Preparation method and application of hydroxyapatite composite material capable of improving biocompatibility and collagen regeneration capacity

Abstract

The invention relates to the technical field of medical cosmetology, and particularly discloses a preparation method and application of a hydroxyapatite composite material capable of improving biocompatibility and collagen regeneration capacity. The invention relates to a preparation method of a hydroxyapatite composite material capable of improving biocompatibility and collagen regeneration capacity. The preparation method comprises the following steps: I, preparing hydroxyapatite; iI, carrying out capacity expansion on hydroxyapatite; and III, preparing the composite material, wherein the used raw materials comprise polylactic acid, polycaprolactone, tranexamic acid and the expanded hydroxyapatite obtained in the step II. The product can be blended with a collagen aqueous solution to obtain an injectable collagen stimulant, the injectable collagen stimulant has excellent biocompatibility, can effectively promote generation and deposition of in-vivo collagen and resist pigmentation, and after the stimulant is injected into a target area, a more lasting curative effect can be maintained by regulating and controlling a local microenvironment, so that the injectable collagen stimulant is suitable for clinical application. The immunological rejection reaction possibly caused by the traditional preparation is reduced.

Description

Technical Field

[0001] This application relates to the technical field of medical aesthetics, and particularly to a preparation method and application of a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability. Background Art

[0002] Hydroxyapatite (HA) is an important calcium phosphate and a natural mineral of calcium phosphate. Because it is similar to the main inorganic components of human bones and teeth, it has good bioactivity and osteoconductivity. Hydroxyapatite has thus achieved a firm bond with bone tissue and can be used for bone defect filling and soft tissue injection filling. It has the effects of stimulating collagen regeneration, tightening and lifting, and the effect lasts longer than traditional fillers. It is widely used in the medical aesthetics industry and is usually used as a bone repair and replacement material. On this basis, researchers have conducted new research on its biocompatibility, collagen-stimulating regeneration ability, and other aspects of functional expansion. Summary of the Invention

[0003] In order to improve the utilization value of hydroxyapatite, this application provides a preparation method and application of a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability.

[0004] In the first aspect, this application provides a preparation method of a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability, including the following steps: I. Preparation of hydroxyapatite: Degrease, steam, dry, crush, screen, polish and sinter the biological hard tissue, and then cool to obtain hydroxyapatite; II. Expansion of hydroxyapatite: Mix and disperse the hydroxyapatite obtained in step I and an expanding agent in water according to a weight ratio of 5:(1.2 - 1.8), stir evenly, then perform spray drying and calcination to obtain expanded hydroxyapatite; III. Preparation of the composite material: Disperse polylactic acid and polycaprolactone with a weight ratio of 10:(5 - 8) in an organic solvent to obtain a phase A mixed solution with a total concentration of polylactic acid and polycaprolactone of 0.05 - 0.06 g / L. Disperse an emulsifier in water to obtain a phase B mixed solution with a concentration of 0.05 - 0.06 g / L, and mix the phase A mixed solution and the phase B mixed solution according to a volume ratio of 1:(2 - 2.3). Then add tranexamic acid and the expanded hydroxyapatite obtained in step II to the system, and control the weight ratio of tranexamic acid, the expanded hydroxyapatite obtained in step II, and the polylactic acid in the system to be (0.5 - 1):100:500. Stir at room temperature for 5 - 6 h, let stand overnight, filter to obtain a precipitate, dry the precipitate, and grind it to obtain the hydroxyapatite composite material.

[0005] By adopting the above technical solution, the present application places biological hard tissues in hydrogen peroxide for fat removal, and then obtains hydroxyapatite after a series of treatments. It is a natural material and has better bioactivity and collagen-stimulating ability compared with commercially available synthetic hydroxyapatite. The natural hydroxyapatite is blended with an expanding agent according to a certain weight ratio, and after drying, expanded hydroxyapatite is obtained, and the particle size is controlled between 45 and 60 nm; the expanding agent can assist in forming an internally porous and connected internal structure within the hydroxyapatite, improving the loading capacity of the hydroxyapatite. Subsequently, the expanded hydroxyapatite, polylactic acid, polycaprolactone, and tranexamic acid are blended. Among them, both the expanded hydroxyapatite and polycaprolactone have the ability to stimulate the surrounding self-tissues to produce collagen, and can achieve the effects of thickening the dermis, improving skin texture, and increasing tissue volume. Tranexamic acid can play a role in preventing or treating pigmentation, inhibiting tyrosinase activity, reducing the number of active melanocytes, reducing the formation of active melanin, and improving local skin dullness. Polylactic acid can greatly improve the biocompatibility, drug-loading capacity, and sustained-release capacity of hydroxyapatite. The adsorption capacity of the expanded hydroxyapatite is well supported, and its binding degree with tranexamic acid is improved. The above substances with various specified weight ratios cooperate with each other to give full play to the synergistic effect, and finally a composite material with the ability to resist pigmentation and stimulate collagen regeneration is obtained.

