【作者】 赵宇；

【导师】 柏树令；

【作者基本信息】 中国医科大学 ， 组织工程学， 2018， 博士

【摘要】 目的:软组织缺损的修复重建一直是整形外科所面临的难题和挑战,目前临床常用的软组织修复方式主要是应用自体的皮瓣移植、游离脂肪移植和异体材料填充等,但这些方法均存在弊端。利用种子细胞和支架材料所构建的组织工程脂肪,被认为更适用于软组织的修复重建。细胞外基质(Extracellular matrix ECM)是由组织细胞分泌到细胞外间隙中的大分子物质,在细胞间起连接支持的作用,并能调节细胞的各类生理活动。目前已能从多种组织中分离提取出不同的细胞外基质,如猪小肠黏膜下层和脱细胞真皮,其衍生的生物制品已被应用于临床。但这些生物制品大多取自动物或人的尸体,难以避免病原体传播及免疫排斥反应的发生。脂肪组织作为一种疏松的结缔组织,除含有多种的细胞成分外,还富含大量细胞外基质,这些细胞外基质在维持脂肪组织的结构和功能上发挥重要的作用。脂肪组织可以通过吸脂手术的方式从人体获取,吸脂手术是当前应用最为广泛的整形外科手术之一,对患者造成的创伤小,恢复快,易于被接受。支架材料是组织工程技术的核心和关键,为细胞生长和组织再生提供空间构架,同时还参与种子细胞的黏附、增殖、迁移及新生血管的形成。已有研究者将不同组织来源的细胞外基质构建成具有三维立体形态的生物活性支架,但这些支架多需要外科手术的方式将其植入体内,无法避免植入时的创伤。软组织缺损的形态多不规则,可注射的水凝胶支架形态更适用于软组织缺损的修复。本实验将吸脂手术获取人体脂肪,通过物理,化学及酶制剂相结合的脱细胞方法,获取到脱细胞的脂肪组织(Decellularized adipose tissue DAT),并将其构建成可注射的水凝胶支架。对脂肪脱细胞水凝胶支架的成分,理化性质及体内外生物相容性等进行检测,探讨其用于脂肪组织工程支架的可行性,从而为软组织缺损的修复重建提供新的解决方案。研究方法:1、采用物理,化学和酶学相结合的脱细胞方法,为吸脂手术获取到的人体脂肪组织脱细胞,对提取到的脱细胞脂肪组织分别进行HE染色,DAPI染色、油红O染色及DNA定量检测,评估其脱细胞的有效性;进行胶原纤维、网状纤维、弹力纤维的特异性染色及I型、IV型胶原蛋白,层连蛋白,纤连蛋白的免疫组织化学染色,及胶原蛋白和糖胺聚糖(glycosaminoglycans GAG)的定量测定,评估DAT中ECM不同成分的保留情况。2、利用胶原酶消化法,分离出人脂肪来源干细胞(adipose-derived stem cells ADSCs),进行传代培养,测定细胞增殖的情况,绘制生长曲线,进行成脂成骨定向诱导分化。3、将提取的脱细胞脂肪组织,经冻干后研磨成粉,在HCl和胃蛋白酶混合溶液作用下溶解,经体外调节pH值中性后,制备出DAT水凝胶支架。对水凝胶的超微结构,可注射性及体外的凝胶化过程进行观察,同时制备鼠尾腱I型胶原水凝胶支架与其进行对比,进行凝胶浊度动力学检测和SDS-聚丙烯酰胺凝胶电泳,并观察DAT水凝胶植入大鼠体内后的凝胶化过程。4、将第3代ADCSs在体外接种到DAT水凝胶支架,观察细胞在支架表面的活性及增殖情况,并对复合支架的ADCSs进行成脂诱导,观察支架对ADCSs成脂能力的影响。再将DAT水凝胶支架注射进大鼠皮下后,不同时间段取材,观察植入的水凝胶支架在体内的大体形态变化,将组织切片进行HE染色,masson染色及CD45、CD68的免疫组织化学染色。结果:1、DAT的提取及鉴定:吸脂手术获取的人体脂肪组织,经一系列脱细胞方法处理后,提取出的脱细胞脂肪组织,外观呈不透明的乳白色,质地柔软,无固定形态;HE及DAPI染色无可见细胞核,残留的DNA含量极其微少;组织学染色可见胶原纤维,网状纤维,弹力纤维的存在,免疫组化染色结果显示DAT中的I、IV型胶原蛋白,层连蛋白,纤连蛋白均得以保留;胶原蛋白和GAG定量检测提示这两种成分均存在于脱细胞的组织中,但含量略有减少。2、ADSCs的分离培养鉴定:利用胶原酶消化法分离出ADSCs,通过贴壁生长去除未黏附细胞,ADSCs的生长曲线呈S型,细胞具有自我更新能力,传到第10代仍保持良好的增殖状态。细胞在一定条件下,能定向诱导成脂肪细胞和成骨细胞。3、DAT水凝胶支架的构建及性能检测:构建的DAT水凝胶支架在体外4℃时能顺利通过18G针头,在体内外37℃条件下,能自主聚合形成凝胶。形成的凝胶具有多孔三维结构,含有包括I型胶原蛋白在内的多种肽类成分,凝胶的特性随浓度不同而不同,高浓度的凝胶具有更牢固的外形,开始形成凝胶的时间更早,成胶的速率也更快。4、DAT水凝胶支架体内外生物相容性检测:DAT水凝胶支架,体外复合ADSCs培养对细胞无毒性作用,能促进ADSCs的生长增殖,并能自发诱导ADSCs向脂肪细胞的转化。植入体内后,早期可引起轻微炎症反应,随时间延长,炎症反应逐渐消失,宿主细胞开始向支架内部迁徙,并伴有新生血管和脂肪组织形成。结论:1、采用物理、化学和酶学结合的脱细胞方法,能够从吸脂来源的人体脂肪中提取DAT,提取的DAT中有效去除了脂肪组织中的细胞及脂质成分,保存脂肪ECM中的绝大部分成分。2、DAT能被构建成可注射的水凝胶支架,在体内外一定条件下能自主聚合形成凝胶。形成的凝胶支架具有多孔的三维结构,具有更为复杂的的蛋白成分,凝胶的理化性质随浓度的不同而改变。3、DAT水凝胶支架具有良好的生物相容性,在体外能促进ADSCs的增殖与成脂,植入大鼠体内能促进宿主细胞的迁徙及新生脂肪和血管形成,有望成为脂肪组织工程的理想的支架和软组织缺损的最佳填充材料。更多还原

【Abstract】 Objectve: Repair and reconstruction of soft tissue defects is the difficult problem and challenge for plastic surgery.The traditional repair methods mainly include autologous flap transplantation,free fat graft and allograft material filling,etc,but all of these methods have drawbacks.The adipose tissue engineering constructed by seed cells and scaffolds is more suitable for soft tissue repair and reconstruction.Extracellular matrix(ECM)is some macromolecules secreted by cells into the extracellular space,which plays an important role between the cells and tissues.Intact ECM has been derived from a variety of tissues,such as porcine small intestinal submucosa and decellularized dermis matrix,which