| In recent years, with the development of living standards and transforms of lifestyle, the incidence of myocardial infarction is increasing in China. Myocardial infarction becomes one of the most common causes of death and disability. Although great progress has been made in the diagnosis and treatment to myocardial infarction, such as implement of percutaneous coronary intervention (PCI), which significantly declined the mortality of myocardial infarction. However, these treatments do not resolve the fundamental issue, which is absolute lost of cardiomyocytes caused by myocardial infarction. Heart transplantation is the last resort treatment of advanced heart disease, but it is greatly limited because of high medical costs and the scarcity of donor.Stem cells transplantation is an potential therapeutic means of myocardial infarction. However, clinical trials showed that stem cells transplantation had little significantly long-term benefit, and the endpoint of all-cause death was not better than the compared group. The main reason to these are the lost of stem cell due to the blood flow and peroxide damage produced by necrosis cell after myocardial infarction, making the number of the transplanted stem cell retention and survival veru low. If a temporary "shelter place" could be provided to the transplanted stem cells, or the function of stem cells could be regulated, or their resistance to local microenvironment peroxidation damage could be enhanced, then it is a great hope to improve the survival and retention of stem cells.Injectable myocardial tissue engineering means that the scaffold could treat heart disease (mainly myocardial infarction) by injecting into the damaged myocardium through endocardial or epicardial. Generally refers to a three-dimensional network-like structure of the hydrogel, which has a certain toughness and viscosity, rich in water more than 95%, and capable of carrying drugs or cells. Studies have shown that injectable hydrogel for myocardial tissue engineering can provide a mechanical support for the heart wall, thereby reducing ventricular tension, improving ventricular remodeling and heart function; In addition, as an cellular transport carrier, hydrogels could encapsulate stem cells, therefore, reducing cell leakage caused by blood flow and increasing cell retention time and cell numbers in targeted sites.Biologically functional material can be further optimized based on clinical needs. Such as anti-cancer drugs carrying hydrogels, angiogenesis promoted functional polypeptide hydrogel, biological nano-materials with site-targeting effect, hydrogel with antioxidant effects etal.. After coronary infarction, the myocardial cells and fibroblasts would produce a series of ROS (Reactive oxygen species, ROS) in the metabolic process dued to localized myocardial ischemia and hypoxia, or reperfusion injury caused by blood flow recanalization. The ROS includs:O2-, H2O2 and HO2·,·OH, etc., which could cause seriously injury to myocardial cells and transplanted stem cells. Therefore, hydrogel with antioxidant effects may hold great potential of not only providing mechanical support, but also being able to effectively reduce the local ROS levels in the infarct zone, thus protecting cells from damage and improving survival after transplantation.Puerarin (chemical name:8-β-D-glucose pyranose-7,4’-hydroxy isoflavone) is extracted from the main active ingredient which was isolated from the dried root of kudzu. Based on the chemical structure, puerarin is classified as an isoflavone compounds. Studies have shown that:puerarin can scavenge superoxide anion and ·OH, suppress red blood cell hemolysis and lipid oxidation caused by hydrogen peroxide, and protect peroxidation induced endothelial cells and PC 12 cell from death and injury. Researches had showed that these effects may be related to the following mechanisms:(1) puerarin can provide proton in vivo, which can bind excess ROS and cause direct damage to H2O2, decrease intracellular ROS levels, reduce the damage to the cells; (2) puerarin can improve endogenous antioxidant enzymes in MSCs, such as activities of SOD enzyme and GSH-PX, which could enhance the antioxidant defense system to the ROS scavenging ability; (3)puerarin could directly participate in the repair of the DNA of damaged cells. Thus, facing with local microenvironment peroxide damage after myocardial infarction, puerarin is expected to be able to regulate the survival of the transplanted stem cells.Therefore, developing a type of hydrogel with puerarin for the therapy of myocardial tissue, could not only be able to provide mechanical support for the infarcted ventricular wall and reducing transplanted cells loss by encapsulation, but also play a significant role in cellular protection by its antioxidant effects and improve transplanted cells retention and survival.Drugs-carrying hydrogels generally conducted by the way of chemically bonding or physical encapsulation. In the former type the drug always treated as the object of both the drug and part of the vector, which can significantly improve drug entrapping rate, but it always requires certain type of materials and complex process. When we tried to construct a hydrogel by bonding puerarin to a polypeptide, we found that puerarin can self-assemble into a hydrogel by heating and cooling process without any chemical modification. Considering the chemical structure of puerarin and the principle of other hydrogel-forming systems, we hypothesized that puerarin hydrogel forming principle may be as follow:puerarin structure containing hydrophilic glucose pyranose structure and have relatively hydrophobic structure of isoflavones. Puerarin is slightly soluble substance at room temperature, under heating can be completely dissolved, the benzene ring can be stacked by intermolecular forces, hydrogen bonding forces and hydrophobic to self-assemble into a three-dimensional colloidal structure when cooling. Puerarin hydrogel is the first natural medicines that capable of forming self-assembled hydrogel by its own discovered so far. The discovery of puerarin hydrogel was found to have obvious advantages:because the ingredient is only puerarin itself, so its drug entrapping rate is 100%; it is a easy way to form a gel without complex process; the gel can be formed within a few minutes after heating and cooling, the time interval allows it to be as injectable. Then, we do a extensive research for puerarin hydrogel, including physical and chemical properties and biocompatibility testing, the ability of MSCs protection under peroxide environment in vitro and the function of puerarin hydrogels carrying MSCs