3D Culture Promotes Secretion Of Extracellular Matrix Structure Fat Flap With Lipoaspirates In Vitro

Background and objectivesLarge soft-tissue defects remain challenges for plastic and reconstructive surgery.Autologous fat grafting is considered to be the preferred method of repairing soft tissue defects because of its advantages of satisfactory cosmetic outcomes,easy access,simple operation,abundant sources and high level of safety.However,the unpredictability of graft outcomes,with retention rates ranging from 20%to 90%,limits autologous fat grafting’s clinical application.Adipose tissue engineering(ATE)represents a possible solution for large-volume soft-tissue reconstruction.Although there have been several reports on the construction of tissue-engineered fat(TEF)in vivo and in vitro,each condition has various limitations.Existing ATE methods require the implantation of a scaffold in vivo.These strategies not only require complex surgical operations,but also cause donor site deformity.And foreign scaffold implantation is needed,which severely reduces patient compliance.In vitro ATE construction is small in size,complex in operation,and requires long construction time,which limits its clinical application.In this study,we developed an in vitro ATE strategy.With lipoaspirates,a larger volume functionalized fat tissue can be constructed in vitro in a shorter time without causing donor site deformities.Methods and resultsFirstly,we explored the optimal filling density of lipoaspirates to construct tissue engineered fat in vitro.We set up four experimental groups:30%,50%,80%,and 100%volume/space ratios.After filling tissue engineering chambers(TECs)with lipoaspirates,we cultured tissues for 7 days to observe the formation of tissues and assessed their viability using the live/dead test.The results showed that lipoaspirates of 80%and 100%volume/space ratios groups could spontaneously form adipose-flaplike tissues.But the results of live/dead test and perilipin,CD31 immunofluorescence staining showed that the activity of adipose tissues in the 100%volume/space ratio group was significantly reduced compared with other groups.Having clarified that 80%volume/space ratio is the optimal fill ratio of lipoaspirates for constructing tissue engineered fat in vitro,we explore the optimal time of construction.We selected the 80%volume/space ratios for the next experiment.The adipose tissue was structurally stable at 7 days and 14 days in vitro.Ki67 immunofluorescence staining showed that the tissue cultured at 7 days in vitro had stronger proliferative activity,and scanning electron microscopy showed that the tissue cultured at 7 days in vitro contained more small adipocytes with proliferative activity,which was consistent with immunofluorescence staining results.We refer to this spontaneous lipoaspirates formed structural and functional adipose tissue as "self-assembled" fat(SAF).To probe the principles of SAF construction,we performed Masson staining of 80%volume/space ratio fat cultured in vitro for different times and showed that the extracellular matrix(ECM)content increased over time.The q-PCR assay for Col 1,3,4,and 6 showed the greatest change in Col 4 expression levels,with the most pronounced increase in expression levels on day 7,so we performed western blot analysis of Col 4 protein expression levels,which showed that Col 4 also increased over time,and the protein increase slowed down after 7 days.We also performed Col 4 and CD34 immunofluorescence staining and found a spatiotemporal correlation between the expression of Col 4 and CD34-positive stem cells.Finally,we grafted SAF into mice to observe its retention,and the results showed that SAF was better retained than the fat of control group.HE staining results showed that SAF had fewer oil droplets at 3 months after grafted.Masson trichrome staining results showed that SAF had less fibrosis at 3 months after grafted.Perilipin and CD31 immunofluorescence staining showed SAF vascularization was earlier than the fat of control group.Conclusion80%volume/space ratio of lipoaspirates cultured for 7 days in vitro can construct SAF.Self-assembly of lipoaspirates was correlated with the secretion of ECM.SAF had higher retention rate and better grafting results than directly grafted lipoaspirates.