Hydrogel capable of resisting scars and promoting healing as well as preparation method and application of hydrogel

Abstract

The invention discloses hydrogel capable of resisting scars and promoting healing as well as a preparation method and application of the hydrogel. The hydrogel is loaded with BMP4 protein and nanoparticles, the nanoparticles are nanoparticles formed by wrapping lactic acid with PLGA (poly (lactic acid-glycolic acid)); according to the anti-scar and healing-promoting hydrogel disclosed by the invention, photo-crosslinking hydrogel GelMA is used as a delivery carrier, and is combined with the PLGA-coated lactic acid nanoparticles and the BMP4 protein to realize anti-inflammatory and anti-scar binocular effects, so that both symptoms and root causes are treated, wound healing is accelerated, and scar formation is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of medical materials, and in particular to an anti-scar healing-promoting hydrogel and a preparation method and application thereof. Background Art

[0002] Wound healing is a complex and orderly biological process, which is usually divided into four stages: hemostasis, inflammation, proliferation and remodeling. After the wound is formed, the body will start the coagulation mechanism, and the platelets at the wound will aggregate and form a blood clot to stop the wound from bleeding continuously. After that, a variety of pro-inflammatory cells will be recruited at the wound: macrophages, neutrophils, etc., to destroy bacteria and remove necrotic tissue fragments, providing a good biological environment for the subsequent wound repair. The inflammatory period is about 4-6 days, and it will be accompanied by redness, swelling, fever and pain in the early stage. Shortly after the onset of inflammation, skin cells will proliferate in large numbers, including fibroblasts, endothelial cells, etc., as well as the new capillaries, which together form granulation tissue to fill and cover the wound. The proliferation period varies with different degrees of injury, mostly 4-24 days. Finally, the remodeling period is mainly the process of scar formation. A large number of fibroblasts are activated to differentiate into myofibroblasts and deposit a large amount of extracellular matrix to form scars. Scars are ubiquitous consequences of wound healing and surgical operations. The impact of excessive scars goes beyond the physical realm, causing emotional distress and functional limitations, and significantly reducing the quality of life. Hypertrophic scars and keloids, in particular, present considerable challenges as they tend to extend beyond the original wound boundaries. As a result, there is growing interest in developing effective strategies to prevent and manage excessive scarring.

[0003] The essence of scar is an imperfect tissue that does not have normal skin tissue structure and physiological function, loses normal tissue vitality, and even causes deformity. It not only destroys the beauty of the skin, but also hinders the physiological function of related tissues or organs, and even causes deformity. Among them, hypertrophic scar is a skin disease characterized by excessive skin fibrosis. It is more likely to occur after burns and surgery. After the skin injury heals, the scar continues to proliferate and develops into hypertrophic scars, which not only seriously affects the patient's body appearance and mental health, but also may further deteriorate into scar cancer. Scar cancer often occurs in contracture scars caused by burns, often near joints. Scar tissue is fragile and is constantly damaged by joint activity, eventually leading to cancer. Therefore, it is very necessary to fully understand the formation mechanism of scars and reduce scar formation after trauma, both for the benefit of human health and to meet people's requirements for skin beauty. In recent years, significant progress has been made in clarifying the potential mechanism of scar formation. Multiple factors, including inflammation, fibroblast proliferation and collagen deposition, have a significant impact on scar formation. The understanding of these mechanisms has also promoted the exploration of new treatments to alleviate scar formation. A simple, effective and low-cost scar inhibition treatment is urgently needed.

[0004] At present, traditional wound dressings mainly include gauze, bandages and patches, which are mainly used to protect wounds and prevent infection. However, these traditional dressings have limited effects in promoting healing and reducing scars. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-scar and pro-healing hydrogel and a preparation method and application thereof. The anti-scar and pro-healing hydrogel of the present invention can achieve the slow release of drugs and simultaneously achieve the dual effects of anti-inflammatory and anti-scar, that is, it can accelerate wound healing and reduce scar formation.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] A first aspect of the present invention provides a use of lactic acid in the preparation of a product for promoting wound healing and / or reducing scar formation.

[0008] The second aspect of the present invention provides an application of lactic acid combined with BMP4 protein in the preparation of a product for promoting wound healing and / or reducing scar formation.

[0009] The third aspect of the present invention provides a hydrogel for resisting scars and promoting healing, wherein the hydrogel is loaded with BMP4 protein and nanoparticles;

[0010] The nanoparticles are PLGA-encapsulated lactic acid nanoparticles.

[0011] Preferably, the hydrogel is a photo-crosslinked hydrogel GelMA.

[0012] Preferably, the nanoparticles are prepared by the following method:

[0013] (a) adding lactic acid to a dichloromethane solution of PLGA under stirring and stirring to obtain a mixed solution;

[0014] (b) adding the PVA solution to the mixed solution and performing primary emulsification to obtain a primary emulsion, and then adding the PVA solution to the primary emulsion and performing secondary emulsification to obtain a W / O / W nanoparticle suspension;

[0015] (c) removing dichloromethane from the W / O / W nanoparticle suspension by volatilization, and then centrifuging, washing, and drying to obtain the nanoparticles.

[0016] Preferably, in step (a), the concentration of the PLGA dichloromethane solution is 5-15 mg / ml.

[0017] Preferably, in step (a), the mass ratio of PLGA to lactic acid is (8-12):1.

[0018] Preferably, in step (b), the mass volume concentration of the PVA solution is 0.5% to 1.5%;

[0019] The volume ratio of the added amount of PVA solution to the mixed solution during the primary emulsification process is (0.8-1.2):1;

[0020] The volume ratio of the added amount of PVA solution to the mixed solution during the secondary emulsification process is (8-12):1.

[0021] The fourth aspect of the present invention provides a method for preparing the above-mentioned anti-scar and healing-promoting hydrogel, the preparation method comprising the following steps:

[0022] (1) adding a photoinitiator to the GelMA solution and stirring to obtain a mixed solution;

[0023] (2) The nanoparticles and BMP4 protein are dispersed in a mixed solution, and then photo-crosslinked to obtain the anti-scar healing hydrogel.