have been used in clinical.However,all of these biological products derived from animals or carcasses,it is very difficult to avoid the risk of immune rejection and pathogen transmission.Adipose tissue is loose connective tissue,which contains a lot of ECM components in addition to various cells.Adipose tissue can be obtained by liposuction from healthy human body.Liposuction is one of the most fashionable plastic surgery with the minimally invasive and fastest recovery.Scaffolds is the key part for tissue engineering,which can provide space support for tissue regeneration and involved in the adhesion,proliferation,migration,and angiogenesis of cells.Extracellular matrix derived from different tissues can be fabricate a variety of bioactive scaffolds,some have been used clinically.But These scaffolds are solid forms,which require surgical implantation and increase the surgical trauma.Soft tissue defects have more superficial location and irregular shape,therefore,fabrication of an injectable hydrogel scaffold is very important for the treatment of soft tissue defects.In this experiment,we use a decellularization method that combines physics,chemical reagents and enzymes to obtain decellularized adipose tissue(DAT)from human lipoaspirate.And then,we fabricate DAT into injectable hydrogel scaffolds and assess the completeness of decellularization,reserved ECM components,physical and chemical properties of the gel,its compatibility with cells and tissues and other related properties.To investigate whether the scaffolds can be used for adipose tissue engineering,and to provide new biological scaffolds for soft tissue repair and reconstruction.Methods: 1.Using decellularization methods combined of physical,chemical and enzymatic,we extracted the adipose tissue ECM from healthy human lipoaspirate.The ECM extracted from adipose tissue was operated HE staining,DAPI staining,oil red O staining,the specific staining of collagen fibers,reticular fibers and elastic fibers,the immunohistochemical staining of collagen I,IV,laminin,fibronectin,the quantitative detection of residual DNA and preserved collagens and GAGs.2.With collagenase digestion method,we isolated human adipose-derived stem cells(hADSCs)and subcultured.The hADSCs were determined proliferation and growth curve,induced adipogenic and osteogenic differentiation.3.DAT is dissolved in HCl and pepsin after freeze-dried and ground into powder,and the DAT solution forms gel when the pH was adjusted to neutral by NaOH.Hydrogel ultrastructure,injectability and gel process in vitro was observed.Turbidity kinetic and SDS-polyacrylamide gel electrophoresis was operated,and DAT hydrogel formed gel in rats was observed.Rat tail collagen I hydrogel scaffolds was prepared by contrast in this experiment.4.We examined the proliferation 、 toxicity and adipogenic of ADCSs after compositing with DAT hydrogel scaffold in vitro.Specimens drawn at different time periods were implemented HE staining,masson staining and immunohistochemical staining of CD45,CD68 to observe the inflammatory response