| Background and Objective:Autologous fat transplantation is widely used in plastic surgery.A large amount of fat obtained at one time is cryopreserved for multiple treatments,which has great advantages in saving human resource costs and reducing clinical complications.However,the biological activity and clinical safety of frozen fat have been controversial.A large number of previous studies have shown different results.So far,there is no standardized cryopreservation method for adipose tissue.Adipose tissue acquisition,purification,cryopreservation methods,injection methods,injection volume,laboratory testing methods,any difference in these links may have an impact on the outcome of transplantation.The aim of this study was to evaluate the biological value of cryopreserved fat in clinical use by observing the changes of biological activity of adipose tissue and the survival rate of transplants at different cryopreserved times.In order to provide theoretical reference and basic experimental basis for further optimization of cryopreservation scheme and clinical use of frozen fat.Methods:In the first part,adipose tissue particles obtained by liposuction were purified and stored at low temperature(-80 C).Fresh fat and rewarned fat were detected after 1 week,4 weeks,8 weeks,12 weeks,16 weeks,20 weeks and 24 weeks.The contents include:gross morphological observation,HE staining histological observation,immunofluorescence staining analysis of living cell area ratio,CCK-8 activity detection,SVF cell culture.To evaluate the changes of biological activity of frozen adipose tissue.In the second part,32 SPF BALB/c-nu nude mice(females)were randomly divided into four groups:fresh control group,1 week group,4 weeks group,8 weeks group,12 weeks group,16 weeks group,20 weeks group and 24 weeks group.Fresh fat and frozen fat were injected subcutaneously into the back of nude mice,each volume was about 0.2 ml,and fat grafts were harvested after 8 weeks.The following tests were performed on the grafts:gross morphological observation and retention rate analysis,histological evaluation of paraffin section HE staining,and immunofluorescence staining analysis of implant fraction(survival rate).Results:The area of living adipocytes and mitochondrial activity of adipocytes in 1 week group were significantly lower than those in fresh adipose group(P<0.01),while those in 4 week group were significantly lower than those in 1 week group(P<0.05).but there was no significant difference among the other adjacent groups.The number of adherent cells derived from frozen fat was significantly lower than that from fresh fat(P<0.01).In the early stage of cryopreservation(1 week group,4 weeks group),the biological activity of adipose tissue decreased earlier than its morphological changes.With the prolongation of cryopreservation time,the graft survival rate,histological score and implant score(survival rate)decreased gradually.Compared with the fresh control group,the 1 week group and the 4 week group decreased significantly(P<0.01).The difference between the 1 week group and the 4 week group was significant(P<0.05).but there was no significant difference between the other adjacent groups.The survival rate of frozen adipose tissue transplantation was lower than that of graft retention.Conclusion:The biological activity of frozen fat decreased earlier than its morphological changes.Most adipocytes are inactivated during one freeze-preservation,but there is no significant correlation with the length of freeze-preservation.Adherent cells can be isolated from frozen fat,but the number is very limited.Frozen fat grafts are mostly preserved as oil cysts,fibrous tissues or necrotic tissues,and there is a risk of serious clinical complications.The clinical value of adipose tissue directly frozen without cryoprotectant is limited and it is not recommended for clinical use. |
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