Application of nano silicate in assisting fat transplantation

Abstract

The invention discloses an application of nano silicate in assisting fat transplantation. The application comprises the following steps: preparing the nano silicate into a silicate ion solution by adopting a serum-free culture medium, mixing the silicate ion solution with a fat aspirator, and performing transplantation injection, meanwhile, the invention discloses a fat graft obtained by mixing a silicate ion solution prepared from the nano silicate by adopting a serum-free culture medium with a fat aspirator, a preparation method of the fat graft, and application of the fat graft as a soft tissue filler. Compared with traditional fat transplantation, the fat graft provided by the invention has the advantages that by synergistically promoting angiogenesis, inhibiting inflammation / fibrosis and protecting early excessive apoptosis of fat cells due to ischemia and hypoxia, the long-term survival quality of transplanted fat can be remarkably optimized, the fat retention rate is increased to 60% or above, and the survival rate of the transplanted fat is increased to 60% or above. Wide application prospects are realized in the aspect of clinical fat transplantation.

Description

Technical Field

[0001] This invention relates to the field of bioengineering materials technology, specifically to the application of nano-silicates in assisted fat grafting. Background Technology

[0002] Fat grafting is a plastic surgery technique that involves extracting and processing fat from one area (such as the abdomen or thighs) where fat accumulates, and then injecting it into areas that require filling or shaping (such as the face, breasts, or buttocks). It can be used for both cosmetic shaping and reconstructive treatments (such as for trauma or post-operative defects).

[0003] Autologous fat grafting, as a soft tissue filler, is increasingly valued by plastic surgeons both domestically and internationally due to its advantages such as abundant sources, easy harvesting, simple operation, good filling and appearance, and no rejection reaction. However, the survival rate of transplanted fat is only 30-50%, requiring some patients to undergo multiple fillings, especially in areas such as the bridge of the nose and chin, where the local tissue volume is small, the vascular network is not rich, and the skin tension is high, resulting in a low survival rate of transplanted fat and making it difficult to achieve the desired effect.

[0004] The low survival rate after fat grafting is mainly due to insufficient blood supply re-establishment, leading to necrosis and apoptosis of transplanted fat cells. The survival of transplanted adipose tissue is closely related to the re-establishment of blood supply within the adipose tissue. Currently, the main methods to improve the vascularization of fat grafts include cell-assisted methods, such as adding SVF, ADSCs, and BMSCs. Stem cells can secrete pro-angiogenic factors (such as VEGF and HIF-1α) to stimulate new blood vessel formation, but these methods have short-term effects, are expensive, and may pose a tumorigenic risk while improving fat grafting. Therefore, they are not suitable for widespread application.

[0005] Therefore, how to overcome the shortcomings of traditional fat grafting, such as insufficient vascularization leading to cell apoptosis and high absorption rate, is an urgent problem to be solved. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides the application of nano-silicates in assisted fat grafting. The aim is to significantly enhance vascularization in fat through the innovative application of silicate materials, activate the vascular-related HIF / 1α / VEGF pathway, and ultimately increase the fat retention rate to >60%.

[0007] In a first aspect, this invention provides the application of nano-silicates in assisted fat grafting. The nano-silicates are prepared into a silicate ion solution using a serum-free culture medium, mixed with aspirated fat, and then injected for transplantation.

[0008] In a second aspect, the application further provides a fat graft, which is prepared by mixing nanosilicate ionsolution prepared from nanosilicate and serum-free medium with fat aspirate.

[0009] In a third aspect, the application further provides a preparation method of the fat graft of the second aspect, which comprises soaking nanosilicate in serum-free medium to prepare nanosilicate ionsolution with a concentration of 0.001-0.002 g / mL, mixing fat aspirate with the nanosilicate ionsolution at a volume ratio of 2-4:1, and then oscillation incubation to obtain the fat graft.

[0010] As an implementation manner of the third aspect, the preparation method of the nanosilicate ionsolution comprises the following steps: soaking 1 g of nanosilicate in 5 mL of serum-free medium, shaking uniformly in a humidified incubator at 37℃ / 5% CO2 for 18-36 h, collecting supernatant, sterilizing, diluting with serum-free medium to a concentration of 0.001-0.002 g / mL, and then storing at 0-4℃ to obtain the nanosilicate ionsolution.

[0011] As an implementation manner of the third aspect, the method for obtaining fat aspirate comprises the following steps: using a 3.0 mm blunt needle to aspirate fat, and centrifuging at 1800-3000 rpm for 1-5 min to remove oil droplets and blood water.

[0012] As an implementation manner of the third aspect, the oscillation incubation method comprises the following steps: oscillation incubation at 37℃ and 100 rpm for 30 min.

[0013] As an implementation manner of the first aspect or the second aspect, the nanosilicate is nanocalcium silicate, and the particle size of the nanosilicate is 200-500 mesh.

[0014] In a fourth aspect, the application further provides application of the fat graft of the second aspect, which is used as a filler for filling concave parts of face (such as tear grooves), chest or buttocks, or for filling skin photoaging and scars.

[0015] Compared with the prior art, the application has the following beneficial effects:

[0016] The application innovatively prepares nanosilicate ionsolution from nanosilicate and serum-free medium, and then mixes the nanosilicate ionsolution with fat aspirate to form a nanosilicate ion functionalized fat graft, which forms a biomimetic composite system for fat transplantation and soft tissue filling. In vitro experiments have verified that the nanosilicate ion functionalized fat graft is nontoxic to human umbilical vein endothelial cells, has a significant proliferative effect in a suitable concentration range, can promote the secretion of endothelial growth factor (VEGFA), significantly increases the migration speed of HUVECs (healing rate increase ≥ 30%), and enhances the vascularization and tissue survival mechanism by promoting the migration ability of HUVECs.

