藻酸钙载体制备及复合软骨细胞修复关节软骨缺损的研究
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摘要
关节软骨损伤后有限的自身修复能力及其对人体功能的严重影响，使其成
为骨科基础和临床研究中的热点和难点课题。近十几年来，组织工程方法为软
骨修复提供了新的途径，并已取得了一定进展，但仍有很多问题亟待解决。本
课题就是根据这种需求，在软骨细胞培养的基础上，制备柱形藻酸钙载体并复
合兔关节软骨细胞进行体外培养、体内异位成软骨及修复兔膝关节软骨缺损的
实验研究，为软骨组织工程的进一步研究提供实验依据。
1．兔关节软骨细胞体外培养
目的：掌握细胞培养方法，观察兔关节软骨细胞体外生长情况，为组织
工程软骨的研究提供种子细胞和相关技术支持。方法：酶消化法分离幼兔关节
软骨细胞，进行原代及传代培养，观察细胞形态变化及体外生长增殖情况。结
果：原代细胞经培养3天后进入对数生长期，6代之内细胞形态及功能良好，
以3～5代细胞生长活力最为旺盛。未经传代连续培养3wk的细胞，形态趋向
于成纤维细胞，但胞外基质分泌明显，甲苯胺蓝染色阳性。结论：兔关节软骨
细胞增殖快，生长稳定，可作为软骨组织工程的种子细胞。
2．藻酸钙柱形载体的制备及复合细胞前后的结构观察
目的：制备新型细胞载体，为组织工程研究提供材料支持。方法：藻酸钠
溶液与葡萄糖酸钙溶液以不同比例混合均匀，置特殊模具中，经冷冻、脱水等
第四罕医大学博士学位 文
处圳获得杜形藻酸钙载体村料。以其复合兔片竹软忖驯胞，斤对衬本IV介汕胞
前后的结构进行纵织学和扫描电镜观察。结果：藻酸钙具何厂放0W网抢状g．‘巾J，
网眼呛上1！。＂j见人小小等的微几，网！IR中IIJ吸咐·定贝JIu兔片u软刀邻fi地。f．’i
论：经初步观察，藻酸钙DZ形材料可试用作兔关曹软骨驯胞的8划个，Q介则胞
进行软骨组织＿卜程的研究。
3．藻酸钙－软骨细胞复合物植入兔背肌成软骨的实验研究
R的：通过观察关节软骨纲胞复合干载体后莉下州叼J物休内刑）十卜］的州
及成软骨惰况，为组织丁程方法修复关节软骨缺损的研究捉供进 步的实验伙
排。方法：将藻酸钙载体复合兔关节软骨细胞，手术植入兔背剖汕KQ内，汁训
厂术后 ZWk、4Wb 时取材进行组织学观察。结果：软骨绷胞n｛FjJ种动物f个内进
·步’卜长增殖，4W卜卜1可见新生软骨织织形成。结论：藻酸例－－软们哪！胞V介物
Illl‘异体动物休内形成软骨组织，可川厂软骨织织卜程的研4乙
4．藻酸钙载体复合软骨细胞修复兔关节软骨缺损
目的：观察藻酸钙－软骨细胞复合物在兔股骨滑车关节币i个层软竹缺损修
复中的作用。方法：新西兰白兔共26只，取其中以）只，】’骸股关V们股竹附
车面造成直径0．3cm的全厚层关节软骨缺损，人侧为藻酸钙复合软什圳胞川，
植入藻酸钙－软骨细胞细胞复合物，右侧为单纯藻酸钙纠，枪入＜丫I藻刑叭5划A、
作为对照。另外6只，缺损处不加任何处理，作为标准宁门组。术们4wk、Hwk、
12Wb、24Wb、28Wb分别取材行组织学观察。结果：藻酸钙复合软骨绷胞纲形成
软骨组织修复：单纯藻酸钙组获得部分纤维性修复，完全空白织没行修复，缺
损底部方少V纤雏红织，医骁组
Repairing articular cartilage defect has been a difficult task because of the poor ability of intrinsic cartilage repair. The concept of tissue engineering provided new methods for cartilage repair. Many problems still exist although great progresses have been achieved in various fields. This study aimed at obtaining experimental data for tissue engineering based on chondrocyte culture in vitro, preparation of calcium alginate scaffold and its combination with chondrocytes, ectopic neo-cartilage formation and joint cartilage defects repair in New Zealand rabbits. 1 Growth characteristics of rabbit chondrocytes cultured in vitro Aim: To investigate the growth characteristics of cultured rabbit articular cartilage chondrocytes and master the method of cell culture, provide experimental basis for the research on tissue-engineered cartilage. Method: Chondrocytes were harvested from slices of the young rabbit articular cartilage which were digested with enzymes for primary and passage culture in vitro. The following data were obtained: morphology, growth curve, adhesive rate of living chondrocytes. Result: Chondrocytes proliferated rapidly in vitro. The primary cells started rapid growth 72 hours after planting and kept satisfactory shape and function within six passages. The third to fifth passages seemed the most active. After long-term culture cell density increased evidently and secreted matrix which helped to form a membrame on the
culture dish. Conclusion: Chondrocytes kept rapid proliferation rate and stable growth characteristics in vitro which might be useful for articular cartilage defect repair by tissue engineering method.
