Cytokine Release Microspheres Combined With Bone Marrow Mesenchymal Stem Cell Transplantation For The Treatment Of Ischemic Heart Disease Clinical Evaluation And Mri In The Tracer Experiment Study

Part OneThe study of the sustained release performance of gelatin microspheres containing vascular endothelial growth factorObjective The present study was aimed to investigate whether gelatin microspheres containing VEGF could release VEGF persistently.Methods Gelatin microspheres were prepared through glutaraldehyde crosslinking of gelatin in aqueous solution dispersed in an oil phase.The gelatin microspheres were sterilized by ⁶⁰Co radiation.The mean diameter of these microspheres was measured after swelling in phosphate buffer saline for 24h.The in vitro release test of ¹²⁵â… -VEGF from ¹²⁵â… -VEGF-impregnated gelatin microspheres was carred out on a shaker at 37℃. The VEGF-impregnated microspheres were placed in phosphate buffer solution and the buffer was changed periodically.The amount of ¹²⁵â… -VEGF was measured by a gamma counter.The release character of ¹²⁵â… -VEGF-incorporating gelatin microspheres was also assessed in vivo.Gelatin microspheres were incorporating ¹²⁵â… -labeled VEGF was subcutaneously injected into the mouse hind limb.As a control,the same amount of ¹²⁵â… -VEGF in aqueous solution was subcutaneously.The mice were killed at intervals, and the limbs with the microspheres injection sites were cut and measured on a gamma counter to evaluate the time profile of in vivo degradation of the gelatin microspheres.Results The diameter of the gelatin microspheres was 202.2±44.7μm.The remaining radioactivity of microspheres decreased with time in vitro and in vivo.In vitro, ¹²⁵â… -VEGF was released from the gelatin microspheres within 96 hours up to 48.73%. The residual radioactivity of ¹²⁵â… -VEGF at 144 hours remained 46.98%.From then on, the remaining radioactivity of ¹²⁵â… -VEGF did nearly not decrease with time.The remaining radioactivity of ¹²⁵â… -VEGF in vivo decreased with time.At 96 hours,144 hours,and 288 hours after gelatin microspheres transplantation,the residual radioactivity of ¹²⁵â… -VEGF remained 51.9%,41.89,and 9.98%,respectively.Conclusions Gelatin microspheres containing VEGF could release VEGF persistently. Part TwoGelatin microspheres containing vascular endothelial growth factor benefits to therapy of myocardial infarctionObjective The present study was aimed to investigate whether gelatin microspheres contained VEGF could benefit to myocardial infarction.Methods Gelatin microspheres were prepared through glutaraldehyde crosslinking of gelatin in aqueous solution dispersed in an oil phase.The gelatin microspheres were lable by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarboc yanine perchlorate(DiI).The release character of VEGF-incorporating gelatin microspheres was assessed in vitro and in vivo. Eighteen Chinese mini swines were subjected to open-chest experimental myocardial infarction and were randomized into three groups.Each group received intramyocardial injection of phosphate buffered saline(control group),vacant gelatin microspheres(MS group),and gelatin microspheres incorporating VEGF(VEGF-MS group),respectively. Twenty-four hours and three weeks later,left ventricular function was assessed by MRI. After final MRI examination,animals were humanely killed and the hearts were removed for histologic study.Results All the gelatin microspheres were labled by Dil and showed red fluorescence. Twenty-four hours and three weeks after transplantation,there was no statistically significant difference in infarct size,left ventricular ejection fraction,end-diastolic volume,end-systolic volume,and anterior left ventricular wall thickening among three groups.At 3 weeks,severe fibrosis was observed in gelatin microspheres injection sites of the control group and the MS group.In contrast,there was less fibrosis in microspheres injection sites with more surviving myocardium in the VEGF-MS group. Histologically,the micro vascular density of VEGF-MS group was higher than that of the MS group and the control group(9.8±1.5/HPF vs 4.1±0.9 and 3.7±1.1/HPF,P<0.05).Conclusions VEGF incorporated gelatin microspheres can induce