Autologous Transplantation Of Cryopreserved Bone Marrow–Derived Endothelial Progenitor Cells To Prevent And Treat Multiple Organs Dysfunction In Porcine

Multiple organ dysfunction syndrome (MODS) is one of the most common causes of death in clinical treatment for severe trauma cases, and it has been the most frequent cause of death in patients admitted to intensive care units (50% - 80%). In recent years, most of researchers have made a great development about MODS, but its mortality rate maintained highly. Therefore, How to prevent MODS is still a serious problem in clinical medicine. At present, the pathogenesis of MODS is not clear and there are not satisfactory therapic methods in clinic.With the development of clinical technologies of progenitor cells, the study will improve in pathogenesis reaserchs and clinical therapy of MODS. Some studies indicate that the progenitor cells have the ability to proliferate, migrate, differentiate and repair the damaged organs after trauma. The endothelial cells are not only the participant of inflammatory response, but also the first damaged target cells. The damage of capillary and dysfunction of microcirculation may be the earliest part of MODS. The massive animal and clinical experiments have already confirmed that when tissues were damaged or especially ischemia, the proliferation, migration, differentiation of EPCs were reinforced into peripheral blood and turned into endothelial cells and replaced the damaged endothelial cells in tissue. The denuded damaged vessel is repaired by new endothelial cells. EPCs can migrate to ischemia or damaged tissue and improve the function of ischemia organ. If the proliferation, migration, and differentiation of EPCs are seriously disturbanced after trauma, they would make the microcirculation unrepaired and MODS come bad to worse.Recent studies show that EPCs can play an important role in the ischemia disease of heart and brain, angiogenesis of tumor, and concrescence of trauma. Furthermore, EPCs can treat ischemic disease by transplanting. But quantity of EPCs is finite no matter what EPCs are coming from marrow, blood of hilum, liver of embryo and peripheral blood. It can not fulfil the need to clinical application. This is the most reason to restrict to further apply. Furthermore, the number and function of circulating EPCs will be reduced in the event of infection, shock and organ dysfunction. These patients may need more EPCs to achieve the therapeutic effect. Cryopreservation can conserve for long time to obtain a large number of EPCs. It provides a better method in clinical application. In this study, the isolated mononuclear cells were amplified to the fourth generation of EPCs, and the EPCs were frozen, and then the cells were transplanted into animals after cell recovery. The proliferation, migration, adhesion and angiogenesis of EPCs were determined in vitro after cryopreservation, and the distribution of cryopreserved EPCs was observed in vivo after transplantation, then the prevention and treatment of MODS were evaluated. There were three parts in our study. In the first part, our study was to replicate a porcine model of MODS. In the second part, we used the standard method of isolation, culture, cryopreservation and identification of EPCs in vitro and compared the function between fresh and cryo-EPCs. In the last part, the fresh and cryo-EPCs were transplanted into animal model and the distribution of these cells was observed in pigs in vivo, and then we investigated the mobidity and mortality of MODS animals.Part 1 Establishment a porcine model of MODSObjective: Our study was to replicate a porcine model of MODS which was characterized by the development of delayed two-phase process, and it was the foundation of our investigation of autologous transplantation of cryopreserved EPCs to prevent and treat MODS.Methods: Twenty healthy male minipigs weighing 25.22±3.17 Kg were randomly divided into two groups. One group was subjected to hemorrhagic shock plus endotoxiemia (group M, n=10). Another group was normal control only with anesthesia and sham operation (group C, n=10). Blood specimens were collected every 24 hours for the detection of serum WBC, GRAN, SALT, SAST, Cr, BUN and arterial blood gas analysis during the seven-day observation,which were used to evaluate MODS of animal by compared with the initial value of itself.Results: The numbers of WBC, GRAN, SALT, SAST, Cr, and BUN were significantly increased in the experimental group (M group), the death rates were significantly higher than control group, but PaO₂ was significantly decreased. The pathological changes were mainly manifested by inflammation, organ failure, and non-specific changes.The incidence of MODS was significantly higher in the experimental group (80%) than the control group (0) (P < 0.017), and the mortality of animal was significantly