Effect And Mechanisms Of Intramyocardial Delivery Of BFGF And AD-MSC Via A Novel Biodegradable And Thermosensitive Hydrogel After Rat Myocardial Infarction

Background Many studies have shown that intramyocardial injection of biomaterials improves cardiac function after implantation because of its potential to stimulate angiogenesis.These hydrogels are of great significance in biomedical applications because they can expand in situ under physiological conditions and are easy to use.The necrotic tissue extends and thins with the degradation of collagen matrix,and the necrotic cardiomyocytes separate from each other.After a few weeks,the injured cardiomyocytes were gradually replaced by fibrous tissue,the infarct area gradually expanded and the left ventricular dilated,which may eventually lead to the thinning of the wall,the formation of scar tissue,the dilatation of the left ventricle and the decrease of cardiac function.Heart transplantation has traditionally been used to treat refractory severe heart failure,but its use is limited due to donor shortages.Cardioplasty seems to reduce the size and fibrosis of infarct scars,limit poor remodeling after myocardial infarction,and improve diastolic function.Clearly,there is an urgent need for new therapies for treatment after myocardial infarction.In order to replace the diseased myocardium and extracellular matrix and restore cardiac function,cardiac tissue engineering has become the main research focus of postmyocardial infarction treatment.Intramyocardial injection of biomaterials after myocardial infarction provides a theoretical method to reduce the stress of ventricular wall after myocardial infarction and can actively change the process of ventricular wall remodeling.At present,biological derivatives,synthetic and mixed injection materials have been used in various studies and designed for this purpose,although the optimal design parameters have yet to be clarified.By injecting bone marrow mononuclear cells into the stroma,the researchers demonstrated the beneficial effects of injectable fibrin glue scaffolds.By injecting these cells into the infarcted myocardium,it was found that the injectable fibrin stents could reduce the infarct size,improve the survival rate of the transplanted cells,and induce the formation of neovascularization in the ischemic myocardium.The highly purified collagen was injected into the infarcted area of rats,which showed that collagen significantly increased the thickness of infarct wall,inhibited abnormal contraction and protruded,and improved cardiac function.Thermally sensitive hydrogel is a member of hydrogel family and a biomaterial with structure similar to extracellular matrix.After injection in vivo,at a certain temperature(critical solution temperature),the thermosensitive hydrogel can be changed from liquid to solid(gel),which not only makes injection simple,but also forms gel on local myocardium.At present,they are widely used in tissue filling and tissue repair and regeneration.The hydrogel of interest to our team is a new biodegradable injectable thermosensitive hydrogel dextran-polycaprolactone-2-hydroxyethyl methacrylate-poly-N-isopropyl acrylamide copolymer(Dex-PCLHEMA/PNIPAAm).Current theories and analyses still lack solid foundation and clinical evidence.Therefore,we hope to explain the mechanism of injectable biomaterials in inhibiting ventricular remodeling after myocardial infarction from physical and biological aspects in future work.We reviewed the requirements and challenges of injectable synthetic materials and the possible mechanism of injection thermo-sensitive hydrogel in the treatment of myocardial infarction.Objective The aim of this study was to study the effect of Dex-PCL-HEMA/PNIPAAm hydrogel carrying basic fibroblast growth factor(bFGF)combined with encapsulated Adipose mesenchymal stem cells(ADMSCs)compared with bFGF or ADMSCs injected alone on the peripheral area of myocardial infarction in rats.To observe whether the combined injection group has stronger angiogenesis and