Accelerated Expansion of Epidermal Keratinocytes and Improved Dermal Reconstruction Achieved by Engineered Amniotic Membrane
|School||Second Military Medical University|
|Keywords||Amniotic membrane decellularization dermal substitute EDC cross-linking epidermal keratinocyte basement membrane skin equivalent animal model woundhealing|
Background: The development of favorable dermal substitute has always been amain focus in skin tissue engineering research and the basis of composite skin equivalents.Dermal substitutes can serve as the structural template for wound healing by introducingdermal reconstruction, regulating the proliferation and differentiation of keratinocytes, andpromoting the formation of an intact and functional basement membrane (BM). Largenumbers of dermal substitutes have already been derived either from natural materials suchas allogeneic/xenogeneic acellular dermis, collagen, and hyaluronic acid, or from syntheticmaterials such as polymers and electrospun nanomaterials, and development of many othernew dermal substitutes is also in progress. Among all these currently available dermalsubstitutes, Integra and Tegaderm have already been successfully used in clinic. Althoughmost dermal substitutes can mimic the structural and fuctional characteristics of normaldermis, nearly all of them lack the component of basement membrane, except foraccellular dermis. Even in accellular dermis, since the removal of epidermal layer andfibroblasts and vascular endothelial cells in the matrix requires strong cell removaldetergents, severe damage can be caused to the basement membrane.Basement membrane is a very important structure in normal skin, located at theepidermal-dermal junction. It plays a vital role in maintaining the fine function of skin. Innormal skin, the epidermal stem cells attached closely to the basement membrane byhemidesmosomes, which can regulate the proliferation, migration and differentiation ofepidermal stem cells. Epidermal stem cells are deprived of the attachment and support ofbasement membrane when cultured in vitro, and gradually lose their ability of continousproliferation, and finally differentiate into epidermal keratinocytes. There are abundantstudies showing that by adding natural or artificial basement membrane components ontothe dermal substitutes, the in vitro proliferation rate of epidermal keratinocytes wassignificantly enhanced, and the function of the epidermal layer was also improved.Human amniotic membrane (AM) is the innerest layer of the placeta. It comprises asingle layer of epithelial cells, a dense BM and an avascular stromal matrix rich in collagen. The amniotic matrix is composed of type I, type IV and type VII collagen, fibronectin andglycosaminoglycans, similar with human dermis. There are fibroblasts in the matrixwithout containing any capillaries or vascular endothelial cells. The basement membranecompsists of laminin, type IV collagen and type VII collagen, similar with the basementmembrane of skin and cornea, and is the thickest basement membrane in the body. Theamniotic membrane also possesses properties of promoting epithelialization, alleviatinginflammation and inhibiting scarring. With very low immunogenicity, it has been widelyused in the treatment of various corneal diseases, and tissue banks of amniotic membranehave also been established in some countries. Many studies reported that the amnioticmembrane had good histocompatibility for xenografting/allografting and did not inducesignificant immune rejection response. The amniotic membrane matrix with a basementmembrane structure has been proved to be a favorable substrate for in vitro expansion ofhuman limbal stem cells, mesenchymal stem cells and epidermal stem cells, preventing celldifferentiation and maintaining the properties of stem cells, thus it could be a good carriercandidate for quick proliferation and grafting of epidermal keratinocytes. The intactamniotic membrane has plenty of growth factors and is very helpful in maintaining theproliferating ability of stem cells, while the acelluar amniotic membrane (AAM) is moresuitable for the adhesion, proliferation and migration of stem cells.In the present study, we used repeated freeze-thaw cycles and DNase digestion toprepare an acellular amniotic membrane with an intact BM structure, and cross-linked itwith soluble1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to improve itsmechanical strength