Thermo-responsive, Hydrogen Bonding Assembled Hydrogels And Stem Cell Delivery Application

Stem cell therapy has received widespread attention in modern biomedical science due to its functional tissue restoration,diverse physiological regulation,and extremely low immunogenicity.Conventional stem cell therapy is achieved by directly injecting a saline suspension of stem cells into a lession.Implantation is facile for clinical translation.However,during the injection process,stem cells are subjected to severe physical damage due to the lack of protection of the buffer medium,resulting in extremely low transport efficiency.At the same time,due to the lack of extracellular matrix（ECM）microenvironment in the lesion after injection,cell retention rate are very low.As a kind of stem cell deliverer,injectable hydrogel combines the injectability with the inherent advantages of hydrogels to facilely construct an ECM-like microenvironment.It can effectively improve stem cell survival rate and has become a hotspot in stem cell research.The injectability of hydrogels is achieved by the different responsiveness of their intrinsic polymer systems.Among them,the response mode of thermo-responsive injectable hydrogel based on lower critical solution transition temperature（LCST）has attracted much attention due to its mild delivery,rapid gelation and facile operation.When at room temperature,the polymer system is at sol state ready for injection;in a body temperature environment,the polymer system gels immediately and provides a biomimetic protective barrier.However,since the cross-linking of such thermo-responsive hydrogels are mostly only driven by hydrophobic aggregation,the mechanical strength and mechanical stability are not ideal.In this study,two methods of enhancement,synergistic enhancement of thermo-responsive hydrophobic aggreagation and quadruple hydrogen bond self-assembly and synergistic enhancement of the multiple cross-linking,are employed to regulate the mechanical properties of the injectable hydrogel and the delivery efficiency of stem cells.First,A one-step copolymerization of three monomers of methacrylated gelatin（GelMA）,2-methyl-2-acrylic acid-2-（2-methoxyethoxy）ethyl ester（MEO₂MA）,2-（3-（6-methyl-）4-oxo-1,4-dihydropyrimidin-2-one）-ureido)ethyl methacrylate（UPyMA）was carried out to synthesize the thermo-responsive polymer Gel-MEO-UPy,an injectable hydrogel based on the temperature sensitive polymer was then prepared.Injecting test,rheological tests and confocal laser scanning microscopy prove that Gel-MEO-UPy hydrogel has stable linear viscoelasticity,thermo-responsive injectability and self-healing ability;UPyMA self-assembled quadruple hydrogen bond remarkably enhances the hydrophobic aggregation of PMEO₂MA.In vitro cell experiments demonstrated that Gel-MEO-UPy polymer system has good biocompatibility for bone marrow mesenchymal stem cells（BMSCs）.Gel-MEO-UPy sol provide significantly much more protection to BMSC during delivery through injection than salt solution.In-vivo cell tracking showed that 10 wt%Gel-MEO-UPy hydrogel could still retain about 30%of BMSC in the subcutaneous part of the back of the nude mice in the third week,while the saline solution control group lost more than 90%of the cells within one week.The significantly increased cell retention rate of BMSC within Gel-MEO-UPy hydrogels has thus been demonstrated.Secondly,We fabricated a thermo-responsive injectable hydrogel system enhanced by multiple crosslinking networks based on the study above to further optimize the degradation rate and mechanical stability.The thermo-responsive polymer Gel-MEO-UPy and the macromonomer methacrylated hyaluronic acid（HAMA）were simply physically blended to obtain a series of photocurable precursors HG.HGU hydrogel was prepared by UV curing of HG.A series of rheological experiments show that the photocuring precursor H0.5G5containing 5 wt%Gel-MEO-UPy and 0.5 wt%HAMA is a thermo-responsive injectable hydrogel with a LCST of 30°C.The dynamic physical properties of H0.5G5 hydrogel is comparable to that of 10 wt%Gel-MEO-UPy hydrogel.Moreover,the self-healing efficiency of H0.5G5 hydrogel is significantly improved compared to 10 wt%Gel-MEO-UPy hydrogel,while the mechanical strength and stability of H0.5G5U are further enhanced by the formation of multiple crosslinked networks.The multi-crosslinked network hydrogel H0.5G5U has a much slower degradation rate than the 10 wt%Gel-MEO-UPy hydrogel.In vitro cell experiments showed that both HG and HGU polymer systems have good biocompatibility for BMSC.BMSCs adhere and proliferate within the H0.5G5U hydrogel.Among the HG polymer systems,H0.5G5 has the best BMSC delivery efficiency of about 60%,which is equivalent to 10 wt%Gel-MEO-UPy sol.Finally,two kinds of thermo-responsive injectable hydrogels were obtained via two different kinds of enhancements,both of which have good biocompatibility and biodegradability.Compared with the traditional medium of salt solution during injection and delivery,the protective effect of stem cells has been significantly improved using those thermos-responsive injectable hydrogels as deliverers.