【作者】 孙锦章；

【导师】 韩春茂；

【作者基本信息】 浙江大学 ， 外科学， 2007， 硕士

【摘要】 背景资料临床上，烧伤、创伤、肿瘤、手术、慢性溃疡等各种原因引起的皮肤缺损是一种常见的疾病。大面积严重烧伤更是医务人员面临的棘手问题。传统的治疗方法便是皮肤移植，例如自体皮移植，同种异体皮移植、异种皮移植。其中，自体皮移植由于没有排斥反应，疗效肯定，是首选的方法。但是却又常常碰到自体皮来源不足的难题，尤其对于大面积烧伤病人来说，这种供需矛盾就更加突出了。此外，自体皮移植还存在另外一个缺点，即造成新的损伤，可能会引起供皮区创面的疤痕形成或色素沉着。近20年来，伴随着材料科学和生命科学的发展，组织工程学迅速兴起，为临床上各种组织缺损的修复、重建提供了一种崭新的，更加理想的治疗策略。因此它越来越受到医学界的关注。皮肤组织工程是这一新兴学科中发展最早、最快的领域之一。第一种经FDA批准上市的组织工程产品就是组织工程皮肤产品。目前已经有诸如Integra，AlloDerm，Transcyte，Apligraf等多种产品问世，并获得FDA批准用于临床。这些商品化的组织工程皮肤在用于烧伤以及慢性溃疡等患者的创面修复中，显示出了很好的治疗效果，有效的促进了皮肤组织的再生。然而，国外的这些组织工程皮肤产品，价格均十分昂贵，远远超过了我国普通老百姓的承受能力，难以满足我国老百姓的需求。因此研制一种价格低廉，又能满足临床要求的，具有自我知识产权的组织工程皮肤产品对于我国的广大患者来说就显得尤为重要。胶原是哺乳动物体内含量最多的蛋白质，它具有生物相容性好、易降解、低抗原性的特性，是组织工程支架中广泛使用的一种生物材料。壳聚糖由在自然界广泛分布的甲壳素经脱乙酰化处理制得，这两者都属于天然生物可降解材料，在自然界分布广泛，制取比较容易，所需成本也较低。将这两种成分按一定比例混合，再通过胶联处理，制备的胶原-壳聚糖多孔材料，微观下成蜂窝状的多孔结构，结构较为均一，孔径在80-150μm范围之内，研究表明，此种支架适合真皮成纤维细胞的迁移和生长，可以作为一种诱导真皮再生的良好支架。但是，完全意义上的组织工程皮肤应当包括表皮和真皮的完整结构，我们要制备具有更大临床应用价值的组织工程复合皮肤，就不仅需要有真皮结构，还要有多层的表皮结构。而构成真皮结构的成纤维细胞和形成表皮结构的表皮细胞在支架中具有不同的分布和生长状态。胶原-壳聚糖均一的多孔结构，似乎不能很好地满足这种要求。我们需要一种具备更佳微观结构的生物支架，既能适应成纤维细胞的接种生长，又能适应表皮细胞生长融合的需要。Wang等利用明胶、6-硫酸软骨素、透明质酸，采用冷冻—冻干的方法制成一种不对称的双面膜，两面具有不同孔径结构，上面一层较薄，孔径较小（20-50μm），底下一层较厚，孔径较大（平均150μm），体外构建时，在上面小孔层培养角朊细胞，而在底下的大孔中接种真皮成纤维细胞。此种支架表层的小孔既可防止下层成纤维细胞向上生长，也可防止表层角质形成细胞落入成纤维细胞层面。研究表明它能很好的适应组织工程复合皮肤构建需求。纤维蛋白由纤维蛋白原在凝血酶的催化下聚合而来，具有良好的组织和细胞相容性，在体内可降解，临床上主要被用作手术过程中的止血剂和组织粘合剂。研究表明它具有促进细胞黏附、增殖和迁移的作用，目前亦被作为一种良好的载体材料用于组织工程的创伤修复。此外纤维蛋白在微观下呈多孔网状结构，而且其结构与纤维蛋白原以及凝血酶的浓度和混合比例有关。于是，在本实验中，我们尝试将纤维蛋白复合到胶原—壳聚糖多孔支架上，从而制备出一种更能适应组织工程皮肤构建的不对称细胞支架。目的利用高分子生物材料胶原-壳聚糖和纤维蛋白胶制备适应组织工程皮肤构建的不对称支架，并探讨其用于体外组织工程皮肤构建的可行性。材料和方法一．皮肤细胞的分离培养1．人真皮成纤维细胞（Fbs）的培养取包皮环切术后的皮肤块，以组织块培养法分离出人真皮成纤维细胞，并传代培养，选择3—8代细胞作为工作细胞，用于组织工程皮肤构建。2．人表皮细胞的培养选择由本院皮肤科馈赠的人角质形成细胞系（HaCaT），解冻复苏后加入DMEM（高糖，含10％胎牛血清，100U／ml青霉素，100U／ml链霉素）培养基，置于37℃，5％CO₂的饱和湿度孵箱中培养，2天换次液，传代培养。二．胶原-壳聚糖／纤维蛋白胶不对称支架的制备将由牛跟腱中提取出的胶原和壳聚糖分别溶于0.5mol／L的乙酸溶液中，配制浓度为0.5％的胶原溶液和壳聚糖溶液，再将胶原溶液和壳聚糖溶液按质量比9：1混合均匀，注入模具；采用冷冻一冻干法，—20℃冷冻2小时后，置冻干机中冻干，得到胶原／壳聚糖，再将其置105℃真空干热处理24小时，再置于浓度比为2：1的1—乙基—3—3（二甲基胺丙基）—碳化二亚胺（EDAC）和N-羟基丁二酰亚胺（NHS）混合溶液中交联处理24小时，用三蒸水反复漂洗后，再次冷冻—冻干便获得EDAC-NHS交联的胶原-壳聚糖多孔支架。将此种多孔支架置于75％乙醇中浸泡消毒过夜，磷酸盐缓冲液（PBS）反复漂洗5次以上；将配好浓度的纤维蛋白原溶液以50μl／cm²的量均匀涂覆在其表面，再以等量的凝血酶滴加在其上，室温或37℃温箱静置30分钟，获得胶原-壳聚糖／纤维蛋白胶不对称支架。三．胶原-壳聚糖／纤维蛋白胶不对称支架的组织学和微结构观察将纤维蛋白原（原浓度为80mg／ml）用DMEM培养基配成浓度为40mg／ml和8mg／ml，将凝血酶（原浓度为600U／ml）用DMEM培养基配成浓度为300U／ml和60U／ml；将纤维蛋白原溶液以50μl／cm²的量均匀涂覆在已消毒的胶原-壳聚糖多孔支架表面，再以相应浓度等量（体积）的凝血酶滴加在其上，室温或37℃温箱静置30分钟，待纤维蛋白原聚合成胶后，获得胶原-壳聚糖／纤维蛋白胶不对称支架。将制备的胶原-壳聚糖／纤维蛋白胶不对称支架置于10％中性福尔马林溶液中固定，石腊包埋，HE染色。同时留取标本，戊二醛和锇酸双固定后，临界点干燥，用作扫描电镜标本。四．胶原-壳聚糖／纤维蛋白胶不对称支架用于体外组织工程皮肤构建用上述方法制备胶原-壳聚糖／纤维蛋白胶不对称支架，将培养的表皮细胞以5×10⁵／cm²密度接种到此支架的表面，加入含10％胎牛血清，100U／ml青、链霉素的DMEM高糖培养液浸没材料，置37℃，5％CO₂饱和湿度孵箱中培养，2～3天换次液，3～7天后改为气液界面培养。同时在没有纤维蛋白胶层的胶原-壳聚糖多孔支架上接种相同浓度的表皮细胞，相同条件下培养作为对照。五．