| Severe skin injury or loss,excessive loss of body fluids,cause significant complications such as shock,wound and systemic infections and even death.The most effective method for repairing large areas of skin defects is still autologous skin transplantation.However,the severe shortage of transplantable skin limited the application of the treatments.Construction of suitable skin substitutes is an effective way to solve this problem.Using tissue engineering principles and techniques to construct autologous skin substitutes to achieve personalized treatment is the focus of skin regenerative medicine.The three core problems of the construction of tissue engineering skininclude seeding cells,scaffold materials,and three-dimensional construction in vitro.Seeding cells are the foundation of tissue engineered skin construction,and the ideal seeding cells must be safely,and massively amplified to meet the needs of skin tissue regeneration.Epidermal stem cells have the potential of high proliferative ability,self-renewal and differentiation to various layers of epidermal cells,and are the source of good seeding cells for tissue engineering skin.Epidermal stem cells are located in the basal layer,the number of which is limited,so how to quickly promote the proliferation of epidermal stem cells is the primary task.In 2009,Hans Clevers successfully established "Mini-gut"(organoid)in 3D(three-dimensional)culture,followed by organoid culture techniques widely used in other tissues.The core of the organoid culture system is the stem cells with self-renewal ability,which can be differentiated by directional differentiation to produce the organ.Therefore,organoid culture system lay the foundation to build the tissue engineering skin with autologous source.Scaffold material is the basis of structure construction.The ideal tissue engineering scaffold material should have three-dimensional structure,which can provide a good space for cell migration and growth,and tightly adhesion to the wound,promote cell accurate localization,non-toxic side effects,and good biological compatibility.The interaction between extracellular matrix(ECM)and cells can affect the fate of cells.ECM is mainly composed of three core protein components like collagen,proteoglycan,glycoprotein,as well as ECM-related regulatory factors,growth factors,cytokines,enzymes and other components,which are necessary for cell physiological activities.The ECM structure of acellular skin is very similar to that of natural tissue,therefore providing three-dimensional scaffold materialfor cell growth,adhesion,migration and differentiation,meanwhile have no immunogenicity,degradation,excellent mechanical properties and biocompatibility.The acellular strategy determines the retention of ECM components and directly influences the biological characteristics of the ECM after cells decellularization.Our laboratory own independent intellectual property rights of the "four-step" acellular system,which can be used to rapidly and efficiently get the acellular liver matrix with the integrity of collagen and the retention of almost all ECM coupling cytokines,and the biological activity is excellent,which laid a solid foundation to the construction of acellular skin matrix materials.We divided our study into three parts.In the first part,we successfully prepared acellular porcine skin flap and identified the biological characteristics;in the second part,we established and optimized the culture system of human epidermal organs and fibroblasts,identifying the phenotype of the skin organoids.And finally,we completed the construction of the whole skin of tissue engineering using skin organoids and acellular skin matrix,and evaluated its function to promote wound healing in mice.Part ? Preparation and identification of acellular porcine skin matrixWe successfully prepared acellular porcine skin matrix(APSM).The combination of PLA2 and SDC for decellularization was shortest,highly efficient,and fully decellularized.Electron microscopic observations revealed that dermal collagen fibers and basement membranes remained intact.The phenotypic identification of ECM components showed that the dermis mainly expressed collagen,glycoprotein,and proteoglycans.The basement membranes such as Collagen IV,Laminin,Nidogen,and Perlecan expressed positively.Quantitative analysis of collagen and proteoglycans further demonstrated that the structural collagen of APSM was fully retained,and proteoglycans could also be effectively retained.The APSM has low SDC and DNA residues,low toxicity and immunogenicity.We also carried out Matrisome analysis of APSM.The matrix proteins of APSM were abundantly enriched.The mass spectrometry results showed 149 ECM proteins,among which collagen,glycoproteins,and proteoglycans were rich in the “core matrisome”,“matrisome-Associated” regulatory factors,secreted factors,and related proteins are more likely to be lost." The decellularized matrix is enriched in 79 non-matrisome or cellular proteins that may be potential stromal histone proteins.Therefore,we confirmed that the “four-step decellularization method” is stable and reliable,and its advantage is that it has a short operation time.While the amount of genetic material residues is small,ECM components are retained to the utmost,and can be used as a scaffold material for constructing tissue engineering skin.Part ? Establishment and identification of culture system of human epidermal organoids and fibroblastsWe successfully established the isolation of human keratinocytes(hKCs)and 3D Organoids culture methods,which are more viable than those obtained by conventional overnight isolation methods;and has also established a new method for the separation of hFbs.The 2D-cultured hKCs showed typical polygonal paving-like morphology,expressed integrin? 6,ck14 and transcription factor P63 and other basal layer stem/progenitor cell markers,and the proliferative ability sign Ki67 expression positive;a few cells expressed the mature cell marker Involucrin.The cultured hFbs were spindle-shaped,expressed fibroblast cell markers Vimentin,and fewer number of polygonal cells expressing the endothelial cell marker VWF.Organoids were cultured through a preliminary use of a combination of 10 factors: N-2 supplement + B-27 supplement + N-Acetyl-L-cysteine + EGF + FGF10 + Noggin + R-spondin1 + Wnt3 a + A83-01 + Forskolin to successfully culture the epidermis Organoids.The culture system was optimized to determine the optimal culture combination was Nac+B27+EGF+Wnt3a+Fsk+A83-01,and the efficiency of Organoids formation was close to 4% under NaBEWFs+A83-01(day2)conditions.After 24-28 days,the cultured cells remain stable and can be expanded by about 4000-5000 times.Organoids are solid round,the outer layer corresponding to the basal layer expresses the stem/progenitor cell markers Integrin ?6,Integrin ?4,transcription factors P63,SOX9,and CK5,CK14,the inner layer expresses the basal mature cell markers Involucrin,CK1,CK10,a small amount of cells Expression of the melanocyte marker gp100 was confirmed as an epidermis Organoids.Moreover,we show that Organoids are reprogrammed from Integrin ?6+++ cells.Part ? Construction of TE skin with organoids,fibroblasts,acellular porcine skin matrix and transplanted TE skin to repair full-thickness skin defectFirstly,we verified that APSM micronized powder can promote the adhesion,proliferation,and maintenance of stem cell characteristics of hKCs without cytotoxicity and good biocompatibility.Optimized 3D medium(NaBEWFs+A83-01)can effectively support the formation of epidermis of the epidermis Organoids.We then inoculated fibroblasts and epidermis Organoids cells onto APSM for gas-liquid interface culture.We successfully constructed a tissue-engineered full-thickness skin containing dermal fibroblasts and epidermis complexes.The construction time was short and the ultrastructure was similar to normal skin tissue under transmission electron microscope.APSM and its constructed TE skin can significantly speed up the skin repair process in injured mice.Repair early to reduce inflammatory response,promote the surface of the wound and the surface of the blood vessel formation,dermal remodeling,and hair follicles and other skin accessories regeneration repair.In summary,our acellular method can be a gentle,rapid and effective preparation of excellent performance of extracellular matrix scaffold materials,optimization of epidermal cells and dermal fibroblasts separation methods,the establishment and optimization of epidermal 3d-organoids culture system,so that autologous cell construction tissue engineering skin is no longer just a dream,Construction of tissue engineered skin that can promote wound healing. |
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