Novel Thermoresponsive Injectable Hydrogel Based On Collagen And N-isopropylacrylamide Copolymers To Deliver Mesenchymal Stem Cells For Cardiac Repair

Part1A Novel Thermoresponsive Injectable Hydrogel Based on Collagen and N-isopropylacrylamide CopolymersObjective:We sought to construct a novel thermoresponsive injectable hydrogel based on N-isopropylacrylamide copolymer and collagen to increase the stem cell retention, decrease the cell redistribution to other organs, and then enhance heart function of infarcted animals.Methods:In this study, the novel biodegradable thermosensitive hydrogel was synthesized by free radical polymerization with N-isopropylacrylamide (NIPAAm), acrylic acid (AAc) and macromer2-hydroxylethyl methacrylate-poly(ε-caprolactone)(HEMAPCL). After the synthesis of NIPAAm/AAc/NHS/HEMAPCL copolymer, the hydrogel and collagen bioconjugate was prepared by mixing hydrogel copolymer solution with type I collagen. The NIPAAm/AAc/HEMAPCL feed ratio of88/9.6/2.4copolymer was used to conjugate type I collagen. The composition of the copolymer was confirmed by1H NMR and the number average molecular weight was measured using gel permeation chromatography (GPC). The LCST, cloud point and rheological property were determined by DSC, UV via spectrometer and rotational rheometer respectively. Scanning electron microscopy (SEM) was also used to study the morphology of the hydrogel. It was found that LCST of the hydrogel ascended remarkably with the increasing NIPAAm content and hydrogel with higher AAc/HEMAPCL ratio exhibited better storage modulus, water content and injectability. The hydrogel degradation experiment was conducted in PBS for2weeks and demonstrated a less than20%weight loss in14days.Results:The novel hydrogel had good characteristics of thermoresponsitivity, injectability, and low cytotoxicity, which are suitable for stem cell transplantation. With the NIPAAm/AAc/HEMAPCL feed ratio of88/9.6/2.4, the synthesis procedure of poly(NIPAAm-co-AAc-co-HEMAPCL) copolymer was optimized. After being conjugated with collagen I, the novel copolymer had a lower lower critical solution temperature transition temperature (LCST) of29.7℃, are rather suitable for injecting transplantation of cells. Moreover, low cytotoxicity is very important for stem cells therapy. In our study, poly(NIPAAm-co-AAc-co-HEMAPCL)-type I collagen and its degradation showed excellent cell viability in vivo. The novel hydrogel show the storage modulus over80000Pa at37℃, which means that the hydrogel is tough enough to be applied as cell scaffold material in the myocardial tissue. The copolymer was soluble at low temperature and exhibited sol-gel transition within30sec when heated above LCST. The formed gel with higher AAc/HEMAPCL ratio showed higher water content and degradation rate but lower storage modulus. Degradation experiment revealed that it took at least14days for the hydrogels to achieve a weight loss less than15%. Cell viability test in vitro demonstrated that this kind of copolymer was an injectable material with promising biocompatibility for stem cell encapsulation whether before or after degradation.Conclusions:The novel hydrogel, with excellent characterization of thermosensitivity, injectability, and low cytotoxicity, would be a favorable vehicle for cell transplantation, which improved the cell retention and survival in the infarcted myocardium, and decreased the distribution to other organs, and therefore improved the heart functions. Part2The biological characteristics of mice bone marrow-derived mesenchymal stem cells isolated and cultured with attachment methodObjective:To establish a simple and effective method of isolation, purification and cultivation of C57/BL6mice bone marrow-derived mesenchymal stem cells (MSCs) in vitro, and explore their biological characteristics.Methods:MSCs were isolated and purified for culture in vitro and serial passage by the attachment culture method. The morphological changes of the MSCs were continuously observed under the inverted microscope. The cell growth curve was measured by MTT method, and membrane antigens were detected with flow cytometry (FCM).Results:The MSCs in primary culture which were adhered to plastic surface within48h after displacement took the oval, asteroid, short and fusiform shapes.7to12days after culture in DMEM/F12medium containing15%FBS, the cells reached90%confluence in single layers, taking a