胶原-壳聚糖／纤维蛋白胶不对称支架用于组织工程复合皮肤的构建将制备的胶原-壳聚糖多孔材料置于75％乙醇中浸泡消毒30分钟，磷酸盐缓冲液（PBS）反复漂洗后，将培养的真皮成纤维细胞消化、离心后计数板计数，将细胞浓度调整到2×10⁶／ml，将细胞悬液接种到胶原／壳聚糖多孔材料中，然后将材料-细胞复合物置于6孔板中，加入含10％胎牛血清和青、链霉素各100U／ml的DMEM高糖培养基（每孔3ml），浸没材料，置37℃，5％CO₂饱和湿度孵箱中培养1周，每2～3天换次液。1周后将此材料-细胞复合物取出，转入另-6孔板中，置孵箱中放置30分钟使其表面略干燥，然后在其表面依次涂上预先配置好浓度的纤维蛋白原和凝血酶，再将其置于孵箱中放置30分钟，待纤维蛋白胶体形成后，将培养的表皮细胞消化、计数，以5×10⁵／cm²密度接种到此不对称支架的表面，再放回孵箱静置半小时，然后加入培养基浸没材料，继续培养，3～7天后转到自制的不锈钢网架上，作气液界面培养，2～3天换次液。六．不对称支架中真皮成纤维细胞的活性检测将接种了真皮成纤维细胞的不对称支架，置于DMEM（高糖，含10％胎牛血清，100U／ml青、链霉素）培养基中培养，隔2～3天换液，1周后取材做二乙酸荧光素（FDA）和碘化丙啶（PI）双染色，2周后取材做扫描电镜，观察细胞的形态变化、生长增殖、细胞外基质的分泌以及与支架材料的黏附情况。七．构建的组织工程皮肤的组织学观察将体外构建的组织工程皮肤予不同的时间点取材，10％中性福尔马林固定，乙醇梯度脱水，二甲苯透明，浸腊，石蜡包埋，5μm切片，常规HE染色，光镜下观察并拍照。八．免疫组化染色将构建的组织工程皮肤气液界面培养2周后取材，10％中性福尔马林固定，做石蜡切片，经脱腊、水化后，按迈新免疫组化试剂盒（Kit-9710）说明，做角蛋白10（CK-10）和广谱角蛋白（Pan-CK）免疫组化染色。结果一．皮肤细胞的分离培养1．人真皮成纤维细胞（Fbs）组织块培养1周后，可以看见许多梭形Fbs从组织块周围长出，细胞成漩涡状生长，再经过1周时间，细胞铺满瓶底，行传代培养。2．HaCaT细胞用DMEM培养基（高糖，含10％胎牛血清，100U／ml青、链霉素）培养，细胞生长状态良好，2天换一次液，接种后4～5天，细胞融合，呈典型的铺路石样外观。二．胶原-壳聚糖／纤维蛋白胶不对称支架的组织切片和扫描电镜观察胶原-壳聚糖／纤维蛋白胶不对称支架，HE染色，镜下见此支架具备不对称的双层结构，由纤维蛋白胶所构成的表皮面层厚度较薄，结构较致密，而由胶原-壳聚糖构成的真皮面层厚度较厚，结构疏松，呈网状结构，孔径较大，且在局部，纤维蛋白胶嵌入其下面的胶原-壳聚糖内部，形成类似钉突样锚定结构。扫描电镜下观察，见胶原-壳聚糖成蜂窝状多孔结构，孔径大小在80—150μm范围，纤维蛋白胶也呈多孔的网状结构，但孔径较小约在5-20μm范围。三．人皮肤成纤维细胞在不对称支架中的活性检测接种了成纤维细胞的不对称支架，体外培养1周后做FDA-PI染色，荧光显微镜下观察，可见支架中的细胞形态呈梭形，生长状态良好。2周后的扫描电镜，可见细胞贴附材料纤维生长，并且在细胞周围有大量细胞外基质分泌。四．组织工程皮肤的组织学观察1．胶原-壳聚糖／纤维蛋白胶不对称支架用于体外组织工程皮肤构建在胶原-壳聚糖／纤维蛋白胶不对称多孔支架上，气液界面培养2周可见表皮层复层明显，由3～4层细胞构成。而无纤维蛋白胶的胶原壳-聚糖多孔支架上气液界面培养3周，表皮细胞复层分化仍不明显。2．胶原-壳聚糖／纤维蛋白胶不对称多孔支架用于体外组织工程复合皮肤构建构建的组织工程复合皮肤，在不同的时间点取材，气液界面培养1周后，HE染色可见材料内部有较多成纤维细胞生长，支架表面，表皮细胞融合成片，有2～3层细胞。3周时，支架表面有7～10层表皮细胞，形成明显的双层结构，形态类似正常皮肤。五．免疫组化染色用不对称支架构建的组织工程皮肤，气液界面培养2周后，表皮层的分化已十分明显，呈多层结构，Pan-CK和CK-10染色阳性。结论我们将胶原-壳聚糖多孔支架和纤维蛋白胶相结合成功地制备出一种不对称支架，此种不对称支架具有的微观结构适合种子细胞的有序分布和生长，且生物相容性好，适应了组织工程皮肤构建的需要，有潜在的应用前景。更多还原

【Abstract】 BackgroundIn clinical practice, skin defects caused by all kinds of factors such as burns, trauma,and chronic ulcers are common, and their traditional therapy method is skin transplantation. The main skin sources are autogenous skin, allogenous skin, or xenoskin. Among these, autogenous skin is the best choice because it has no rejection. However, autogenous skin transplantation has its deficiencies either. First there is not enough skin for the patients with extensive burns. Second, injury in the donor site is a problem, because of its possible scarring or pigmentation. In recent 20 years, tissue engineering rised quickly with the development of biomaterial and life science. Tissue engineering offers us a better method to repair and reconstruct tissue defects. Tissue engineered skin is a field that developed most quickly in tissue engineering. At present, many commercial products such as Integra, AlloDerm, Transcyte, Apligraf have been developed and obtained the permissions