Macrophage‐mediated extracellular matrix remodeling after fat grafting in nude mice

The FASEB JournalVolume 36, Issue 10 e22550 RESEARCH ARTICLEOpen Access Macrophage-mediated extracellular matrix remodeling after fat grafting in nude mice Xiangdong Zhang, Xiangdong Zhang Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorXiaoxuan Jin, Xiaoxuan Jin Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorYibao Li, Yibao Li Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorMimi Xu, Mimi Xu Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorYao Yao, Yao Yao Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorKaiyang Liu, Kaiyang Liu Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorChijuan Ma, Chijuan Ma Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorYuchen Zhang, Yuchen Zhang Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorJiangjiang Ru, Jiangjiang Ru Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorYunfan He, Corresponding Author Yunfan He [email protected] Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China Correspondence Jianhua Gao and Yunfan He, Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, 1838, Guangzhou North Road, Guangzhou 510515, Guangdong, China. Email: [email protected] and [email protected]Search for more papers by this authorJianhua Gao, Corresponding Author Jianhua Gao [email protected] Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China Correspondence Jianhua Gao and Yunfan He, Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, 1838, Guangzhou North Road, Guangzhou 510515, Guangdong, China. Email: [email protected] and [email protected]Search for more papers by this author Xiangdong Zhang, Xiangdong Zhang Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorXiaoxuan Jin, Xiaoxuan Jin Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorYibao Li, Yibao Li Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorMimi Xu, Mimi Xu Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorYao Yao, Yao Yao Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorKaiyang Liu, Kaiyang Liu Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorChijuan Ma, Chijuan Ma Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorYuchen Zhang, Yuchen Zhang Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorJiangjiang Ru, Jiangjiang Ru Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, ChinaSearch for more papers by this authorYunfan He, Corresponding Author Yunfan He [email protected] Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China Correspondence Jianhua Gao and Yunfan He, Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, 1838, Guangzhou North Road, Guangzhou 510515, Guangdong, China. Email: [email protected] and [email protected]Search for more papers by this authorJianhua Gao, Corresponding Author Jianhua Gao [email protected] Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China Correspondence Jianhua Gao and Yunfan He, Department of Plastic Surgery, Nanfang Hospital, Southern Medical University, 1838, Guangzhou North Road, Guangzhou 510515, Guangdong, China. Email: [email protected] and [email protected]Search for more papers by this author First published: 13 September 2022 https://doi.org/10.1096/fj.202200037R Xiangdong Zhang and Xiaoxuan Jin contributed equally to this work and share first authorship. Jianhua Gao and Yunfan He contributed equally to this work and co-corresponding authors. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Clinical unpredictability and variability following fat grafting remain non-negligible problems due to the unknown mechanism of grafted fat retention. The role of the extracellular matrix (ECM), which renders cells with structural and biochemical support, has been ignored. This study aimed to clarify the ECM remodeling process, related cellular events, and the spatiotemporal relationship between ECM remodeling and adipocyte survival and adipogenesis after fat grafting. Labeled Coleman fat by the matrix-tracing technique was grafted in nude mice. The ECM remodeling process and cellular events were assessed in vivo. The related cytokines were evaluated by qRT-PCR. An in vitro cell migration assay was performed to verify the chemotactic effect of M2-like macrophages on fibroblasts. The results demonstrated that in the periphery, most of the adipocytes of the graft survived or regenerated, and the graft-derived ECM was gradually replaced by the newly-formed ECM. In the central parts, most adipocytes in the grafts died shortly after, and a small part of the graft-derived and newly-formed ECM was expressed with irregular morphology. Adipose ECM remodeling