Intelligent response type hydroxyapatite medical beauty gel as well as preparation method and application thereof

Abstract

The invention relates to the field of medical beauty industry, and particularly discloses intelligent response type hydroxyapatite medical beauty gel as well as a preparation method and application thereof. The intelligent response type hydroxyapatite medical beauty gel is prepared from the following raw material components: 90 to 95 parts of hydroxyapatite, 35 to 40 parts of regenerated silk fibroin composite magnetic microspheres, 50 to 55 parts of hyaluronic acid, 20 to 25 parts of recombinant III type humanized collagen and 15 to 20 parts of basic fibroblast growth factor sustained release microspheres. The preparation method comprises the following steps: uniformly dispersing all the components in hyaluronic acid, and stirring to obtain the medical beauty gel. The intelligent response type hydroxyapatite medical beauty gel belongs to an important gel material in orthopedic repair in the medical beauty industry, can well repair a bone defect part, and has excellent osteogenesis performance, biocompatibility and biological safety in vivo; and the product has the great advantages of high safety and good beautifying effect in medical beautifying bone shaping and bone repair.

Description

Technical Field

[0001] This application relates to the technical field of the medical aesthetics industry, and in particular to a smart responsive hydroxyapatite medical aesthetic gel, its preparation method, and its application. Background Technology

[0002] Hydroxyapatite (HA) is an important calcium phosphate, a natural mineralization of calcium apatite. Due to its similarity to the main inorganic components of human bones and teeth, it possesses excellent bioactivity and osteoconductivity. This allows hydroxyapatite to bond firmly with bone tissue, making it suitable for filling bone defects and soft tissue injections. It stimulates collagen regeneration, tightens and lifts the skin, and its effects last longer than traditional fillers. It is widely used in the medical aesthetics industry, typically as a bone repair and replacement material.

[0003] Currently, traditional hydroxyapatite materials have been widely used in orthopedic repair, but their application is limited by problems such as poor distribution uniformity, limited biocompatibility and slow bone regeneration rate. Especially when dealing with open or multi-segment complex fractures, it is difficult to achieve precise and stable distribution in specific locations, and there is a lack of effective means to accelerate the bone tissue regeneration process, resulting in a prolonged healing period, increased risk of infection and patient suffering. Summary of the Invention

[0004] To address the aforementioned technical issues, this application provides a smart responsive hydroxyapatite medical aesthetic gel, its preparation method, and its application.

[0005] In a first aspect, this application provides a smart responsive hydroxyapatite medical aesthetic gel, the raw materials used comprising the following components by weight: 90-95 parts of hydroxyapatite; 35-40 parts of regenerated silk fibroin composite magnetic microspheres; 50-55 parts hyaluronic acid; 20-25 parts of recombinant type III humanized collagen; 15-20 parts of basic fibroblast growth factor sustained-release microspheres.

[0006] Preferably, the raw materials used, by weight, include the following components: 93 parts of hydroxyapatite; 38 portions of regenerated silk fibroin composite magnetic microspheres; 52 parts hyaluronic acid; 22.5 portions of recombinant type III humanized collagen; 17.5 portions of basic fibroblast growth factor sustained-release microspheres.

[0007] Preferably, the basic fibroblast growth factor sustained-release microspheres are made from basic fibroblast growth factor and polylactic acid-glycolic acid polymer, wherein the LA / GA ratio in the polylactic acid-glycolic acid polymer is 3:1.

[0008] Preferably, the regenerated silk fibroin composite magnetic microspheres are made from regenerated silk fibroin and Fe3O4 nanoparticles.

[0009] By adopting the above technical solution, this application utilizes hydroxyapatite, regenerated silk fibroin composite magnetic microspheres, hyaluronic acid, recombinant type III humanized collagen, and basic fibroblast growth factor sustained-release microspheres mixed in a certain weight ratio to obtain a hydroxyapatite medical aesthetic gel with intelligent response function. This gel can solve the problem that traditional bone repair materials are difficult to accurately fill irregular defect areas, improve the dynamic regulation capability of bone repair materials, achieve on-demand filling and controllable release, and enhance the synergy of multi-stage repair functions, thereby improving repair efficiency and effect.

[0010] Specifically, hydroxyapatite is uniformly dispersed in the gel, providing osteoblast adhesion sites, accelerating extracellular matrix deposition, promoting early mineralization in bone defect areas, releasing ions to stimulate osteogenic differentiation of bone marrow mesenchymal stem cells, and hydroxyapatite is in a slow dissolution state in the physiological environment. The calcium and phosphorus ions it releases participate in new bone mineralization, which can prevent mechanical collapse in the later stages of repair. It can also induce vascular endothelial cell migration and promote the growth of new blood vessels into the gel.

[0011] Secondly, this application utilizes Fe3O4 nanoparticles to impart magnetism to regenerated silk fibroin composite magnetic microspheres. Under the guidance of an external magnetic field, the resulting regenerated silk fibroin composite magnetic microspheres can achieve the directional migration of microsphere clusters to the fracture site. The regenerated silk fibroin has excellent biocompatibility and biodegradability, and is easily absorbed by the human body. The recombinant type III humanized collagen is similar to human collagen, which can minimize immune risks and batch variations. The bioactive adhesion fragments in its structure enable it to promote cell adhesion and regulate cell phenotype, thereby accelerating the repair and regeneration of damaged skin. The synergistic effect of regenerated silk fibroin and recombinant type III humanized collagen provides an effective cartilage differentiation environment, thereby effectively promoting the repair of cartilage defects. Specifically, it promotes the migration of bone marrow mesenchymal stem cells to the cartilage defect site, increases cell density, and promotes cartilage differentiation of bone marrow mesenchymal stem cells, thus creating conditions for tissue regeneration. Good anti-inflammatory ability provides a suitable immune environment for cartilage repair.

