Injectable Chitosan-based Hydrogel For Cardiac Tissue Engineering

Myocardial infarction(MI) is associated with a great loss of cardiomyocytes, andlikely to develop into heart failure. MI is a serious threat to human health, which is ofhigh morbidities and mortalities. Available clinical therapies for MI, such as drugtherapy, interventional therapy, have certain limitations and cannot meet the needs ofclinical requirement. Therefore, to find a novel and effective therapy of MI hasbecome one of the urgent problems in the clinical.In recent years, the rise of the stem cells and tissue engineering has brought newopportunities for the treatment of MI. In earlier studies, researchers found thattransplantation of stem cells into the ischemic myocardium will lead to certainimprovement of cardiac function. With further research, it was found that the purestem cell transplantation is challenged by low cell retention and survival, leading tolittle improvement of heart function in long term. It was shown that injectable cardiactissue engineering was likely to overcome the above problems and was paid more andmore attention. The seed cells, injectable scaffold and in vivo transplantation therapyare what mainly investigated in injected cardiac tissue engineering.In seed cell research, there are a variety of stem cells that have been used forcardiac tissue engineering, including pluripotency, directed potent, and totipotent stemcells. However, no consensus has been yet researched about the ideal seed cells forcardiac tissue engineering. Bone marrow mesenchymal stem cells (BMSC) is one ofthe pluripotent stem cells that were the most widely investigated in cardiac tissueengineering, with potential for clinical application. Recently, it was found that whiteadipose mesenchymal stem cells (ADSC) have similar properties with BMSC,possessing potentials for myocardial repair. In addition, the adipose tissues existextensively and the cells are easily isolated, thus ADSC are valuable in research ofmyocardial therapy. Cardiac stem cells (CSC) are of orient potency to cardiovascularlineages and are also important seed cells in cardiac tissue engineering. The CSC weretraditionally isolated from heart tissues, which is limited in the research andapplication. It has been found that brown adipose tissue is a novel source of the CSC.Brown adipose CSC have high cardiac differentiation potential like heart-derivedCSC, possessing the great value in myocardial regeneration. However, the availablemethod for brown adipose CSC isolation is of low efficiency, which greatly limited their application in research. An efficient method for brown adipose CSC isolationwill be of great significance in promoting the application of brown adipose CSC.Induced pluripotent stem cells (iPSC) are highly similar to the ESC, but they avoidthe ethical problems faced by ESCs. Research has demonstrated the therapeuticpotential of iPSCs for MI. However, as one of the totipotent stem cells, thetumorigenicity and immunogenicity are also concerns for iPSC application in thecardiac tissue engineering, requiring further clarification.In area of stem cell therapy, a reliable method for cell labeling would crucial fortracking of cell fate and evaluation of therapeutic effect. Traditional method based onhistology cannot dynamically track the same animal at different time, whose resultswere also easily influenced by investigators' bias. The bioluminescence imagingbased on the Fluc is a non-invasive method, which could dynamically track celltransplant on the living animals. Further, the imaging is real-time, quantative and ofsensitivity. It has been widely used in stem cell and tissue engineering research, whichis valuable in tracking and evaluating stem cell transplantation for MI.In scaffold research, there have also been a variety of injectable scaffoldssuccessfully used for cardiac tissue engineering, including natural biomaterials andsynthetic biological materials. In the past years, cardiac tissue engineering based onthe injectable scaffold has made a series of progress. It has shown that injectablescaffold for stem cell transplantation may have multiple roles, including improvingcell retention and survival, regulating stem cell function, et al. However, previousstudies paid little attention to the mechanism that injectable scaffolds acted. In ourprevious studies, we have successfully applied thermosensitive chitosan hydrogel asESC carrier to the injected myocardial tissue engineering, and confirmed its roles inenhancing the retention and survival of transplanted ESCs, improving the MImicroenvironment as well as promoting myocardial repair. However, the underlyingmechanisms that chitosan hydrogel functioned have not been elucidated, no relevantreport is available.Based on the current advances and the problems in injectable cardiac tissueengineering, this study is mainly focused on the following three aspects:Part Ⅰ: The isolation, cultivation, labeling and biological characterization ofdifferent seed cells in cardiac tissue engineeringExperiment Ⅰ: The isolation, cultivation and differentiation of different stem cells.White and brown adipose tissues were isolated from rat inguinal and shoulder regions, respectively; ADSC were isolated by collagenase and trypsin digestion. Afterexpansion, they were identified by flow cytometry and multiple differentiation; brownadipose CSC were isolated by optimize the combination of collagenase IV, dispase IIand trypsin. They