Application of anti-angiogenic drugs in improving the retention rate of soft tissue grafts

Abstract

The present invention discloses the use of anti-angiogenic drugs in improving the retention rate of soft tissue grafts. By using different types of anti-angiogenic drugs to inhibit the early vascularization of soft tissue grafts, the body's immune response to the soft tissue grafts is weakened, thereby reducing inflammation and immune cells, and improving the retention rate of soft tissue grafts.

Description

Technical Field

[0001] The present invention belongs to the technical field of plastic surgery, and particularly relates to the application of anti-angiogenesis drugs in improving the retention rate of soft tissue grafts. Background Art

[0002] Soft tissue defects and volume loss caused by aging, trauma, or iatrogenic causes are common diseases in plastic surgery. Autologous soft tissue transplantation is currently one of the main treatments for plastic surgery, but the unstable long-term retention rate of the graft is the biggest drawback limiting its application.

[0003] The retention rate of soft tissue grafts is related to multiple factors, with vascularization considered a key factor. According to the classic "three-zone theory" of fat grafting, fat grafts can be divided into an outermost survival zone, a central regeneration zone, and an inner necrotic zone, based on the degree of vascular ingrowth. Traditional theory holds that during tissue transplantation, blood vessels, as pathways for nutrient transport, play a crucial role in promoting the retention and survival of transplanted tissue. Therefore, traditional theory suggests that promoting vascularization is generally necessary during tissue transplantation. For example: Ling Xiyue et al. (Ling Xiyue, Dong Hai. Research progress on improving the survival rate of autologous fat particle transplantation [J]. Journal of Yanbian University of Medicine, 2017, 40(2): 147-149.) believe that "the key to improving the survival rate of fat transplantation is to accelerate the reconstruction of blood vessels after transplantation and promote the differentiation of pre-adipocytes, which is closely related to the action of many factors. Research results in recent years have shown that vascular endothelial growth factor (VEGF), leptin, stromal vascular component cells (SVF), platelet-rich plasma (PRP), basic fibroblast growth factor (b-FGF), insulin and insulin-like growth factor-1, epidermal growth factor, platelet-derived growth factor and adrenaline can all promote the survival of transplanted fat." Li Zheng et al. (Li Zheng, Yu Panzhou, Liang Qiguo. Research progress on methods to improve the survival rate of autologous fat transplantation [J]. "Chinese Journal of Medical Cosmetology", 2021, 11(10): 87-90.) believe that "the survival rate of fat transplantation is not only related to the separation and purification of fat, but also depends on factors such as the recipient site after transplantation, local blood supply, extracellular matrix, mature fat cells, etc. After fat transplantation, sufficient blood supply and tissue perfusion are required to ensure that the transplanted cells obtain the oxygen and nutrients required for their survival. If the blood supply is insufficient, nutrients are lacking, and metabolic products are easily accumulated, the survival rate of fat transplantation in the recipient site will be greatly reduced. Therefore, promoting the differentiation of pre-adipocytes, accelerating the reconstruction of blood supply after transplantation, and improving tissue perfusion are of great significance to improving the survival rate of transplanted fat.

[0004] In addition, patent publication number CN107185046A discloses a method for increasing the survival rate of fat cell transplants, by treating a fat cell population with a sterile stabilizing reagent in vitro, adding autologous high-concentration platelet plasma PRP and recombinant human vascular endothelial growth factor VEGF-C to the fat cell population, stimulating the release of multiple cytokines, improving angiogenesis of adipose tissue and preventing cell apoptosis of fat grafts, thereby allowing the transplanted fat cells to survive during the production period.

[0005] However, the inventors' long-term clinical practice has revealed that, contrary to conventional wisdom, vascular ingrowth in soft tissue transplants can negatively impact graft retention. In the early stages of transplantation, the large number of immune and inflammatory cells brought by the blood vessels often leads to graft phagocytosis, disintegration, and necrosis. Therefore, minimizing or preventing the negative effects of vascular ingrowth on grafts remains a pressing technical challenge in the field of plastic surgery. Summary of the Invention

[0006] In order to solve the above-mentioned technical problems, improve the retention rate of soft tissue grafts, and thus ensure the surgical effect of plastic surgery, the present invention proposes the following technical solutions.

