Absorbable bone wax for promoting bone healing and preparation method thereof

Abstract

The invention relates to absorbable bone wax for promoting bone healing and a preparation method thereof, and belongs to the technical field of bone healing materials, the bone wax comprises a degradable copolymer, PTMC, magnesium-calcium silicate-biological peptide composite microspheres, mesoporous silica loaded panax notoginseng saponins and hydrogenated castor oil. The degradable copolymer is prepared by reacting PEG (Polyethylene Glycol), stannous octoate, D, L-lactide and epsilon-caprolactone and precipitating. The magnesium-calcium silicate-biological peptide composite microspheres are prepared by loading biological peptide on porous magnesium-calcium silicate microspheres and then coating the biological peptide with polydopamine. The biological peptide is a peptide fragment between 1kDa and 5kDa obtained by carrying out step-by-step enzymolysis on urechis unicinctus through papain and trypsin. All the components are compounded according to a specific proportion to prepare the bone wax, the limitation that traditional bone wax is poor in degradability and single in function is broken through, and the integrated functions of hemostasis, degradation and efficient bone induction are achieved. Through cooperation of the controllable degradation base material and the high-activity functional component, precise matching of the degradation rate and the bone regeneration period is achieved.

Description

Technical Field

[0001] This invention belongs to the field of bone healing materials technology, specifically relating to an absorbable bone wax that promotes bone healing and its preparation method. Background Technology

[0002] Bone wax is a key consumable used in orthopedic and neurosurgical procedures to control diffuse bleeding on the bone surface. Traditional bone wax, with beeswax and paraffin as its core components, has an immediate hemostatic effect, but because it is non-degradable, its residue in the body can form a physical barrier, hindering bone cell migration and bone tissue remodeling, and easily causing nonunion, chronic inflammation and infection risks. Its application is limited in surgical scenarios that require bone healing, such as spinal fusion and bone defect repair.

[0003] To overcome the limitations of traditional bone wax, absorbable bone wax technology has been gradually developed. For example, related patent CN105816905B uses polyoxypropylene and polyoxyethylene block copolymers and random copolymers as base materials, and combines them with chitosan to achieve absorbability and basic healing promotion functions; CN109125791B introduces strontium-doped hydroxyapatite and microcrystalline cellulose into the copolymer base material to enhance bone repair activity; CN109432487B further optimizes the formula, using block copolymers and random copolymers as carriers, and combining them with alkylated chitosan and strontium-doped carbon-containing nano-hydroxyapatite to improve the compatibility of the material with bone tissue.

[0004] While existing technologies have achieved the absorbability and preliminary bone healing promotion capabilities of bone wax, significant technical bottlenecks remain: First, the active ingredients have limited healing efficiency and a weak induction effect on osteoblast proliferation and differentiation, resulting in a relatively long bone defect healing cycle; second, the synergy between the substrate and the active ingredients is insufficient, making it difficult to precisely match the degradation rate with the bone tissue regeneration rate, which can easily lead to hemostasis failure due to excessively rapid degradation or hindering bone growth due to excessively slow degradation; and third, the function is singular, focusing only on hemostasis and basic repair.

[0005] With the development of precision orthopedic surgery, the clinical demand for bone wax has evolved from "hemostasis and absorbability" to an integrated approach of "hemostasis, degradation, and efficient bone induction." Therefore, it is necessary to screen highly active bone-inducing components, optimize the ratio of substrate and active ingredients, and refine the compounding process to achieve a precise match between degradation rate and bone regeneration, thereby enhancing bone repair and overcoming the limitations of existing technologies. Summary of the Invention

[0006] To address the problems of low healing efficiency, insufficient synergy between the substrate and active ingredients, mismatched degradation rates, and limited functionality in existing bone wax technologies, this invention provides an absorbable bone wax for promoting bone healing and its preparation method. The method involves preparing a biodegradable copolymer, magnesium-calcium silicate-biopeptide composite microspheres, and mesoporous silica-supported Panax notoginseng saponins, which are then combined with PTMC and hydrogenated castor oil to prepare the bone wax. This overcomes the limitations of traditional bone waxes, such as poor degradability and limited functionality, achieving integrated functions of hemostasis, degradation, and efficient bone induction. Through the synergy of a controllable degradable substrate and highly active functional components, a precise match between the degradation rate and the bone regeneration cycle is achieved. The specific technical solution is as follows:

[0007] An absorbable bone wax that promotes bone healing comprises a biodegradable copolymer in a mass ratio of (25-30):(8-10):(9-13):(3-5):(2-4), PTMC, magnesium-calcium silicate-biopeptide composite microspheres, mesoporous silica-supported Panax notoginseng saponins and hydrogenated castor oil. The biodegradable copolymer is prepared by reacting PEG, stannous octoate, D,L-lactide, and ε-caprolactone at a mass ratio of 100:(0.3-0.6):(115-135):(115-135) at 125℃-135℃ and precipitating in n-hexane; the PEG comprises PEG-400 and PEG-1000 at a mass ratio of (2-4):(5-7); The magnesium-calcium silicate-biopeptide composite microspheres are prepared by porous magnesium-calcium silicate microspheres and biopeptides adsorbed in phosphate buffer at a mass ratio of (10-15):(0.8-1.2), centrifuged to obtain solid, dispersed in Tris-HCl buffer containing dopamine at pH 8.2-8.5, stirred to prepare polydopamine coating layer, and the solid is obtained. The bioactive peptides are obtained by hydrolyzing a pulverized monocyclic urchin in deionized water with 1.5%–2% papain by mass of the pulverized material at pH 6.2–6.5 and 50–55°C for 2–2.5 hours, followed by hydrolysis with 1%–2% trypsin by mass of the pulverized material at pH 7.8–8.2 and 35–40°C for 1.5–2 hours, yielding peptide fractions between 1 kDa and 5 kDa. The porous magnesium-calcium silicate microspheres were prepared by sol-gel of Ca(NO3)2·4H2O, Mg(NO3)2·6H2O and TEOS in a mass ratio of (46-52):(7-9):(20-25), spray-dried, calcined at 650℃-750℃ for 3.5-4.5 hours, and ground through a 150-200 mesh sieve.

[0008] Furthermore, the preparation method of the biodegradable copolymer includes the following steps: PEG:stannous octoate:D,L-lactide:ε-caprolactone = 100:(0.3-0.6):(115-135):(115-135) by mass ratio; under argon protection, PEG, stannous octoate, D,L-lactide and ε-caprolactone monomers are stirred and reacted to obtain a reaction solution; anhydrous dichloromethane is added and stirred to obtain a diluted solution; under stirring, the diluted solution is added dropwise to n-hexane to precipitate, centrifuged, the solid is taken, washed with n-hexane, and vacuum dried to obtain the biodegradable copolymer.

[0009] In the above-mentioned method for preparing the biodegradable copolymer, the PEG comprises PEG-400 and PEG-1000 in a mass ratio of (2-4):(5-7); the stirring reaction is carried out at 125℃-135℃ for 22-26 hours; the amount of anhydrous dichloromethane is 8-12 times the mass of the reaction solution; the amount of n-hexane is 8-12 times the volume of the diluent; and all n-hexane is pre-cooled n-hexane at 2℃-6℃.

[0010] Furthermore, the preparation method of the magnesium-calcium silicate-biopeptide composite microspheres includes the following steps: The porous magnesium-calcium silicate microspheres are dispersed in phosphate buffer at a mass ratio of (10-15):(120-180):(0.8-1.2). The biopeptide is added, the mixture is shaken to adsorb, centrifuged, and the solid is collected. The solid is then dispersed in Tris-HCl buffer (pH 8.2-8.5) containing 1 mg / mL-1.5 mg / mL dopamine. The mixture is stirred and centrifuged at room temperature in the dark. The solid is then washed with deionized water and freeze-dried to obtain the magnesium-calcium silicate-biopeptide composite microspheres.

[0011] In the above-mentioned method for preparing magnesium-calcium silicate-biopeptide composite microspheres, the preparation method of the biopeptide includes: pulverizing *Ulva monocyclicis* into a slurry, adding deionized water, adjusting the pH to 6.2–6.5, adding 1.5%–2% papain by weight of the slurry, enzymatically hydrolyzing at 50℃–55℃ for 2–2.5 h, inactivating the enzyme, centrifuging at 4000 rpm–5000 rpm for 15 min–20 min, taking the supernatant, adjusting the pH to 7.8–8.2, adding 1%–2% trypsin by weight of the slurry, enzymatically hydrolyzing at 35℃–40℃ for 1.5 h–2 h, inactivating the enzyme, centrifuging at 8000 rpm–8500 rpm for 10 min–15 min, taking the supernatant, separating by ultrafiltration membrane, collecting peptide fractions with molecular weights between 1 kDa and 5 kDa, and freeze-drying to obtain the biopeptide.