2. Preparation of calcium alginate column and investigation of the structure Aim: Preparation of a novel cell scaffod which might act as a material carrier in cartilage tissue engineering research. Method: Calcium alginate column was obtained by thorough mixture of sodium alginate and calcium gluconate in certain ratio which experienced freezing and dehydration afterwards. Some of the columns were combined with chondrocytes and investigated for histological and SEM structure as well as those without chondrocytes. Result: The calcium alginate column showed open mesh structure of micropores with different sizes in the wall. Certain amount of chondrocytes could be found in the material meshes. After twenty-three-day's culture the material-chondrocyte construct kept original frame and structure. Round living cells existed among the nets. Conclusion: Calcium alginate column could be used as carrier of chondrocytes in the research on tissue-engineered cartilage formation.
3 Ectopic cartilage formation using CaAlg-chondrocyte construct in the muscle pockets of rabbits
Aim: The scaffold-chondrocyte constructs were implanted into the back muscle pouch of rabbits as allograft to investigate the ectopic cartilage formation, aiming at providing further supporting data on tissue engineering research. Method: The carrier-chondrocyte construct was implanted into the back muscles of rabbits by surgical technique. Histological examinations of the samples were performed at 2wk and 4 wk respectively after the operation. Result: Chondrocytes proliferated actively in the living body and new cartilage tissue could be found at the fourth week postoperatively. Conclusion: The calcium alginate combined with chondrocytes might play an active role in the research on cartilage tissue engineering.
4 Animal experiment of full-thickness articular cartilage defect repair Aim: To observe the effect of CaAlg-chondrocyte construct on the repair of animal articular cartilage defect. Method: Twenty-six New Zealand rabbits were selected as experimental animals and full-thickness articular cartilage defects were created in the femur trochlea facing to patella. The defects were covered with chondrocyte-calcium alginate constructs, calcium alginate without chondrocytes, or nothing at all respectively. The result was investigated grossly and histologically at 4 > 8 > 12 ^ 24 and 28wk postoperatively. Resul
为骨科基础和临床研究中的热点和难点课题。近十几年来，组织工程方法为软
骨修复提供了新的途径，并已取得了一定进展，但仍有很多问题亟待解决。本
课题就是根据这种需求，在软骨细胞培养的基础上，制备柱形藻酸钙载体并复
合兔关节软骨细胞进行体外培养、体内异位成软骨及修复兔膝关节软骨缺损的
实验研究，为软骨组织工程的进一步研究提供实验依据。
1．兔关节软骨细胞体外培养
目的：掌握细胞培养方法，观察兔关节软骨细胞体外生长情况，为组织
工程软骨的研究提供种子细胞和相关技术支持。方法：酶消化法分离幼兔关节
软骨细胞，进行原代及传代培养，观察细胞形态变化及体外生长增殖情况。结
果：原代细胞经培养3天后进入对数生长期，6代之内细胞形态及功能良好，
以3～5代细胞生长活力最为旺盛。未经传代连续培养3wk的细胞，形态趋向
于成纤维细胞，但胞外基质分泌明显，甲苯胺蓝染色阳性。结论：兔关节软骨
细胞增殖快，生长稳定，可作为软骨组织工程的种子细胞。
2．藻酸钙柱形载体的制备及复合细胞前后的结构观察
目的：制备新型细胞载体，为组织工程研究提供材料支持。方法：藻酸钠
溶液与葡萄糖酸钙溶液以不同比例混合均匀，置特殊模具中，经冷冻、脱水等
第四罕医大学博士学位 文
处圳获得杜形藻酸钙载体村料。以其复合兔片竹软忖驯胞，斤对衬本IV介汕胞
前后的结构进行纵织学和扫描电镜观察。结果：藻酸钙具何厂放0W网抢状g．‘巾J，
网眼呛上1！。＂j见人小小等的微几，网！IR中IIJ吸咐·定贝JIu兔片u软刀邻fi地。f．’i
论：经初步观察，藻酸钙DZ形材料可试用作兔关曹软骨驯胞的8划个，Q介则胞
进行软骨组织＿卜程的研究。
3．藻酸钙－软骨细胞复合物植入兔背肌成软骨的实验研究
R的：通过观察关节软骨纲胞复合干载体后莉下州叼J物休内刑）十卜］的州
及成软骨惰况，为组织丁程方法修复关节软骨缺损的研究捉供进 步的实验伙
排。方法：将藻酸钙载体复合兔关节软骨细胞，手术植入兔背剖汕KQ内，汁训
厂术后 ZWk、4Wb 时取材进行组织学观察。结果：软骨绷胞n｛FjJ种动物f个内进
·步’卜长增殖，4W卜卜1可见新生软骨织织形成。结论：藻酸例－－软们哪！胞V介物
Illl‘异体动物休内形成软骨组织，可川厂软骨织织卜程的研4乙
4．藻酸钙载体复合软骨细胞修复兔关节软骨缺损
目的：观察藻酸钙－软骨细胞复合物在兔股骨滑车关节币i个层软竹缺损修
复中的作用。方法：新西兰白兔共26只，取其中以）只，】’骸股关V们股竹附
车面造成直径0．3cm的全厚层关节软骨缺损，人侧为藻酸钙复合软什圳胞川，
植入藻酸钙－软骨细胞细胞复合物，右侧为单纯藻酸钙纠，枪入＜丫I藻刑叭5划A、
作为对照。另外6只，缺损处不加任何处理，作为标准宁门组。术们4wk、Hwk、
12Wb、24Wb、28Wb分别取材行组织学观察。结果：藻酸钙复合软骨绷胞纲形成
软骨组织修复：单纯藻酸钙组获得部分纤维性修复，完全空白织没行修复，缺
损底部方少V纤雏红织，医骁组