[0007] In one aspect, the present invention provides the use of an anti-angiogenesis drug in improving the retention rate of fat grafts.

[0008] Another aspect of the present invention provides the use of an anti-angiogenesis drug in the preparation of a product for improving the retention rate of soft tissue grafts.

[0009] Preferably, the anti-angiogenesis drug or the product is used in an animal transplanted with a soft tissue graft, or the anti-angiogenesis drug or the product is used to treat a soft tissue graft to be transplanted.

[0010] Preferably, the anti-angiogenic drug comprises one or more of VEGF monoclonal antibody, soluble VEGFR, humanized VEGFR-2 monoclonal antibody or small molecule tyrosine kinase inhibitor.

[0011] Furthermore, the VEGF monoclonal antibody includes bevacizumab, the soluble VEGFR includes aflibercept, the humanized VEGFR-2 monoclonal antibody includes ramucirumab, and the small molecule tyrosine kinase inhibitor includes etanercept.

[0012] Preferably, the dosage of the anti-angiogenesis drug is 1 mL of the soft tissue graft combined with 0.5-10 mg of the anti-angiogenesis drug.

[0013] Furthermore, 1 mL of the soft tissue graft is combined with 1.25-6.25 mg of bevacizumab, for example, 1.25 mg, 2.5 mg, 3.75 mg, 5 mg, or 6.25 mg. For another example, 1 mL of the soft tissue graft is combined with 0.05-0.25 mL of bevacizumab at a concentration of 25 mg / mL.

[0014] 1 mL of the soft tissue graft is combined with 2-10 mg of the aflibercept, for example, 2 mg, 4 mg, 6 mg, 8 mg, or 10 mg. For another example, 1 mL of the soft tissue graft is combined with 0.05-0.25 mL of the aflibercept at a concentration of 40 mg / mL.

[0015] 1 mL of the soft tissue graft is combined with 0.5-2.5 mg of ramucirumab, for example, 0.5 mg, 1 mg, 1.5 mg, 2 mg, or 2.5 mg. For another example, 1 mL of the soft tissue graft is combined with 0.05-0.25 mL of ramucirumab at a concentration of 10 mg / mL.

[0016] 1 mL of the soft tissue graft is combined with 1.0-2.5 mg of the orally administered Etan, for example, 1.0 mg, 1.25 mg, 1.50 mg, 1.75 mg, 2.0 mg, 2.25 mg, or 2.5 mg.

[0017] Preferably, the soft tissue graft comprises one or more of a skin graft, a fat graft, a tendon graft, a muscle graft, a ligament graft, a joint capsule graft, a synovial capsule graft, a corneal graft or a nerve graft.

[0018] Preferably, the soft tissue graft is fat, further preferably autologous fat or allogeneic fat, and more preferably autologous fat.

[0019] Anti-angiogenic drugs can inhibit the early vascularization of fat grafts and reduce the body's immune response to fat grafts. Similar to their mechanism of action for fat grafts, anti-angiogenic drugs can also inhibit the early vascularization of other soft tissue grafts and reduce the body's immune response, such as skin grafts, tendon grafts, muscle grafts, ligament grafts, joint capsule grafts, synovial grafts, corneal grafts, and nerve grafts.

[0020] In one embodiment of the present invention, the anti-angiogenic drug and the soft tissue graft are applied to animals, including humans and non-human mammals.

[0021] Preferably, the non-human mammals include mice, rabbits, cats, dogs, pigs, etc.

[0022] Beneficial effects of the present invention:

[0023] This invention uses anti-angiogenic drugs to inhibit the early vascularization of soft tissue grafts, weakening the body's immune response to the graft, thereby reducing inflammation and immune cells, and improving the graft's retention rate. This invention provides new insights and technical means for plastic surgery, improving graft retention after soft tissue transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the immunohistochemical staining result of the fat graft in Example 1 of the present invention, wherein the dark part is the stained blood vessel;

[0025] Figure 2 The statistical results of the number of blood vessels in the fat graft in Example 1 of the present invention are as follows;

[0026] Figure 3 The volume change of the fat graft in Example 1 of the present invention is shown;

[0027] Figure 4 This is the volume change curve of the fat graft in Example 1 of the present invention.