[0006] In step II, the present application controls the weight ratio of hydroxyapatite, expanding agent, and thickening agent to obtain expanded hydroxyapatite with good internal structure connectivity and evenly distributed pores. If the dosage of the expanding agent is too large, it will affect the structural stability of the hydroxyapatite and is prone to fragmentation, completely losing the loading and adsorption capacity; if the dosage of the expanding agent is too small, it cannot achieve an effective expansion effect. In step III, the present application also controls the weight ratio of tranexamic acid and the expanded hydroxyapatite obtained in step II, and a composite material with both anti-pigmentation and good structural stability can be obtained without incurring additional costs.

[0007] Preferably, in the said step II, the weight ratio of the hydroxyapatite obtained in step I to the expanding agent is 5:1.5.

[0008] By adopting the above technical solution, the present application strictly controls the weight ratio of hydroxyapatite, expanding agent, and thickening agent. At this time, it can ensure that the obtained expanded hydroxyapatite has good internal structure connectivity and evenly distributed pores. This ratio can not only avoid the problems of structural instability and fragmentation caused by excessive dosage of the expanding agent, but also prevent the expansion effect from being affected due to insufficient dosage of the expanding agent, thus effectively improving the loading capacity and adsorption performance of the hydroxyapatite.

[0009] Preferably, in the step II, the swelling agent is prepared by the following method: at an ambient temperature of 50 - 75 °C, resorcinol, formaldehyde, and sodium carbonate with a weight ratio of 100:50:(0.5 - 0.7) are dispersed in an aqueous surfactant solution with a concentration of 3 - 6 g / L, stirred at a rotation speed of 200 - 400 rpm, then heated to 80 - 90 °C and left standing, filtered to obtain a solid substance, washed, dried, and blended with camphor terpene in a weight ratio of 1:(1 - 1.2) to obtain the swelling agent.

[0010] By adopting the above technical solution, the present application controls the ratio of resorcinol, formaldehyde, and sodium carbonate, and also utilizes the blending process with camphor terpene. The formed swelling agent can effectively improve the loading capacity and porous connectivity of hydroxyapatite, promote the formation of a uniform and stable internal pore structure of hydroxyapatite in the subsequent steps, thereby enhancing its adsorption performance and biocompatibility. This improvement significantly improves the comprehensive performance of the final composite material, including better collagen regeneration ability and anti-pigmentation effect.

[0011] Preferably, the weight ratio of resorcinol, formaldehyde, and sodium carbonate is 100:50:0.55.

[0012] By adopting the above technical solution, the present application strictly controls the weight ratio of resorcinol, formaldehyde, and sodium carbonate, making the prepared swelling agent have better physical and chemical properties. The swelling agent under this ratio can better assist hydroxyapatite to form a porous connectivity structure, thereby effectively improving its loading capacity and adsorption performance, and enhancing the overall functionality and stability of the composite material.

[0013] Preferably, the concentration of the aqueous surfactant solution is 5 g / L.

[0014] By adopting the above technical solution, when the concentration of the aqueous surfactant solution is optimized to 5 g / L, it can better promote the dispersion effect of resorcinol, formaldehyde, and sodium carbonate in the aqueous solution, thereby improving the uniformity and stability of the subsequent reaction. This helps to generate a swelling agent with a more uniform structure, further enhancing the porous connectivity and loading capacity of the swelling hydroxyapatite. The selection of this parameter ensures that the finally prepared hydroxyapatite composite material has more excellent biocompatibility and collagen regeneration performance.

[0015] Preferably, in the step III, the weight ratio of tranexamic acid, the swelling hydroxyapatite obtained in the step II, and polylactic acid in the system is 0.8:100:500.

[0016] By adopting the above technical solution, the present application optimizes the proportion relationship among tranexamic acid, expanded hydroxyapatite, and polylactic acid in the system. Specifically, when the weight ratio of the four is set to 0.8:100:500, the function of tranexamic acid can be fully exerted. It can not only effectively inhibit the activity of tyrosinase, reduce melanin production, and improve the problem of dull skin tone, but also enhance the modification effect on the surface of expanded hydroxyapatite, further improving the overall stability and biocompatibility of the composite material, thereby more effectively promoting collagen regeneration, achieving the purpose of improving skin texture and tissue repair, ensuring the mutual cooperation among the components, and maximizing the functional characteristics of the composite material.

[0017] Preferably, in the step III, the expanded hydroxyapatite is also subjected to hydrophilic treatment before being added to the system. Specifically, the expanded hydroxyapatite is immersed in an ethanol solution, tris(hydroxymethyl)aminomethane is added and stirred evenly, vacuum-treated at a pressure of 0.1 - 0.12 MPa for 6 - 8 h, then dopamine hydrochloride is added until its concentration reaches 0.2 - 0.3 g / mL, stirred for 12 - 14 h, and then the solid substance is taken out, washed, and freeze-dried to obtain hydrophilic expanded hydroxyapatite.

