Self-Assembling Hydrogel Of D-Amino Acids Enhances Therapeutic Effects Of Mesenchymal Stem Cells For Hindlimb Ischemia

BackgroundLower extremity arterial disease are the most common peripheral artery diseases,mainly due to arterial ischemia and insufficient blood perfusion to meet the needs of limbs,thus causing a series of symptoms.Human placental-derived mesenchymal stem cells（h PMSCs）have a broad application prospect in the treatment of ischemic diseases because of their angiogenesis promoting effect.However,when transplanted into the ischemic site,the low survival rate and retention rate of cells limit its therapeutic effect.Biological materials can improve the survival of stem cells in vivo by providing suitable survival microenvironment,so they can be used as scaffolds to deliver cells and are widely used in stem cell-based therapy strategies.ObjectiveTo study whether the self-assembling hydrogel of D-Amino Acids designed in this research can significantly improve the survival rate in vivo and angiogenesis of human placenta-derived mesenchymal stem cells,and further enhance the therapeutic effect in the model of hindlimb ischemia in mice.Methods1.Designed and synthesized the self-assembling hydrogel of D-Amino Acids（NapDFDFKGRGD）with good biological stability and its enantiomer L-configuration selfassembled hydrogel（Nap-FFKGRGD）.2.Characterization of the self-assembling hydrogel of L/D-Amino Acids,included observation of microstructure by transmission electron microscope,testing of mechanical properties by rheometer,and biological stability experiment of resistance to protease K degradation in vitro.3.Experiments of the biocompatibility and anti-apoptosis ability of the self-assembling hydrogel of L/D-Amino Acids with h P-MSCs.4.Established the model of hindlimb ischemia in mice,sham-operated group（Sham group）,ischemia group（PBS group）,h P-MSCs group（h P-MSCs for treatment）,h PMSCs/L-Gel group（h P-MSCs and L-Gel combined transplantation）,h P-MSCs/D-Gel group（h P-MSCs and D-Gel combined transplantation）.5.Real-time monitored the retention rate of transplanted h P-MSCs in vivo by BLI.6.Three-dimensional imaged of angiogenesis of collateral vessels in hindlimbs of mice by High-resolution Micro-CT.7.Molecular mechanism of angiogenesis,included quantitative RT-PCR and immunofluorescence staining.8.Histopathological methods evaluated the tissue repair and functional recovery,included HE staining and Masson staining.Results1.The self-assembling hydrogel of D-Amino Acids had good biocompatibility,biological stability and anti-apoptosis ability in vitro.2.BLI and Micro-CT analysis conformed the co-transplantation system of h P-MSCs and D-Gel could significantly improve the survival and retention of cells in vivo and enhance their ability to promote collateral angiogenesis.3.The self-assembling hydrogel of D-Amino Acids could improve the angiogenesis ability of h P-MSCs,included upregulating the expression of VEGFA,VEGFR2,Ang-1 and Ang-2 genes and CD31 protein.4.Histopathological and functional analysis showed the self-assembling hydrogel of DAmino Acids could enhance the therapeutic effect of h P-MSCs to the model of hindlimb ischemia in mice.ConclusionThe self-assembling hydrogel of D-Amino Acids had excellent biological stability,which could significantly improve the survival rate of transplanted h P-MSCs and the ability of promoting angiogenesis,and further enhance the therapeutic effect of h P-MSCs to the model of hindlimb ischemia in mice.Meanwhile this hydrogel provided a promising biomaterial scaffold for regenerative medicine and tissue engineering.