Study on promoting the regeneration of grafted fat by cell-assisted lipotransfer

Abstract

Background: Cell-assisted lipotransfer (CAL), a modified adipose-derived stromal/stem cells (ADSCs)-based approach for autologous fat grafting that is an ideal option for soft tissue augmentation, has many shortcomings in terms of retention and adverse effects. The objective of our study was to improve the treatment efficacy of CAL by adding fibroblasts.

Methods: ADSCs and fibroblasts were isolated from human adipose and dermal tissues, with fibroblasts identified by immunofluorescence and ADSCs identified by the multilineage differentiation method. We performed cell proliferation, apoptosis, migration, adipogenic, and hemangioendothelial differentiation experiments, qPCR and Western blotting analysis in co-cultures of fibroblasts and ADSCs. Subsequently, we conducted animal experiments with BALB/c nude mice. Masson's staining, immunofluorescence staining and ultrasound were used to analyze the occurrence of adverse reactions of the grafted fat, and CT and three-dimensional reconstruction were used to accurately evaluate the volume of the grafted fat.

Results: We found that the co-culture of fibroblasts and ADSCs promoted their mutual proliferation, adipogenic differentiation, hemangioendothelial differentiation and proliferation and migration of HUVECs. Fibroblasts inhibit the apoptosis of ADSCs. Moreover, in animal experiments, the autografted adipose group combined with ADSCs and fibroblasts had the least occurrence of oily cysts, and fat had the best form of survival.

Conclusions: We enhanced adipocyte regeneration and angiogenesis in ADSCs and fibroblast cells after adding fibroblasts to conventional CAL autologous fat grafts. In turn, the volume retention rate of the grafted fat is improved, and the adverse reactions are reduced.

Keywords: Adipose-derived stromal/stem cells; Autologous fat grafting; Cell-assisted lipotransfer; Fibroblasts.

Fig. 1

Effects of ADSCs and fibroblasts on the proliferation and apoptosis of each other. A: Representative scatter plots of flow cytometry analysis to measure apoptosis by staining ADSCs with Annexin V-APC and 7-AAD. The apoptosis rate of ADSCs in ADSCs co-cultured with fibroblasts was lower than that in ADSCs cultured alone. B: ADSCs and fibroblasts promote each other's proliferation. a: ADSCs promote the proliferation ability of fibroblasts. b: Fibroblasts promote the proliferation ability of ADSCs. c: The proliferation ability of fibroblasts and ADSCs when co-cultured is greater than their respective proliferation abilities when they are cultured separately. Student's t test was used to analyze the data, and the asterisks represent significant differences between the groups (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, n = 3).

Fig. 2

Adipogenic and hemangioendothelial cells differentiation of ADSCs and fibroblasts. A: The capacity of ADSCs and fibroblasts to differentiate into adipocytes was improved when they were co-cultured. B: The capacity of ADSCs and fibroblasts to differentiate into hemangioendothelial cells was improved when they were co-cultured (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, n = 3).

Fig. 3

A Co-culture system of ADSCs combined with fibroblasts promotes angiogenesis, proliferation, and migration capabilities of HUVECs. A: Co-culture supernatant of ADSCs combined with fibroblasts can considerably improve the angiogenesis of HUVECs when compared to fibroblast or ADSCs supernatant. B: In the Transwell filter assay, compared with fibroblasts or ADSCs supernatant, the co-culture supernatant of ADSCs combined with fibroblasts significantly promoted the migration of HUVECs. C: Co-culture supernatant of ADSCs combined with fibroblasts can considerably promote the proliferation of HUVECs when compared to fibroblast or ADSCs supernatant. D: In the wound healing assay, compared with the fibroblasts or ADSCs supernatant, the co-culture supernatant of ADSCs combined with fibroblasts significantly promoted the migration of HUVECs (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, n = 5).

Fig. 4

Process of fat grafting. A: Fibroblasts, ADSCs (and expanded in vitro) and adipose tissue (purified) were isolated from human tissues and divided into the following groups: fibroblasts combined with ADSCs mixed with adipose group, ADSCs mixed with adipose group, fibroblasts mixed with adipose group, and fat group. The four groups were subcutaneously grafted into BALB/c nude mice. B: The purified adipose tissue is divided into three layers: the top layer is the oil layer, the middle layer is the purified fat layer, and the bottom layer is the blood and normal saline. C: Determination of ADSCs (94% viability) and fibroblast viability (89% viability) before fat grafting.

Fig. 5

Micro-CT + 3D reconstruction for assessment of grafted fat volume. At the 12th week after fat grafting, micro-CT + 3D reconstruction was used to measure the fibroblasts combined with ADSCs mixed with fat group, the ADSCs mixed with purified fat group, the fibroblasts mixed with purified fat group, and the fat group with transplanted fat volume. (n = 5).

Fig. 6

A: Ultrasound showing the occurrence of oily cysts (red arrows) at 12 weeks after fat grafting. B: Masson's staining. (n = 5).

Fig. 7

Immunofluorescence staining. Immunofluorescent staining of adipocytes with perilipin (orange), macrophages with F4/80 (green), and hemangioendothelial cells (red) in 4 different subgroups at week 12 after fat grafting. Abundant hemangioendothelial cells can be seen in adipocyte-rich areas, and oil droplets or dead adipocytes are encapsulated by macrophages to form oily cysts. (n = 5).

Supplementary Fig. 1

Identification of primary ADSCs from human tissues. A: ADSCs were identified by CD73+, CD90+, CD105+, CD31−, CD34− and CD45−. B: ADSCs were identified by their ability to differentiate via adipogenesis (oil red O stains adipocyte lipid droplets), chondrogenesis (alcian blue stains chondrocyte glycosaminoglycans), and osteogenesis (alizarin red stains bone mineralized nodules).

Supplementary Fig. 2

Identification of fibroblasts from human tissues. A: The primary fibroblasts were identified by CD26+, CD10+ and CD106− flow cytometry. B: Vinmentin + immunofluorescence chemical staining.

Supplementary Fig. 3

In ADSCs co-cultured with fibroblasts, compared with ADSCs cultured alone, qPCR results showed that the mRNA expression levels of VEGF, C/EBPα, and IGF1 genes were increased.