[0012] The preparation method of the above-mentioned magnesium-calcium silicate-biopeptide composite microspheres includes the following steps: Ca(NO3)2·4H2O:Mg(NO3)2·6H2O:TEOS = (46-52):(7-9):(20-25) by mass ratio; Ca(NO3)2·4H2O and Mg(NO3)2·6H2O are dissolved in deionized water to obtain solution A; TEOS is dissolved in anhydrous ethanol to obtain solution B; solution B is added to solution A under stirring to form a sol, which is then spray-dried to obtain precursor powder; the precursor powder is heated to 650℃-750℃, calcined for 3.5h-4.5h, cooled, and ground through a 150-200 mesh sieve to obtain porous magnesium-calcium silicate microspheres.

[0013] In the above preparation method of magnesium-calcium silicate-biopeptide composite microspheres, the oscillation adsorption is performed at 35℃~40℃ for 22h~26h; the stirring is performed at 150rpm~200rpm for 24h~28h.

[0014] Furthermore, the preparation method of the mesoporous silica-loaded total saponins of Panax notoginseng includes the following steps: CTAB:deionized water:sodium hydroxide aqueous solution:TEOS in a mass ratio of (1-1.2):(500-550):(3.5-4.5):(4-5); CTAB is dissolved in deionized water, sodium hydroxide aqueous solution is added, TEOS is added under stirring, the mixture is stirred and reacted, centrifuged, the solid is collected, washed, and calcined to obtain mesoporous silica nanoparticles; the mesoporous silica nanoparticles are dispersed in phosphate buffer, total saponins of Panax notoginseng are added, the mixture is loaded by shaking in the dark, centrifuged, the solid is collected, rinsed, and freeze-dried to obtain mesoporous silica-loaded total saponins of Panax notoginseng.

[0015] In the above method for preparing total saponins of Panax notoginseng supported on mesoporous silica, the concentration of the sodium hydroxide aqueous solution is 1.8M to 2.0M; the reaction is stirred for 1.5h to 2.5h after the addition of TEOS; the washing is performed by alternating washing with anhydrous ethanol and deionized water 2 to 3 times each; the calcination is performed by heating to 550℃ to 600℃ at a rate of 1℃ / min and calcining for 5h to 6h; the total saponins of Panax notoginseng are added at a ratio of 100μg to 120μg of total saponins of Panax notoginseng per milligram of mesoporous silica nanoparticles; the light-protected shaking loading is performed at 25℃ to 30℃ and 80rpm to 100rpm for 12h to 16h in the dark; the rinsing is performed with phosphate buffer solution pre-cooled to 4℃ to 8℃ with a pH of 7.0 to 7.4.

[0016] The preparation method of the above-mentioned absorbable bone wax that promotes bone healing includes the following steps: According to the formula mass ratio, the biodegradable copolymer and PTMC are mixed evenly at 60℃~65℃ to form a base material. The temperature is then lowered to 45℃~50℃, and hydrogenated castor oil, magnesium-calcium silicate-biopeptide composite microspheres, and mesoporous silica-supported Panax notoginseng saponins are added in sequence and mixed evenly to form a paste. The paste is then injected into a mold at 45℃~50℃ and cooled and shaped at 2℃~6℃ to obtain bone wax.

[0017] This invention provides an absorbable bone wax that promotes bone healing and its preparation method, with the following beneficial effects: I. This invention's bone wax overcomes the limitations of traditional bone waxes, which suffer from poor degradability and limited functionality, achieving integrated functions of hemostasis, degradation, and efficient bone induction. Through the synergy of a controllable degradable substrate and highly active functional components, it achieves a precise match between the degradation rate and the bone regeneration cycle. Simultaneously, through the sequential synergy of anti-inflammatory, angiogenesis-promoting, and osteogenic effects, it improves the bone healing microenvironment and enhances bone regeneration dynamics. Furthermore, it possesses excellent biocompatibility and manipulability, meeting the multifunctional needs of bone waxes.

[0018] II. In the preparation of biodegradable copolymers, PEG-400 and PEG-1000 are compounded in a specific ratio to optimize the hydrophilicity and swelling properties of the substrate, ensuring fluid wetting and release of active ingredients. D,L-lactide and ε-caprolactone are copolymerized in a specific ratio to form an amorphous-crystalline interwoven structure. The product is controlled by adjusting the reaction temperature and time to ensure the degradation rate and avoid excessively rapid collapse or long-term retention. An appropriate amount of stannous octoate is used as a catalyst to precisely control the degree of polymerization, ensuring the mechanical properties and degradation stability of the substrate.