is associated with increased infiltration of macrophages and fibroblasts, as well as up-regulated expression of cytokines in the adipose tissue. To sum up, our results describe the various preservation mode of fat grafts after transplantation and underscore the importance of macrophage-mediated ECM remodeling in graft preservation after fat grafting. The appreciation and manipulation of underlying mechanisms that are operant in this setting stand to explore new therapeutic approaches and improve clinical outcomes of fat grafting. Abbreviations Arg1 arginase-1 bFGF basic fibroblast growth factor ECM extracellular matrix Fizz1 found in inflammatory zone 1 IL-10 interleukin-10 IL-13 interleukin-13 IL-4 interleukin-4 MCP-1 monocyte chemoattractant protein-1 MIP-1α/β macrophage inflammatory protein-1α/β MMP matrix metalloproteinase TGF-β transforming growth factor-β 1 INTRODUCTION Fat grafting is a widespread and well-established standard procedure for soft-tissue revolumization and rejuvenation.1-4 Although the technique has been optimized, clinical unpredictability and variable long-term volume retention remain non-negligible problems due to the unknown mechanism of grafted fat retention.3 The most well-known theories to explain the variations and mechanism of grafted fat retention are the three zones survival theory, host replacement theory, and cell survival theory. The three-zone survival theory states that the transplanted fat is divided into three zones, from the periphery to the center5: the surviving area (adipocytes survived); the regenerating area (adipocytes died, adipose-derived stromal cells survived); necrotic area (both adipocytes and adipose-derived stromal cells died). Cell survival theory focuses on the viability of the adipocytes being grafted, citing that grafted living adipocytes can survive in the recipient site and are incorporated into adjacent vascularized tissue.6 The host replacement theory states that grafted adipocytes are replaced by new adipocytes through the process of stem cell differentiation.6-10 Based on the widespread belief that the number of cells in grafts correlates with the ultimate volume of persisting fat, cell components (such as adipocytes, stem cells, and SVF cells) have become of major concern to current research projects.10, 11 Additionally, various lipoaspirate processing methods have emerged regarding the most important factors in preserving grafted cell viability, improving host-derived stem cell regenerative capacity, and maximizing its long-term durability in clinics, such as those in cell-assisted lipotransfer, telfa gauze processing techniques, and Tissu-Trans Filtron inline filtration system.12, 13 These have gained widespread clinical acceptance and have resulted in higher fat graft retention rates.14 Current studies have focused on cellular events, and little research has been done on the role of extracellular matrix (ECM) in fat grafting. The majority of cells after fat grafting inevitably undergo apoptosis and necrosis due to exposure to ischemia, hypoxia, or excessive stress in the survival environment.14, 15 Moreover, although cryopreserved fat loses up to 92.7% of cellular metabolic activity and contains massive cell necrosis,16 the volume and morphology of the grafts after the transplantation of cryopreserved fat are similar to those of transplanted fresh fat grafts.17 This suggests that the effect of ECM on grafted fat retention should not be ignored. The ECM is a highly specialized and dynamic three-dimensional network in which cells reside in tissues.18 The core constituents of the ECM are collagen scaffolds and scaffold-bound bioactive substances.19 ECM, with its multitude of molecules, comprises the microenvironment of cells,17 rendering them with structural and biochemical support.20, 21 ECM also supplies cells with proper mechanical and chemical signals to dictate cell survival, proliferation, differentiation, and migration to adjust tissue functions and homeostasis.22, 23 After fat transplantation, the grafted fat is exposed to an acute hypoxic environment with high levels of macrophage infiltration.24 Researches have proven that macrophages are capable of secreting MMPs, which are associated with the degradation of collagen components of ECM and the subsequent release of bound bioactive substances (such as nanovesicles, growth factors, and cryptic peptides).25-27 The released bioactive substances trigger signaling events that affect inflammation progression, matrix remodeling, cell function, and prime pre-metastatic niches.19 Some animal experiments and clinical studies28-31 have