[0018] By adopting the above technical solution, the present application uses dopamine hydrochloride to perform hydrophilic treatment on the expanded hydroxyapatite. Dopamine hydrochloride can deposit on the surface of the expanded hydroxyapatite in a granular form in this environment, and significantly improve the surface hydrophilicity of the expanded hydroxyapatite, which helps it to infiltrate in the aqueous solution, improve the adsorption effect of the expanded hydroxyapatite, and further improve the adsorption degree of tranexamic acid when the expanded hydroxyapatite is blended with tranexamic acid, achieving the purpose of improving skin texture and giving full play to the functional characteristics of the composite material.

[0019] Preferably, the concentration of the dopamine hydrochloride is 0.25 g / mL.

[0020] By adopting the above technical solution, the present application uses dopamine hydrochloride at a specific concentration to significantly increase the density of active sites on the surface of the expanded hydroxyapatite, thereby enhancing the interaction force between it and the surrounding tissues and drug molecules, effectively improving the overall hydrophilicity of the composite material, further optimizing its biocompatibility and collagen regeneration efficiency, and ensuring more uniform and stable tissue integration and long-term functional maintenance effects in practical applications.

[0021] In the second aspect, the present application provides a hydroxyapatite composite material prepared by the preparation method of the above hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability.

[0022] By adopting the above technical solutions, the hydroxyapatite forms a porous connected structure after expansion treatment, significantly improving the loading capacity and adsorption performance. The expanded hydroxyapatite and polycaprolactone act synergistically to effectively stimulate the surrounding tissues to generate collagen, achieving the effects of thickening the dermis, improving skin texture, and increasing tissue volume. The introduction of tranexamic acid not only inhibits the activity of tyrosinase, reduces the number and activity of melanocytes, but also significantly improves skin dullness, prevents pigmentation. Poly(lactic acid) greatly improves the overall biocompatibility of the composite material, enhances the drug-loading capacity and drug sustained-release characteristics at the same time, optimizes the adsorption performance of the expanded hydroxyapatite, strengthens the binding strength between it and tranexamic acid, and endows the composite material with better mechanical properties. In summary, the composite material comprehensively exhibits the advantages of anti-pigmentation, promoting collagen regeneration, and multi-functional improvement.

[0023] In a second aspect, the present application provides an injectable collagen stimulant, which is prepared by the following method: blending the hydroxyapatite composite material with an aqueous collagen solution to obtain the injectable collagen stimulant.

[0024] By adopting the above technical solutions, the injectable collagen stimulant prepared in the present application not only has excellent biocompatibility, but also can effectively promote the generation and deposition of collagen in vivo, resist pigmentation. After co-culturing with human skin fibroblasts for 7 days, the content of type I collagen can reach more than 14.25 ng / mL, which is increased by more than 1.40 ng / mL compared with the blank group. At the same time, it can significantly improve the change rates of the L * value (representing skin brightness), a * value (representing skin red-green saturation), and b * value (representing skin blue-yellow saturation).

[0025] Due to the synergistic effect among the components in the hydroxyapatite composite material, especially the existence of the specially treated expanded hydroxyapatite and functional additives, the overall stability and tissue integration ability of the material can be significantly enhanced. In addition, after the stimulant is injected into the target area, it can achieve more persistent efficacy maintenance by regulating the local microenvironment, while reducing the immune rejection reaction that may be caused by traditional preparations, thereby providing a safe and efficient new solution for the fields of medical aesthetics and orthopedic repair.

[0026] In summary, the present application has the following beneficial technical effects: 1. The hydroxyapatite of the present application forms a porous connected structure after expansion treatment, and then is blended with poly(lactic acid), polycaprolactone, and tranexamic acid to finally obtain a composite material with the ability to resist pigmentation and stimulate collagen regeneration; 2. During the preparation process of this application, the ratios of each component and the process conditions are strictly controlled to ensure that the composite material has excellent biocompatibility and stable physical and chemical properties; 3. In the application in the field of medical aesthetics, the collagen stimulant prepared from the composite material of this application can effectively stimulate collagen regeneration, and at the same time has the effect of whitening, comprehensively improving the skin health status. Detailed implementation manners

[0027] Material sources Unless otherwise specified, the raw materials used in this application are all commercially available products, specifically: The pig bone slices are purchased from Anhui Shenhua Meat Products Co., Ltd.; Polylactic acid is purchased from NatureWorks of the United States, with the product number 4032D; Polycaprolactone is purchased from Perstorp of Sweden, with the product number Capa 6500; Tranexamic acid is purchased from Shanxi Pude Pharmaceutical Co., Ltd.; Collagen, with a molecular weight of 30000 Da; Human skin fibroblasts are purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.; DMEM high-glucose medium is purchased from Thermo Fisher Scientific; Human type I collagen ELISA kit is purchased from Wuhan Eiaab Science Co., Ltd.

