Injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres, preparation method and application

Abstract

The application discloses a kind of injectable crosslinking hyaluronic acid gel containing hydroxyapatite microspheres, preparation method and application, comprising the following steps: step 1: in hyaluronic acid HA solution, crosslinking agent and crystallinity 60~90% hydroxyapatite microspheres are added, and the composite gel is obtained after crosslinking, and the composite gel is cut by 60~100 mesh screen to obtain gel large particles;Step 2: in HA solution, crosslinking agent and crystallinity 30~60% hydroxyapatite microspheres are added, and the composite gel is obtained after crosslinking, and the composite gel is cut by 140~200 mesh screen to obtain gel small particles;Step 3: in non-crosslinking HA solution, gel large particles and gel small particles are added, and mixed evenly to obtain the required injectable crosslinking hyaluronic acid gel containing hydroxyapatite microspheres;The composite gel obtained by the application has sustained collagen neogenesis efficiency, the in-vivo effect maintenance time is prolonged, and has excellent soft tissue filling repair effect.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology for medical devices, specifically to an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres, its preparation method, and its application. Background Technology

[0002] Facial deformities or changes in appearance due to aging significantly impact a person's physical and mental health and quality of life. With social progress and increased disposable income, people have higher aspirations for facial aesthetics, driving the booming development of the cosmetic medicine market. Currently, facial soft tissue filling materials mainly fall into two categories: natural biodegradable polymers such as hyaluronic acid (HA) and collagen (Col). As major components of the human extracellular matrix, hyaluronic acid possesses excellent water retention and physical support functions, while collagen exhibits superior bioactivity. However, both have the drawback of relatively short-lasting effects in vivo, and excessive or frequent use may lead to other adverse reactions. In recent years, with the development of biomaterials science and technology, some medical synthetic polymers such as poly-L-lactic acid (PLLA), polymethyl methacrylate (PMMA), and polycaprolactone (PCL), as well as inorganic bioceramics such as hydroxyapatite (HAP), have been approved for clinical application. Due to their exhibiting certain biostimulatory effects, they are gradually gaining attention from consumers and the market in the field of facial soft tissue filling and repair.

[0003] HAP (hydroxyapatite) is a major inorganic component of human bones and teeth, possessing excellent biocompatibility and osteoconductivity, and has been widely used in orthopedics, dentistry, ophthalmology, and plastic surgery. Studies have found that HAP also exhibits excellent soft tissue compatibility, significantly stimulating fibroblast proliferation and collagen matrix secretion. Therefore, facial soft tissue fillers prepared by combining HAP with other polymer materials, possessing bioactive, semi-permanent, and even permanent shaping effects, are highly favored and valued in the medical aesthetics field. For example, the marketed Radiesse product is an injectable carboxymethyl cellulose (CMC) gel containing approximately 30% HAP particles, widely used for lip augmentation, nasolabial fold filling, facial fat malnutrition, wrinkles, hand repair, and contouring after liposuction. It has a relatively long duration of action in vivo, although some complications have been reported. Because the degradation behavior and soft tissue repair and regeneration properties of HAP are closely related to its physicochemical properties—for example, studies have found that nanoscale hydroxyapatite has a better effect on promoting collagen matrix secretion—further research has shown that HAP has better effects on promoting collagen matrix secretion. Most of the HAP products currently on the market have only single physicochemical properties, and their microstructure and degradation rate are fixed, making it difficult to effectively exert the effect of HAP in stimulating collagen fiber regeneration in vivo for a long time. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres, its preparation method, and its application.

[0005] The technical solution adopted in this invention is:

[0006] A method for preparing an injectable crosslinked hyaluronic acid gel containing hydroxyapatite microspheres includes the following steps:

[0007] Step 1: Add cross-linking agent and hydroxyapatite microspheres (HAP) with a crystallinity of 60-90% to hyaluronic acid (HA) solution, degas and mix thoroughly, and obtain a composite gel after cross-linking. Cut the composite gel through a 60-100 mesh sieve to obtain large gel particles L-HA / HAP.

[0008] Step 2: Add crosslinking agent and hydroxyapatite microspheres (HAP) with a crystallinity of 30-60% to HA solution, mix thoroughly and uniformly, and obtain composite gel after crosslinking. Cut the composite gel through a 140-200 mesh sieve to obtain gel particles S-HA / HAP.