[0017] The fat graft provided by the application can significantly accelerate the construction of a functional blood vessel network after fat transplantation by activating the angiogenesis pathway, and improve the excessive apoptosis caused by insufficient blood supply in the early stage of fat transplantation; the rapidly released bioactive ions of the fat graft can immediately stimulate local host cells (especially endothelial cells), rapidly up-regulate pro-angiogenic factors, and create a favorable "start" microenvironment for subsequent repair.

[0018] The application innovatively uses a completely soluble ion solution to replace the solid particles of the prior art, which can effectively avoid the complication of granuloma / calcification (histological foreign body reaction rate < 2%), and has zero risk of foreign body residue.

[0019] The application can realize one-step mixing in 30 minutes during the operation (without cell separation / exogenous growth factors), the cost is reduced to 1 / 10 of the traditional technology (single consumable < $100), and the clinical operation process is revolutionarily simplified.

[0020] The application mixes silicate and fat particles as a soft tissue filler, and the core advantage is that the silicate can significantly improve the survival rate of fat cells and reduce postoperative absorption by stimulating angiogenesis and collagen deposition, achieving more durable volume maintenance; at the same time, the silicate particles can provide necessary structural support to make up for the defect of pure fat texture being too soft, and enhance the shaping stability and contour clarity of the filled area; in addition, the composite has the advantages of convenient source, low cost, and mature and simple operation, which not only promotes graft integration and reduces the risk of complications, but also realizes soft tissue augmentation with high cost performance and natural effect.

[0021] In summary, the fat graft provided by the application significantly optimizes the long-term survival quality of transplanted fat (fat retention rate of more than 60% at 3 months) by synergistically promoting angiogenesis (microvessel density increased by more than 2 times compared with traditional fat transplantation), inhibiting inflammation / fibrosis, and protecting fat cells, solving the pain point of high fat absorption rate in the prior art fat transplantation, and having a broad application prospect in clinical fat transplantation. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Fig. A, B and C correspond to the results of the first day, the third day and the fifth day, respectively, in the toxicity results of different concentrations of CS on human umbilical vein endothelial cells in the CCK-8 experiment of experimental example 1;

[0023] Figure 2 The counting results of human umbilical vein endothelial cells in each concentration group under fluorescence microscope in the EDU cell proliferation experiment of experimental example 1 are shown in the following table:

[0024] Figure 3Statistical results of the percentage of EDU positive cells in each concentration group in the EDU cell proliferation experiment of experimental example 1;

[0025] Figure 4 Statistical results of the concentration (pg / mL) of vascular endothelial growth factor (VEGFA) in each concentration group in experimental example 2;

[0026] Figure 5 The photographs of the scratch area of the CS experimental group and the blank control group at 0h, 12h and 24h in the endothelial cell scratch experiment of experimental example 3;

[0027] Figure 6 Statistical results of the scratch healing rate of the CS experimental group and the blank control group at 12h and 24h in the endothelial cell scratch experiment of experimental example 3;

[0028] Figure 7 The photographs of the in vitro blood vessel formation of human umbilical vein endothelial cells in different groups under the inverted microscope in the endothelial cell tube formation experiment of experimental example 4;

[0029] Figure 8 Statistical results of the number of tube lumen branch nodes of human umbilical vein endothelial cells in the endothelial cell tube formation experiment of experimental example 4;

[0030] Figure 9 The photographs of the general observation at different time points after transplantation in the fat transplantation experiment of experimental example 5;

[0031] Figure 10 Statistical results of the fat volume retention rate in the fat transplantation experiment of experimental example 5;

[0032] Figure 11 The photographs of the H&E staining of fat in the fat transplantation experiment of experimental example 5;

[0033] Figure 12 Statistical results of the fat cell integrity, cystic / vacuolar cells, inflammatory cells and fibrotic cells in the fat transplantation experiment of experimental example 5, wherein A, B and C correspond to the results of the 2nd week, the 4th week and the 12th week, respectively;

[0034] Figure 13 The photographs of the detection of the density of blood vessel neogenesis by CD31 immunohistochemistry in the fat transplantation experiment of experimental example 5;

[0035] Figure 14 Statistical results of the number of cells of CD31 + in the fat transplantation experiment of experimental example 5;

[0036] Figure 15 The expression results of VEGF and HIF-1α stained by immunohistochemistry and immunofluorescence, respectively, in the fat transplantation experiment of experimental example 5;

[0037] Figure 16 Figure 9 is a fluorescence image of TUNEL staining for detecting apoptotic cells in the fat grafting experiment of Example 5;

[0038] Figure 17 Figure 10 is a statistical result of the percentage of apoptotic cells in total cells detected by TUNEL staining in the fat grafting experiment of Example 5;

[0039] Figure 18 Figure 11 is a photograph of the tissue and implant in the soft tissue filling experiment of Example 6 at 2 weeks and 12 weeks;

[0040] Figure 19 Figure 12 is a statistical result of the volume retention rate of the filler in the soft tissue filling experiment of Example 6 at 2 weeks and 12 weeks;

[0041] Figure 20 Figure 13 is a HE staining image in Example 6;

[0042] Figure 21 Figure 14 is a statistical result of the histological evaluation after HE staining in Example 6;

[0043] Figure 22 Figure 15 is a Masson staining image in Example 6;

[0044] Figure 23 Figure 16 is a statistical result of the collagen area fraction after Masson staining in Example 6;

[0045] Figure 24 Figure 17 is a CD31 immunohistochemical staining image in Example 6;

[0046] Figure 25 Figure 18 is a statistical result of the number of capillaries in each field of view after CD31 immunohistochemical staining in Example 6. DETAILED DESCRIPTION

[0047] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with specific examples. It should be understood that the specific examples described herein are only used to explain the present application and should not be used to limit the present application.