[0019] III. In the preparation of magnesium-calcium silicate-biopeptide composite microspheres, calcination at 650℃~750℃ and passing through a 150-200 mesh sieve to form porous magnesium-calcium silicate microspheres can provide high specific surface area, achieving efficient loading of biopeptides and Mg. 2+ The sustained release of silicate ions provides continuous active stimulation for osteogenic formation. Monocyclic urticaria undergoes a two-step enzymatic hydrolysis with papain and trypsin to directionally obtain highly efficient active peptides ranging from 1kDa to 5kDa, providing anti-inflammatory, proliferative, differentiation-promoting, mineralization-enhancing, and angiogenic effects. A dopamine coating strengthens the interfacial binding of microspheres to the substrate and bone tissue, ensuring stable release of the bioactive peptides.

[0020] IV. In the mesoporous silica-loaded total saponins of Panax notoginseng, calcination at 550℃~600℃ removes the template agent to form mesoporous silica, thereby achieving physical protection and controlled sustained release of total saponins of Panax notoginseng, avoiding rapid release, delaying oxidation and degradation inactivation, and ensuring the sequential functions of early anti-inflammatory and mid-to-late-stage angiogenesis promotion.

[0021] 5. PTMC degrades slowly through surface erosion, and its overall degradation rate is synergistically regulated by a specific ratio with degradable copolymers.

[0022] VI. Hydrogenated castor oil optimizes the adhesiveness and plasticity of the ointment, ensuring bone surface adhesion and immediate hemostasis during surgery, and its degradation products are biocompatible.

[0023] VII. In the preparation method of bone wax, biodegradable copolymers are blended with PTMC to form a hydrophilic-hydrophobic interwoven three-dimensional network, providing a stable carrier for the subsequent dispersion of functional components; hydrogenated castor oil is added first to optimize the rheological properties of the paste, and then solid functional components are added to avoid particle agglomeration and ensure uniform dispersion; low-temperature mixing avoids thermal inactivation of bioactive peptides and total saponins of Panax notoginseng, and low-temperature setting ensures the morphological stability of bone wax.

[0024] In summary, the compound system of biodegradable copolymers and PTMC in bone wax of this invention, matched with the sustained-release properties of composite microspheres and Panax notoginseng saponins, achieves a match between the degradation rate and the bone regeneration rate, avoiding hemostatic failure due to excessively rapid degradation or bone growth inhibition due to excessively slow degradation. Hydrogenated castor oil provides rapid hemostasis, while Panax notoginseng saponins offer early anti-inflammatory effects. The composite microspheres continuously promote bone healing, and the sustained release of Panax notoginseng saponins continuously promotes angiogenesis; each function precisely exerts its effect at different stages of bone healing. Bioactive peptides and Mg... 2+ A synergistic osteogenic network is formed by the activation of silicate ions and the formation of total saponins from Panax notoginseng, enhancing activity and optimizing the environment. A dopamine coating and mesoporous loading ensure stable release and efficacy of functional components during degradation. All components work synergistically to ensure biocompatibility and cell adaptability. Detailed Implementation

[0025] The present invention will be further described below with reference to specific implementation examples, but the present invention is not limited to these embodiments.

[0026] Definitions: PTMC stands for polytrimethylene carbonate; PEG stands for polyethylene glycol; CTAB stands for hexadecyltrimethylammonium bromide; TEOS stands for tetraethyl orthosilicate.

[0027] Example 1 An absorbable bone wax that promotes bone healing comprises a biodegradable copolymer in a mass ratio of 27:9:11:4:3, PTMC, magnesium-calcium silicate-biopeptide composite microspheres, mesoporous silica-supported total saponins of Panax notoginseng, and hydrogenated castor oil.

[0028] The preparation method of the biodegradable copolymer includes the following steps: Prepare materials according to the mass ratio of PEG:stannous octoate:D,L-lactide:ε-caprolactone = 100:0.5:125:127, wherein the PEG comprises PEG-400 and PEG-1000 in a mass ratio of 3.5:5.5. Under a micro-positive pressure protection of 0.09 MPa argon gas, the PEG, stannous octoate, D,L-lactide, and ε-caprolactone monomers are stirred at 125℃~135℃ and 320 rpm for 24 h to obtain a reaction solution; cool to 55℃, add 11 times the mass of the reaction solution in anhydrous dichloromethane, and stir to obtain a diluted solution; under stirring at 400 rpm, add the diluted solution dropwise to 10 times the volume of the diluted solution in 4℃ pre-cooled n-hexane to precipitate, centrifuge at 3200 rpm for 18 min, collect the solid, wash three times with 4℃ pre-cooled n-hexane, and vacuum dry at 42℃ to constant weight to obtain the biodegradable copolymer.