[0009] Step 3: Add the L-HA / HAP obtained in Step 1 and the S-HA / HAP obtained in Step 2 to the non-crosslinked HA solution, and mix evenly to obtain the desired injectable crosslinked hyaluronic acid gel containing hydroxyapatite microspheres; wherein the volume ratio of L-HA / HAP to S-HA / HAP is 1 to 2:1.

[0010] Furthermore, in step 1, the HA has a molecular weight of 1400kDa to 2000kDa, the HAP has a particle size of 20-50μm, and the mass ratio of HA to HAP is 1:6 to 10.

[0011] Furthermore, in step 1, the crosslinking agent is 1,4-butanediol glycidyl ether (BDDE), wherein the molar ratio of BDDE to HA structural units is 0.6–1.0:1.

[0012] Furthermore, in step 2, the HA used has a molecular weight of 500kDa to 1050kDa, the HAP particle size is 20-50μm, and the mass ratio of HA to HAP is 1:3 to 7.

[0013] Furthermore, in step 2, the crosslinking agent is 1,4-butanediol glycidyl ether (BDDE), wherein the molar ratio of BDDE to HA structural units is 0.4–0.6:1.

[0014] Furthermore, in steps 1 and 2, the crosslinking reaction temperature is 40–50°C, and the reaction time is 4–24 hours.

[0015] Furthermore, in step 3, the molecular weight of HA is 1400kDa to 2000kDa, the concentration is 2 to 4mg / mL, and the volume ratio of HA to HAP is 1:9 to 19.

[0016] Furthermore, in step 3, lidocaine is added to the mixed solution, with a final lidocaine concentration of 0–3 mg / mL.

[0017] An injectable cross-linked hyaluronic acid gel.

[0018] An application of an injectable cross-linked hyaluronic acid gel, wherein the injectable cross-linked hyaluronic acid gel is used to prepare tissue filler materials and drug carrier materials.

[0019] The beneficial effects of this invention are:

[0020] (1) The gel provided by the present invention contains HAP microspheres with different crystallinity, including HAP with medium-high crystallinity (60-90%) and HAP with medium-low crystallinity (30-60%), which form composite gel particles with cross-linked HA. First, it improves the problem of uneven mixing and easy sedimentation caused by direct physical mixing of HAP with HA solution or gel, which leads to poor injectability after long-term storage. Second, HAP with different crystallinity can play a role in the body at different times, which is beneficial to continuously stimulate collagen production in the body.

[0021] (2) The gel provided by this invention contains two types of HA / HAP composite gel particles with different degrees of cross-linking and sizes. The large-sized L-HA / HAP composite gel particles enhance elasticity, hardness, and anti-enzymatic ability due to the addition of HAP, thereby improving facial shaping ability and effect. The small-sized S-HA / HAP composite gel particles have a relatively low degree of HA cross-linking, which is conducive to the rapid release of medium-low crystallinity (30-60%) HAP from the composite gel particles to directly contact the cells, ensuring the promotion of collagen production in the early and middle stages after implantation. The medium-high crystallinity (60-90%) HAP encapsulated inside the L-HA / HAP composite gel particles can be slowly degraded, which is conducive to the continuous effect of promoting collagen production in the later stage of implantation, thereby achieving its long-term shaping and filling repair effect.

[0022] (3) The gel obtained by the present invention can be directly injected to achieve the purpose of soft tissue filling or facial contour correction. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the gel obtained in Example 1 of the present invention. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0025] A method for preparing an injectable crosslinked hyaluronic acid gel containing hydroxyapatite microspheres, characterized by comprising the following steps:

[0026] Step 1: Add crosslinking agent BDDE and medium-high crystallinity (60-90%) HAP to HA solution, degas and mix thoroughly, and obtain composite gel after crosslinking. Cut the composite gel into irregular gel particles of uniform size (L-HA / HAP).

[0027] The specific process is as follows:

[0028] Hyaluronic acid (HA) is dissolved in sodium hydroxide solution under mechanical stirring to obtain a hyaluronic acid solution; the specific amount of sodium hydroxide is determined according to the actual situation. Generally, the concentration of HA is 80–120 mg / mL.

[0029] The crosslinking agent BDDE and HAP with a crystallinity of 60-90% were sequentially added to the above HA solution and mixed evenly by mechanical stirring. The molar ratio of BDDE to HA structural units was 0.6-1.0:1; the molecular weight of HA was 1400-2000 kDa; and the concentration of HAP in the mixed solution was 600-1000 mg / mL.

