Composition for fat grafting

Abstract

The present invention relates to a composition for fat grafting containing adipose tissue treated with nervonic acid. According to the present invention, during fat tissue graft surgery, the survival rate and engraftment rate of fat included in grafted adipose tissue may be increased, thereby improving efficiency of fat tissue grafting.

Description

Composition for fat grafting

[0001] The present invention relates to a composition for fat transplantation, and more particularly, to a composition for fat transplantation containing adipose tissue treated with nervonic acid.

[0002]

[0003] Fat grafting is a widely used method to improve skin elasticity. Fat grafting involves extracting a patient's own fat using a syringe and transplanting it to areas of skin that appear sagging. Fat grafting has shown promising results in improving wrinkles that develop with age, as well as correcting lip lines, jawlines, and forehead lines, rhinoplasty, and filling in sunken skin caused by burns or wounds or areas lost after cancerous tissue excision. However, it has been reported that 40-60% of the grafted volume is reabsorbed by the body after fat grafting (S Eremia et al., Dermatol. Surg., 26:1150, 2000; Fulton JE et al., Dermatol. Clin., 19:523, 2001). Fat grafting in areas with thin skin and minimal subcutaneous fat can lead to problems such as palpable clumps of fat. Furthermore, the survival rate and duration of the grafted fat often fall short of expectations, necessitating reimplantation.

[0004] The basic structure of the skin is maintained by subcutaneous fat tissue, and this subcutaneous fat tissue plays a role in determining the volume and strength of the skin. Therefore, as explained above, increasing the volume of fat tissue may be a better solution for maintaining skin volume and improving wrinkles than the existing method of providing elasticity to the dermis or epidermis of the outer layer of the skin. Recently, studies related to this have been published (Kim W Set al., J. Dermatol. Sci., 53:96, 2009; Trottier Vet al., Stem Cells., 26:2713, 2008; Park B Set al., Dermatol. Surg., 34(10):1323-1326, 2008).

[0005] Accordingly, the inventors of the present invention have made great efforts to develop a method for increasing the survival efficiency of transplanted fat tissue after fat grafting surgery, and as a result, have confirmed that when the transplanted fat tissue is treated with nervonic acid, the survival rate of transplanted fat cells increases, inflammation decreases, and angiogenesis in the transplanted fat tissue increases, thereby completing the present invention.

[0006]

[0007] Summary of the invention

[0008] The purpose of the present invention is to provide a composition for fat transplantation with increased fat transplantation efficiency.

[0009] Another object of the present invention is to provide a composition that increases fat transplantation efficiency.

[0010] Another object of the present invention is to provide a method for increasing fat transplantation efficiency.

[0011] In order to achieve the above purpose, the present invention provides a fat transplant composition containing adipose tissue, characterized in that the adipose tissue is treated with nervonic acid.

[0012] The present invention also provides a composition for increasing fat transplantation efficiency containing nervonic acid as an effective ingredient.

[0013] The present invention also provides a method for transplanting adipose tissue, comprising the step of treating the adipose tissue to be transplanted with nervonic acid.

[0014]

[0015] Figure 1 is a schematic diagram showing the method of experiment and analysis of allogeneic fat transplantation in a mouse model using neural acid.

[0016] Figure 2a shows the results of H&E staining to determine the area of ​​vacuoles (cysts) in the transplanted fat 5 weeks after transplantation of adipose tissue derived from a mouse model treated with Nervonic acid.

[0017] Figure 2b shows the results of quantifying the area of ​​vacuoles (cysts) in the transplanted fat, as confirmed by H&E staining, 5 weeks after transplantation of adipose tissue derived from a mouse model treated with Nervonic acid.

[0018] Figure 3a shows the results of observing lipid accumulation in the transplanted fat using Oil Red O staining 5 weeks after transplanting the mouse model-derived fat tissue treated with Nervonic acid.

[0019] Figure 3b shows the results of quantifying lipid accumulation in transplanted fat, as confirmed by Oil Red O staining, 5 weeks after transplantation of adipose tissue derived from a mouse model treated with Nervonic acid.

[0020] Figure 4a shows the results of immunohistochemical staining to confirm the expression of Perilipin-1 in the transplanted fat 5 weeks after transplantation of adipose tissue derived from a mouse model treated with Nervonic acid.

[0021] Figure 4b shows the results of quantifying the area of ​​fat cells expressing Perilipin-1 in the transplanted fat, as confirmed by immunohistochemical staining, 5 weeks after transplantation of adipose tissue derived from a mouse model treated with Nervonic acid.

[0022] Figure 5 shows the results of confirming the expression level of inflammation-related markers in the transplanted fat at the mRNA level using qRT-PCR 5 weeks after transplantation of adipose tissue derived from a mouse model treated with Nervonic acid.