[0012] Furthermore, regarding the basic fibroblast growth factor sustained-release microspheres, this application utilizes polylactic acid-glycolic acid polymer to provide a sustained-release effect for the basic fibroblast growth factor sustained-release microspheres, thereby achieving continuous drug delivery. Moreover, the polylactic acid-glycolic acid polymer and hydroxyapatite can achieve a good synergistic effect. The polylactic acid-glycolic acid polymer provides a flexible matrix, while hydroxyapatite enhances osteogenic activity. The combined use of the two can improve the compressive strength of the repaired structure to a level close to that of cancellous bone.

[0013] This application also strictly controls the LA / GA ratio in the polylactic acid-glycolic acid polymer to be 3:1. At this ratio, the polylactic acid-glycolic acid has high strength and slow degradation rate, achieving a balance between mechanical stability and continuous drug delivery. If the LA ratio is too high, the degradation rate will be too slow, which may lead to new damage to the bone defect. If the GA ratio is too high, the strength will be insufficient, and it will not be able to achieve a synergistic reinforcement effect with hydroxyapatite.

[0014] Basic fibroblast growth factor sustained-release microspheres can continuously release active factors, stimulate the proliferation of bone marrow mesenchymal stem cells, and upregulate the expression of osteogenic-related genes (such as ALP and BMP-2), accelerating osteogenic differentiation. At the same time, they can induce vascular endothelial cell migration, promote angiogenesis, and provide oxygen and nutrients to the bone defect area. Since basic fibroblast growth factor is easily degraded in the physiological environment, this application formulates it into sustained-release microspheres to increase the cumulative release rate. When used in combination with regenerated silk fibroin and recombinant type III humanized collagen, the regenerated silk fibroin and recombinant type III humanized collagen can rapidly reduce inflammation, increase cell density, and promote differentiation in the early stage of repair, while the basic fibroblast growth factor sustained-release microspheres can stably and continuously release active factors, enhancing long-term repair capabilities.

[0015] Secondly, this application also provides a method for preparing a smart responsive hydroxyapatite medical aesthetic gel, comprising the following steps: S1. Preparation of regenerated silk fibroin composite magnetic microspheres: Aminated Fe3O4 nanoparticles were obtained by amination treatment. Then, they were mixed and dispersed with regenerated silk fibroin in a solvent at a weight ratio of 5:(12-15). After stirring, they were added to the oil phase for homogenization and emulsification. Then, EDC / NHS composite crosslinking agent was added for crosslinking reaction. After filtration, washing, drying, and pH shell coating, regenerated silk fibroin composite magnetic microspheres were obtained. S2. Preparation of basic fibroblast growth factor sustained-release microspheres: Basic fibroblast growth factor was dispersed to form an inner aqueous phase, polylactic acid-glycolic acid polymer was dispersed to form an oil phase, and PVA was dispersed to form an outer aqueous phase. The inner aqueous phase and the oil phase were blended at the condition that basic fibroblast growth factor / polylactic acid-glycolic acid polymer = 1: (20-30), ultrasonically emulsified, and then blended with the outer aqueous phase to form a W / O / W emulsion. The emulsion was filtered to obtain microspheres, dried, and obtained basic fibroblast growth factor sustained-release microspheres. S3. Preparation of hydroxyapatite / recombinant type III humanized collagen scaffold: Recombinant type III humanized collagen and hydroxyapatite were blended, and after adding a cross-linking agent, the cross-linking reaction was carried out at 25-35℃ for 15-20h. The mixture was then filtered, washed, and dried to obtain a hydroxyapatite / recombinant type III humanized collagen scaffold. S4. Preparation of medical aesthetic gel: The regenerated silk fibroin composite magnetic microspheres, basic fibroblast growth factor sustained-release microspheres, and hydroxyapatite / recombinant type III humanized collagen scaffold were all dispersed in hyaluronic acid and stirred to obtain a medical aesthetic gel.

[0016] By employing the above-mentioned technical solutions, this application prepared regenerated silk fibroin composite magnetic microspheres, basic fibroblast growth factor sustained-release microspheres, and hydroxyapatite / recombinant type III humanized collagen scaffolds, respectively. Subsequently, all of these substances were thoroughly dispersed in hyaluronic acid to obtain a medical aesthetic gel. The scaffold composed of hydroxyapatite and recombinant type III humanized collagen exhibits a stable 3D network structure within the gel. Furthermore, the hydroxyapatite / recombinant type III humanized collagen scaffold synergistically interacts with the polylactic acid-glycolic acid polymer portion of the basic fibroblast growth factor sustained-release microspheres and the regenerated silk fibroin portion of the regenerated silk fibroin composite magnetic microspheres. Overall, the preparation method of this application can most fully utilize the individual advantages of each substance and the synergistic effect between them.

[0017] Preferably, in step S1, the specific steps for pH-coating the regenerated silk fibroin composite magnetic microspheres are as follows: The regenerated silk fibroin composite magnetic microspheres were sequentially immersed in polyacrylic acid solution and chitosan solution, and then dried.

[0018] By adopting the above technical solution, this application has coated the regenerated silk fibroin composite magnetic microspheres with a pH shell. When the ambient pH is below 7.0, the pH shell automatically dissolves and releases the internal active components, accelerating drug release. When the pH exceeds 7.0, it shrinks and delays the release of the active components, achieving targeted and controlled release to the lesion. It can also isolate the magnetic part from direct contact with human tissue during the initial injection period, acting as a "flexible buffer layer" to alleviate the stress mismatch between the magnetic core and the regenerated silk fibroin, avoid phase separation, and improve structural integrity. Therefore, the composite magnetic microspheres coated with the pH shell have both pH response and magnetic targeting, enabling more precise drug release and achieving bone repair effects.

[0019] Preferably, in step S2, the concentration of polylactic acid-glycolic acid polymer in the oil phase is 45-55 g / L.