transplantation in vivo rat model of myocardial infarction. Study was divided into the following three parts:Part Ⅰ:study of the physical and chemical properties and biocompatibility of puerarin hydrogelThe minimum gelation concentration of puerarin hydrogel is 1%, the higher concentration of the gels, the less transparency of the gels, when concentration of the gel is 6%, it appears like milky opaque hydrogel; Results of transmission electron microscope showed that the topography of puerarin hydrogel is a three-dimensional network,1%,2% and 4% concentration of hydrogel are formed as long nanofibers, the diameter of the fibers were from 10 nm to 50 nn, the fiber length and diameter of 1%,2% and 4% gels were similarl; Mechanical characterization found that puerarin hydrogel is a stress-dependent gel instead of non-frequency-dependent gel, and the mechanical strength of 2% and 4% hydrogel in stability state is about 10 KPa, which is similar to the strength of myocardial tissue, the similarity of the mechanical strength between gel and cardiac tissue would make it suitable for cardiac tissue engineering; In vitro drug release study found that:2% and 4% concentration hydrogel release puerarin at a faster rate in former 12 h in a physiological environment; Cytocompatibility experiments showed that:puerarin hydrogel have good biocompatibility with myocardial cells and cardiac fibroblasts; Histocompatibility experiment showed that, puerarin hydrogel do not appear significant inflammation when injected in normal myocardial.Part II:Function and mechanism of puerarin hydrogel for the MSCs protection under peroxide environment in vitroIn this study,100μmol/L H2O2 intervention for 1 hours was served as exogenous ROS, which could simulate peroxide environment of myocardial infarction in vivo. The content of intracellular ROS was detected by SOD activity and levels of MDA and fluorescent probe DHE. Compared with the control group, MDA level, SOD activity and the intensity of DHE red fluorescence increased significantly in the H2O2 group, and 2%,4% and 6% concentration of puerarin hydrogel can significantly reduced SOD activity and MDA level, and decreased intensity of DHE red fluorescence; The cell survival and proliferation was detected by CCK-8 assay, the concentration of LDH in cell culture medium reflects cytotoxicity; AO/EB staining could observe survival and apoptotic of the cells from cell morphology; Flow cytometry Annexin V-FITC/PI double staining could detect the apoptosis of cells quantitatively. The results show that rate of cell survival decreased and apoptosis increased after H2O2 induction, and different concentrations of puerarin hydrogels could decrease the rate of MSC apoptosis under oxidative stress; Western blot analysis displayed that different concentrations of puerarin hydrogel could increase the expression of anti-apoptosis protein Bcl-2 and reduced expression of pro-apoptotic protein Bax and Caspase-3. These results indicated that H2O2 was able to induce ROS production in cells, reduce cell survival and increase cell apoptosis. While different concentrations of puerarin hydrogel could inhibit the production of ROS, protect cells from injury and decrease cell apoptosis. The mechanism of which may be attributed to increase of expression of anti-apoptotic Bcl-2 protein and reduce the expression of pro-apoptotic protein Bax and Caspase-3.Part Ⅲ:Study of puerarin hydrogel carrying MSCs transplantation in the treatment of myocardial infarctionIn this part, we will study of the injectable hydrogel of puerarin carrying MSCs transplantation in the treatment of myocardial infarction in rat mode to observe its effect on cell survival and retention, cardiac function and cardiac structure. Echocardiography was used to evaluate the changes of cardiac function after transplantation; HE staining, Masson staining was used to evaluate heart tissue morphological changes after transplanting, including wall thickness, infarct size and myocardial fibrosis; and immunofluorescence evaluation of myocardial infarction site and neo-angiogenesis, western blot detection of the expression of anti-apoptotic and pro-apoptotic proteins. The results showed that:four weeks after cell transplantation, puerarin combined with MSCs group increased cell retention and survival significantly compared with PBS group; 4 weeks after transplantation, cardiac ultrasound tests showed that puerarin hydrogel group, MSCs group and puerarin hydrogel combined with MSCs group could improve left ventricular ejection fraction (LVEF) and fractional shortening (FS), but the improvement of puerarin hydrogel combined with MSCs group were more significant; Histological analysis showed that puerarin hydrogel group, MSCs group and pueraria hydrogel combined MSCs group could improve post-MI ventricular wall thickness, decrease infarct size, reduce infracted zone fibrosis, reduce myocardial injury, amiliorate angiogenesis, increase the number of myocardial tissue. The protective effects of puerarin hydrogel combined with MSCs treatment group are more apparent.In summary, the present study successfully prepared puerarin hydrogel. It is the first natural medicine that can be able to self-assemble into hydrogel without any chemical modifications. The mechanical strength of the puerarin hydrogel is close to that of myocardial tissue, and could release puerarin for more than 12 hours. The gel also holds the characteristics with excellent cardiac cells and cardiac fibroblasts compatibility. The in vitro experiments revealed that H2O2 could simulate peroxide environment after myocardial infarction. Puerarin hydrogel is a powerful antioxidant that can reduce the level of ROS within cells, increase cell survival, decrease apoptosis rate and cell pro-apoptotic proteins, which may be related to inhibition of cell apoptosis; The in vivo experiments in rat mode proved that puerarin hydrogels can improve the survival and retention of transplanted cells in MI zone, and further improve heart function and ameliorate ventricular remodeling. The gel-injected infarcted myocardium exhibits a reduced cardiomyocyte apoptosis, enhanced infarct neovascularization, diminished cardiac fibrosis, along with improvements in global cardiac function.In this study, we developed an effective and translation approach to enhance MSCs delivery to the heart. Based on the great benefit of this study, it may have great potential for clinic translation. Nonetheless, this study highlights the beneficial effects of incorporating cells into biomaterials in order to advance regenerative medicine applications with stem cells. |
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