鼻中隔软骨细胞细胞组织工程法构建预定形态软骨

目的 探讨利用人算中隔软骨细胞组织工程方法构建预定形态软骨的可能性。方法 将人鼻中隔软骨细胞播种在聚乙醇酸（polyglycoli acid,PGA)无纺网支架材料上，制成片状和管状结构，埋入裸裸鼠体内，经4、6、8周后取材作大体及组织学观察。结果 大体观察见裸 本内形成了预定的片状和管状软骨。组织学观察：6周时软骨细胞基本成熟，Masson三色染色显示胶原形成，番红花－“O”染色证实其基质中存在糖多糖。对照组于6周时PGA纤维基本消失。结论 人鼻中软骨细胞与PGA无纺网复合可在裸鼠体内形成预定形态软骨。     

软骨组织工程进展

组织工程软骨的研究目前仍处于实验室阶段，本文就组织工程软骨的概念，生物支架材料，体外和体内培养方法及组织工程软骨性能等方面作一简述。     

定向诱导人骨髓间质干细胞向软骨细胞分化的初步研究

目的 采用组织工程方法，将人骨髓间质干细胞（mesenchymal stem cells,MSC）在体外培养后诱导后，诱导为软骨细胞，为软骨的修复和重建提供条件。方法 根据5例的项实验经验，采用12例开胸手术所取正常肋骨分别实验。将肋骨剪开，用含双抗的不完全培养基反复冲洗骨髓腔得到骨髓液，经密度梯度离心获得单个核细胞。体外培养、分离获得人内髓MSC，经无血清培养诱导液进行诱导培养，透射电镜观察细胞外基质形成及超微结构，免疫组化检测MSCⅡ型胶原表达。结果 12例的肋骨骨髓MSC体外培养2周后见细胞融合，传代培养并进行诱导。部分细胞开始由梭形转变为圆形，并分泌基质，甲苯胺蓝染色呈阳性；MSCⅡ型胶原表达阳性。结论 人MSC在体外能诱导为软骨细胞，可用于细胞工程软骨组织的构建，是一种有重要意义的“种子细胞”。     

组织工程化喉软骨的构建

OBJECTIVE: To explore the method of fabricating tissue engineered laryngeal cartilage. METHODS: The rib and articular cartilage of infant New Zealan white rabbits were harvested in sterile condition. The chondrocytes were separated by collagenase digestion and cultured in vitro for 3 passage. Serial steps of solution casting, extrusion molding and particulate leaching were used to make larynx-shaped biomaterial models with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHH). The chondrocytes were seeded onto PHBHH scaffolds to form cell-PHBHH composites, which were subsequently in vitro for one week. After that, the measure of filling inner space of cell-PHBHH composites together with wrapping total composites using either greater omentum (n = 9) or fascia flap and muscle (also n = 9) in experimental groups was taken to implant the larynx-shaped biomaterial models seeded with chondrocytes into the belly and the back of adult New Zealan white rabbits. Control groups (every group n = 3) were the same measure as experimental groups but without chondrocyte on PHBHH scaffolds. Finally, morphological observation, HE staining & special staining and immunohistochemical test were conducted to assess cartilage regeneration and its shape at different period following implantation. RESULTS: The rate of viable cell in the final cell suspension was (93 +/- 2)% after well-controlled prolongation of digestion trysin. Similar to that by traditional procedures (94 +/- 2)% (P > 0.05). The larynx-shaped PHBHH models with edges and corners of laryngeal cartilage made by us appeared to be hollow half-trumpet shape and its porosity was more than 90%. It showed that chondrocytes equally attached to the surface of porous PHBHH and filled within porousness with scanning electron microscopic examination. Tissue engineered larynx-shaped specimens could alternatively be harvested with the above mentioned two different implantation measures. The specimens presented to be similar to that before implantation in gross shape. It was demonstrated to be cartilaginous tissue through histological and immunohistochemical examination. Furthermore, There was nearly no difference between two kinds of tissue engineered laryngeal cartilage with two measures of implantation in morphology and histology. CONCLUSIONS: The regeneration of tissue engineered cartilage in vivo is not influenced by the chondrocytes harvested by improvement of well-controlled prolonged digestion with trysin during in vitro cell culture. It seems that PHBHH may be used as scaffold in cartilage tissue engineering and wrapping together with filling method with either greater omentum or fascia flap and muscle is appropriate for fabricating tissue engineered laryngeal cartilage.     

