| Background and Aims:Ischemic heart disease(IHD)is ‘the leading killer’ of human health,and 7.25 million people die each year from IHD worldwide,accounting for 12.8% of the total deaths.Although treatment of IHD has made great progress with the innovation of vascular recanalization technology and devices and the emergence of new drugs,the incidence of heart failure from IHD is still as high as 40% and the mortality rate is as high as 6%.After acute myocardial infarction(AMI),a large number of myocardial cells were lost due to ischemia and hypoxia and gradually replaced by scar tissue without systolic and diastolic function,which led to IHD eventually progressing to irreversible heart failure(HF).Stem cell therapy has been developed into one of the most prospective strategies for the treatment of IHD.Accumulating preclinical studies have shown that mesenchymal stem cells(MSCs)have strong paracrine cardiac repair potential,and have become ideal candidate for clinical stem cells because of their safety and effectiveness.However,the low engraftment and the poor survival of transplanted MSCs in the myocardial ischemia microenvironment limit their therapeutic efficiency and become a bottleneck for further clinical transformation of MSCs-based therapy.Transglutaminase cross-linked Gelatin(Col-Tgel)is a novel enzyme-crosslinked hydrogel,which has a hydrophilic three-dimensional network structure formed by the cross-linking of linear gelatin molecular chains under the action of Transglutaminase(TG).Col-Tgel is easy for cell adhesion and nutrients penetration and has good biocompatibility and tailorable biomechanical properties.Col-Tgel is becoming an excellent biomaterial scaffold for cellular delivery in vivo.Here,we tested the hypothesis that Col-Tgel increases retention of intramyocardially-injected MSCs,and thereby reduces post-MI cardiac injury.Methods:1.Preparation of Col-Tgel and detection of its morphological characteristicsThe gelatin solution of type A 300 Bloom was prepared as component A of Col-Tgel,and the purified TG crosslinker solution was prepared as component B of Col-Tgel.Component A and component B were mixed at 20:1 ratio to generate Col-Tgel hydrogel.Morphology and microstructure of Col-Tgel were observed by a scanning electron microscope.2.Isolation,culture and identification of adipose tissue-derived stem cells(ADSCs)Rat inguinal subcutaneous adipose tissue was excised,and ADSCs were isolated by enzymatic digestion.ADSCs were cultured with DMEM/F12-10% FBS.Cell morphology was determined by light microscope with phase contrast,and surface characteristic markers of ADSCs were identified by flow cytometry.(CD105、CD31、CD45 和 CD90.2)3.Establishment and morphological detection of Col-Tgel/ADSCs in vitro 3D cultureOnce ADSCs were mixed with Col-Tgel component A,component B was added to form Col-Tgel/ADSCs suspension.A 3D culture system was established by the hanging drop method.The morphology of ADSCs in Col-Tgel was directly observed with phase-contrast microscope.The microstructure of Col-Tgel/ADSCs was examined by electron microscope,and the spatial distribution of ADSCs in Col-Tgel was observed by confocal microscope.4.Biological behavior of ADSCs in Col-Tgel culture systemPropidium iodide(PI)staining was used to detect ADSCs cell viability,MTT staining and CCK-8 assay(Cell Counting Kit-8 assays)were used to test ADSCs proliferation ability,MTT staining and Transwell assay were used for ADSCs migration ability testing.5.The establishment of mouse acute myocardial infarction model and intramyocardial injectionC57BL/6j mice were divided into following groups: 1)SHAM,anterior descending coronary artery was passed through but not ligated;2)MI + PBS,ligating the anterior descending coronary artery of the mouse to establish an AMI model,and injecting PBS into the myocardial ischemia area;3)MI + PBS-ADSCs,establishing an AMI model,and injecting PBS-ADSCs suspension into myocardial ischemic area;4)MI + Tgel,establishing an AMI model,and injecting Col-Tgel into myocardial ischemic area;5)MI +Tgel-ADSCs,establishing an AMI model,injecting Col-Tgel/ADSCs into the ischemic area;6.Detection of dynamic retention of ADSCs implanted in the myocardiumImmunofluorescent staining of heart tissue sections was used to dynamically track CM-Di I-labeled transplanted ADSCs at the 3rd,7th,14 th,28th,and 42 nd day after surgery.7.Assessment of survival rate,cardiac function,myocardial fibrosis and ventricular remodeling in mice post MIThe survival and death of the mice were recorded within 4 weeks after the operation,and the postoperative survival analysis was performed.The cardiac function of the mice was measured at the 1st,14 th,and 28 th day using the high-resolution small animal ultrasound system.The degree of myocardial fibrosis in mice at 4 weeks after operation was evaluated through the Masson tricolor method.Polymerase chain reaction(PCR)was used to detect the ventricular remodeling gene expression levels of the atrial natriuretic peptide(BNP)and brain natriuretic peptide(BNP).Results:1.Col-Tgel was a three-dimensional network formed by glutamine residues and lysine residues on the gelatin molecular chain under the acyl transfer reaction of transglutaminase(TG).Scanning electron microscope showed that Col-Tgel has a highly connected pore structure and a well-developed network structure.Col-Tgel mechanical strength was about 15.42±3.11 k Pa,and the porosity is about 47%.2.Under light microscope,rat