External Volume Expansion: Timing and Effects on the Rate of Fat Graft Retention in BALB/c Nude Mice

Abstract

Background/aim: Fat grafting has been widely used for soft-tissue augmentation. External volume expansion (EVE) is a favorable tool for improvement in the rate of fat graft retention. However, few studies have focused on the most appropriate time for its implementation. In this study, BALB/c nude mice were used to investigate the effective time for the implementation of external volume expansion to improve the rate of fat retention.

Materials and methods: Sixteen mice were divided into four groups, and EVE was performed at different time points before or both before and after fat grafting. Fat tissue from a human donor was injected into the mice following EVE. Visual assessment, micro-computed tomography analysis, and histopathological evaluation were used to assess fat retention.

Results: After 10 weeks, the group that underwent EVE 5 days before fat grafting demonstrated a significantly higher preserved fat volume, as determined by micro-computed tomography (p<0.05). Moreover, the group that received additional EVE after fat grafting exhibited a higher retention rate compared to the groups receiving EVE only before grafting (p<0.05). Histopathological analysis indicated that swelling, edema, and inflammation were more pronounced in the group with EVE immediately before grafting, while angiogenesis and lipogenesis were more active in the group with additional EVE after grafting.

Conclusion: EVE is a safe and effective approach for improving the rate of fat graft retentions. Furthermore, the timing of external tissue expansion plays a crucial role in fat retention. Based on our animal study, performing EVE immediately before and after fat grafting may be an effective strategy for enhancing the rate of fat graft retentions.

Keywords: BALB/c nude mice; external volume expansion; fat graft; fat retention rate.

Figure 1. Schematic flow of the experiment. Group B underwent external volume expansion (EVE) 2 weeks before fat grafting and group C 5 days before fat grafting. Group D underwent EVE 5 days before fat grafting and 5 days immediately after it. The degree of fat retention was confirmed visually, via micro-computed tomography (micro-CT) and by histopathological assessments.

Figure 2. External volume expansion (EVE) procedure. A, B: Appearance of the EVE device, with a diameter of about 1 cm, used in this study. C: Appearance of the EVE device attached to a to BALB/c mouse. Negative pressure of −55 mmHg was applied with the EVE device connected to a suction pump.

Figure 3. Visual evaluation of fat graft (images taken using a WB50F digital camera). Fat was grafted at a site nearly 5 cm in the cephalad direction from the tail and 3 cm in the lateral direction of the midline spine in mice of group A (control), group B receiving external volume expansion (EVE) for 2 weeks before fat grafting, group C receiving EVE for 5 days before fat grafting, and group D which received EVE for 5 days before and 5 days after fat grafting. The most prominent decrease in fat volume was observed in group A (control group), and fat retention was observed to be best in groups C and D, while some degree of fat retention was also observed in group B.

Figure 4. Three-dimensional imaging of fat graft in mice of the control group (group A), group B receiving external volume expansion (EVE) for 2 weeks before fat grafting, group C receiving EVE for 5 days before fat grafting, and group D which received EVE for 5 days before and 5 days after fat grafting. using micro-computed tomography. The volume of each graft was delineated in imaging. The rate of change in the volume was calculated by dividing the mean for mice measured at 10 weeks after initiation of the experiment by that measured immediately after fat grafting.

Figure 5. Fat retention rate after fat graft in mice of the group A (control), group B receiving external volume expansion (EVE) for 2 weeks before fat grafting, group C receiving EVE for 5 days before fat grafting, and group D which received EVE for 5 days before and for 5 days after fat grafting. Best preservation of volume was observed in group D (68.4%) and then in group C (57%). Least preservation was noted in group A (39.3%). Significantly different at: *p<0.05 **p<0.01, and ****p<0.0001.

Figure 6. Representative images of hematoxylin and eosin-stained samples taken at 10 weeks from mice of the control group (A), group B receiving external volume expansion (EVE) for 2 weeks before fat grafting (B), group C receiving EVE for 5 days before fat grafting (C), and group D which received EVE for 5 days before and 5 days after fat grafting (D). Tissue swelling and edema were not evident in group A, whereas these were observed in groups B, C and D. These histological changes might have occurred after external volume expansion (EVE); edematous changes were prominent in the deep dermis and hypodermis (arrows). Swelling and edema were more evident in groups C and D than in group B.

Figure 7. Representative images from evaluation of angiogenesis using CD31 antibody to stain samples taken at 10 weeks from mice of the control group (A), group B receiving external volume expansion (EVE) for 2 weeks before fat grafting (B), group C receiving EVE for 5 days before fat grafting (C), and group D which received EVE for 5 days before and 5 days after fat grafting (D). The degree of staining was low in group A, which had not undergone EVE; however, it was high in groups B, C and D. Stronger staining was noted in group D than in groups B and C (arrows). Original magnification, ×20.

Figure 8. Representative images from evaluation of inflammation using CD45 antibody to stain samples taken at 10 weeks from mice of the control group (A), group B receiving external volume expansion (EVE) for 2 weeks before fat grafting (B), group C receiving EVE for 5 days before fat grafting (C), and group D which received EVE for 5 days before and 5 days after fat grafting (D). When examining the tissues around the grafted fat on a slide stained for the marker CD45, the degree of staining was low in group A, which had not undergone EVE; however, groups B, C and D showed deep staining. Staining was increased in groups C and D compared with that in group B (arrows). Original magnification, ×20.

Figure 9. Representative images from evaluation of adipogenesis using antibody to vimentin to stain samples taken at 10 weeks from mice of the control group (A), group B receiving external volume expansion (EVE) for 2 weeks before fat grafting (B), group C receiving EVE for 5 days before fat grafting (C), and group D which received EVE for 5 days before and 5 days after fat grafting (D). Evaluating the tissues around the grafted fat on a slide stained with a human vimentin antibody, the degree of staining was low in group A, which had not undergone EVE; however, groups B, C and D, which had undergone EVE, showed strong staining. Group D showed a stronger degree of staining compared with that in groups B and C (arrows). Accordingly, we found that adipogenesis occurred when EVE was performed. Original magnification, ×20.