[0029] The preparation method of magnesium-calcium silicate-biopeptide composite microspheres includes the following steps: S1, Preparation of bioactive peptides: *Ulva monocyclicis* was pulverized into a slurry, and 9 times its weight of deionized water was added to adjust the pH to 6.3. 1.8% of the slurry weight of papain was added, and the mixture was enzymatically hydrolyzed at 52℃ for 2 hours. The temperature was then raised to 88℃ to inactivate the enzyme for 12 minutes, cooled to room temperature, and centrifuged at 4500 rpm for 18 minutes. The supernatant was collected. The pH was adjusted to 8.0 using 0.3M sodium hydroxide aqueous solution, and 1.5% of the slurry weight of trypsin was added. The mixture was enzymatically hydrolyzed at 37℃ for 1.5 hours, and then raised to 88℃ to inactivate the enzyme for 12 minutes. The temperature was then cooled to room temperature, centrifuged at 8200 rpm for 12 minutes, and the supernatant was collected. Ultrafiltration membranes with molecular weight cutoffs of 1 kDa and 5 kDa were used for separation, and peptide fractions with molecular weights between 1 kDa and 5 kDa were collected. These fractions were then freeze-dried to obtain bioactive peptides. S2, Preparation of porous magnesium-calcium silicate microspheres: Ca(NO3)2·4H2O:Mg(NO3)2·6H2O:TEOS = 49:8:23 mass ratio was used. Ca(NO3)2·4H2O and Mg(NO3)2·6H2O were dissolved in 5.5 times the total mass of deionized water to obtain solution A. TEOS was dissolved in 3.5 times the mass of anhydrous ethanol to obtain solution B. Solution B was added to solution A at a rate of 10 mL / min under stirring at 500 rpm to form a uniform sol. Spray drying was then performed with the following parameters: inlet temperature 200℃, outlet temperature 90℃, feed rate 8 mL / min, and atomization pressure 0.3 MPa, to obtain precursor powder. The precursor powder was heated to 700℃ at a rate of 5℃ / min, calcined for 4 h, cooled, and ground through a 150-mesh sieve to obtain porous magnesium-calcium silicate microspheres. S3, Loading and Surface Modification: The porous magnesium-calcium silicate microspheres were dispersed in a pH 7.3 phosphate buffer solution at a mass ratio of 13:150:1. The bioactive peptide was added, and the mixture was shaken at 90 rpm for 24 h at 35℃–40℃. After centrifugation at 4800 rpm for 18 min, the solid was collected and dispersed in a pH 8.3 Tris-HCl buffer solution containing 1.2 mg / mL dopamine. The mixture was stirred at 180 rpm for 26 h at room temperature in the dark, and then centrifuged at 4800 rpm for 18 min. The solid was collected, washed twice with deionized water, and freeze-dried to obtain magnesium-calcium silicate-bioactive peptide composite microspheres with a bioactive peptide loading of not less than 40 wt%.

[0030] The preparation method of mesoporous silica-supported total saponins of Panax notoginseng includes the following steps: CTAB:deionized water:sodium hydroxide aqueous solution:TEOS = 1.1:520:4:4.5 by mass ratio; CTAB is dissolved in deionized water at 62℃, and 1.9M sodium hydroxide aqueous solution is added. The system temperature is maintained between 75℃ and 80℃. TEOS is added dropwise at 0.8mL / min while stirring at 380rpm. The reaction is continued for 2 hours, cooled to room temperature, centrifuged at 8200rpm for 20 minutes, and the solid is collected. It is washed twice alternately with anhydrous ethanol and deionized water to remove residual CTAB and impurities. The solid is then calcined at 580℃ for 5.5 hours in air at 1℃ / min to completely remove the template agent CTAB, forming a regular mesoporous structure. After cooling, mesoporous silica nanoparticles are obtained. The nanoparticles were dispersed in a pH 7.0 phosphate buffer solution at 13 times their mass (13 times the mass of the mesoporous silica nanoparticles) and ultrasonically dispersed at 150W for 35 min to ensure uniform dispersion and expose more mesoporous channels. Total Panax notoginseng saponins were added at a ratio of 110 μg of total Panax notoginseng saponins per milligram of mesoporous silica nanoparticle (the total Panax notoginseng saponins were diluted with pH 7.0 phosphate buffer to a concentration of 8 mg / mL before addition to improve loading uniformity). The mixture was loaded for 14 h at 25℃–30℃ with light-protected shaking at 90 rpm (low temperature and light protection reduce oxidative degradation of total Panax notoginseng saponins, while gentle shaking promotes adsorption of total Panax notoginseng saponins into the mesoporous channels). The mixture was then centrifuged at 8200 rpm for 20 min below 8℃. The solid was collected, washed once with pre-cooled pH 7.0 phosphate buffer solution at 6℃, and freeze-dried to obtain mesoporous silica-loaded total Panax notoginseng saponins with a loading rate of not less than 30%.