[0020] By adopting the above technical solution, this application strictly controls the concentration of polylactic acid-glycolic acid polymer in the oil phase, and optimizes the surface morphology and drug loading stability of basic fibroblast growth factor sustained-release microspheres. If the concentration is too low, it will lead to incomplete encapsulation of basic fibroblast growth factor, resulting in insufficient strength of the oil-water interface film, drug leakage and precipitation, surface crystallization, and a surge in degradation rate. If the concentration is too high, it will cause the solvent in the oil phase to evaporate too quickly, forming surface pores, accelerating drug burst release, and easily forming irregular porous structures, increasing the surface defect rate. It may also cause polymer chain entanglement, inhibiting drug dispersion.

[0021] Preferably, in step S2, the drying process specifically involves: The obtained microspheres were flash-frozen in liquid nitrogen and then freeze-dried in a vacuum environment of -80℃ and 8Pa for 24 hours to obtain basic fibroblast growth factor sustained-release microspheres.

[0022] By adopting the above technical solution, this application performs liquid nitrogen flash freezing on the obtained microspheres, which instantly freezes the water inside the microspheres, inhibits ice crystal growth, avoids large ice crystals from piercing the microsphere wall or squeezing the drug carrier, maintains the internal porosity, and makes the solution inside the microspheres form an amorphous solid to prevent phase separation. Subsequently, vacuum freeze-drying is performed, and the ice crystals directly sublimate into water vapor, avoiding the surface tension of liquid water from damaging the microsphere structure. The low temperature operation throughout the process can inhibit the thermal degradation of basic fibroblast growth factor, while the vacuum environment can block the oxidation reaction, and the activity retention rate is close to 100%.

[0023] Thirdly, this application also provides an application of a smart responsive hydroxyapatite medical aesthetic gel, comprising the following steps: The intelligent responsive hydroxyapatite medical aesthetic gel is placed at the bone defect site. After suturing the periosteum, an external magnetic field is applied to the bone defect site to position the intelligent responsive hydroxyapatite medical aesthetic gel before suturing the subcutaneous tissue and skin.

[0024] Preferably, the magnetic field strength of the external magnetic field is 150-200 Gs.

[0025] By adopting the above technical solution, this application places the intelligent responsive hydroxyapatite medical aesthetic gel at the bone defect site, sutures the periosteum, and then applies an external magnetic field to the bone defect site. The magnetic field strength is controlled at 150-200 Gs, which can generate appropriate magnetic response force, achieving millimeter-level positioning accuracy of the medical aesthetic gel at the bone defect site. In addition, the magnetic field strength can also stimulate osteoblast activity, accelerate new bone formation, generate bioelectric effects, improve local blood circulation, and reduce inflammation. Under this field strength, hydroxyapatite can better release calcium and phosphorus ions, activate osteoblasts, and accelerate bone repair. After inducing precise positioning of the medical aesthetic gel, the subcutaneous tissue and skin are sutured, which can achieve good repair of the bone defect, significantly increase the volume of new bone and the number of trabeculae, reduce trabecular separation, promote bone mass accumulation, optimize the three-dimensional spatial arrangement of trabeculae, and significantly improve the mechanical properties of bone tissue. It has excellent osteogenic properties, biocompatibility, and biosafety in vivo.

[0026] In summary, this application has the following beneficial technical effects: 1. This application utilizes hydroxyapatite, regenerated silk fibroin composite magnetic microspheres, hyaluronic acid, recombinant type III humanized collagen, and basic fibroblast growth factor sustained-release microspheres mixed in a certain weight ratio to obtain a hydroxyapatite medical aesthetic gel with intelligent response function. This gel can solve the problem that traditional bone repair materials are difficult to accurately fill irregular defect areas, improve the dynamic regulation ability of bone repair materials, realize on-demand filling and controllable release, and enhance the synergy of multi-stage repair functions, thereby improving repair efficiency and effect. 2. This application prepared regenerated silk fibroin composite magnetic microspheres, basic fibroblast growth factor sustained-release microspheres, and hydroxyapatite / recombinant type III humanized collagen scaffolds, respectively. These substances were then thoroughly dispersed in hyaluronic acid to obtain a medical aesthetic gel. The scaffold composed of hydroxyapatite and recombinant type III humanized collagen exhibits a stable 3D network structure within the gel. Furthermore, the hydroxyapatite / recombinant type III humanized collagen scaffold synergistically interacts with the polylactic acid-glycolic acid polymer portion of the basic fibroblast growth factor sustained-release microspheres and the regenerated silk fibroin portion of the regenerated silk fibroin composite magnetic microspheres. Therefore, the preparation method of this application can most fully utilize the individual advantages and synergistic effects of each substance. 3. This application places a smart responsive hydroxyapatite medical aesthetic gel at the bone defect site. After suturing the periosteum, an external magnetic field is applied to the bone defect site. A certain magnetic field strength can generate an appropriate magnetic response force, achieving millimeter-level positioning accuracy of the medical aesthetic gel at the bone defect site. Furthermore, this magnetic field strength can also stimulate osteoblast activity, accelerate new bone formation, generate bioelectric effects, improve local blood circulation, and reduce inflammation. Under this field strength, hydroxyapatite can better release calcium and phosphorus ions, activate osteoblasts, and accelerate bone repair. After inducing precise positioning of the medical aesthetic gel, the subcutaneous tissue and skin are sutured, which can achieve good repair of the bone defect, significantly increase the volume of new bone and the number of trabeculae, reduce trabecular separation, promote bone mass accumulation, optimize the three-dimensional spatial arrangement of trabeculae, and significantly improve the mechanical properties of bone tissue. It has excellent osteogenic properties, biocompatibility, and biosafety in vivo. Detailed Implementation

[0027] Material source Unless otherwise specified, all raw materials used in this application are commercially available products, specifically: Hydroxyapatite was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., model number H875578, nano-grade; Regenerated silk fibroin was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. Recombinant type III humanized collagen was purchased from Guangdong Mingtong Biotechnology Co., Ltd. Basic fibroblast growth factor was purchased from Shanghai Maclean Biotechnology Co., Ltd.

