【作者】 郑必祥；

【导师】 彭代智；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 硕士

【摘要】 烧伤治疗的一个核心问题就是创面问题。对于大面积烧伤患者而言,及时有效地覆盖创面对于提高患者的生存率至关重要。采用自体皮肤移植是治疗皮肤缺损的传统办法,但这些植皮方法均不同程度地缺乏真皮成分,对愈后外观和功能存在较大的影响。真皮移植和理想真皮组织替代物的研制是皮肤组织工程研究人员的首要和重要任务。由于猪皮来源广泛,与人皮肤在结构和功能上有些相似,异种脱细胞真皮基质(ADM)通常由猪皮制备,并受到人们的广泛关注。但其在临床应用中仍存在着下列缺点:(1)免疫原性较强,局部长期存在炎症免疫反应;(2)现有的脱细胞真皮较致密,对营养的渗透性差,血管化速度慢,导致移植后存活率低。本研究针对目前异种ADM所存在的问题,首先在真皮动物来源种属选择上,探讨了猪真皮、大鼠真皮与人真皮组织学上的差异,为脱细胞真皮动物来源的选择提供实验依据;然后对现有的制备ADM的方法进行了改进研究,使脱细胞更彻底,免疫原性更低;在此基础上,通过兔皮下植入ADM的实验来评价ADM的体内生物相容性。最后,又进行了ADM复合肝素的初步研究,为复合型ADM的研制提供了初步的实验数据。目的1.通过比较研究巴马小型猪、SD大鼠与人皮肤在真皮组织结构、胶原类型和生物力学性能方面的差异,为脱细胞真皮动物来源的选择提供组织学上的实验依据。2.改良本实验室已有制作SD大鼠ADM的方法,并通过HE染色法评估其效果,通过兔皮下植入实验来评价ADM的体内生物相容性。3.探索将肝素和SD大鼠脱细胞真皮支架复合的简易方法,为临床推广应用创造条件。方法1.收集巴马小型猪、SD大鼠与人的背部全层皮肤样本,采用大体观察、HE染色、Masson三色染色、天狼猩红染色、VVG染色、扫描电镜及透射电镜等方法,显微照相后进行形态学观察和图像分析软件测量,同时利用材料试验机检测真皮生物力学性能。2.在酶消化法的基础上选取不同浓度和处理时间制备异种脱细胞真皮,HE染色法观察脱细胞效果;对比有无低渗处理的酶消化法加去污剂法制备的ADM的脱细胞效果,HE染色法观察并用图像分析软件测量计算脱细胞程度。3.新西兰大白兔随机分为4组,分别为A组:有基底膜DM组;B组:有基底膜ADM组;C组:无基底膜网状层ADM组;D组:假手术组。每组6只。在兔的脊背中线切2.0 cm切口,向切口两侧游离制备皮下囊,囊的大小为4 cm×2 cm,囊间距2 cm,每侧4个囊;然后将ADM剪成2 cm×2 cm大小的小片,植入皮下囊内。术后1周、2周、4周、8周和12周分别取出皮下埋藏的植入物,大体观察真皮周围组织有无明显炎症反应。真皮植入物样本用4%多聚甲醛固定,HE染色,光学显微镜下观察炎性细胞浸润。4.将ADM浸泡肝素溶液不同时间,并通过甲苯胺蓝分光光度法测量肝素含量,并与未脱细胞的DM比较。结果1.与人皮肤比较,光镜下可见巴马小型猪皮肤的真皮胶原纤维束更粗[(39.29±10.17) m vs (17.21±5.20) m] (P=0.0003),而其间隙率更低[(19.01±1.55)% vs (32.36±1.28)%] (P=0.029),SD大鼠的胶原纤维束稍粗[(19.27±0.36) m] (P=0.049),间隙率接近[(30.54±3.50)%] (P=0.049)。巴马小型猪真皮弹性纤维分布、Ⅰ型和Ⅲ型胶原含量及其比值也与人真皮有明显差异(P<0.05),SD大鼠则与人真皮类似。2.扫描电镜显示巴马小型猪真皮的胶原纤维束排列紧密、束间孔隙小,而SD大鼠和人真皮胶原纤维束比较纤细,排列松散,束间散在大小不一的相互连通的孔隙;透射电镜显示巴马小型猪真皮的胶原原纤维较细,横纹的周期更长,而SD大鼠胶原原纤维较粗,横纹的周期相近。3.生物力学检测显示巴马小型猪真皮的最大应变[(0.52±0.05) mm/mm]低于人真皮[(0.80±0.06) mm/mm] (P=0.003),而弹性模量[(69.65±12.16) MPa]和最大应力[(20.72±1.49) MPa]高于人真皮[分别为(44.23±4.63) MPa、(11.85±0.19) MPa] (P=0.028,P=0.001),SD大鼠真皮的最大应变[(0.63±0.08) mm/mm]也低于人真皮(P=0.042);而弹性模量[(35.60±7.29) MPa] (P=0.158)和最大应力[(8.60±2.16) MPa] (P=0.120)与人真皮无明显差异。4.每1 cm×1 cm的真皮片加入2 ml的酶消化液所制备的ADM细胞成分残余少,作用2小时不会出现消化过度的情况。这个方案为酶消化法的最佳处理方案。联合低渗处理所制的ADM均未见细胞成分残余,而且纤维束完整,排列规则,达到了既去除细胞成分而又保持纤维三维结构的目的,与未增加低渗处理组比较,有明显的统计学差异(P=0.000)。5.与有基底膜的DM组比较,ADM组的动物大体观察均未见皮肤坏死的征象,其组织切片提示早期以单个核细胞侵入为主,术后2~3月,成纤维细胞与毛细血管逐渐长入植入物,未见明显的炎性与免疫排斥反应征象。6.大鼠真皮的肝素含量从脱细胞前的(4.34±0.83) g/cm2降为脱细胞后的(2.36±0.13) g/cm2 (P=0.000),脱细胞处理显著的降低了真皮的肝素含量。浸泡肝素后ADM的肝素含量,从浸泡15 min以后,其肝素含量[(4.25±0.77) g/cm2]就达到了未经酶消化的DM的肝素含量的水平,并以浸泡12小时为高峰[(8.43±1.43) g/cm2]。结论1.猪真皮在组织形态学方面与人真皮有较大差异,而SD大鼠的真皮却与人真皮有更多的相似之处,因此,以SD大鼠作为异种脱细胞真皮动物来源具有较为可靠的组织学依据。2.根据真皮组织的抗原性分布特点设计了去除基底膜的物理方法,在改良胰酶消化法的基础上结合低渗处理显著减低了异种真皮组织的免疫原性,植入体内仅见到轻微的炎症反应。3.脱细胞处理显著降低了真皮基质内的肝素含量,短时间浸泡肝素(15~30 min)就能使ADM恢复消化前肝素含量。这一异种ADM复合肝素的简易方法将为临床的推广应用创造条件。更多还原

【Abstract】 Background: One of the most important treatment in severely burned patients is appropriate burn wound management for rapid skin repair and regeneration. Timely and effective coverage on burn wound is vital for the patients suffering from burn injury. The classic therapies for skin loss are auto-transplantations, but these methods of dermoplasty more or less lack the component of dermis, would seriously affect the appearance and function of reconstructive skin. The research of ideal dermal graft and dermis transplantation is the chief and important responsibility for the skin tissue engineering researchers and burn surgeons. Xenogenic acellular dermal matrix (ADM) which is usually derived from swine is attracting widespread attention because it is easily available and has some similarities with the structure and function of human dermis. However, the following deficiencies still exist in clinical applications: (1) the strong immunogenicity, long-term presence of local inflammatory and immune response; (2) the current acellular dermal matrix is very dense and has poor nutrition