and biostability. The appropriately cross-linked acellular amnioticmembrane thus obtained had improved mechanical strength and enhanced biostability, andit was able to promote the adhesion and proliferation of epidermal keratinocytesremarkably, while retaining its good in vivo histocompatibility. Epidermal keratinocyteswere cultured on the cross-linked acellular amniotic membrane for2weeks to form acomposite skin equivalent, and transplanted onto full-thickness skin defects in nude mice.The results showed that the composite skin equivalent survived well and covered thewound in3weeks. New epidermis formation and dermal reconstruction during woundhealing in the composite skin equivalent transplantation group is apparently better than inthe epidermal sheet transplantation and blank control groups.Materials and Methods:(1) Fresh amniotic membranes obtained from15different donors were divided into three groups: one was treated with repeatd freeze-thaw and DNase digestion, one wastreated with Dispase II digestion and scraping, and the untreated one served as control. Thedecellularization effect in different groups was compared by the following examinations.1) H&E surface and section staining.2) Hoechst DNA staining.3) Immunohistochemical staining against laminin, type IV, type VI, and type VIIcollage, MHC-I, MHC-II, and vimentin.4) Scanning and Transmission Eelectronic Microscopy.(2) EDC cross-linking was used to improve the the mechanical strength andbiostability of acellular amniotic membrane, and then the histocompatibility of thecross-linked acellular amniotic membrane was examined. Briefly, the acellular amnioticmembrane was corss-linked with EDC at a concentration of0.05mmol per mg AAM for5min,30min and6h, resulting in acellular amniotic membranes with different cross-linkingdegrees. Epidermal keratinocytes were cultured either in extraction medium fromcross-linked AAM or cultured directly on cross-linked AAM to test the cytotoxicity of thecross-linked AAM. Biocompatibility of the cross-linked AAM was further evaluated in asubcutaneous model in immunocompetent rats. The following examinations wereperformed.1) The ninhydrin method was used to determine the cross-linking index.2) The uniaxial tension meter was used to measure the maximum tension andmaximum stretch length of acellular amniotic membrane.3) The in vitro degradation rate was measured by type I collagenase digestion method.4) CCK-8assay and live/dead staining were used to observe the viability of cells.5) H&E and Masson’s trichrosome stainings were used to observe the inflammatoryresponse and in vivo degradation condition of the scaffold after subcutaneous implantation.6) Immunohistochemical staining against CD31, CD11b, CD4, CD8, CD68andvimentin was used to detect the types of cells infiltrating the scaffold.(3) The EDC cross-linked AAM was used as a substrate for expansion of epidermalkeratinocytes, and compared with the conventional cell culture dish. Compostite skinequivalents constructed with keratinocytes and the cross-linked AAM were transplantedonto full-thickness skin defects in nude mice, and transplantation of epidermal sheet andsham transplantation groups served as control. The following tests were performed todetermine the proliferation rate of epidermal keratinocytes and to evaluate the effect of wound healing after transplantation.1) CCK-8assay and Hochest nucleus staining were used to determine the proliferationrate of cultured epidermal keratinocytes.2) H&E staining was performed to observe the structure of composite skinequivalents.3) Immunohistochemical staining against P63was performed to dertermine theproliferating status of epidermal keratinocytes.4) Gross appearance of the wound was recorded daily.5) H&E staining and immunohistochemical staining against laminin were used toexplore the histological structure of the healed wounds.Results:(1) The method of repeated freeze-thaw+DNase digestion was capable of removingboth the epithelial cells and mesenchymal cells in amniotic membranes clearly, while afterDispase II digestion and scraping there were still some epithelial cells residual, and themesenchymal cells were rarely removed. The cleavage rate of DNA amount between twodecellularization groups was not significantly different (p＞0.05), but the remain of totalprotein amount in the repeated freeze-thaw+DNase digestion group was significantlyhigher than that in the Dispase II+scraping