[0023] Figure 6a shows the results of H&E staining and immunohistochemical staining to confirm the vascular structure and CD31 expression in the transplanted fat 5 weeks after transplantation of adipose tissue derived from a mouse model treated with Nervonic acid.

[0024] Figure 6b shows the results of quantifying the area of ​​CD31 expression in the transplanted fat, as confirmed by immunohistochemical staining, 5 weeks after transplantation of adipose tissue derived from a mouse model treated with Nervonic acid.

[0025] Figure 6c shows the results of confirming the expression level of angiogenesis-related markers in the transplanted fat at the mRNA level using qRT-PCR 5 weeks after transplantation of adipose tissue derived from a mouse model treated with Nervonic acid.

[0026]

[0027] The invention is described in detail and has preferred embodiments.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, unless otherwise defined herein. Generally, the nomenclature used herein is well known and commonly used in the art.

[0029] In the present invention, nervonic acid was treated to the adipose tissue to be transplanted during a fat tissue transplantation surgery, and the survival rate of fat cells, vacuole (cyst) area, expression of inflammation markers, and expression of angiogenesis markers within the transplanted adipose tissue were confirmed. It was confirmed that nervonic acid treatment increased the survival rate of fat cells, decreased the formation of vacuoles within the transplanted tissue, decreased inflammation, increased angiogenesis, and thus increased the efficiency of fat transplantation.

[0030] Therefore, from one aspect, the present invention relates to a fat transplant composition containing adipose tissue, characterized in that the adipose tissue is treated with nervonic acid.

[0031] In the present invention, the composition for fat transplantation may be characterized as being for fat tissue transplantation surgery, filler, or fat redistribution surgery.

[0032] In the present invention, the nervonic acid may be characterized by being treated at a concentration of 10 to 1000 μM, more specifically, it is preferable to treat at a concentration of 50 to 500 μM, more specifically, it is preferable to treat at a concentration of 80 to 240 μM, and even more specifically, it is preferable to treat at a concentration of 120 to 200 μM.

[0033] From another perspective, the present invention relates to a composition for increasing fat transplantation efficiency containing nervonic acid as an active ingredient.

[0034] In the present invention, the nervonic acid may be contained at a concentration of 10 to 1000 μM, more specifically, it is preferably contained at a concentration of 50 to 500 μM, more specifically, it is preferably contained at a concentration of 80 to 240 μM, and even more specifically, it is preferably contained at a concentration of 120 to 200 μM.

[0035] In another aspect, the present invention relates to a method for transplanting adipose tissue, comprising the step of treating the adipose tissue to be transplanted with nervonic acid.

[0036] In the present invention, the nervonic acid may be characterized by being treated at a concentration of 10 to 1000 μM, more specifically, it is preferable to treat at a concentration of 50 to 500 μM, more specifically, it is preferable to treat at a concentration of 80 to 240 μM, and even more specifically, it is preferable to treat at a concentration of 120 to 200 μM.

[0037] The composition of the present invention can provide the efficacy of increasing the transplantation efficiency of adipose tissue, thereby providing volume and elasticity to the skin and improving wrinkles.

[0038]

[0039] [Example]

[0040] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0041]

[0042] Example 1. Confirmation of the effect of reducing fear of transplanted fat after performing allogeneic fat transplantation by treating mouse-derived adipose tissue with nervonic acid.

[0043] Vacuoles (cysts) in transplanted fat occur due to necrosis of adipocytes within the transplanted fat after fat grafting, and the degree of vacuolation is considered an important indicator of the outcome of fat grafting. Therefore, in this example, we treated adipose tissue extracted from a mouse with nervonic acid and transplanted it into a syngeneic mouse, and confirmed the efficacy of reducing vacuole (cyst) formation in the transplanted fat.

[0044] The C57BL / 6NCrlOri mice used in the experiment were purchased from Orient Bio Co., Ltd., and all experiments were conducted using 5-month-old mice.

[0045] Fat for transplantation was obtained from the peritoneal fat of mice, and the obtained fat was cut into very fine pieces and centrifuged at 3000 rpm for 15 minutes to isolate only pure adipose tissue.

[0046] The isolated adipose tissue for transplantation was mixed with 300 μl of adipose tissue per sample and 160 μM nervonic acid or an equivalent amount of DMSO (control), and then transplanted subcutaneously on both sides of the back of syngeneic mice. Five weeks after transplantation, the transplanted adipose tissue was harvested and fixed with 4% paraformaldehyde (PFA).

