Printing Three-dimensional Skin Tissue Analogues With Acellular Dermal Matrix-Gelatin Methacrylate Composite Hydrogel

Research background and objectiveTissue regeneration has become a promising strategy for repairing damaged skin tissues.Current treatments such as tissue engineered skin(TES)or skin substitutes are limited due to their rigorous conditions of preservation,transportation and a lengthy preparation period.Bioprinting provides an advanced platform that facilitates the highly specific deposition of multiple types of skin cells and biomaterials.In skin tissue bioprinting,a major challenge is to seek for a suitable source of bioink capable of promoting wound healing.However,current bioinks for skin printing such as collagen and gelatin have several shortcomings such as insufficient mechanical properties and recapitulation of microenvironment.The acellular dermal matrix(ADM)is obtained by decellularizing the skin tissue.It has similar components and microenvironment compared with the natural ECM,and is suitable for cell adhesion,growth and proliferation.Methacrylic acid gelatin(GelMA)is obtained by chemical modification of gelatin and has widely adjustable mechanical properties,and after exposure to blue light for a few minutes or even seconds a highly crosslinked polymer can be formed.Here,we use 3D bioprinting technology to construct tissue engineered skin,which is made with ADM-GelMA composite hydrogel loaded with fibroblasts.We have prepared ADM and determinated its composition.The aim of this study is to construct TES with ADM-GelMA composite hydrogel using 3D bioprinting technology,and compare the 3D model characteristic,rheological properties,microstructure and cell proliferation evaluation of ADM-GelMA composite hydrogel with those of ADM hydrogel and GelMA hydrogel.MethodsWe decellularizated the pig skin using the method combining with low temperature freezing,enzymatic digestion and chemical reagents(0.25%Trypsin/1%Triton X-100),then determinated the composition of the ADM.GelMA was provided by the Polymer Laboratory of Zhejiang University with a 70%substitution rate.ADM hydrogel and GelMA hydrogel were used as the control groups,and ADM-GelMA composite hydrogel was used as the experimental group.The fibroblast suspension was uniformly mixed with the composite hydrogel,and an extrusion type three-dimensional printer was used for bioprinting skin substitutes.The proliferation of Fbs in each group of hydrogels was measured using a Cell Counting Kit-8,and the viability of Fbs in the tissue construct after printing was evaluated using live/dead staining.The microstructure of the scaffolds was observed by a scanning electron microscope.Student-t test was used to analyze the data.Results(1).Hematoxylin and eosin(HE)staining showed that there were no cell residues visible in the ADM after decellularization.As decellularization process removed all the cellular components,the content of collagen increased to 131.5%.(2).Three dimensional bioprinting resulted in the printed construct of GelMA hydrogel and ADM-GelMA composite hydrogel with good structural fidelitycan due to its rapid cross-linking during printing.While the printed construct of ADM hydrogel collapses gradually during the printing process,bacause of its long cross-linking time(half an hour).(3).SEM showed porous and interconnected networks of hydrogel scaffolds.(4).Cell proliferation assays showed that relatively lower concentrations of ADM and GelMA bioinks resulted in a high cell viability,and the biocompatibility of ADM hydrogel and ADM-GelMA composite hydrogel was batter than that of 5%GelMA hydrogel.(5).Live/dead cell viability assays showed that Fbs grew well in the hydrogel during a 7-day period,indicating that the damage to the cells during the printing process was almost negligible.ConclusionIn this study,Fbs loaded ADM-GelMA composite hydrogel was designed for bioprinted dermal substitutes.The results showed that ADM-GelMA composite hydrogel is biocompatible and has good mechanical properties.It has good structural fidelity bacause of its rapid cross-linking,We confirmed that ADM-GelMA composite hydrogel is a promising bio-ink for three-dimensional bioprinting,and it is expected to apply as a dermal substitute for skin wound repair in the future.