Injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres as well as preparation method and application of injectable cross-linked hyaluronic acid gel

Abstract

The invention discloses injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres as well as a preparation method and application of the injectable cross-linked hyaluronic acid gel containing the hydroxyapatite microspheres, and the preparation method comprises the following steps: step 1, adding a cross-linking agent and the hydroxyapatite microspheres with the crystallinity of 60-90% into a hyaluronic acid HA solution, cross-linking to obtain composite gel, and cutting the composite gel through a 60-100-mesh screen to obtain large gel particles; 2, a cross-linking agent and hydroxyapatite microspheres with the crystallinity being 30%-60% are added into the HA solution, composite gel is obtained after cross-linking, and the composite gel is cut through a 140-200-mesh screen to obtain small gel particles; and step 3, adding the large gel particles and the small gel particles into the non-crosslinked HA solution, and uniformly mixing to obtain the injectable crosslinked hyaluronic acid gel containing the hydroxyapatite microspheres. The composite gel obtained by the invention has a continuous collagen regeneration promoting effect, the in-vivo effect maintenance time is prolonged, and the composite gel has an excellent soft tissue filling and repairing effect.

Description

Technical Field

[0001] The present invention relates to the technical field of medical device biomaterials, and in particular to an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres, a preparation method and applications thereof. Background Art

[0002] Facial deformities or changes in appearance caused by aging greatly affect people's physical and mental health and quality of life. With the progress of society and the increase in disposable income, people have a higher pursuit of facial beauty, which has promoted the vigorous development of the medical beauty market. At present, there are two main types of facial soft tissue filling materials, the natural biodegradable polymer materials hyaluronic acid (HA) and collagen (Col). As the main components of the human extracellular matrix, the former has good water retention and physical support functions, and the latter has excellent biological activity. However, both have the defect of short maintenance time in the body, and excessive and frequent use may cause 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), polycaprolactone (PCL), and inorganic bioceramics such as hydroxyapatite (HAP) have been approved for clinical use. Because they show certain biostimulation effects, they have gradually attracted the attention of consumers and the market in the field of facial soft tissue filling and repair.

[0003] HAP, a major inorganic component of human bones and teeth, possesses excellent biocompatibility and osteoconductivity, and has been widely used in orthopedics, dentistry, ophthalmology, and plastic surgery. Studies have shown that HAP also exhibits excellent soft tissue compatibility, significantly stimulating fibroblast proliferation and collagen matrix secretion. Consequently, bioactive, semi-permanent, or even permanent facial soft tissue fillers prepared from HAP and other polymeric materials have attracted considerable attention in the aesthetic medical field. For example, the marketed Radiesse product is an injectable carboxymethylcellulose (CMC) gel containing approximately 30% HAP particles. It has been widely used for lip augmentation, nasolabial fold soft tissue augmentation, facial lipodystrophy, wrinkles, hand contour defects, and post-liposuction contour correction. While its long-lasting effect in vivo has been associated with some reported complications, the degradation behavior and soft tissue repair and regeneration properties of HAP are closely linked to its physical and chemical properties. For example, studies have shown that nanoscale hydroxyapatite exhibits enhanced collagen matrix secretion. Most of the HAPs in existing marketed products have single physical and chemical properties, and their microscopic morphology and degradation rate are fixed, making it difficult for HAP to effectively stimulate collagen fiber regeneration in the body for a long time. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres, a preparation method and application.

[0005] The technical solution adopted in the present invention is:

[0006] A method for preparing an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres comprises the following steps:

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

[0008] Step 2: Add a crosslinking agent and hydroxyapatite microspheres HAP with a crystallinity of 30-60% to the HA solution, mix thoroughly, and obtain a composite gel after crosslinking. Cut the composite gel through a 140-200 mesh sieve to obtain small 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-cross-linked HA solution and mix them evenly to obtain the desired injectable cross-linked 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, the molecular weight of HA used in step 1 is 1400 kDa to 2000 kDa, the particle size of HAP is 20-50 μm, and the mass ratio of HA to HAP is 1:6-10.

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

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

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

[0014] Furthermore, in step 1 and step 2, the cross-linking reaction temperature is 40 to 50° C., and the reaction time is 4 to 24 hours.

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

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

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

[0018] The invention discloses an application of an injectable cross-linked hyaluronic acid gel, wherein the injectable cross-linked hyaluronic acid gel is used for preparing tissue filling materials and drug carrier materials.