[0024] Preferably, in step (1), the concentration of the GelMA solution is 8 to 12 mg / ml;

[0025] The final concentration of the photoinitiator added is 0.8% to 1.2%.

[0026] Preferably, in step (2), the final concentration of the added nanoparticles is 5 to 15 mg / ml;

[0027] The final concentration of BMP4 protein added was 10-50 ng / ml.

[0028] A fifth aspect of the present invention provides a use of the above-mentioned anti-scar and pro-healing hydrogel in the preparation of a product that promotes wound healing and / or reduces scar formation.

[0029] A fourth aspect of the present invention provides a product for promoting wound healing and / or reducing scar formation, the product comprising the anti-scar and healing-promoting hydrogel and a PLA scaffold;

[0030] The anti-scar and healing-promoting hydrogel adheres to the PLA scaffold.

[0031] Compared with the prior art, the beneficial effects of the present invention include at least:

[0032] The anti-scar and pro-healing hydrogel of the present invention uses the photo-crosslinked hydrogel GelMA as a delivery carrier, combined with PLGA-encapsulated lactic acid nanoparticles and BMP4 protein to achieve the dual purposes of anti-inflammatory and anti-scar, treating both the symptoms and the root cause, accelerating wound healing and reducing patient pain, and reducing scar formation to maximize the appearance of the wound after healing.

[0033] In addition, the materials involved in the present invention are all biomaterials with good biocompatibility, which can reduce the immune rejection reaction during treatment; the present invention uses photo-cross-linked hydrogel GelMA as a delivery carrier, and uses PLGA to encapsulate lactic acid to make nanoparticles, so as to achieve controllable drug release time and rate, improve the stability and sustainability of drug release, and prolong the effective time of the drug in the body.

[0034] In addition, the anti-scar and pro-healing hydrogel of the present invention can regulate the wound microenvironment and the immune system and inhibit fibrosis in the process of tissue regeneration, and can achieve the dual effects of anti-inflammatory and anti-scar at the same time. It also promotes the regeneration of skin appendages at the wound site, including hair follicles, sebaceous glands, etc., by regulating the immune system, so that the tissue at the defect can achieve functional regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0036] Figure 1 It is an electron microscope scanning image of PLGA-encapsulated lactic acid nanoparticles and anti-scar and pro-healing hydrogel in the embodiment of the present invention;

[0037] Figure 2 The experimental results of the effect of lactic acid on T cells in the embodiment of the present invention are as follows;

[0038] Figure 3The experimental results of the effect of lactic acid on macrophages in the embodiments of the present invention;

[0039] Figure 4 The experimental results of the animal experiment Ctrl on the anti-scar and pro-healing hydrogel promoting wound healing in the embodiment of the present invention;

[0040] Figure 5 The experimental results of the animal experiment PG-PLGA@LA-BMP4 on the anti-scar and pro-healing hydrogel promoting wound healing in the embodiment of the present invention;

[0041] Figure 6 The experimental results of PG in animal experiments on the anti-scar and pro-healing hydrogels in the embodiments of the present invention for promoting wound healing;

[0042] Figure 7 The experimental results of PG-BMP4 in animal experiments on the anti-scar and pro-healing hydrogel promoting wound healing in the embodiments of the present invention;

[0043] Figure 8 The experimental results of the animal experiment of PG-PLGA@LA on the anti-scar and pro-healing hydrogel promoting wound healing in the embodiment of the present invention;

[0044] Fig. 9 The flow cytometry analysis results of the control group in the study of the effects of the anti-scar and pro-healing hydrogel on T cells and macrophages in the embodiments of the present invention;

[0045] Fig.10 The flow cytometry analysis results of the experimental group in the study of the effects of the anti-scar and pro-healing hydrogel on T cells and macrophages in the embodiments of the present invention;

[0046] Fig.11 The experimental results of Ctrl in the effect of the anti-scar and pro-healing hydrogel on hair follicles and sebaceous glands in the embodiments of the present invention;

[0047] Fig.12 The experimental results of PG in the effect of the anti-scar and pro-healing hydrogel on hair follicles and sebaceous glands in the embodiments of the present invention;

[0048] Fig.13 The experimental results of PG-BMP4 in the effect of the anti-scar and pro-healing hydrogel on hair follicles and sebaceous glands in the embodiments of the present invention;

[0049] Fig.14 The experimental results of PG-PLGA@LA in the effect of the anti-scar and pro-healing hydrogel on hair follicles and sebaceous glands in the embodiments of the present invention;

[0050] Fig.15The experimental results of PG-PLGA@LA-BMP4 in the effect of the anti-scar and pro-healing hydrogel on hair follicles and sebaceous glands in the embodiments of the present invention;

[0051] Fig.16 The experimental results of the control group in the effect of the anti-scar and pro-healing hydrogel on type I collagen and type III collagen in the examples of the present invention;

[0052] Fig.17 The experimental results of PG in the effect of the anti-scar and pro-healing hydrogel on type I collagen and type III collagen in the embodiment of the present invention;

[0053] Fig.18 The experimental results of PG-BMP4 in the effect of the anti-scar and pro-healing hydrogel on type I collagen and type III collagen in the embodiment of the present invention;

[0054] Fig.19 The experimental results of PG-PLGA@LA in the effect of the anti-scar and pro-healing hydrogel on type I collagen and type III collagen in the embodiments of the present invention;

[0055] Fig. 20 The experimental results of PG-PLGA@LA-BMP4 in the effect of the anti-scar and pro-healing hydrogel on type I collagen and type III collagen in the embodiments of the present invention;

[0056] Fig.21 The experimental results of Ctrl in the effect of the anti-scar and pro-healing hydrogel on myofibroblasts in the examples of the present invention;

[0057] Fig. 22 The experimental results of PG in the effect of the anti-scar and pro-healing hydrogel on myofibroblasts in the examples of the present invention;

[0058] Fig.23 The experimental results of PG-BMP4 in the effect of the anti-scar and pro-healing hydrogel on myofibroblasts in the embodiments of the present invention;

[0059] Fig.24 The experimental results of PG-PLGA@LA in the effect of the anti-scar and pro-healing hydrogel on myofibroblasts in the examples of the present invention;