[0048] In the embodiments of the present application, the words such as “exemplary” or “for example” are used to represent an example, illustration or description. Any embodiment or design scheme described as “exemplary” or “for example” in the embodiments of the present application should not be interpreted as more preferred or more advantageous than other embodiments or design schemes. In fact, the words such as “exemplary” or “for example” are intended to present the relevant concept in a specific manner.

[0049] Unless otherwise specified, the raw materials in the examples are commercially available.

[0050] Calcium silicate (CS) powder (200 mesh, 400 mesh, 500 mesh) was purchased from Kunshan Huaxia Science and Technology New Material Co., Ltd.

[0051] The serum-free medium was a commercially available Gibco DMEM medium.

[0052] BMSCs were purchased from Saiye Biotechnology Co., Ltd.

[0053] Human umbilical vein endothelial cells were commercially available HUVEC.

[0054] Example 1: The example describes one of the methods for preparing a fat graft, which includes the following steps:

[0055] S1, prepare a silicate ion solution: immerse 1 g of nano calcium silicate (CS) powder (400 mesh) in 5 mL of serum-free medium, shake uniformly in a humidified incubator at 37°C / 5% CO2 for 24 h, collect the supernatant, sterilize with a filter (Millipore, 0.22um), obtain the stock solution, dilute the stock solution with serum-free medium, prepare a CS ion solution with a concentration of 0.0015 g / mL, and store at 4°C;

[0056] S2, obtain fat aspirate: use a 3.0 mm blunt needle to aspirate fat, centrifuge at 2000 rpm for 3 min to remove oil droplets and blood water, and obtain fat aspirate;

[0057] S3, mix the fat aspirate and the CS ion solution at a volume ratio of 3:1, incubate at 37°C with 100 rpm shaking for 30 minutes, then stand for 15 minutes, and obtain the fat graft.

[0058] Example 2: This example describes a second method for preparing a fat graft, which includes the following steps:

[0059] S1, prepare a silicate ion solution: immerse 1 g of nano calcium silicate (CS) powder (200 mesh) in 5 mL of serum-free medium, shake uniformly in a humidified incubator at 37°C / 5% CO2 for 18 h, collect the supernatant, filter with a filter (Millipore, 0.22um), obtain the stock solution, dilute the stock solution with serum-free medium, prepare a CS ion solution with a concentration of 0.001 g / mL, and store at 0°C;

[0060] S2, obtain fat aspirate: use a 3.0 mm blunt needle to aspirate fat, centrifuge at 1800 rpm for 1 min to remove oil droplets and blood water, and obtain fat aspirate;

[0061] S3, mix the fat aspirate and the CS ion solution at a volume ratio of 4:1, incubate at 37°C with 100 rpm shaking for 30 minutes, then stand for 10 minutes, and obtain the fat graft.

[0062] Example 3: This example describes a third method for preparing a fat graft, comprising the following steps:

[0063] S1, preparing a silicate ion solution: 1 g of nano calcium silicate (CS) powder (500 mesh) was soaked in 5 mL of serum-free medium, shaken in a humidified incubator at 37°C / 5% CO2 for 36 h, the supernatant was collected, sterilized and filtered with a filter (Millipore, 0.22 um), 0.22 um, to obtain a stock solution, which was then diluted with serum-free medium to prepare a CS ion solution with a concentration of 0.002 g / mL, which was stored at 2°C;

[0064] S2, obtaining fat aspirate: using a 3.0 mm blunt needle for liposuction, centrifuging at 3000 rpm for 5 min to remove oil droplets and blood water, obtaining fat aspirate;

[0065] S3, mixing the fat aspirate with the CS ion solution at a volume ratio of 2:1, incubating at 37°C for 30 min, and then standing for 20 min to obtain the fat graft.

[0066] Experimental Example 1: This experimental example studies the effect of the concentration of silicate on the proliferation of human umbilical vein endothelial cells, including CCK-8 experiment and EDU cell proliferation experiment.

[0067] First, on the basis of Example 1, a series of gradient concentrations of calcium silicate (CS) were prepared by the following method: 1 g of CS (400 mesh) was soaked in 5 mL of serum-free medium, shaken in a humidified incubator at 37°C / 5% CO2 for 24 h, the supernatant was collected, sterilized with a filter (Millipore, 0.22 um), and a stock solution was obtained, which was then diluted with serum-free medium to prepare gradient-diluted CS ion solutions (1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128 and 1 / 256 dilutions), i.e. a fresh medium containing a series of gradient concentrations of calcium silicate (CS) (1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256) was obtained, which was stored at 4°C and used in subsequent experimental examples.

[0068] The CCK-8 experiment procedure is as follows:

[0069] ①Human umbilical vein endothelial cells (HUVECs, commercially available product) were inoculated at a density of 5×103 cells per well in a 96-well plate, incubated in a standard human umbilical vein endothelial cell culture medium (commercially available product) in an incubator (37°C, 5% CO2) overnight to adhere to the wall;

[0070] ②The next day, the old medium was aspirated, and the experimental group was replaced with fresh medium containing a series of gradient concentrations of calcium silicate (CS) (1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256). The cells were treated with fresh medium containing CS for a specific period of time (e.g., 1 day, 3 days, 5 days), and a blank control group containing only the medium (without cells) was set up.

[0071] ③After the treatment was completed, about 100 μL of medium was carefully aspirated from each well (to avoid disturbing the cells), and then 100 μL of fresh medium and 10 μL of CCK-8 reagent (commercial product) were directly added to each well. The 96-well plate was returned to the incubator and incubated for 2 hours in the dark.

[0072] ④After the incubation was completed, the absorbance (OD value) of each well was immediately measured at 450 nm using a microplate reader. The cell proliferation rate was calculated by comparing the OD value of the experimental group with that of the control group (usually the 0 concentration group), and the blank well background value was subtracted. The results are shown in Figure 1 , where a higher OD value indicates stronger metabolic activity and stronger cell proliferation ability.