[0031] The preparation method of the above-mentioned absorbable bone wax that promotes bone healing includes the following steps: According to the formula mass ratio, the biodegradable copolymer and PTMC are mixed evenly at 62°C to form a base material. The temperature is then lowered to 47°C, and hydrogenated castor oil, magnesium-calcium silicate-biopeptide composite microspheres, and mesoporous silica-supported Panax notoginseng saponins are added sequentially and mixed evenly to form a paste. The paste is then injected into a mold at 47°C and cooled and shaped at 4°C to obtain bone wax.

[0032] Example 2 An absorbable bone wax that promotes bone healing comprises a biodegradable copolymer in a mass ratio of 25:10:9:5:2, PTMC, magnesium-calcium silicate-biopeptide composite microspheres, mesoporous silica-supported total saponins of Panax notoginseng, and hydrogenated castor oil.

[0033] The preparation method of the biodegradable copolymer includes the following steps: Prepare materials according to the mass ratio of PEG:stannous octoate:D,L-lactide:ε-caprolactone = 100:0.6:115:135, wherein PEG comprises PEG-400 and PEG-1000 in a mass ratio of 4:5. Under a micro-positive pressure protection of 0.08 MPa argon gas, PEG, stannous octoate, D,L-lactide, and ε-caprolactone monomers are stirred at 125℃~135℃ and 350 rpm for 22 h to obtain a reaction solution; cool to 50℃, add 12 times the mass of the reaction solution in anhydrous dichloromethane, and stir to obtain a diluted solution; under stirring at 300 rpm, add the diluted solution dropwise to 12 times the volume of the diluted solution in 2℃ pre-cooled n-hexane to precipitate, centrifuge at 3500 rpm for 15 min, collect the solid, wash four times with 2℃ pre-cooled n-hexane, and vacuum dry at 40℃ to constant weight to obtain the biodegradable copolymer.

[0034] The preparation method of magnesium-calcium silicate-biopeptide composite microspheres includes the following steps: S1, Preparation of bioactive peptides: *Ulva monocyclicis* was pulverized into a slurry, and 8 times its weight of deionized water was added to adjust the pH to 6.5. 1.5% of the slurry weight of papain was added, and the mixture was enzymatically hydrolyzed at 55℃ for 2 hours. The temperature was then raised to 90℃ to inactivate the enzyme for 10 minutes, cooled to room temperature, and centrifuged at 5000 rpm for 15 minutes. The supernatant was collected. The pH was adjusted to 7.8 using 0.5M sodium hydroxide aqueous solution, and 2% of the slurry weight of trypsin was added. The mixture was enzymatically hydrolyzed at 35℃ for 2 hours, and the temperature was raised to 85℃ to inactivate the enzyme for 15 minutes. The temperature was then cooled to room temperature, centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected. Ultrafiltration membranes with molecular weight cutoffs of 1 kDa and 5 kDa were used for separation, and peptide fractions with molecular weights between 1 kDa and 5 kDa were collected. These fractions were then freeze-dried to obtain bioactive peptides. S2, Preparation of porous magnesium-calcium silicate microspheres: Ca(NO3)2·4H2O:Mg(NO3)2·6H2O:TEOS = 46:9:20 mass ratio was used. Ca(NO3)2·4H2O and Mg(NO3)2·6H2O were dissolved in deionized water at a mass of 6 times the total solute to obtain solution A. TEOS was dissolved in anhydrous ethanol at a mass of 3 times the TEOS to obtain solution B. Solution B was added to solution A at a rate of 8 mL / min under stirring at 550 rpm to form a uniform sol. Spray drying was then performed with the following parameters: inlet temperature 210℃, outlet temperature 85℃, feed rate 10 mL / min, and atomization pressure 0.25 MPa, to obtain precursor powder. The precursor powder was heated to 650℃ at a rate of 6℃ / min, calcined for 4.5 h, cooled, and ground through a 150-mesh sieve to obtain porous magnesium-calcium silicate microspheres. S3, Loading and Surface Modification: The porous magnesium-calcium silicate microspheres were dispersed in a pH 7.2 phosphate buffer at a mass ratio of 15:120:1.2. The bioactive peptide was added, and the mixture was incubated at 35℃–40℃ with shaking at 100 rpm for 22 h. After centrifugation at 5000 rpm for 15 min, the solid was collected and dispersed in a pH 8.5 Tris-HCl buffer containing 1 mg / mL dopamine. The mixture was stirred at 200 rpm for 24 h at room temperature in the dark, and then centrifuged at 5000 rpm for 15 min. The solid was collected, washed three times with deionized water, and freeze-dried to obtain magnesium-calcium silicate-bioactive peptide composite microspheres with a bioactive peptide loading of not less than 40 wt%.

