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.

Fat grafting has been widely used for soft-tissue augmentation after its recognition as a convenient and safe procedure (1). Records of fat grafting began with a case by Neuber, who used autologous fat grafts to treat a depressed scar on a patient’s face in 1893 (2). Currently, fat grafting is used in various fields of surgery, including breast augmentation, depressed scar correction and cosmetic procedures. Fat grafting is generally known to exert effects superior in terms of safety and practicality to those of surgical procedures using artificial implants; the former is also associated with a low risk of side-effects, such as inflammatory responses and infection due to grafting materials (3).

In some cases of autologous fat grafting, the injected fat does not settle and thus results in autolysis (4,5), often resulting in unsatisfactory outcomes. The retention rates of autologous fat grafting vary and have been reported to be approximately 30-80% (1,6). However, to the best of our knowledge, there is currently no consensus regarding the most optimal approach for improving the rate. Therefore, various relevant studies are being conducted worldwide (7).

Several studies are currently focusing on factors that affect the retention rates after fat grafting. The rates of fat graft retention are typically affected considerably by the condition of the surrounding soft tissues in the recipient site. The grafted fat settles while new blood vessels grow after receiving nourishment via nutrient diffusion in the recipient site (1). If the recipient site is not in a healthy condition (e.g. poor blood circulation, severe underlying conditions, and history of radiotherapy), the grafted fat may not settle properly, leading to the loss of a large volume of the graft (8).

Recently, some studies have indicated that external volume expansion is effective for improving the retention rates of grafted fat (1,6). During external volume expansion, transient ischemia occurs, which is the state of hypoxic injury that elicits responses, such as swelling and inflammation. If external volume expansion is stopped, cell proliferation, angiogenesis and lipogenesis proceed at the recipient site due to the injury sustained. The consequent change in tissue condition leads to favorable conditions for the retention of the fat to be grafted. In this context, one previous study has reported that the retention rate of grafted fat in patients who underwent external volume expansion is higher than that of those who did not (1).

Evidence shows that external volume expansion increases rate of fat graft retentions via a preconditioning effect before fat grafting which lasts for nearly 2 weeks (1). Another study indicated that when performing fat grafting on tissues that have been subjected to radioactive irradiation, external volume expansion helps improve the retention rate (8). Several studies have reported the effectiveness of external volume expansion; however, few studies have focused on the most effective 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

Animal model. Animal experiments were performed after seeking approval from the Institutional Animal Care and Use Committee of Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF; approval number: DGMIF-20040201-00). All applicable institutional and/or national guidelines for the care and use of animals were followed. We followed guidelines of the Institutional Animal Care and Use Committee of Daegu-Gyeongbuk Medical Innovation Foundation during this study. A total of 33 male BALB/c nude mice (age, 6 weeks; weight ³ 20 g) were purchased from the Yeongnam branch of ORIENT BIO Inc. and were bred at the Laboratory Animal Center Return Animal Zone of DGMIF. In total, 16 male mice were selected and randomly divided into four groups with four mice in each group; the average weights of each group were equalized. Each animal was allocated a unique ID, which was marked on its tail using a permanent marker. Each mouse cage was classified according to the mouse ID, experiment number, and experimental group. For observation, indoor settings were maintained at 22±1˚C, with 50%±10% relative humidity, 10-20 air changes/h and a 12-h light/dark cycle (lights on, 07:00 h; lights off, 19:00 h). The illuminance of the breeding rooms was maintained at 150-300 Lux. In this controlled environment, the mice were randomized into four groups of four mice per cage (IVC rack to breed, 395×346×213 mm) for breeding.

Experimental models were developed using the 16 male BALB/c nude mice (age, 16 weeks; weight, 21.87-25.22 g; average weight, 23.84 g). To facilitate the implementation of external volume expansion, the fat-grafting site was selected at a distance of nearly 5 cm in the cephalad direction of the head from the tail of an experimental mouse, where there is abundance of soft tissues, and at nearly 3 cm in the lateral direction of the midline spine. The experimental animals were divided into four groups to perform the experiments (Figure 1).

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.

Human fat harvest. The Kyungpook National University Chilgok Hospital Institutional Review Board (No. 2023-03-036) approved this study. After obtaining consent for the use of fat tissue for research and experimental purposes from a 36-year-old female donor with no underlying medical conditions who was scheduled to undergo liposuction on the abdomen, an aspiration cannula connected to a 10 ml syringe was used to aspirate fat cells from the lower abdomen in accordance with the principles of the Coleman technique. Adipocytes were obtained from the lower abdomen, and fat aspirate was centrifuged at 112×g for 3 min to remove the separated supernatant and serum. Pure fat samples were then extracted to be used directly for the in vivo animal studies.