[0030] The mixed solution was placed in a vacuum stirrer to remove air bubbles and further stirred until homogeneous.

[0031] The degassed mixture was placed in a water bath and heated to crosslink the mixture to obtain a composite gel; the water bath crosslinking reaction temperature was 40-50℃ and the reaction time was 4-24 hours.

[0032] The composite gel was soaked in deionized water for 5 days, with the water changed 3 times a day to remove the residue of alkali and cross-linking agent. Then it was soaked in PBS for 1 day to obtain the composite gel product.

[0033] The composite gel product was cut into uniformly sized, irregular L-HA / HAP particles through a stainless steel sieve with a mesh size of 60–100.

[0034] Step 2: Add crosslinking agent BDDE and HAP with a crystallinity of 30-60% to the HA solution, mix thoroughly and evenly, and obtain a composite gel after appropriate crosslinking. Cut the composite gel into irregularly sized gel particles (S-HA / HAP).

[0035] The specific process is as follows:

[0036] Hyaluronic acid (HA) is dissolved in sodium hydroxide solution under mechanical stirring to prepare a hyaluronic acid solution; the specific amount of sodium hydroxide is determined according to the actual situation. Generally, the concentration of hyaluronic acid is 80–120 mg / mL.

[0037] The crosslinking agent BDDE and HAP with a crystallinity of 30-60% were sequentially added to the above HA solution and mixed evenly by mechanical stirring. The molar ratio of BDDE to HA structural units was 0.4-0.6:1; the molecular weight of HA was 500-1050 kDa; and the concentration of HAP in the mixed solution was 300-700 mg / mL.

[0038] The mixed solution was placed in a vacuum stirrer to remove air bubbles and further stirred until homogeneous.

[0039] The degassed mixture was placed in a water bath and heated to crosslink the mixture to obtain a composite gel; the water bath crosslinking reaction temperature was 40-50℃ and the reaction time was 4-24 hours.

[0040] The composite gel was soaked in deionized water for 5 days, with the water changed 3 times a day to remove the residue of alkali and cross-linking agent. Then it was soaked in PBS for 1 day to obtain the composite gel product.

[0041] The composite gel product was cut into uniformly sized, irregular S-HA / HAP particles through a stainless steel sieve with a mesh size of 140–200.

[0042] Step 3: Add the L-HA / HAP obtained in Step 1 and the S-HA / HAP obtained in Step 2 to the non-crosslinked HA buffer solution (i.e., the HA buffer solution without any modification), and mix well to obtain the desired injectable composite gel.

[0043] The specific process is as follows:

[0044] A buffer solution with an HA concentration of 2–4 mg / mL was prepared, and then mixed with L-HA / HAP and S-HA / HAP in a certain proportion to obtain the desired hyaluronic acid gel. The volume ratio of HA to L-HA / HAP and S-HA / HAP gel particles was 1:9–19, and the volume ratio of L-HA / HAP to S-HA / HAP was 1–2:1.

[0045] Example 1

[0046] An injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres was prepared according to the following steps:

[0047] Step 1: Add crosslinking agent BDDE and HAP with 82% crystallinity to HA solution, degas and mix thoroughly, and obtain composite gel after crosslinking. Cut the composite gel to obtain irregular L-HA / HAP gel particles of uniform size.

[0048] Weigh 2.0g of hyaluronic acid with a molecular weight of 1500kDa and dissolve it in 20mL of 0.8% sodium hydroxide solution under mechanical stirring to obtain a 100mg / mL HA solution.

[0049] 0.7 mL of crosslinking agent BDDE and 15 g of HAP particles with 82% crystallinity were added to the above solution and mixed evenly by mechanical stirring.

[0050] The above mixture was placed in a vacuum stirrer and centrifuged at 1000 rpm for 10 min to remove air bubbles and then further stirred to mix evenly. After degassing, the mixture was placed in a water bath and heated at 40°C for 24 h to crosslink the mixture and obtain a composite gel.

[0051] The composite gel was soaked in deionized water for 5 days, with the water changed 3 times a day to remove the residue of alkali and cross-linking agent, and the composite gel product was obtained. The composite gel product was cut into uniform particles through a 100-mesh stainless steel sieve.