[0028] The present application will be further described in detail below with reference to embodiments and comparative examples.

[0029] Example 1.1 A method for preparing a smart responsive hydroxyapatite medical aesthetic gel includes the following steps: S1. Preparation of regenerated silk fibroin composite magnetic microspheres: 20g of Fe3O4 nanoparticles were placed in ethanol and thoroughly dispersed. Then, 1g of the amination reagent 3-aminopropyltriethoxysilane was added, and the mixture was stirred at 70℃ for 2h to obtain aminated Fe3O4 nanoparticles. Subsequently, 15g of the aminated Fe3O4 nanoparticles and 36g of regenerated silk fibroin were co-dispersed in a 9.3mol / L lithium bromide solution. After magnetic stirring for 30min, the lithium bromide was removed by dialyzing. The entire mixture was then added dropwise to a cyclohexane-Span 80 mixture (cyclohexane to Span 80 volume ratio 100:1), and homogenized and emulsified at 8000rpm for 5min. Finally, 0.5% of the solution was added... The v / v EDC / NHS composite crosslinking agent was crosslinked at 40℃ for 2 hours. After filtration, washing, and drying, microspheres with a particle size of 5-10 μm were obtained. Subsequently, they were successively immersed in polyacrylic acid solution (0.1 g / L, pH=3.0) and chitosan solution (0.1 g / L, pH=5.0), each time for 10 min, and repeated 3 times. After removal and drying, regenerated silk fibroin composite magnetic microspheres were obtained. S2. Preparation of basic fibroblast growth factor sustained-release microspheres: Basic fibroblast growth factor (BGF) was dispersed in PBS containing 0.5% trehalose to ensure a BGF concentration of 0.01 g / L, forming an inner aqueous phase. Polylactic acid-glycolic acid (LA / GA) polymer (LA / GA = 3:1) was then dispersed in dichloromethane to obtain an oil phase with a LA / GA polymer concentration of 45 g / L. PVA solution was used as the outer aqueous phase. The inner aqueous phase and oil phase were then mixed according to a ratio of 10 g BGF to 200 g PVA. The lactic acid-glycolic acid polymer was designed for blending and ultrasonically emulsified at 100W for 30s to form a W / O emulsion. An external aqueous phase was then added until the PVA concentration reached 1wt%. The mixture was homogenized at 1000rpm for 2min to form a W / O / W emulsion. Microspheres were obtained by filtration and then subjected to liquid nitrogen flash freezing. Subsequently, the microspheres were placed in an environment of -80℃ and 8Pa vacuum for 24h to obtain basic fibroblast growth factor sustained-release microspheres. S3. Preparation of hydroxyapatite / recombinant type III humanized collagen scaffold: 20g of recombinant type III humanized collagen was dispersed in 167mL of lithium bromide solution with a concentration of 12mol / L, and then 95g of hydroxyapatite was added. After stirring for 30min, 10mL of 1,4-butanediol glycidyl ether crosslinking agent was added, and after stirring for 20min, the crosslinking reaction was carried out at 25℃ for 20h. After filtration, washing, and drying, hydroxyapatite / recombinant type III humanized collagen scaffold was obtained. S4. Preparation of medical aesthetic gel: All of the hydroxyapatite / recombinant type III humanized collagen scaffold, 35g of regenerated silk fibroin composite magnetic microspheres, and 20g of basic fibroblast growth factor sustained-release microspheres were dispersed in 55g of hyaluronic acid and stirred to obtain a medical aesthetic gel.

[0030] Example 1.2 A method for preparing a smart responsive hydroxyapatite medical aesthetic gel includes the following steps: S1. Preparation of regenerated silk fibroin composite magnetic microspheres: 20g of Fe3O4 nanoparticles were placed in ethanol and thoroughly dispersed. Then, 1g of the amination reagent 3-aminopropyltriethoxysilane was added, and the mixture was stirred at 70℃ for 2h to obtain aminated Fe3O4 nanoparticles. Subsequently, 15g of the aminated Fe3O4 nanoparticles were co-dispersed with 45g of regenerated silk fibroin in a 9.3mol / L lithium bromide solution. After magnetic stirring for 30min, the lithium bromide was removed by dialyzing. The entire mixture was then added dropwise to a cyclohexane-Span 80 mixture (cyclohexane to Span 80 volume ratio 100:1), and homogenized and emulsified at 8000rpm for 5min. Finally, 0.5% of the solution was added... The v / v EDC / NHS composite crosslinking agent was crosslinked at 40℃ for 2 hours. After filtration, washing, and drying, microspheres with a particle size of 5-10 μm were obtained. Subsequently, they were successively immersed in polyacrylic acid solution (0.1 g / L, pH=3.0) and chitosan solution (0.1 g / L, pH=5.0), each time for 10 min, and repeated 3 times. After removal and drying, regenerated silk fibroin composite magnetic microspheres were obtained. S2. Preparation of basic fibroblast growth factor sustained-release microspheres: Basic fibroblast growth factor (BGF) was dispersed in PBS containing 0.5% trehalose to ensure a BGF concentration of 0.01 g / L, forming an inner aqueous phase. Polylactic acid-glycolic acid (PLA) polymer (LA / GA = 3:1) was then dispersed in dichloromethane to obtain an oil phase with a PLA concentration of 55 g / L. PVA solution was used as the outer aqueous phase. The inner aqueous phase and oil phase were then mixed according to a ratio of 10 g BGF to 300 g PVA. The lactic acid-glycolic acid polymer was designed for blending and ultrasonically emulsified at 100W for 30s to form a W / O emulsion. An external aqueous phase was then added until the PVA concentration reached 1wt%. The mixture was homogenized at 1000rpm for 2min to form a W / O / W emulsion. Microspheres were obtained by filtration and then subjected to liquid nitrogen flash freezing. Subsequently, the microspheres were placed in an environment of -80℃ and 8Pa vacuum for 24h to obtain basic fibroblast growth factor sustained-release microspheres. S3. Preparation of hydroxyapatite / recombinant type III humanized collagen scaffold: 25g of recombinant type III humanized collagen was dispersed in 208mL of lithium bromide solution with a concentration of 12mol / L, and then 90g of hydroxyapatite was added. After stirring for 30min, 10mL of 1,4-butanediol glycidyl ether crosslinking agent was added. After stirring for 20min, the crosslinking reaction was carried out at 35℃ for 15h. The mixture was filtered, washed, and dried to obtain a hydroxyapatite / recombinant type III humanized collagen scaffold. S4. Preparation of medical aesthetic gel: All of the hydroxyapatite / recombinant type III humanized collagen scaffold, 40g of regenerated silk fibroin composite magnetic microspheres, and 15g of basic fibroblast growth factor sustained-release microspheres were dispersed in 50g of hyaluronic acid and stirred to obtain a medical aesthetic gel.

