Relationship Between Inorganic Phosphorus And Calcified Nodules Following Fat Transplantation

Background:Autologous fat transplantation was currently widely used in the field of plastic and aesthetic surgery,and had become one of the mainstream filling methods.Postoperative calcified nodule was one of the common complications.Such nodules were particularly prone to occur in large-volume fat transplantation,such as breast augmentation with fat grafting.These nodules might interfere with the diagnosis of certain tumors in addition to reduce patient satisfaction,becoming one of the most restrictive factors for further development of fat transplantation.Although some technical improvements had been performed to prevent the occurrence of such nodules to a certain extent,at present,the formation mechanism of such calcified nodules was still unclear.Inorganic phosphorus was one of the products of fat metabolism.It had been reported that inorganic phosphorus played a crucial important role in inducing other forms of pathological calcification,but the correlation between inorganic phosphorus and the formation of calcified nodules after fat transplantation was unclear.Objective:1.To verify whether there is a higher phosphorus environment in the central area of calcified nodules after fat transplantation in clinical study.2.To confirm whether inorganic phosphorus could induce calcification of adipose-derived stem cells.3.Establishment of an animal model of calcified nodules after fat transplantation.(1)To verify the presence of a local high concentration of phosphorus environment in the central necrosis area after fat transplantation.(2)To verify whether the local high phosphorus environment can accelerate the formation of calcified nodules in the fat transplantation area.Methods:1.In clinical:(1)Clinically,10 samples of necrotic fluids in the central area of calcified nodules after fat transplantation were collected.Another 10 lipoaspirates,as a control group,were collected from them who underwent cosmetic liposuction.The inorganic phosphorus concentrations of the two groups of samples were tested and compared.2.In vitro:(1)Human adipose-derived stem cells were isolated and cultured in vitro and were cultured in osteogenic induction medium containing various concentrations of phosphorus.Degree of calcification of each group was tested and compared.(2)Human adipose-derived stem cells were cultured with osteogenic induction solution containing high concentration of phosphorus for different time periods.Degree of calcification was tested and compared.(3)Human adipose-derived stem cells were cultured in osteogenic induction medium in addition with or without an inhibitor of inorganic phosphorus generation.Degree of calcification was tested and compared.3.In vivo:(1)4ml of human-derived lipoaspirates were injected into the back of nude mice in a single spot,and one group of mice were killed and the grafts were taken out after 2 and 7 months respectively to detect the degree of calcification.(2)Nude mice were injected with 4ml human lipoaspirates in a single point on the back,and the fluid in the central necrosis area was extracted 1 week,2 weeks,1 month,2 months,and 7 months after fat transplantation.The concentration of inorganic phosphorus of fluid was tested and compared.(3)A single-point bolus injection of 1ml,2ml,and 4ml on the back of nude mice was performed and the concentration of inorganic phosphorus in the necrotic fluid in the central area was detected and compared after 2 months.The grafts were taken out after 7 months to detect the degree of calcification.(4)4ml human lipoaspirates were injected into the dorsum of nude mice,while high-phosphorus solution was injected locally in the fat transplantation area(three times a week).The mice in control group were injected with the same amount of normal saline.The specimens were removed 2 months later and the degree of calcification was tested and compared.Results:1.In clinical:(1)The concentration of inorganic phosphorus in the central necrosis area of specimens with calcified nodules was significantly higher than that of normal lipoaspirates.2.In vitro:(1)After culturing human adipose-derived stem cells with osteogenic induction fluids containing different phosphorus concentrations,it was found that the higher the phosphorus concentration,the higher the degree of calcification of the cells.(2)After culturing human adipose-derived stem cells with osteogenic induction solution of the same phosphorus concentration for different times,it was found that the longer the induction time,the higher the degree of cell calcification.(3)Tetramizole could significantly inhibit the process of calcification of the cells induced by osteogenic induction medium in a dose-dependent manner.3.In vivo:(1)4ml human lipoaspirates were injected into nude mice 2 months later without obvious occurrence of calcified nodules,but after 7 months,obvious calcified nodules were detected.(2)It was found that the necrotic fluid in the central area was clear in two weeks of injection.But the necrotic fluid in the central area became muddy after one month and two months,and it became semi-solid with little liquid in seven months that it is impossible to accurately measure the concentration of inorganic phosphorus after 7 months.The concentration of inorganic phosphorus in the necrotic fluid in the central area did not change significantly in the first month after the operation,but the concentration of inorganic phosphorus in the necrotic fluid in the central area increased significantly after 2 months.(3)The greater the injection volume of fat grafts,the higher the concentration of inorganic phosphorus in the central necrosis fluid 2 months after surgery,and the more obvious the degree of calcification after 7 months.(4)Significant calcification nodules appeared after 2 months in the experimental group that high-phosphorus solution was injected 3 times a week after 4 ml of fat grafting,but no obvious calcification was observed in the control group that normal saline was injected locally.Conclusion:1.Clinically,the concentration of inorganic phosphorus in the necrotic fluid of the calcified nodules after fat transplantation was significantly higher than that of normal lipoaspirates.2.Inorganic phosphorus could promote the calcification of fat-derived stem cells in a time-and concentration-dependent manner,and tetramizole could inhibit this calcification process.3.There was no significant change in the concentration of inorganic phosphorus in the central necrotic area within 1 month after large-volume fat transplantation,but the concentration of inorganic phosphorus increased significantly after 2 months.4.After large-volume fat transplantation,there would be an increase in the local inorganic phosphorus concentration in the central necrosis area,and the greater the injection volume,the higher the inorganic phosphorus concentration in the central necrosis area,and the more obvious the degree of calcification.5.The local high-phosphorus environment caused by exogenous injection could accelerate the calcification process of the tissue around the fat transplantation area.