shown that adipogenesis can be induced by simple decellularized adipose tissue, indicating an important role of adipose tissue ECM in directing stem cell differentiation and adipogenesis.28-30 Therefore, we speculated that ECM remodeling after adipose grafting is involved in graft retention after adipose tissue transplantation. This study aimed to clarify the ECM remodeling process and related cellular events after fat grafting. First, the matrix-tracing technique was employed to label the ECM constituents of the grafting fat with FITC NHS ester. Second, fat was grafted in nude mice, and the ECM remodeling process (graft-derived ECM degradation and newly-formed ECM synthesis) in grafts was evaluated. Third, cellular events, including macrophage infiltration, matrix metalloproteinase (MMP) secretion, and fibroblast recruitment, which may contribute to ECM remodeling, were assessed. Last, the spatiotemporal relationship between ECM remodeling and adipocyte survival and adipogenesis in fat grafts was preliminarily investigated. 2 MATERIALS AND METHODS Fat harvesting and processing Human abdominal region adipose tissue was obtained from females who underwent liposuction. All clinical procedures performed in this study were approved by the Institutional Review Board of Nanfang Hospital, Southern Medical University, and written informed consent was obtained from the tissue donors. The harvested adipose tissue was centrifuged to generate standard Coleman fat (1200 × g, 3 min). Coleman fat labeling and animal models The ECM of Coleman fat was labeled according to a previous study.32 Briefly, 100 μM Alexa Fluor 647 NHS Ester was used to incubate with 7.5 ml of Coleman fat for 1 h at 25°C (A20006, Thermo Fisher Scientific, United States). All experiments were approved by the Nanfang Hospital Animal Ethics. The study was conducted in accordance with the National Health and Medical Research Council of China; female BALB/c nude mice, 4 to 6 weeks old (n = 25), were purchased from Southern Medical University. Coleman fat (0.3 ml) was injected subcutaneously into the dorsal region of the mouse. Each mouse had two injection sites on the dorsum. Animals were sacrificed after 1 and 3 days, and after 1, 4, and 12 weeks (n = 5 mice per time point), the subcutaneous fat grafts were photographed, and their volumes were measured using the liquid overflow method. Only half of the fat grafts were fixed in paraformaldehyde, with the remainder preserved at −80°C for further analysis. Graft tissue liquid extract The grafts at weeks 1 and 4 were first cut into pieces, and the samples were mixed well with PBS solution and centrifuged at 1000 × g for 3 min. The liquid portion above the centrifuge tube was collected and passed to remove cell and tissue debris through a 0.20-μm syringe filter (Xiboshi, DIONEX, USA). In vitro culture of cells and transwell system Murine L929 fibroblasts and M2-macrophages (Thermo Fisher Scientific Inc., Waltham, MA, USA) were cultured in a complete medium at 37°C with 5% CO2. 4 × 104 L929 fibroblasts were maintained in a complete medium were pipetted onto each transwell insert with a polyethylene terephthalate membrane pore size of 8 μm in 24-well plates. The inserts were placed in diverse media in a 24-well plate at 37°C in 5% CO2: M2-macrophage suspension; graft tissue liquid extract at week 1; graft tissue liquid extract at week 4; and complete medium. The media within the transwell inserts were removed after 12 h, 24 h, or 48 h, respectively. Cells that did not migrate across the polyethylene terephthalate membrane were wiped with a cotton swab. Cells were then stained with crystal violet (Sigma-Aldrich) and observed under a microscope (BX 51, Olympus). Quantitative analysis was performed using Image Pro Plus software (version 6.0; Media Cybernetics, Silver Spring, MD, USA). Macrophage depletion in the grafted fat Female BALB/c nude mice, 4 to 6 weeks old (n = 15), were purchased from Southern Medical University. NHS-labeled Coleman fat (0.3 ml) was injected subcutaneously into the dorsal region of the mouse. Each mouse had two injection sites on the dorsum. The two flanks where the fat graft was transplanted of each mouse were respectively injected with liposome-encapsulated clodronate (left) and PBS-Liposome (right) (ClodronateLiposome, 283 539; Amsterdam, Netherlands; 0.5 ml/100 g body weight) for every other day, right after lipotransfer, for a period of 1 week. Animals were sacrificed after 1, 4, and 12 weeks (n = 5 mice per time point). Histology and immunofluorescence staining The graft samples were embedded in paraffin, sliced, and cut into 