[0019] The beneficial effects of the present invention are:

[0020] (1) The gel provided by the present invention contains HAP microspheres of different crystallinity, including HAP with medium-high crystallinity (60-90%) and HAP with medium-low crystallinity (30-60%), all of which form composite gel particles with cross-linked HA. Firstly, the problems of uneven mixing and easy sedimentation caused by direct physical mixing of HAP and HA solution or gel, which lead to poor injectability, are improved. Secondly, HAP with different crystallinity can act at different time periods in the body, which is conducive to the continuous stimulation of collagen production in the body.

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

[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. BRIEF DESCRIPTION OF THE DRAWINGS

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

[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 cross-linked hyaluronic acid gel containing hydroxyapatite microspheres, characterized by comprising the following steps:

[0026] Step 1: Add the crosslinker BDDE and medium-high crystallinity (60-90%) HAP to the HA solution, degas and mix thoroughly, obtain a composite gel after crosslinking, and cut the composite gel into uniformly sized irregular gel particles (L-HA / HAP);

[0027] The specific process is as follows:

[0028] HA is dissolved in a 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] Crosslinker BDDE and HAP with a crystallinity of 60-90% are sequentially added to the HA solution and mixed uniformly by mechanical stirring. The molar ratio of BDDE to HA structural units is 0.6-1.0:1; the molecular weight of HA is 1400-2000 kDa; and the concentration of HAP in the mixed solution is 600-1000 mg / mL.

[0030] The mixed solution is placed in a vacuum stirring degassing machine to remove bubbles in the mixture and further stirred to mix evenly;

[0031] The defoamed mixture is placed in a water bath for heating and reaction to cross-link the mixture to obtain a composite gel; the water bath cross-linking reaction temperature is 40-50° C., and the reaction time is 4-24 hours.

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

[0033] The composite gel product is cut into irregular L-HA / HAP particles of uniform size by passing through a stainless steel sieve with a certain mesh size. The composite gel product is passed through a stainless steel sieve with a mesh size of 60 to 100.

[0034] Step 2: Add the crosslinking agent BDDE and HAP with a crystallinity of 30-60% to the HA solution, mix thoroughly, and obtain a composite gel after moderate crosslinking. The composite gel is cut to obtain irregular gel particles of uniform size (S-HA / HAP);

[0035] The specific process is as follows:

[0036] HA is dissolved in a 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] Crosslinker BDDE and HAP with a crystallinity of 30-60% are sequentially added to the HA solution and mixed uniformly by mechanical stirring. The molar ratio of BDDE to HA structural units is 0.4-0.6:1; the molecular weight of HA is 500-1050 kDa; and the concentration of HAP in the mixed solution is 300-700 mg / mL.

[0038] The mixed solution is placed in a vacuum stirring degassing machine to remove bubbles in the mixture and further stirred to mix evenly;

[0039] The defoamed mixture is placed in a water bath for heating and reaction to cross-link the mixture to obtain a composite gel; the water bath cross-linking reaction temperature is 40-50° C., and the reaction time is 4-24 hours.

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

[0041] The composite gel product is cut into irregular S-HA / HAP particles of uniform size through a stainless steel sieve with a mesh size of 140 to 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-cross-linked HA buffer solution (i.e., the HA buffer solution without any modification), and mix them evenly 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 is prepared and then mixed with a certain ratio of L-HA / HAP and S-HA / HAP to obtain the desired hyaluronic acid gel. The volume ratio of HA to L-HA / HAP and S-HA / HAP gel particles is 1:9-19, and the volume ratio of L-HA / HAP to S-HA / HAP is 1-2:1.

[0045] Example 1

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

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

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

[0049] 0.7 mL of cross-linking agent BDDE and 15 g of HAP particles with a crystallinity of 82% were successively added to the above solution and mixed uniformly by mechanical stirring.

[0050] The mixture was placed in a vacuum stirring degassing machine, centrifuged at 1000 rpm for 10 minutes to remove bubbles in the mixture, and further stirred and mixed to be uniform; after degassing, the mixture was placed in a water bath and heated at 40°C for 24 hours to cross-link the mixture to obtain a composite gel;

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

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

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

[0054] 0.50 mL of cross-linking agent BDDE and 10 g of HAP particles with a crystallinity of 47% were successively added to the above solution and mixed evenly by mechanical stirring.

