Experimental Research On The Cell Biology And Constructing Tissue-engineered Fat Tissue Of Dedifferentiated Adipocyte

Background:Free fat tissue transplantation has a broad application for clinical use,but it is limited by the shortcomings such as high absorption ratio after adipose tissue transplantation,as well as resulting in secondary deformities on the donor site. However,the development of adipose tissue engineering technology,pointing out the prospect for free fat tissue transplantation.Previous studies have identified a putative stem cell population within adipose stromal compartment.This cell population,termed adipose-derived stem cells(ASCs),can be isolated from adipose tissue.ASCs can differentiate toward the multiple lineages like adipocyte,chondrocyte, and osteoblast.But ASCs is a mixed population residing within adult adipose tissue. Only about 40%of them can differentiate into adipocytes.So it can not satisfy the development of adipose tissue engineering,neither.It is still a hot spot of finding a better kind of cell which is conveniently isolated,highly expandable in culture and highly differentiatable to adipocytes.Adipose tissue is heterogeneous.It contains mature adipocytes,vascular endothelial cells,fibroblasts,histiocytes and stem cells et al.Large lipid accumulations make mature adipocytes naturally buoyant and therefore difficult to culture.Little research has been conducted on the nature of them.They have also been considered to be in the terminal stage of differentiation and lacking proliferative activity.Studies,concluded by foreign researchers,suggested that mature adipocytes could be induced to undergo lipolysis,reinitiate the proliferative mechanisms and begin to proliferate in vitro.This physiological transition has been termed adipocyte dedifferentiation.Liao has proved that dedifferentiated adipocytes could differentiate toward the multiple lineages such as adipocyte,chondrocyte,and osteoblast,and had a stronger adipogenic capacity than ASCs.Therefore,DA has a probability to become a kind of more excellent seed cells source for fatty tissue engineering.However,the researches on how to purify the mature fat cells with a high efficiency and then obtain DA,as well as the multilineage differentiation potential of DA were very few both at home and abroad at present.There is no vivo study in which DA is used as seed cells for the construction of tissue engineered adipose tissue neither.The present study will explore these issues.OBJECTIVE:1,To find a method which can be used for isolation,abstraction and cultivation of mature adipocytes from liposuction,to induce the dedifferentiation phenomenon of human mature adipocytes cultured in vitro,so we can get dedifferentiated adipocytes(DA).2,To provide the laboratory evidence for DA to be used extensively in tissue engineering by charactering DA in the following aspects:morphology, differentiation capability.3,The DA - FG mixture was implanted in the nude mice by subcutaneous injection for exploring the feasibility and potential of DA to be the seed cells of fat tissue engineering.METHODS:Mature adipocytes and ASCs were harvested from human fat aspirates via liposuction.Mature adipocytes were cultured and dedifferentiated to DA by ceiling adherent culture method,cell morphology were observed during the whole process. The adipogenic,chondrogenic and osteogenic ability of DA were assessed by oil red O staining,alcian blue staining and alizarin bordeaux staining,respectively.The compatibility of FG scaffold and cells was detected by Light microscope and scanning electron microscopy.DiI fluorescent dye marked DA-FG mixture(Group A, n = 8,cell volume 4×10~6) and ASCs-FG mixture(Group B,n = 8,cell volume 4×10~6),as well as blank FG stent(Group C,n = 8) were injected subcutaneously in nude mice.After 8 weeks of implantation,the specimens were harvested for general observation,histologic analysis,oil red O staining analysis and fluorescent microscope analysis.RESULTS:Human mature adipocytes can dedifferentiate into fibroblast-shaped DA.After two weeks of adipogenic differentiation,lipid droplets can be displayed by oil red O staining.After two weeks of chondrogenic differentiation,matrix of cartilage cells can be detected by alcian blue staining.After three weeks of osteogenic differentiation,calcium salts mineralization can be detected by alizarin bordeaux staining.The formation of new organizations were found subcutaneously on the back of nude mice at 8 weeks in both group A and group B,the(?)±S of the wet weight of the newly formed tissue were 0.1249±0.0190(g) and 0.0971±0.0166(g) (F=12.350,P=0.01) respectively.The(?)±S of fibrosis ratios were 14.20%±2.625%and 25.89%±5.314%(F=34.678,P=0.001) respectively.No new organizations formed in group C,and the stents were completely absorbed.New organization was confirmed to be mature fat tissue and forming from the implanted seed cells by detections.CONCLUSIONS:1,Mature adipocytes could be isolated and extracted from the fatty portions of autologous liposuction aspirates on human.They can dedifferentiate into DA in vitro.2,DA showed fibroblast-like morphology,with strong proliferative activity.It also had adipogenic,chondrogenic and osteogenic ability.3,Tissue-engineered fat was formed after DA-FG mixture injection in vivo,the wet weight of the newly formed tissue was more that of ASCs-FG group with the same cell volume,and the fibrosis ratio was less than the latter.DA is expected to be a promising seed cell for adipose tissue engineering.