vascularization in ischemic myocardium.And sustained VEGF release by gelatin microspheres could benefit to myocardial infarction. Part ThreeGelatin microspheres containing vascular endothelial growth factor enhances the benefits of bone marrow mesenchymal stem cells transplantationObjective The present study was to investigate the efficacy of transplantation of mesenchymal stem cells(MSCs) with gelatin microspheres containing vascular endothelial growth factor in ischemic myocardium.Methods Eighteen Chinese mini swines were subjected to open-chest experimental myocardial infarction and were randomized into three groups.Each group received intramyocardial injection of phosphate buffered saline(control group),autogenetic mesenchymal stem cells(MSCs group),and MSCs with gelatin microspheres incorporating VEGF(VEGF-MSCs group) in the pefi-infarction area of left ventricular wall,respectively.Three weeks later,left ventricular function was assessed by means of magnetic resonance imaging(MRI).The contrast of the MSCs hypointense lesion was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region.Results The mean diameter of the microspheres is(104±22.6)μm.At 24 hours,injection sites of MSCs were identified by MRI as large intramyocardial signal voids that persisted at 3 weeks.Between two groups,there were no significant difference in the contrast of the lesions and in the size of the lesions at 24 hours.At 3 weeks after injection,the size of the lesions diminished(P<0.0001) and the contrast of the lesion decreased(P<0.0001). Histology(at 3 weeks) revealed the iron inclusion from Prussian Blue staining matches DAPI fluorescent dyes on adjacent histological sections at×400 magnification at 3 weeks after MSCs injection,indicating partial ferumoxide particles is still contained within original MSCs.In the MSCs-VEGF microsphere group,the capillary density of the injection site was significantly more than that in MSCs group(42.2±13.9/HPF vs 29.5±15.4/HPF,P<0.0001).There were much more dense fluorescently labled MSCs per high power fields in injection sites of MSCs-VEGF microsphere group than that in injection sites of MSCs group(354±83/HPF vs 278±97/HPF,P<0.0001).Moreover,the apoptosis rate of MSCs of MSCs-VEGF microsphere group is more than that of MSCs group[(6.4±4.1)%vs(11.9±4.8)%,P<0.0001].Conclusion VEGF incorporated microspheres benefits to the survival of MSCs transplanted to ischemic myocardium.Microenvironment if one of the most important factors that influence the survival of transplanted MSCs Part Four Dynamic MRI of ferumoxide-labeled bone mesenchymal stem cells after transplantation in myocardiumObjective To investigate the potential ability of magnetic resonance imaging(MRI) in tracking magnetically labeled mesenchymal stem cells(MR-MSCs) in a swine myocardial infarction(MI) model.Methods Adult Chinese mini pigs(n=6) were subjected to open-chest experimental MI. Their autogeneic bone marrow-derived mesenchymal stem cells(MSCs) was cultured and doubly labled with ferumoxides and DAPI.At the 14^th day after MI,labeled MSCs were injected intramyocardially into peri-infarct zone and normal myocardium.The contrast and the volume of the MR-MSCs hypointense lesion from the FGRE images, acquired at 24 and 3 weeks after injection,was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region.After final MRI examination,animals were humanely killed and the hearts were removed for histologic study.The heart was excised and histology corresponding to MRI slices that demonstrated MR-MSCs lesions was performed. Repeated-measures ANOVA and a paired t test were used for comparison of the contrast and the volume of the MR-MSCs hypointense lesion at different time points. Comparisons between independent groups were performed with the standard Student t test.Results Before tranlplantation,Prussian Blue staining of ferumoxides-labeled MSCs revealed 100%of MSCs were labled by ferumoxides particles.And all the MSCs were also labeled by DAPI.At the 14^th day after MSCs transplantation,DE-MRI showed the infarct in all animals.The contrast of the MR-MSCs hypointense lesion from the FGRE images,acquired at 24 hours and 3 weeks after injection,was determined using the difference