[0047] A schematic diagram of the fat transplantation and analysis method using nervonic acid is shown in Fig. 1. 160 μM nervonic acid was treated in adipose tissue derived from a mouse model, and fat was transplanted into a syngeneic mouse. Five weeks later, the degree of vacuole formation and the area occupied by the vacuoles in the transplanted fat tissue were observed through H&E staining. Specifically, the fixed transplanted fat tissue obtained through the above-mentioned process was embedded in paraffin, sectioned into 4 μm thick sections, and the area occupied by the vacuoles in the transplanted fat was observed and photographed through hematoxylin & eosin staining (H&E staining). At this time, the vacuole selection criteria were based on the criteria of Hu et al. (Hu Yet et al., Dermatol. Surg., 44(7):976-984, 2018), and a part with a diameter exceeding 120 μm was selected as a vacuole (cyst).

[0048] As a result, as shown in Fig. 2a, it was confirmed that the number and area of ​​vacuoles (cysts) marked with "*" in the transplanted fat were reduced in the Nervonic acid treatment group compared to the control group (DMSO).

[0049] The area of ​​vacuoles (cysts) in the transplanted fat was quantified by H&E staining 5 weeks after treating 160 μM Nervonic acid in adipose tissue derived from a mouse model and transplanting the fat into the same mouse, and the results are shown in Fig. 2b. Specifically, the sum of the areas of vacuoles in the transplanted fat measured using QuPath v0.5.0 (Bankhead Pet et al., Sci Rep., 7(1):16878, 2017) was divided by the area of ​​the total transplanted fat, and the area of ​​the Nervonic acid-treated group was compared to the area of ​​the control group (DMSO).

[0050] As a result, as shown in Fig. 2b, the fear area in the Nervonic acid treatment group was reduced by 0.63±0.07 times compared to the control group.

[0051]

[0052] Example 2. After performing allogeneic fat transplantation by treating mouse-derived adipose tissue with nervonic acid, the fat cells in the transplanted fat and the fat transplantation efficiency were confirmed.

[0053] To determine the effect of nervonic acid treatment on the transplantation efficiency of adipocytes after fat grafting, 160 μM nervonic acid was treated in adipose tissue derived from a mouse model and transplanted into a syngeneic mouse. 5 weeks later, lipid accumulation in the transplanted fat was confirmed through Oil Red O staining and photographed. Specifically, the transplanted fat tissue collected 5 weeks after transplantation was frozen into 12 μm thick sections, and staining was performed using the NovaUltra Special Stain Kit (IHC world, MD, USA) according to the method provided by the vendor. The stained tissue was photographed using Scanscope AT2.

[0054] As a result, as shown in Fig. 3a, it was confirmed that lipid accumulation in red-stained adipocytes increased in the Nervonic acid treatment group compared to the control group (DMSO).

[0055] Five weeks after transplanting 160 μM Nervonic acid-treated adipose tissue derived from a mouse model into a syngeneic mouse, lipid accumulation in the transplanted fat was quantified using Oil Red O staining.

[0056] As a result, as shown in Fig. 3b, fat accumulation increased 1.89±0.31 times in the Nervonic acid treatment group compared to the control group (DMSO).

[0057]

[0058] Example 3. Confirmation of the efficacy of nervonic acid treatment on increasing the survival rate of fat cells in transplanted fat after performing allogeneic fat transplantation on mouse-derived adipose tissue.

[0059] To confirm the efficacy of nervonic acid in increasing the survival rate of adipocytes after fat transplantation, 160 μM nervonic acid was treated in adipose tissue derived from a mouse model and transplanted into a syngeneic mouse. Five weeks later, the amount of viable adipocytes expressing Perilipin-1 in the transplanted fat was confirmed and photographed through immunohistochemical staining of the Perilipin-1 marker.

[0060] Specifically, the transplanted adipose tissues, sectioned into 4 μm thick, were rehydrated in graded concentrations of ethanol, and then cleared with xylene. The cleared tissues were retrievaled in Dako Target Retrieval Solution pH6 Citrate for 7 minutes and then washed three times with 1X PBS. The washed samples were treated with Dako Real Peroxidase-blocking solution and MOM blocking solution for 15 and 30 minutes, respectively, and washed three times with 1X PBS. After treatment with the primary antibody against Perilipin-1 (Cell Signaling Technology, MA, USA) overnight at 4℃. The following day, the secondary antibody (HRP-Labelled Polymer Anti-rabbit) was treated for 30 minutes. After washing three times with PBS, the tissues were treated with Dako Lipid DAB and Substrate chromogen system, and then washed in the same manner and stained with hematoxylin. After washing, dehydration, and mounting, they were photographed and observed.

[0061] As a result, as shown in Fig. 4a, it was confirmed that the number of living fat cells expressing Perilipin-1 in the cell membrane of the transplanted fat increased in the Nervonic acid treatment group compared to the control group (DMSO).

[0062] Five weeks after the adipose tissue derived from a mouse model was treated with Nervonic acid and transplanted into a syngeneic mouse, the area occupied by adipocytes expressing Perilipin-1 in the transplanted fat was quantified using Perilipin-1 immunohistochemical staining.

