Silk Fibroin/Chitosan Nanofibrous Patch Based Adipose Tissue-derived Mesenchymal Stem Cells Promotes Cardiac Repair Post Myocardial Infarction In Rats

Background and Aims:According to the latest update from World Health Organization(WHO), ischemic heart disease(IHD) ranks first in the leading causes of death worldwide with 7.4 million deaths, accounting for 13.2% of all-cause mortality in 2012. During acute myocardial infarction(AMI), large numbers of cardiomyocytes are lost and replaced by non-contractile scar tissue because of the extremely limited regenerative ability of cardiomyocytes, thus leading to cardiac structural and functional remodeling, finally resulting in congestive heart failure(HF). Conventional revascularization therapies such as application of thrombolytic agents, percutaneous coronary intervention(PCI) and coronary artery bypass grafting(CABG) could successfully reduce the mortality of AMI. However, apparent decline of the incidence of HF after AMI has not been seen. In this case, novel therapeutic strategy to replace damaged tissue and restrain ventricular remodeling is urgently needed.Stem cell transplantation has emerged as a promising therapeutic strategy for IHD. And a variety of stem cell types have been shown to be beneficial in cardiac repair. However, the harsh MI microenvironment induced by local inflammation and ischemia leads to low retention and poor survival of transplanted stem cells, which has become major obstacles to achieve intended therapeutic effect. In this case, how to improve the survival rate of delivered stem cells becomes the key problem of heart regeneration. Studies show that just mechanical support for ventricular expansion by cardiac patches restrains ventricular remodeling. Under these circumstances, improving the efficacy of cell therapy for MI hearts, developing new materials serve as cardiac patches to mimic the natural extracellular matrix(ECM) and providing 3-D scaffold for cell surviving deserve further investigating.The tissue-engineered cardiac patch, which not only provides mechanical support and appropriate elasticity but also has good biocompatibility, is a promising strategy for heart regeneration after AIM. Electrospun cellulose nanofibers, which have 3-D structure with sizes in nano to micrometers, could provide suitable space for cell growth. However, the stable structure and poor hydrophilia make cellulose not a good choice for colonization and growth of attached cells. Layer-by-layer(LBL) coating technique, with alternating absorption of oppositely charged polyelectrolytes, particles, and ions on solid substrates, which can construct bioactive surface, would solve such problem. Silk fibroin(SF)with good elasticity and chitosan(CS) as hydrophile can greatly enhance the biocompatibility by assembling onto the cellulose nanofibers via LBL method.Adipose tissue-derived mesenchymal stem cells(AD-MSC), with no ethical controversy and low immunogenicity, can be easily obtained from adipose tissue, and have demonstrated good viability in vitro and the strong capability of paracrine and differentiation. Additionally, transplantation of AD-MSCs would not induce teratomas in vivo. As a result, AD-MSCs are ideal seed cells for tissue regeneration, with good prospects for clinical application.The present study was aimed to fabricate novel biocompatible SF/CS nanofibers via LBL and electrospinning technology. The nanofibers, serving as cardiac patches, were seeded with AD-MSCs constitutively expressing both firefly luciferase(Fluc) and green fluorescent protein(GFP) and may become AD-MSCs carrier and meanwhile provide reliable ventricular mechanical support. This study was to assess if cardiac patches could promote heart regeneration and to explore the mechanism and provide a theoretical basis for further clinical research.Methods:1. Cellulose acetate(CA, Mn=3×104 Da) were used to fabricate electrospun nanofibrous mats. CS and SF were assembled onto the cellulose electrospun mat via LBL method. ζ-potentials of CA, CS and SF were measured to determine the force of LBL coating process. X-ray photoelectron spectroscopy(XPS) detected the surface layer of LBL coated mats. And the micro-structure of the nanofibers was characterized by scanning electron microscope(SEM). 