5-μm-thick sections. Hematoxylin and eosin (HE) and Masson staining were used to assess the content of the grafts. For immunofluorescent staining, the following primary antibodies were used: anti-perilipin antibody (Progen, Heidelberg, Germany) to stain viable adipocytes; anti-Mac2 (Cedarlane Corp, Burlington, Ontario, Canada) and anti-CD86 antibody to stain macrophages; anti-CD206 antibody to stain anti-inflammatory M2 macrophages; anti-MMP1 antibody to stain the matrix-degrading enzyme; anti-vimentin antibody to stain fibroblasts; anti-procollagen antibody to stain neocollagen (Abcam, Cambridge, UK). DAPI (Sigma, St. Louis, MO, USA) was used to stain the nuclei. The sections were then observed under microscopes (LSM 980, Zeiss Axioscope, Oberkochen, Germany; BX 51, Olympus, Japan). Qualification analyses were performed using Image Pro Plus software (version 6.0; Media Cybernetics, Silver Spring, MD, USA) and Image J software. Flow cytometry analyses For graft analysis, the graft samples were minced into 0.1 cm3 pieces, homogenized, and digested for 30 min with 0.125% collagenase (Sigma, Missouri, USA) in a shaking water bath at 37°C. After centrifugation (800 g, 3 min) and re-suspended in fresh DMEM, the cells were stained with the following monoclonal antibodies: CD11b-Pacific Blue, CD11c-PE/Cy7, F4/80-PE, CD206-FITC (BioLegend, San Diego, USA). An irrelevant control monoclonal antibody was included for each fluorochrome. Finally, the cells were analyzed on a BD LSR-II flow cytometer (Becton Dickinson, CA, USA). Cell Quest software (Becton Dickinson) was employed for data acquisition and analysis. Gates were set to ensure no more than 0.1% of cells showed a reaction with the control antibodies. CD11b+/F4/80+ cells were identified as macrophages. F4/80+, CD11b+, CD11c+, CD206low/F4/80+, CD11b+, CD11c+, CD206high were classified M1-macrophages and M2-macrophages respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) Isolation of total RNA and synthesis of complementary DNA were performed according to the manufacturer's protocol. Relative mRNA expression was calculated with β-actin as the reference gene, and the resulting complementary DNA was subjected to PCR analysis using gene-specific primers as follows: monocyte chemoattractant protein-1(Mcp-1); forward 5′-GCTGGAGCTGAGGAGATTATTG-3′ and reverse 5′-AGAACCTCTGTCCGTGATGA-3′; macrophage inflammatory protein-1α/β (Mip -1α/β); forward 5′-GCTTCAGACACCAGAAGGATAC-3′ and reverse 5′-TGATGTTGAGCAGGTGACAG-3′; Mmp1; forward 5′-GTTGACAGGCTCCGAGAAAT-3′ and reverse 5′-CAT CAGGCACTCCACATCTT-3′; interleukin-4 (Il-4); forward 5′-GACGGCACAGAGCTATTGAT-3′ and reverse 5′-GGATATGGCTCCTGGTACATTC-3′; interleukin-10 (Il-10); forward 5′-CCCTTTGCTATGGTGTCCTTTC-3′ and reverse 5′-AGGATCTCCCTGGTTTCTCTTC-3′; basic fibroblast growth factor (bFgf) forward 5′-GAGAGTTCA GAGTGCTAATGGG-3′ and reverse 5′-CTCCTGAGTGCT GGGATTAAAG-3′; transforming growth factor β (Tgf-β) forward 5′-GGTGGTATACTGAGACACCTTG-3′ and reverse 5′-CCCAAGGAAAGGTAGGTGATAG-3′; collagen 1; forward 5′-GAGGGCCAAGACGAAGACATC-3′ and reverse 5′-CAGATCACGTCATCGCACAAC-3′; collagen 6; forward 5′-ACAGTGACGAGGTGGAGATCA-3′ and reverse 5′-GATAGCGCAGTCGGTGTAGG-3′; Arginase-1 (Arg1); forward 5′-CCAGGGACTGACTACCTTAAAC-3′ and reverse 5′-GAAGGCGTTTGCTTAGTTCTG-3′; Ym1; forward 5′-CCCTATGCCTATCAGGGTAATG-3′ and reverse 5′-ACGGCACCTCCTAAATTGT-3′; found in inflammatory zone 1 (Fizz1); forward 5′-CCCAGTGA ATACTGATGAGACC-3′ and reverse 5′-GGAGGGATAG TTAGCTGGATTG-3′; β-actin; and forward 5′-GAGGTAT CCTGACCCTGAAGTA-3′ and reverse 5′-CACACGCAG CTCATTGTAGA-3′. Statistical analysis Data are expressed as mean ± SEM. Results were analyzed using SPSS software (version 26.0; IBM Corp., Armonk, NY, United States). Data were analyzed by statistical methods. An independent t test and least significant difference post hoc analysis were used to compare two and three groups at a single time point, respectively, and a one-way analysis of variance (ANOVA) was used to compare groups at all time points. Values of p < .05 were considered statistically significant. 