[0073] The steps of the EDU cell proliferation experiment are as follows:

[0074] ①Human umbilical vein endothelial cells (HUVECs, commercial product) were inoculated in a 24-well plate, and the experimental group was replaced with medium containing a series of gradient concentrations of CS (1 / 64, 1 / 128, 1 / 256) for 24 hours of treatment.

[0075] ②In the last 2-4 hours before the treatment was completed (key step), EDU working solution was added to the medium of all wells (including the control group);

[0076] ③After the incubation was completed, the medium containing EDU was completely aspirated, and the cells were gently washed with pre-cooled PBS for 2-3 times. Then, the cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes and washed with PBS for 3 times;

[0077] ④The cells were then permeabilized with 0.3% Triton X-100 in PBS at room temperature for 10-15 minutes and washed with PBS for 3 times;

[0078] ⑤Click reaction mixture containing Click reaction buffer, CuSO4, fluorescently labeled azide (such as Alexa Fluor 488 Azide), and reaction accelerator was added, and the mixture was incubated at room temperature in the dark for 30 minutes to allow specific click chemistry reaction between the fluorescent dye and EDU. The cells were thoroughly washed with PBS for 3 times to remove unbound dye;

[0079] ⑥The cells were observed and counted under a fluorescence microscope (see Figure 2), the percentage of EDU positive cells of each concentration group was calculated (see Figure 3 to evaluate the direct effect of CS on cell proliferation.

[0080] Experimental result analysis: From the results of Figure 1 , CS had no cytotoxicity to HUVECs at dilution ratios of 1 / 4 to 1 / 256. Among them, there was no statistical difference in the proliferation results between the concentration group with a dilution ratio of 1 / 256 and the concentration group with a dilution ratio of 1 / 128. From the results of Figure 2 and Figure 3 , the counting results of the concentration group with a dilution ratio of 1 / 256 and the concentration group with a dilution ratio of 1 / 128 were both higher, but the percentage of EDU positive cells of the concentration group with a dilution ratio of 1 / 128 was the highest.

[0081] The above experiments proved that CS had no cytotoxicity to HUVECs, and within a certain dilution range, CS showed a stimulating effect on cell proliferation, and the effective concentration of stimulation was different for different cell types. This experiment took human umbilical vein endothelial cells as an example to illustrate that CS had a significant stimulating effect on endothelial cell proliferation.

[0082] At the same time, since the concentration group with a dilution ratio of 1 / 128 had the strongest stimulating effect on the proliferation of human umbilical vein endothelial cells, we selected the concentration group with a dilution ratio of 1 / 128 as the best dilution concentration, which corresponded to a CS concentration of about 0.0015 g / mL.

[0083] Experimental Example 2: This experimental example studies the effect of different concentrations of silicate on the secretion of endothelial growth factor (VEGF).

[0084] Through the preliminary screening of CCK-8 experiment and EDU cell proliferation experiment in the above experimental example 1, we selected 1 / 56, 1 / 128 and 1 / 256 concentrations for qPCR experiment to study the expression amount of endothelial growth factor (VEGFA) under different concentrations of silicate.

[0085] Experimental method: Endothelial cells were inoculated in culture plates and replaced with culture medium containing gradient concentration of CS (1 / 64, 1 / 128, 1 / 256) after adhesion for 24 hours. After treatment, the cell supernatant was collected aseptically and centrifuged at 12,000xg at 4°C for 10 minutes to remove cell debris. The VEGFA ELISA kit was used according to the instructions: the standard and sample were added to the pre-coated antibody 96-well plate, and then biotinylated detection antibody, horseradish peroxidase (HRP) labeled streptavidin and TMB substrate were added in turn. After termination of the reaction, the OD value was measured at 450 nm. The VEGFA concentration (pg / mL) in the supernatant was quantified by standard curve, and each group had more than 3 replicate wells.

[0086] The experimental results are shown in Figure 4 Fig. 1, it was found that VEGFA was expressed in all three proportions. Based on the results of Experimental Example 1, we finally selected 1 / 128 for subsequent experiments.

[0087] Experimental Example 3: This experimental example studies the effect of silicate on the scratch healing of human umbilical vein endothelial cells.

[0088] The HUVECs monolayer scratch model was used for cell scratch experiment to compare the cell migration ability of the silicate-containing culture medium and the control group.

[0089] Experimental method: Human umbilical vein endothelial cells were inoculated in a 6-well plate. When the cells were 90% confluent, a straight line scratch was made perpendicular to the marker line with a sterile gun head. The detached cells were washed away with PBS. The serum-free medium containing 1 / 128 concentration of CS was replaced, and a blank control group containing only the medium (without CS) was set. At 0h, 12h, and 24h, the scratch area was photographed at the same position (results shown in Figure 5 ). ImageJ software was used to quantify the scratch area, calculate the migration rate (%), and statistically analyze the results (see Figure 6 ) to evaluate the effect of CS on migration.

[0090] As can be seen from Figure 5 , the scratch area of the CS group is smaller than that of the blank control group after 12h and 24h. As can be seen from Figure 6 , the migration rate of the CS group is higher after 12h and 24h. This indicates that CS (1 / 128) significantly improves the migration speed of HUVECs (healing rate increase ≥ 30%), and clarifies its mechanism of enhancing vascularization and tissue survival by promoting the migration ability of HUVECs, providing key experimental evidence for silicate-assisted fat transplantation.

[0091] Experimental Example 4: This experimental example studies the effect of silicate on angiogenesis.

[0092] The HUVECs / Matrigel in vitro model was used for cell tube formation experiment to compare the tube formation ability of the CS treatment group and the control group.

