Adipose Tissue Transplant Impregnated with Collagen Increases Engraftment by Promoting Cell Proliferation, Neovascularization, and Macrophage Activity in a Rat

Objectives: To clarify the effect of impregnating transplanted adipose tissue with collagen on angiogenesis, cell proliferation, and tissue remodeling process and to reveal whether collagen impregnation contributes to improving the engraftment of transplanted adipose tissue in rats. Methods: Adipose tissue was harvested from the inguinal and injected into the back of the rat, in addition to collagen. Engraftment tissue was harvested, semi-quantitatively evaluated and underwent HE or Perilipin staining. Moreover, we evaluated viable adipocyte counts and neovascularization. Macrophages were evaluated using ow cytometry, and the adiponectin or VEGF mRNA was detected using real-time PCR. Results: By impregnating transplanted adipose tissue with collagen, higher engraftment rate semi-quantitatively and a greater number of new blood vessels histologically were identied. Perilipin staining revealed a higher adipocyte number. The total cell, M1 macrophage, and M2 macrophage count were higher. There was increased adiponectin mRNA signicantly at week 4 compared to that at week 1 after transplantation. Note that the expression levels of VEGF mRNA increased. Discussion: In rats, collagen impregnation enhanced cell proliferation, induced M2 macrophages, which are involved in wound healing, and promoted adipocytes and neovascularization. Therefore, impregnating transplanted adipose tissue with collagen could increase the engraftment rate of adipose tissue.


Introduction
Adipose tissue transplantation is a relatively minimal invasive procedure; because it can be performed relatively easily and does not require advanced techniques such as for vascular anastomosis, it is widely performed in various settings such as in reconstruction after craniofacial surgery and following mastectomy [1][2][3][4][5][6]. However, as per the current method, the amount per transplant is limited; because there is no blood ow in transplanted adipose tissue, the tissue might become ischemic. If this happens, the majority of cases develop necrosis, thus making graft survival unstable and making multiple transplants necessary in multiple cases [2,[7][8][9]. Many studies have indicated that there is considerable discrepancy with regard to the related effects and complications of adipose tissue transplantation, which vary as per the level of skill of the surgeon. Thus, the current method still has room for improvement [8,10]. Transplanted adipose tissue is engrafted through the penetration of capillaries from surrounding tissue. However, this process requires time and when blood ow cannot be maintained, it becomes fat necrosis and scar tissue [2]. Therefore, to stabilize and achieve engraftment of adipose tissue transplants, the reperfusion of blood ow to the adipose tissue needs to be achieved more quickly.
Furthermore, the tolerance to ischemia of the actual transplanted adipose tissue needs to be improved.
Previously, we reported that adding lipid fraction obtained from adipose tissue to arti cial dermis induces early neovascularization in the arti cial dermis [11]. When the arti cial dermis contains collagen, this collagen induces neovascularization. Therefore, based on this empirical data, it is possible that while the mechanism is unclear, the interaction between collagen and adipocytes strongly enhances neovascularization. The purpose of this study is to clarify the effect of collagen impregnation to transplanted adipose tissue on angiogenesis, cell proliferation, and tissue remodeling process, as well as to reveal whether the collagen impregnation contributes to the improvement of transplanted adipose tissue in rats and nally to contribute to establishing a more effective fat transplantation method.

Results
Semiquantitative evaluation of the engraftment rate of the transplanted adipose tissue At one week and at 4 weeks after lipoinjection, the engraftment rate of the transplanted adipose tissue expressed by engrafted tissue length/transplanted tissue length was higher in the group with added collagen (control group vs. collagen group: 1 week (0.27 ± 0.35 vs. 0.52 ± 0.37), 4 weeks (0.27 ± 0.41 vs. 0.60 ± 0.39) ( Table 1).  (Fig. 1). Furthermore, on Perilipin staining of viable adipocytes, additional Perilipin-positive cells were reported in the collagen group (Fig. 2). These results suggested that in the group with collagen, greater angiogenesis and viable adipocytes were induced.
Changes in cell count, and macrophage count caused by collagen impregnation Sliced control group and collagen group adipose tissue for transplantation were both injected into the back of an individual rat, and then engrafted tissue in the animal's back was extracted one week later (n = 12). In the extracted tissue, the total cell count, M1 macrophage count, and M2 macrophage count were analyzed using ow cytometry by uorescence-activated cell sorting (FACS). Assuming the control group was 1.0, the collagen group had a signi cantly higher total cell count of 1.89 (p < 0.05), M1 macrophage count of 1.69 (p < 0.05), and M2 macrophage count of 2.19 (p < 0.05). These results suggest that collagen impregnation induces additional M1 and M2 macrophages (Fig. 3).
Gene expression of adiponectin and VEGF in grafted adipose tissue To investigate whether collagen treatment improves the survival of transplanted adipose tissue, the expression of adiponectin mRNA, as a marker for functional adipocytes, was compared ( Fig. 4a). At 1 week after transplantation, there was no signi cant difference between control-and collagen-treated grafts. The expression of adiponectin mRNA was similar at 1 week and 4 weeks after transplantation in control conditions. However, collagen treatment signi cantly increased adiponectin mRNA at 4 weeks compared to that at 1 weeks after transplantation (p < 0.05). To evaluate the effects of collagen on angiogenesis in grafts, the expression levels of VEGF mRNA were investigated (Fig. 4b). There was no signi cant difference between control and collagen-treated grafts, although the expression levels of VEGF mRNA seemed to be increased by collagen treatment at 4 weeks after transplantation. These results suggested that collagen treatment improve the functional adipocytes in the grafts at 4 weeks after transplantation, possibly via angiogenesis.