[0028] In the above figures, Bev represents the bevacizumab-treated group, Afl represents the aflibercept group, Ram represents the ramucirumab group, and Control represents the control group. DETAILED DESCRIPTION

[0029] The present invention is further described below with reference to the accompanying drawings and specific examples. These examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental methods in the following examples, where specific conditions are not specified, are generally performed in accordance with conventional conditions in the art or the conditions recommended by the manufacturer. Unless otherwise specified, conventional methods are used. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art.

[0030] In the present invention, the term "soft tissue" refers to the parts of the animal body other than bones, including skin, fat, tendons, muscles, ligaments, joint capsules, synovial capsules, corneas and nerves.

[0031] In the present invention, the term "fat graft" refers to a substance derived from autologous or allogeneic fat tissue that is processed and used for filling.

[0032] In the present invention, the term "anti-angiogenic drug" refers to a drug that reduces or inhibits angiogenesis.

[0033] Example 1 Fat transplantation experiment in mice

[0034] 1.1 Preparation of fat graft

[0035] C57BL / 6 mice were killed by intraperitoneal injection of the anesthetic sodium pentobarbital (50 mg / kg), and the subcutaneous fat pads in the bilateral inguinal regions of the mice were removed and cut into very small pieces, which were collected into a syringe to obtain fat grafts.

[0036] 1.2 Fat graft retention experiment

[0037] C57BL / 6 male mice aged 8 weeks and older were selected as recipient mice and randomly divided into a bevacizumab-treated group (Bev group), an aflibercept-treated group (Afl group), a ramucirumab-treated group (Ram group), and a control group (Control group), with seven C57BL / 6 mice in each group. The prepared fat graft was injected subcutaneously into the back of each C57BL / 6 recipient mouse using an 18G syringe, with 0.3 mL of fat graft injected at a single site per mouse.

[0038] Each mouse in the Bev group received a 0.05 mL injection of 25 mg / mL bevacizumab into the back; each mouse in the Afl group received a 0.05 mL injection of 40 mg / mL aflibercept into the back; each mouse in the Ram group received a 0.05 mL injection of 10 mg / mL ramucirumab into the back; and each mouse in the Control group received a 0.05 mL injection of normal saline into the back. Seven mice in each Bev, Afl, Ram, and Control group were sacrificed on days 3, 7, 14, 21, 30, 60, and 90, respectively, and the fat grafts were removed.

[0039] 1.2.1 Immunohistochemical staining of vascular marker CD31 was performed. The results are shown in Figure 1 、 Figure 2 and Table 1.

[0040] Table 1 Number of blood vessels in fat grafts (mean)

[0041]

[0042] Depend on Figure 1 As can be seen, within the half-life of bevacizumab (21 days), the number of blood vessels in the fat grafts of the bevacizumab group was significantly lower than that of the control group. However, as the mice were kept for longer than the half-life of bevacizumab after fat transplantation, the difference in blood vessel number between the bevacizumab group and the control group gradually narrowed, indicating that bevacizumab has a significant inhibitory effect on angiogenesis in fat grafts.

[0043] During the half-life of aflibercept (7 days), the number of blood vessels in the fat grafts of the aflibercept group was significantly lower than that of the control group. However, as the mice were maintained for longer than the half-life of aflibercept after fat transplantation, the difference in blood vessel number between the aflibercept and control groups gradually narrowed, indicating that aflibercept has a significant inhibitory effect on angiogenesis in fat grafts.

[0044] During the half-life of ramucirumab (14 days), the number of blood vessels in the fat grafts of the ramucirumab group was significantly lower than that of the control group. However, as the mice were maintained for longer than the half-life of ramucirumab after fat transplantation, the difference in blood vessel number between the ramucirumab group and the control group gradually narrowed, indicating that ramucirumab has a significant inhibitory effect on angiogenesis in fat grafts.