[0093] The experimental method is as follows:

[0094] ① Matrigel plating: Pre-cooled Matrigel gel was evenly plated in a 96-well plate (50μL / well), and polymerized at 37℃ for 30 minutes. Human umbilical vein endothelial cells (HUVECs) suspension (5×10 4 / mL) was mixed with serum-free medium containing 1 / 128 concentration of CS, and inoculated on the Matrigel surface (100μL / well). A blank control group containing only the medium (without CS) was also set.

[0095] ②Culture observation: 37℃ culture for 3-6 hours, randomly take multiple fields under an inverted microscope (see Figure 7 );

[0096] ③Quantitative analysis: ImageJ software (Angiogenesis Analyzer plug-in) quantifies the number of lumen branch nodes (see Figure 8 ) to evaluate the effect of silicate on promoting angiogenesis.

[0097] As can be seen from Figure 7 and Figure 8 , the number of branch points formed by endothelial cells in the CS-containing medium within 3-6 hours is increased by ≥25% (p<0.05), which confirms that CS significantly accelerates the construction of functional vascular networks after fat transplantation by activating the angiogenesis pathway.

[0098] Example 5: Fat transplantation experiment one

[0099] Fat grafts were prepared according to the method and parameters of Example 1, and fat transplantation experiments were performed using the prepared fat grafts, respectively, to explore the effect of silicate-assisted fat transplantation on fat retention in nude mice, the pathological results of silicate-assisted fat transplantation survival quality, and the improvement of silicate-assisted fat transplantation in fat vascularization and cell apoptosis in nude mice.

[0100] The experimental method is as follows:

[0101] ①Fat pretreatment: Collect waste fat from plastic liposuction surgery, wash with PBS for 3 times to remove blood cells, centrifuge at 1000g for 5 minutes, and obtain middle layer fat particles;

[0102] ②Take the CS ion solution prepared in Example 1 with a CS concentration of 0.0015g / mL (corresponding to the dilution ratio of 1 / 128 in the CS-containing fresh culture medium in Test Example 1), mix the above middle layer fat particles with the CS ion solution at a volume ratio of 3:1, and mix at 37℃ for 30min to form a fat graft;

[0103] ③Nude mouse transplantation: Select 6-8 week old BALB / c nude mice (n≥5 / group), prepare subcutaneous compartments on both sides of the dorsal midline after anesthesia;

[0104] ④Inject 200μL of fat grafts (CS experimental group: fat+CS; stem cell positive control group: fat+BMSCs; blank control group: fat+culture medium group) on each side, and close the incision with 5-0 suture;

[0105] ⑤Postoperative management: Apply antibiotics to the wound to prevent infection;

[0106] ⑥Kill and take samples in batches at 2, 4, and 12 weeks (see Figure 9) ; volume retention was calculated using the drainage method: the whole grafts at different time points were immersed in a graduated cylinder, the volume was calculated according to the water level rise, and the data were recorded. Subsequently, the volume retention was calculated (see Figure 10 ), and the calculation formula was as follows:

[0107] Volume retention rate (%) = (final volume of the experimental group / initial injection volume) x 100%;

[0108] ⑦ Histological analysis: the adipocyte survival / liquefaction cysts were evaluated by H&E staining (see Figure 11 , Figure 12 ), the vascular neogenesis density at 2 and 12 weeks was detected by CD31 immunohistochemistry (see Figure 13 , Figure 14 ), the proportion of apoptotic cells in the adipose graft at 1 week was detected by TUNEL staining, and the expression of VEGF and HIF-1α was respectively stained by immunohistochemistry and immunofluorescence.

[0109] Experimental results and analysis:

[0110] (1) The effect of silicate-assisted fat transplantation on fat retention in nude mice

[0111] As can be seen from the results of Figure 9 and Figure 10 , at 4 weeks after the operation, the fat volume retention rate of the blank control group was (43.68+14.20)%, that of the stem cell positive control group was (61.88+14.2)%, and that of the CS experimental group was (70.58+18.18)%, among which the CS experimental group was much higher than the blank control group (P=0.0015);

[0112] At 12 weeks after the operation, the fat wet weight retention rate of the blank control group was (41.30+13.86)%, that of the stem cell positive control group was (57.16+16.88)%, and that of the CS experimental group was (62.18+3.020)%. The results suggest that CS can improve the retention rate of transplanted fat, and the effect is similar to that of stem cells.

[0113] (2) Pathological results of the survival quality of silicate-assisted fat transplantation

[0114] As can be seen from Figure 11 , at 2 weeks after the operation, there were a large number of inflammatory cell infiltration and partial fibrosis in the adipocytes of the blank control group, and the fat integrity was better after CS treatment compared with the blank control group (P<0.05).

[0115] We analyzed that in the early stage of fat grafting (acute inflammation stage), the main role of CS is usually to initiate or enhance the necessary pro-inflammatory response, which is a key mechanism for subsequent promotion of vascularization and tissue integration. It activates immune cells and endothelial cells by releasing bioactive ions (Si, Ca), driving the expression of anti-inflammatory factors and angiogenic factors.

[0116] By Figure 12 It can be seen that at 4 weeks postoperatively: compared with the blank control group, the CS experimental group had less vacuoles / cysts and fibrosis (P=0.0004; P=0.0011), and at 12 weeks, the CS experimental group had significantly reduced inflammation (P=0.0298), fibrocytes (P=0.0004), and cysts / vacuoles (P=0.0013); and the integrity of adipocytes was significantly better (P=0.0009). The results suggest that silicate can improve the quality of fat graft retention and survival.