permeability with slow vascularization, subsequently leads to low survival rate after transplantation. This research is focused on the problems in xenogenic ADM. We firstly compared the histological differences of dermis among three species including human, swine and rat, and obtained the morphological evidences for selecting the animal species on xenogenic acellular dermal matrix. Then the methods of preparing acellular dermal matrix were compared between their effects of decellularization. The xenogenic acellular dermal matrix was implanted underneath the skin of rabbits for evaluating the inflammatory response of ADM. Subsequently, the effect of decellularization on the heparin level of ADM was investigated, and the methods of restoring the heparin level of ADM to normal dermis were explored.Objectives1. To perform a comparative study of dermal tissue structure, content of difference types of collagen and biomechanical properties between Bama miniature pig, Sprague-Dawley(SD) rat and human skin, obtain experimental information of resource selection on xenogenic acellular dermal matrix.2. To improve the preparation of acellular dermal matrix (ADM), and evaluate the decellular effects by HE staining, then assess the in vivo biocompatibility by subcutaneous implantation.3. To find a simple method of combining the heparin on the ADM, establish the conditions for further clinical application.Methods1. The dorsal full-thickness skin samples were collected from Bama miniature pig, SD rat and human, and observed grossly. The sections of these samples were stained by hematoxylin and eosin (HE) staining, Masson’s trichrome staining, sirius red staining and Verhoeff’s Van Gieson (VVG) staining, and were examined under light microscopy. These samples were also observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The relevant data of histological images were measured with image analysis software. In the meantime, these samples were detected by material testing machine for biomechanical properties.2. ADM was prepared by using various concentration of enzymatic digestive solution in various reaction times.The decellular effect was evaluated by HE staining. The decellularization of two methods (with or without hypotonic solution) was also compared. The relevant data of histological images were measured with image analysis software.3. New-Zealand rabbits were divided into four groups randomly. They were cellular dermal matrix (DM) with basement membrane (group A); ADM with basement membrane (group B); ADM without basement membrane (group C); sham operation (group D). Each group had six rabbits. A 2 cm midline incision was made on the dorsum of rabbit, prepared of subcutaneous capsule on both sides of the incision. The size of the subcutaneous capsule was 4 cm×2 cm. The space between two capsules was 2 cm. There were four capsules on each side. The ADMs were cut into 2 cm×2 cm in each piece. Each subcutaneous capsule implanted a piece of ADM. At 1, 2, 4, 8 and 12 weeks after implantation operation, the implants were taken for evaluating the inflammatory reaction. The implant samples were fixed by 4% paraformaldehyde, stained by HE staining, then were examined under light microscopy for observing the inflammatory cells.4. The ADMs were soaked in heparin solution at different times, then measured the content of heparin by toluidin blue spectrophotometry and compared with that of DM.Results1. Compared with human dermis by light microscope, Bama miniature pig had much thicker dermal collagen bundles [(39.29±10.17) m vs (17.21±5.20) m] (P=0.0003), and had significant decrease in the gap rate between these bundles [(19.01±1.55)% vs (32.36±1.28)%] (P=0.029), and SD rats had slight thicker