[0060] Fig.25 The experimental results of PG-PLGA@LA-BMP4 in the effect of the anti-scar and pro-healing hydrogel on myofibroblasts in the embodiments of the present invention;

[0061] Fig.26 The experimental results of Ctrl in the effect of the anti-scar and pro-healing hydrogel on TGF-β and hair follicle cells in wound tissue in the embodiments of the present invention;

[0062] Fig. 27 The experimental results of PG in the effect of the anti-scar and pro-healing hydrogel on TGF-β and hair follicle cells in wound tissue in the embodiment of the present invention;

[0063] Fig.28 The experimental results of PG-BMP4 in the effect of the anti-scar and pro-healing hydrogel on TGF-β and hair follicle cells in wound tissue in the embodiments of the present invention;

[0064] Fig.29 The experimental results of PG-PLGA@LA in the effect of the anti-scar and pro-healing hydrogel on TGF-β and hair follicle cells in wound tissue in the embodiments of the present invention;

[0065] Fig.30 The experimental results of PG-PLGA@LA-BMP4 in the effect of the anti-scar and pro-healing hydrogel on TGF-β and hair follicle cells in wound tissue in the embodiments of the present invention;

[0066] Fig.31 A histogram showing the effect of the anti-scar and pro-healing hydrogel on TGF-β and hair follicle cells in wound tissues according to an embodiment of the present invention;

[0067] Fig.32 The experimental results of Ctrl in the effect of the anti-scar and pro-healing hydrogel on Treg cells and inflammatory cells in wound tissue in the embodiments of the present invention;

[0068] Fig.33 The experimental results of PG in the effect of the anti-scar and pro-healing hydrogel on Treg cells and inflammatory cells in wound tissue in the embodiment of the present invention;

[0069] Fig.34 The experimental results of PG-BMP4 in the effect of the anti-scar and pro-healing hydrogel on Treg cells and inflammatory cells in wound tissue in the embodiments of the present invention;

[0070] Fig.35 The experimental results of PG-PLGA@LA in the effect of the anti-scar and pro-healing hydrogel on Treg cells and inflammatory cells in wound tissue in the embodiments of the present invention;

[0071] Fig.36 The experimental results of PG-PLGA@LA-BMP4 in the effect of the anti-scar and pro-healing hydrogel on Treg cells and inflammatory cells in wound tissue in the embodiments of the present invention;

[0072] Fig.37 It is a fluorescence intensity bar graph of the effect of the anti-scar and pro-healing hydrogel on Treg cells and inflammatory cells in wound tissue in an embodiment of the present invention. DETAILED DESCRIPTION

[0073] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0074] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0075] An embodiment of the present invention provides an application of lactic acid in preparing a product for promoting wound healing and / or reducing scar formation.

[0076] Lactic acid is a metabolic product of the human body and has good biosafety. The early stage of the wound is an acidic environment. Lactic acid can lower the pH value of the environment. The acidic environment helps to inhibit the growth of pathogenic microorganisms and reduce the risk of infection, thereby reducing the inflammatory response and facilitating the formation and recovery of the early wound microenvironment. Lactic acid can also polarize T cells into Tregs. Tregs have the following functions: 1. Reduce inflammation by inhibiting the activation of inflammatory cells and promoting the proliferation of anti-inflammatory immune cells. By secreting IL-10 and TGF-β, it induces macrophages to polarize from pro-inflammatory M1 to anti-inflammatory M2. 2. Promote wound epithelialization. Tregs secrete a variety of growth factors (such as EGF, VEGF and FGF) to promote keratinization. 1. Promote the proliferation and migration of skin cells, fibroblasts and endothelial cells, accelerate epidermal coverage and angiogenesis, and promote the formation of new tissue; 2. Promote stem cell activity. Treg activates the proliferation and differentiation of skin stem cells and other tissue stem cells by secreting factors related to the Wnt signaling pathway, thereby enhancing the regeneration capacity of tissues and promoting wound closure; 3. Inhibit the activation and proliferation of fibroblasts. Treg inhibits the activation and proliferation of fibroblasts by secreting TGF-β and IL-10, reduces excessive extracellular matrix (ECM) synthesis, and reduces the formation of fibrosis and scar tissue; 4. Promote the regeneration of skin appendages: hair follicles and sebaceous glands, restore the normal structure and function of the skin, and reduce the possibility of scar formation.

[0077] Another embodiment of the present invention provides an application of lactic acid combined with BMP4 protein in the preparation of a product for promoting wound healing and / or reducing scar formation.

[0078] BMP4 can inhibit the proliferation and activation of fibroblasts, thereby reducing the formation of scar tissue. It inhibits the proliferation and differentiation of fibroblasts by regulating the cell cycle and cell signal transduction pathways. In addition, BMP4 can promote the degradation and clearance of collagen, reduce the deposition of collagen in scar tissue, thereby promoting the remodeling and reduction of scar tissue. Moreover, BMP4 can regulate the synthesis and deposition of extracellular matrix, promote the regeneration and remodeling of normal extracellular matrix, and reduce the formation of scar tissue. In addition, BMP4 has anti-inflammatory effects, can regulate inflammatory responses, and reduce the release of inflammatory factors, thereby reducing the damage of inflammation to tissue structure, promoting wound healing and reducing scar formation.

[0079] Another embodiment of the present invention provides a hydrogel for resisting scars and promoting healing, wherein the hydrogel is loaded with BMP4 protein and nanoparticles;

[0080] The nanoparticles are PLGA-encapsulated lactic acid nanoparticles.

[0081] In the present invention, lactic acid can be protected and its release rate can be controlled by encapsulating lactic acid with PLGA. PLGA is a biodegradable polymer that can be hydrolyzed into lactic acid and glycol in the body and finally metabolized and excreted from the body without causing long-term toxic side effects on tissues and organs. PLGA can form a stable microsphere or nanoparticle structure, effectively protecting the drug from the influence of the external environment, preventing the drug from being degraded or inactivated, and prolonging the retention time of the drug. In addition, the release rate and mode of the drug in the carrier can be regulated by changing parameters such as the molecular weight and copolymerization ratio of PLGA, so as to achieve sustained release or sustained release and improve the stability and efficacy of the drug in the body. PLGA is not easy to melt or decompose at high temperatures, has good heat resistance and stability, and is suitable for various preparation methods and storage conditions. The degradation rate of PLGA will be accelerated under acidic conditions, and the release of lactic acid by PLGA will reduce the pH value of the wound, thereby accelerating the release of lactic acid and forming a virtuous cycle of increasing the degradation rate.