3 RESULTS Preservation mode after transplantation To observe the spatiotemporal relationship between ECM and mature adipocytes after adipose tissue transplantation, the ECM component of the grafts was labeled prior to transplantation using FITC NHS ester. In the early post-transplant period, the grafts were soft and showed a blue-green color characteristic of NHS dye labeling (Figure 1). The graft was harder in the late post-transplant period than in the early period, appearing light green and dark yellow. With time, the volume of the graft continuously decreased, and its blue-green color continuously diminished. FIGURE 1Open in figure viewer Macroscopic observation of fat grafts on day 1 (above, left), day 3 (above, middle), week 1 (above, right), week 4 (below, left), and week 12 (below, right). Immunofluorescence results showed that on day 1 after transplantation, many areas that were strongly positive for NHS and perilipin were visible within the graft (Figure 2A). The grafted ECM and adipocytes, both at the periphery and the center of the graft, were viable. On day 3, NHS positivity in the whole areas of the graft and perilipin positivity in the periphery of the graft was still strongly expressed. However, the expression level of perilipin positivity in the central parts was decreasing. At week 1 after transplantation, NHS positivity in the periphery of the graft was sharply diminished, whereas perilipin positivity was still strongly expressed. In contrast, perilipin positivity was sharply diminished in the central parts, and NHS positivity was stronger than that in the periphery (Figure 2B,C) (p < .05). At the late post-transplant period (weeks 4–12), the periphery of the graft retained the majority of perilipin positivity, with minor NHS positivity. However, the central part of the graft showed little perilipin positivity, while the expression of NHS positivity was irregular, missing, and fractured. In a very small number of graft samples, we observed that a small amount of NHS-positive tissues that were expressed in the center of the graft at week 12 post-transplantation showed an intact ECM network structure (Figure S1). These results suggest a different mode of graft preservation in distinct parts and at different time points after fat grafting. Multiple spatiotemporal relationships exist between ECM remodeling and adipocyte survival after adipose tissue transplantation. FIGURE 2Open in figure viewer Immunohistology of the surviving adipocytes and preserved grafted ECM in grafts. (A) Immunofluorescence staining of NHS (red), perilipin (green), and DAPI (blue) of the fat grafts at all time points. Scale bar = 200 μm. (B) Semi-quantitative analysis of the expression of NHS positivity (left) and perilipin positivity (right) at different time points. *p < .05, peripheral area vs. central area at the same time point. N = 5. An independent t test was used to compare two groups at a single time point. Nucleated cells gradually increased from the periphery to the center HE and immunofluorescence results showed tightly arranged adipocytes within the graft during the early post-transplant period, while vacuoles and fibrous-like tissue were seen within the graft during the late post-transplant period (Figures 2 and 3A). Numerous nucleated cells appeared around the graft in the early post-transplant period (days 1–3 and week 1), and an upward tendency was found in the amount of nucleated cells with time both in the periphery and center of the graft (Figure 3B). By the late post-transplant period, nucleated cells spread throughout the entire graft, and the level of cell number peaked at week 4. At the same time, small adipocytes could be seen in the late post-transplant graft. FIGURE 3Open in figure viewer HE staining of fat grafts. (A) Adipocytes tightly arranged within the graft during the early post-transplant period, while vacuoles and fibrous-like tissue were seen within the graft during the late post-transplant period. Numerous nucleated cells could be observed in the grafts. Scale bar = 200 μm. (B) Semi-quantitative analysis of the number of nucleated cells. Over time, the number of nucleated cells gradually increased and peaked at week 4 both the periphery and center of the graft, then decreased. *p < .05, peripheral area vs. central area at the same time point. N = 5. An independent t test was used to compare two groups at a single time point. CD86-positive CD206-negative cells infiltration was involved in graft-derived ECM degradation To further observe the relationship between infiltrating nucleated cells and ECM remodeling in the graft, CD86, and CD206 antibodies were used for immunofluorescent staining of macrophages within the graft (Figure 4A). Soon after transplantation, the number of CD86-positive CD206-negative cells gradually increased and peaked at week 1 in the periphery of the graft, with fewer CD86-positive CD206-negative cells in the central parts (Figure 4B). By weeks 1–4 post-transplantation, numerous CD86-positive CD206-negative cells had infiltrated the periphery and central parts of the graft and CD86 expression was reduced from week 4 to week 12 after transplantation. Quantitative