【作者】 吕荣晖；

【导师】 曹成波；

【作者基本信息】 山东大学 ， 化学工艺， 2007， 硕士

【摘要】 大面积烧伤、创伤和皮肤溃疡等皮肤缺损会导致皮肤功能障碍，严重时会危及患者生命。治疗皮肤缺损不仅需要及时和永久的覆盖创面而且要能最大限度地恢复其功能和外观，传统的治疗方法不能同时满足上述两个要求。近年来皮肤组织工程学的出现为改善创面修复质量，解决皮源不足等问题提供了崭新的思路和方法。运用皮肤组织工程学构建的复合皮是一种新型的永久性创面覆盖物，具有组织弹性好、耐磨、拉伸力和外观质地近似正常皮肤等优点，是目前研究的热点，其中真皮支架不仅影响表皮细胞的生物学行为和培养效率，而且决定着移植效果，是制约复合移植的一个瓶颈。在现有的真皮支架中，脱细胞真皮基质因其更接近正常真皮结构，成为近年来研究热点之一。但由于制备思路的局限与制备工艺的不足，目前脱细胞真皮基质存在着免疫原性较强、降解速度慢、生物活性低和渗透性差等缺点。针对以上不足，本论文以组织工程学原理为指导，综合运用化学化工、皮革科学与工程、生物医学工程以及材料学等知识，采用多种化学、生物与物理综合方法处理乳猪皮，在前期研究基础上，重点研究交联、复合对新型胶原支架性能的影响，优选了关键制备技术和方法，确定了复合移植用胶原真皮支架的制备工艺，并对其主要性能进行了表征。其主要研究内容和结果如下：（1）优化戊二醛和碳化二亚胺（EDC）的交联条件，并研究其对材料收缩温度、拉伸强度和降解性等性能的影响，寻求适当的交联剂及交联方法。实验结果显示：随着交联剂用量的增加，收缩温度不断提高；经EDC交联试样的拉伸强度没有明显变化，而经戊二醛交联的有小幅度提高；用EDC交联试样的体外降解时间增加幅度略高于经戊二醛交联的；经两种交联剂交联试样的溶胀度都有大幅度的下降；成纤维细胞在EDC交联试样上的增殖情况明显好于戊二醛交联的。（2）优化硫酸软骨素等的复合工艺，并研究其对支架材料性能的影响。实验结果显示：复合的硫酸软骨素等多糖可以在支架中保持较长时间，保证其可以在支架中较好的发挥作用；硫酸软骨素等多糖的复合可以增加材料的拉伸强度以及延长体外降解时间等，但效果不明显；在材料中复合壳寡糖、硫酸软骨素和透明质酸均能有效增强支架材料的细胞黏附性；肝素在增强材料细胞黏附性方面效果不明显。（3）以组织工程原理为指导，依据（1）、（2）的研究结果，结合现有材料的优点以及理想真皮支架材料的要求，构建了一种复合移植用胶原真皮支架，并检测了其主要性能。结果显示：复合移植用胶原真皮支架表面较光滑，纤维编织紧密；其拉伸强度介于10.01-10.87MPa之间，具有较好的拉伸强度；体外降解时间处于20-50h之间，并可根据需要采取不同的处理工艺进行调整；孔隙率处于87-93％之间；透水汽性在3000g·m^-2·24h^-1左右；复合移植用胶原真皮支架中的纤维间质去除干净；扫描电镜微观结构观察显示胶原纤维束保持了其原有结构并得到松散，材料内部具有丰富的多孔结构；具有良好的生物活性，能有效促进细胞的黏附、增殖和生长。本文制备的复合移植用胶原真皮支架充分去除了毛、表皮及纤维间质，弱化胶原的抗原性，并用EDC适度交联以及复合硫酸软骨素、壳寡糖和成纤维细胞生长因子等活性生物材料，使其具有了高渗透性、适宜的降解时间、良好的生物活性和拉伸强度，满足理想真皮支架的要求，且有利于新生皮肤的血管化，加快创面愈合，有望应用于复合移植等领域。更多还原