in vivo when the DAT hydrogel scaffold was implanted subcutaneously in rats.Results: 1.Extraction and identification of DAT: Decellularized adipose tissue(DAT)showed soft variable shape with opaque milky white after human adipose tissue derived from liposuction operated by a series of decellularization methods.The cell components were not observed in the HE and DAPI staining and only with tiny residual DNA.Collagen fibers,reticular fibers,elastic fibers were observed in histological staining and collagen I,IV,laminin,fibronectin were observed in immunohistochemistry staining.Quantitative detection of collagens and GAGs contents showed that both components were present in the decellularized tissue with a slight decrease.2.Isolation and identification of ADSCs: ADSCs were isolated by using collagenase digestion and non-adherent cells were removed by adherent growth.ADSCs had strong self-renewal capacity so that they can keep good proliferative states when passaged to 10 generations.Isolated cells can be induced into adipocytes and osteoblasts under certain conditions.3.Fabrication of DAT hydrogel scaffolds and the detection of properties: DAT was dissolved in HCl and pepsin after freeze-dried and ground into powders,and the DAT solution formed gel when the pH was adjusted to neutral by NaOH.DAT hydrogels can pass 18 G needles at 4℃,and automatically formed gels in vivo and in vitro at 37℃.Gel was porous scaffold with different protein contents compared with type I collagen.Properties of the hydrogel scaffolds differ with the concentration,the higher concentration of the gel had the firmer appearance,earlier start time and faster rate of gelation.4.The biocompatibility testing in vitro and in vivo of DAT hydrogel: DAT hydrogel scaffolds had been verified that it was non-toxic to ADSCs,can promote the growth and proliferation of ADSCs,and induced spontaneous conversion of ADSCs to adipocytes in vitro.After being implanted subcutaneously in rats,the scaffolds can cause some early inflammation,and the inflammation gradually disappears after some weeks.DAT hydrogel can stimulate the host cells migration,promote the formation of new blood vessels and adipose tissue.Conclusion: 1.By using decellularization method combined of physical,chemical and enzymatic,we can extract DAT from healthy human lipoaspirateS.Cellular components and the grease were removed and most of the ECM components were retained in the obtained DAT.2.DAT can be fabricated to injectable hydrogel scaffolds which can automatically form gels in vivo and in vitro.Gel was porous scaffolds with complex protein contents compared with type I collagen.The properties of the hydrogel scaffolds differed with the concentration.3.DAT hydrogel scaffolds have good biocompatibility,they can promote the proliferation and adipogenesis of cells in vitro and promote the infiltration and angiogenesis of host cells by being implanted in the body.The human DAT hydrogel scaffolds were the ideal scaffolds and filling materials for soft tissue defects and adipose tissue engineering.更多还原