fluorescein-conjugated mouse IgG and three monoclonal antibodies (MAbs 2G2, 2E9, and 6C9) portraying differing fine antigenic specificity for rabbit cartilage proteoglycan monomer were utilized to detail alterations in cartilage proteoglycan. In normal and IgG-immune animals, fluorescein isothiocynate (FITC)-conjugated MAbs 2G2 and 2E9 stained cellular/pericellular (C/PC) region intensely. FITC-MAb 2G2 stained cartilage interterritorial matrix as well. FITC-MAb 6C9 stained only C/PC area lightly but did not stain matrix. A marked decrease in staining intensity with FITC-MAb 2G2 was noted in cartilage sections derived from animals with immune synovitis. A corresponding increase in staining of cartilage was noted with FITC-MAb 6C9. The augmented staining of articular cartilage with FITC-MAb 6C9 was most prominent in femoral condyle tissue sections, which corresponded to the cartilaginous area, with the greatest severity in gross pathology. There was a slight augmentation of staining with FITC-MAb 2E9, especially in the C/PC area of medial/femoral cartilage. In addition, the animals with immune synovitis showed abortive cartilage repair exemplified by the presence of chondrocyte cloning (up to 20 cells) which correlated with increased FITC-MAb 2G2 staining. The differential MAb staining patterns of cartilaginous tissues obtained utilizing FITC-conjugated monoclonal antibodies with known fine antigenic specificity indicates a modulation of proteoglycans involving predominantly core protein epitopes in the articular cartilage of animals with chronic immune synovitis.     

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