Objective:To investigate the effectiveness of new cryoprotective agents in preserving and transplanting human adipose tissue.Methods:The adipose tissue samples were obtained from healthy adult females who underwent liposuction at the Department of Plastic Surgery of Peking University Third Hospital from January to March 2022. The adipose tissue samples were centrifuged and then randomly divided into 9 groups. These groups were cryopreserved in liquid nitrogen using different cryoprotective agents [group A, group B, and dimethyl sulfoxide (DMSO) group] and cryopreservation times (1-month, 2-month, and 3-month groups), respectively. The cryoprotective agent formulation in group A was dextrose glycoside 40 (DEX), amino acids, vitamins, and inorganic salts. In group B, the formulation included DMSO and DEX. The ratio of cryoprotective agent in the DMSO group was 10% DMSO, 20% fetal bovine serum (FBS), and 70% DMEM-12. For cryopreservation, 5 ml cryogenic tubes were used with a fat to cryoprotective agent ratio of 3∶2, and each group contains 6 tubes for cryopreservation. After thawing the adipose tissue, HE staining was used to observe the histological morphology. Immunohistochemical staining was employed for the quantitative analysis of lipid droplet-encapsulated protein (Perilipin), and the Perilipin positivity rate was calculated by the ratio of the number of positive cells to the total number of cells. Adipocyte viability was assessed using the CCK-8 method. Thirty-eight healthy, clean nude mice were selected and divided into 3 groups of 12 mice each according to the use of different cryoprotective agents (groups A, B, and DMSO), while the other 2 mice were used as the day 0 control group. The mean fat freezing duration for all groups was 3 months. After nude mice were anesthetized intraperitoneally, 0.9 ml of thawed cryopreserved fat was injected into the dorsum bilaterally. The rate of adipose tissue retention was calculated by MRI scanning and three-dimensional software at 1, 2, and 3 months after transplantation, and compared between the groups. The fat grafts were explanted from the mice after they were sacrificed, and then subjected to histological morphology and quantitative analysis of Perilipin by using HE staining and immunohistochemical staining. GraphPad Prism 8.0 software was used for statistical analysis of the data. The data that conformed to a normal distribution were expressed as Mean ± SD. The overall comparison between multiple groups used analysis of variance for repeated measures. The comparison of data between groups at the same time point used Tukey’s multiple comparison test.Results:The morphology of adipose tissue in different cryoprotective agent groups closely resembled that of normal fresh adipose tissue after being cryopreserved in liquid nitrogen for 1-3 months. The difference in the proportion of Perilipin-stained positive cells in each group was not statistically significant ( P>0.05). The CCK-8 method indicated that the effect of the DMSO group was superior to groups A and B at 1 and 3 months of cryopreservation ( P<0.01), and that the DMSO group and group B were superior to group A at 2 months of cryopreservation ( P<0.01). In the animal experiments, there was no statistically significant difference between the groups in the volume retention rate 1-3 months after cryopreserved fat transplantation ( P>0.05). Additionally, the adipose tissues in each group exhibited varying degrees of localized necrosis accompanied by an inflammatory reaction 1-3 months after transplantation. There was no statistically significant difference in the Perilipin staining positivity between the groups ( P>0.05). Conclusion:The use of new cryoprotective agents for cryopreserving adipose tissue does not show a significant difference compared to the traditional cryoprotective agent. However, it is theoretically safer as it avoids the potential toxic effects of using DMSO or FBS on the human body.

Objective:To investigate the effectiveness of new cryoprotective agents in preserving and transplanting human adipose tissue.Methods:The adipose tissue samples were obtained from healthy adult females who underwent liposuction at the Department of Plastic Surgery of Peking University Third Hospital from January to March 2022. The adipose tissue samples were centrifuged and then randomly divided into 9 groups. These groups were cryopreserved in liquid nitrogen using different cryoprotective agents [group A, group B, and dimethyl sulfoxide (DMSO) group] and cryopreservation times (1-month, 2-month, and 3-month groups), respectively. The cryoprotective agent formulation in group A was dextrose glycoside 40 (DEX), amino acids, vitamins, and inorganic salts. In group B, the formulation included DMSO and DEX. The ratio of cryoprotective agent in the DMSO group was 10% DMSO, 20% fetal bovine serum (FBS), and 70% DMEM-12. For cryopreservation, 5 ml cryogenic tubes were used with a fat to cryoprotective agent ratio of 3∶2, and each group contains 6 tubes for cryopreservation. After thawing the adipose tissue, HE staining was used to observe the histological morphology. Immunohistochemical staining was employed for the quantitative analysis of lipid droplet-encapsulated protein (Perilipin), and the Perilipin positivity rate was calculated by the ratio of the number of positive cells to the total number of cells. Adipocyte viability was assessed using the CCK-8 method. Thirty-eight healthy, clean nude mice were selected and divided into 3 groups of 12 mice each according to the use of different cryoprotective agents (groups A, B, and DMSO), while the other 2 mice were used as the day 0 control group. The mean fat freezing duration for all groups was 3 months. After nude mice were anesthetized intraperitoneally, 0.9 ml of thawed cryopreserved fat was injected into the dorsum bilaterally. The rate of adipose tissue retention was calculated by MRI scanning and three-dimensional software at 1, 2, and 3 months after transplantation, and compared between the groups. The fat grafts were explanted from the mice after they were sacrificed, and then subjected to histological morphology and quantitative analysis of Perilipin by using HE staining and immunohistochemical staining. GraphPad Prism 8.0 software was used for statistical analysis of the data. The data that conformed to a normal distribution were expressed as Mean ± SD. The overall comparison between multiple groups used analysis of variance for repeated measures. The comparison of data between groups at the same time point used Tukey’s multiple comparison test.Results:The morphology of adipose tissue in different cryoprotective agent groups closely resembled that of normal fresh adipose tissue after being cryopreserved in liquid nitrogen for 1-3 months. The difference in the proportion of Perilipin-stained positive cells in each group was not statistically significant ( P>0.05). The CCK-8 method indicated that the effect of the DMSO group was superior to groups A and B at 1 and 3 months of cryopreservation ( P<0.01), and that the DMSO group and group B were superior to group A at 2 months of cryopreservation ( P<0.01). In the animal experiments, there was no statistically significant difference between the groups in the volume retention rate 1-3 months after cryopreserved fat transplantation ( P>0.05). Additionally, the adipose tissues in each group exhibited varying degrees of localized necrosis accompanied by an inflammatory reaction 1-3 months after transplantation. There was no statistically significant difference in the Perilipin staining positivity between the groups ( P>0.05). Conclusion:The use of new cryoprotective agents for cryopreserving adipose tissue does not show a significant difference compared to the traditional cryoprotective agent. However, it is theoretically safer as it avoids the potential toxic effects of using DMSO or FBS on the human body.