[0031] Example 1.3 A method for preparing a smart responsive hydroxyapatite medical aesthetic gel includes the following steps: S1. Preparation of regenerated silk fibroin composite magnetic microspheres: 20g of Fe3O4 nanoparticles were placed in ethanol and thoroughly dispersed. Then, 1g of the amination reagent 3-aminopropyltriethoxysilane was added, and the mixture was stirred at 70℃ for 2h to obtain aminated Fe3O4 nanoparticles. Subsequently, 15g of the aminated Fe3O4 nanoparticles were co-dispersed with 40g of regenerated silk fibroin in a 9.3mol / L lithium bromide solution. After magnetic stirring for 30min, the lithium bromide was removed by dialyzing. The entire mixture was then added dropwise to a cyclohexane-Span 80 mixture (cyclohexane to Span 80 volume ratio 100:1), and homogenized and emulsified at 8000rpm for 5min. 0.5% of the solution was then added. The v / v EDC / NHS composite crosslinking agent was crosslinked at 40℃ for 2 hours. After filtration, washing, and drying, microspheres with a particle size of 5-10 μm were obtained. Subsequently, they were successively immersed in polyacrylic acid solution (0.1 g / L, pH=3.0) and chitosan solution (0.1 g / L, pH=5.0), each time for 10 min, and repeated 3 times. After removal and drying, regenerated silk fibroin composite magnetic microspheres were obtained. S2. Preparation of basic fibroblast growth factor sustained-release microspheres: Basic fibroblast growth factor (BGF) was dispersed in PBS containing 0.5% trehalose to ensure a BGF concentration of 0.01 g / L, forming an inner aqueous phase. Polylactic acid-glycolic acid (PLA) polymer (LA / GA = 3:1) was then dispersed in dichloromethane to obtain an oil phase with a PLA concentration of 50 g / L. PVA solution was used as the outer aqueous phase. The inner aqueous phase and oil phase were then mixed according to a ratio of 10 g BGF to 250 g PVA. The lactic acid-glycolic acid polymer was designed for blending and ultrasonically emulsified at 100W for 30s to form a W / O emulsion. An external aqueous phase was then added until the PVA concentration reached 1wt%. The mixture was homogenized at 1000rpm for 2min to form a W / O / W emulsion. Microspheres were obtained by filtration and then subjected to liquid nitrogen flash freezing. Subsequently, the microspheres were placed in an environment of -80℃ and 8Pa vacuum for 24h to obtain basic fibroblast growth factor sustained-release microspheres. S3. Preparation of hydroxyapatite / recombinant type III humanized collagen scaffold: 22.5 g of recombinant type III humanized collagen was dispersed in 187 mL of lithium bromide solution with a concentration of 12 mol / L, and then 93 g of hydroxyapatite was added. After stirring for 30 min, 10 mL of 1,4-butanediol glycidyl ether crosslinking agent was added, and after stirring for 20 min, the crosslinking reaction was carried out at 30 °C for 17.5 h. The mixture was filtered, washed, and dried to obtain a hydroxyapatite / recombinant type III humanized collagen scaffold. S4. Preparation of medical aesthetic gel: All the hydroxyapatite / recombinant type III humanized collagen scaffold, 38g of regenerated silk fibroin composite magnetic microspheres, and 17.5g of basic fibroblast growth factor sustained-release microspheres were dispersed in 52g of hyaluronic acid and stirred to obtain a medical aesthetic gel.

[0032] Example 2.1 A method for preparing a smart responsive hydroxyapatite medical aesthetic gel differs from Example 1.3 in that, in S2, the polylactic acid-glycolic acid polymer (LA / GA=3:1) is replaced with a polylactic acid-glycolic acid polymer (LA / GA=1:1), while the rest is the same as in Example 1.3.

[0033] Example 2.2 A method for preparing a smart responsive hydroxyapatite medical aesthetic gel differs from Example 1.3 in that, in S2, the polylactic acid-glycolic acid polymer (LA / GA=3:1) is replaced with a polylactic acid-glycolic acid polymer (LA / GA=17:3), while the rest is the same as in Example 1.3.

[0034] Example 3.1 A method for preparing a smart responsive hydroxyapatite medical aesthetic gel differs from Example 1.3 in that, in S2, the concentration of polylactic acid-glycolic acid polymer in the oil phase is 40 g / L, while the rest are the same as in Example 1.3.

[0035] Example 3.2 A method for preparing a smart responsive hydroxyapatite medical aesthetic gel differs from Example 1.3 in that, in S2, the concentration of polylactic acid-glycolic acid polymer in the oil phase is 60 g / L, while the rest are the same as in Example 1.3.

[0036] Comparative Example 1.1 The difference from Example 1.3 is that S3 is removed, and in S4, 22.5g of recombinant type III humanized collagen scaffold, 38g of regenerated silk fibroin composite magnetic microspheres and 17.5g of basic fibroblast growth factor sustained-release microspheres are dispersed in 52g of hyaluronic acid. The rest is the same as in Example 1.3.