analysis of macrophage (CD11b+/F4/80+) infiltration in the grafts showed a significant upward pattern from day 1 to week 4. The proportion of macrophages reached a peak at week 4 and then declined during the period from week 4 to week 12 (Figure 4C). The M2/M1 macrophage (F4/80+, CD11b+, CD11c+, CD206high/F4/80+, CD11b+, CD11c+, CD206low) ratio was significantly the highest at week 4 compared to the other time points and kept a high level for up to week 12. The protein MMP1, secreted by macrophages, was subsequently labeled. MMP1 surrounded CD86-positive macrophages and the NHS fluorescence expression in the areas of MMP1 positivity had almost disappeared, and the remaining NHS-positive ECM structure was fractured, folded, or even missing (Figure 5). FIGURE 4Open in figure viewer Immunohistology of the CD86-positive and CD206-positive cells in grafts. (A) Immunofluorescence staining of CD86-positive cells (red), CD206-positive cells (green), and DAPI (blue) of the fat grafts at all time points. Scale bar = 100 μm. (B) Semi-quantitative analysis of the number of CD86-positive (left) and CD206-positive (right) cells. *p < .05, peripheral area vs. central area at the same time point. N = 5. An independent t test was used to compare two groups at a single time point. (C) Quantification of macrophage proportion in the grafts at all time points (left). Quantification of M2/M1 macrophage (F4/80+, CD11b+, CD11c+, CD206high/F4/80+, CD11b+, CD11c+, CD206low) ratio at all time points (right). *p < .05 vs. 1d, ^p < .05 vs. 3d, +p < .05 vs. 1w, #p < .05 vs. 4w, −p < .05 vs. 12w. N = 5. One-way analysis of variance (ANOVA) was used to compare groups at all time points. FIGURE 5Open in figure viewer Immunohistology of preserved grafted ECM, CD86-positive cells, and MMP1 in grafts. Immunofluorescence staining of NHS (white), CD86-positive cells (red), MMP1 (green), and DAPI (blue) of the fat grafts. MMP1 surrounded CD86-positive cells and the NHS fluorescence expression in the areas of MMP1 positivity had almost disappeared, and the remaining NHS-positive ECM structure was fractured, folded, or even missing. Scale bar = 200 μm. qRT-PCR was used to detect the expression levels of human-derived Collagen I and VI at each time point. Collagen I transcription level was significantly decreased in the given time points (Figure 6A). Similarly, there is a dramatic drop in the expression level of Collagen VI during the period from week 1 to week 12, with an insignificant difference between day 1 and day 3 (Figure 6B). The expression levels of Mcp1 and Mip1α peaked on day 1 after transplantation and then gradually declined (Figure 6C,D). The expression levels of Mmp1 gradually increased on days 1–3 post-transplantation, peaked on day 3, and then gradually decreased (Figure 6E). FIGURE 6Open in figure viewer Quantitative reverse transcription PCR analysis of the expression levels of Collagen I, Collagen VI, Mcp-1, Mip-1α, and Mmp1. (A) Collagen I transcription level was significantly decreased in the given time points. (B) The expression level of Collagen VI was dropped during the period from week 1 to week 12, with an insignificant difference between day 1 to day 3. (C and D) The expression levels of Mcp-1 and Mip-1α peaked on day 1 after transplantation and then gradually declined. (E) The expression levels of MMP1 gradually increased on days 1–3 post-transplantation, peaked on day 3, and then gradually decreased. *p < .05 vs. 1d, ^p < .05 vs. 3d, +p < .05 vs. 1w, #p < .05 vs. 4w, −p < .05 vs. 12w. N = 5. One-way analysis of variance (ANOVA) was used to compare groups at all time points. Host-derived ECM within the graft Masson's staining results showed that in the late period, an abundant quantity of collagenous components in the intercellular matrix could be observed within the grafts, and small nascent adipocytes were visible within some of the collagen components (Figure 7). NHS expression around perilipin-positive adipocytes within the graft was negative in the late post-transplant period (Figure 2), whereas Masson's staining results showed significant expression of the collagen component at the periphery of the adipocytes in the graft. FIGURE 7Open in figure viewer Histological evaluation of collagen content. Masson staining of graft samples at weeks 4 and 12. An abundant quantity of collagenous components in the intercellular matrix could be observed within the grafts, and small nascent adipocytes were visible within some of the collagen components. Fibroblast infiltration and newly-formed ECM synthesis Figure 8A shows that fibroblasts were observed in both th