External volume expansion. BALB/c nude mice were administered breathing anesthesia using 2-3% isoflurane, and an external volume expander was attached to the point about 5 cm in the cephalad direction from the back and about 3 cm in the lateral direction of the midline spine. The experiment was conducted using a dome-shaped expander (CGBIO Co., Seoul, Republic of Korea) with a diameter of approximately 1 cm connected to an aspiration pump that was created to enable external volume expansion. A negative pressure of −55 mmHg was applied using a device for external tissue expansion (Figure 2). Group A comprised a control group without external volume expansion. For groups B and C, external volume expansion was performed 3 h a day for a total of 2 weeks and for 5 days before fat grafting, respectively, and for group D, 3 h a day for 5 days before and for 5 days after fat grafting. External volume expansion was performed without breathing anesthesia (Figure 1).

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.

Visual assessment. To visually determine the preserved condition of grafted fat, close-up photographs were taken of the dorsal side of the experimental animals using a WB50F digital camera (Samsung, Seoul, Republic of Korea). As the experiment progressed, the state of fat retention in each group was visually compared (Figure 3).

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.

Quantum FX micro-computed tomographic (micro-CT) assessment. Quantitative measurement of the rates of fat graft retention in each group was performed using Quantum FX micro-CT (PerkinElmer, Waltham, MA, USA) at DGMIF Laboratory Animal Center a total of three times: Immediately after fat grafting (2 weeks after experiment initiation) as well as at 6 and at 10 weeks after experiment initiation. The values set for performing Quantum FX micro-CT were as follows: 90 kVp and 180 μA, with a field of view of 40×40×40 mm for 2 min. Analyze 12.0 software (Analyze Direct, Stillwell, KS, USA) was used to reproduce the CT scans; back scan sizes were set at 512 matrix and 0.078 mm voxel. Data were analyzed using Analyze 12.0 to create images. The volume of fat was measured by manually delineating the two-dimensional images within the threshold range of 1,000 to 2,100 of interest, and then extrapolating to obtain the three-dimensional volume. The mice were placed under anesthesia using carbon dioxide gas before scanning them via Quantum FX micro-CT (Perkin Elmer) (Figure 1).

Histopathological evaluation. All animals were euthanized and subjected to autopsy 10 weeks after the initiation of the experiments (Figure 1). To observe the histopathological changes in the soft tissues that were subjected to external volume expansion, tissue samples were prepared after euthanizing the mice. The skin and soft tissues of the mice were harvested from the fat-grafted area and then fixed using 10% neutral buffered formalin before being imbedded in paraffin. Tissue sections with a thickness of 4 μm were made from the blocks for all samples and then stained with hematoxylin and eosin (H&E) so that the histopathological changes could be identified. They were also stained with antibodies against CD31 (3528S, Cell Signaling Technology, Danvers, MA, USA) for angiogenesis assessment, CD45 (ab10559, Abcam, Cambridge, UK) for inflammatory response assessment, and human vimentin (ab92547, Abcam) for lipogenesis assessment. Images of the stained tissues were taken using a Nikon electric drip fluorescence microscope (Nikon, Tokyo, Japan). The images were analyzed using NIS-Elements software (Nikon).

Statistical analysis. Statistical analyses of all collected data were performed using GraphPad Prism 8 (GraphPad, San Diego, CA, USA). The mean differences in individual fat volumes were compared for each group using two-way analysis of variance to validate the statistical significance of the experimental group, in which the level of statistical significance was set to <5% (p<0.05).

Results

While performing external volume expansion, a decrease in grafted fat volume was visually confirmed in all groups. In group A, where external tissue expansion was not performed, the most significant reduction in volume was observed visually, while the best preservation of transplanted fat was visually confirmed in groups C and D. (Figure 3).

A total of three micro-CT scans were performed: Immediately after fat grafting (2 weeks after experiment initiation) and at 6 and 10 weeks after experiment initiation. Quantitative assessment was performed on the volume of grafted fat. At 10 weeks after experiment initiation, the average volume of the preserved fat was compared in each group; the results were as follows: group A, 225.71±30.98 cc; group B, 250.32±21 cc; group C, 303.86±49.68 cc; and group D, 443.89±64.63 cc. Considering errors in volume estimation during fat grafting, the rates of change in the volume were calculated by dividing the mean volumes for each group measured at 10 weeks after the experiment by those measured immediately after fat grafting (Figure 4). The calculated changes were noted to be 39.3%, 46.6%, 57.0%, and 68.4% for groups A, B, C, and D, respectively. Statistical analysis was performed using the mean changes for each mouse of each group, and the results revealed significantly higher rates of retention in groups C and D at 10 weeks than in group A (p<0.05 and p<0.0001, respectively), as well as a significant difference between groups D and B (p<0.01) (Figure 5).

