双层鱼胶原膜作为微粒皮移植覆盖物的实验研究

发布时间：2018-04-21 08:35

本文选题：烧伤 + 微粒皮移植　；参考：《第二军医大学》2017年硕士论文

【摘要】：研究背景创面修复是烧伤领域永恒的命题,尤其对于大面积烧伤患者,创面修复尤其关键,直接关系到患者的存活率及生存质量。大面积创面引起机体应激严重,消耗过度,并且容易引细菌定植、进而引起全身感染,导致全身脏器损伤。尽早的覆盖创面是烧伤患者治疗的关键,因而创面覆盖物的选择与进展是贯穿烧伤发展始终的,其中包括临时覆盖物与永久的覆盖物。永久的覆盖主要指真皮替代物和字体皮,大面积救治治疗过程中应用的主要植皮方式有:Meek植皮术、自异体混合植皮术、微粒皮移植术以及自体表皮细胞培养移植等,然而,任何一种植皮方式都需要合适的临时覆盖物来覆盖间隙以及保证永久覆盖物存活。临时覆盖物的发展也对创面修复起到了重要的作用,临时覆盖从传统的纱布、凡士林纱布、阴离子辅料等到胶原膜或胶原支架覆盖物,应用越来越广泛。因此,创面覆盖物的更新发展,也是创面修复技术的发展的重要支持。在大面积烧伤封闭创面的过程中,各种植皮方式应用各有利弊。其中,Meek植皮术多应用于早起切痂后覆盖创面,实现了扩展比例可为1:3至1:9,并且实现了机械化,但是扩展比例有限;自异体混含植皮术,植皮存活率稳定,但存在手术重复操作多,耗时长等缺点;自体表皮细胞培养移植,理论可扩展1000倍,但是存在创面愈合质量差的问题;微粒皮移植术理论上可扩展100倍,临床应用可扩展10～20倍,但是存在覆盖物选择、方向性及均一性等临床难题。本研究着眼于微粒皮移植,覆盖物的选择一直是影响微粒皮移植成活率的重要因素,活性高的异体皮是理想的微粒皮移植覆盖物,但是异体皮来源受限往往限制了大面积烧伤患者的微粒皮移植开展,因而,寻找一种可以模拟皮肤功能的覆盖物对微粒皮移植十分关键。在诸多覆盖物材料的发展中,其中在创面修复领域,胶原材料的使用越来越广泛。胶原自身为创面修复过程中所需要的重要成分,并且在皮肤及创面修复中起重要作用。并且,胶原的获取途径越来越多,提取方式及加工方式越来越成熟。在创面修复方面,胶原的应用也越来越广泛,已有的产品包括真皮替代物及外用敷料。近年来,鱼乘源的胶原材料越来越多的应用在医学领域,其中包括止血材料及创面修复领域,如何利用鱼胶原材料促进创面愈合具有重大临床意义。本研究旨在寻找一种可替代异体皮肤功能的生物敷料,结合鱼胶原的应用发展,构建一种模拟皮肤结构功能的双层鱼胶原膜,并将之应用于微粒皮移植覆盖物,为微粒皮移植提供一种来源广泛的覆盖物选择,从而使微粒皮移植在大面积烧伤患者的创面修复中应用更广,创面修复效率更高。第一部分双层鱼胶原膜(FCM-B)的构建研究目的:制备一种模拟皮肤结构,包含表皮真皮,并且在功能上模拟皮肤的双层胶原敷料,外层可以模拟表皮致密并且韧性高具备保护及隔绝功能;内层疏松柔软,具备良好的贴附功能。用于创面可以达到吸收渗液,并且可以保护创面。研究方法:利用食用废料的罗非鱼鱼皮与鱼鳞,经酸联合酶消化提取技术,提取并纯化鱼胶原。在不添加具有毒性的交联剂的基础上,应用不同的交联方法可以获得理化特性差别明显的胶原膜。利用脱水热处理(DHT,dehydrothermaltreatment)交联的方法得到外层鱼胶原膜(FCM-O,Fish Collagen Membrane Outer-layer),利用真空冷冻干燥(FC,vacuum Frozen drying)技术得到内层鱼胶原膜(FCM-I,Fish Collagen Membrane Inner-layer)。将制备好的FCM-O提前铺在制备FCM-I的模具底部,再次利用真空冷冻干燥技术,制备成膜。研究结果:成功制备具有双层结构,外层致密柔韧,内层疏松柔软的双层鱼胶原膜,从外观上和质地上与人皮肤具有相似性。第二部分双层鱼胶原膜(FCM-B)的特性研究研究目的:通过FCM-B的结构特性及理化特性检测来验证双层鱼胶原膜与皮肤结构和功能上的相似性。研究方法:1.结构观察1.1组织学观察利用FCM-B冰冻切片,行HE染色、Massion染色,观察其结构与胶原排列情况,并与正常皮肤组织切片做对比,观察其结构的相似性;1.2电镜扫描观察并对FCM-B进行扫描电镜扫描,观察胶原膜的结构,观察各层次的表征,并测量各层次的厚度。2.物理特性2.1力学性能取FCM-O,FCM-I及FCM-B哑铃型样本,利用Instron5567材料测试系统(Instron,USA),进行力学测试,获得杨氏模量、断裂伸长率及断裂强度等指标,重复测量3遍。2.2 水蒸气透过率(WVTR,Water Vapor Transmission Rate)取直径3.5cm的FCM-O,FCM-I及FCM-B样本,使用水蒸气透过率检测仪(W3/030,Labthink,中国)获得结果,测量重复3次。2.3吸收容量(C%)将已经制备好的三种胶原膜用双蒸水浸泡,在相同环境浸泡相同时间直至胶原膜充分吸收水分,通过测量浸泡前后胶原膜质量变化计算C%。3.生物化学特性3.1生物可降解性将制备的FCM-B、FCM-O及FCM-I用PBS处理,浸泡前后的干重计算降解速率,并计算半量降解时间t1/2。3.2细胞毒性将FCM浸提液按一定浓度剃度加入等量人成纤维细胞悬液中,使用CCK-8试剂盒,通过检测吸光度(OD)值,计算成纤维细胞的相对增值率。统计方法:FCM的各项指标(计量资料)结果均用平均值±标准差表示;两组值之间应用t检验;多组组比较应用oneway-ANOVA分析,实验组与各组间比较应用SNK检验。研究结果:1.结构观察组织学切片结果显示FCM-B冰冻切片与正常皮肤组织切片在双层结构和胶原排列上具有结构相似性;电镜扫描结果显示FCM-B在厚度与孔隙上上与皮肤表皮真皮具有相似性。2.物理学特性通过检测测得FCM-I机械性能显著FCM-O(p0.05);FCM-I的WVTR显著高于FCM-O(p0.05),FCM-B的WTVR接近正常皮肤水平。FCM-I吸收容量显著高于FCM-O(p0.05)。3.生物化学性能检测体外降解时间T1/2(FCM-O)约为15天;T1/2(FCM-I)约为21天,FCM-O的生物降解时间明显长于FCM-I(p0.05)。细胞毒性试验表明FCM浸提液对人成纤维细胞增值率无显著影响。第三部分双层鱼胶原膜(FCM-B)覆盖SD大鼠微粒皮移植实验研究目的:为了验证双层胶原膜在微粒皮移植过程中能起到异体皮的作用,能够促进微粒皮移植创面的愈合。研究方法:SD大鼠背部做直径2cm的圆形全层皮肤缺损创面,根据1:10的比例将取下的皮肤取薄皮制备成微粒,回植在创面上,根据覆盖物的不同分为空白对照组、异体皮覆盖组及FCM-B、FCM-I、FCM-O覆盖组,观察不同覆盖物对微粒皮移植存活的影响,计算创面愈合率及收缩率;留取组织标本,分别进行胶原(Massion)染色、HE染色,观察表皮细胞生长情况及创面的胶原分布情况。统计方法:多组组比较应用oneway-ANOVA分析,实验组与各组间比较应用SNK检验。研究结果:实验早期发现FCM-I、FCM-O创面愈合不佳,在3周时FCM-B覆盖组与异体皮覆盖组的创面愈合率差异无统计学意义(p0.05),创面收缩率异体皮覆盖组低于FCM-B(p0.05),组织学观察发现上皮形成厚度FCM-B组大于异体皮覆盖组(p0.05)。研究结论本研究构建的双层胶原膜,在结构上与皮肤的表皮真皮具有高度的相似性,并且在理化特性上接近理想创面覆盖物要求,通过动物实验结果证实,FCM-B替代同种异体皮作为微粒皮移植覆盖物具有可能性。