[0117] (3) Results of silicate-assisted fat grafting to improve fat vascularization in nude mice

[0118] By Figure 13 And Figure 14 It can be seen that in the early stage of vascularization: 2 weeks: the number of CD31+ cells in the stem cell positive control group and the CS experimental group was significantly higher than that in the blank control group (blank control group: 5.00±1.08%; stem cell positive control group: 9.00±2.962; CS experimental group: 9.00±3.962%).

[0119] Late stage of fat grafting: 12 weeks: the number of CD31+ cells in the stem cell positive control group and the CS experimental group was significantly higher than that in the blank control group (blank control group: 7.667±1.08%; stem cell positive control group: 11.67±2.962; CS experimental group: 9.00±3.962%). The results suggest that silicate can enhance fat graft vascularization.

[0120] We analyzed that the rapid release of bioactive ions in the CS experimental group can immediately stimulate local host cells (especially macrophages and endothelial cells), rapidly up-regulate pro-angiogenic factors, and create a favorable "start" microenvironment for subsequent repair. This is crucial in situations that require rapid establishment of initial blood supply (such as ischemic areas, early stage of transplantation).

[0121] (4) Effect of silicate on fat graft retention rate

[0122] HIF-1a is the core transcription factor of cell response to hypoxia, and its activation is the key signal starting point of early ischemic stage of transplanted fat; VEGF, as a direct downstream target gene of HIF-1a, is the core effector molecule driving angiogenesis. Both directly determine the efficiency of blood revascularization of the graft. Therefore, we take samples after one week after transplantation, and by immunofluorescence staining HIF-1a and immunohistochemical staining VEGF expression, Figure 15 A, C can be seen that, compared with the blank control group HIF-1a high expression (17.13±5.820), stem cell positive control group HIF-1a (11.31±6.910); compared with the control group and stem cell group, HIF-1a of CS experimental group were significantly reduced (4.397±1.652; p<0.05); this phenomenon shows that stem cells and silicate can improve the hypoxic microenvironment of fat graft, but silicate can be evenly mixed in fat graft, and the effect is better; from Figure 15 B, D can be seen: compared with the control group (6.78±3.42), the VEGF of CS experimental group was highly expressed (19.60±2.98), which was statistically significant; in addition, the stem cell positive control group (16.84±4.29) and the control group also had significant difference. The results suggest that silicate can change the retention rate of fat transplantation by activating HIF-1a-VEGF signaling axis.

[0123] (5) the effect of silicate on the apoptosis of the graft

[0124] After one week after transplantation, the apoptosis of adipocytes was detected by TUNEL method, and Figure 16 and Figure 17 It can be seen that the blank control group has a high peak of apoptosis (36.7±15.7%), mainly due to ischemia and hypoxia; the stem cell positive control group significantly inhibited apoptosis (21.0±5.2%); the CS experimental group reduced the apoptosis rate to 22.4±4.7% by silicate promoting vascularization, which had no statistical difference with the stem cell positive control group (p>0.05); both of them effectively improved the survival of fat, but the CS experimental group avoided the risk of cell therapy, and had more transformation potential. This study confirmed that silicate promoting vascularization can significantly reduce the apoptosis of the graft.

[0125] Example 6: soft tissue filling experiment two

[0126] Based on the preparation method of fat graft described in example 1, the effect of the ratio of fat aspirate to CS ionic solution in fat graft on fat retention rate was explored.

[0127] Experimental materials:

[0128] Thymus gland nude mice (BALB / c nude), 6-8 weeks old, 18-22 g in weight; the feeding condition is SPF level environment; totally 25, no significant difference between individuals.

[0129] Experimental grouping:

[0130] 25 nude mice were randomly divided into 5 groups, 5 in each group.

[0131] Based on the preparation method of fat grafts described in Example 1, the volume ratio of fat aspirate to CS ionic solution in Example 1 was 3:1, denoted as experimental group 1; at the same time, two different volume ratios of fat aspirate to CS ionic solution, 2:1 and 4:1, were set as experimental group 2 and experimental group 3, respectively (experimental group 2 and experimental group 3 had the same preparation method and parameters as experimental group 1 except for the different volume ratios of fat aspirate to CS ionic solution); and control group 1 (fat control group: Fat) and control group 2 (positive control group: hyaluronic acid (HA)) were set.

[0132] Injection method:

[0133] After the nude mice in each group were anesthetized, 0.2 mL of the corresponding injection material in each group was injected subcutaneously at multiple points on the back of the nude mice.

[0134] Among them, the anesthesia method: inhalation anesthesia (such as isoflurane);

[0135] Injection site: subcutaneous injection on the back;

[0136] Injection method: use of syringe specifications (such as 1 mL syringe) and needle specifications (such as 27G);

[0137] Injection volume: accurate volume of each point (200 μL), 4 points per animal;

[0138] Postoperative care: suture and erythromycin application to prevent wound infection.

[0139] The corresponding injection materials in each group were: experimental group 1 injected with fat grafts prepared by Fat+CS (3:1); experimental group 2 injected with fat grafts prepared by Fat+CS (2:1); experimental group 3 injected with fat grafts prepared by Fat+CS (4:1); control group 1 injected with pure fat (Fat); and control group 2 injected with hyaluronic acid (HA).

[0140] After the above operation injection was completed, macroscopic observation was performed, inflammation was observed on the 3rd day after injection, blood vessels were observed on the 14th day, and long-term retention rate was observed on the 90th day; at the same time, local reactions such as redness, ecchymosis, induration, abscess, necrosis, and skin discoloration were observed.

[0141] Experimental results and analysis:

[0142] (1) Retention rate of the graft

[0143] At 2W and 12W postoperatively, the animals were sacrificed by intraperitoneal injection of an overdose of sodium pentobarbital, the implantation area was completely exposed and dissected carefully, and the implant was removed together with a small amount of surrounding connective tissue (see photos in Figure 18 ).