dermal collagen bundles [(19.27±0.36) m] (P=0.049), and was similar in the gap rate between these bundles [(30.54±3.50)%,P=0.837]. The distribution of elastic fiber, the contents of type I and III collagen and their ratio also had significant differences between human and porcine dermis (P<0.05), but they were similar between human and SD rat dermis.2. The results from SEM showed that the dermal collagen bundles of Bama miniature pig were densely arranged and had smaller pores. The dermal collagen bundles of human and SD rat were porous arranged and had bigger pores. The collagenous fibrils of Bama miniature pig were thinner and had longer cyclical transverse striation than human by TEM. The collagenous fibrils of SD rat were thicker but had similar cyclical transverse striation than human by TEM.3. The results of biomechanical testing showed that the maximum strain of Bama miniature pig dermis [(0.52±0.05) mm/mm] was smaller than that of human dermis [(0.80±0.06) mm/mm] (P=0.003) while the elastic modulus [(69.65±12.16) MPa] and maximum stress [(20.72±1.49) MPa] of Bama miniature pig dermis were stronger than that of human dermis [(44.23±4.63) MPa, (11.85±0.19) MPa] (P=0.028, P=0.001). The maximum strain of SD rat dermis [(0.63±0.08) mm/mm] (P=0.042) was also smaller. But the elastic modulus [(35.60±7.29) MPa] (P=0.158) and maximum stress [(8.60±2.16) MPa] (P=0.120) of SD rat dermis were similar with that of human dermis.4. The ADM made by using 2 ml enzymatic digestive solution per cm2 flap of reticular layer of SD rat dermis with 2 h digestion had less residual cell components without over-digestion. This was the best protocol for preparating ADM by enzymatic digestion. The method of preparing ADM with hypotonic solution could reach thoroughly decellularization and maintain the three-dimensional construction of collagen fibers. 5. Compared with the rabbits implanted cellular DM, the rabbits implanted ADM had no skin necrosis, The histological changes showed that the main infiltrating cells were mononuclear cells in the early period after implantation, the fibroblasts and capillaries could be found inside of the implants after 2 or 3 months. There was no apparent inflammatory reaction and immunologic rejection in rabbits implanted with ADM.6. The content of heparin in the rat cellular dermis was (4.34±0.83) g/cm2, that in ADM was (2.36±0.13) g/cm2 (P=0.000). The decellularized process obviously reduced the content of heparin in the dermis. The content [(4.25±0.77) g/cm2 ] of heparin in the ADM after soaking in heparin solutions for 15 min could reach the level of the content of heparin in cellular DM, the content [(8.43±1.43) g/cm2] of heparin in the ADM reached to peak after soaking in heparin solutions for 12 h.Conclusion1. The morphology characteristics of pig and human dermis are quite different, while that of SD rat and human are more similar, therefore using SD rat as the animal resource of xenogenic ADM has reliable and solid histological fundament.2. We design the physical method to remove the basement membrane according to the distribution of the antigenicity of dermis, and apparently decreased immunogenicity of xenogenic ADM could be achieved by improved enzymatic digestion method following the treatment with hypotonic solution. This xenogenic ADM might cause slight inflammation after implantation.3. The decellularized process could significantly reduce the content of heparin in the dermis. The content of heparin in soaking the ADM in heparin solutions for short time (15 -30 min) could reach the level of heparin content in cellular DM. This simple method with the combination of the heparin on the ADM could provide the conditions for further clinical application.更多还原