[0052] Step 2: Add crosslinking agent BDDE and HAP with 47% crystallinity to HA solution, degas and mix thoroughly, and obtain composite gel after crosslinking. Cut the composite gel to obtain irregular S-HA / HAP gel particles of uniform size.

[0053] Weigh 2.0g of hyaluronic acid with a molecular weight of 500kDa and dissolve it in 20mL of 0.8% sodium hydroxide solution under mechanical stirring to obtain a 100mg / mL HA solution.

[0054] Add 0.50 mL of crosslinking agent BDDE and 10 g of HAP particles with a crystallinity of 47% to the above solution in sequence, and mix them evenly by mechanical stirring.

[0055] The above mixture was placed in a vacuum stirrer and centrifuged at 1000 rpm for 10 min to remove air bubbles and then further stirred to mix evenly. After degassing, the mixture was placed in a water bath and heated at 40°C for 12 h to crosslink the mixture and obtain a composite gel.

[0056] The composite gel was soaked in deionized water for 5 days, with the water changed 3 times a day to remove the residue of alkali and cross-linking agent, and the composite gel product was obtained. The composite gel product was cut into uniform particles through a 200-mesh stainless steel sieve.

[0057] Step 3: Thoroughly mix 0.6 mL of L-HA / HAP, 0.3 mL of S-HA / HAP, and 0.1 mL of 2 mg / mL non-crosslinked HA buffer solution to obtain the injectable composite gel.

[0058] Example 2

[0059] An injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres was prepared according to the following steps:

[0060] Step 1: Add crosslinking agent BDDE and HAP with 90% crystallinity to HA solution, degas and mix thoroughly, and obtain composite gel after crosslinking. Cut the composite gel to obtain irregular L-HA / HAP gel particles of uniform size.

[0061] Weigh 2.0g of hyaluronic acid with a molecular weight of 1500kDa and dissolve it in 20mL of 0.8% sodium hydroxide solution under mechanical stirring to obtain a 100mg / mL HA solution.

[0062] 0.7 mL of crosslinking agent BDDE and 15 g of HAP particles with 90% crystallinity were added to the above solution and mixed evenly by mechanical stirring.

[0063] The above mixture was placed in a vacuum stirrer and centrifuged at 1000 rpm for 10 min to remove air bubbles and then further stirred to mix evenly. After degassing, the mixture was placed in a water bath and heated at 40°C for 24 h to crosslink the mixture and obtain a composite gel.

[0064] The composite gel was soaked in deionized water for 5 days, with the water changed 3 times a day to remove the residue of alkali and cross-linking agent, and the composite gel product was obtained. The composite gel product was cut into uniform particles through a 100-mesh stainless steel sieve.

[0065] Step 2: Add crosslinking agent BDDE and HAP with 30% crystallinity to HA solution, degas and mix thoroughly, and obtain composite gel after crosslinking. Cut the composite gel to obtain irregular S-HA / HAP gel particles of uniform size.

[0066] Weigh 2.0g of hyaluronic acid with a molecular weight of 500kDa and dissolve it in 20mL of 0.8% sodium hydroxide solution under mechanical stirring to obtain a 100mg / mL HA solution.

[0067] 0.40 mL of crosslinking agent BDDE and 10 g of HAP particles with 55% crystallinity were added to the above solution in sequence and mixed evenly by mechanical stirring.

[0068] The above mixture was placed in a vacuum stirrer and centrifuged at 1000 rpm for 10 min to remove air bubbles and then further stirred to mix evenly. After degassing, the mixture was placed in a water bath and heated at 40°C for 24 h to crosslink the mixture and obtain a composite gel.

[0069] The composite gel was soaked in deionized water for 5 days, with the water changed 3 times a day to remove the residue of alkali and cross-linking agent, and the composite gel product was obtained. The composite gel product was cut into uniform particles through a 200-mesh stainless steel sieve.

[0070] Step 3: Thoroughly mix 0.5 mL of L-HA / HAP, 0.4 mL of S-HA / HAP, and 0.1 mL of 2 mg / mL non-crosslinked HA buffer solution to obtain the injectable composite gel.

[0071] Example 3

[0072] An injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres was prepared according to the following steps:

[0073] Step 1: Add cross-linking agent BDDE and HAP with 60% crystallinity to HA solution, degas and mix thoroughly, and obtain composite gel after cross-linking. Cut the composite gel to obtain irregular L-HA / HAP gel particles of uniform size.