Discussion
Fat transplantation is extensively used as a postoperative reconstruction method such as craniofacial surgery and mastectomy; however, most of the transplanted adipose tissue is necrotic, engraftment is unstable, and developing more stable fat transplantation method is awaited. The engraftment instability of adipose tissue transplantation arises when the adipose tissue for transplantation is placed in an ischemic environment, and the transplanted adipose tissue developed necrosis [2, 7-9, 12, 13]. To improve adipose tissue engraftment, we believe that it is important to reduce the cell count with necrosis arising from such ischemic environment, i.e., neovascularization and cell proliferation within the transplanted adipose tissue is required [13][14][15]. This study's results suggest that in rat adipose tissue, collagen impregnation to transplanted adipose tissue increases angiogenesis and improves the fat engraftment rate via the number of adipocytes and the proliferation of macrophages. In this study, adipose tissue transplanted into rats histologically promoted angiogenesis following collagen impregnation, thereby improving the engraftment rate. The tissue remodeling mechanism appeared to be involved in the increase in engraftment rate. After transplantation, the adipose tissue is placed in an ischemic environment, which causes tissue necrosis and tissue remodeling [16]. Macrophage migration is strongly involved in the process of such remodeling [17,18]. Macrophages are broadly divided into M1 and M2 macrophages. M1 macrophages induce the in ammatory reaction required for tissue engraftment, and they are involved in the scavenging of necrotic adipose tissue, whereas M2 macrophages are involved in tissue repair, and both M1 and M2 macrophages carry out an important role in the process of adipose tissue remodeling, and engraftment [18][19][20][21][22][23]. Furthermore, macrophages improve the survival of adipose tissue grafts by inducing neovascularization and activated stem cells [18,24]. In this study, we reported that collagen impregnation to adipose tissue transplants signi cantly increased M1, and M2 macrophages in the transplanted tissue. In particular, M2 macrophages considerably increased, and we believe that this result re ects a state whereby impregnating transplanted adipose tissue with collagen induces the in ammatory reaction required for tissue remodeling and induces a potent tissue repair action [19,24,25].
In this study, no signi cant difference was observed in the expression level of vascular endothelial growth factor (VEGF) mRNA, which is a factor that promotes neovascularization at 1 week and 4 weeks after transplantation; moreover, the level tended to be higher in the collagen group. This result might re ect the fact that impregnating transplanted adipose tissue with collagen serves as a platform for neovascularization and helps give rise to abundant neovascularization in the transplanted tissue. It is possible that the ligation and clustering of integrin receptors α1β1/α2 β1 on the surface of endothelial cells (EC) via the GFPGER (502-507) sequence of the collagen bers is important for neovascularization activity [26]. While there is still room for debate, to summarize the results of the histological evaluation, we can draw the conclusion that collagen impregnation to transplanted adipose tissue produced additional neovascularization [25][26][27]. Recently, attention has been drawn to the effectiveness of adipose-derived stem cells (ASC) as a means to promote neovascularization in transplanted adipose tissue. ASC induce differentiation to adipocytes by co-culturing with mature adipocytes [28], and they contribute to neovascularization by differentiating into vascular EC [14,[29][30][31][32]. Furthermore, it has been reported that hypoxia results in the ASC secretion of neovascularization growth factors such as VEGF, and therefore induces neovascularization in an ischemic environment after transplantation [33]. ASC presents in subcutaneous adipose tissue can differentiate into adipocytes [34]; it has been reported that when ASC are cultured from collagen, more lipids are produced, which is a marker of adipose differentiation [35]. Therefore, collagen impregnation could support ASC differentiation into adipocytes. In this study, on comparing the level of adiponectin mRNA expression at 1 week and 4 weeks after transplantation; the expression level tended to be higher, although not signi cantly higher, in the control group. In the collagen group, a signi cant increase in the level of adiponectin mRNA expression was observed at 4 weeks. Furthermore, while there was no signi cant difference observed, at 4 weeks, the level of adiponectin mRNA expression tended to be higher in the collagen group than in the control group.
Adiponectin is an adipocytokine produced particularly in adipose tissue and that produces and secretes various bioactive factors [36]. In humans, it is the gene with the most abundant expression in adipose tissue [37]. This study's results show that when collagen was added, there was a greater viable adipocyte count at 4 weeks after transplantation, which supports the higher engraftment rate in the collagen group indicated by semi-quantitative evaluation. Moreover, in the collagen group, there was a greater change over time from 1 to 4 weeks after transplantation. This suggests that collagen impregnation leads to greater viable adipocyte proliferation. Our results suggested that collagen impregnation increases the adipocyte count and increases neovascularization in the transplanted adipose tissue. It is possible that a greater effect can be anticipated by combining ASC with collagen, and therefore this needs to be studied further in future. Harvesting adipose tissue and modi cation of adipose specimens for transplantation