[0028] The following further details this application in combination with preparation examples, implementation examples, application examples and comparative examples.

[0029] Preparation example 1.1 The preparation method of the swelling agent includes the following steps: At an environmental temperature of 75 °C, 100 g of resorcinol, 143 g of 35 wt% formaldehyde aqueous solution, 6 L of liquid paraffin, and 0.5 g of sodium carbonate are dispersed in Span-80 (concentration of 6 g / L) aqueous solution. The amount of Span-80 aqueous solution used is 1 L. Stir at a speed of 400 rpm for 1 h, then heat to 90 °C and let stand for 2 d. Take out the solid substance and wash it three times by centrifugation with 1 wt% Tween-80 solution at a speed of 10000 rpm, dry it, and blend it with camphor terpene in a weight ratio of 1:1 to obtain the swelling agent.

[0030] Preparation example 1.2 The preparation method of the swelling agent includes the following steps: At an ambient temperature of 50 °C, 100 g of resorcinol, 143 g of 35 wt% aqueous formaldehyde solution, 6 L of liquid paraffin, and 0.7 g of sodium carbonate were dispersed in an aqueous solution of Span-80 (concentration 3 g / L), with the amount of the Span-80 aqueous solution being 1 L. Stir for 1 h at a rotation speed of 200 rpm, then heat to 80 °C and let stand for 2 d. Take out the solid substance and centrifuge and wash it three times with a 1 wt% Tween-80 solution at a rotation speed of 10,000 rpm, dry it, and blend it with camphor terpene in a weight ratio of 1:1.2 to obtain the swelling agent.

[0031] Preparation Example 2.1 The preparation method of the swelling agent is different from that of Preparation Example 1.1 in that the amount of sodium carbonate used is 0.55 g, and the rest are the same as those in Preparation Example 1.1.

[0032] Preparation Example 2.2 The preparation method of the swelling agent is different from that of Preparation Example 1.1 in that the amount of sodium carbonate used is 0.6 g, and the rest are the same as those in Preparation Example 1.1.

[0033] Preparation Example 2.3 The preparation method of the swelling agent is different from that of Preparation Example 1.1 in that the amount of sodium carbonate used is 0.65 g, and the rest are the same as those in Preparation Example 1.1.

[0034] Preparation Example 3.1 The preparation method of the swelling agent is different from that of Preparation Example 2.1 in that the concentration of Span-80 in the Span-80 aqueous solution is 4 g / L, and the rest are the same as those in Preparation Example 2.1.

[0035] Preparation Example 3.2 The preparation method of the swelling agent is different from that of Preparation Example 2.1 in that the concentration of Span-80 in the Span-80 aqueous solution is 5 g / L, and the rest are the same as those in Preparation Example 2.1.

[0036] Example 1.1 A preparation method of a hydroxyapatite composite material for improving biocompatibility and collagen regeneration ability, comprising the following steps: I. Hydroxyapatite preparation: The pig bone slices are defatted with 30% by volume hydrogen peroxide for 8 h, dried at 80 °C for 3 h, steamed for 24 h and then air-dried, crushed and ground, sieved with a standard sieve to obtain particles, and polished and sintered at 1100 °C for 2 h, and cooled to room temperature to obtain hydroxyapatite; II. Hydroxyapatite swelling: 500 g of the hydroxyapatite obtained in step I, 120 g of the swelling agent prepared in Preparation Example 1.1, and 40 g of sodium carboxymethylcellulose are mixed and dispersed in 1500 g of water, stirred for 1 h, then spray-dried at a spray temperature of 200 °C, and calcined at a temperature of 1100 °C for 3 h to obtain swollen hydroxyapatite with a particle size of 45 ± 0.1 nm; III. Preparation of composite material: 100 g of polylactic acid and 80 g of polycaprolactone were dispersed in 3 L of dichloromethane to obtain a mixed solution of phase A with a total concentration of 0.06 g / L of polylactic acid and polycaprolactone. Then, 345 g of Tween-80 was dispersed in 6.9 L of water to obtain a mixed solution of phase B with a concentration of 0.05 g / L. The mixed solution of phase A was stirred and emulsified at 30 °C for 10 min. Subsequently, 6 L of the mixed solution of phase B was slowly poured into 3 L of the emulsified mixed solution of phase A and thoroughly blended. Then, 0.1 g of tranexamic acid and 20 g of the expanded hydroxyapatite obtained in step II were added to the system. After stirring evenly, it was ultrasonically treated for 10 min and stirred at room temperature for 5 h, left standing overnight, the supernatant was extracted, filtered, and the precipitate was obtained. The precipitate was washed with deionized water until there was no residue on the surface, dried at 60 °C for 6 h, and ground to obtain the hydroxyapatite composite material.

