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全外生长的影响。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     

兔自体耳软骨膜复合游离移植形成工程化软骨的实验研究

目的：探求用软骨腹复合移植的方法形成自体软骨的可行性。方法：将兔自体耳软骨膜包裹异体耳软骨脱细胞基质材料复合游离移植于兔背部皮下（I组），并在移植物附近皮下游离移植双层耳软骨膜（Ⅱa组）及单纯脱细胞的异体耳软骨基质（Ⅱb组）作为对照。在术后3周、6周分别取标本进行研究。结果：在3周、6周时，I组与Ⅱa组成软骨细胞的增殖变化情况差异有显性（P＜0．05），I组比Ⅱa组成软骨细胞的增殖明显旺盛，有的已经向软骨细胞转化，并有少量细胞外基质分泌，基质内软骨囊出现。Ⅱb组无成软骨细胞及软骨囊出现。结论：自体耳软骨膜与异体耳软骨脱细胞后的基质材料复合移植促进自体软骨生成。提示该基质材料有促进软骨膜细胞分化和生长的作用。     

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。     

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。     

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。     

利用脂肪干细胞构建软骨修复兔膝关节软骨缺损的初步研究

目的 利用兔同种异体软骨脱细胞基质支架和脂肪干细胞体外构建组织工程软骨,探讨其修复关节软骨损伤的可行性.方法 将新西兰大白兔的脂肪干细胞与软骨脱细胞基质支架复合,于软骨细胞方向诱导培养基中培养两周,构建组织工程软骨.兔24只随机分为A、B、C 3组, A组关节软骨缺损处置入经诱导的脂肪源干细胞复合软骨基质支架, B组缺损处只置入软骨基质支架, C组软骨缺损处不做任何处理.分别于术后第12周处死动物,修复处行大体、组织学、Ⅱ型胶原免疫组化染色和透射电镜检测.结果 A组软骨缺损处被类软骨组织填充,修复区表面光滑;Ⅱ型胶原免疫组化染色和甲苯胺蓝染色阳性;电镜下可见软骨陷窝内有细胞结构存在,且有大量均匀颗粒状细胞分泌基质成分存在,细胞周围大量胶原纤维.B组软骨缺损处为纤维组织状物填充,C组软骨缺损处无修复组织填充.结论 脂肪干细胞与软骨脱细胞基质复合并向软骨诱导后可良好地修复关节软骨缺损,具有替代正常软骨的潜力.     

三维多孔生物降解活性支架材料在骺软骨组织工程中的应用

目的:探讨以生物活性可降解吸收材料聚己内酯/聚乳酸共聚物(PCL/PLA)为载体的兔骺软骨细胞体外培养的可行性,从而为开辟治疗骨骺早闭的新方法奠定基础。方法:应用具有三维多孔立体结构的生物活性可降解吸收支架材料为细胞外基质替代物,进行单纯骺软骨细胞体外培养及以生物材料为载体的体外降解实验,检测细胞生长情况。结果:兔骺软骨细胞生长良好,增殖活跃,合成大量软骨基质,并与材料形成细胞-材料复合体,细胞充满于材料孔隙内。结论:生物活性可降解吸收材料具有良好的组织相容性及表面活性,它所提供的三维立体空间结构有利于细胞生长繁殖。     

多孔左旋聚乳酸-脱细胞软骨基质支架性能研究

目的 研究不同实验条件下制备的多孔左旋聚乳酸-脱细胞软骨基质支架的结构与性能,分析其作为软骨组织工程支架材料的可行性.方法 在三种实验温度下,应用热致相分离法将左旋聚乳酸与猪脱细胞软骨基质按不同比例混合,制备生物多孔复合物支架,并对各支架材料孔径、孔隙率、亲水性、生物力学强度和降解率进行研究.结果 多孔左旋聚乳酸-脱细胞软骨基质支架孔径为100～300μm.形态自然圆润,取向规则,脱细胞软骨基质在左旋聚乳酸三维结构中均匀分布,其结构和性能均符合软骨组织工程支架材料的要求.结论 左旋聚乳酸-脱细胞软骨基质支架可用于软骨组织工程研究.     