[Abstract]:Background wound repair is an eternal topic in the field of burn. Especially for patients with large area burns, the repair of the wound is particularly critical, which is directly related to the survival rate and quality of life of the patients. Large area wounds cause severe stress, excessive consumption, and easy to induce bacterial colonization, and then cause systemic infection, resulting in systemic organ damage. Early coverage of the wound is the key to the treatment of burn patients, so the selection and progress of the wound cover is throughout the development of the burn, including the temporary cover and permanent cover. The permanent cover mainly refers to the dermis and the skin. The main skin grafting methods used in the process of large area treatment are: Meek skin grafting, self grafting. Allograft skin grafting, particle skin grafting and autologous epidermal cell culture, however, any kind of skin grafting method requires suitable temporary cover to cover the gap and guarantee the survival of the permanent cover. The development of the temporary cover also plays a vital role in the wound repair, temporarily covering from the traditional gauze, the petticic. Bayashi Sabu, the anionic excipients wait until the collagen membrane or the collagen scaffold covers more and more widely. Therefore, the renewal and development of the wound cover is also an important support for the development of the wound repair technology. In the process of large area burns, the application of various skin grafting methods has advantages and disadvantages. Among them, Meek skin grafting should be used for early eschar cutting. After covering the wound, the expansion ratio can be 1:3 to 1:9, and the mechanization is realized, but the expansion ratio is limited; the survival rate of skin grafting is stable from the allograft containing skin grafting, but there are many disadvantages of repeated operation and time consuming, and the theory of autoepidermal cell culture and transplantation can be extended 1000 times, but the quality of wound healing is poor. The particle skin grafting can be expanded 100 times in theory, and the clinical application can be expanded by 10~20 times, but there are clinical problems such as selection of cover, orientation and uniformity. This study focuses on the skin grafting of particles. The selection of cover has always been an important factor affecting the survival rate of particle skin transplantation, and the high active skin allograft is the ideal skin grafting. Cap, but the limitation of the source of allograft often restricts the skin grafting of large area burn patients. Therefore, it is critical to find a kind of skin graft that can simulate skin function. In the development of many covering materials, the use of collagen is more and more widely used in the field of wound repair. The important components of the wound repair process are important and play an important role in the repair of skin and wound. Moreover, more and more ways of obtaining collagen are obtained. The methods of extraction and processing are becoming more and more mature. In the field of wound repair, the application of collagen is becoming more and more extensive, and the existing products include dermal substitute and external dressing. The collagen material of fish multiplying source is more and more applied in the field of medicine, including the field of hemostat and wound repair. How to use the raw material of fish glue to promote the healing of the wound is of great clinical significance. This study aims to find a biological dressing that can replace the function of the skin allograft, and combine the application and development of fish collagen to construct a kind of simulated skin. The skin structure function of the double layer fish gum membrane, which