of FDA. These tissue engineered products have displayed a good therapeutic efficacy in wound repair and they can promote the regeneration of skin. However, the price of these products abroad is very high and beyond the endurance of common people of our country. It can’t meet our people’s demand. So, it is important to develop a tissue engineered-skin product by ourselves which not only meet the clinic requirement but also low in price. Collagen is a protein which occupies the highest content in mammal animals, with20—30%, and it has many characteristics: Good biocompatibility, degradation and low in antigenicity. So it has been widely used as an ideal scaffold in tissue engineering. Chitosan is extracted from chitin which distributes extensively in natural environment, and the cost price is low. The collagen-chitosan has good biocompatibility, proper degradation and mechanical intension, and it displays a porous structure with a pore size at 80-150μm which is suitable for the migration and proliferation of dermal fibroblasts. However, the complete tissue engineered-skin should contain epidermis and dermis. So if we want to develop composite skin substitutes, dermis and epidermis should be constructed simultaneously. But it seems that the homogeneous larger pore size of collagen-chitosan is not suitable for the construction of composite skin substitutes, so we must develop a scaffold which has a better microstructure.Fibrin glue is a polymer of fibrinogen in the presence of thrombin, and it has a good biocompatibility and degradation in vivo, and it was used widely in surgical operations as a hemostat and tissue adhesive. Researches have indicated that it could promote attachment, migration and proliferation of cells. At present, it is applied in the repair of the wound with tissue engineering methods. In addition, fibrin glue has a porous net-like microstructure which is related to the concentration of fibrinogen as well as thrombin.In this study, we successfully prepared a kind of asymmetric scaffold which possess a bilayer structures with different pore size in either side by combining fibrin glue to collagen-chitosan porous scaffold, and then we used it to construct tissue engineered composite skin, we found it can provide a good environment for cell growth, and has a potential prospect.ObjectiveThe aim of this study was to prepare an asymmetric scaffold which is fit for the construction of tissue engineered- skin with collagen-chitosan and fibrin glue, and then investigate the feasibility of using it to fabricate skin substitutes in vitro.Material and methods1. Isolation and culture of skin cells1.1 Cultures of human dermal fibroblastsPrimary human dermal fibroblasts were isolated by tissue culture