[0037] Example 1.2 A preparation method of a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability, comprising the following steps: I. Preparation of hydroxyapatite: The pig bone slices were degreased with 30% (volume fraction) hydrogen peroxide for 8 h, dried at 80 °C for 3 h, cooked for 24 h and then air-dried, crushed and ground, sieved with a standard sieve to obtain particles, and polished and sintered at 1100 °C for 2 h, and cooled to room temperature to obtain hydroxyapatite; II. Expansion of hydroxyapatite: 500 g of the hydroxyapatite obtained in step I, 180 g of the expander prepared in Preparation Example 1.2, and 45 g of sodium carboxymethylcellulose were mixed and dispersed in 1500 g of water, stirred for 1 h, and then spray-dried at a spray temperature of 200 °C, and calcined at 1100 °C for 3 h to obtain expanded hydroxyapatite with a particle size of 60 ± 0.1 nm; III. Preparation of composite material: 100 g of polylactic acid and 50 g of polycaprolactone were dispersed in 3 L of dichloromethane to obtain a mixed solution of phase A with a total concentration of 0.05 g / L of polylactic acid and polycaprolactone. Then, 414 g of Tween-80 was dispersed in 6.9 L of water to obtain a mixed solution of phase B with a concentration of 0.06 g / L. The mixed solution of phase A was stirred and emulsified at 30 °C for 10 min. Subsequently, 6.9 L of the mixed solution of phase B was slowly poured into 3 L of the emulsified mixed solution of phase A and thoroughly blended. Then, 0.2 g of tranexamic acid and 20 g of the expanded hydroxyapatite obtained in step II were added to the system. After stirring evenly, it was ultrasonically treated for 10 min and stirred at room temperature for 6 h, left standing overnight, the supernatant was extracted, filtered, and the precipitate was obtained. The precipitate was washed with deionized water until there was no residue on the surface, dried at 60 °C for 6 h, and ground to obtain the hydroxyapatite composite material.

[0038] Example 1.3 A preparation method of a hydroxyapatite composite material for improving biocompatibility and collagen regeneration ability, the difference from Example 1.1 lies in that: in Step III, the dosage of tranexamic acid is 0.16 g, and the rest are the same as in Example 1.1.

[0039] Example 1.4 A preparation method of a hydroxyapatite composite material for improving biocompatibility and collagen regeneration ability, the difference from Example 1.1 lies in that: in Step III, the dosage of tranexamic acid is 0.12 g, and the rest are the same as in Example 1.1.

[0040] Example 1.5 A preparation method of a hydroxyapatite composite material for improving biocompatibility and collagen regeneration ability, the difference from Example 1.1 lies in that: in Step III, the dosage of tranexamic acid is 0.17 g, and the rest are the same as in Example 1.1.

[0041] Example 2.1 A preparation method of a hydroxyapatite composite material for improving biocompatibility and collagen regeneration ability, the difference from Example 1.3 lies in that: in Step II, the dosage of the swelling agent prepared in Preparation Example 1.1 is 140 g, and the rest are the same as in Example 1.3.

[0042] Example 2.2 A preparation method of a hydroxyapatite composite material for improving biocompatibility and collagen regeneration ability, the difference from Example 1.3 lies in that: in Step II, the dosage of the swelling agent prepared in Preparation Example 1.1 is 150 g, and the rest are the same as in Example 1.3.

[0043] Example 2.3 A preparation method of a hydroxyapatite composite material for improving biocompatibility and collagen regeneration ability, the difference from Example 1.3 lies in that: in Step II, the dosage of the swelling agent prepared in Preparation Example 1.1 is 170 g, and the rest are the same as in Example 1.3.

[0044] Examples 3.1 - 3.3 A preparation method of a hydroxyapatite composite material for improving biocompatibility and collagen regeneration ability, the difference from Example 2.2 lies in that: in Step II, the swelling agents prepared in Preparation Examples 2.1 - 2.3 are respectively used to replace the swelling agent prepared in Preparation Example 1.1, and the rest are the same as in Example 2.2.

[0045] Examples 4.1 - 4.2 A method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability, which is different from Example 3.1 in that: the swelling agents prepared in Preparation Example 2.1 in Step II are respectively replaced with the swelling agents prepared in Preparation Examples 3.1-3.2, and the rest are the same as Example 3.1.