【Abstract】 Extensive skin burn, traumatic injury, skin ulcer and other skin defects would cause skin dysfunction and even threaten to patient’s life if they were severe. It should not only be timely and permanent coverage, but also reach the goal of functional and cosmetic restoration of skin to treat skin defects. It was difficult to achieve both of them by traditional treatment methods. The establishment of skin tissue engineering supplied a new clue and method to improve the wound healing quality, solve the shortage of skin source and so on. Composite skin was a new permanent wound dressing, which had many advantages, such as good elasticity, fine wear resistance, good tensile strength, skin texture likely and so on, so it became a research hotspot recently. The dermal substitutes which were the base of the composite skin and the bottleneck of composite grafting influenced not only the biological effect and efficacy of keratinocytes, but also the outcome of repair. In recent years many kinds of dermal substitutes had been developed, in which acellular dermal matrix （ADM） had been the research focus. Because the limitation of ideas and the defects of preparing technology, ADM had many shortcomings, such as strong immunogenicity, bad permeability, low biological activity and so on.The thesis was guided by the principle of tissue engineering, combined with chemistry and chemical, biomedical engineering, leather science and engineering, material science. On the base of previous research, the study was focused on the influence of cross-linking and compounding on the performance of new type collagen scaffold, and then the method and technique of the preparation was optimized to determine the preparation process of a new composite transplant collagen dermal scaffold, which was developed by adopting various chemical, biological and physical comprehensive methods to deal with suckling pigskin. The major research contents in this paper were listed as follow:（1） The cross-linking conditions of glutaraldehyde （GA） and EDC were optimized, and the influences of cross-linking on the degradation, tensile strength and so on were studied. Results show that: The shrinkage temperature rises with the increase of crosslinker dosage; The tensile strength of EDC-treated samples does not change significantly, and that of GA-treated samples has a light increase; The degradation time of EDC-treated samples increases lightly higher than that of GA-treated samples; The swelling of samples cross-linked by the two cross-linkers declines significant; The fibroblast proliferation is significantly better in the EDC-treated samples than that in the GA-treated samples.（2） The compounding conditions were optimized, and the compounding effects on the performance of scaffold were studied. Results show that: The polysaccharide, such as chondroitin sulfate can maintain a long time to ensure that they can play their role better in scaffold; The compounding can increase the tensile strength and extend the degradation time, but the effect is not significant; The chitooligosaccharides, chondroitin sulfate and hyaluronic acid compounding can effectively enhance the cell adhesion, but the heparin compounding can not.（3） According to the principle of tissue engineering and the research results of （1）, （2）, combining with the advantages of existing materials and the requirements of ideal dermal scaffold, a new composite transplant collagen dermal scaffold was prepared, and its major performance including physical property, biocompatibility and so on were studied systematically. Results show that: The surface of sample is smooth and glossy, and the fibers woven closely; Its tensile-strength is between 10.65MPa and 10.87MPa,which demonstrates it has better tensile strength; The degradation time is between 20h and 50h, which can be controlled by changing treatment process; The permeability is around 3000 g·m^-2·24h^-1 ; The porosity is between 87% and 93%; The collagenous interstitial fibers of composite transplant collagen dermal scaffold has been removed completely; SEM results illustrate that collagen fiber bundles maintains its original structure and be appropriate loose, and the scaffold has rich internal porous structure; It has good bioactivity, and can accelerate cells adhesion. The composite transplant collagen dermal scaffold, whose interfibrillary substance among the dermis, such as epidermis, fat cell, mucoprotein ect. was removed to weaken the antigen of the collagen, was cross-linked by EDC and compounded with biological active substance, such as chondroitin sulfate, chitooligosaccharides, fibroblast growth factor and so on. And it has high permeability, good biological activity and suitable degradation time, so it is expected to be used as auto-dermal complex transplant dermal scaffolds.更多还原