[0045] As shown in Table 1 and Figure 2 As shown in the results, on day 3 after fat grafting, the average number of blood vessels in the fat grafts of the bevacizumab group was 1.55, while that of the control group was 2.45. The number of blood vessels in the bevacizumab group was less than that of the control group, and the difference between the two groups reached a significant level (p < 0.05). On day 7 after fat grafting, the average number of blood vessels in the fat grafts of the bevacizumab group was 1.1, while that of the control group was 2.5. The number of blood vessels in the bevacizumab group was less than that of the control group, and the difference between the two groups was significant (p < 0.05). On day 14 after fat grafting, the average number of blood vessels in the bevacizumab group was 1.35, while that of the control group was 4.75. The number of blood vessels in the bevacizumab group was less than that of the control group, and the difference between the two groups was significant (p < 0.05). On day 21 after fat grafting, the average number of blood vessels in the bevacizumab group was 1.9, while that of the control group was 3.9. The number of blood vessels in the bevacizumab group was less than that of the control group, and the difference between the two groups was significant (p < 0.05). On the 30th day after fat transplantation, the average number of blood vessels in the Bevacizumab group was 2.2, while that in the Control group was 2.3. The number of blood vessels in the Bevacizumab group was less than that in the Control group, and the difference between the two groups was not significant (p>0.05). As the time of fat transplantation was further prolonged, the number of blood vessels in the Bevacizumab group gradually decreased, while the number of blood vessels in the Control group gradually increased. On the 90th day after fat transplantation, the average number of blood vessels in the Bevacizumab group was 2.0, while that in the Control group was 2.66. The number of blood vessels in the Bevacizumab group was less than that in the Control group, and the difference between the two groups was not significant (p>0.05). Table 1 and Figure 2 The results showed that in the first 30 days after fat transplantation, the amount of angiogenesis in the fat grafts of the bevacizumab group was less than that of the control group. As the feeding time of the mice after fat transplantation prolonged, the difference in angiogenesis between the two groups gradually increased (before 14 days), and then gradually decreased (after 14 days), until the difference between the two groups gradually became less obvious 30 days after fat transplantation, indicating that bevacizumab can effectively inhibit the angiogenesis of fat grafts.

[0046] On day 3 after fat grafting, the average number of blood vessels in the fat grafts of the aflibercept group was 0.7, while that of the control group was 2.45. The number of blood vessels in the aflibercept group was significantly lower than that of the control group (p < 0.05). On day 7 after fat grafting, the average number of blood vessels in the fat grafts of the aflibercept group was 1.2, while that of the control group was 2.5. The number of blood vessels in the aflibercept group was significantly lower than that of the control group (p < 0.05). On day 14 after fat grafting, the average number of blood vessels in the aflibercept group was 1.15, while that of the control group was 4.75. The number of blood vessels in the aflibercept group was significantly lower than that of the control group (p < 0.05). On day 21 after fat grafting, the average number of blood vessels in the aflibercept group was 1.4, while that of the control group was 3.9. The number of blood vessels in the aflibercept group was significantly lower than that of the control group (p < 0.05). On the 30th day after fat transplantation, the average number of blood vessels in the aflibercept group was 1.9, while that in the control group was 2.3. The number of blood vessels in the aflibercept group was less than that in the control group, but the difference between the two groups gradually decreased and did not reach a significant level (p>0.05). As the time of fat transplantation continued to increase, the number of blood vessels in the aflibercept group and the control group gradually increased, and the difference further decreased. Table 1 and Figure 2 The results showed that in the first 30 days after fat transplantation, the amount of angiogenesis in the fat grafts of the aflibercept group was less than that of the control group. As the mice were kept for longer periods of time after fat transplantation, the difference in angiogenesis between the two groups gradually increased (before 14 days) and then gradually decreased (after 14 days). The difference between the two groups was still significant 30 days after fat transplantation, indicating that aflibercept can effectively inhibit the angiogenesis of fat grafts.

