Effects of age on the fat grafts with adipose-derived stem cells for fat survival: an experimental study
10.3760/cma.j.cn114453-20200505-00262
- VernacularTitle:不同年龄脂肪来源干细胞辅助脂肪移植效果的实验研究
- Author:
Yaping QU
1
;
Jie LUAN
;
Dali MU
;
Su FU
;
Qian WANG
;
Zifei LI
;
Tong LIU
Author Information
1. 中国医学科学院北京协和医学院整形外科医院乳房整形与美容中心 100144
- Keywords:
Adipose tissue;
Stem cells;
Adipose-derived stem cells;
Age;
Cell-assisted lipotransfer;
Survival, fat;
Neovascularization
- From:
Chinese Journal of Plastic Surgery
2020;36(8):905-915
- CountryChina
- Language:Chinese
-
Abstract:
Objective:The aim of this study is to compare the effects of age on the biological properties of adipose-derived stem cells(ASCs) and fat survival of ASC-assisted lipotransfer. To identify the effect of age factors on the biological characteristics of human ASCs and compare the effects of ASCs-assisted subcutaneous fat transplantation/lipotransfer on nude mice at different ages.Methods:Human lipoaspirates were obtained from 30 healthy female patients (aged from 18 to 65 years) acquiring the abdominal liposuction. Samples were divided into three groups according to donor age: group A, 18-29 years; group B, 30-49 years; group C, 50-65 years. Stromal vascular fraction cells were isolated from the harvested adipose tissue using collagenase. The yield and cell viability of SVF were tested using the Muse cell count and viability assay. ASCs were cultured and harvested at the second passage. MSC surface markers of ASCs were examined by the flow cytometry. The cell proliferation of ASCs from different age was determined by the CCK-8 assay. The scratch test was used for assessing the ASCs migration ability. The adipogenic differentiation potential of ASCs was analyzed by induction of lipid formation in vitro. The expression levels of PPAR-γ and CEBP-α genes were detected by RT-PCR assay. The survival of adipocytes in the grafts was analyzed by perilipin-A immunofluorescence staining. The fat survival of ASCs-enriched grafts from different age was measured in animal models. The weight and residual volume of fat grafts were compared in different groups after three months. The histologic analysis was evaluated by cell integrity and necrosis tissue in fat grafts. The vessel density was measured using the CD31 immunohistochemical staining. Data were analyzed by SPSS software version 21.0 with one-way ANOVA to compare the difference of multiple groups. A value of P< 0.05 was considered statistically significant. Results:The yield and cell viability of SVF isolated from lipoaspirates were: group A, (7.06±1.28)×10 5/ml and 82.46%±2.81%; group B, (6.90±0.32)×10 5/ml and 82.01%±3.85%; group C, (6.40±0.62)×10 5/ml and 77.82%±3.45%, respectively. No significant difference was found in different age groups. SVF viability was decreased with increasing age. The expression of positive surface markers CD90, CD44, CD105 and CD73 of ASCs in each group was above 95%, and the expression of negative surface markers was below 2%, all of which met the criteria for the expression level of mesenchymal stem cell surface markers. Moreover, there was a decline in cell proliferation and migration of ASCs with increasing age. No significant difference was found in the adipogenic differentiation of ASCs in three groups. The fat grafts were harvested three months after cell-assisted lipotransfer. The graft weight was(0.18±0.02) g in group A, (0.17±0.02) g in group B, (0.15±0.01) g in group C, (0.13±0.03) g in control group, respectively; F=9.274, P<0.001. The residual volume of grafts was(262.88±17.69)/mm 3 in group A, (263.83±25.96)/mm 3 in group B, (240.06±25.08)/mm 3 in group C, (201.81±31.48)/mm 3 in the control group; F=12.95, P<0.001. There were significant differences in the weight and residual volume of fat grafts in different age groups( F=5.231, P=0.012; F=3.364, P=0.049). HE staining result showed that compared with the blank control group, ASC-assisted groups had uniform distribution of adipocytes, less fibrous connective tissue and necrotic tissue. There was a statistically significant difference in the proportion of fat integrity and necrotic tissue between the groups ( F=3.434, P=0.027; F=9.314, P<0.001). Results of the histologic analysis showed no significant difference in the proportion of fat cell integrity and necrotic tissue in each group( F=0.282, P=0.756; F=0.421, P=0.661). Immunofluorescence staining result showed that, compared with the control group, a higher number of perilipin-positive adipocytes were observed in ASCs-assisted fat grafting from different age groups, with uniform distribution. The vessel density of fat grafts was (15.70±4.16)/mm 2 in group A, (17.03±8.30)/mm 2 in group B; (16.68±6.71)/mm 2 in group C, (11.50±4.04)/mm 2 in control group; F=3.523, P=0.019. Conclusions:The proliferation and migration of human ASCs decreased with age, but age did not affect the adipogenic differentiation potential of ASCs. ASCs from different ages effectively improved the fat survival of grafts. ASCs-assisted fat grafting was more effective in young people than in elder.
