Alternatively activated macrophages at the recipient site improve fat graft retention by promoting angiogenesis and adipogenesis

Abstract The inflammatory response mediated by macrophages plays a role in tissue repair. Macrophages preferentially infiltrate the donor site and subsequently, infiltrate the recipient site after fat grafting. This study aimed to trace host‐derived macrophages and to evaluate the effects of macrophage infiltration at the recipient site during the early stage on long‐term fat graft retention. In our novel mouse model, all mice underwent simulated liposuction and were divided into 2 groups. The fat procurement plus grafting (Pro‐Grafting) group was engrafted with prepared fat (0.3 ml). The pro‐Grafting+M2 group was engrafted with prepared fat (0.3 ml) mixed with 1.0 × 106 GFP+M0 macrophages, and then, 2 ng IL‐4 was injected into the grafts on Day 3. In addition, 1.0 × 106 GFP+M0 macrophages were injected into the tail vein for tracing in the Pro‐Grafting group. As a result, GFP+macrophages first infiltrated the donor site and subsequently infiltrated the recipient site in the Pro‐Grafting group. The long‐term retention rate was higher in the Pro‐Grafting+M2 group (52% ± 6.5%) than in the Pro‐Grafting group (40% ± 3.5%). CD34+ and CD31+ areas were observed earlier, and expression of the adipogenic proteins PPAR‐γ, C/EBP and AP2 was higher in the Pro‐Grafting+M2 group than in the Pro‐Grafting group. The host macrophages preferentially infiltrate the donor site, and then, infiltrate the recipient site after fat grafting. At the early stage, an increase in macrophages at the recipient site may promote vascularization and regeneration, and thereby improve the fat graft retention rate.


| INTRODUC TI ON
Autologous fat transplantation is a method of transferring subcutaneous fat from a donor site to a recipient site. 1,2 Its major drawback is an unpredictable retention rate. 3,4 Many studies have focused on the mechanism underlying adipose tissue repair after fat grafting to increase the retention rate. 5,6 Inflammation occurs in adipose tissue immediately after grafting. 7 This is mainly mediated by macrophages, which have been detected in transplanted grafts around oil droplets. 8 M1-polarized macrophages first appear in the early stages of the inflammatory response and remove free oil and phagocytize dead cells. 5,9 Then, M1-polarized macrophages shifts into M2-polarized macrophages to promote vascularization and regeneration. 10 Early interference with infiltration of macrophages into fat grafts significantly affects graft retention. 11 Thus, macrophage, especially M2-polarized macrophages, infiltration during the early stage is very important for fat graft retention.
Our previous study showed that macrophages were first observed at the donor site and then at the recipient site. 12 Repair processes and the long-term retention rate are optimal when adipose tissue is grafted after the donor site no longer requires macrophages for repair. 12 Thus, we hypothesized that host-derived macrophages preferentially infiltrate the donor site and subsequently infiltrate the recipient site, and that an increased level of macrophages at the recipient site during the early state improves the fat graft retention rate.
To investigate this hypothesis, we used our novel mouse model, in which simulated liposuction and fat grafting were performed, and traced macrophages. In addition, we investigated the effect of macrophage injection on fat grafting in this mouse model.

| Animals
All experiments were approved by the Nanfang Hospital Animal Ethics Committee Laboratory and were conducted according to the guidelines of the National Health and Medical Research Council of China. A total of 231 8-week-old male C57/BL6 mice and 10 8-week-old male GFP mice, obtained from Southern Medical University, were housed in individual cages with a 12 h light/dark cycle and provided standard food and water ad libitum. One hundred and twenty-six of these mice were anaesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). To prepare fat grafts, bilateral subcutaneous inguinal fat pads were separated and removed from each mouse and placed in culture dishes on ice.
Using corneal scissors, these fat pads were gently cut into very small pieces, similar to the size of aspirated fat tissue used clinically. A volume of 0.3 ml of prepared adipose tissue served as the fat graft baseline for each mouse. The graft volume was measured using the liquid overflow method.

| Pro-Grafting group and tracing
Twenty-one C57/BL6 mice underwent a simulated liposuction operation by incising the skin of the groin, cutting the subcutaneous fat pad of the groin and crushing it with clips, and then sealing the skin with 7-0 nylon sutures. A 1 ml syringe and an 18-gauge needle were used to subcutaneously inject 0.3 ml of the prepared fat into each mouse within 30 min after the tissue was harvested. At the same time, 1.0 × 10 6 GFP+M0 macrophages were injected into the tail vein ( Figure 1). The recipient and donor sites were harvested and carefully separated from the surrounding tissues on Days 7, 14 or 30. Each harvested sample was evaluated by immunofluorescence analysis.

| Pro-Grafting and Pro-Grafting+M2 groups
Using IBM SPSS version 21.0 (IBM Corp.,), 84 C57/BL6 mice were randomly divided into 2 groups (42 mice per group). The random number table method was used to determine which mice were assigned to which group. Body weight did not significantly differ between the 2 groups. All mice underwent a simulated liposuction operation by incising the skin of the groin, cutting the subcutaneous fat pad of the groin and crushing it with clips, and then sealing the skin with 7-0 nylon sutures. A 1 ml syringe and an 18-gauge needle were used to subcutaneously inject 0.3 ml of the prepared fat into each mouse within 30 min after the tissue was harvested. In the Pro-Grafting+M2 group, 1.0 × 10 6 -prepared GFP+M0 macrophages were immediately injected into grafts ( Figure 1) and 2 ng IL-4, which was used to promote shifting to M2 macrophages, was injected into the grafts on Day 3. As control, 1.0 × 10 6 -prepared GFP+M0 macrophages and PBS were injected into grafts in the Pro-Grafting group ( Figure 1).
Grafts were harvested and carefully separated from the surrounding tissues on Days 3,7,14,30,60 or 90 in the 2 groups. Each harvested sample was evaluated by histology, immunofluorescence and Western blot analysis.