group, as77.2±4.72%VS48.5±4.16%(p＜0.05). Laminin, type IV collagen, type VI collagen and type VII collagen were seen presentat the basement membrane area of the intact amniotic membrane, and after repeatedfreeze-thaw+DNase digestion treatment they were still there, similar with the intactamniotic membrane. In the Dispase II+scraping treated group, nearly all these basementmembrane components were gone. TEM results showed that the fibrous collagen structureof the matrix in the repeated freeze-thaw+DNase digestion group was barely influenced,while the collagen fibers in the Dispase II+scraping group became apparently loose. Boththe epithelial cells and mesenchymal cells in the aminotic membrane expressed MHC-Iantigen and did not express MHC-II antigen, and only the mesenchymal cells expressedvimentin, but after repeated freeze-thaw+DNase digestion, the staining of MHC-I,MHC-II and vimentin in the acellular amniotic membrane were all negative.(2) The fresh AAM appeared soft and smooth, and it was difficult to handle it as itwas easy to coil up. Appropriate EDC cross-linking made the AAM become a bit harderwith a flat and smooth surface, and it was easy for handling. SEM showed that EDCcross-linking made the originally uniform and dense mesh collagen fibers in the matrix of fresh AAM associate with each other into thicker fibrous bundles or even cords. Themechanical strength of AAM increased and its stretch length decreased with the extent ofcross-linking increasing. EDC cross-linking also significantly increased the biostability ofAAM. Epidermal keratinocytes cultured for7days using the extracted culture mediumfrom cross-linked AAM did not show any difference from normal culture medium group.Direct cytotoxicity test showed that there was no significant difference in the cell viabilityof epidermal keratinocytes between the fresh AAM and5min-AAM groups when culturedfor7days (p＞0.05), but the OD value of the30min-AAM and6h-AAM groups wassignificantly lower than that of the fresh AAM and5min-AAM groups at day7(1.27±0.30%and10.02±1.43%VS0.42±0.14%and0.44±0.18%, p<0.05). The5min-AAMretained the flat and smooth morphology and the ability of supporting the growth ofepidermal keratinocytes as did fresh AAM. In vivo histocompatibility observation resultsshowed that the5min-AAM degraded completely in4months, forming a thicksubcutaneous tissue, and without obvious acute and chronic inflammatory response,indicating that the5min-AAM possessed good biocompatibility.(3) CCK-8assay results showed that the relative cell viability of the5min-AAM groupat day7and14was367±33%and631±43%, respectively, significantly higher than294±30%and503±41%of the conventional cell culture dish (CCD) group (p<0.05). Theepidermal keratinocytes on the5min-AAM formed a2-3layer epidermis structure aftercultured for14days. On day7, the percentage of P63positive cells was54.32±4.27%,significantly higher than33.32±3.18%of the CCD group (p<0.05). When composite skinequivalents constructed with epidermal keratinocytes and5min-AAM were transplanted tofull-thickness skin defects in nude mice, the cells survived well and covered the wounds byforming a new epidermis in3weeks, similar to normal skin. The wound healing effect inthe composite skin equivalent transplantation group was apparently better than in theepidermal sheet transplantation and control group. Histological observation results showedthat dermal reconstruction in the healed wounds was good, with an intact and thickbasement membrane.Conclusions:(1) Repeated freeze-thaw+DNase digestion method is capable of removing theepithelial and mysenchymal cells clearly in aminotic membrane, better than theconventional Dispase II digestion+scraping method. More importantly, the basementmembrane of the amniotic membrane can be intactly retained after decellularization by repeated freeze-thaw+DNase digestion.(2) Appropriate cross-linking by EDC can improve the mechanical strength andanti-collagenase degradation ability of the acellular amniotic membrane, and maintain itsgood in vitro and in vivo biocompatibility.(3) As a culture substrate, the cross-linked acellular amniotic membrane can increasethe expansion rate of human epidermal keratinocytes by retaining their proliferating ability.It can also improve the healing of full-thickness skin defects by promoting dermalreconstruction and new basement membrane regeneration after grafted, thus is an idealdermal substitute.