is applied to the skin graft covering, provides a wide selection of cover for the skin grafting of particles, so that the skin grafting is more widely used and the wound healing efficiency is higher in the wound repair of the large area burned patients. The construction of the first part of the first part of the double layer fish glue film (FCM-B) Objective: to prepare a kind of skin structure, including epidermis dermis, and the double layer collagen dressings that mimic the skin in function. The outer layer can simulate the compact and high tenacity of the epidermis and have the function of protection and isolation; the inner layer is loose and soft and has good adhesion function. It can be used in the wound surface and can protect the wound and protect the wound. Methods: the fish collagen was extracted and purified by the acid combined enzyme digestion and extraction technology using the fish skin and fish scales of edible waste. On the basis of no toxic crosslinking agent, different cross-linking methods could be used to obtain the collagen membrane with obvious differences in physicochemical properties. The cross linking of DHT (dehydrothermaltreatment) was used. The original membrane (FCM-O, Fish Collagen Membrane Outer-layer) was obtained by means of vacuum freeze drying (FC, vacuum Frozen drying) technology to obtain the original film of inner layer of fish gum (FCM-I, Fish Collagen). Research results: the double layer fish glue original film with double structure, dense and flexible outer layer and soft and soft inner layer is similar to human skin in appearance and texture. The research aim of the characteristics of the second part double layer fish gelatin original film (FCM-B) is to verify the double layer of the original film by the structure and physicochemical properties of FCM-B Similar to skin structure and function. Research methods: 1. structure observation 1.1 histology observation using FCM-B frozen section, HE staining, Massion staining, observe the structure and collagen arrangement, and compare with normal skin tissue sections, observe the structure of the similarity, 1.2 scanning electron microscope and scanning electron microscope for FCM-B, Observe the structure of the collagen membrane, observe the characterization of various levels, and measure the physical properties of the thickness.2. of each level 2.1 mechanical properties of FCM-O, FCM-I and FCM-B dumbbell type samples, using the Instron5567 material test system (Instron, USA) to carry out mechanical testing, obtain the young's modulus, breaking elongation and fracture strength and so on, repeated measurement of the 3 times.2.2 water evaporation. The gas transmittance (WVTR, Water Vapor Transmission Rate) takes the FCM-O, FCM-I, and FCM-B samples of the diameter 3.5cm, and uses the water vapor transmittance detector (W3/030, Labthink, China) to obtain the results. The repeated 3 times.2.3 absorption capacity is measured to soak the prepared three kinds of collagen membranes in double water and soak the same time in the same environment until collagen. The membrane fully absorbs moisture and calculates the C%.3. biochemical properties 3.1 biodegradability by measuring the quality changes of the collagen membrane before and after soaking. The FCM-B, FCM-O and FCM-I are treated with PBS, the dry weight before and after soaking is calculated and the degradation time is calculated, and the t1/2.3.2 cytotoxicity of the half quantity is calculated by the FCM extract