[0144] Statistical analysis of volume retention rate using the drainage method: The entire graft at different time points was immersed in a graduated cylinder, the volume was calculated according to the water level rise, and the data were recorded. Subsequently, the volume retention rate was calculated, and the formula was as follows:

[0145] Volume retention rate (%) = (final volume of the experimental group / initial injection volume) x 100%;

[0146] The statistical results are shown in Figure 19 From the statistical results, it can be seen that the retention rate of the experimental group 1 (Fat + CS (3:1) group) was as high as 68.3% at 12 weeks, which was significantly better than that of the control group 1 (Fat group (34.5%)) and the control group 2 (HA group (51.6%)), and the advantage was due to the continuous release of ions from silicate to promote functional angiogenesis.

[0147] And the retention rate of the experimental group 1 (Fat + CS (3:1) group) was 22.5% higher than that of the HA group at 12 weeks, and it avoided the risk of late degradation and collapse of HA and pathological wrapping of highly cross-linked HA, confirming its unique value as a new generation of fillers in long-acting volume maintenance.

[0148] (2) HE staining experiment and results

[0149] After constructing the nude mouse implantation model, sample collection was first performed. The tissue block at the implantation site was removed completely and immediately fixed in 4% paraformaldehyde for more than 24 hours to ensure sufficient fixation. Subsequently, the fixed tissue was routinely paraffin-embedded: sequentially dehydrated in gradient ethanol (70%, 80%, 90%, and 100% ethanol for 1 hour each), transparentized in xylene (twice, 30 minutes each), and finally immersed in wax (60°C paraffin, twice, 1 hour each) and embedded into a wax block. Continuous sectioning was performed using a paraffin microtome with a thickness of 4 μm, and the sections were attached to glass slides treated with polylysine, and dried in a 60°C oven overnight. Before staining, deparaffination and hydration were performed: xylene deparaffination (twice, 10 minutes each), gradient ethanol (100%, 95%, 85%, and 70%) hydration for 5 minutes each, and distilled water washing. Subsequently, hematoxylin staining (5 minutes), hydrochloric acid alcohol differentiation (1 second), water return to blue (15 minutes), and eosin staining (2 minutes) were performed, followed by gradient ethanol dehydration, xylene transparency, and neutral gum sealing. The tissue morphology, inflammatory response, and fibrosis degree were observed under a microscope, and the HE staining images are shown in Figure 20Histological evaluation results are shown in Figure 21 (especially including Integrity, Cysts / Vacuoles, Inflammation, Fibrosis).

[0150] HE staining results showed that the histocompatibility of different proportions of materials had significant differences. Compared with the control group 1 (Fat group), a small amount of inflammatory cell infiltration persisted in the experimental group 3 (Fat+CS(4:1) group), indicating a lower foreign body reaction. In the experimental group 2 (Fat+CS(2:1) group), abnormal hyperplasia of collagen fiber bundles was observed between tissues, forming obvious fibrotic partitions, and the tissue structure was densified, which may not be flexible. The experimental group 1 (Fat+CS(3:1) group) showed the most ideal morphological structure, with well-surviving adipocytes, only accompanied by mild and loose fibrous tissue hyperplasia and a small amount of inflammatory cells, indicating that the graft and host tissue achieved good integration. And the inflammatory cells in the experimental group 1 (Fat+CS(3:1) group) were significantly reduced compared with the control group 2 (HA group) (P<0.05).

[0151] (3) Masson staining experiment and results

[0152] Masson staining was performed on the paraffin sections after dewaxing and hydration (the same as HE staining). First, Regaud hematoxylin or Weigert hematoxylin was used to stain the nucleus for 5-10 minutes, and then the blue color was returned after washing with water for 15 minutes. Subsequently, Biebrich scarlet-acid fuchsin solution was used to stain the cytoplasm and muscle fibers for 5-10 minutes. After the dye was poured out, without water washing, phosphomolybdic acid solution was used for treatment for about 10 minutes to differentiate and extract the red dye. After that, without water washing, aniline blue solution was immediately used for collagen fiber recoloring for 3-5 minutes. Finally, 1% glacial acetic acid was used for short-term treatment (1-2 minutes) to remove excess dye and enhance color contrast. Routine gradient alcohol dehydration, xylene transparency, and neutral resin sealing were performed. Masson staining and collagen area fraction statistics results are shown in Figure 22 and Figure 23 .

[0153] Masson staining results showed that in the control group 1 (Fat group), collagen replaced a large area of adipose tissue (accounting for 36.6 ± 4.3%). In the experimental group 2 (Fat + CS (2:1)), collagen fibers (blue) were deposited in large quantities, accounting for 40.2 ± 3.8%, which was significantly different from the control group 1 (Fat group) p < 0.05, and the fat vacuoles were wrapped in dense intervals, suggesting that excess silicate triggered a foreign body reaction, which promoted fibrosis. In the experimental group 1 (Fat + CS (3:1)), collagen was only distributed around blood vessels (accounting for 16.7 ± 2.1%), and fat vacuoles were complete and fused into pieces, with microvessels penetrating between fat lobules. This indicates that this ratio synergistically promotes vascularization and inhibits fibrosis. In the experimental group 3 (Fat + CS (4:1)), collagen was diffusely distributed (accounting for 28.9 ± 2.7%), and some fat vacuoles were atrophic, and the blood vessel density was lower than that in the experimental group 1 (Fat + CS (3:1)).

