| Ischemic heart disease has a high morbidity and mortality.Clinical treatment for myocardial infarction,especially end-stage ischemic heart disease with ventricular aneurysm and heart failure were drug therapy,percutaneous coronary intervention and coronary artery bypass graft,all of which cannot recover myocardial tissue.The only assured method,heart transplant,is very expensive and suffered with few donors.In recent decades,research in cardiac regenerative medicine shows that stem cells transplantation therapy can improve heart function,lead to myocyte regenerate.But effect of stem cells was restricted because of the very low retention and survival rate of cells in the process of transplant.As a result,a variety of methods such as magnetic function,genetic modification,chemical management and biological materials have been tested to improve cell retention and survival in local cardiac tissue[1].Biological materials technology has made great breakthroughs in recent years,that laid a solid foundation for the development of cardiac regenerative medicine.Previous biological materials to cure heart disease are made into epicardial patches.So it is difficult for stem cells to communicate with host cardiac tissue.Other materials formed as injectable hydrohydrogels were made of fibrin or matrigel,both were difficult to manufacture.Collagen,the predominant protein in mammalian extracellular matrix(ECM),provides structural support for maintaining tissue integrity and contributes to the specificity of ECM microenvironments.Several optimal properties,such as being biocompatible,adhesive,porous and readily combined with other materials,have made collagen appropriate for use as a natural scaffold in tissue engineering applications.Injectable collagen gel extracted and purified from bovine tissue,has been successfully applied for nerve injury in mice,but there is no cardiovascular system applications.We hypothesized that combining human umbilical cord mesenchymal stem cells(h UMSCs)with collagen hydrogel produces greater infarct size reduction compared with cell administered alone after myocardial infarction(MI).Chinese Bama mini pigs underwent ligation of the left anterior descending coronary artery to make myocardial infarction.Intramyocardial combination gel/hUMSCs(1.5 milliliter/100 million cells,n=20),hUMSCs alone(100 million cells,n=20),or placebo(phosphate-buffered saline;n=20)was injected into the infarct border zones at 60 days after MI.Cardiac function and scar size were assessed with ultrasonic cardiogram and cardia magnetic resonance images.Scar tissue were collected at different time and managed with histology method.MethodsCell Isolation and CulturingTo obtain hUMSCs,fresh umbilical cord was harvested from obstetrics department of Nanjing Drum Tower Hospital.Blood vessels were excluded and the umbilical cord were cultured in the human mesenchymal stem cell medium.Cells were amplifed,harvested,and cryopreserved.The ability of differentiation into bones,cartilage and adipose were tested in vitro.In vitro co-culture of hUMSCs and collagen hydrogelsThe hUMSCs and collagen hydrogels were co-cultured in vitro and absorbancy of 492 nm wave length light were tested with MTT technology in 0,1,3,5 and 7 days.Myocardial InfarctionAll animal protocols were reviewed and approved by the Nanjing University Drum Tower Hospital Institutional Animal Use and Care Committee.Chinese Bama mini pig(17-22kg)were adopted.Anesthesia was induced with droperidol and maintained with propofol.A left minithoracotomy was created with a small 4-to 5-cm incision in the fifth anterior/lateral intercostal space.The pericardium was opened.The left anterior descending coronary artery were ligated with 5-0 sutures.Lung was expanded with manual resuscitator bag.All incisions were closed.Animals were recovered and provided adequate postoperative analgesia with a transdermal fentanyl patch(75 μg/h)for 3 days.Intramyocardial InjectionAt 60 days after MI,animals underwent stem cell(collagen hydrogels/hUMSCs,n=20;hUMSCs,n=20)or placebo(n=20)injection.A right minithoracotomy was created with a small 4-to 5-cm incision in the fifth anterior/lateral intercostal space.The pericardium was opened,and the infarct area was identified by wall motion abnormalities,tissue color and correlation with coronary anatomy.A 5ml syringe was used and 10 separate injections were administered to the infarct and border zone.Lung was expanded with manual resuscitator bag.All incisions were closed.Animals were recovered and provided adequate postoperative analgesia with a transdermal fentanyl patch(75 μg/h)for 3 days.On the morning of stem cell injection,cells were thawed,washed,and resuspended in phosphate-buffered saline(PBS).For hUMSC alone injections,100 million hUMSCs were suspended in 3 mL PBS;For the combination collagen hydrogels/hUMSCs injections,1.5 milliliter collagen hydrogels and 100 million hUMSCs were suspended in 1.5 mL PBS and mixed before injection.Animals were euthanized at 1,3,6 and 12 months after MI.CMR and UCG StudiesCMR studies were conducted on a Siemens Trio 1.5-T Tim(Erlangen,Germany)scanner with Syngo magnetic resonance software using a 16-channel body surface coil with ECG gating and short breath-hold acquisitions.All animals underwent CMR at baseline,2 months after MI(preinjection),3 months after MI,6 months after MI and 12 months after MI.Steady-state free-precession cine images in 2-chamber short-axis planes(slice thickness,8 mm;field of view,280-300 mm;matrix,192×100;repetition time,40 milliseconds;echo time,1-2 milliseconds;number of averages,2;bandwidth,965 kHz;flip angle,60°)were obtained.At end diastole and end systole,semi-automated epicardial and endocardial borders were drawn in contiguous short-axis cine images covering the apex to mitral valve plane with QMass Software(Medis,Leiden,Netherlands)to calculate LV mass,end-diastolic volume(EDV),end-systolic volume,stroke volume,and EF.Short-axis and 2-chamber long-axis delayed-enhancement images(slice thickness,8 mm with no gap;feld of view,280-300 mm;matrix,256×100;repetition time/echo time,500 milliseconds;bandwidth,250 kHz;and flip angle,20°)were acquired 8 minutes after intravenous infusion of gadolinium 0.3 mmol/kg.Scar size was calculated from the short-axis delayed-enhancement images covering the apex to the mitral valve plane.Using QMass software,we drew epicardial and endocardial contours of the LV with a semi-automated tool.The signal intensity of normal and scarred myocardium was calculated by the software,and infarct size was quantified by full-width at half-maximum intensity.HistologyHearts were fxed for>24 hours in 10%buffered formalin and sliced transversely into 4-mm-thick slices with a commercial meat cutter.Slices 4 through 8 were embedded in paraffn(formalin fxed)and processed for confocal analysis.Engraftment of human stem cells in swine hearts was assessed with qPCR for the human Alu repetitive DNA elements.Immunofluorescence costaining was performed with sca-1 antibody.Representative samples were selected from the infarct zone,border zones containing infarct and nonscarred tissue,and remote zone obtained from the posterior noninfarcted LV wall for engraftment quantification.The total number of positively stained cells was quantified per slide to calculate the number of cells per unit volume(cm3)from each sample.Morphometric analyses were performed with a custom research package(Image J;National Institutes of Health,Bethesda,MD).Microscopic evaluations and image acquisitions were performed with a Zeiss LSM-710 confocal microscope(Carl Zeiss MicroImaging,Thomwood,NY).ResultsAlthough cell therapy group had reduced MI size relative to placebo(P<0.05),the MI size reduction was greater in combination versus cell therapy alone(P<0.05).Accompanying enhanced MI size reduction were ejection fraction improvement in cell-treated pigs,whereas placebo pigs had persistently maintained left ventricular function and combined pigs have most improvement in left ventricular function(P<0.05).Immunohistochemistry showed that treatment with collagen hydrogels combined with hUMSCs pursuit the angiogenesis and cardiac myocyte regeneration(P<0.05). |
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