Cardiac Tissue Engineering Based On Injectable Fibrin As Scaffolds

Objective:The research is aimed at studying the usefulness of the combination of fibrin glue (as scaffold) and adiposederived stem cells (ADSCs) to treat myocardial infarction. The present study focuses on the following issues:1) To investigate a method of culture and isolation of rat adipose tissue-derived stem cells in vitro and explore their biological properties, and to discuss the feasibility of ADSCs as seed cells in injectable myocardial tissue engineering.2) To investigate the effects of the fibrin glue on the proliferation and viability of adipose tissue-derived stem cell of rat, and to evaluate the biocompatibility and biodegradability of the fibrin glue using histopathology staining in vivo, which provided the experiment evidence of injectable myocardial tissue engineering.3) Injectable cardiac tissue engineering aims to support cell-based therapies and enhance their efficacy for cardiac diseases. No research is devoted to studying the usefulness of the combination of fibrin glue (as scaffold) and adipose-derived stem cells (ADSCs) to treat myocardial infarction.Methods:1) Firstly, the isolated ADSCs were cultured in vitro. The cell phenotype and generation cycle were detected by flow cytometry;growth curves of the cells were drawn with help of MTT assay;the characteristics of osteogenic and adipogenic and cardiomyocytes differentiation potential were investigated.2) Fibrin glue were prepared by combining fibrinogen with thrombin of different concentration for exploring suitable concentration and time. To Culture adipose tissue-derived stem cell and fibrin glue in vitro, the suitable concentration of fibrinogen was explored. The proliferation was tested by MTT assay and cell viability was assessed with LIVE/DEAD stain between ADSCs culture alone and ADSCs seeded fibrin glue. Fibrin glue was injected into the cardiac apex of 8 Rats, histopathology staining was performed to evaluate the biocompatibility and biodegradability of fibrin glue at different point in vivo.3) Fibrin glue was co-injected with ADSCs into the left ventricular wall of rat infarction models. Myocardial infarction was induced in 90 female rats using coronary artery ligation. Surviving rats that underwent ligation were randomly divided into 4 equal group, injections were performed along the border zone. 100?L of PBS was injected into the ischemic LV in control group (n=10).50?L of fibrinogen and 50?L of thrombin were simultaneously injected into ischemic myocardium in Fibrin group (n=10).5×106ADSCs in 100?L of PBS were injected into the infract in ADSCs group (n= 18,8 for 24-h cell retention observation).5×106ADSCs in 100uL of fibrin glue were injected into the infract in Fibrin+ADSCs group (n=18,8 for 24-h cell retention observation). Four weeks after the injection, the surviving rats underwent examination of heart functions by the echocardiography and hemodynamics. The rats were killed and their hearts were taken out to undergo immunohistochemistry with 4'6-diamidino-2-phenylindole (DAPI) and actin to measure the area of cardiac infarction. The heart infarcted size and wall thickness were calculated by masson trichrome staining. The differentiation of ADSCs in the infarcted heart was identified using cardiac troponin T (cTnT), vWAg antibody and smooth muscle a-actin (SMA). Blood vessel formation was detected by vWAg antibody.Results:1) The ADSCs were positive for CD90 and CD29, but negative for CD34 and CD45;The ADSCs could be expanded for 15 passages, and showed active proliferation on generation 7 (P7), more than 80%cells of ADSCs were found at G0-G1 phase. The ADSCs were differentiate into osteogenic and adipogenic cells. However, ADSCs may not have the ability to differentiate into cardiomyocytes in vitro.2) The fibrin glue prepared by using l0mg/mL fibrinogen and 50iu/mL thrombin is suitable for the application in cardiac tissue engineering. ADSCs showed fast proliferation in fibrin glue group than the control group (P<0.05);ADSCs were successfully cultured in fibrin glue in vitro. The viability of ADSCs maintain>90%and the mortality of ADSCs maintain<7%at 24h and 72h. The biocompatibility and biodegradability of fibrin glue are good in the myocardium.3) After injection, both the.24h-cell retention and 4-week graft size were significantly higher and larger in the Fibrin+ADSCs group than those of the ADSCs group alone (P<0.1). The heart function improved significantly in the Fibrin+ ADSCs group compared with that of the ADSCs group 4 weeks after transplantation (P<O.01). The heart infarcted size of the Fibrin+ADSCs group was significantly less than those of the ADSCs group and fibrin group (both, P<0.01). The co-transplantation of ADSCs with fibrin glue had the best effect to preserve the infarcted wall thickness compared with ADSCs or fibrin glue treatment. The ADSCs could differentiate into cardiomyocyte-like, endothelial and vascular smooth muscle cells in vivo. In addition, the arteriole densities within the infarcted area improved significantly in the Fibrin+ADSCs group compared with those in the ADSCs group 4 weeks after transplantation (P<0.01). Conclusion:1) The Rat ADSCs was easy to isolate and culture in vitro. The proliferation ability of ADSCs was strong, especially before 7th passage. The ADSCs have the potential for osteogenic and adipogenic differentiation. The ADSCs would be a promising source of stem cells for tissue engineering.2) The proliferation and viability of ADSCs with fibrin glue were good. The biocompatibility and biodegradability of fibrin glue are good in the myocardium. Fibrin glue may work as a vehicle in deliverying ADSCs for injectable myocardial tissue engineering.3) The result from this study indicate that fibrin glue was potential injectable scaffolds that can be used to deliver stem cells to infarcted myocardium. The ADSCs with fibrin glue has the therapeutic potential to improve the function of infarcted hearts. The method of in situ injectable tissue engineering combining fibrin glue with ADSCs is promising clinically.