[0046] Example 5.1 A method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability, which is different from Example 1.3 in that: in Step III, the swollen hydroxyapatite is also subjected to hydrophilic treatment before being added to the system. Specifically, all the swollen hydroxyapatite is immersed in a 40 wt% ethanol solution, tris(hydroxymethyl)aminomethane is added to make its concentration reach 1.2 mg / mL, and then it is stirred evenly. After vacuum treatment at a pressure of 0.12 MPa for 6 h, dopamine hydrochloride is added until its concentration reaches 0.3 g / mL, and after stirring for 14 h, the solid substance is taken out. Under the condition of oscillation, it is washed 5 times with a large amount of deionized water (the washing duration is not less than 100 min) until the washing liquid is clear, and then freeze-dried to obtain hydrophilic swollen hydroxyapatite.

[0047] Example 5.2 A method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability, which is different from Example 1.3 in that: in Step III, the swollen hydroxyapatite is also subjected to hydrophilic treatment before being added to the system. Specifically, all the swollen hydroxyapatite is immersed in a 40 wt% ethanol solution, tris(hydroxymethyl)aminomethane is added to make its concentration reach 1.2 mg / mL, and then it is stirred evenly. After vacuum treatment at a pressure of 0.1 MPa for 8 h, dopamine hydrochloride is added until its concentration reaches 0.2 g / mL, and after stirring for 12 h, the solid substance is taken out. Under the condition of oscillation, it is washed 5 times with a large amount of deionized water (the washing duration is not less than 100 min) until the washing liquid is clear, and then freeze-dried to obtain hydrophilic swollen hydroxyapatite.

[0048] Example 5.3 A method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability, which is different from Example 5.1 in that: the concentration of dopamine hydrochloride is controlled to be 0.25 g / mL, and the rest are the same as Example 5.1.

[0049] Comparative Example 1.1 It is different from Example 1.3 in that: in Step II, the dosage of the swelling agent prepared in Preparation Example 1.1 is 100 g, and the rest are the same as Example 1.3.

[0050] Comparative Example 1.2 The difference from Example 1.3 lies in that: in Step II, the dosage of the swelling agent prepared in Preparation Example 1.1 is 200 g, and the rest are the same as in Example 1.3.

[0051] Comparative Example 2.1 The difference from Example 1.3 lies in that: in Step III, the dosage of polycaprolactone is 40 g, and the rest are the same as in Example 1.3.

[0052] Comparative Example 2.2 The difference from Example 1.3 lies in that: in Step III, the dosage of polycaprolactone is 100 g, and the rest are the same as in Example 1.3.

[0053] Application Examples 1 - 20 An injectable collagen stimulant is prepared by the following method: the hydroxyapatite composites obtained in Examples 1.1 - 5.3 and Comparative Examples 1.1 - 2.2 are respectively blended with sodium alginate and an aqueous collagen solution at a weight ratio of 10:1:80 to obtain an injectable collagen stimulant, wherein the collagen concentration in the aqueous collagen solution is 5 wt%.

[0054] Performance Detection 1. Inoculate human skin fibroblasts into a 48 - well plate, add 0.4 mL of high - glucose DMEM medium to each well, and the cell density is 1×10 4 / well. Cultivate in 3 groups, and add natural hydroxyapatite particles, SYN1, and SYN2 at 5 mg / mL respectively. Each group has 3 replicate wells. After culturing for 2 d and 7 d, aspirate the supernatant, and detect the content of type I collagen (ng / mL) with reference to the instruction manual of the human type I collagen ELISA kit. Record the results in Table 1 (Since the collagen content in human skin is about 70%, mainly divided into type I (accounting for 85%), type III, and type V, therefore, type I collagen is selected for detection in the experiment); 2. Sixty-three patients with melasma were grouped according to the random number table method, with 3 patients in each group. Before treatment, all 21 groups of patients had their faces cleaned and photographed. Then, compound lidocaine cream (National Medicine Approval Number: H20063466, specification: 25 mg prilocaine + 25 mg / g lidocaine) was topically applied to the face for 20 - 30 minutes. On this basis, 20 groups of patients received intradermal injection of injectable collagen stimulant, and another group of patients received injection of 5 wt% collagen aqueous solution. After routine disinfection, intradermal injection treatment was performed at intervals of 0.5 cm on the melasma lesion sites. A disposable hydrodermabrasion needle 32G was used, and the automatic microneedle instrument (Dermapenworld, model: Dermapen 4) was set to mode 2. The needle insertion depth was 0.8 - 1 mm, the point spacing was 0.5 - 0.7 mm, the interval time was 1 - 2 s, the single injection volume was 0.020 - 0.027 mL, and 100 sites were injected on the entire face. The treatment was performed once a month for 15 minutes each time. After the treatment, a medical repair facial mask was used for cold compress for 15 - 20 minutes. The patients were instructed not to get their skin wet within 24 hours after the operation and to do a good job in moisturizing and sun protection. All 21 groups of patients were treated for 3 months. Before and after the treatment, a facial image analyzer system (Canfield Scientific, Inc., model: VISIA - CR) was used to measure facial image parameters, including the L * value (representing skin brightness), a * value (representing skin red - green saturation), and b * value (representing skin blue - yellow saturation). The changes in the L * value, a * value, and b * value of the 21 groups of patients before and after treatment were recorded, and the results were recorded in Table 1.