[0082] The anti-scar healing hydrogel of the present invention can realize the slow release of drugs by limiting the above raw material components, and simultaneously achieve the dual effects of anti-inflammatory and anti-scar, that is, it can realize the acceleration of wound healing and the reduction of scar formation.

[0083] In one embodiment, the hydrogel is a photo-crosslinked hydrogel GelMA.

[0084] GelMA in the present invention is a biomaterial of natural origin, has good biocompatibility, is not easy to cause immune response and tissue rejection, and can effectively reduce the toxic and side effects of drugs in the body; and the gel structure of GelMA can protect the drug from the influence of the external environment, has strong stability, can prolong the residence time of the drug in the body, and improve the efficacy; in addition, GelMA can enhance the effectiveness of the drug by changing the structure and adding functional monomers, such as adding nanoparticles, bioactive factors and other substances, which can improve the targeting and therapeutic effect of the drug; in addition, GelMA has a porous structure, which is conducive to the adhesion and proliferation of cells in the skin defect site, and provides a good living environment for skin cells.

[0085] The present invention does not specifically limit the preparation method of the nanoparticles, and those skilled in the art can prepare them using conventional preparation methods in the art; in one embodiment, the nanoparticles are prepared by the following method:

[0086] (a) adding lactic acid to a dichloromethane solution of PLGA under stirring and stirring to obtain a mixed solution;

[0087] (b) adding the PVA solution to the mixed solution and performing primary emulsification to obtain a primary emulsion, and then adding the PVA solution to the primary emulsion and performing secondary emulsification to obtain a W / O / W nanoparticle suspension;

[0088] (c) removing dichloromethane from the W / O / W nanoparticle suspension by volatilization, and then centrifuging, washing, and drying to obtain the nanoparticles.

[0089] In one embodiment, in step (a), the concentration of the PLGA dichloromethane solution can be any value between 5 and 15 mg / ml, specifically 5 mg / ml, 10 mg / ml or 15 mg / ml.

[0090] In one embodiment, in the step (a), the mass ratio of PLGA to lactic acid may be (8-12):1, specifically 8:1, 10:1 or 12:1.

[0091] In one embodiment, in step (b), the mass volume concentration of the PVA solution may be 0.5% to 1.5%, specifically 0.5%, 0.8%, 1% or 1.5%;

[0092] The volume ratio of the added amount of the PVA solution to the mixed solution during the primary emulsification process can be (0.8-1.2):1, specifically 0.8:1, 1:1 or 1.2:1;

[0093] The volume ratio of the added amount of the PVA solution to the mixed solution during the secondary emulsification process can be (8-12):1, specifically 0.8:1, 1:1 or 1.2:1.

[0094] In one embodiment, in the step (a), the stirring treatment is stirring at 10000 rpm for 10 to 12 minutes.

[0095] In one embodiment, in the step (b), the emulsification method is ultrasonic treatment, the ultrasonic treatment power is 200 W, and the time is 4 to 6 minutes.

[0096] In one embodiment, in step (c), the centrifugation is performed at 8000-12000 rpm for 8-12 min.

[0097] In one embodiment, washing is performed by resuspending with ultrapure water, centrifuging, and repeating the above operation 2 to 4 times.

[0098] Another embodiment of the present invention provides a method for preparing the above-mentioned anti-scar and healing-promoting hydrogel, the preparation method comprising the following steps:

[0099] (1) adding a photoinitiator to the GelMA solution and stirring to obtain a mixed solution;

[0100] (2) The nanoparticles and BMP4 protein are dispersed in a mixed solution, and then photo-crosslinked to obtain the anti-scar healing hydrogel.

[0101] In one embodiment, in step (1), the concentration of the GelMA solution may be 8 to 12 mg / ml;

[0102] The final concentration of the photoinitiator can be 0.8% to 1.2%.

[0103] In one embodiment, in step (2), the final concentration of the added nanoparticles may be 5 to 15 mg / ml;

[0104] The final concentration of BMP4 protein added can be 10 to 50 ng / ml.

[0105] In one embodiment, the solvent of the GelMA solution is a PBS solution with a pH value of 7.4.

[0106] In one embodiment, the photoinitiator is Irgacure 2959.

[0107] The present invention does not specifically limit GelMA, and it can be purchased from the market or prepared. In one embodiment, GelMA is prepared by the following method:

[0108] Methacrylic anhydride was slowly added to the gelatin aqueous solution, and the reaction was stirred at 50°C. After the reaction was completed, an equal amount of PBS solution was added to terminate the reaction; then, the reaction solution was dialyzed (molecular weight cutoff 1000Da) for 48 hours to remove unreacted methacrylic anhydride and other small molecule impurities; then, the dialyzed solution was freeze-dried to obtain purified GelMA.

[0109] Another embodiment of the present invention provides a use of the above anti-scar healing-promoting hydrogel in the preparation of a product that promotes wound healing and / or reduces scar formation.

[0110] Another embodiment of the present invention provides a product for promoting wound healing and / or reducing scar formation, the product comprising the anti-scar and healing-promoting hydrogel and a PLA scaffold;

[0111] The anti-scar and healing-promoting hydrogel adheres to the PLA scaffold.

[0112] The technical solution of the present invention is further described in detail below through specific embodiments.

[0113] Example 1

[0114] This embodiment is a method for preparing PLGA-encapsulated lactic acid nanoparticles, and the preparation method comprises the following steps:

[0115] 1. Solution Preparation

[0116] 1.1 Preparation of 1% (w / v) PVA solution:

[0117] Weigh 1 g of PVA, dissolve it in 100 ml of ultrapure water, stir and heat until dissolved, and cool to room temperature for use.