[0154] (4) CD31 immunohistochemical staining experiment and results

[0155] Before immunohistochemical staining, paraffin sections need to be deparaffinated and hydrated (steps same as HE staining), then antigen retrieval: place the sections in sodium citrate buffer at pH 6.0, heat to boiling in a microwave oven at high heat, then reduce the heat to maintain for 15 minutes, and cool naturally to room temperature. Rinse with PBS (pH 7.4) 3 times for 5 minutes each. To block endogenous peroxidase activity, add 3% H2O2 solution and incubate at room temperature for 10 minutes, then rinse with PBS. Add 5% BSA blocking solution and incubate at room temperature for 30 minutes, then add primary antibody (rabbit anti-mouse CD31 monoclonal antibody, diluted 1:200), incubate overnight at 4°C in a humidified box. The next day, warm for 30 minutes, rinse with PBS, then add HRP-labeled secondary antibody (goat anti-rabbit IgG), incubate at room temperature for 1 hour. Rinse with PBS, then use DAB developing solution (freshly prepared) to develop color, control the developing time under a microscope (about 1-3 minutes), and stop the reaction with distilled water. Lightly restain the cell nuclei with hematoxylin (30 seconds), dehydrate and transparentize, then mount with neutral resin. Observe the CD31-positive vascular structure under a microscope, and calculate the microvessel density (MVD) results are shown in Figure 24 and Figure 25 .

[0156] CD31 immunohistochemical staining results showed that the control group 1 (Fat) had weak CD31 positive signals and a small number of new microvessels, indicating that a low concentration of silicate environment was not conducive to vascular growth, which might lead to a lack of nutrients in the center of the graft. The experimental group 2 (Fat + CS (2:1)) exhibited strong pro-angiogenic ability, with a large number of dense brown microvessel networks visible, and the microvessel density was significantly the highest (11.00 ± 3.9), demonstrating the excellent pro-vascularization characteristics of high-proportion fat particles. However, the blood vessels were mostly present near the fibrous beaded, and in combination with the above Masson staining, it was known that this group was accompanied by a large amount of fibrosis; the experimental group 1 (Fat + CS (3:1)) also exhibited good vascularization (9.00 ± 1.97), although the microvessel density was slightly lower than that of the experimental group 2 (Fat + CS (2:1)), but the newly formed blood vessels were evenly distributed throughout the graft area, forming an efficient functional blood network sufficient to support the survival and metabolism of adipocytes.

[0157] Based on the above retention rate determination results, HE staining results, Masson staining results, and CD31 immunohistochemical staining results, we made the following comprehensive analysis:

[0158] At week 12, the experimental group 2 (Fat + CS (2:1)) had the highest retention rate, but in combination with HE staining (severe inflammation and fibrosis) and CD31 results (poor vascularization), it was known that this was an undesirable, non-functional high retention rate, which was clinically manifested as a hard nodular mass;

[0159] Although the experimental group 3 (Fat + CS (4:1)) had the best biological performance (better fat survival and less fibrosis), its low volume retention rate was a major shortcoming in its application as a filler;

[0160] The comprehensive data of the experimental group 1 (Fat + CS (3:1)) was the most valuable for application, as it not only exhibited good biocompatibility and integration histologically, but also maintained an acceptable volume retention rate, demonstrating its optimal trade-off between effectiveness (effect) and durability (stability) as a soft tissue filler.

[0161] At the same time, it was reflected that in the embodiments of the present application, the Fat + CS (3:1) of embodiment 1 could be the best embodiment.

[0162] The above experimental data confirmed that silicate could significantly optimize the long-term survival quality of transplanted fat by synergistically promoting angiogenesis, inhibiting inflammation / fibrosis, and protecting adipocytes. And it could achieve:

[0163] ①Survival breakthrough: Activate endogenous angiogenesis by silicate ions, significantly improve fat graft retention rate (vs traditional pure fat grafting), and solve the industry problem of >60% mortality rate;

[0164] ②Clinical safety: No exogenous growth factor / serum addition, avoiding immune rejection and ethical risks;

[0165] ③High conversion efficiency: Raw material cost reduced by 80%, compatible with existing liposuction equipment, creating a ready-to-use long-acting soft tissue filler, achieving "transplantation regeneration".

[0166] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions described in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. The application of nano-silicates in assisted fat grafting, characterized in that, Nano-silicates were prepared into a silicate ion solution using a serum-free culture medium, mixed with fat aspirate, and then injected for transplantation.

2. A fat graft, characterized in that, It is obtained by preparing a silicate ion solution from nano-silicates using a serum-free culture medium, and then mixing it with fat aspirate.

3. The method for preparing a fat graft as described in claim 2, characterized in that, Nano-silicates were soaked in serum-free culture medium to prepare a silicate ion solution with a concentration of 0.001-0.002 g / mL. The fat aspirate was mixed with the silicate ion solution at a volume ratio of 2-4:1, and after shaking and incubation, it was allowed to stand for 10-20 min to obtain the fat graft.

4. The method for preparing a fat graft as described in claim 3, characterized in that, The silicate ion solution is prepared as follows: 1g of nano-silicate is soaked in 5mL of serum-free culture medium, and shaken in a humidified incubator at 37℃ / 5%CO2 for 18-36h. The supernatant is collected and then diluted with serum-free culture medium to a concentration of 0.001-0.002g / mL to obtain the silicate ion solution. The solution is then sterilized and stored at 0-4℃.

5. The method for preparing a fat graft as described in claim 3, characterized in that, The method for obtaining the fat aspirate is as follows: liposuction is performed using a 3.0mm blunt needle, followed by centrifugation at 1800-3000 rpm for 1-5 minutes to remove oil droplets and blood.

6. The method for preparing a fat graft as described in claim 3, characterized in that, The oscillation incubation method is as follows: oscillate and incubate at 37°C and 100 rpm for 30 minutes.

7. The application as described in claim 1 or a fat graft as described in claim 2, characterized in that, The nano-silicate is nano-calcium silicate, and the particle size of the nano-silicate is 200-500 mesh.

8. The application of a fat graft as described in claim 2, characterized in that, The fat grafts are used as fillers to fill depressions in the face, chest, or buttocks, or to fill skin photoaging and scars.

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