were identified by flow cytometry and cardiac differentiation; iPSCwere expanded in feeder system. The ascorbic acid (Vc) was used to induce iPSCdifferentiation into cardiomyocytes and the optimal dose was investigated.Through this part of the study, we established platforms for ADSC, brownadipose CSC and iPSC cultivation and differentiation. The result demonstrated:(1)0.1%collagenase IV+0.1%dispase II+0.05%trypsin digestion can significantlyimprove the issolating efficiency of the brown adipose CSC, thus establishing a noveland effective method for brown adipose CSC isolation;(2) ascorbic acid cansignificantly improve the iPSC differentiation into myocardial cells.Experiment Ⅱ: Labeling of stem cells by lentiviral particles. Lentivirus carryingFluc-mRFP-tTK were constructed by co-transfection of plasmids into293T cells;ADSC and iPSC were labeled by lentiviral infection and the optimal MOI wasexplored.Through this part of the study, we established platform for preparation oflentivirus and mastered key technologies for lentiviral transduction of different stemcells. The results show that, at a MOI of10-15,the infection efficiency of ADSCcould be upto70-80%; at a MOI of15-20, the infection efficiency of iPSC could beupto25-30%.Experiment3: iPSC tumorigenicity and immunogenicity. Undifferentiated iPSCs,induced differentiated but not purified iPSC-derived cells (iPSC-derivates) andiPSCs-CMs were subcutaneously transplanted into nude mice. Bioluminescenceimaging was used to dynamically track the survival, proliferation and teratomaformation of implanted cells; iPSC and iPSC-CMs were intramyocardiallytransplanted both into immunocompetent and immunosuppressed mice of MI.Bioluminescence imaging was used to dynamically track the survival, proliferationand exclusion of implanted cells. Combined with host lymphocyte infiltrationdetection, the immunogenicity of iPSC was revealed.The experiment in nude mice show that differentiation status significantlyinfluence their tumorigenicity, undifferentiated iPSC have higher tumorigenic risk;theexperiment in MI mice showed that the tumorigenicity of intramyocardialtransplanted iPSC increased with their differentiation and development in the myocardium, leading to the increase of the host immune rejection.Part Ⅱ: The study on the preparation and biological evaluation of injectablechitosan-based hydrogel as well as the mechanism of their CytoprotectionExperiment Ⅰ: Preparation of Thermosensitive chitosan-based hydrogel andBiological Evaluation. Temperature-sensitive chitosan hydrogel was prepared. Itscyto-compatibility was enaluated by coculture with the ADSC. After injection intoischemic myocardium,the tissue compatibility,biodegradation and ROS scanvengingwere evaluated by histology.The results showed that chitosan-based hydrogel has good cyto-compatibilityand tissue compatibility;In MI microenvironment, the chitosan-based hydrogels havea suitable degradable rate and can effectively scavenge ROS.Experiment Ⅱ: The study of chitosan degradation products in promoting theADSC adhesion and survival in ROS environment. Medium was supplemented withH2O2to simulate ROS environment, in which the role of ROS impairing ADSC werestudied. On this basis, the mechanism of chitosan degradation products (D-Glu andN-AC-Glu) scavenging ROS and protecting ADSC were investigated.The results showed that ROS induced ADSC damage by downregulatingadhesion-related proteins. D-Glu and N-AC-Glu could protect ADSC throughscanvenging ROS, which would promote ADSC adhesion and survival.Part Ⅲ: Transplantation of ADSC in temperature-sensitive chitosan-basedhydrogel for myocardial infarction.MI rats were randomly divided into four groups. They were injected with100μLPBS (PBS group),100μL of chitosan hydrogels (Chitosan group),100μLADSC/PBS (ADSC/PBS group),100μLADSC/Chitosan (ADSC could/groupof Chitosan), respectively. After transplantation, non-invasive bioluminescenceimaging was performed to track the retention and survival of ADSC at differenttime; the mechanism of chitosan-based hydrogel acting was investigated byhistological and molecular means;4w after transplantation, the effect of myocardialrepair was systematically evaluated by histology, echocardiographymap,hemodynamic, et al.The results confirm that chitosan-based hydrogel could significantly enhance therentention and survival of transplanted ADSC when used as myocardial scaffold. Therole of chitosan-based hydrogel enhancing stem cell survival, homing and therapeuticpotentials was mediated by scanvenging of ROS, thus promoting myocardial repair. In summary, this study established platforms for isolation, cultivation anddifferention of ADSC, brown-adipose CSC and iPSC; developed a novel and effectivemethod for brown adipose isolation based on―Cocktail‖enzymes. We mastered thekey technologies for lentiviral transduction of the different stem cells and establishednon-invasive imaging platform; the tumorigenicity and immunogenicity of iPSC invivo were primarily revealed based on the imaging platform. More importantly, theroles of chitosan-based hydrogel improving MI microenvironment, promoting stemcell survival, homing and therapeutic potential were confirmed in vivo and in vitromodels. In this study, the biological evaluation of stem cells, establishment ofisolating and labeling methods for different stem cells, as well as the finding on themechanisms, will be instructive for seed cell selection, scaffold design and clinicaltherapy of MI.