higher in the experimental group (70%) than the control group (10%). (P < 0.017)Conclusion: The two-hit model of MODS is a successful animal model which conforms to clinical course and has high mobidity and mortality. And the model is easy to duplicate and stable.Part 2 Comparison of characteristics and function of cryopreserved and fresh endothelial progenitor cells from porcine bone marrowObjective: The purpose of this part was to detect characteristic and function of endothelial progenitor cells of cryopreservation from porcine bone marrow; it would be the foundation to treat MODS for transplantation.Methods: The isolated BMMCs were cultured with specific culture solution for EPCs. After 17 days of culture, the P4-EPCs were identificated by taking up Dil-ac-LDL and FITC-UEA-1, flow cytometry, immunohistochemistry, ultramicrostructural organization, and function of angiogenesis testing. And the P4-EPCs were frozen, these cells were determined by same methods of identification, and then the function of fresh and cryopreserved cells were compared.Results: The attaching cells were emerged after 48h of culture, and the cells would be clustering after 5 days of culture. The Weibel-Palade body was appeared in fresh and cryo-EPCs. And more than 88% EPCs could take up Dil-Ac-LDL and FITC-UEA-1. CD133 (+), CD34 (++), CD31 (+++), KDR (+++) and CD133 (+), CD34 (++), CD31 (+++), KDR (+++) were determined by immunohistochemistry testing and the positivity of CD133 was 17.24±2.87%, 3.93±0.74%; the positivity of CD34 was 37.21±9.32 %, 35.71±7.35 %; the positivity of CD31 was 72.07±14.14%, 72.23±16.69%; the positivity of KDR was 89.09±15.06%, 87.28±17.34% by flow cytometry testing in the fresh and cryo-EPCs. There were no significant difference in the angiogenesis (25.42±4.38 / HP versus 23.79±3.54 / HP, p > 0.05), adherence rate (42.7±2.1% versus 39.5±1.7%, p > 0.05), migration rate (15±0.71% versus 14.2±0.63%, p > 0.05), proliferation (25.06±2.82×104 versus 21.64±2.34×104, p > 0.05), and content of NO (83.21±3.54umol/L versus 81.59±3.17 umol/L, p > 0.05) between fresh and cryo-EPCs.Conclusion: There were no significant differences between the fresh and cryopreserved EPCs in cell morphology, characteristics and functions. Therefore, this method can obtain sufficient homogeneous EPCs before treatment, it ensure the therapeutic effects of EPCs transplantation.Part 3 Autologous transplantation of cryopreserved endothelial progenitor cells to prevent and treat MODS Objective: The aim of last section was to investigate the effect of transplantation fresh and cryopreserved EPCs from porcine bone marrow for post-traumatic multiple organ dysfunction treatment, and determined the best dose and time of EPCs transplantation. Methods: The experimental animals were randomly divided into three sections and every section was randomly divided into three groups. The cell dose of transplantation (n = 21): 1×10⁵ / Kg (group A), 1×10⁶ / Kg (group B), 1×10⁷ / Kg (group C). The time of cell transplantation (n = 21): T2 (group A), T3 (group B), T4 (group C). Cryopreserved cell transplantation (n = 21): PBS transplantation (group A), fresh EPCs transplantation (group B), cryopreserved EPCs transplantation (group C). The incidence of MODS and survival rate of animals were compared in different experimental groups after EPCs transplantation.Results: The cell dose: the incidence of MODS was 71.43%, 42.86%, 42.86% (P < 0.017), and the survival rate was 28.71%, 57.14%, 42.86% (P < 0.017) in group A, B, C. The time of transplantation: the incidence of MODS was 57.14%, 42.86%, 71.43% (P < 0.017), and the survival rate was 42.86%, 57.14%, 14.29% (P < 0.017) in group A, B, C. Cry- EPCs transplantation: the incidence of MODS was 85.71%, 42.86%, 42.86% (P < 0.017), the survival rate was 14.29%, 57.14%, 57.14% (P < 0.017) in group A, B, C.Conclusion: Autologous transplantation of fresh and cryopreserved endothelial progenitor cells can improve the function of organs with post-traumatic ischemia and hypoxia. Therefore, it can decrease the mobidity and mortality of MODS and prevent the development of the MODS. The optimal cell dose of transplantation is 1×10⁶ / Kg, the best time is T3, and there were no significant differences between fresh and cryo-EPCs in the efficacy and security of transplantation.Summary: The reduction and dysfunction of EPCs may be the key to the development of MODS, and autologous transplantation of endothelial progenitor cells in vivo can migrate to different organizations and repaire the lesions. EPCs could be frozen after amplification in vivo, and there were no significant differences between fresh and cryo-EPCs in cell morphology, characteristics and functions. Therefore, autologous transplantation of cryo-EPCs can improve the function of organs with post-traumatic ischemia and hypoxia and decrease the mobidity and mortality of MODS.