[0035] The preparation method of mesoporous silica-supported total saponins of Panax notoginseng includes the following steps: CTAB:deionized water:sodium hydroxide aqueous solution:TEOS = 1:550:3.5:5 by mass ratio; CTAB is dissolved in deionized water at 60℃, and a 2.0M sodium hydroxide aqueous solution is added. The system temperature is maintained in the range of 75℃ to 80℃. TEOS is added dropwise at a rate of 1 mL / min while stirring at 350 rpm. The reaction is continued for 1.5 h, cooled to room temperature, and centrifuged at 8500 rpm for 15 min. The solid is collected and washed three times each with anhydrous ethanol and deionized water to remove residual CTAB and impurities. The solid is then calcined at 550℃ for 6 h in air at a rate of 1℃ / min to completely remove the template agent CTAB, forming a regular mesoporous structure. After cooling, mesoporous silica nanoparticles are obtained. The mesoporous silica nanoparticles are then separated into... Dispersed in phosphate buffer at pH 6.8, 12 times the mass of mesoporous silica nanoparticles, the nanoparticles were ultrasonically dispersed at 200W for 30 min to ensure uniform dispersion and expose more mesoporous channels. Total Panax notoginseng saponins were added at a ratio of 120 μg of total Panax notoginseng saponins per milligram of mesoporous silica nanoparticles (the total Panax notoginseng saponins were diluted with pH 6.8 phosphate buffer to a concentration of 5 mg / mL before addition to improve loading uniformity). The mixture was loaded for 12 h at 25℃–30℃ and 100 rpm in the dark (low temperature and darkness reduce the oxidative degradation of total Panax notoginseng saponins, while gentle shaking promotes adsorption of total Panax notoginseng saponins into the mesoporous channels). The mixture was then centrifuged at 8500 rpm for 15 min below 8℃. The solid was collected, washed once with pre-cooled pH 6.8 phosphate buffer at 8℃, and freeze-dried to obtain mesoporous silica-loaded total Panax notoginseng saponins with a loading rate of not less than 30%.

[0036] The preparation method of the above-mentioned absorbable bone wax that promotes bone healing includes the following steps: According to the formula mass ratio, the biodegradable copolymer and PTMC are mixed evenly at 65°C to form a base material. The temperature is then lowered to 50°C, and hydrogenated castor oil, magnesium-calcium silicate-biopeptide composite microspheres, and mesoporous silica-supported Panax notoginseng saponins are added sequentially and mixed evenly to form a paste. The paste is then injected into a mold at 50°C and cooled and shaped at 6°C to obtain bone wax.

[0037] Example 3 An absorbable bone wax that promotes bone healing comprises a biodegradable copolymer in a mass ratio of 30:8:13:3:4, PTMC, magnesium-calcium silicate-biopeptide composite microspheres, mesoporous silica-supported total saponins of Panax notoginseng, and hydrogenated castor oil.

[0038] The preparation method of the biodegradable copolymer includes the following steps: Prepare materials according to the mass ratio of PEG:stannous octoate:D,L-lactide:ε-caprolactone = 100:0.3:135:115, wherein PEG comprises PEG-400 and PEG-1000 in a mass ratio of 3:6. Under a micro-positive pressure protection of 0.1 MPa argon gas, PEG, stannous octoate, D,L-lactide, and ε-caprolactone monomers are stirred at 125℃~135℃ and 300 rpm for 26 h to obtain a reaction solution; cool to 48℃, add 8 times the mass of the reaction solution in anhydrous dichloromethane, and stir to obtain a diluted solution; under stirring at 500 rpm, add the diluted solution dropwise to 8 times the volume of pre-cooled n-hexane at 6℃ to precipitate, centrifuge at 3000 rpm for 20 min, collect the solid, wash three times with pre-cooled n-hexane at 6℃, and vacuum dry at 45℃ to constant weight to obtain the biodegradable copolymer.