[0063] As a result, as shown in Fig. 4b, the area occupied by living fat cells in the transplanted fat increased by 2.69±0.56 times in the Nervonic acid treatment group compared to the control group (DMSO).

[0064]

[0065] Example 4. Confirmation of the efficacy of treating mouse-derived adipose tissue with nervonic acid and performing allogeneic fat transplantation on reducing inflammation in the transplanted fat.

[0066] To confirm the efficacy of nervonic acid in reducing inflammation in adipose tissue after fat transplantation, 160 μM nervonic acid was treated in adipose tissue derived from a mouse model and transplanted into syngeneic mice. Five weeks later, the gene expression levels of markers related to inflammation were compared at the mRNA level using qRT-PCR.

[0067] Specifically, the transplanted adipose tissue collected 5 weeks after transplantation was frozen and ground using liquid nitrogen, and then extracted using Trizol (Invitrogen, MA, USA) according to the method provided by the vendor. cDNA was synthesized using SuperScriptTM IV Reverse Transcriptase (Invitrogen, MA, USA). qRT-PCR was performed using the primers specified in Table 1, and 2 -ΔΔCt It was quantified through analysis.

[0068]

[0069] As a result, as shown in Fig. 5, compared to the control group (DMSO), the expression level of the inflammatory marker TNF-a decreased by 0.38±0.16 times in the Nervonic acid treatment group, while the expression level of the anti-inflammatory marker IL-10 increased by 1.22±0.04 times, confirming that inflammation in the transplanted fat was reduced.

[0070]

[0071] Example 5. Confirmation of the efficacy of nervonic acid treatment on angiogenesis in transplanted fat after transplantation of allogeneic fat tissue derived from mice.

[0072] To confirm the effect of nervonic acid on angiogenesis in adipose tissue after fat transplantation, 160 μM nervonic acid was treated in adipose tissue derived from a mouse model and transplanted into a syngeneic mouse. Five weeks later, the vascular structure in the transplanted adipose tissue was photographed and confirmed through H&E staining and immunohistochemical staining for CD31, a vascular endothelial factor.

[0073] Specifically, immunohistochemical staining was performed in the same manner as described above, using a primary antibody against CD31 (Cell Signaling Technology, MA, USA). As a result, as shown in Figure 6a, the vascular structures indicated by arrows in the H&E staining and the vascular structures expressing CD31 were confirmed to increase in the nervonic acid-treated group compared to the control group (DMSO).

[0074] Five weeks after fat transplantation into a syngeneic mouse model, which was treated with neuronic acid, the area occupied by vascular structures in the transplanted fat was quantified using CD31 immunohistochemical staining.

[0075] As a result, as shown in Fig. 6b, the area of ​​blood vessel structures expressing CD31 increased 1.91±0.17 times in the Nervonic acid treatment group compared to the control group (DMSO).

[0076] Furthermore, to further confirm the effect of neuronic acid on angiogenesis in transplanted fat at the genetic level, adipose tissue derived from a mouse model was treated with 160 μM neuronic acid and transplanted into syngeneic mice. Five weeks later, the gene expression levels of markers related to angiogenesis were compared at the mRNA level via qRT-PCR. Specifically, the RNA extraction, cDNA synthesis, and result analysis methods used for qRT-PCR were identical to those described above, and qRT-PCR was performed using the primers specified in Table 2.

[0077]

[0078] As a result, as shown in Fig. 6c, it was confirmed that VEGFA, Angpt-1, and TEK, which are markers for angiogenesis, increased by 1.27±0.04 times, 2.02±0.40 times, and 1.13±0.01 times, respectively, in the Nervonic acid treatment group compared to the control group (DMSO).

[0079] Through the above results, it was confirmed that Nervonic acid can be used as an adjuvant for fat transplantation by improving the efficiency of fat transplantation through increasing the survival rate of fat cells, reducing inflammation, and promoting angiogenesis during fat transplantation.

[0080]

[0081] According to the present invention, when performing a fat tissue transplantation surgery, the survival rate and engraftment rate of fat included in the transplanted fat tissue can be increased, thereby improving the efficiency of fat tissue transplantation.

[0082]

[0083] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0084]

[0085] Electronic file attached.

Claims

1. A composition for fat transplantation containing adipose tissue, characterized in that the adipose tissue is treated with nervonic acid.

2. A composition according to claim 1, characterized in that the nervonic acid is treated at a concentration of 10 to 1000 μM.

3. A fat grafting composition according to claim 1, characterized in that the fat grafting composition is for fat tissue transplant surgery, filler, or fat repositioning surgery.

4. A composition for increasing the efficiency of fat transplantation containing nervonic acid as an active ingredient.

5. A composition characterized in that the nervonic acid in the fourth paragraph is contained at a concentration of 10 to 1000 μM.

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