[0118] 1.2 Preparation of phosphate buffered saline (PBS):

[0119] Prepare 0.01 M PBS, pH 7.4, and cool to room temperature for use.

[0120] 2. Primary emulsion preparation

[0121] 2.1 Dissolving PLGA:

[0122] Weigh 100 mg of PLGA, dissolve it in 10 ml of dichloromethane (DCM), and stir thoroughly until it is completely dissolved.

[0123] 2.2 Add lactic acid:

[0124] L-lactic acid (10 mg) was added to the PLGA-DCM solution which was stirred by a magnetic stirrer, and the solution was stirred at a high speed (10000 rpm) for 10 minutes.

[0125] 2.3 Emulsification:

[0126] The above solution was slowly added dropwise to 10 ml of 1% (w / v) PVA solution, and ultrasonic treatment was performed using an ultrasonic crusher (200 W, 5 minutes) to form a primary emulsion.

[0127] 3. Secondary emulsification and solvent evaporation

[0128] 3.1 Secondary emulsification:

[0129] The primary emulsion was added to 100 ml of 1% (w / v) PVA solution and sonicated using an ultrasonicator (200 W, 5 minutes) to form a W / O / W (secondary emulsification) nanoparticle suspension.

[0130] 3.2 Solvent evaporation:

[0131] The emulsified suspension was magnetically stirred at room temperature for 4 hours to evaporate the dichloromethane.

[0132] 4. Collection and Cleaning of Nanoparticles

[0133] 4.1 Centrifugal collection:

[0134] The suspension was placed in a centrifuge and centrifuged at 10,000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was collected.

[0135] 4.2 Cleaning:

[0136] The nanoparticles were resuspended in ultrapure water and centrifuged again, and repeated three times to remove excess PVA and unencapsulated lactic acid.

[0137] 4.3 Drying:

[0138] The washed nanoparticles are suspended in a small amount of ultrapure water and placed in a vacuum drying oven for drying to obtain dry PLGA-encapsulated lactic acid nanoparticles.

[0139] Example 2

[0140] This embodiment is a method for preparing a hydrogel for resisting scars and promoting healing, and the preparation method comprises the following steps:

[0141] 1. Synthesis of GelMA

[0142] 1.1 Dissolve gelatin:

[0143] Weigh 10 g of gelatin, dissolve it in 100 ml of ultrapure water, control the temperature at 50°C, and stir until it is completely dissolved.

[0144] 1.2 Reaction:

[0145] 10 ml of methacrylic anhydride (MA) was slowly added, and the mixture was stirred at 50° C. for 3 hours.

[0146] 1.3 Termination reaction:

[0147] After the reaction was completed, an equal amount of PBS solution was added to terminate the reaction.

[0148] 1.4 Dialysis:

[0149] The reaction solution was dialyzed (molecular weight cutoff 1000 Da) for 48 hours to remove unreacted methacrylic anhydride and other small molecular impurities.

[0150] 1.5 Freeze drying:

[0151] The dialyzed solution was freeze-dried to obtain purified GelMA.

[0152] 2. Preparation of GelMA hydrogel loaded with nanoparticles and BMP4 protein:

[0153] 2.1 Dissolve GelMA:

[0154] Weigh 100 mg of GelMA and dissolve it in 10 ml of PBS solution. Control the temperature at 37°C and stir until it is completely dissolved.

[0155] 2.2 Add photoinitiator:

[0156] 1% (w / v) photoinitiator (Irgacure 2959) was added to the GelMA solution and stirred evenly.

[0157] 2.3 Add nanoparticles and proteins:

[0158] The PLGA-encapsulated lactic acid nanoparticles prepared in Example 1 were dispersed in the GelMA solution at a concentration of 10 mg / ml;

[0159] Add BMP4 protein at a final concentration of 30 ng / ml to the mixed solution to ensure that the protein is evenly dispersed.

[0160] 2.4 Photo-cross-linking molding:

[0161] The mixed solution is poured into a pre-designed mold and irradiated with ultraviolet light equipment for photo-crosslinking to form a stable anti-scar and pro-healing hydrogel.

[0162] Example 3

[0163] This embodiment is a product for promoting wound healing and / or reducing scar formation (denoted as PG-PLGA@LA-BMP4), which comprises the anti-scar healing-promoting hydrogel of Example 2 and a PLA scaffold;

[0164] The anti-scar and pro-healing hydrogel of Example 2 adheres to the PLA scaffold.

[0165] Example 4

[0166] This example is a verification of the efficacy of the hydrogel in the present application in preventing scars and promoting healing:

[0167] 1. The PLGA-encapsulated lactic acid nanoparticles prepared in Example 1 and the anti-scar healing-promoting hydrogel prepared in Example 2 were scanned by electron microscope. The scanning results are shown in FIG. Figure 1 As shown;

[0168] Figure 1 In the figure, from left to right, the first picture is a solid picture of the hydrogel, and the second to fourth pictures are electron microscope scanning pictures of the nanoparticles;

[0169] Depend on Figure 1 It can be seen that:

[0170] The hydrogel has the ability to maintain its shape and can be arranged in a regular grid after 3D printing. The diameter of the nanoparticles is about 100nm. The core-shell structure can be observed under an electron microscope and has good dispersion and uniformity.

[0171] 2. Study on the effect of lactic acid on T cells:

[0172] T cells were extracted from mouse spleen and cultured in well plates;

[0173] The control group did not receive any treatment. The experimental group culture medium was added with 10 mM lactic acid. After stimulating the cells for 3 days, the cells were collected for staining. The antibodies used were: CD3, CD4, CD8, CD25, FOXP3. After staining, flow cytometry analysis was performed. The experimental results are shown in the figure. Figure 2 As shown, Figure 2 In the figure, the top three pictures are the control group (from left to right, they are the flow cytometry results of CD8+T cells, CD4+T cells and Treg cells), and the bottom three pictures are the experimental group (from left to right, they are the flow cytometry results of CD8+T cells, CD4+T cells and Treg cells).

[0174] Depend on Figure 2 It can be seen that

[0175] After lactic acid stimulation, the proportion of Treg cells in the experimental group was greater than that in the control group. It can be seen that lactic acid promotes the polarization of T cells into Treg cells and has the effect of reducing inflammation.