ADSCs grew adherently,and were spindle-shaped,and the cytoplasm was transparent and clear.Under fluorescent microscope,CM-Di I-labeled ADSCs emitted red fluorescence.Flow cytometry showed that the surface maker CD105 and CD90.2 were positive,and the expression of CD31 and CD45 were negative,indicating that ADSCs have characteristic stem cell markers.3.Col-Tgel/ADSCs in vitro 3D culture system was hemispherical.After 3 days culture,under scanning electron microscope,ADSCs colonized and survived in the three-dimensional structure of Col-Tgel,which was spherical or spindle-shaped.As showed in the confocal microscope 3D synthesized photos,red fluorescent ADSCs filled almost all areas inside Col-Tgel.4.PI staining showed that Col-Tgel was non-cytotoxic and had no effect on the viability of ADSCs,after Col-Tgel and ADSCs co-culturing for 3 days.After continuous 28 days culture,dynamic MTT staining and CCK-8 experiments showed that during the initial 7days,there was no statistical difference in the proliferation rate of ADSCs in the 2D and3 D culture systems.However,as the incubation time prolonged to day 14 and 28,the proliferation rate of ADSCs in the 3D system was significantly higher than that in the 2D system.In terms of detecting the migration speed of ADSCs,MTT staining showed that from the third day after inoculation,ADSCs began to migrate outward from the edge of hemispherical Col-Tgel system,and the number of ADSCs that migrated outward gradually increased and the migration distance also gradually increased with the extension of time.Compared with the 2D culture system,the number of migrated ADSCs in the 3D culture system was significantly few on the 3rd,7th and 14 th days after inoculation.The Transwell test also confirmed that compared with PBS and Mtrigel,Col-Tgel significantly slowed down the rate of migration of ADSCs into the down-chamber after 3rd,7th,14 th and 21 st day inoculation.5.After intramyocardial implantation of ADSCs,HE staining of heart tissue sections showed that the implanted Col-Tgel was a light red stripe,and CM-Di I labeled ADSCs emitted red fluorescence under a fluorescence microscope.The synthetic picture of them showed Col-Tgel co-localized with ADSCs in the ischemic area of myocardium.Interesting,it was found that Tgel-ADSCs were strip-shaped after implantation,which was more limited,and PBS-ADSCs were patch-shaped after implantation,which was more diffused.6.Immunofluorescence staining of tissue sections on the 3rd,7th,14 th,28th,and 42 nd day after ADSCs myocardial implantation showed that there was no significant statistical difference in the retention rate of ADSCs between the Tgel-ADSCs group and the PBS-ADSCs group on the 3rd day after implantation.However,the amounts of ADSCs in the Tgel-ADSCs group on day 7,14,28,and 42 were significantly more than those in the PBS-ADSCs group.ADSCs in the PBS-ADSCs group decreased rapidly on the 3rd to 7th days after implantation,and were undetectable by the 28 th day,while the ADSCs in the Tgel-ADSCs group decreased significantly less on the 3rd to 7th days,the retention of ADSCs could still be detected on the 42 nd day.7.The survival curve data post-MI showed that administration of PBS-ADSCs,Col-Tgel,and Tgel-ADSCs had a tendency to improve the survival rate of mice after surgery,but there was no statistical difference between them.Ultrasound examination of the mouse heart at 1st day,2nd week,and 4th week after surgery showed that : compared with the control group MI + PBS,the administration of PBS-ADSCs or Col-Tgel alone increased the left ventricular ejection fraction(LVEF)and decreased the left ventricular end-systolic diameter(LVESD)and the left ventricular end-diastolic diameter(LVEDD),but,there was no statistical difference between them.However,compared with the control group MI +PBS,the MI+Tgel-ADSCs group significantly increased LVEF and decreased LVESD at the 4th week after operation with statistical difference.The results of Masson-Tricolor staining indicated that compared with the MI+PBS group,the group given ADSCs or Col-Tgel alone had a tendency to reduce myocardial fibrosis,but there was no statistical difference between them;However,the MI + Tgel-ADSCs group significantly reduced the area of myocardial fibrosis with statistical difference,comparing with the MI+PBS group.Real-time quantitative PCR showed that compared with the MI + PBS group,the PBS-ADSCs group,Col-Tgel group,and Tgel-ADSCs group significantly down-regulated the m RNA level of ANP in myocardial tissue,all with statistical differences;At the same time,compared with the MI + PBS group,the administration of ADSCs or Col-Tgel alone decreased the m RNA level of BNP,but there was no statistical difference between them.However,compared with the MI + PBS group,the MI + Tgel-ADSCs transplantation group significantly reduced the m RNA level of BNP,and there was a statistical difference.Conclusion:In an in vitro 3D culture system,Col-Tgel provides sufficient proliferation space for ADSCs.At the same time,Col-Tgel limits the ability of ADSCs to migrate,which is conducive to the slow release of cells.After Col-Tgel and ADSCs were implanted into the myocardium,Col-Tgel increased the long-term retention rate of ADSCs,and enhanced ADSCs Cardioprotective effect.Therefore,the co-implantation of Col-Tgel and ADSCs is a clinically promising method to improve the efficiency of stem cell treatment of IHD. |
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