shaving at a certain concentration and adding the same amount of people. In fibroblast suspension, CCK-8 kit was used to calculate the relative value of fibroblasts by detecting the value of absorbance (OD). Statistical methods: the results of FCM were expressed with mean standard deviation; t test was used between the two groups; oneway-ANOVA analysis was used in multiple groups. The experimental group and each group should be compared. The results of SNK test. The results of the study: 1. the histological section of the structure observation showed that the frozen section of FCM-B and the normal skin tissue were similar to the structure of the double layer structure and the collagen arrangement, and the scanning electron microscope showed that the FCM-B was similar to the skin epidermis and the skin epidermis was similar to the skin epidermis. The physical properties of the skin epidermis were detected by the FCM-I machine. The performance of the weapon was significantly higher than that of FCM-O (P0.05), and the WVTR of FCM-I was significantly higher than that of FCM-O (P0.05). The WTVR of FCM-B was close to the normal skin level, and the.FCM-I absorption capacity was significantly higher than that of FCM-O (P0.05) for about 15 days of biodegradation in vitro. The experiment showed that the FCM extract had no significant effect on the rate of human fibroblast increment. Third part of the double layer fish gelatin original membrane (FCM-B) covered the experimental study of SD rat skin grafting. In order to verify the effect of the double collagen membrane on the skin graft in the process of particle skin transplantation, it can promote the healing of the skin graft. The method of research: SD A round full layer skin defect wound on the back of 2cm was made on the back of the rat. According to the 1:10 proportion, the skin of the skin was prepared to make the particles and replanted on the wound. According to the different coverage, the skin was divided into blank control group, the allograft skin cover group and the FCM-B, FCM-I, and FCM-O cover group. The effects of different cover on the survival of the skin graft were observed and the wound was calculated. The healing rate and the contraction rate of the tissue specimens were left with collagen (Massion) staining and HE staining. The growth of epidermal cells and the distribution of collagen in the wound were observed. Statistical methods were compared with oneway-ANOVA analysis in groups of groups. The experimental group and each group were compared with SNK. The results of the study were that FCM-I, FCM-O wound healing was not healed in the early stage of the experiment. There was no significant difference in the wound healing rate between the FCM-B group and the allogenic skin cover group at 3 weeks (P0.05). The wound shrinkage rate of the skin allograft group was lower than FCM-B (P0.05). The histological observation found that the epithelial thickness FCM-B group was larger than the allogeneic skin cover group (P0.05). Conclusion the double layer collagen membrane constructed in this study was structured with the skin. The epidermis of the epidermis is highly similar and close to the requirements of the ideal wound cover in physical and chemical properties. The results of animal experiments have proved that the replacement of the allogenic skin by FCM-B as a skin graft cover is possible.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R644

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