[0176] 3. Study on the effect of lactic acid on macrophages:

[0177] After flushing the mouse peritoneal cavity with saline or cell culture medium, extract the peritoneal lavage fluid to obtain macrophages, and culture the cells in a well plate;

[0178] The culture medium of the control group was not treated in any way. The culture medium of the experimental group was added with 10mM lactic acid. After stimulating the cells for 3 days, the cells were collected for staining. The antibodies used were: CD11B, CD86, CD206. After staining, flow cytometry analysis was performed. The experimental results are shown in the figure. Figure 3 As shown, Figure 3 The upper figure is the control group (from left to right are the flow cytometry results of M1 macrophages and M2 macrophages), and the lower figure is the experimental group (from left to right are the flow cytometry results of M1 macrophages and M2 macrophages).

[0179] Depend on Figure 3 It can be seen that after lactic acid stimulation, the proportion of M2 and M1 cells in the experimental group was greater than that in the control group. It can be seen that lactic acid promotes the polarization of M0 cells to M2 cells and has the effect of reducing inflammation.

[0180] 4. Animal experiments on the promotion of wound healing by anti-scar and pro-healing hydrogels:

[0181] Six BALB / c female mice aged about 4 weeks were selected in the control group (denoted as Ctrl), experimental group (denoted as PG-PLGA@LA-BMP4), PLA+hydrogel group (denoted as PG), PLA+hydrogel+protein group (denoted as PG-BMP4) and PLA+hydrogel+lactic acid group (denoted as PG-PLGA@LA). Full-thickness skin defects with a diameter of about 7 mm were cut on the back skin of the mice. The control group was rinsed with normal saline, and PG-PLGA@LA-BMP4, PG, PG-BMP4 and PG-PLGA@LA were fixed on the wound with transparent medical tape. The wounds were observed for 30 days and photographed for record. The results are shown in the figure. Figures 4 to 8 As shown, Figures 4 to 8 From left to right are pictures of the wounds of mice on days 0, 3, 7, 14, and 28 of wound healing. Figures 4 to 8 They are Ctrl, PG-PLGA@LA-BMP4, PG, PG-BMP4 and PG-PLGA@LA, respectively.

[0182] Depend on Figures 4 to 8 It can be seen that the wound healing rates of mice in the experimental group and other groups are continuously greater than those in the control group, and the wound healing rates of mice in other groups are all lower than those in the experimental group but higher than those in the control group. Moreover, the scars finally formed by mice in the experimental group are shallower than those in the control group and other groups. It can be seen that the anti-scar and pro-healing hydrogel of the present invention can accelerate wound healing and reduce scars, and both lactic acid and BMP4 protein have the effects of accelerating wound healing and reducing scars, and the combined therapeutic effect of lactic acid and BMP4 protein is greater than the therapeutic effect of using them alone.

[0183] 5. Study on the effect of anti-scar and pro-healing hydrogel on T cells and macrophages:

[0184] Six BALB / c female mice of about 4 weeks old were selected in the control group and the experimental group. Full-thickness skin incisions with a diameter of about 7 mm were cut on the back skin of the mice. The control group was rinsed with physiological saline, and the product of Example 3 was fixed to the wound with transparent medical tape in the experimental group. On the seventh day of wound healing, three mice were randomly selected from each group, and the skin tissues at the wounds were taken. After digestion with collagenase, cells were collected and divided into two batches of cell suspensions, which were then stained with antibodies. The antibodies used were: the first batch: CD3, CD4, CD8, CD25, FOXP3, the second batch: CD11B, CD86, CD206, and finally flow analysis was performed; the analysis results are shown in the figure. Figures 9-10 As shown;

[0185] Fig. 9 This is the flow cytometry analysis result of the control group. Fig.10 This is the loss analysis result of the experimental group. Figures 9-10 From left to right in the figure are M1 macrophages, M2 macrophages and Treg cells.

[0186] Depend on Figures 9-10 It can be seen that the content of Treg cells and the ratio of M2 to M1 macrophages in the wounds of mice in the experimental group were greater than those in the control group. It can be seen that the anti-scar and pro-healing hydrogel of the present invention promotes the polarization of T cells to Treg cells, promotes the polarization of M2 macrophages, and has the effect of reducing inflammation.

[0187] 6. Effects of anti-scar and pro-healing hydrogels on hair follicles and sebaceous glands;

[0188] Six BALB / c female mice aged about 4 weeks were included in the control group (denoted as Ctrl), experimental group (denoted as PG-PLGA@LA-BMP4), PLA+hydrogel group (denoted as PG), PLA+hydrogel+protein group (denoted as PG-BMP4) and PLA+hydrogel+lactic acid group (denoted as PG-PLGA@LA). A full-thickness skin defect with a diameter of about 7 mm was cut on the back skin of the mice. The control group was rinsed with normal saline, and PG-PLGA@LA-BMP4, PG, PG-BMP4 and PG-PLGA@LA were fixed on the wound with transparent medical tape. On the seventh day of wound healing, three mice were randomly selected from each group, and the skin tissue at the wound was taken. The tissue was embedded in paraffin and then the paraffin in the slices was removed using xylene.

[0189] From high concentration to low concentration alcohol, and finally into distilled water, the slices are placed in a hematoxylin aqueous solution for staining, separated in acid water and ammonia water, rinsed with running water, placed in distilled water for a while, and dehydrated in 70% and 90% alcohol. Stain in alcohol eosin staining solution for 2 to 3 minutes.

[0190] The experimental results are as follows Figures 11 to 15 As shown, Figures 11 to 15Ctrl, PG, PG-BMP4, PG-PLGA@LA, and PG-PLGA@LA-BMP4; Figures 11 to 15 The first to third pictures from left to right are slices of wound healing at three time points, namely the 7th, 14th, and 21st days. The fourth picture is an enlarged picture of the wound slice on the 21st day.