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.

These tissues were stained with H&E for microscopic observation. Although swelling and edema in the tissues was hardly observed in the mice belonging to group A, it was evident in the tissues of groups B, C, and D. In a comparison of the groups that had undergone external volume expansion, swelling and edema were more prominent in groups C and D compared with group B. These were considered histological changes that occurred after external volume expansion. Edema was observed prominently in the deep dermal layer and below the dermal layer (Figure 6).

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.

Endothelial cells in blood vessels were stained with CD31 antibodies to observe angiogenesis under a microscope. The degree of staining was found to be increased in groups B, C and D compared with that in group A; however, a slight degree of staining was noted in group A. In comparison of the groups that had undergone external volume expansion, a greater degree of staining was observed in group D than in groups B and C (Figure 7).

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.

Tissues were stained for the pan-leukocyte antigen CD45 to observe the degree of inflammation under a microscope. The density of the inflammatory cells deeply stained with antigen CD45 was observed in the surrounding area of the grafted fat under the microscope; a higher density of inflammatory cells was noted in groups B, C, and D than in group A, which had not undergone external volume expansion. In the comparison of the groups that had undergone external volume expansion, an increase in the degree of staining was observed in groups C and D compared with that in group B (Figure 8).

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.

Fat cells were stained with human vimentin antibody to identify the degree of lipogenesis. Although no large difference in the degree of staining was observed in the deep dermal layer and below the dermal layer, in the area surrounding the grafted fat, tissues obtained from groups B, C and D were more deeply stained than those obtained from group A, which had not undergone external volume expansion. In a comparison of the groups that had undergone external volume expansion, the degree of staining was noted to be increased in group D compared with that in groups B and C (Figure 9).

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.

Discussion

Fat grafting is a relatively less invasive surgical technique with high recognition in plastic surgery for its high cosmetic and functional utilities; its safety and efficiency have been established (8,9). A previous study reported that after fat grafting, patient satisfaction with the cosmetic effect reached nearly 80%; its efficacy in resolving deformity and congenital or acquired transformation in the body has been widely demonstrated (10).

The rate of fat graft retention is an important factor for evaluating the effectiveness of the fat grafting technique (11). However, retention rates are highly variable after fat grafting (12). Studies are currently being conducted to improve the retention rates of grafted fat (1,6). Various studies are in progress to develop fat collecting methods, fat processing methods, and fat grafting with additives to improve fat retention rates. However, to the best of our knowledge, no consensus has yet been reached regarding the most optimal method (4,5,8).

Grafted fat tissues rely on nutrients diffused from blood vessels into the surrounding area (12). To improve angiogenesis, efforts are being made to develop various techniques involving the use of growth factors, biomaterials, and stem cells (13-19). However, the literature reports few cases of successful clinical implementation to promote angiogenesis in soft tissues (20-23).

Several studies have identified external volume expansion as a prominent tool for increasing the retention rate in fat grafting (3,4,6). However, studies on the effective time for the clinical implementation of external volume expansion are scarce. Therefore, this study aimed to explore this topic.

Although quantitative evaluation of plastic surgery techniques and procedures is essential, visual assessment is the best means of evaluating cosmetic aspects. In this experiment, close-up images to determine the degree of fat graft retention were taken using a WB50F digital camera (Samsung) and we visually confirmed that fat retention was higher in groups C and D than in groups A and B.

Based on the results of a comparison of the average volume change in each group, the best preservation of volume was observed in group D (68.4%) and then in group C (57%); the least preservation was noted in group A. This finding suggests that external volume expansion is advantageous in fat preservation when performed in addition to fat grafting. Statistical analysis confirmed significantly higher survival rates in groups C and D compared to group A, with group D showing significance over group B. For all of the above mentioned results, the retention rates were higher in groups B, C and D, which had undergone external volume expansion, than in group A, which had not. Among the groups that had undergone external volume expansion, the retention rates were higher in groups C and D, which had undergone expansion 5 days before fat grafting, than in group B, which had undergone expansion 2 weeks before fat grafting.