Animals
The rats were anesthetized by iso urane inhalation with an intra-abdominal injection of a combination of three anesthetic agents, including medetomidine at 0.75 mg/kg, midazolam at 4.0 mg/kg, and butorphanol at 5.0 mg/ kg. Note that adipose tissue harvested from the inguinal region of the rats was thinly sliced using surgical scissors; after washing with physiological saline, centrifugal separation was performed (1,800 rpm for 3 min at 12 °C). As the adipose specimen for transplant, we harvested the portion after removing the oil component in the supernatant and the uid in the underlay, which was de ned as the control group (Fig. 5). Bovine collagen suspension adjusted to a collagen concentration of 0.25% (Olympus Terumo Biomaterials) was added to the harvested adipose specimen for transplantation and used as the collagen group.

Tissue transplant
The adipose specimen for transplantation was then injected beneath the brous membrane on the back of the same individual rat, in addition to an adipose specimen for transplantation containing collagen using an 18 G cannula to infuse 0.1 cc/1 cm (Fig. 6).
The adipose specimen for transplantation was injected to beneath the brous membrane on the back of the same individual rat, in addition to an adipose specimen for transplantation containing collagen using an 18 G cannula to infuse 0.1 cc/1 cm.
Endogenous peroxidase activity was blocked by incubating in 0.3% hydrogen peroxidase and 0.1% sodium azide containing 0.01 M phosphate-buffered saline, and a Histo ne® simple staining system (Nichirei Biosciences) was used for secondary detection. The nal product was visualized using 3,3diaminobenzidine.

Isolation of mononuclear cells in ltrating to implanted fat tissue
Implanted fat tissue was removed, washed with cold PBS, sliced with scissors into small pieces, and digested in PBS containing 1 mg/ml collagenase (Sigma-Aldrich) with gentle shaking at 37 °C for 30 min. After ltering through a 100-µm-sized nylon cell strainer, the single cells were collected and centrifuged, and then erythrocytes were lysed in the ammonium-chloride-potassium lysing buffer. After centrifugation, the cells were collected for ow cytometry analysis.

Quantitative real-time PCR
Total RNA of fat grafts was extracted as previously described [38]. High-capacity RNA to cDNA kit (Applied Biosystems, Life Technologies) was used for reverse transcriptase reactions. The expression levels of adiponectin and VEGF were determined using quantitative real-time PCR using ABI 7500 Real-Time PCR system (Life Technologies). The expression level of the house-keeping gene GAPDH was used as an internal control. Primers and probes used for target genes were TaqMan Gene Expression Assays (Rn01775763_g1 for GAPDH; Rn00595250_m1 for adiponectin; Rn01511602_m1 for VEGF) purchased from Applied Biosystems, Life Technologies.

Statistics
Differences between multiple groups were analyzed using the Mann-Whitney U test. Statistical signi cance was set as p < 0.05 and analyzed using StatMate V (ATMS Co., Ltd., Tokyo, Japan).

Conclusions
On impregnating transplanted adipose tissue with collagen, we report that the level of adiponectin mRNA increased, and the histological viable adipocyte count increased in the rats. Furthermore, the level of VEGF mRNA tended to increase, and histologically an increase in neovascularization was observed, which suggested that collagen impregnation might contribute to increased neovascularization in transplanted adipose tissue in the rats. Moreover, we reported that collagen impregnation increased M1 and M2 macrophages in transplanted adipose tissue in the rats, and therefore might contribute to tissue remodeling. It was suggested that impregnating transplanted adipose tissue with collagen increases the engraftment rate of transplanted adipose tissue by increasing the viable adipocyte count, promoting neovascularization, and inducing macrophages.

Declarations
Acknowledgments: None Author Contributions: C.S. designed the research, conducted experiments, and wrote the manuscript. H.I. contributed to perform the quantitative real-time PCR. T.Y. contributed to perform ow cytometry. T.K. have made substantial contribution to study design, interpretation of data, and made critical comments on the study. H.M. organized this research as project managers. All authors reviewed the manuscript.
Con ict of Interest: There are no con icts of interest to declare.
Funding: This study received funding from Japan Society for the Promotion of Science grant number 16K11381.
Data availability: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Consent to participate/Consent to publish: Not Applicable