| Isolation and culture of GFP+M0 macrophages
Brewer thioglycollate is a well-established sterile agent that increases the migration of monocytes into the peritoneum, allowing the isolation of large numbers of resident, nonmanipulated macrophages. GFP mice were intraperitoneally injected with 2.5 ml of 4% Brewer thioglycollate (Sigma-Aldrich Chemical Co.,). Four days later, macrophages were collected by peritoneal lavage with 4 ml phosphate-buffered saline (PBS). Lavage fluid was centrifuged, and cells were plated in Gibco RPMI-1640 medium (Life Technologies, Inc., Burlington) at a density of 3 × 10 6 cells per 100 mm polystyrene dish (BD Biosciences, Mississauga,). The macrophage population was allowed to adhere to the plate for 4 h at 37°C in 5% CO2, and then non-adherent (non-macrophage) cells were eliminated by aspiration of the supernatant. GFP+M0 macrophages were cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum (Thermo Fisher Scientific, Inc.,) and 10 mg/ml Gibco penicillin/streptomycin.

| Histological examination
Tissue samples were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin for haematoxylin-eosin staining. Tissue blocks were sectioned, examined under an Olympus BX51 microscope and photographed using an Olympus DP71 digital camera.

| Statistical analysis
Data are expressed as mean ± standard deviation and were ana-

| Comparison of fat graft retention between the 2 groups
The texture of grafts was better in the Pro-Grafting+M2 group than in the Pro-Grafting group on Day 90 ( Figure 3A). significantly higher in the Pro-Grafting+M2 group (52% ± 6.5%) than in the Pro-Grafting group (40% ± 3.5%) ( Figure 3B).

| Comparison of vascularization between the 2 groups
Immunofluorescence analysis detected more CD31+ areas in the Pro-Grafting+M2 group than in the Pro-Grafting group on Day 14 ( Figure 4). Quantitative analysis showed that the number of

| Comparison of adipogenic protein expression between the 2 groups
Expression of the adipogenic proteins PPARγ, C/EBP and AP2 on Day 30 was higher in the Pro-Grafting+M2 group than in the Pro-Grafting group ( Figure S2).

| Comparison of regeneration between the 2 groups
Histological analysis showed that the structure of grafts was better in the Pro-Grafting+M2 group than in the Pro-Grafting group on After fat grafting, the inflammatory response, which involves infiltration of M1 and M2 macrophages, is initiated in adipose tissue, followed by the regenerative response, which involves infiltration of CD34+ cells and adipogenesis ( Figure 6D).

| DISCUSS ION
This study showed that the host-derived macrophages preferentially infiltrate the donor site rather than the recipient site during early period after fat grafting, and that an increased level of macrophages at the recipient site improves the fat graft retention rate.
The number of host-derived macrophages are limited, which leads to insufficient local macrophage infiltration when more or larger areas need to be repaired after fat grafting. [13][14][15] Since the donor site can induce the infiltration of most host-derived macrophages, the number of macrophage infiltration at the recipient site is not enough to initiate an inflammatory response. 12 A similar finding was made in this study by cell tracing. Macrophages can be recruited by inflammatory factors, including IL-1β, IL-6 and TNFα. [16][17][18] Our previous study demonstrated that the levels of IL-1β, IL-6 and TNFα are higher at donor sites than at recipient sites after fat grafting, which may cause the different infiltration of macrophages. 12 Thus, we suggest that donor sites immediately release IL-1β, IL-6 and TNFα into the blood and recruit host-derived macrophages, resulting in a lower level of macrophages at recipient sites during the inflammatory stage after fat grafting.
Macrophages have been reported to play a significant role in tissue inflammation and regeneration. 11,12,19 In the early stage of the inflammatory response, high levels of inflammatory factors induce M1 macrophages polarization and initiate the inflammatory response. 20 Then, M2 macrophages polarization and produces antiinflammatory factors to solve the inflammatory response and promote tissue repair. 21,22 M2 can promote vascularization and tissue regeneration, 11,23 but delayed infiltration of M2 may result in poorer vascularization 24 and long-term infiltration of M2 can also promote tissue fibrosis. 25 Thus, matched infiltrating macrophages during the inflammatory stage are important for tissue vascularization and regeneration after grafting. [11][12][13] In this study, we observed that infiltration of macrophages was delayed at recipient sites; thus, we used macrophages to assist fat grafting in the mouse model. Vascularization, regeneration and consequently, the long-term retention rate were increased in the Pro-

| CON CLUS IONS
Host-derived macrophages preferentially infiltrate the donor site and subsequently infiltrate the recipient site after fat grafting.
During the early stage, an increased level of macrophages at the recipient site promotes vascularization and regeneration and thereby increases the fat graft retention rate.