应用改性聚乳酸为基质网架构建组织工程皮肤的实验研究
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摘要
皮肤是覆盖与保护体表的重要组织器官,可以因为各种原因造成皮肤缺损与异常。对于这种缺损目前多采用自体皮肤移植,但是这样的治疗可以引起诸多的不便。近年来,随着组织工程技术的发展,研究人工皮肤替代物的报道逐渐增多,其核心是应用工程学的原理和方法与生命科学相结合,发展能恢复、保持或改善功能的生物替代品。聚乳酸作为一种高分子聚合物,具有良好的生物降解性、生物相容性。因而聚乳酸在组织工程中的应用极为广泛。实验表明聚乳酸可吸收材料不仅符合组织工程支架所具备的基本要求,而且与传统的生物支架材料相比,具有来源丰富,易加工成型等优点。但是聚乳酸也有它的缺点,例如:具有疏水基团,对细胞的低亲和性影响了它的进一步利用与开发,还有柔韧性差等。本实验首先成功获得了大量最佳生长状态的作为皮肤组织工程种子细胞的角质形成细胞(KC)和真皮成纤维细胞(FB),在此过程中深入探讨关于KC和FB的分离、培养以及传代的实验技术,并且分析了几种维生素及辅酶对KC、FB增殖的影响。另外还利用部分机械性能已经得到改进的分别添加了十二烷基苯磺酸钠、双醛淀粉、聚乙烯醇、甘油的聚乳酸及纯聚乳酸网架构建大鼠真皮
Title: Reconstruction Studies on Tissue Engineering Artificial Skin With ModifiedPolylactic Acid Scaffold.
Tissue engineering is the product of co-development and combination of modern cell biology,biomaterial science, and engineering. The aim of tissue engineering is to investigate and restoretissue and organ substitutes. In this study, we use PLA which is no-toxic, no-hot, biocompatibilityand biodegradability , and modified PLA as scaffold construct tissue engineering corium and tissueengineering artificial skin. Details as following:
Ⅰ. Isolation, cultures and subcultures of KC and FB.
KC isolated from new born rat by treatment of trypsin-dispase was cultured,subcultured, and adopted with techniques of containing serum . It not only isolated theepidermal and dermal, but also obtained all cell of basal stratum.
The method pre-digest with trypsin and the method pre-digest with EDTA are thebest of methods in the subculturing KC.Both of them could avoide the damage of KCcoming from trypsin and advanced the efficiency of subculturing.KC grows very wellafter subcultured.
FB was isolated and cultured by the normal methods. According to the principles of plantingwith high cell density(1.0×105cell/ml) and refeed in short time established, the FB was purifiedafter subcultures.
Ⅱ. Effects of some kinds of vitamin and coenzyme on the proliferation of KC and FB
The effects of some kinds of vitamin and coenzyme on KC and FB proliferation were studiedby means of the MTT -method. The results showed that VC,CoA, ATP had the promotional effectson proliferation of KC. VB6 had the restraining effects on proliferation of KC. VB6, VC, CoA, ATPhad the promotional effects on proliferation of FB.
Ⅲ Biocompatibility of five different modified PLA scaffold and reconstruction of tissueengineering artificial coriumA kind of method that salt out scaffold preparation is used to make scaffold.Scaffold is made from PLA combined with linear alkyl benzene sulfonate(LAS),dialdehyde starch, polyvinylalcohol (PVA) and glycerin.
The artificial corium was reconstructed by planting subcultured FB separatelyinto(onto) five kinds of modified Polylactic Acid sponge, cultured for two week. Theresults were studied by means of the MTT -method. There are four kinds of modifiedpolylactic acid sponge witch FB have grown very well except LAS.
Ⅵ Reconstruction of tissue engineering artificial skin.The artificial skin was reconstructed by planting subcultured FB and KCseparately into(onto) the sponge, cultured for one week.The artificial skin wascomposed of epidermis and dermis under light microscope, the skin is consisted of KCand FB .KC and FB have grown very well in the scaffold.
In all,the research bases on the successful isolation and culture of KC and FB,andstudies the best subculture method of KC and the effect of some kinds of vitamine andcoenzyme on the proliferation of KC and FB.What's more,it explores thebiocompatibility of cells with the modofied PLA ( PLA-DAS , PLA-PVA ,PLA-glycerin) and PLA scaffold.The result shows that FB grow well in the scaffoldand it convinced that the scaffold could be used for new scaffold material of artificialskin.
Title: Reconstruction Studies on Tissue Engineering Artificial Skin With ModifiedPolylactic Acid Scaffold.
Tissue engineering is the product of co-development and combination of modern cell biology,biomaterial science, and engineering. The aim of tissue engineering is to investigate and restoretissue and organ substitutes. In this study, we use PLA which is no-toxic, no-hot, biocompatibilityand biodegradability , and modified PLA as scaffold construct tissue engineering corium and tissueengineering artificial skin. Details as following:
Ⅰ. Isolation, cultures and subcultures of KC and FB.
KC isolated from new born rat by treatment of trypsin-dispase was cultured,subcultured, and adopted with techniques of containing serum . It not only isolated theepidermal and dermal, but also obtained all cell of basal stratum.
The method pre-digest with trypsin and the method pre-digest with EDTA are thebest of methods in the subculturing KC.Both of them could avoide the damage of KCcoming from trypsin and advanced the efficiency of subculturing.KC grows very wellafter subcultured.
FB was isolated and cultured by the normal methods. According to the principles of plantingwith high cell density(1.0×105cell/ml) and refeed in short time established, the FB was purifiedafter subcultures.
Ⅱ. Effects of some kinds of vitamin and coenzyme on the proliferation of KC and FB
The effects of some kinds of vitamin and coenzyme on KC and FB proliferation were studiedby means of the MTT -method. The results showed that VC,CoA, ATP had the promotional effectson proliferation of KC. VB6 had the restraining effects on proliferation of KC. VB6, VC, CoA, ATPhad the promotional effects on proliferation of FB.
Ⅲ Biocompatibility of five different modified PLA scaffold and reconstruction of tissueengineering artificial coriumA kind of method that salt out scaffold preparation is used to make scaffold.Scaffold is made from PLA combined with linear alkyl benzene sulfonate(LAS),dialdehyde starch, polyvinylalcohol (PVA) and glycerin.
The artificial corium was reconstructed by planting subcultured FB separatelyinto(onto) five kinds of modified Polylactic Acid sponge, cultured for two week. Theresults were studied by means of the MTT -method. There are four kinds of modifiedpolylactic acid sponge witch FB have grown very well except LAS.
Ⅵ Reconstruction of tissue engineering artificial skin.The artificial skin was reconstructed by planting subcultured FB and KCseparately into(onto) the sponge, cultured for one week.The artificial skin wascomposed of epidermis and dermis under light microscope, the skin is consisted of KCand FB .KC and FB have grown very well in the scaffold.
In all,the research bases on the successful isolation and culture of KC and FB,andstudies the best subculture method of KC and the effect of some kinds of vitamine andcoenzyme on the proliferation of KC and FB.What's more,it explores thebiocompatibility of cells with the modofied PLA ( PLA-DAS , PLA-PVA ,PLA-glycerin) and PLA scaffold.The result shows that FB grow well in the scaffoldand it convinced that the scaffold could be used for new scaffold material of artificialskin.
引文
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