Objective To explore the feasibility of our method on assessing the three-dimensional facial morphological features based on three-dimensional surface imaging techniques.Methods According to the admission criteria,three-dimensional facial images of 34 attractive young Chinese women and 172 healthy reference young women were selected from the three-dimensional facial images database of our department.For those images,anthropometric facial measurements including facial height,width,the degree of convexity and concavity were computed using three-dimensional surface imaging techniques combined with three-dimensional coordinate system.The variance between attractive group and reference group was analyzed and the facial morphological features of each group were preliminarily summarized.Results Both groups met the criteria of mesoprosopy,not the "vertical facial trisection and rule of fifths".The attractive group had a relatively narrow anterior facial frame,a more rounded and full upper face compared with the reference group.They also had smaller intercan thai width compared with the reference group.Moreover,the attractive group showed more prominent nose,more full and prominent medial cheek.In addition,the attractive group shared less protruded upper lips and less retruded chin compared with the reference group.Conclusions Assessing facial morphology using three-dimensional surface imaging techniques is a convenient and feasible method.The evaluation of sagittal facial convexity and concavity is an indispensable part of facial morphology features assessment.

Objective To compare the differences of mitochondrial functions between keloid fibroblasts and normal skin fibroblasts and explore its relationship with cell proliferation.Methods Keloid fibroblasts (KFb) and normal skin fibroblasts (NFb) were isolated by explants culture method.KFb and NFb were cultured under normoxia or hypoxia (2％ O2).Differences of cell proliferation were detected by CCK-8.Flow cytometer was used to detect the content of mitochondria and reactive oxygen species (ROS) in KFb and NFb.Ultra-structures of mitochondria in KFb and NFb were observed by transmission electron microscope (TEM).Mitochondria fusion/fission related genes MFN1,MFN2 and FIS1 were detected by RT-PCR.Oxygen consumption rate,lactate production and ATP contents were determined by spectrophotometry.Results KFb showed a higher proliferation rate compared with NFb,especially under hypoxia.The oxygen consumption rate,ATP content,lactate production and ROS of KFb were lower than NFb under normoxia.After incubated under hypoxia,there was a significant increase in oxygen consumption,ATP content,lactate production and ROS in KFb,while NFb showed less increase compared with KFb.KFb had 15.33％ more mitochondrion than NFb,and expressions of MFN1,MFN2,FIS1 in KFb were 33.27％,113.39％ and 20.34％ higher compared with NFb.Under TEM,KFb showed an increase of enlarged mitochondrion,with disrupted inner membrane and loss of cristae.Conclusions KFb may have dysfunctions of mitochondrion which lead to changes of cell metabolism and continuous proliferation of KFb.

Objective To compare the differences of mitochondrial functions between keloid fibroblasts and normal skin fibroblasts and explore its relationship with cell proliferation.Methods Keloid fibroblasts (KFb) and normal skin fibroblasts (NFb) were isolated by explants culture method.KFb and NFb were cultured under normoxia or hypoxia (2％ O2).Differences of cell proliferation were detected by CCK-8.Flow cytometer was used to detect the content of mitochondria and reactive oxygen species (ROS) in KFb and NFb.Ultra-structures of mitochondria in KFb and NFb were observed by transmission electron microscope (TEM).Mitochondria fusion/fission related genes MFN1,MFN2 and FIS1 were detected by RT-PCR.Oxygen consumption rate,lactate production and ATP contents were determined by spectrophotometry.Results KFb showed a higher proliferation rate compared with NFb,especially under hypoxia.The oxygen consumption rate,ATP content,lactate production and ROS of KFb were lower than NFb under normoxia.After incubated under hypoxia,there was a significant increase in oxygen consumption,ATP content,lactate production and ROS in KFb,while NFb showed less increase compared with KFb.KFb had 15.33％ more mitochondrion than NFb,and expressions of MFN1,MFN2,FIS1 in KFb were 33.27％,113.39％ and 20.34％ higher compared with NFb.Under TEM,KFb showed an increase of enlarged mitochondrion,with disrupted inner membrane and loss of cristae.Conclusions KFb may have dysfunctions of mitochondrion which lead to changes of cell metabolism and continuous proliferation of KFb.