Preparation Of Multifunctional Gelatin Hybrid Hydrogels And Study On The Construction Of Engineered Cardiac Tissues In Vitro

The development of myocardial tissue engineering has given new impetus to the restoration of injured myocardium and has also played a key role in the development of new cardiac medications and toxicity testing.Great progress has been made in the construction of engineered cardiac tissue in vitro with similar structure and physiological function to natural myocardial tissue using hydrogel scaffold.However,the myocardial microtissues constructed by previous hydrogel scaffolds lack the contraction-excitation coupling and electrical conduction characteristics similar to those found in natural myocardium,which cannot provide a good conductive microenvironment for cardiomyocytes.Meawhile,natural muscle fibers are arranged in an anisotropic manner,and the mechanical and electrical conductive properties of cardiomyocytes are regulated by intercalated disc structure,so that cardiomyocytes can adapt to the physical,electrical,and mechanical stimuli from microenvironment,for now,most of the synthetic hydrogel scaffolds are isotropic.Based on this,this paper mainly uses the methods of nanomaterials and tissue engineering to prepare several suitable conductive hydrogel scaffolds using gold-and carbon-based nanomaterials and introduce anisotropic structures to promote the directional growth and maturation of cardiomyocytes under the action of an external electrical/magnetic field,to obtained the engineered cardiac tissues in vitro.In addition,potential scaffolds suitable for cardiomyocytes embedding and three-dimensional（3D）culture are also explored,finally,it is expected to be applied them as cardiac patch or injection to the myocardial repair and regeneration of ischemic heart disease such as myocardial infarction.The specific research contents are as follows:（1）For the first time,water dispersible ultra-high aspect ratio gold nanowires（Au NWs）were produced and integrated into a methacrylic gelatin（GelMA）hydrogel matrix to stimulate cardiomyocyte development by increasing the hydrogel’s electrical conductivity and mechanical capabilities.The influence of hybrid hydrogels with different structures of gold nanomaterials（particles,rods,and lines）on the cardiomyocyte function was investigated.Among these,Au NWs hybrid group significantly promoted the cell retention and excitation threshold,tissue actinin organization,expression of connexin and calcium-regulated maturation,and the up-regulation of related mature genes,indicating that Au NWs with ultra-high aspect ratio have much potential in the field of cardiac tissue engineering.（2）GelMA-PDA-rGO conductive hybrid hydrogel scaffolds were made with dopamine-reduced graphene oxide（PDA-rGO）as a conductive filler.The cardiomyocytes inoculated on GelMA-PDA-rGO conductive hybird hydrogel scaffold showed more mature phenotypes regardless of the presence or absence of external electrical stimulation than those on pure GelMA hydrogel.Meanwhile,we found that applying electrical stimulation on the conductive hydrogel scaffolds had a synergistic enhancement effect on cardiomyocyte function and maturity.（3）An electromagnetic bi-functional graphene oxide-ferric oxide nanocomposite reduced by dopamine（PDA-rGO-Fe₃O₄）and an anisotropic and electromagnetic multifunctional hybrid hydrogel scaffold（GelMA-PDA-rGO-Fe₃O₄）were developed.The cardiomyocytes cultured on the surface of this anisotropic hybrid hydrogel displayed directional growth and spontaneous beating,whereas cardiomyocytes cultured on the isotropic and GelMA groups distributed and grew in any direction.The electrophysiological behavior,cardiac related protein expression,and calcium treatment ability of cardiomyocytes were considerably improved with external electrical stimulation.A magnetic field stimulation apparatus was built in vitro to explore the effects of alternating magnetic stimulation on cardiomyocyte function.Beating behaviors and RT-PCR results exhibited that the anisotropic hydrogel scaffolds with magnetic stimulation synergistically promoted the maturation of cardiomyocytes.（4）The double-network hybrid hydrogel（HAMA-P-GelMA）with enzyme and light cross-linking was developed based on GelMA and hyaluronic acid（HA）.The possibility of3D culture of embedded endothelial cells and cardiomyocytes has been preliminarily studied.The high concentrations of SA cross-linked HAMA-P hydrogels are injectable due to their rapid gel kinetics,enabling them to embed cells for 3D printing and cardiac repair applications.3D cultured HUVECs had better cell survival,adhesion,and spreading behavior thanks to the hybrid hydrogel,which also improved the physicochemical features of the integrated scaffold.Furthermore,3D co-culture of cardiomyocytes and HUVECS in HAMA-P-GelMA hydrogels could improve the spreadability of cardiomyocytes.