Injectable Alginate-chitosan Composite Hydrogel For The Treatment Of Rats With Myocardial Infarction

Objective:For years people have been working hard to repair or reconstruct the damaged cardiac tissue. The major repair methods include stem cell transplantation and heart transplantation. The former is confronted with limited cell sources, low survival rates and low retention, uncontrolled differentiation in vivo, inducing tumors and other issues. The latter has immune rejection, lifelong medication and other shortcomings. With the continuous development and application of new biological materials, Thus, cardiac tissue engineering may provide a novel approach for repairing damaged myocardium. Sodium alginate (SA)-chitosan (CS) composite hydrogel can be manufactured simply and are effective in various models of tissue regeneration. However,its potential application in cardiac tissue engineering has not been tested. This study sought to examine whether SA-CS composite hydrogel can treat the myocardial infarction (MI) effectively and explore the relevant mechanism by which injectable materials affect ventricular remodeling, which can provide novel strategy and theoretical evidence for the clinical application of biomaterials in the treatment of ischemic disease such as MI.Methods:SA-CS composite hydrogel was prepared by mixing SA solution with the temperature-sensitive sodium glycerophosphate(GP)-CS solution,using the physical cross-linking ways. The physical and chemical properties of the composite hydrogel such as gelation time, structure morphology, equilibrium swelling,compressive strength,and degradation in vitro etc was characterized. After the establishment of acute MI model of male rats, the animals were randomly divided into PBS group,0.8% SA group,6%(GP-CS) group, GP-CS/SA composite hydrogel group. Thirty minutes later, the animals were subjected to intramyocardial injections with 150μl the above four kinds of solution at three points in the infarct border zone. At 4 and 8 weeks after injection, the cardiac function were detected by M-mode echocardiography. Heart tissue slices were prepared and subjected to Hematoxylin and Eosin (H&E) Staining and Masson trichrome staining for the tracking the biomaterial distribution and collagen visualization. Serial 5μm-thick sections were immuno-labeled with the a-SMA, c-kit, CD68, Ki67 antibodies. Immunohistochemical staining and TUNEL-positive cells in the infarcted area or border zone were counted. Scar area, collagen content, infarcted wall thickness and heart expansion index (El) were measured using image analysis system (Image J). MMP-9, MMP-2, TIMP-2, Collagen Ⅰ, Collagen Ⅲ and TGF-β1, p-Smad2 mRNA and protein expression levels were detected by Real time RT-PCR and Western blot to detect the myocardial tissue of the border zone.Results:The physical and chemical properties of GP-CS/SA composite hydrogel depends on the relative quality of SA solution, such as gelation time, structure, equilibrium swelling, compressive strength, storage modulus and in vitro degradation. Furthermore, GP-CS/SA composite hydrogel formed by using physical cross-linking were improved in the mechanical strength and degradation compared to SA or CS hydrogel. GP-CS/SA composite hydrogel can promote angiogenesis, reduce inflammation, attenuate tissue apoptosis, recruit endogenous repair and induce endogenous myocardial regeneration. In addition, GP-CS/SA composite hydrogel group exhibited preservation of cardiac function and attenuation of adverse ventricular remodeling over the 8 weeks following time course. Importantly, this therapy suggested a potential for cell cycle re-entry in adult cardiomyocytes and possible cardiac stem cells recruitment-two important processes in endogenous regeneration of cardiac muscle.Compared with the the control group,real time-PCR showed the expression levels of TGF-β1, TIMP-2, Collagen I, Collagen III were significantly decreased, while the MMP-9, MMP-2 expression were upregulated by the GP-CS/SA composite hydrogel injection. Western blot analysis also showed the expression levels of TGF-β1, TIMP-2, p-Smad2 were down regulated, while the MMP-9, MMP-2 expression were upregulated.Conclusions:GP-CS/SA composite hydrogel retains low immunogenicity, good biocompatibility, certain compressive strength and biodegradability rate. Intramyocar-dial injection of GP-CS/SA composite hydrogel in situ is an effective treatment for MI, whichcan attenuate adverse ventricular remodeling, preserve cardiac function and induce endogenous myocardial repair. These beneficial effects may be related to the inhibition of TGF-β1/pSmad2 signaling pathway and modulation of MMPs/TIMPs expression. The striking functional benefits, the simplicity of manufacturing, and the natural of this biomaterial provide impetus for further translation to the clinic.