myocardial protection,and to explore the possible mechanism.Methods Part ?50 male SD rats were included in this part.Rat models of myocardial infarction were induced by coronary artery ligation.All the surgeried mice were radomally divided into four groups with an equal volume of 100?l PBS without bFGF(PBS group),100?l hydrogel without bFGF(Gel group),100?l PBS containing 2?g bFGF(bFGF group)or 100?l hydrogel containing2?g bFGF(Gel+bFGF group)respectively intramyocardially injected into the peri-infarction areas.The first day and 30 days after the MI operations,all the mice were tested on cardiac function with transthoracic echocardiography examination.HE and Masson staining were used to detect the myocardial infarct size and collagen deposition.Histological TUNEL staining was used to test the cell apoptosis.Agiogenesis was investigated with ?SMA and CD31 immumohistochemical staining.The expression of bFGF was tested with real-time RT-PCR and western blot analysis 30 days after myocardial infarction.Part ? Rat models of myocardial infarction were induced by coronary artery ligation.The rats in PBS,Gel,ADMSC and Gel+ADMSC group underwent thoracotomy operations with LAD ligation and,respectively,received intramyocardial injections of 100 ?L PBS without ADMSCs(PBS group),100 ?L hydrogel without ADMSCs(Gel group),100 ?L PBS containing ADMSCs(PBS+ADMSCs group),or 100 ?L hydrogel containing ADMSCs(hydrogel+ADMSCs group).Each group had a minimum of eight animals available for testing.Ten rats in sham group underwent thoracotomy operations without LAD ligation.The first day and 30 days after the MI operations,all the mice were tested on cardiac function with by transthoracic echocardiography.HE and Masson staining were used to detect the myocardial infarct size and collagen deposition.Histological TUNEL staining was used to test the cell apoptosis.Agiogenesis was investigated with ?SMA and CD31 immumohistochemical staining.The expression of TGF-?1,Bax,Bcl-2,MMP-2,MMP-9,TIMP-1,TIMP-2,collagen I,collagen ? were tested with real-time RT-PCR and western blot analysis 30 days after myocardial infarction.Part ?50 male SD rats were included in this part.Rat models of myocardial infarction were induced by coronary artery ligation.All the surgeried mice were radomally divided into four groups with an equal volume of 100?l PBS without bFGF(PBS group),100?l hydrogel containing 2?g bFGF(Gel+bFGF group),100?l hydrogel containing ADMSCs(hydrogel+ADMSCs group)or 100?l hydrogel containing 2?g bFGF and ADMSCs(bFGF+ADMSCs group)respectively intramyocardially injected into the peri-infarction areas.The first day and 30 days after the MI operations,all the mice were tested on cardiac function with transthoracic echocardiography examination.HE and Masson staining were used to detect the myocardial infarct size and collagen deposition.Histological TUNEL staining was used to test the cell apoptosis.Agiogenesis was investigated with ?SMA and CD31 immumohistochemical staining.The expression of p-Smad2,p-Smad3,p-Smad7 and P-38 signaling pathways were tested with western blot analysis 30 days after myocardial infarction.Results Part ? On the 30 th day after myocardial infarction,echocardiography showed that the left ventricular end systolic diameter(LVESD)and left ventricular end diastolic diameter(LVEDD)in Gel or bFGF groups were significantly lower than those in PBS group,but the left ventricular ejection fraction(LVEF)was significantly decreased(P<0.05).The improvement of cardiac function in Gel+bFGF group was better than that in Gel or bFGF group(P<0.05).The myocardial injury fraction,myocardial infarction area,collagen deposition,myocardial apoptosis index and fibrosis degree in Gel+bFGF group were significantly lower than those in Gel or bFGF group(P<0.05),accompanied with stronger arterial and capillary formation in Gel+bFGF group(P<0.05).Compared with PBS or Gel group,the expression of bFGF gene and protein increased significantly in bFGF group and Gel+bFGF group on the 30 th day after myocardial infarction(P<0.05),and