[0055] Table 1 Performance Detection Data analysis: As can be seen from Table 1, for the hydroxyapatite composite materials obtained in Examples 1.1 - 1.5 of the present application, after co - culturing with human skin fibroblasts for 2 days, the content of type I collagen can reach 14.25 - 14.40 ng / mL, which is 0.029 - 0.040 higher than that of the blank group. After treating the patients with the injectable collagen stimulant obtained in Application Examples 1 - 5 of the present application for 3 months, the change rate of the L * value in the facial image parameters of the treated patients can be increased to 8.61 - 8.89%, the change rate of the a * value can be increased to 1.58 - 1.68, and the change rate of the b * value can be increased to 20.04 - 20.36%. This proves that the composite materials of the present application indeed have good anti - pigmentation and collagen regeneration stimulation capabilities.

[0056] In Examples 1.1 - 1.5, the content of type I collagen in Example 1.3 is the highest, and the change rate of the L * value, the change rate of the a * value, and the change rate of the b * value are also the highest, proving that by strictly controlling the ratio among tranexamic acid, expanded hydroxyapatite, and polylactic acid in the system, the present application can fully exert the effect of tranexamic acid, not only effectively inhibit the activity of tyrosinase, reduce melanin production, and improve the problem of dull skin tone, but also further enhance the overall stability and biocompatibility of the composite material, thereby more effectively promoting collagen regeneration, achieving the purpose of improving skin texture and tissue repair, ensuring the mutual cooperation among the components, and maximizing the functional characteristics of the composite material.

[0057] Examples 2.1 - 2.3 are different from Example 1.3 in that the dosage of the swelling agent is different. Among them, the content of type I collagen in Example 2.2 is the highest, and the L * value change rate, the a * value change rate, and the b * value change rate are also the largest, proving that by strictly controlling the weight ratio of hydroxyapatite, swelling agent, and thickening agent, the present application can ensure that the expanded hydroxyapatite with good internal structure connectivity and uniform pore distribution is obtained, effectively improving the loading capacity and adsorption performance of hydroxyapatite.

[0058] Examples 3.1 - 3.3 are different from Example 2.2 in that the dosage of sodium carbonate is different when preparing the swelling agent. Among them, the content of type I collagen in Example 3.1 is the highest, and the L * value change rate, the a * value change rate, and the b * value change rate are also the largest, proving that this dosage of sodium carbonate can effectively improve the loading capacity and porous connectivity of hydroxyapatite, promoting the formation of a uniform and stable internal pore structure of hydroxyapatite in subsequent steps, thereby enhancing its adsorption performance and biocompatibility.

[0059] Examples 4.1 - 4.2 are different from Example 3.1 in that the concentration of span - 80 in the span - 80 aqueous solution is different when preparing the swelling agent. Among them, the content of type I collagen in Example 4.2 is higher, and the L * value change rate, the a * value change rate, and the b * value change rate are also larger, proving that when the concentration of the surfactant aqueous solution is optimized to 5 g / L, it can better promote the dispersion effect of resorcinol, formaldehyde, and sodium carbonate in the aqueous solution, thereby improving the uniformity and stability of subsequent reactions.

[0060] Examples 5.1 - 5.3 are different from Example 1.3 in that the expanded hydroxyapatite is also subjected to hydrophilic treatment before being added to the system. The results show that the content of type I collagen is higher, and the change rate of the L * value, the change rate of the a * value, and the change rate of the b * value are also greater. It is proved that the hydrophilic treatment significantly improves the surface hydrophilicity of the expanded hydroxyapatite, which helps it to infiltrate in the aqueous solution, improves the adsorption effect of the expanded hydroxyapatite, and can further improve the adsorption degree of tranexamic acid when the expanded hydroxyapatite is blended with tranexamic acid, achieving the purpose of improving the skin texture and giving full play to the functional characteristics of the composite material.

[0061] Example 5.3 is different from Examples 5.1 - 5.2 in that the concentration of dopamine hydrochloride is different, and the content of type I collagen is higher, and the change rate of the L * value, the change rate of the a * value, and the change rate of the b * value are also greater. It is proved that dopamine hydrochloride at a specific concentration can significantly increase the density of active sites on the surface of the expanded hydroxyapatite, thereby enhancing the interaction force between it and the surrounding tissues and drug molecules, effectively improving the overall hydrophilicity of the composite material, further optimizing its biocompatibility and collagen regeneration efficiency, and ensuring more uniform and stable tissue integration and long-term functional maintenance effects in practical applications.