组织工程化兔耳廓软骨的实验研究

目的 探讨采用表面修饰过的壳聚糖（chitosan)-聚乳酸（polylacticacid,PLA)－聚乙内酯（polycrylactone,PLC)及牛跟腱胶原海绵作为支架培养组织工程化耳廓软骨的可行性，比较动态与静肪两种不同培养方法对软骨生长的影响。方法 将4周龄新西兰大耳白兔耳廓软骨细胞接种在20个壳聚－PLA－PCL及牛跟腱I型胶原海绵支架上，将细胞支架复合物分为2组，动态组（样本量为10）采用旋转培养，静态组（样本量为10）采用静置培养。所有标本体外培养1周，裸鼠或兔皮下培养8周。分别于体外1周，体内4周及8周时取样行扫描电镜检查，大体及组织学观察以及免疫组化分析，测定软骨细胞数及细胞外基质中的Ⅱ型胶原含量。结果 表面修饰过的壳聚糖支架上软骨细胞贴壁率高，细胞分化与增殖好；每个时段动态组生成的软骨细胞量及Ⅱ型胶原均较静态组多，两组间差异有显著性（P＜0．05）；2种生物支架上均长出了组织工程软骨；裸鼠皮下较兔皮下形成的软骨更接近天然软骨。结论 壳聚糖－PLA－PCL及牛跟腱胶原海绵是较理想的组织工程耳廓软骨的支架材料，动态培养更有利于新生软骨组织的生成，兔皮下仅能形成软骨样组织。     

自体组织工程软骨研究方法及其进展

“组织工程学”这一名称于１９８７年被正式提出,近年来已取得很大进展。软骨组织由于结构简单,成分单一,非常适合组织工程研究。耳、鼻、喉和气管是富于软骨的器官,组织工程软骨可以用于这些器官损伤后的修复与重建。本文简介组织工程软骨的研究方法及其进展。     

内衬上皮的管状组织工程软骨构建的实验研究

目的研究以聚丙交酯-乙交酯共聚物[poly（DL-lactide-co-glycolide）,PLGA]包埋甲壳胺无纺布为细胞支架,在有免疫力的动物体内构建内衬上皮的管状组织工程软骨的可行性,为喉、气管缺损的修复探索新方法。方法取1月龄兔耳廓软骨,收集体外培养第3～4代的软骨细胞,接种于经多聚赖氨酸处理的以PLGA包埋甲壳胺无纺布的片状细胞支架材料上,体外培养7～10d后包裹于兔腹部缝制的皮管上并深埋于体内,分别于6、12、18周取材行大体和组织学观察以及HE染色、甲苯胺蓝染色和Masson＇s三色染色。结果同种异体软骨细胞/PLGA包埋甲壳胺无纺布的细胞支架复合物体外培养1周时有基质产生;植入体内6周后,复合物中有基质分泌,软骨细胞不成熟;12周时,软骨细胞接近成熟并形成凹陷,内衬上皮的管状组织工程软骨形成;18周时,新生软骨基本接近正常软骨。结论以PLGA包埋甲壳胺无纺布为细胞支架,同种异体软骨细胞与细胞支架所形成的复合物在有免疫力的动物体内可构建出内衬上皮的管状组织工程软骨。     