利用脂肪干细胞构建组织工程软骨修复兔膝关节软骨缺损

目的探讨以脂肪于细胞（ADSCs）复合脱细胞软骨基质支架构建组织工程软骨修复兔膝关节软骨缺损的效果。方法以人关节软骨脱细胞基质为支架，复合经诱导的兔ADSCs，体外分别经静态培养和生物反应器培养，构建组织工程软骨。对膝全厚关节缺损进行修复，并与单支架组、空白对照组比较，其中空白对照组12个关节，脱细胞软骨支架组16个关节，静态培养细胞支架组24个关节，生物反应器培养细胞支架组8个关节。分别于术后3、6个月对修复关节进行大体、组织学及免疫组化观察。结果实际完成观察的关节数为44个，其中空白对照组9个，脱细胞软骨支架组11个，静态培养细胞支架组18个，生物反应器培养细胞支架组6个。空白对照组全为纤维组织或纤维软骨样修复；单支架组5个关节为未成熟透明软骨，无成熟透明软骨形成；静态培养细胞支架组83．3％为透明软骨，其中3个关节为成熟透明软骨，12个关节为未成熟透明软骨；生物反应器培养细胞支架组100％为透明软骨，其中2个为成熟透明软骨，4个为未成熟透明软骨。Wakitani评分各组差异有统计学意义（P〈0．05）。结论ADSCs复合脱细胞软骨基质支架能良好地修复兔膝关节全厚软骨缺损，应用生物反应器技术有助于构建组织工程软骨，促进软骨缺损的修复。     

同种脱细胞软骨基质与软骨细胞复合物修复兔甲状软骨缺损的实验研究

目的 探讨脱细胞软骨基质(acelluar cartilaginous matrix，ACM)与软骨细胞复合后植入兔体内，能否形成软骨修复甲状软骨缺损。方法 分离培养免甲状软骨细胞，与ACM体外复合培养，形成ACM-软骨细胞复合物，进行组织学分析及用于修复兔甲状软骨缺损。新西兰大白兔18只，制成两侧甲状软骨缺损模型，随机分为3组，每组6只。对照组：只制备缺损，不作修复；ACM修复组：采用单纯ACM修复缺损；实验组：用ACM-软骨细胞复合物修复。术后8周处死动物，取出标本，进行大体和组织学观察。结果 体外培养时，软骨细胞能在ACM表面生长，未长入基质内。动物实验结果，对照组：甲状软骨缺损处被肌肉、结缔组织充填；ACM修复组：ACM内炎性细胞浸润，轻度吸收变形；实验组：复合物植人体内后8周未形成软骨，甲状软骨缺损修复不理想。结论 ACM体外培养和体内植入均未能为软骨细胞生长提供支持，作为一种天然细胞培养支架，尚有待进一步改进与完善。     

用含活性细胞的胶原支架复合皮修复小鼠全层皮肤缺损的研究

目的构建具有活性细胞成分的复合皮，观察其在修复小鼠全层皮肤缺损中的作用。方法将异种成纤维细胞种植于脱软骨细胞胶原支架上培养3d，形成真皮替代物。再将其放置于培养的上皮细胞膜上共同培养10d，组成含活性细胞成分的复合皮。将复合皮移植于小鼠的全层皮肤缺损处，记录其生长情况并于术后定期活检，进行组织学观察。结果胶原支架中可见生长良好的成纤维细胞和复层表皮细胞。移植术后1周，复合皮与小鼠缺损创面粘连紧密，可见明显的血管化。术后6周，创面愈合良好，移植物与创缘融合，未见明显排斥反应。结论以脱软骨细胞胶原为支架构建的复合皮，可作为皮肤替代物修复全层皮肤组织缺损。     

Feasibility of Autologous Bone Marrow Mesenchymal Stem Cell–Derived Extracellular Matrix Scaffold for Cartilage Tissue Engineering

Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell–derived ECM (aBMSC‐dECM) membrane was collected and fabricated into a three‐dimensional porous scaffold via cross‐linking and freeze‐drying techniques. Articular chondrocytes were seeded into the aBMSC‐dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC‐dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC‐dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC‐dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC‐dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering.     

Tissue engineering: chondrocyte cultures on type I collagen support. Cytohistological and immunohistochemical study

Cartilaginous tissue has limited capacity for regeneration after damage, since the natural repair process leads to the formation of fibrocartilaginous tissue which does not have the resistance and capability of deformation under load, typical of hyaline cartilage which covers the articular surfaces. The possibility of transplanting human chondrocytes for cartilage reconstruction has been demonstrated in orthopaedics. The scope of our study was to evaluate the possibility of cultivating and expanding human chondrocytes seeded on a pure equine type I collagen support. Human articular cartilaginous cells multiplied and grew on a type I collagen substrate with production of extracellular matrix. This chondrocyte culture showed a correct morphology and phenotype as shown by alcian-PAS staining to indicate the presence of mucopolysaccharides and by immunohistochemical methods to identify type II collagen. The use of scaffolds may lead to improvement in the surgical technique, by making it possible to hold the cells physically in the area to be repaired and by allowing optimum spatial adaptation inside injuries of all shapes.     