technique from skin pieces that is obtained from donors after circumcision. Subculture was carried out. The 3-8 generation of cells were used as work cells.1.2 Culture of human epidermal cellsHunan keratinocyte cell line （HaCaT） was chosen in the study. The cells were cultured in DMEM （high glucose） medium supplemented with 100U/ml penicillin, 100U/ml streptomycin and 10% fetal bovine serum（FBS）, at 37 ℃, 5%CO₂. The medium was changed every 3days.2. Preparation of collagen-chitosan/fibrin glue asymmetric scaffoldCollagen and chitosan were dissolved in 0.5 M acetic acid solution to form a 0.5% （w/v） solution respectively, and then these two kinds of solution were mixed in a mass ratio of 9:1 After deaeration under reduced pressure to evolve entrapped air bubbles, the collagen/chitosan composite was injected into a mold, frozen in a refrigerator at — 20℃ for 2h and then lyophilized for 24h to obtain a porous collagen/chitosan scaffold , and then this scaffold was crosslinked at 105℃ under reduced pressure for 24 h and then was further treated with EDAC/NHS solution for another 24h. After washed with double-distilled water （10min x 5times）, the scaffold were freeze-dried again to obtain the EDAC/NHS treated collagen/chitosan scaffold. The scaffolds were immersed in 75% ethanol for over night for sterilization, followed with solvent exchange by PBS for 5 times at least. The fibrinogen solution was smoothly spread on the surface of collagen/chitosan scaffold （50μl/cm² ）, and then the scaffold was put into incubator at 37℃ for 30min to make fibrinogen polymerize after thrombin was dropped onto it .When fibrin glue formed the asymmetric scaffold was obtained.3. Histology and microstructure observation of the asymmetric scaffoldFibrinogen solution was dilute to 40mg/ml and 8mg/ml with DMEM medium at the same time, thrombin solution was dilute to 300U/ml and 60U/ml with DMEM medium. Fibrinogen was spread onto the outside surface of collagen -chitosan porous scaffold which was sterilized , and then corresponding thrombin was drop onto it, and then the scaffold was put into incubator at 37℃ for 30min .The asymmetric scaffold was prepared after the fibrin glue was formed on the surface. Some collagen-chitosan/fibrin glue asymmetric scaffolds were fixed in 10% formaldehyde, embedded in paraffin, sections were stained with haematoxylin-eosin（HE）. At the same time other scaffolds were fixed in glutaraldehyde and osmic acid, dried at critical vertex, and then observed under scanning electron microscope （SEM）.4. Construction of tissue engineered skin with collagen -chitosan/fibrin glue asymmetric scaffoldThe HaCaT cells was digested from the flasks, and then were seeded onto the upper surface of the asymmetric scaffolds with a density