[0037] Comparative Example 1.2 The difference from Example 1.3 is that S3 is removed, and in S4, 93g of hydroxyapatite, 38g of regenerated silk fibroin composite magnetic microspheres and 17.5g of basic fibroblast growth factor sustained-release microspheres are dispersed in 52g of hyaluronic acid. The rest is the same as in Example 1.3.

[0038] Comparative Example 1.3 The difference from Example 1.3 is that in S4, the regenerated silk fibroin composite magnetic microspheres are removed, while the rest are the same as in Example 1.3.

[0039] Comparative Example 1.4 The difference from Example 1.3 is that in S4, the basic fibroblast growth factor sustained-release microspheres are removed, while the rest are the same as in Example 1.3.

[0040] Comparative Example 2.1 The difference from Example 1.3 is that S1 is removed, and in S4, 22.5g of recombinant type III humanized collagen, 93g of hydroxyapatite, 27.6g of regenerated silk fibroin composite magnetic microspheres and 17.5g of basic fibroblast growth factor sustained-release microspheres are dispersed in 52g of hyaluronic acid. The rest is the same as in Example 1.3.

[0041] Comparative Example 2.2 The difference from Example 1.3 is that S2 is removed, and in S4, 22.5g of recombinant type III humanized collagen, 93g of hydroxyapatite, 38g of regenerated silk fibroin composite magnetic microspheres and 0.6g of basic fibroblast growth factor are dispersed in 52g of hyaluronic acid. The rest is the same as in Example 1.3.

[0042] Comparative Example 2.3 The difference from Example 1.3 is that S3 is removed, and in S4, 22.5g of recombinant type III humanized collagen, 93g of hydroxyapatite, 26g of regenerated silk fibroin and 17.5g of basic fibroblast growth factor sustained-release microspheres are dispersed in 52g of hyaluronic acid. The rest is the same as in Example 1.3.

[0043] Comparative Example 2.4 The difference from Example 1.3 is that in S1, the regenerated silk fibroin composite magnetic microspheres are not coated with a pH shell, while the rest is the same as in Example 1.3.

[0044] Performance testing One hundred and fifty male SD rats (a total of 300 bilateral skull defect models) were randomly assigned to fourteen groups (examples and comparative examples) and a control group, with ten rats in each group. Each group was further divided into two subgroups based on the observation time: a 4-week group and an 8-week group, with five rats in each subgroup. After accurate weighing, the SD rats were anesthetized via intraperitoneal injection of 3% sodium pentobarbital solution (at a dose of 40 mg / kg). Once the SD rats reached deep anesthesia, a standard cranial preparation procedure was performed. The prepared area was disinfected with 1% povidone-iodine, followed by deiodination and further disinfection with 75% alcohol. Finally, a sterile drape was applied. Using a scalpel, an incision of approximately 15 mm was made starting from the midpoint of the line connecting the inner corners of the eyes, along the midsagittal crest of the skull. The skin and periosteum were then sequentially cut. The periosteum was then dissected using blunt dissection techniques to ensure adequate exposure of the skull in the surgical area. A 5mm diameter scalpel was used at approximately 1000 rpm. Two 5 mm bone defects were drilled on both sides of the sagittal suture of the skull using a millimeter trephine. During the operation, sterile saline was used for intermittent irrigation (drip rate maintained at 20 d / min) to effectively avoid thermal damage to bone tissue caused by frictional heat. When the trephine was about to reach the meninges, the dental elevator was replaced immediately. Layered prying was applied along the edge of the bone window to completely detach the pre-set circular bone flap, so that the dura mater could be fully exposed. According to the grouping, the medical aesthetic gel of each group was placed at the corresponding skull defect site. The periosteum was sutured. An external magnetic field with a magnetic field strength of 150-200 Gs was applied to the bone defect site to position the intelligent responsive hydroxyapatite medical aesthetic gel before suturing the subcutaneous tissue and skin. The skin of the suture area was disinfected with 1% povidone-iodine. Penicillin (20000 IU / 100g) was injected into the thigh muscle to prevent infection. Records were made and the rats were marked. The SD rats were placed on a 37°C heat pad. After they regained consciousness, they were returned to the SPF environment for continued feeding. After surgery, SD rats were fed for 4 and 8 weeks respectively, and then euthanized by carbon dioxide asphyxiation. The entire experimental process strictly followed the ethical guidelines for animal experiments. The skull of the SD rats was dissected, the skull base bone tissue was removed, and soft tissue was cleared to fully expose the bone defect area. Subsequently, the samples were fixed in 4% paraformaldehyde solution and stored at 4°C in the dark. Then, micro-computed tomography (Micro-CT) was performed. The data obtained from the micro-CT scan were quantitatively analyzed using Cruiser software to obtain the number of trabeculae (Tb.N), trabecular separation (Tb.Sp), and new bone volume (BV).

[0045] Table 1 Data Record Table

[0046] Data Analysis: As shown in Table 1, the medical aesthetic gels obtained in Examples 1.1-1.3 were able to reduce the number of trabeculae in the bone defect areas of SD rats from 0.61 mm to [amount missing] within 8 weeks. -1 Increased to 1.26-1.31mm -1 In SD rats, the trabecular separation in the bone defect area decreased from 1.70 mm to 1.06-1.16 mm in 4 weeks and from 1.64 mm to 0.34-0.39 mm in 8 weeks. The newly formed bone volume in the bone defect area of ​​SD rats also decreased from 0.65 mm in 4 weeks. 3 Increased to 6.07-6.24mm 3 8 weeks from 2.20mm 3 Increased to 24.07-25.65mm 3 It is evident that the apatite medical aesthetic gel of this application can enhance the dynamic regulation capability of bone repair materials and improve repair efficiency and effectiveness.