带蒂肌筋膜瓣充填与包裹构建喉支架形态组织工程软骨

目的 探讨带蒂肌筋膜组织瓣构建组织工程喉支架形态软骨方法,为肌筋膜瓣复合组织工程化软骨修复重建喉软骨支架功能提供实验依据.方法 采用溶剂浇铸、模压成形和颗粒滤沥方法制备喉支架形态聚羟基丁酸酯与聚羟基己酸酯共聚物[poly(3-hydroxybutyrate-co-3-hydroxyhexanoate),PHBHH]生物材料塑形物,接种软骨细胞形成细胞-PHBHH复合物,体外共同培养1周后用于体内植入.12只新西兰白兔,以其脊背部一侧骶棘肌及其筋膜制备肌筋膜组织瓣,采用筋膜衬里方法充填与包裹软骨细胞-PHBHH喉支架形态复合物(实验组9只),原位植入.设空白对照组(3只).分别于术后6、12和18周取材,行大体形态观察,组织学和免疫组化检测评估喉支架形态组织工程化软骨成形与再生情况.结果 制备的PHBHH多孔生物材料塑形物呈中空半面喇叭状,形似喉支架形态,乙醇静态容积测定孔隙率＞90％.筋膜衬里的带蒂肌筋膜组织瓣血运丰富,可有效充填与包裹喉支架形态塑形物.不同时间点均获取大体形态维持良好的喉支架形态组织工程软骨,组织学和免疫组化检测均证实体内植入6周即可形成软骨组织,12周及18周软骨组织进一步成熟.单纯PHBHH喉支架作为对照组体内植入未检测到软骨组织.结论 带蒂肌筋膜组织瓣可保障血运,采用筋膜衬里的肌筋膜组织瓣充填与包裹方法可构建喉支架形态组织工程软骨.     

鼻中隔软骨翻瓣法加鼻甲粘膜修补鼻中隔穿孔

1994年 2月～ 2 0 0 0年 6月 ,我们利用残余的鼻中隔软骨以翻瓣法加鼻甲粘膜移植修补鼻中隔穿孔 1 7例 ,效果满意 ,现介绍如下。1　资料与方法1 .1　临床资料鼻中隔穿孔 1 7例 ,男 1 2例 ,女 5例 ;年龄 2 5～5 0 (平均 38.2 )岁。主要临床表现为不同程度鼻出血、干鼻痂、干燥感、不适感等 ,4例有哨音和精神压抑感。引起穿孔原因 :鼻外伤 2例 ,鼻中隔手术 7例 ,鼻部冷冻、微波治疗不当 5例 ,长期溃疡感染 1例 ,不明原因 2例。穿孔形状 :圆形 4例 ,椭圆形 1 1例 ,裂隙状 2例。穿孔直径 0 .3～ 1 .5ｃｍ。1 .2　手术方法术前充分估计残余的鼻中…     

软骨组织工程管状泡沫支架的研制

目的研制管状泡沫支架,为气管软骨组织工程提供合适的支架.方法以PDLLA(Mw=4.23×104)为原材料,氯化钠颗粒(直径50～200μm)为致孔剂,通过"溶剂浇铸-颗粒滤沥"法制备一定孔径,不同孔隙率的管状泡沫支架,经过大体及扫描电镜观察、孔隙参数的测定及分析,选择软骨组织工程合适的支架.结果支架外观为白色、管柱状泡沫,内径8mm,外径12mm.各型支架具有不同的特点.孔径80～250μm、孔隙率90.6%的支架具有一定的强度及韧性,适于气管软骨组织工程.结论通过"溶剂浇铸-颗粒滤沥"法制备的管状泡沫支架是气管软骨组织工程合适的支架.     

Fabrication of cartilage in predetermined shapes from human nasoseptal chondrocytes with tissue engineering method]

OBJECTIVE: To investigate the feasibility of fabricating a new cartilage in predetermined shapes with tissue engineering methods. METHODS: Human nasoseptal chondrocytes were seeded onto a nonwoven mesh of polyglycolic acid(PGA) to form a cell-PGA construct. The construction was then configured in sheet and tube shapes, and implanted subcutaneously into the dorsa of 11 athymic mice. The specimens were harvested 4, 6, 8 weeks after implantation and subjected to gross morphologic and histologic analysis. RESULTS: Gross observation showed that the predetermined sheet and tube shapes of new cartilage were formed. Histological observation demonstrated that new mature cartilages were formed in 6-week. A Masson's trichrome stain showed the interwining bands of collagen at the periphery of the cartilage. Staining of Safranin O evaluated that the new cartilage was bound of glycosaminoglycan. In the control group, the PGA of the specimens were completely absorbed at 6 weeks. CONCLUSION: Human nasoseptal chondrocytes-PGA construct could develop into a new cartilage in predetermined shapes in athymic mice.     