[0047] On day 3 after fat grafting, the mean number of blood vessels in the fat grafts of the ramucirumab group was 1.05, while that of the control group was 2.45. This was significantly lower in the ramucirumab group than in the control group (p < 0.05). On day 7 after fat grafting, the mean number of blood vessels in the fat grafts of the ramucirumab group was 1.2, while that of the control group was 2.5. This was significantly lower in the ramucirumab group than in the control group (p < 0.05). On day 14 after fat grafting, the mean number of blood vessels in the ramucirumab group was 1.35, while that of the control group was 4.75. This was significantly lower in the ramucirumab group than in the control group (p < 0.05). On day 21 after fat grafting, the mean number of blood vessels in the ramucirumab group was 1.5, while that of the control group was 3.9. This was significantly lower in the ramucirumab group than in the control group (p < 0.05). On the 30th day after fat transplantation, the average number of blood vessels in the Ramucirumab group was 1.35, while that in the Control group was 2.3. The number of blood vessels in the Ramucirumab group was less than that in the Control group, and the difference between the two groups was significant (p < 0.05). As the fat transplantation time continued, the number of blood vessels in the Ramucirumab group and the Control group gradually increased, and the difference further decreased. On the 90th day after fat transplantation, the average number of blood vessels in the Ramucirumab group was 2.15, while that in the Control group was 2.66. The number of blood vessels in the Ramucirumab group was less than that in the Control group, but the difference between the two groups was not significant (p > 0.05). Table 1 and Figure 2 The results showed that in the first 30 days after fat transplantation, the amount of angiogenesis in the fat grafts of the ramucirumab group was less than that of the control group. As the feeding time of the mice after fat transplantation prolonged, the difference in angiogenesis between the two groups gradually increased (before 14 days) and then gradually decreased (after 14 days). The difference between the two groups was still significant 30 days after fat transplantation, indicating that aflibercept can effectively inhibit the angiogenesis of fat grafts.

[0048] 1.2.2 The volume of fat graft was measured by drainage method and the fat graft volume retention rate was calculated. The results are shown in Figure 3 、 Figure 4 and Table 2.

[0049] Table 2 Results of fat graft volume changes

[0050]

[0051] Depend on Figure 3As can be seen, during the first three days of fat grafting, no significant changes in fat graft volume were observed in the bevacizumab, aflibercept, ramucirumab, or control groups. After the seventh day, the changes in fat graft volume in the bevacizumab, aflibercept, and ramucirumab groups were smaller than those in the control group. This indicates that the fat graft retention rates in the bevacizumab, aflibercept, and ramucirumab groups were higher than those in the control group. This suggests that as blood vessels intrude into the fat grafts on the third day, the angiogenesis-inhibiting effects of bevacizumab, aflibercept, and ramucirumab begin to manifest, inhibiting angiogenesis in the bevacizumab, aflibercept, and ramucirumab groups. Consequently, the fat graft retention rates in the bevacizumab, aflibercept, and ramucirumab groups are higher than those in the control group. When the mice were fed for more than 7 days, the differences in fat graft retention rates among the bevacizumab group, aflibercept group, ramucirumab group and control group reached a significant level (p < 0.05), and bevacizumab, aflibercept and ramucirumab improved the fat graft retention rate.

[0052] like Figure 4 As shown in Table 2, during the first 7 days of fat transplantation, the fat graft volume in the bevacizumab and control groups did not change significantly. The reason for the fat graft volume exceeding 100% is that, in the very early stages after fat transplantation, the fat graft absorbs water due to inflammation, causing slight swelling and a slight increase in volume. On the 14th day after fat transplantation, the fat graft volume in the bevacizumab group was 95% of its initial volume, while that in the control group was 83.3%. The volume change in the bevacizumab group was smaller than that in the control group, and the difference in fat graft volume between the two groups reached a significant level (p < 0.05). On the 21st day after fat transplantation, the fat graft volume in the bevacizumab group was 87% of its initial volume, while that in the control group was 81%. The volume change in the bevacizumab group was smaller than that in the control group. As the mice were housed for longer periods of time, the difference in fat graft volume change between the bevacizumab and control groups remained significant (p < 0.05). On day 30 of fat transplantation, the fat retention rate in the bevacizumab group did not fall below 81%. On day 60, the fat retention rate in the bevacizumab group remained as high as 68.9%. At day 90, the fat retention rate in the bevacizumab group remained as high as 64.4%, significantly higher than the 54.2% in the control group. These experimental results clearly demonstrate that bevacizumab effectively improves fat graft retention by inhibiting angiogenesis.