When performing external volume expansion, inflammation occurs through responses, such as cell distortion, ischemia, and edema as well as cell proliferation, angiogenesis, and lipogenesis (1). These responses were observed on histological examination, and swelling or edema were evident in the H&E-stained slides prepared using samples from the groups that had undergone external volume expansion. Furthermore, in CD31 staining, via which angiogenesis can be assessed, the same groups showed deep staining. We also evaluated the degree of inflammation using the antigen CD45, which was deeply stained in the groups that had undergone external volume expansion. Assessing lipogenesis using human vimentin antibodies, the groups that had undergone external volume expansion showed deep staining. Inflammation occurs when performing external volume expansion, and angiogenesis and lipogenesis actively take place. This is supported by reports of a series of reactions that occur when external tissue expansion is performed, which in turn have been shown to result in an increased rate of fat graft retention (1,6).

In the comparison of groups which had undergone external volume expansion, group with benign disease exhibited less intense H&E staining than groups C and D. We attribute this finding to normalization of histological changes in group B, which had undergone earlier external volume expansion (2 weeks before fat grafting).

In CD31 antibody staining, groups C and D exhibited deeper staining than group B; group D showed the deepest staining. This might be because the duration of external volume expansion was the longest in group D, and, consequently, the angiogenic responses lasted the longest. This appears to be consistent with the finding of the highest fat retention rate in group D.

In CD45 antigen staining, it was evident that inflammatory cells were clustered in groups C and D. It is speculated that the series of reactions induced by external tissue expansion performed immediately prior to fat grafting, along with the resulting histological changes before normalization, would likely lead to an increased rate of fat graft retention (3,4,12).

In human vimentin antibody staining, group D showed the deepest and most widespread staining. This result shows the effect of external volume expansion on the generation of new fat. Based on histological changes due to external volume expansion being performed both before and after fat grafting, the rate of fat retention might have increased as lipogenesis was promoted in the grafted fat.

The histological findings of swelling, edema, angiogenesis, inflammation, and lipogenesis appeared to support the results of the quantitative evaluation conducted via Quantum FX micro-CT.

Our study has some limitations. During external volume expansion in one case, the expander often became detached because pressure was not applied, requiring its reattachment. Because this did not occur to the same degree in all cases, there is a possibility that this gave rise to errors. Because the epidermis of the experimental mice was very fragile, we were very conservative about increasing the pressure. However, despite the installation of a structure for fixation of the device to the surrounding area, it was not possible to prevent detachment. Thus, further studies are warranted to determine the level of negative pressure that is appropriate on the site where additional fat grafting will be performed or has been already performed before this technique is implemented clinically.

Regarding the existing studies on patients who underwent external volume expansion, one study stated that when it was performed on female patients who did not undergo breast augmentation, the breasts became firmer and exhibited growth in size (24). However, side-effects were also reported in some patients who underwent external volume expansion, such as itchiness and skin irritation, which are regarded as factors that must be considered before the clinical implementation of external volume expansion (24).

Autologous fat grafting is known to have effects superior in terms of safety and practicality to surgery that uses artificial implants and is thus widely used in various fields of plastic surgery. It is also used in other surgical fields, including breast augmentation, revision for scar tissues where the depressed skin is observed, and other esthetic purposes; in many cases, it is also used as augmentation for insufficient volume after a reconstructive surgery. Accordingly, several studies are currently being conducted to improve the rate of fat retention. The present study is one such study where we investigated the effects of external volume expansion at different time points. We determined the effective time for the clinical implementation of external volume expansion based on fat retention rates at each period; its effects per period were determined based on histopathological changes. As this study was conducted on animal models, further research would be necessary before direct clinical application. However, based on the results of this study, it has been demonstrated that performing external volume expansion both immediately before and after fat grafting is the most effective approach for enhancing fat retention. Our findings show that the clinical implementation of external volume expansion improves the rate of fat graft retention.

Conclusion

Based on the results of the visual, quantitative, and histological assessments performed in this study, the fat retention rates were found to be higher in the groups that had undergone external volume expansion 5 days before fat grafting than in the group that had undergone the procedure 2 weeks before fat grafting. Furthermore, the rates were observed to be higher in the groups that had undergone external volume expansion simultaneously with fat grafting. Thus, performing external volume expansion immediately before and after fat grafting may be most effective.

Conflicts of Interest

The Authors declare no conflicts of interest exist.

Authors’ Contributions

Conceptualization: Yang JD. Data curation: Lee JS and Kim YH. Formal analysis: Ryu JY and Choi KY. Methodology: Chung HY and Chang YJ. Supervision: Cho BC. Writing -original draft: Chang YJ. Kim YH. Writing - review and editing: Yang JD.

Acknowledgements

This work was supported by Biomedical Research Institute grant, Kyungpook National University Hospital (2020) (No. 2020-NE-08).