long and fusiform shape. Further purification was achieved by expansion at serial passages. MSCs were in latency for2days, converted into growth period on the3rd day and entered the stationary phase on the5th day. FCM results indicated that the positive rate of CD45, CD44、CD29and CD54was2.4%、83.2%、95.1%and94.1%, respectively.Conclusion:The attachment culture method can be effectively used to isolate and purify C57/BL6mice MSCs. The cultured MSCs are stable in growth with active proliferation and share the general biological characteristics of MSCs, which will further make them ideal seed cells for tissue engineering. Part3Preparetion of C57/BL6mice myocardial infarction model and method of improving survival rateObjective:To investigate how to develop a credible, steady and repeatable rat model of acute myocardial infarction with a low mortality, improve the method and decreased those factors that influence death rate of creating the model.Methods:40C57/BL6mice were anesthetized by intraperitoneal injection of pentobarbital sodium. After tracheotomy intubations, respiration machine was linked. Lefe anterior thoracotomy through3,4intercostals region was performed, and then the left anterior descending coronary arteries were ligated to produce acute myocardial infarction. During the operation, some details to affect rat mortality were analyzed and improved, which involving anesthesia, artificial respiration, thoracotomy and area of ligating the left anterior descending coronary artery, etc.Results:The model of acute myocardial infarction was established successfully. Following the details were highlighted, survival rate was95%after operation and92.5%beyond three weeks. The left ventricle became larger and the endocardium of the survival rats showed fibrosis. Histopathology study could see the scar and the proliferation of the fibrous tissue.Conclusion:C57/BL6mice acute myocardial infarction model can be made quickly by this way and the model’s survival rate was improved effectively. The factors, including Correct applying of anesthetic, scientific using of artificial respiration, decreasing of lung injury, exact ligating of left anterior descending coronary arteries, form the basis for making C57BL6mice model with myocardial infarction. Part4Novel Thermoresponsive Injectable Hydrogel Based on Collagen and N-isopropylacrylamide Copolymers to Deliver Mesenchymal Stem Cells for Cardiac RepairBackground:Although stem cell transplantation improves heart function in animal models of myocardial infarction, limited retention of transplanted stem cells in the myocardium and massive redistribution to other organs may hinder its clinical application. Objective:We sought to construct a novel thermoresponsive injectable hydrogel based on N-isopropylacrylamide copolymer and collagen to increase the stem cell retention, decrease the cell redistribution to other organs, and then enhance heart function of infarcted animals.Methods and Results:The thermoresponsive hydrogel was constructed. Bone marrow mesenchymal stem cells (MSCs) were isolated and propagated from male C57/BL6mice. The infarction model was created by ligating the left coronary artery of female C57/BL6mice. Ten minutes after myocardial infarction, F12/Dulbecco’s Modified Eagle’s Medium (group1), poly(NIPAAm-co-AAc-co-HEMAPCL)-type I collagen solution (group2, n=16),1×106MSCs (group3), or1×106MSCs mixed with poly(NIPAAm-co-AAc-co-HEMAPCL)-type I collagen solution (group4), were injected into the peri-infarct region. Two hours,1,7and28days later, optical bioluminescence imaging (BLI) in live animals showed that there were more MSCs located on heart in group4than group3. Otherwise, there were a lot of MSCs distributed to other organs in group3. After the animals were sacrificed, real time PCR showed that in group-4, more cells survived at each time spot as compared to group-3(p<0.01), especially at2hours after cell transplantation, the cell number in group4were almost as6times as that in group3. Finally, at28days, echocardiographic analysis shown that EF and FS improved significantly in group4as compared with group3(p<0.001), but no functional improvement in group1and2were observed (p>0.05).Conclusions: The novel hydrogel could be a favorable stem cell.scaffold for cell transplantation, which could improve the cell retention and survival in the infarcted myocardium, and decreased the distribution to other organs.