[0055] The mixture was placed in a vacuum stirring degassing machine, centrifuged at 1000 rpm for 10 minutes to remove bubbles in the mixture, and further stirred and mixed to be uniform; after degassing, the mixture was placed in a water bath and heated at 40°C for 12 hours to cross-link the mixture to obtain a composite gel;

[0056] The composite gel was immersed in deionized water for 5 days, and the water was changed 3 times a day to remove the residue of alkali solution and cross-linking agent to obtain a composite gel product; the composite gel product was cut into particles of uniform size 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-cross-linked HA buffer solution to obtain an 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 cross-linking agent BDDE and HAP with 90% crystallinity to the HA solution, deaerate and mix well, and obtain a composite gel after cross-linking. Cut the composite gel to obtain irregular L-HA / HAP gel particles of uniform size.

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

[0062] Add 0.7 mL of cross-linking agent BDDE and 15 g of HAP particles with 90% crystallinity to the above solution in sequence, and mix well by mechanical stirring.

[0063] Place the above mixture in a vacuum stirring deaerator, centrifuge at 1000 rpm for 10 min to remove bubbles in the mixture and further mix well by stirring. After deaeration, place it in a water bath, heat at 40°C for 24 h to cross-link the mixture to obtain a composite gel.

[0064] Soak the composite gel in deionized water for 5 days, change the water 3 times a day to remove the residual alkali and cross-linking agent, and obtain the composite gel product. Cut the composite gel product into uniform particles through a 100 mesh stainless steel screen.

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

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

[0067] Add 0.40 mL of cross-linking agent BDDE and 10 g of HAP particles with 55% crystallinity to the above solution in sequence, and mix well by mechanical stirring.

[0068] Place the above mixture in a vacuum stirring deaerator, centrifuge at 1000 rpm for 10 min to remove bubbles in the mixture and further mix well by stirring. After deaeration, place it in a water bath, heat at 40°C for 24 h to cross-link the mixture to obtain a composite gel.

[0069] The composite gel was immersed in deionized water for 5 days, and the water was changed 3 times a day to remove the residue of alkali solution and cross-linking agent to obtain a composite gel product; the composite gel product was cut into particles of uniform size 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-cross-linked HA buffer solution to obtain an injectable composite gel.

[0071] Example 3

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

[0073] Step 1: Add crosslinker BDDE and HAP with a crystallinity of 60% to the HA solution, degas and mix thoroughly, obtain a composite gel after crosslinking, and cut the composite gel into irregular L-HA / HAP gel particles of uniform size;

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

[0075] 0.7 mL of cross-linking agent BDDE and 15 g of HAP particles with a crystallinity of 60% were successively added to the above solution and mixed uniformly by mechanical stirring.

[0076] The mixture was placed in a vacuum stirring degassing machine, centrifuged at 1000 rpm for 10 minutes to remove bubbles in the mixture, and further stirred and mixed to be uniform; after degassing, the mixture was placed in a water bath and heated at 40°C for 24 hours to cross-link the mixture to obtain a composite gel;

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

[0078] Step 2: Add a cross-linking agent and HAP with a crystallinity of 30% to the HA solution, degas and mix thoroughly, obtain a composite gel after cross-linking, and cut the composite gel into irregular S-HA / HAP gel particles of uniform size;

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

[0080] 0.40 mL of cross-linking agent BDDE and 12 g of HAP particles with a crystallinity of 30% were successively added to the above solution and mixed uniformly by mechanical stirring.

[0081] The mixture was placed in a vacuum stirring degassing machine, centrifuged at 1000 rpm for 10 minutes to remove bubbles in the mixture, and further stirred and mixed to be uniform; after degassing, the mixture was placed in a water bath and heated at 40°C for 24 hours to cross-link the mixture to obtain a composite gel;

[0082] The composite gel was immersed in deionized water for 5 days, and the water was changed 3 times a day to remove the residue of alkali solution and cross-linking agent to obtain a composite gel product; the composite gel product was cut into particles of uniform size 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-cross-linked HA buffer solution to obtain an injectable composite gel.

[0084] Comparative Example 1

[0085] The remaining steps of this comparative example were identical to those of Example 1, except that Step 2 was omitted, i.e., the low- to medium-crystallinity HAP and small-particle composite gel were not present. The performance of the facial filler materials of the examples and comparative examples was tested using the following tests (in vitro degradation and in vivo animal experiments).

