Fat extract improves fat graft survival via proangiogenic, anti-apoptotic and pro-proliferative activities

Abstract

Background: Our previous study proved that nanofat could enhance fat graft survival by promoting neovascularization. Fat extract (FE), a cell-free component derived from nanofat, also possesses proangiogenic activity.

Objectives: The aim of this study was to investigate whether FE could improve fat graft survival and whether FE and nanofat could work synergistically to promote fat graft survival. The underlying mechanism was also investigated.

Methods: In the first animal study, human macrofat from lipoaspirate was co-transplanted into nude mice with FE or nanofat. The grafts were evaluated at 2, 4 and 12 weeks post-transplantation. In the second animal study, nude mice were transplanted with a mixture of macrofat and nanofat, followed by intra-graft injection of FE at days 1, 7, 14, 21 and 28 post-transplantation. The grafts were evaluated at 12 weeks post-transplantation. To detect the mechanism by which FE impacts graft survival, the proangiogenic, anti-apoptotic and pro-proliferative activities of FE were analysed in grafts in vivo and in cultured human vascular endothelial cells (HUVECs), adipose-derived stem cells (ADSCs) and fat tissue in vitro.

Results: In the first animal study, the weights of the fat grafts in the nanofat- and FE-treated groups were significantly higher than those of the fat grafts in the control group. In addition, higher fat integrity, more viable adipocytes, more CD31-positive blood vessels, fewer apoptotic cells and more Ki67-positive proliferating cells were observed in the nanofat- and FE-treated groups. In the second animal study, the weights of the fat grafts in the nanofat+FE group were significantly higher than those of the fat grafts in the control group. In vitro, FE showed proangiogenic effects on HUVECs, anti-apoptotic effects on fat tissue cultured under hypoxic conditions and an ability to promote ADSC proliferation and maintain their multiple differentiation capacity.

Conclusions: FE could improve fat graft survival via proangiogenic, anti-apoptotic and pro-proliferative effects on ADSCs. FE plus nanofat-assisted fat grafting is a new strategy that could potentially be used in clinical applications.

Keywords: Anti-apoptotic; Fat extract; Fat transplantation; Nanofat; Pro-proliferative; Proangiogenic.

Fig. 1

FE and nanofat improved fat graft survival. a The design of the first animal study. b Macroscopic images of the fat grafts at 2, 4 and 12 weeks post-transplantation. c Weights of the fat grafts at 2 weeks post-transplantation (*p < 0.05). d Weights of the fat grafts at 4 weeks post-transplantation (*p < 0.05). e Weights of the fat grafts at 12 weeks post-transplantation (*p < 0.05)

Fig. 2

Histological evaluation of grafts. a HE staining of the harvested fat grafts at 12 weeks post-transplantation. Scale bars = 100 μm. b Histologic analysis of the grafts at 12 weeks post-transplantation. The percentage areas of integrity, vacuoles and fibrosis were calculated (*p < 0.05). c Perilipin staining of the harvested fat grafts at 12 weeks post-transplantation. Scale bars = 50 μm. d The percentage area of viable adipocytes in the grafts at 12 weeks post-transplantation was calculated (*p < 0.05)

Fig. 3

Proangiogenic, anti-apoptotic and pro-proliferative effects of FE on fat grafts. a Anti-CD31 staining of grafts at 12 weeks post-transplantation. Scale bars = 50 μm. b The number of CD31-positive vessels per field in the grafts (*p < 0.05). c TUNEL analysis of grafts harvested at 2 weeks post-transplantation. Scale bars = 50 μm. d The number of apoptotic cells per field in the grafts (*p < 0.05). e Anti-Ki67 staining of grafts at 2 weeks post-transplantation. Scale bars = 50 μm. f The number of Ki67-positive cells per field in the grafts (*p < 0.05)

Fig. 4

FE promotes endothelial cell proliferation, migration and tube formation. a Cell proliferation was assessed using a cell counting kit, and the percentage of optical density values relative to the control was calculated (*p < 0.05). b HUVEC migration was evaluated using a cell migration assay. c The relative percentage of wound healing (24 h) was quantified (*p < 0.05). d Tube formation of HUVECs was performed on solidified Matrigel and stained using Calcein-AM. e The assessment of the number of junctions/mm² in each group is shown (*p < 0.05). f–h FE was digested with PK or RNase before incubation with HUVECs. f Cell proliferation was assessed using a cell counting kit, and the percentage of optical density values relative to the control was calculated (*p < 0.05). g The relative percentage of wound healing (24 h) was quantified (*p < 0.05). h The assessment of the number of junctions/mm² in each group is shown (*p < 0.05)

Fig. 5

The anti-apoptotic effects of FE on fat tissues cultured under hypoxic conditions. a Immunofluorescence analysis of adipocyte viability in fat tissue cultured under hypoxic conditions. Scale bars = 50 μm. b The percentage area of viable adipocytes of fat tissue at different time points was calculated (*p < 0.05)

Fig. 6

FE promotes ADSC proliferation and maintains their multipotent differentiation capacity. a ADSCs were treated with FE at the indicated concentrations. Cell proliferation was assessed by counting the cell numbers at each passage. b The cell surface marker expression of ADSCs at passage 2 was analysed. c The cell surface marker expression of ADSCs at passage 5 was analysed. d The differentiation capacity of human ADSCs into the adipogenic, osteogenic and chondrogenic lineages was analysed. Differentiation was detected with Oil Red O, von Kossa and Alcian Blue staining. Scale bars = 100 μm

Fig. 7

Nanofat and FE work synergistically to promote fat graft survival. a The design of the second animal study. b Macroscopic images of the fat grafts at 12 weeks post-transplantation. c Weights of the fat grafts at 12 weeks post-transplantation (*p < 0.05). d HE staining of the harvested fat grafts at 12 weeks post-transplantation. Scale bars = 100 μm. e Histologic analysis to evaluate the grafts at 12 weeks post-transplantation. The percentage areas of integrity, vacuoles and fibrosis were calculated (*p < 0.05)

Fig. 8

A new FE plus nanofat-assisted fat grafting strategy for future clinical application