[0062] Comparative Examples 1.1 - 1.2 are different from Example 1.3 in that the dosage of the swelling agent is different. The results show that the content of type I collagen decreases, and the change rate of the L * value, the change rate of the a * value, and the change rate of the b * value also becomes smaller. It is proved that by strictly controlling the weight ratio of hydroxyapatite, swelling agent and thickener in this application, it is possible to ensure that the expanded hydroxyapatite with good internal structure connectivity and uniformly distributed pores is obtained, effectively improving the loading capacity and adsorption performance of hydroxyapatite.

[0063] Comparative Examples 2.1 - 2.2 are different from Example 1.3 in that the dosage of polycaprolactone is different, and its ratio with polylactic acid and hydroxyapatite is also different. The results show that the content of type I collagen decreases, and the change rate of the L * value, the change rate of the a * value, and the change rate of the b * value also becomes smaller. It is proved that polycaprolactone has the ability to stimulate the surrounding self-tissues to produce collagen, and can achieve the effects of dermal thickening, skin texture improvement and tissue volume increase. And the substances with various specified weight ratios cooperate with each other to give full play to the synergistic effect.

[0064] The embodiments of this specific implementation manner are all preferred embodiments of this application, and do not limit the protection scope of this application. Therefore, all equivalent changes made according to the structure, shape, and principle of this application shall be covered within the protection scope of this application.

Claims

1. A method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability, characterized in that: The following steps are involved: I. Preparation of hydroxyapatite: Degreasing, steaming, drying, crushing, screening, polishing and calcining of biological hard tissues, and cooling to obtain hydroxyapatite; II. Hydroxyapatite expansion: The hydroxyapatite obtained in step I and the expander are dispersed in water at a weight ratio of 5:(1.2-1.8), stirred evenly, spray dried, and calcined to obtain expanded hydroxyapatite; III. Preparation of composite materials: Disperse polylactic acid and polycaprolactone in an organic solvent at a weight ratio of 10:(5-8) to obtain a phase A mixed solution with a total concentration of polylactic acid and polycaprolactone of 0.05-0.06 g / L, disperse an emulsifier in water to obtain a phase B mixed solution with a concentration of 0.05-0.06 g / L, and mix the phase A mixed solution with the phase B mixed solution at a volume ratio of 1:(2-2.3), then add tranexamic acid and the expanded hydroxyapatite obtained in step II to the system, control the weight ratio of tranexamic acid, the expanded hydroxyapatite obtained in step II and the polylactic acid in the system to be (0.5-1):100:500, stir at room temperature for 5-6 hours, let stand overnight, filter to obtain a precipitate, dry the precipitate, and grind to obtain a hydroxyapatite composite material.

2. The method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability according to claim 1, characterized in that: In step II, the weight ratio of the hydroxyapatite obtained in step I to the volume expander is 5:1.

5.

3. The method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability according to claim 1, characterized in that: In the step II, the expander is prepared by the following method: At an ambient temperature of 50-75°C, resorcinol, formaldehyde and sodium carbonate in a weight ratio of 100:50:(0.5-0.7) are dispersed in a surfactant aqueous solution with a concentration of 3-6 g / L, stirred at a speed of 200-400 rpm, then heated to 80-90°C and allowed to stand, the solid matter is filtered, washed, dried, and blended with camphor terpene in a weight ratio of 1:(1-1.2) to obtain a volume expander.

4. The method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability according to claim 3, characterized in that: The weight ratio of resorcinol, formaldehyde and sodium carbonate is 100:50:0.

55.

5. The method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability according to claim 3, characterized in that: The concentration of the surfactant aqueous solution is 5 g / L.

6. The method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability according to claim 1, characterized in that: In the step III, the weight ratio of tranexamic acid, the expanded hydroxyapatite obtained in the step II, and the polylactic acid in the system is 0.8:100:

500.

7. The method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability according to claim 1, characterized in that: In step III, the expanded hydroxyapatite is also subjected to a hydrophilic treatment before being added to the system, specifically: The expanded hydroxyapatite is immersed in an ethanol solution, tris(hydroxymethyl)aminomethane is added and stirred evenly, and vacuum treated at a pressure of 0.1-0.12MPa for 6-8h, and then dopamine hydrochloride is added until its concentration reaches 0.2-0.3g / mL. After stirring for 12-14h, the solid matter is taken out, washed, and freeze-dried to obtain the hydrophilic expanded hydroxyapatite.

8. The method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability according to claim 7, characterized in that: The concentration of the dopamine hydrochloride is 0.25 g / mL.

9. A hydroxyapatite composite material obtained by the method for preparing a hydroxyapatite composite material with improved biocompatibility and collagen regeneration ability according to any one of claims 1 to 8.

10. An injectable collagen stimulator, characterized in that The method is as follows: the hydroxyapatite composite material according to claim 9 is mixed with a collagen aqueous solution to obtain an injectable collagen stimulator.

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