Study On Fat Transplantation Using PFGF2-loaded HBC@TACS Particles

Objective The defect of human soft tissue defect caused by trauma or congenital defect is a difficult problem in plastic surgery.Autologous adipose transplantation is thefirst choice to fill the graft,but there are complications such as high absorption rate and liquefaction after transplantation.How to improve the survival rate of fat transplantationand reduce the absorption of fat after transplantation is the challenge faced by plastic surgery and tissue reconstruction surgery.Basic fibroblast growth factor(FGF2)has thedual role of promoting vascularization and adipogenic differentiation of adipose stem cells and is often used in the study of adipose tissue transplantation.However,the half-life of the protein preparation is short and easy to be degraded,it cannot play a biological role for a long time.We design a kind of slow-release FGF2 plasmid temperature sensitive chitosan microspheres,putamen-"nuclear" is a thiol alkylated chitosan(TACS)and FGF2 plasmid complexes(TACS-p FGF2),owns gene transfection characteristics;The "shell" is the hydroxybutyl chitosan(HBC),which covers the "nuclear" structure.The aim of the study is to explore the feasibility of the sustained-release gene carrier in improving the survival rate of fat transplantation.Methods Thiol alkylated chitosan(TACS)and p FGF2-EGFP plasmid by positive and negative electricity function package p FGF2-EGFP plasmid form the nuclear structure(TACS-p FGF2-EGFP),hydroxyl butyl chitosan(HBC)mixed with nuclear structure formed the HBC @ TACS-p FGF2-EGFP sustained release microspheres.Microspheres were transfected into cells in vitro and the expression of immunoglobulins was tested by western-blot.In vitro release of nuclear shell microspheres and intramuscular injection of microspheres in skeletal muscle of mice were observed,and the sustained release in vivo was observed by fluorescence microscopy.We stripped lacuna under the skin of rabbit's ears for fat transplantation,,3 cm in diameter.The experimental group were transplanted 2 ml fat particles(AG)and 200 ul HBC @ TACS-p FGF2-EGFP sustained release microspheres,the control group were grafted by 2 ml implanted fat particles(AG)and 200 ul normal saline(NS).Samples were collected at week 4,8 and 12 weeks after the surgery,and the biological characteristics of the graft were observed by the volume of the specimens,including the fat survival rate,HE staining,immunohistochemistry and frozen sections.Results Western blot showed that HBC @ TACS-p FGF2-EGFP successfully transfected cells and expressed protein,and in vitro sustained release experimental results showed that HBC @ TACS-p FGF2-EGFP had the characteristics of sustained release gene.The sustained release results in KM mice showed that HBC @TACS-p FGF2-EGFP could slowly release gene in the body and successfully expression protein after transfected cells.Postoperative survival rates were 89.50 ± 1.86%,79.94 ± 1.61% and 75.98 ±2.66%.The survival rate of the control group was 72.56 ± 1.98%,64.54 ± 3.58% and 57.98 ± 4.84%.After statistical analysis,the survival rates of adipose tissue in both the experimental group and the control group were different(P < 0.05).HE staining: in the fourth week after the transplantation of the experimental group,the new blood vessels were seen.The 12 th week after surgery,the fat tissue was arranged in a relatively orderly way,and a small number of fat was replaced by fibrous tissues.In the control group,adipose tissue was disordered in the 4th week and 12 th week after surgery,and more fibrous tissue was seen.Immunohistochemistry: in the experimental group,the obvious FGF2 protein was stained brown at the time point.Conclusion HBC @ TACS-p FGF2-EGFP microspheres can be gene carrier,and the addition of the microsphere during fat transplantation can improve the fat survival rate.