2. AD-MSCs were isolated from Fluc-GFP transgenic mice constitutively expressing both Fluc and GFP. The cell type was characterized by flowcytometry. A microscope was applied to observe the cellular morphology. Bioluminescence imaging(BLI) was used to determine the correlation between Fluc activity and cell number. 3. AD-MSCs of 2~5 generations during logarithmic phase were co-cultured with nanofibrous membrane. Cell morphology was observed by a confocal microscope(CFM) and micro-structure was observed by SEM. MTT assay was done to assess the viability of AD-MSCs. 4. Rat AMI model was induced by ligation of the left anterior descending coronary artery. SF/CS nanofibrous patches(9×9 ㎜2) seeded with or without AD-MSCs were adhered onto the epicardium of the infarcted region. Wild type Sprague Dawley rats were randomized into four groups(n=20 in each group): Sham group, MI group, MI+patch group and MI+Patch/AD-MSCs group. Evans blue-TTC staining detected the sizes of MI. 5. 24 hours post-operation, cell apoptosis was determined by TUNEL staining. The engrafted AD-MSCs was tracked by using BLI and cardiac function was measured by transthoracic echocardiography(TTE) on day 1, 7, 14 and 28 post-operation. 28 days after AMI, Masson’s Trichrom was performed to evaluate the scar size and myocardium fibrosis. CD31 immunofluorescence staining was performed to assess neovascularization.Results1. The CS/SF nanofibrous membrane was successfully fabricated via LBL and electrospinning technology. ζ-potentials of CA, CS, SF were-21.3, +25.4 and-10.2(m V) at p H value 6.04,6.44 and 6.84 respectively. Meanwhile, XPS measurement confirmed SF/CS nanofibrous membrane was successfully constructed. SEM showed nanofibers were uniform in diameter with 3-D porous structure. 2. AD-MSCs cultured in vitro emerged fusiform and transparent in bright light under microscope and uniform green under fluorescent microscopy. Fluc signal intensity showed a linear correlation with the number of AD-MSCs. Flowcytometry indicated that AD-MSCs were positive for CD44, CD90 and negative for CD34, CD45. 3. SEM revealed that AD-MSCs co-cultured with SF/CS nanofibrous membrane proliferated actively, and grew in good morphology. CFM and MTT assay suggested that AD-MSCs co-cultured with SF/CS nanofibrous membrane proliferated in good condition with 3-D distribution. By comparison among different(CS-SF) layers of nanofibrous membrane, 10.5 layers of(CS-SF) nanofibrous membrane was of the best biocompatibility. So we chose 10.5 layers of(CS-SF) nanofibrous membrane for cardiac patches in our later studies. 4. Rat AMI model was successfully established by ligation of the left anterior descending coronary artery. The AMI model was identified by the color change of myocardium below the ligature and ECG that showed ST-T segment elevation. The cardiac patches were successfully adhered onto the epicardium during the operation, and were still adhered on the MI zones intactly 4 weeks after AMI. Infarcted sizes of each group 24 hours after MI showed no significant difference by Evans blue-TTC staining. 5. TTE showed that cardiac patches could improve the heart systolic function and restrain the enlargement of left ventricle. BLI revealed that AD-MSCs were still detectable until 4 weeks after transplantation. The cardiomyocyte apoptotic index decreased and the expression of CD 31 improved, meanwhile the scar size was reduced in both MI+patch group and MI+Patch/AD-MSCs group. The combined use of patches and AD-MSCs showed synergistic action in the therapeutic efficiency.Conclusions1. This study demonstrated that the SF/CS nanofibrous membrane provided microenvironments and 3-D scaffold to support the retention and viability of engrafted AD-MSCs. By comparison of different(CS-SF) layers of nanofibrous membrane, 10.5 layers of(CS-SF) nanofibrous membrane possessed of the best biocompatibility. 2. Cardiac patches could not only have the therapeutic efficiency to improve the cardiac function, attenuate ventricular remodeling and reduce the scar size, but also increase the survival rate of engrafted AD-MSCs, to further improve the therapeutic effect synergistically. 3. The beneficial effect of cardiac patches based AD-MSCs cell therapy in heart repair after AMI may attribute to the inhibition of cardiomyocyte apoptosis, and promotion of angiogenesis.