[0074] Weigh 2.0g of hyaluronic acid with a molecular weight of 1500kDA and dissolve it in 20mL of 0.8% sodium hydroxide solution under mechanical stirring to obtain a 100mg / mL HA solution.

[0075] 0.7 mL of crosslinking agent BDDE and 15 g of HAP particles with 60% crystallinity were added to the above solution and mixed evenly by mechanical stirring.

[0076] The above mixture was placed in a vacuum stirrer and centrifuged at 1000 rpm for 10 min to remove air bubbles and then further stirred to mix evenly. After degassing, the mixture was placed in a water bath and heated at 40°C for 24 h to crosslink the mixture and obtain a composite gel.

[0077] The composite gel was soaked in deionized water for 5 days, with the water changed 3 times a day to remove the residue of alkali and cross-linking agent, and the composite gel product was obtained. The composite gel product was cut into uniform particles through a 100-mesh stainless steel sieve.

[0078] Step 2: Add crosslinking agent and HAP with 30% crystallinity to HA solution, degas and mix thoroughly, and obtain composite gel after crosslinking. Cut the composite gel to obtain irregular S-HA / HAP gel particles of uniform size.

[0079] Weigh 2.0g of hyaluronic acid with a molecular weight of 500kDa and dissolve it in 20mL of 0.8% sodium hydroxide solution under mechanical stirring to obtain a 100mg / mL HA solution.

[0080] 0.40 mL of crosslinking agent BDDE and 12 g of HAP particles with a crystallinity of 30% were sequentially added to the above solution and mixed evenly by mechanical stirring.

[0081] The above mixture was placed in a vacuum stirrer and centrifuged at 1000 rpm for 10 min to remove air bubbles and then further stirred to mix evenly. After degassing, the mixture was placed in a water bath and heated at 40°C for 24 h to crosslink the mixture and obtain a composite gel.

[0082] The composite gel was soaked in deionized water for 5 days, with the water changed 3 times a day to remove the residue of alkali and cross-linking agent, and the composite gel product was obtained. The composite gel product was cut into uniform particles through a 200-mesh stainless steel sieve.

[0083] Step 3: Thoroughly mix 0.6 mL of L-HA / HAP, 0.3 mL of S-HA / HAP, and 0.1 mL of 2 mg / mL non-crosslinked HA buffer solution to obtain the injectable composite gel.

[0084] Comparative Example 1

[0085] The other steps in this comparative example are the same as in Example 1, except that step 2 is omitted, i.e., it does not contain low-to-medium crystallinity HAP and small-particle composite gel. The performance of the facial filler materials in the examples and comparative examples was tested through the following tests (in vitro degradation and in vivo animal experiments).

[0086] 1. In vitro degradation experiment

[0087] The materials from the examples and comparative examples were mixed with hyaluronidase (HAase) solution and then placed in a 37°C constant temperature water bath. The morphological changes of the composite gel were observed at different time points. At the same time, samples of the supernatant were taken to test the HA degradation rate and the dissolution rate of calcium and phosphorus ions.

[0088] 2. In vivo animal experiments

[0089] The materials from the examples and comparative examples were implanted into the subcutaneous tissue of rats, and samples were taken at different times for histological sectioning and staining to observe the growth of new tissue inside the materials.

[0090] The results are as follows:

[0091] 1. In vitro degradation rate and calcium and phosphorus ion dissolution: Under the same concentration of hyaluronidase, the degradation rate of the comparative example composite gel was significantly slower than that of the example. The composite gel of the example was completely degraded within 24 hours, releasing the HAP embedded in the composite gel particles, while the comparative example took 48 hours to completely degrade. The comparative example did not contain HAP with low to medium crystallinity or small S-HA / HAP particles, and its calcium and phosphorus ion dissolution rate was significantly lower than that of the example. In summary, the composite gel of the example has a faster degradation rate and calcium and phosphorus ion dissolution than the comparative example, which is more conducive to stimulating fibroblast proliferation, adhesion, and collagen matrix secretion.

