| Background and ObjectiveAdipose tissue is considered to be the first choice for repairing large volumes of soft tissue defects.However,most of the researchers have only attributed adipose tissue regeneration to adipose-derived mesenchymal stem cells(ASCs),while ignoring the role of mature adipocytes.They have been considered as terminally differentiated cells,and do not have the ability to proliferate and differentiate.In the 1980s,researchers found that mature adipocytes would lose their lipid droplets,and became into fibroblast-like cells when culturing in vitro.These cells are named as dedifferentiated adipocytes(DA),and they regain the ability to proliferate and re-differentiate.We have confirmed that the mature adipocytes in rat inguinal fat flap would undergo dedifferentiation when being expanded by a soft tissue expander.The objective in this research is to reveal the effect of DA in adipose tissue regeneration,and to find out the possible mechanism in adipocytes dedifferentiation.Methods1.A soft tissue expander was implanted beneath dossal fat flap of rabbit.And after 7 days of expansion,the morphological changes of the fat flap were observed.And the changes of tissue structure and cell proliferative activity in the adipose flap was observed by H&E slice staining,whole mount staining and immunofluorescence.RT-PCR was used to detect the expression of cytokines.2.After expansion for 7 days,an external suspension device(ESD)was put out side the skin above adipose flaps.The changes of the volume and the tissue structure were observed and the dynamic changes of cytokines in the valve were analyzed.3.The expanded inguinal fat flap in rat was minced into pieces and transplanted to the same side under dorsal skin.The retention rate of the grafts was observed at different time points,and the pattern of fat regeneration in the graft was observed.4.Culturing mature adipocytes in vitro under mechanical force.Observe the morphological changes of adipocytes.Immunofluorescence staining was used to observe the dynamic changes of cytoskeleton during cell culture.The expression of mechanical signal related proteins were detected by Western Blot.Results1.After expansion,the diameter of adipocytes decreased._There were multilocular lipid droplets adipocytes in the flap.The number of Ki67-positive cells increased.The expression of adipokines decreased significantly.The expression of hypoxia inducible factor 1A increased.2.The volume of the experimental group was almost 1 time larger than that of the control group(34.7 ± 4.6 ml in the experimental group and 17.5 ± 2.3 ml in the control group)after 10 weeks with ESD.During the regeneration,the experimental group expressed higher adipokines and had larger vascularized area.3.The pressure acted on the fat flap gradually increased with the volume of expander.After fat graft,the retention rate of the expanded flap was higher(56%in the experimental group and 32%in the control group).At the early stage after fat graft,the expression of vascular endothelial growth factor was much higher in the experimental group.4.The cells were cultured in vitro under mechanical force approximately 520 Pa.At 4 days after culture,the morphology of adipocytes became less rounded and there were cells contained multilocular lipid droplets.At 7 days,the number of cells increased significantly and intracellular lipid droplets became much small.After 14 days,all cells were fibroblast-like in morphology.Actin changed accordingly with the cell morphology,they became thicker and arranged along the long axis of the cell.The expression of mechanical signals related proteins were significantly increased.ConclusionAfter the expansion,mature adipocytes undergo dedifferentiate process and regain proliferation ability.These dedifferentiated adipocytes can be used as a kind of seed cells,participate in adipose tissue regeneration.The dedifferentiation process is possibly promoted by mechanical signals which acted on cells. |
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