[0047] In Examples 2.1-2.2, this application adjusted the ratio of LA and GA in the polylactic acid-glycolic acid polymer. The results showed that the number of trabeculae and the volume of new bone in the bone defect area of ​​SD rats decreased, while the trabeculae separation slightly increased. It can be seen that when the ratio of LA / GA in the polylactic acid-glycolic acid polymer is 3:1, the polylactic acid-glycolic acid has high strength and slow degradation rate, achieving a balance between mechanical stability and continuous administration. If the proportion of LA is too high, the degradation rate will be too slow, which may lead to new damage in the bone defect area. If the proportion of GA is too high, the strength will be insufficient, and it will not be able to play a synergistic reinforcing role with hydroxyapatite.

[0048] In Examples 3.1-3.2, this application adjusted the concentration of polylactic acid-glycolic acid polymer in the oil phase of S2. The results showed that the number of trabeculae and the volume of new bone in the bone defect area of ​​SD rats decreased, while the trabeculae separation slightly increased. It can be seen that this application strictly controls the concentration of polylactic acid-glycolic acid polymer in the oil phase, and optimizes the surface morphology and drug loading stability of basic fibroblast growth factor sustained-release microspheres. If the concentration is too low, it will lead to incomplete encapsulation of basic fibroblast growth factor, resulting in insufficient oil-water interface film strength, drug leakage and precipitation, surface crystallization, and a surge in degradation rate. If the concentration is too high, it will lead to excessively rapid solvent evaporation in the oil phase, forming surface pores, accelerating drug burst release, and easily forming irregular porous structures, increasing the surface defect rate. It may also lead to polymer chain entanglement, inhibiting drug dispersion.

[0049] In Comparative Example 1.1, hydroxyapatite was removed in this application. The results showed that the number of trabeculae and the volume of new bone in the bone defect area of ​​SD rats decreased, while the separation of trabeculae increased. It can be seen that hydroxyapatite is uniformly dispersed in the gel, providing osteoblast adhesion sites, accelerating extracellular matrix deposition, promoting early mineralization in the bone defect area, releasing ions to stimulate osteogenic differentiation of bone marrow mesenchymal stem cells, and hydroxyapatite is in a slow dissolution state in the physiological environment. The calcium and phosphorus ions released by it participate in new bone mineralization, which can avoid mechanical collapse in the later stage of repair. It can also induce vascular endothelial cell migration and promote the growth of new blood vessels into the gel.

[0050] In Comparative Example 1.2, recombinant type III humanized collagen was removed from this application. The results showed that the number of trabeculae and the volume of new bone in the bone defect area of ​​SD rats decreased, while the trabeculae separation increased. It can be seen that recombinant type III humanized collagen can minimize immune risk and batch variation. The bioactive adhesion fragments in its structure enable it to promote cell adhesion and regulate cell phenotype, which can accelerate the repair and regeneration of damaged skin. Furthermore, the synergistic effect of regenerating silk fibroin and recombinant type III humanized collagen provides an effective cartilage differentiation environment, thereby effectively promoting the repair of cartilage defects. Specifically, it promotes the migration of bone marrow mesenchymal stem cells to the cartilage defect site, increases cell density, and promotes the cartilage differentiation of bone marrow mesenchymal stem cells, thus creating conditions for tissue regeneration. Its good anti-inflammatory ability provides a suitable immune environment for cartilage repair.

[0051] In Comparative Example 1.3, the regenerated silk fibroin composite magnetic microspheres were removed in this application. The results showed that the number of trabeculae and the volume of new bone in the bone defect area of ​​SD rats decreased, and the separation of trabeculae increased. It can be seen that under the guidance of an external magnetic field, the regenerated silk fibroin composite magnetic microspheres can achieve the purpose of directional migration of microsphere clusters to the fracture site. Among them, the regenerated silk fibroin has excellent biocompatibility and biodegradability and is easily absorbed by the human body.

[0052] In Comparative Example 1.4, the basic fibroblast growth factor sustained-release microspheres were removed in this application. The results showed that the number of trabeculae and the volume of new bone in the bone defect area of ​​SD rats decreased, while the trabeculae separation increased. It can be seen that the basic fibroblast growth factor sustained-release microspheres can continuously release active factors, stimulate the proliferation of bone marrow mesenchymal stem cells, and upregulate the expression of osteogenic-related genes (such as ALP and BMP-2), accelerating osteogenic differentiation. At the same time, it can induce vascular endothelial cell migration, promote angiogenesis, and provide oxygen and nutrients to the bone defect area. Since basic fibroblast growth factor is easily degraded in the physiological environment, this application formulates it into sustained-release microspheres to increase the cumulative release rate. When used in combination with regenerated silk fibroin and recombinant type III humanized collagen, the regenerated silk fibroin and recombinant type III humanized collagen can rapidly reduce inflammation, increase cell density, and promote differentiation in the early stage of repair. The basic fibroblast growth factor sustained-release microspheres can stably and continuously release active factors, enhancing long-term repair capabilities.

[0053] In Comparative Example 2.1, this application did not prepare a hydroxyapatite / recombinant type III humanized collagen scaffold by combining hydroxyapatite and recombinant type III humanized collagen. Instead, they were separately added to a medical aesthetic gel for dispersion. The results showed that the number of trabeculae and the volume of new bone in the bone defect area of ​​SD rats decreased, and the trabeculae separation increased. It can be seen that the scaffold composed of hydroxyapatite and recombinant type III humanized collagen has a stable 3D network structure in the gel.

[0054] In Comparative Example 2.2, this application did not prepare basic fibroblast growth factor sustained-release microspheres, but instead directly added basic fibroblast growth factor to a medical aesthetic gel for dispersion. The results showed that the number of trabeculae and the volume of new bone in the bone defect area of ​​SD rats at 8 weeks decreased, and the trabeculae separation increased. It can be seen that this application can improve the cumulative release rate by preparing it as sustained-release microspheres.

