Three-Dimensional Bioprinting Of A Full-Thickness Functional Skin Model Using Acellular Dermal Matrix And Gelatine Methacrylamide Bioink

BackgroundSkin is the largest organ of the human body,which has the physiological functions of protecting the body from invasion,perceiving the surrounding environment,adjusting temperature and so on.However,the skin is also vulnerable to injury.Clinically,there are more and more cases of skin defects caused by burn injury,chronic ulcer and surgical injury.So far,the most common method of skin defect treatment is autologous skin transplantation.However,for patients with large skin defects,the limited donor site limits the therapeutic effect.In addition,tissue engineering skin also provides a new way for the treatment of skin defects.However,due to the great differences in the shape and depth of wound between patients,the traditional tissue engineering skin preparation methods are difficult to meet the requirements.3D bioprinting skin can be customized in shape,cell and material composition and distribution,which has certain advantages over traditional preparation methods.To find a suitable bio ink is one of the key problems in 3D bio printing.The ideal bio ink should have good biocompatibility and sufficient mechanical properties.At present,the commonly used bio inks include acellular dermal matrix(ADM),methacrylated gelatin(Gel MA),alginate,collagen and so on.These existing bio inks have their own defects.A large number of studies are working in this direction by modifying existing materials or mixing a variety of materials,but no breakthrough has been made.At present,there are few researches on using the existing bio ink and adopting new construction methods to obtain the optimized skin model.ObjectiveTo explore the physicochemical characterization,mechanical properties and biocompatibility of ADM and Gel MA hydrogels with different concentrations.According to the properties of the two kinds of hydrogels,we choose suitable concentration to construct full-thickness skin model using 3D bioprinting technology,and then evaluate and explore the effect of the full-thickness skin model to promote wound healing and regeneration.MethodFirstly,ADM was obtained by decellularized method and hydrogel was prepared.Quantitative and qualitative methods were used to detect whether ADM successfully completed acellular and evaluate the effect of decellularized process on extracellular matrix components.The Gel MA hydrogel was synthesized and prepared.The degree of substitution of Gel MA was detected by ~1H NMR.The gel-sol temperature transition point of Gel MA and modified gelatin was detected by inverted tube experiment.Then,the physicochemical properties(pore size,degradation,swelling ratio,water retention,etc.)and mechanical properties(storage/loss modulus,shear thinning and temperature composite modulus)of two kinds of hydrogels were evaluated.The biocompatibility of two kinds of hydrogels was evaluated by means of CCK-8 and live/dead cell fluorescence staining,respectively.According to the above properties,we constructed a new full-thickness skin model.The effect of printing process on cell activity,differentiation of epidermal layer and secretion of extracellular matrix were evaluated in vitro.In vivo,the full-thickness skin model was transplanted into the full-thickness skin defect model of nude mice.The survival,vascularization,wound healing speed and quality of cells in the skin model were detected.ResultsFirst,we successfully prepared ADM and Gel MA hydrogels,and evaluated their physicochemical properties.The results show that the two kinds of bioinks have interconnected porous structure and relatively good swelling performance,and 20%Gel MA has water retention and permeability close to skin.Furthermore,we evaluated the mechanical properties and biocompatibility of bioinks with different concentrations.The results showed that different concentrations of Gel MA had good printing performance.20%Gel MA could promote the adhesion and proliferation of Ha Ca Ts.1.5%ADM and10%Gel MA had good biocompatibility.Therefore,we constructed a full-thickness skin model with 20%Gel MA and Ha Ca Ts as the epidermis,1.5%ADM mixed fibroblasts as the dermis,and 10%Gel MA mixed HUVECs as the vascular network.After implantation,the wound healing time,epithelization,secretion of extracellular matrix and angiogenesis of new tissue were increased in nude mice.ConclusionThe functional skin model(FSM)constructed can provide a suitable three-dimensional environment for the growth of cells,and has good formation of epidermis and dermis,and can promote wound healing and improve the quality of healing.Therefore,FSM may become a functional skin substitute,and this construction method may provide a new idea for the construction of more optimized skin model in the future.