[0086] 1. In vitro degradation experiment

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

[0088] 2. In vivo animal experiments

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

[0090] Here are the results:

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

[0092] 2. In vivo space-filling and soft tissue repair-promoting effects: After being implanted into the subcutaneous tissue of rats for one month, tissue section staining and observation revealed that both the comparative example and the example composite gels showed good biocompatibility, with no persistent inflammatory response. More cells and tissues grew into the example material, and the amount and density of newly formed collagen were significantly higher than those of the comparative example. Three months after implantation, tissue ingrowth into the comparative example and example materials continued to increase, with the example material showing better results, demonstrating a sustained ability to promote tissue repair.

[0093] The present invention uses BDDE as a crosslinker to crosslink HA under alkaline conditions, forming a three-dimensional network structure that uniformly encapsulates HAP particles. The interstices formed by the accumulation of large L-HA / HAP particles facilitate cell ingrowth. Smaller S-HA / HAP particles with faster degradation rates rapidly release HAP with medium to low crystallinity in the early and middle stages of implantation, promoting cell attachment, proliferation, and collagen secretion. HAP with slower degradation rates with medium to high crystallinity can function in the middle and late stages of implantation, promoting cell attachment, proliferation, and collagen secretion. Compared to conventional degradable fillers such as HA, the injectable HA gel containing HAP of the present invention exhibits significantly prolonged in vivo retention, demonstrating long-term filling and tissue repair effects. Furthermore, the two HAP particles continuously provide a favorable local microenvironment for cells, promoting cell adhesion and proliferation and stimulating collagen matrix secretion by fibroblasts. Compared to injectable composite gels containing a single HAP, the injectable composite gel of the present invention, containing two HAP particles with different crystallinities and different sizes, ensures excellent injectability while achieving long-term, sustained subcutaneous soft tissue filling and repair effects.

Claims

1. A method for preparing an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres, characterized in that: The following steps are involved: Step 1: Add a crosslinking agent and hydroxyapatite microspheres HAP with a crystallinity of 60-90% to the hyaluronic acid HA solution, degas and mix thoroughly, and obtain a composite gel after crosslinking. Cut the composite gel through a 60-100 mesh sieve to obtain large gel particles L-HA / HAP; Step 2: Add a crosslinking agent and hydroxyapatite microspheres HAP with a crystallinity of 30-60% to the HA solution, mix thoroughly, and obtain a composite gel after crosslinking. Cut the composite gel through a 140-200 mesh sieve to obtain small gel particles S-HA / HAP; Step 3: Add the L-HA / HAP obtained in step 1 and the S-HA / HAP obtained in step 2 to the non-cross-linked HA solution and mix them evenly to obtain the desired injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres; wherein the volume ratio of L-HA / HAP to S-HA / HAP is 1 to 2:

1.

2. The method for preparing an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 1, characterized in that: The molecular weight of HA used in step 1 is 1400 kDa to 2000 kDa, the particle size of HAP is 20 to 50 μm, and the mass ratio of HA to HAP is 1:6 to 10.

3. The method for preparing an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 1, characterized in that: In the step 1, the cross-linking agent is 1,4-butanediol glycidyl ether (BDDE), wherein the molar ratio of BDDE to HA structural unit is 0.6 to 1.0:

1.

4. The method for preparing an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 1, characterized in that: The molecular weight of HA used in step 2 is 500 kDa to 1050 kDa, the particle size of HAP is 20-50 μm, and the mass ratio of HA to HAP is 1:3 to 7.

5. The method for preparing an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 1, characterized in that: In step 2, the cross-linking agent is 1,4-butanediol glycidyl ether (BDDE), wherein the molar ratio of BDDE to HA structural unit is 0.4-0.6:

1.

6. The method for preparing an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 1, characterized in that: The cross-linking reaction temperature in step 1 and step 2 is 40-50° C., and the reaction time is 4-24 hours.

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

8. The method for preparing an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres 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 to 3 mg / mL.

9. The injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres obtained by the preparation method according to any one of claims 1 to 8.

10. The use of an injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres according to claim 9, characterized in that: The injectable cross-linked hyaluronic acid gel containing hydroxyapatite microspheres is used for preparing soft tissue filling materials in the field of medical cosmetology.

Images