同种异体工程化软骨的构建和修复甲状软骨缺损的实验研究

目的探讨同种异体预定形态组织工程化软骨在有免疫功能动物体内构建的可行性及其对甲状软骨缺损的修复能力.方法利用组织工程技术制备同种异体片状和"C”型半管状工程化软骨;将4周形成的片状工程化软骨用于修复12只新西兰大白兔甲状软骨大片缺损;一定时间取材,分别对预定形态工程化软骨的构建情况及其修复效果进行大体和组织学评价.结果①构建4周形成的预定形态工程化软骨呈乳白色,有弹性和支撑力,8周时软骨呈瓷白色,镜下观察显示软骨组织特征;②甲状软骨缺损修复术后4、8、12周观察,修复区愈合良好,组织学检查修复区与正常软骨间的界面区可见软骨细胞生长及软骨基质生成,无免疫排斥迹象.结论在有免疫功能的动物体内可形成同种异体预定形态组织工程化软骨,获取的工程化软骨对甲状软骨缺损有良好的修复效果,无明显免疫排斥反应.     

定向诱导人骨髓间质干细胞向软骨细胞分化的初步研究

目的　采用组织工程方法 ,将人骨髓间质干细胞 (mesenchymalstemcells ,MSC)在体外培养后 ,诱导为软骨细胞 ,为软骨的修复和重建提供条件。方法　根据 5例的预实验经验 ,采用 12例开胸手术所取正常肋骨分别实验。将肋骨剪开 ,用含双抗的不完全培养基反复冲洗骨髓腔得到骨髓液 ,经密度梯度离心获得单个核细胞 ,体外培养、分离获得人骨髓MSC ,经无血清培养诱导液进行诱导培养 ,透射电镜观察细胞外基质形成及超微结构 ,免疫组化检测MSCⅡ型胶原表达。结果　 12例的肋骨骨髓MSC分别体外培养 2周后见细胞融合 ,传代培养并进行诱导 ,部分细胞开始由梭形转变为圆形 ,并分泌基质 ,甲苯胺蓝染色呈阳性 ;MSCⅡ型胶原表达阳性。结论　人MSC在体外能诱导为软骨细胞 ,可用于组织工程软骨组织的构建 ,是一种有重要意义的“种子细胞”。     

聚羟基乙酸负载软骨细胞修复同种异体甲状软骨缺损

目的　探讨聚羟基乙酸 ( polyglycolicacid ,PGA)负载软骨细胞在有免疫力动物修复同种异体甲状软骨缺损的可行性。方法　酶消化法获取 3天龄新西兰乳兔肋软骨和关节软骨细胞 ,采用组织工程技术制备软骨细胞 PGA复合物 ,体外共同培养 1周后用于修复同种异体成年新西兰白兔甲状软骨缺损 (实验组 7只 )。设单纯PGA材料修复组 (对照A组 4只 )和单纯软骨细胞修复组 (对照B组 4只 )作为对照实验。分别于术后不同时间取材 ,对甲状软骨缺损的修复效果进行大体和组织学评价。结果　体外培养阶段可见黏附于PGA纤维表面的细胞分泌出丰富的软骨基质 ,呈蜘蛛网状分布于PGA纤维之间。术后 4周大体观察 :实验组修复区呈淡黄色 ,与正常软骨界限分明 ;组织学检查 :修复区内有软骨细胞生成和基质分泌 ,但与正常软骨间存在界面无细胞区 ,未见明显炎细胞浸润。 8周 :实验组修复区色乳白 ,与正常软骨仍有界限 ;镜下见修复区软骨细胞较成熟 ,软骨基质含量丰富。1 2周 :实验组修复区呈瓷白色 ,界面区软骨细胞不明显 ,但修复区软骨细胞数量、形态和基质与正常软骨相似。各时间点对照组大体观察修复区均呈不同程度的凹陷 ,暗红色 ,部分软组织充填其中 ,质软 ;组织学及特殊染色检查未发现软骨样结构及其分泌的基质成分。新生软骨     

耳鼻咽喉科领域的软骨组织工程

耳、鼻、喉和气管的软骨组织工程研究已有较大进展，已分别在裸鼠和具有免疫力动物体内形成了与耳鼻咽喉有关的组织工程化软骨组织，并有初步应用的实验研究报道。随着胚胎干细胞、骨髓基质干细胞、同种异体软骨细胞和智能生物材料的研发和应用，耳鼻咽喉科软骨组织工程有望获得突破性进展。本文综述了该领域的软骨组织工程研究现状和发展趋势。     

In vitro analysis of matrix proteins and growth factors in dedifferentiating human chondrocytes for tissue-engineered cartilage

Conclusions

With ongoing culture and dedifferentiation of chondrocytes, significant changes in the expression patterns of various collagens and the insulin-like growth factor (IGF) receptor were detected. The latter could play an important role in the differentiation of human chondrocytes.