in signal intensity between the hypointense and normal myocardium divided by signal intensity of the normal region.Images of MR-MSCs injection sites at 24 hours after injection appeared as ovoid hypoenhancing lesions with sharp borders.At 24h after injection,the contrast[(67±5.48)%vs(61.92±7.76)%t=1.65 P=0.1158) and the size [(0.56±0.24)cm² vs(0.52±0.25)cm²,t=0.39,P=0.7044)]of the lesions showed no statistical difference between in peri-infarct zone and in normal myocardium.At 3 weeks after injection,the contrast of the lesions decreased and the size of the lesions diminished both in peri-infarct zone and in normal myocardium.Moreover,the contrast of the lesions in peri-infarct zone decreased rapidly than that in normal myocardium (26.88±7.27 vs 15±4.51,F=20.08,P=0.0003).Post mortem analysis show fluorescently labeled MSCs was demonstrated on histological sections and There were much more dense fluorescently labled MSCs per high power fields in injection sites of normal myocardium than that in injection sites of peri-infarct zone(106±25/HPF vs 143±31/HPF,t=-2.47,P=0.0293).At 3 weeks,severe fibrosis observed in peri-infarct zone in control sites.In MSCs injection sites of the peri-infarct zone,the capillary density was significantly more than that in control sites(13.4±4.0/HPF vs 9.4±3.1/HPF, t=2.49,P=0.0229).When cryopreserved sections with DAPI positive nuclears were stained by hematoxylin-eosine and Prussian Blue,blue ferumoxides particles were detected around partial nuclears of MSCs.Conclusion Magnetic resonance imaging of MR-MSCs represents a method for noninvasively tracking the quantity and location of intramyocardial delivery in myocardium.The microenvironment is one of the most important facors for transplanted MSCs to survive. Part FiveThe study of mechanism of clearing ferumoxide particles by ferumoxide-labled mesenchymal stem cellsObjective The study tried to illustrate how the division and proliferation of mesenchymal stem cells(MSCs) labeled by superparamagnetic iron oxide(SPIO) influence the SPIO labeling rate.Methods Swine bone marrow-derived mesenchymal stem cells were labeled with SPIO by incubation with ferumoxides injectable solution(25μg Fe/mL,Feridex) in culture medium for 48 hours with 375 ng/mL poly-L-lysine(PLL;average MW=275 kDa). Prussian blue staining revealed that all of the cells were efficiently labeled with SPIO nanoparticles.Then,the MSCs was divided into two groups.One group was cultured in high glucose Dulbecco's modified Eagle medium(DMEM) plus 15%fetal calf serum (FCS) at 37℃and 5%CO₂,and the other group was maintained with low glucose DMEM plus 5%FCS.The labeling efficiency was tested through Prussian blue staining at 24 hours after every passage until the forth passage after SPIO labeling.Results Prussian Blue staining revealed the presence of numerous blue ferumoxide particles in the cytoplasm around cell nucleus of all the MSCs after incubation with Feridex and PLL for 48 hours.The SPIO labeling rate of MSCs decreased with time in both groups.The MSCs maintained in high glucose DMEM plus 15%FCS reached 80% confluent at 72 hours after the former passage.At 24 hours after passage,the SPIO labeling rate was(98±4.39)%.MSCs maintained in high glucose DMEM experienced a passage every 72 hours.At 24 hours after each passage,the SPIO labeling rate decreased to(88.67±3.93)%,(44.5±7.74)%,and(6.17±2.64)%,respectively.However,the MSCs maintained in low glucose DMEM plus 5%FCS reached 80%confluent at 96 hours after the former passage.At 24 hours after the former passage,the SPIO labeling rate was (97±0.86)%.Then,MSCs maintained in low glucose DMEM experienced a passage every 120h or 144h.At 24 hours after each passage,the SPIO labeling rate decreased to (87±4.29)%,(46.7±7.06)%.At 24 hours after the forth passage after SPIO labeling,the SPIO labeling rate was(5.17±3.06)%,which was not statistically different from the labeling rate at the 24h after the forth passage after SPIO labeling in the MSCs maintained in high glucose DMEM[(5.17±3.06)%比(6.17±2.64)%,P=0.558].Conclusion Division and proliferation are the main mechanism of clearing SPIO by MSCs.The faster the MSCs divided and proliferated,the faster the SPIO was cleared.