Function Of Adipose-drived Stem Cells During The Repair Of SD Rats Soft Tissues Trauma

Background:Large soft tissue defects resulted from trauma,tumor resection or congenital deformities can hardly heal completely by self-renovation.The reparation methods used in clinical practice nowadays all have inevitable shortages:Tissue flaps bring donor sites deformities.Tissues substitute transplantation leads to Foreign body reaction.Among those multiple factors which influence wound healing,lack of autogenetic stem cells and cytokines at wounded sites as well as poor visualization, mainly affects the results of wound healing.Therefore,through supplement of the number of stem cells or enhancement of their functions.We may find a novel strategy to promote wound healing and cure large tissue defects.Stem cells are a sort of undifferentiated cells which have multiple differentiation potentials.In conditions of tissue injury,it could be specifically induced into mature cell types by internal environment stimulations.Through this mechanism, we may regenerate damaged tissues according to their own tissue types respectively.There have been some reports of research work on this topic, domestically and abroad,which proved that stem cells contribute to wound healing and tissue regeneration.Cell therapy based on stem cells has become a hot spot in the study of regeneration medicine recently.At present there are two types of stem cells under intensive studies:the bone marrow-derived stromal cells(BMSCs) and the adipose-derived stem cells (ADSCs).BMSCs have multi-lineage differentiation potentials undoubtedly,but the disadvantages of bone marrow puncture and aspiration restricted its applications.It has been proved that there are sorts of cells exist in fat tissue which have similar properties as BMSCs.ADSCs have multi-lineage differentiation potentials and can be induced into adipose cells,osteoblasts,cartilage cells,muscle cells and neurocytes in vitro.Compared with BMSCs,ADSCs have many advantages such as easy to harvest,minimal damage to the donor site and so on.It has become new focus in stem cell research after BMSCs.Researches have confirmed that ADSCs used in tissue engineering for reconstruction and some definite tissue damage in cell therapy obtained good effect. But studies for whether exogenous rat ADSCs contribute to wound healing in a soft-tissue injury animal model and how do they migrate and distribute in vivo conditions have no special reports.And compare the effects of two different transplantation method which would be a better method in cell therapy has no certain final conclusion.So this study seems to be more inspiring.OBJECTIVE1,Determine the culture system of rat ADSCs,induce them into adipose,osteoand neuro-lineages and characterize their stem cell features in vitro,prepare for cell transplantation in vivo.2,Study the effect of exogenous ADSCs in wound healing and soft tissue regeneration,and compare the efficacy of systematical engraft with local injection.3,Systematically inject exogenous ADSCs into a rat soft tissue injury model, then investigate the migration and distribution of the injected cells,experimentally provide the foundation of migration agent mechanism of ADSCs in vivo.METHODS:1,Cell culture and differentiationHarvest adipose tissue from inguinalis fat pad of SD rats,digest and isolate ADSCs,culture them in vitro,observe their morphology.Harvest ADSCs at passage 2 to 4 for in vitro differentiation,characterization and in vivo transplantation.Cells are cultured in three different condition medium and differentiated into adipose-, osteo-,and neuro- lineages respectively.Specific cell markers and specific stains are used to identify the differentiation of these cells,such as oil red O stain for adipose-induction,Von Kossa stain for osteo-induction and specific immunofluorescence stain for neuro-induction.2,Animal modelsAfter anesthesia,two round-shaped wounds measuring 1.8cm in diameter were made on the back of SD rats symmetrically,each measured 2cm distant from dorsal column.The wounds contain skin,subcutaneous tissue and part of the gluteus, formed about 0.5cm deep holes,with approximate volume of 1ml.According to experimental design two correlated animal experiments were carried out.In each experiment the model rats were divided into groups randomly.The first experiment had 18 rats which divided into 3 groups equally and the aims were to observe the condition of wound surface healing and histopathology change latterly.The group 1 was local injection ADSCs one and 2 was vein tail injection ADSCs one and 3 was control one.The second had 12 rats which divided into 2 groups equally and the aim was to observe the migration and distribution of ADSCs transplanted by vein toward wound.3,DiI label and in vivo transplantationADSCs of passage 2-4 were labeled with DiI in vitro and resuspended in DMEM,ready for transplantation.Groups were injected with either medium or cell suspension into vena caulis and the left wound beside spine in the same volume.The healing process were observed regularly,the ratio of the wound contraction and the time of healing were recorded.After 24 days and 48 days,specimens of injured site and several internal organs were harvested and proceeded for histological assays.RESULTS:ADSCs from rat inguinal fat pad were fibroblast-like adherent cells.They are easy to culture and proliferate fast which could satisfy the needs for the experiment.In vitro differentiation proved that they have potentials to differentiate into adipose-,osteo- and neuro-lineage cells.After 14 days of adipose-induction,Oil red O stain proved there were lipid droplets in the round cells which still showed nice proliferation.Von Kossa stain was positive after 28 days of osteo-induction,the mineral nodus could be observed under microscope,and cell proliferation at this point was relatively slow.24 hours after neuro- induction,the expression of Nestin in cell plasma could be observed under fluorescence microscope;immunohistochemical stain showed the expression of MAP-2 on the neuro-like cells,and the cell viability was sound.Under fluorescence microscope,DiI labeled rat ADSCs showed red fluorescence at cytomembrane,while the nucleus were negative.The survival rate of labeled cells was over 90%.24 days after cell transplantation,red fluorescent labeled cells could be detected in specimens of group 2.And there were stronger fluorescence around the wounded sites' dermis as well as deep layers of the subcutaneous tissue,compared with the normal skin.48 days after cell transplantation,strong fluorescence could be observed in lungs and spleens.Fewer fluorescence could also be detected in liver, heart and kidneys.Distant from the wounded sites,red fluorescence could hardly be detected in normal skin,internal organs and adipose tissues.The fluorescence at wounded place is obviously stronger than that in normal tissues,and their distribution mainly resided in dermis and gland-like structure,which looks like more regularly. Control group showed no fluorescence.Group 1 showed obvious contraction of the injured area 3 days after injection while group 2 takes 7 days.They all have more plenty of granulation tissues and less effusion than that of group 3 and they take less time to heal completely than group 3 but there is no significant difference between groupl and group 2.Histopathology indicated that immature gland-like structure were easily detected within local injection group and neovascularization were evidently found in vein tail injection group compared to control at postoperative 24 days.CONCLUSION:1.Rat ADSCs have multi-lineage differentiation properties,they are easy to isolate,easy to culture and have strong proliferation abilities,which could be used in vivo transplantation experiments.2.ADSCs obviously helped wound healing,local injection works more quickly and efficiently than systematical administration.3.Wound can probably simulate the migration and accumulation of intravenous transplanted ADSCs which participated wound repair.