increased more significantly in Gel+bFGF group than that in bFGF group(P<0.05).The expression of m RNA of Bcl-2,Bcl-2/Bax,TIMP-1 and TIMP-2 in Gel+bFGF group was significantly higher than that in Gel or bFGF group(P<0.05),but the expression levels of m RNA of TGF-?1,Bax,MMP-2,MMP-9,Collagen ? and Collagen ? were significantly lower than those in Gel or bFGF group(P<0.05).Part ? On the 30 th day after myocardial infarction,echocardiography showed that compared with PBS group,LVEDD and LVESD in Gel group and AD-MSC group decreased significantly and LVEF increased significantly(P<0.05).However,the improvement of cardiac function in Gel+AD-MSC group was better than that in Gel or AD-MSC group(P<0.05).In addition,myocardial injury fraction,myocardial infarction area,collagen deposition,myocardial apoptosis index and fibrosis in Gel and AD-MSC groups were significantly better than those in PBS group(P<0.05),and the combination of hydrogel and AD-MSC further enhanced the cardioprotective effect(P<0.05).The density of artery and capillaries in AD-MSC group was significantly higher than that in Gel and PBS group,and further increased in Gel AD-MSC group(P<0.05).The expression of m RNA of Bcl-2,Bcl-2/Bax,TIMP-1 and TIMP-2 in Gel+AD-MSC group was significantly higher than that in Gel or AD-MSC group(P<0.05),but TGF-?1,Bax,MMP-2,MMP-9,Collagen ? and Collagen ? were significantly lower than those in Gel or AD-MSC group(P<0.05).Part ? On the 30 th day after myocardial infarction,echocardiography showed that compared with PBS group,LVEDD and LVESD decreased and LVEF increased significantly in both bFGF and AD-MSC groups,but the improvement of cardiac function in bFGF+AD-MSC group was significantly better than that in bFGF group or AD-MSC group(P<0.05).In addition,myocardial injury fraction,myocardial infarction area,collagen deposition,myocardial apoptosis index and fibrosis in bFGF and AD-MSC groups were significantly better than those in PBS group(P<0.05).The combined treatment of bFGF and AD-MSC further enhanced the cardioprotective effect(P<0.05),and the density of arteries and capillaries was also significantly stronger than those in bFGF or AD-MSC group(P<0.05).The expression levels of m RNA and protein of Bcl-2,Bcl-2/Bax,TIMP-1 and TIMP-2 in bFGF+AD-MSC group were significantly higher than those in bFGF or AD-MSC group(P<0.05),but the TGF-?1,Bax,MMP-2,MMP-9,Collagen I and Collagen III were significantly lower than those in bFGF or AD-MSC group(P<0.05).The expression level of TGF-?1/Smad7 signal pathway protein in bFGF+AD-MSC group was significantly higher than that in bFGF or AD-MSC group,but the expression level of TGF-?1/Smad2 signal pathway protein was significantly lower than that of bFGF or AD-MSC group(P<0.05).There was no difference in TGF-?1/Smad3 and P38/MAPK signaling protein among those groups(P<0.05).Conclusion In addition to the cardioprotective effect of Dex-PCL-HEMA/PNIPAAm hydrogel itself,the combined injection of bFGF and AD-MSC into the periphery of myocardial infarction in rats produced stronger cardioprotective effect than the injection of bFGF or AD-MSC alone,significantly improved left ventricular remodeling,increased angiogenesis and increased expression of bFGF gene and protein in rats with myocardial infarction.It was proved that the hydrogel as a carrier provided an effective sustained release system for bFGF,and maintained the activity and slow sustained release of bFGF.At the same time,the multi-porous network structure of the hydrogel provides a good microenvironment for the embedded AD-MSC,reduces the necrosis and apoptosis of cardiac myocytes and AD-MSC after transplantation,and achieves the role of cardioprotection.The results showed that the protective effect of the hydrogel carrying bFGF combined with embedding AD-MSC on the heart was significantly stronger than that of carrying bFGF or embedding AD-MSC alone.The mechanism may be related to activating TGF-?1/Smad7 and inhibiting TGF-?1/Smad2 signaling pathway,but not TGF-?1/Smad3 and P38/MAPK signaling pathway.