[0191] Depend on Figures 11 to 15 It can be seen that the wound skin tissue healing speed of the mice in the experimental group was faster than that of the control group and other groups, the wound skin tissue healing speed of the mice in other groups was slower than that of the experimental group but faster than that of the control group, and the wounds of the mice in the experimental group had more new hair follicles and sebaceous glands on the 21st day than those in the control group and other groups. It can be seen that the anti-scar and pro-healing hydrogel of the present invention can better accelerate wound epithelialization and promote the regeneration of hair follicles and sebaceous glands, and both lactic acid and BMP4 protein have the effects of accelerating wound healing and promoting the regeneration of skin appendages, and the combined therapeutic effect of lactic acid and BMP4 protein is greater than the therapeutic effect of using them alone.

[0192] 7. Effects of anti-scar and pro-healing hydrogels on type I collagen and type III collagen:

[0193] Six BALB / c female mice aged about 4 weeks were selected in the control group (denoted as Ctrl), experimental group (denoted as PG-PLGA@LA-BMP4), PLA+hydrogel group (denoted as PG), PLA+hydrogel+protein group (denoted as PG-BMP4) and PLA+hydrogel+lactic acid group (denoted as PG-PLGA@LA). Full-thickness skin defects with a diameter of about 7 mm were cut on the back skin of the mice. The control group was rinsed with normal saline, and PG-PLGA@LA-BMP4, PG, PG-BMP4 and PG-PLGA@LA were fixed on the wound with transparent medical tape. On the seventh day of wound healing, three mice were randomly selected from each group, and the skin tissues at the wounds were taken, fixed in 10% formalin fixative, dehydrated and embedded as usual.

[0194] The slice thickness is 6 μm. Dewax and wash with water as usual. Add Sirius red staining solution and stain for 10 to 60 minutes. For tissues that are easier to stain, stain for 10 to 20 minutes. Rinse with running water to remove the stain on the surface of the slice.

[0195] The experimental results are as follows Figures 16-20 As shown, Figures 16-20 The following are sections of skin tissue at the wound site of Ctrl, PG, PG-BMP4, PG-PLGA@LA and PG-PLGA@LA-BMP4 mice on the 21st day (from left to right are the staining images of sections from different areas of the mouse wound). The red collagen in the figure represents type I collagen, and the green collagen represents type III collagen.

[0196] Depend on Figures 16-20It can be seen that the red collagen accounts for a larger proportion in the control group and other groups, and the green collagen accounts for a larger proportion in the experimental group. The red collagen proportion in other groups is smaller than that in the control group but larger than that in the experimental group, and the green collagen proportion in other groups is larger than that in the control group but smaller than that in the experimental group. It can be seen that the anti-scar and pro-healing hydrogel promotes the regeneration of type III collagen in the skin, and the ratio of type I collagen to type III collagen in scars is greater than that in normal skin. Therefore, it can be explained that the anti-scar and pro-healing hydrogel has the effect of reducing the formation of scars, and both lactic acid and BMP4 protein have the effect of promoting the production of type III collagen and reducing scars. The combined treatment effect of lactic acid and BMP4 protein is greater than that of their separate treatment effects.

[0197] 8. Effects of anti-scar and pro-healing hydrogel on myofibroblasts:

[0198] Six BALB / c female mice aged about 4 weeks were included in the control group (denoted as Ctrl), experimental group (denoted as PG-PLGA@LA-BMP4), PLA+hydrogel group (denoted as PG), PLA+hydrogel+protein group (denoted as PG-BMP4) and PLA+hydrogel+lactic acid group (denoted as PG-PLGA@LA). A full-thickness skin defect with a diameter of about 7 mm was cut on the back skin of the mice. The control group was rinsed with normal saline, and PG-PLGA@LA-BMP4, PG, PG-BMP4 and PG-PLGA@LA were fixed on the wound with transparent medical tape. On the seventh day of wound healing, three mice were randomly selected from each group, and the skin tissue at the wound was taken to make immunofluorescence a-SMA staining sections.

[0199] The experimental results are as follows Figures 21 to 25 As shown, Figures 21 to 25 The figures represent the immunofluorescence sections of Ctrl, PG, PG-BMP4, PG-PLGA@LA and PG-PLGA@LA-BMP4 mice at the 14th (left figure, the two figures below are single-labeled staining images of DAPi and a-SMA) and 21st (right figure, the two figures below are single-labeled staining images of DAPi and a-SMA) day of wound healing, respectively. Red represents a-SMA.

[0200] Depend on Figures 21 to 25 It can be seen that the a-SMA fluorescence intensity of the wound tissue of the mice in the control group was greater than that of the mice in the experimental group, and the a-SMA fluorescence intensity of the wound tissue of the mice in other groups was greater than that of the experimental group but less than that of the control group. a-SMA marks myofibroblasts, which shows that the anti-scar and pro-healing hydrogel has the effect of inhibiting the generation of myofibroblasts and reducing scar formation, and both lactic acid and BMP4 protein have the effect of inhibiting the generation of myofibroblasts and reducing scar formation, and the combined therapeutic effect of lactic acid and BMP4 protein is greater than that of their separate therapeutic effects.

[0201] 9. Effects of anti-scar and pro-healing hydrogel on TGF-β and hair follicle cells in wound tissue:

[0202] Six BALB / c female mice aged about 4 weeks were included in the control group (denoted as Ctrl), experimental group (denoted as PG-PLGA@LA-BMP4), PLA+hydrogel group (denoted as PG), PLA+hydrogel+protein group (denoted as PG-BMP4) and PLA+hydrogel+lactic acid group (denoted as PG-PLGA@LA). A full-thickness skin defect with a diameter of about 7 mm was cut on the back skin of the mice. The control group was rinsed with normal saline, and PG-PLGA@LA-BMP4, PG, PG-BMP4 and PG-PLGA@LA were fixed on the wound with transparent medical tape. On the seventh day of wound healing, three mice were randomly selected from each group, and the skin tissues at the wound were taken to make immunofluorescence TGF-β (red) and KRT17 (green) staining sections.