of 1×10 6 /cm². After 2h , it was cultured in DMEM medium supplemented with 100U/ml penicillin, 100U/ml streptomycin and 10% FBS in a 5%CO₂ incubator, at 37℃ with medium change every 2 or 3 days , and then shifted to air-liquid interface after 3-7 days. At the same time the equivalent density of cells were seeded onto the surface of collagen-chitosan porous scaffold without fibrin glue in the same culture condition as the control.5. Construction of composite skin substitutes with the asymmetric scaffoldThe dermal fibroblast was digested from the flasks and then the density was adjusted to 5×10⁶ /ml. The cell suspension was inoculated into collagen-chitosan porous scaffold which was sterilized previously, and then incubated in DMEM medium supplemented with lOOU/ml penicillin, lOOU/ml streptomycin and 10% FBS under condition of 5%CO₂ 37℃. The medium was changed every 2-3 days. 1 week later , the collagen-chitosan porous scaffold containing Fbs was transferred to a new 6pore- plate and then was put into incubator at 37℃ for 30 minutes. When the surface of the scaffold is a somewhat dry, the fibinogen and thrombin were overlaid onto it. After the polymerization of the glue at 37℃ for about 0.5h, the epidermal cells were inoculated onto the upper surface of this collagen-chitosan porous scaffold containing Fbs with a density of 1×10⁶ /cm². After immersed culture for 3-7 days, the composite skin substitutes were shifted to a self-designed stainless steel mesh and cultured in the air-liquid interface conditions. Medium changes were performed every 2 or 3 days. The cultures were harvested 1, 2, 3 weeks later for paraffin embedded sections.6. Viability of cells in the asymmetric scaffoldThe asymmetric scaffold seeded dermal fibroblasts was cultured in DMEM medium （high glucose, 100U/ml penicillin, 100U/ml streptomycin and 10% FBS）, and medium changes were performed every 2 or 3days. FDA-PI staining and SEM analysis were done after 1 and 2 week’s culture respectively.7. Histological observation of the constructed skinThe cultures were fixed in 4% formaldehyde overnight, dehydrated in ethanol and embedded in paraffin; sections （5μm） were stained with haematoxylin-eosin（HE）.8. Immunohistochemistrical examination2 weeks after transferred to air-liquid interface, the constructed skin were fixed in 4% formaldehyde and embedded in paraffin. After dewaxed, the sections were used to for immunohistochemistrical examination followed the instruction of Maxin’s ultrasensitive SP kit.Results1. Isolation and culture of skin cells1. Dermal fibroblastsA great number of fibroblasts emigrated from the tissue pieces after cultivation for 1week, and they grew to 90% confluence after another week.1.2 HaCaT cellsThe HaCaT cells grew with a