The Use Of Chitosan Hydrogel For Enhancing The Therapeutic Benefits Of Brown Adipose-derived MSCs For Acute Myocardial Infarction

Acute myocardial infarction (AMI) shows high morbidity and mortality in theworld. AMI is relevant with a large loss of cardiomyocytes, and likely to progress intoheart failure easily. The cardiac regeneration ability of adult heart is weak to replace theexcessive loss. Stem cell is the hot spots of the cardiac repair and regeneration with self-renewal and multipotential differentiation in recent years. Research confirmed that stemcells can improve cardiac function and myocardial regeneration.Brown adipose-derived mesenchymal stem cells (BADSC is a new source of seedcells,which has been discovered in recent years. It holds strong potential of spontaneouscardiac differentiation and has similar properties to MSCs, such as paracrine potential,low immunogenicity and autologous transplantation. Therefore it has a tiny space inmyocardial regeneration. Temperature-responsive chitosan hydrogel is a kind of cardiactissue engineering injectable material and shows good property in the biocompatibilityof hydrogels, the degradation of hydrogels. It has been proved to be applied to themyocardial tissue engineering repair of myocardial infarction.Our experiment frist observe the influence and mechanism of BADSCs in vitro addchitosan components (D-Glu and N-AC-Glu) and BADSCs were lentivirally transducedto express both firefly luciferse (Fluc) and monomeric red fluorescent protein(mRFP).On this basis, chitosan hydrogel was co-injected with BADSCs into the ratinfarction section in vivo to test the treatment condition, and to compare the cardiacdifferentiation ability with only injected BADSCs on acute myocardial infarction.This study is focused on the following three aspects:Part I The isolation, sorting，cultivation, characterization and lentiviral transduction of rat BADSCsThe BADSCs were prepared by enzyme digestion from brown adipose tissuesisolated from rat shoulder regions and analyzed by flow cytometry and cardiacdifferentiation ability. BADSCs were lentivirally transduced to test the expression offirefly luciferse (Fluc) and monomeric red fluorescent protein (mRFP). Result:1)Flowcytometric analysis indicated that sorting cells were positive for CD90,CD133,CD34and negative for CD45.2) BADSCs could spontaneously differentiate into myocardialcells, immunofluorescence identify the myocardial cells could express the myocardialmarker.3) The lentiviral transduction of BADSCs could be viewed strong expressionfluc-mrfp reporter gene.Part II The preparation of temperature-responsive chitosan hydrogel and the study ofchitosan degradation products in promoting the BADSCs cardiac differentiation.Medium was added with D-Glu/N-AC-Glu, on this basis, the mechanism ofchitosan degradation products promoting differention of BADSC was investigated.Result: Chitosan degradation products enhanced the cardiac differentiation of BADSCsthrough promoting collagen synthesis.Part III: Transplantation of BADSCs in temperature-sensitive chitosan hydrogel for ratacute myocardial infarctionMyocardial infarction rat were randomly divided into four treatment groups:4×106cells in100μL chitosan hydrogel (BADSCs/chitosan) or PBS (BADSCs/PBS),100μLchitosan hydrogel and PBS alone were injected as control.After transplantation,bioluminescence imaging was used to track the retention and survival of BADSCs;Masson trichrome stain, echocardiographymap were evaluated the effect of myocardialrepair; Immunofluorescence were tested the cardiac differentiation in vivo. Result:Chitosan hydrogel chould significantly improve the rentention and survival of transplanted BADSCs. Chitosan hydrogel as scaffold for BADSCs delivery in vivosignificantly enhanced the heart function, microvessel density and cardiacdifferentiation.