The ribosomal protein QM is expressed differentially during vertebrate endochondral bone development.

Endochondral ossification is a carefully coordinated developmental process that converts the cartilaginous model of the embryonic skeleton to bone with accompanying long bone growth. To identify genes that regulate this process we performed a complementary DNA (cDNA) subtractive hybridization of fetal bovine proliferative chondrocyte cDNA from epiphyseal cartilage cDNA. The subtracted product was used to screen a fetal bovine cartilage cDNA library. Ten percent of the clones identified encoded the bovine orthologue of the human ribosomal protein "QM." Northern and western blot analysis confirmed that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In contrast, no detectable difference in the expression of mRNA for the ribosomal protein S11 was detected. Immunohistochemical analysis of fetal bovine limb sections revealed that QM was not expressed by the majority of the epiphyseal chondrocytes but only by chondrocytes in close proximity to capillaries that had invaded the epiphyseal cartilage. Strongest QM expression was seen in osteoblasts in the diaphyseal region of the bone adjoining the growth plate, within the periosteum covering the growth plate and within secondary centers of ossification. Hypertrophic chondrocytes within the growth plate adjoining the periosteum also were positive for QM as were chondrocytes in the perichondrium adjoining the periosteum. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. The in vivo and in vitro expression pattern of QM suggests that this protein may have a role in cell differentiation before mineralization.     

Growth and integration of neocartilage with native cartilage in vitro.

Poor integration of neocartilage with recipient has been a major obstacle to articular cartilage restoration. An in vitro study was designed to provide insights regarding the integration process. Cartilage explants and chondrocytes were harvested from the distal sternum of 16-day-old chick embryos. Four million chondrocytes and one 1mm(3) explant were centrifuged together in a 0.75ml tube. In the constructs, consisting of cartilage explant and chondrocyte pellet, isolated chondrocytes attached to the surface of the explant at the beginning of the culture, followed by significant chondrocyte death at the interface between chondrocyte pellet and explant. Chondrocyte apoptosis was seen almost exclusively at this interface. Meanwhile, the interface was a zone with active extracellular matrix deposition as demonstrated by immunohistochemistry. By two weeks, the junction of neocartilage and native cartilage explant had formed an acellular zone with collagen fibrils orientated parallel with the surface of the cartilage explant. In conclusion, chondrocyte death leads to acellularity and fibril network reorganization at the neocartilage/explant interface, and impacts the quality of cartilage repair as abnormal matrix remodeling implies.     

S-100 protein in human cartilage lesions

S-100 protein is an acidic calcium-binding protein that was originally isolated from the mammalian central nervous system in 1965. Initially, S-100 protein was thought to be specific to neuroectodermal tissues, but its presence in chondrocytes was recently reported. This study is an analysis of the distribution of S-100 protein in lesions of human cartilage and its possible significance. Several cartilaginous tumors, both benign and malignant, as well as normal epiphyseal growth plates, were examined for S-100 protein by the immunoperoxidase technique. Each cartilaginous lesion that was examined showed immunoreactivity for S-100 protein. The staining product was noted only intracellularly. The highest intensity of staining was seen in the hypertrophic chondrocytes of the zone of provisional calcification in the growth plate and in the large chondrocytes located adjacent to areas of matrix mineralization in cartilaginous tumors. In normal epiphyseal growth plates, the intensity of staining increased in chondrocyte cytoplasm as one moved from the proliferating columnar chondrocytes through the zone of hypertrophic chondrocytes to the hypertrophic, degenerating chondrocytes in the zone of provisional calcification. In cartilaginous tumors, the cells of enchondroma and of the cartilaginous cap of osteochondroma were more immunoreactive than those of chondromyxoid fibroma. In benign chondroblastoma, the chondroblasts were less reactive than the chondrocytes in areas of chondroid matrix production. The latter areas of chondroblastomas showed stronger immunoreactivity in the matrix-enclosed cells adjacent to areas of mineral deposition. Among conventional chondrosarcomas, grade-I tumors showed greater immunoreactivity of the chondrocyte cytoplasm than did those of a higher grade, in which chondroid matrix production was less abundant.(ABSTRACT TRUNCATED AT 250 WORDS)