good state in DMEM medium and after seeded 4-5 days,the cell became confluence with a typical stone-like appearance.2. Histology and SEM observation of collagen-chitosan/fibrin glue asymmetric scaffoldIn HE staining images, the scaffold displayed an asymmetric bilayer structure, the top layer consisted of fibrin glue was a thin layer with a tight structure while the bottom layer consisted of collagen-chitosan scaffold was a thicker layer with a loose and porous structure. In some area, the top layer developed a rete ridge-like structure inserted into the bottom layer. With the SEM examination, a two-layer structure with different pore sizes and porosity was shown on the asymmetric scaffold clearly. The pore size of the bottom layer is at the rang of 80—150μm while the top layer had a smaller pore size, at the range of 5-20 μm.3. Viability of cells in the asymmetric scaffoldThe asymmetric scaffold seeded fibroblasts was stained with FDA and PI and then observed under fluorescence microscope. The image of fluorescence reveals a great number of cells （green fluorescence） grew in the dermal layer of the asymmetric scaffold with shuttle-like or irregular morphology, and the SEM image reveals that the fibroblasts were adhered on the walls of the scaffold with typical shuttle-like morphology, and much extracellular matrix was around.4. Histological observation of tissue engineered skinOn the surface of the asymmetric scaffold, 3-4 layers of cells were observed when cultured on air-liquid interface for 2 weeks, while no obvious multilayer were seen on the surface of collagen-chitosan porous scaffold without fibrin glue layer, even though they had been cultured for 3 weeks. The culturing of tissue engineered composite skin was terminated when cultured inair-liquid interface conditions for 1, 2and 3 weeks. The HE staining images reveal a great many fibroblasts grew in the bottom layer of the asymmetric scaffold while epidermal cells started to differentiate into a multilayer structure on its top layer. 3 weeks later, the epidermal cells developed into a epidermis-like structure with 7-10 layers of cells. The constructed composite skin substitutes possess a histological structure similar to the normal skin.5. Immunohistochemistry analysisAfter 2 weeks of cultivation, the multilayered epithelium of the tissue engineered-skin was obvious, The Pan- CK is positive in the whole layer of epithelium and the CK10 is positive only on the upper part of it.ConclusionsWe successfully developed a kind of asymmetric scaffold with different pore size on both sides by combining biopolymers: collagen-chitosan and fibrin glue, which is fit for the construction of tissue engineered skin in vitro and may have future application prospect.更多还原