[0203] The experimental results are as follows Figures 26 to 31 As shown, Figures 26-30 The figures represent the immunofluorescence sections of Ctrl, PG, PG-BMP4, PG-PLGA@LA and PG-PLGA@LA-BMP4 mice at the 14th (left picture, the picture below the picture is the single-label staining picture of TGF-β and KRT17) and 21st (right picture, the picture below the picture is the single-label staining picture of TGF-β and KRT17) day of wound healing, respectively. TGF-β is a scar marker, and KRT17 marks hair follicle cells. Fig.31 is a bar graph of the fluorescence intensity of each group. Figures 26 to 31 It can be seen that the TGF-β fluorescence intensity of the wound tissue of the mice in the control group and other groups is greater than that of the mice in the experimental group, the KRT17 fluorescence intensity of the mice in the experimental group is greater than that of the control group and other groups, the TGF-β fluorescence intensity of the wound tissue of the mice in other groups is greater than that of the mice in the experimental group, and the KRT17 fluorescence intensity is greater than that of the control group. Therefore, it can be explained that the anti-scar and pro-healing hydrogel has the effect of promoting hair follicle regeneration and reducing scar formation, and both lactic acid and BMP4 protein have the effect of promoting hair follicle regeneration and reducing scar formation, and the combined treatment effect of lactic acid and BMP4 protein is greater than the treatment effect of using them alone.

[0204] 10. Effects of anti-scar and pro-healing hydrogels on Treg cells and inflammatory cells in wound tissue:

[0205] Six BALB / c female mice aged about 4 weeks were included in the control group (denoted as Ctrl), experimental group (denoted as PG-PLGA@LA-BMP4), PLA+hydrogel group (denoted as PG), PLA+hydrogel+protein group (denoted as PG-BMP4) and PLA+hydrogel+lactic acid group (denoted as PG-PLGA@LA). A full-thickness skin defect with a diameter of about 7 mm was cut on the back skin of the mice. The control group was rinsed with normal saline, and PG-PLGA@LA-BMP4, PG, PG-BMP4 and PG-PLGA@LA were fixed on the wound with transparent medical tape. On the seventh day of wound healing, three mice were randomly selected from each group, and the skin tissues at the wound were taken to make immunofluorescence FOXP3 (red) and IL-6 (green) staining sections.

[0206] The experimental results are as follows Figures 32-37 As shown, Figures 26-30 The immunofluorescence sections of Ctrl, PG, PG-BMP4, PG-PLGA@LA and PG-PLGA@LA-BMP4 mice at the 7th day (left figure, the figure below is the single-label staining of FOXP3 and IL-6) and 14th day (right figure, the figure below is the single-label staining of FOXP3 and IL-6) of wound healing, respectively. FOXP3 marks Treg cells, and IL-6 marks inflammatory cells. Fig.37 The fluorescence intensity bar graphs of each group.

[0207] Depend on Figures 32-37 It can be seen that the IL-6 fluorescence intensity of the wound tissue of the mice in the control group and other groups is greater than that of the mice in the experimental group, and the FOXP3 fluorescence intensity of the mice in the experimental group is greater than that of the control group and other groups. The IL-6 fluorescence intensity of the wound tissue of the mice in other groups is greater than that of the mice in the experimental group, and the FOXP3 fluorescence intensity is greater than that of the control group. Therefore, it can be explained that the anti-scar and pro-healing hydrogel has the effect of promoting Treg cell polarization and anti-inflammatory, and lactic acid has the effect of promoting Treg cell polarization and anti-inflammatory, BMP4 protein has an anti-inflammatory effect, and the combined treatment effect of lactic acid and BMP4 protein is greater than the treatment effect of using them alone.

[0208] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. Use of lactic acid in the preparation of products for promoting wound healing and / or reducing scar formation.

2. Application of lactic acid combined with BMP4 protein in the preparation of products for promoting wound healing and / or reducing scar formation.

3. A hydrogel for preventing scars and promoting healing, characterized in that: The hydrogel is loaded with BMP4 protein and nanoparticles; The nanoparticles are PLGA-encapsulated lactic acid nanoparticles; Preferably, the hydrogel is a photo-crosslinked hydrogel GelMA.

4. The anti-scar healing hydrogel according to claim 3, characterized in that: The nanoparticles are prepared by the following method: (a) adding lactic acid to a dichloromethane solution of PLGA under stirring and stirring to obtain a mixed solution; (b) adding the PVA solution to the mixed solution and performing primary emulsification to obtain a primary emulsion, and then adding the PVA solution to the primary emulsion and performing secondary emulsification to obtain a W / O / W nanoparticle suspension; (c) removing dichloromethane from the W / O / W nanoparticle suspension by volatilization, and then centrifuging, washing, and drying to obtain the nanoparticles.

5. The anti-scar healing hydrogel according to claim 4, characterized in that: In the step (a), the concentration of the PLGA dichloromethane solution is 5 to 15 mg / ml; The mass ratio of PLGA to lactic acid is (8-12):1; Preferably, in step (b), the mass volume concentration of the PVA solution is 0.5% to 1.5%; The volume ratio of the added amount of PVA solution to the mixed solution during the primary emulsification process is (0.8-1.2):1; The volume ratio of the added amount of PVA solution to the mixed solution during the secondary emulsification process is (8-12):

1.

6. The method for preparing the anti-scar and healing-promoting hydrogel according to any one of claims 3 to 5, characterized in that: The steps include: (1) adding a photoinitiator to the GelMA solution and stirring to obtain a mixed solution; (2) The nanoparticles and BMP4 protein are dispersed in a mixed solution, and then photo-crosslinked to obtain the anti-scar healing hydrogel.

7. The preparation method according to claim 6, characterized in that: In the step (1), the concentration of the GelMA solution is 8 to 12 mg / ml; The final concentration of the photoinitiator added is 0.8% to 1.2%.

8. The preparation method according to claim 6, characterized in that: In the step (2), the final concentration of the added nanoparticles is 5 to 15 mg / ml; The final concentration of BMP4 protein added was 10-50 ng / ml.

9. Use of the anti-scar healing-promoting hydrogel according to any one of claims 3 to 5 in the preparation of products for promoting wound healing and / or reducing scar formation.

10. A product for promoting wound healing and / or reducing scar formation, characterized in that: Comprising the anti-scar and pro-healing hydrogel as described in any one of claims 3 to 5 and a PLA scaffold; The anti-scar and healing-promoting hydrogel adheres to the PLA scaffold.

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