[0092] 2. Effects of filling space-occupying tissue and promoting soft tissue repair in vivo: One month after implantation into the subcutaneous tissue of rats, tissue section staining observation showed that the composite gel of the comparative example and the example showed good biocompatibility and no persistent inflammatory response. The material of the example had more cells and tissue ingrowth inside, and the number and density of newly formed collagen were significantly higher than those of the comparative example. Three months after implantation, the tissue ingrowth inside the materials of both the comparative example and the example continued to increase, with the example showing better results and demonstrating a sustained ability to promote tissue repair.

[0093] This invention uses BDDE as a crosslinking agent to crosslink HA under alkaline conditions, forming a three-dimensional network structure that uniformly encapsulates HAP particles. The gaps created by the accumulation of large-sized L-HA / HAP particles facilitate cell ingrowth, while the rapidly degrading small-sized S-HA / HAP particles quickly release low-to-medium crystallinity HAP in the early and mid-stages of implantation, promoting cell adhesion, proliferation, and collagen secretion. The slower-degrading, high-to-medium crystallinity HAP plays a role in the mid-to-late stages of implantation, further promoting cell adhesion, proliferation, and collagen secretion. Compared to conventional biodegradable filler materials such as HA, the injectable HA gel containing HAP of this invention has a significantly longer in vivo retention time, providing long-term filling and tissue repair effects. Simultaneously, the two types of HAP particles continuously provide a favorable local microenvironment for cells, promoting cell adhesion and proliferation, and stimulating collagen matrix secretion from fibroblasts. Compared to injectable composite gels containing a single HAP feature, the injectable composite gel of this invention contains two types of HAP with different crystallinities and composite gel particles of different sizes, ensuring excellent injectability of the final product while achieving long-term, continuous subcutaneous soft tissue filling and repair effects.

Claims

1. A process for the preparation of an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres, characterized in that, It comprises the following steps: Step 1: adding a crosslinking agent and hydroxyapatite microspheres HAP with a crystallinity of 60-90% into a hyaluronic acid (HA) solution, defoaming, mixing uniformly, obtaining a composite gel after crosslinking, and cutting the composite gel through a 60-100 mesh screen to obtain gel large particles L-HA / HAP; the molecular weight of HA is 1400 kDa-2000 kDa, the particle size of HAP is 20-50 μm, and the mass ratio of HA to HAP is 1:6-10; Step 2: adding a crosslinking agent and hydroxyapatite microspheres HAP with a crystallinity of 30-60% into a HA solution, mixing uniformly, obtaining a composite gel after crosslinking, and cutting the composite gel through a 140-200 mesh screen to obtain gel small particles S-HA / HAP; the molecular weight of HA is 500 kDa-1050 kDa, the particle size of HAP is 20-50 μm, and the mass ratio of HA to HAP is 1:3-7; Step 3: adding L-HA / HAP obtained in Step 1 and S-HA / HAP obtained in Step 2 into a non-crosslinked HA solution, mixing uniformly to obtain the required injectable crosslinked hyaluronic acid gel containing hydroxyapatite microspheres; the volume ratio of L-HA / HAP to S-HA / HAP is 1-2:

1.

2. A process for the preparation of an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 1, characterized in that, The crosslinking agent in Step 1 is 1,4-butanediol glycidyl ether (BDDE), and the molar ratio of BDDE to HA structural units is 0.6-1.0:

1.

3. A process for the preparation of an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 1, characterized in that, The crosslinking agent in Step 2 is 1,4-butanediol glycidyl ether (BDDE), and the molar ratio of BDDE to HA structural units is 0.4-0.6:

1.

4. A process for the preparation of an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 1, characterized in that, The crosslinking reaction temperature in Steps 1 and 2 is 40-50 ℃, and the reaction time is 4-24 hours.

5. The method for preparing an injectable crosslinked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 1, characterized in that, The molecular weight of HA in Step 3 is 1400 kDa-2000 kDa, the concentration is 2-4 mg / mL, and the volume ratio of HA to HAP is 1:9-19.

6. A process for the preparation of a hydroxyapatite microsphere-containing injectable cross-linked hyaluronic acid gel according to claim 1, characterized in that, In Step 3, lidocaine is added to the mixed solution, and the final concentration of lidocaine is 0-3 mg / mL.

7. The injectable crosslinked hyaluronic acid gel containing hydroxyapatite microspheres prepared by the method of any one of claims 1-6.

8. Use of an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 7, characterized in that, The injectable crosslinked hyaluronic acid gel containing hydroxyapatite microspheres is used for preparing a soft tissue filling material in the field of medical cosmetology.

Images