[0053] During the first 7 days of fat grafting, no significant changes in fat graft volume were observed between the aflibercept and control groups. On day 14 of fat grafting, the fat graft volume in the aflibercept group was 88.3% of its initial volume, while that in the control group was 83.3% of its initial volume. There was no significant difference in volume change between the aflibercept and control groups. On day 21 of fat grafting, the fat graft volume in the aflibercept group was 83.3% of its initial volume, while that in the control group was 81% of its initial volume. There was no significant difference in fat graft volume change between the aflibercept and control groups. As mice were housed for more than 21 days, a difference in fat retention between the aflibercept and control groups gradually emerged. On day 30 of fat grafting, the fat graft volume in the aflibercept group was 80% of its initial volume, while that in the control group was 75%. This difference in fat graft volume change between the aflibercept and control groups was significant. On day 60 after fat grafting, the fat retention rate in the aflibercept group was 72.2%, compared to 56.7% in the control group. At day 90, the fat retention rate in the aflibercept group remained as high as 66.7%, significantly higher than the control group's 54.2%. These experimental results clearly demonstrate that aflibercept effectively improves fat graft retention by inhibiting angiogenesis.

[0054] During the first seven days of fat transplantation, there was no significant change in fat graft volume in the ramucirumab and control groups. The reason for the fat graft volume exceeding 100% is that in the very early stages after fat transplantation, due to factors such as inflammation and water absorption by the fat graft, the fat graft will swell slightly, resulting in a slight increase in volume. As the fat transplantation time exceeded seven days, the difference in fat graft volume change between the ramucirumab and control groups became increasingly significant. On the 14th day after fat transplantation, the fat graft volume in the ramucirumab group was 96.7% of the initial volume, while the fat graft volume in the control group was 83.3% of the initial volume. The difference in fat graft volume change between the two groups reached a significant level (p < 0.05). On the 21st day after fat transplantation, the fat graft volume in the ramucirumab group was 95% of the initial volume, while the fat graft volume in the control group was 81% of the initial volume. The volume change in the ramucirumab group was significantly less than that in the control group. On day 30 of fat transplantation, the fat graft volume in the ramucirumab group was 86.7% of its initial volume, while the fat graft volume in the control group was 75% of its initial volume. On day 60 of fat transplantation, the fat retention rate in the ramucirumab group did not fall below 71%. At day 90, the fat retention rate in the ramucirumab group remained as high as 65.8%, significantly higher than the 54.2% in the control group. These experimental results clearly demonstrate that ramucirumab effectively improves fat graft retention by inhibiting angiogenesis.

[0055] In summary, Example 1 demonstrates that anti-angiogenic drugs improve fat graft retention by inhibiting early vascularization of fat grafts. The inventors have experimentally discovered that, based on a similar mechanism, anti-angiogenic drugs can effectively improve the retention of other soft tissue grafts, including skin grafts, tendon grafts, muscle grafts, ligament grafts, joint capsule grafts, synovial capsule grafts, corneal grafts, and nerve grafts, by inhibiting graft vascularization.

[0056] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. Use of an anti-angiogenic drug in the preparation of a product for improving the retention rate of soft tissue grafts, characterized in that: The anti-angiogenic drug is bevacizumab, aflibercept or ramucirumab, and the soft tissue graft is a fat graft.

2. The use according to claim 1, characterized in that: The dosage of the anti-angiogenic drug is 1 mL of the soft tissue graft combined with 0.5-10 mg of the anti-angiogenic drug.

3. The use according to claim 2, characterized in that: The dosage of bevacizumab is 1.25-6.25 mg per 1 mL of the soft tissue graft; and / or, The dosage of aflibercept is 2-10 mg per 1 mL of the soft tissue graft; and / or, The dosage of ramucirumab is 0.5-2.5 mg per 1 mL of the soft tissue graft.

4. The use according to claim 1, characterized in that: The fat graft is autologous fat or allogeneic fat.

5. The use according to claim 4, characterized in that: The fat graft is autologous fat.

6. The use according to claim 1, characterized in that: The anti-angiogenesis drug is applied to an animal transplanted with a soft tissue graft, or the soft tissue graft to be transplanted is treated with the anti-angiogenesis drug.

7. The use according to claim 6, characterized in that: The animals include humans and non-human mammals.

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