[0055] In Comparative Example 2.3, this application did not prepare regenerated silk fibroin into regenerated silk fibroin composite magnetic microspheres, but instead directly dispersed the regenerated silk fibroin in a medical aesthetic gel. The results showed that the number of trabeculae and the volume of new bone in the bone defect area of ​​SD rats decreased, and the trabeculae separation increased. It can be seen that the regenerated silk fibroin composite magnetic microspheres of this application can achieve the purpose of directional migration of microsphere clusters to the fracture site under the guidance of an external magnetic field.

[0056] In Comparative Example 2.4, this application did not perform pH layer coating on the regenerated silk fibroin composite magnetic microspheres. The results showed that the number of trabeculae and the volume of new bone in the bone defect area of ​​SD rats decreased, and the trabeculae separation was improved. It can be seen that the pH layer coating of this application can realize the automatic dissolution of the pH shell and release of the internal active components, accelerate drug release, achieve targeted and controlled release of the lesion, and isolate the magnetic part from direct contact with human tissue during the initial injection. As a "flexible buffer layer", it can alleviate the stress mismatch between the magnetic core and the regenerated silk fibroin, avoid phase separation, and improve structural integrity. Therefore, the composite magnetic microspheres coated with pH shell have the dual driving force of pH response and magnetic targeting, which can release drugs more accurately and achieve bone repair effect.

[0057] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A smart responsive hydroxyapatite medical aesthetic gel, characterized in that, The raw materials used, by weight, include the following components: 90-95 parts of hydroxyapatite; 35-40 parts of regenerated silk fibroin composite magnetic microspheres; 50-55 parts hyaluronic acid; 20-25 parts of recombinant type III humanized collagen; 15-20 parts of basic fibroblast growth factor sustained-release microspheres.

2. The intelligent responsive hydroxyapatite medical aesthetic gel according to claim 1, characterized in that, The raw materials used, by weight, include the following components: 93 parts of hydroxyapatite; 38 portions of regenerated silk fibroin composite magnetic microspheres; 52 parts hyaluronic acid; 22.5 portions of recombinant type III humanized collagen; 17.5 portions of basic fibroblast growth factor sustained-release microspheres.

3. The intelligent responsive hydroxyapatite medical aesthetic gel according to claim 1, characterized in that, The basic fibroblast growth factor sustained-release microspheres are made from basic fibroblast growth factor and polylactic acid-glycolic acid polymer, wherein the LA / GA ratio in the polylactic acid-glycolic acid polymer is 3:

1.

4. The intelligent responsive hydroxyapatite medical aesthetic gel according to claim 1, characterized in that, The regenerated silk fibroin composite magnetic microspheres are made from regenerated silk fibroin and Fe3O4 nanoparticles.

5. A method for preparing the intelligent responsive hydroxyapatite medical aesthetic gel according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation of regenerated silk fibroin composite magnetic microspheres: Aminated Fe3O4 nanoparticles were obtained by amination treatment. Then, they were mixed and dispersed with regenerated silk fibroin in a solvent at a weight ratio of 5:(12-15). After stirring, they were added to the oil phase for homogenization and emulsification. Then, EDC / NHS composite crosslinking agent was added for crosslinking reaction. After filtration, washing, drying, and pH shell coating, regenerated silk fibroin composite magnetic microspheres were obtained. S2. Preparation of basic fibroblast growth factor sustained-release microspheres: Basic fibroblast growth factor was dispersed to form an inner aqueous phase, polylactic acid-glycolic acid polymer was dispersed to form an oil phase, and PVA was dispersed to form an outer aqueous phase. The inner aqueous phase and the oil phase were blended at the condition that basic fibroblast growth factor / polylactic acid-glycolic acid polymer = 1: (20-30), ultrasonically emulsified, and then blended with the outer aqueous phase to form a W / O / W emulsion. The emulsion was filtered to obtain microspheres, dried, and obtained basic fibroblast growth factor sustained-release microspheres. S3. Preparation of hydroxyapatite / recombinant type III humanized collagen scaffold: Recombinant type III humanized collagen and hydroxyapatite were blended, and after adding a cross-linking agent, the cross-linking reaction was carried out at 25-35℃ for 15-20h. The mixture was then filtered, washed, and dried to obtain a hydroxyapatite / recombinant type III humanized collagen scaffold. S4. Preparation of medical aesthetic gel: The regenerated silk fibroin composite magnetic microspheres, basic fibroblast growth factor sustained-release microspheres, and hydroxyapatite / recombinant type III humanized collagen scaffold were all dispersed in hyaluronic acid and stirred to obtain a medical aesthetic gel.

6. The preparation method of the intelligent responsive hydroxyapatite medical aesthetic gel according to claim 5, characterized in that, In step S1, the specific steps for pH-coating the regenerated silk fibroin composite magnetic microspheres are as follows: The regenerated silk fibroin composite magnetic microspheres were sequentially immersed in polyacrylic acid solution and chitosan solution, and then dried.

7. The preparation method of the intelligent responsive hydroxyapatite medical aesthetic gel according to claim 5, characterized in that, In S2, the concentration of polylactic acid-hydroxyacetic acid polymer in the oil phase is 45-55 g / L.

8. The preparation method of the intelligent responsive hydroxyapatite medical aesthetic gel according to claim 5, characterized in that, In step S2, the specific drying operation is as follows: The obtained microspheres were flash-frozen in liquid nitrogen and then freeze-dried in a vacuum environment of -80℃ and 8Pa for 24 hours to obtain basic fibroblast growth factor sustained-release microspheres.

9. An application of a smart responsive hydroxyapatite medical aesthetic gel, characterized in that, Includes the following steps: The intelligent responsive hydroxyapatite medical aesthetic gel is placed at the bone defect site. After suturing the periosteum, an external magnetic field is applied to the bone defect site to position the intelligent responsive hydroxyapatite medical aesthetic gel before suturing the subcutaneous tissue and skin.

10. The application of the intelligent responsive hydroxyapatite medical aesthetic gel according to claim 9, characterized in that, The magnetic field strength of the applied magnetic field is 150-200 Gs.

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