Objective

Tissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. So far, little is known about the expression of markers for cell proliferation and differentiation in cultured chondrocytes.

Material and methods

Human chondrocytes were isolated from septal cartilage (n=5) and held in primary cell culture. Cells were harvested after 24 h and 6 days. Proliferation was analyzed using an Alamar Blue assay. The differentiation of the cells was investigated using bright field microscopy, the expression patterns of various proteins using immunohistochemistry and the expression of distinct genes using a microarray technique.

Results

The chondrocytes showed strong proliferation (Day 0: 16.7±0.7 fluorescent units; Day 5: 52.4±2.2 fluorescent units) from the third day of cell culture in medium without growth factors. From this point onwards, a dedifferentiation of the chondrocytes could be observed. In cell culture, the chondrocytes expressed collagen 1 and 10 without expression of collagen 3. After 6 days of cell culture, they expressed collagen 2. The chondrocytes showed constant low expression of the fibroblast growth factor-2 receptor, but constant high expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)2 and MMP9. The cells never expressed the epidermal growth factor receptor. The proportion of IGF receptor-expressing cells diminished significantly during cell culture.     

转化生长因子-β1和胰岛素对人鼻中隔软骨细胞增殖和分化影响的研究

目的 了解转化生长因子-β1(transforminggrowth factor-beta 1,TGF-β1)和胰岛素对人鼻中隔软骨细胞增殖和分化的影响。方法 体外培养人鼻中隔软骨细胞,采用四甲基偶氮唑蓝(MTT)代谢和35S-Na2SO4掺入的检测比较不同浓度TGF-β1和胰岛素对人鼻中隔软骨细胞的增殖以及软骨基质蛋白多糖合成的影响,观察TGF-β1和胰岛素对软骨细胞表型的影响。结果在15％血清的培养条件下,TGF-β1和胰岛素均能显著促进软骨细胞增殖,且在各自一定的浓度范围内呈量效关系,联合作用效果累加;TGF-β1、TGF-β1和胰岛素联合作用促使软骨基质蛋白多糖的合成量明显提高;TGF-β1使传代软骨细胞去分化提前。结论一定浓度的TGF-β1、胰岛素和TGF-β1 胰岛素对体外培养的人鼻中隔软骨细胞具有显著的促增殖作用。     

组织工程化喉软骨的构建

目的 研究组织工程化喉支架软骨的构建方法。方法 酶消化法获取幼兔肋和关节软骨细胞并进行体外培养,传代时延长胰蛋白酶作用时间3-5 min,收集第3代培养的软骨细胞用于实验;采用溶液浇铸、模压成形和颗粒滤沥方法制备喉形态聚羟基烷酸酯类[poly(3-hydroxybutyrate-co-3-hydroxyhexanoate,PHBHH)]生物材料塑形物,接种软骨细胞形成细胞-PHBHH复合物,体外共同培养1周后,分别在成兔体内用大网膜充填复合物中空部分与整体包裹、腹内成形(9只动物)和背部筋膜瓣与肌肉组织共同充填与包裹复合物、皮下成形(9只)的方法将负载软骨种子细胞的喉形态材料塑形物植入腹腔和背部,设空白对照组(每批3只)。术后一定时间取材,大体形态观察、组织学和免疫组织化学检测评估工程化喉软骨的成形与再生情况。结果 获取的软骨细胞活性为(93±2)％,传统方法为(94±2)％(P>0.05)。制备的喉形态生物材料塑形物呈中空半面喇叭状,液体(乙醇)静态容积测定孔隙率>90％。两种体内植入法均构建出喉形态组织工程化组织,大体形态维持良好,组织学和免疫组织化学均证实为软骨组织,且形态学和组织学表现两者基本一致。结论 改良培养细胞传代过程中胰酶作用程度未影响软骨细胞的成软骨性能;PHBHH可作为喉形态塑形物的塑形和构建组织工程化软骨的