Repairing articular cartilage defect has been a difficult task because of the poor ability of intrinsic cartilage repair. The concept of tissue engineering provided new methods for cartilage repair. Many problems still exist although great progresses have been achieved in various fields. This study aimed at obtaining experimental data for tissue engineering based on chondrocyte culture in vitro, preparation of calcium alginate scaffold and its combination with chondrocytes, ectopic neo-cartilage formation and joint cartilage defects repair in New Zealand rabbits. 1 Growth characteristics of rabbit chondrocytes cultured in vitro Aim: To investigate the growth characteristics of cultured rabbit articular cartilage chondrocytes and master the method of cell culture, provide experimental basis for the research on tissue-engineered cartilage. Method: Chondrocytes were harvested from slices of the young rabbit articular cartilage which were digested with enzymes for primary and passage culture in vitro. The following data were obtained: morphology, growth curve, adhesive rate of living chondrocytes. Result: Chondrocytes proliferated rapidly in vitro. The primary cells started rapid growth 72 hours after planting and kept satisfactory shape and function within six passages. The third to fifth passages seemed the most active. After long-term culture cell density increased evidently and secreted matrix which helped to form a membrame on the
culture dish. Conclusion: Chondrocytes kept rapid proliferation rate and stable growth characteristics in vitro which might be useful for articular cartilage defect repair by tissue engineering method.
2. Preparation of calcium alginate column and investigation of the structure Aim: Preparation of a novel cell scaffod which might act as a material carrier in cartilage tissue engineering research. Method: Calcium alginate column was obtained by thorough mixture of sodium alginate and calcium gluconate in certain ratio which experienced freezing and dehydration afterwards. Some of the columns were combined with chondrocytes and investigated for histological and SEM structure as well as those without chondrocytes. Result: The calcium alginate column showed open mesh structure of micropores with different sizes in the wall. Certain amount of chondrocytes could be found in the material meshes. After twenty-three-day's culture the material-chondrocyte construct kept original frame and structure. Round living cells existed among the nets. Conclusion: Calcium alginate column could be used as carrier of chondrocytes in the research on tissue-engineered cartilage formation.
3 Ectopic cartilage formation using CaAlg-chondrocyte construct in the muscle pockets of rabbits
Aim: The scaffold-chondrocyte constructs were implanted into the back muscle pouch of rabbits as allograft to investigate the ectopic cartilage formation, aiming at providing further supporting data on tissue engineering research. Method: The carrier-chondrocyte construct was implanted into the back muscles of rabbits by surgical technique. Histological examinations of the samples were performed at 2wk and 4 wk respectively after the operation. Result: Chondrocytes proliferated actively in the living body and new cartilage tissue could be found at the fourth week postoperatively. Conclusion: The calcium alginate combined with chondrocytes might play an active role in the research on cartilage tissue engineering.
4 Animal experiment of full-thickness articular cartilage defect repair Aim: To observe the effect of CaAlg-chondrocyte construct on the repair of animal articular cartilage defect. Method: Twenty-six New Zealand rabbits were selected as experimental animals and full-thickness articular cartilage defects were created in the femur trochlea facing to patella. The defects were covered with chondrocyte-calcium alginate constructs, calcium alginate without chondrocytes, or nothing at all respectively. The result was investigated grossly and histologically at 4 > 8 > 12 ^ 24 and 28wk postoperatively. Resul
引文
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