[0039] The preparation method of magnesium-calcium silicate-biopeptide composite microspheres includes the following steps: S1, Preparation of bioactive peptides: *Ulva monocyclicis* was pulverized into a slurry, and 10 times its weight of deionized water was added to adjust the pH to 6.2. Papain (2% of the slurry weight) was added, and the mixture was hydrolyzed at 50°C for 2.5 h. The temperature was then raised to 85°C to inactivate the enzyme for 15 min, cooled to room temperature, and centrifuged at 4000 rpm for 20 min. The supernatant was collected. The pH was adjusted to 8.2 using 0.1 M sodium hydroxide aqueous solution, and trypsin (1% of the slurry weight) was added. The mixture was hydrolyzed at 40°C for 1.5 h, raised to 90°C to inactivate the enzyme for 10 min, cooled to room temperature, and centrifuged at 8500 rpm for 10 min. The supernatant was collected. Ultrafiltration membranes with molecular weight cutoffs of 1 kDa and 5 kDa were used for separation, and peptide fractions with molecular weights between 1 kDa and 5 kDa were collected. These fractions were then freeze-dried to obtain bioactive peptides. S2, Preparation of porous magnesium-calcium silicate microspheres: Ca(NO3)2·4H2O:Mg(NO3)2·6H2O:TEOS = 52:7:25; Ca(NO3)2·4H2O and Mg(NO3)2·6H2O were dissolved in 5 times the total mass of deionized water to obtain solution A; TEOS was dissolved in 4 times the mass of anhydrous ethanol to obtain solution B; under stirring at 450 rpm, solution B was added to solution A at a rate of 12 mL / min to form a uniform sol, which was then spray-dried with parameters: inlet temperature 190℃, outlet temperature 95℃, feed rate 5 mL / min, and atomization pressure 0.35 MPa to obtain precursor powder; the precursor powder was heated to 750℃ at 4℃ / min, calcined for 3.5 h, cooled, and ground through a 200-mesh sieve to obtain porous magnesium-calcium silicate microspheres; S3, Loading and Surface Modification: The porous magnesium-calcium silicate microspheres were dispersed in a phosphate buffer solution at pH 7.4. The biopeptide was added, and the mixture was incubated at 35℃–40℃ with shaking at 80 rpm for 26 h. After centrifugation at 4500 rpm for 20 min, the solid was collected and dispersed in a Tris-HCl buffer solution at pH 8.2 containing 1.5 mg / mL dopamine. The mixture was stirred at 150 rpm for 28 h at room temperature in the dark, and then centrifuged at 4500 rpm for 20 min. The solid was collected, washed twice with deionized water, and freeze-dried to obtain magnesium-calcium silicate-biopeptide composite microspheres with a biopeptide loading of not less than 40 wt%.

[0040] The preparation method of mesoporous silica-supported total saponins of Panax notoginseng includes the following steps: CTAB:deionized water:sodium hydroxide aqueous solution:TEOS is prepared at a mass ratio of 1.2:500:4.5:4; CTAB is dissolved in deionized water at 65℃, and 1.8M sodium hydroxide aqueous solution is added. The system temperature is maintained in the range of 75℃ to 80℃. TEOS is added dropwise at a rate of 0.5 mL / min while stirring at 400 rpm. The reaction is continued for 2.5 h, cooled to room temperature, and centrifuged at 8000 rpm for 25 min. The solid is collected and washed twice alternately with anhydrous ethanol and deionized water to remove residual CTAB and impurities. The solid is then calcined at 600℃ for 5 h in air at a rate of 1℃ / min to completely remove the template agent CTAB, forming a regular mesoporous structure. After cooling, mesoporous silica nanoparticles are obtained. The nanoparticles were dispersed in phosphate buffer solution at pH 7.2, 15 times their weight in the mesoporous silica nanoparticles, and ultrasonically dispersed at 150W for 40 min to ensure uniform dispersion and expose more mesoporous channels. Total Panax notoginseng saponins were added at a ratio of 100 μg of total Panax notoginseng saponins per milligram of mesoporous silica nanoparticles (the total Panax notoginseng saponins were diluted with pH 7.2 phosphate buffer solution to a concentration of 10 mg / mL before addition to improve loading uniformity). The mixture was loaded for 16 h at 25℃–30℃ with light-protected shaking at 80 rpm (low temperature and light protection reduce oxidative degradation of total Panax notoginseng saponins, while gentle shaking promotes adsorption of total Panax notoginseng saponins into the mesoporous channels). The mixture was then centrifuged at 8000 rpm for 25 min below 8℃. The solid was collected, washed twice with pre-cooled pH 7.2 phosphate buffer solution at 4℃, and freeze-dried to obtain mesoporous silica-loaded total Panax notoginseng saponins with a loading rate of not less than 30%.

[0041] The preparation method of the above-mentioned absorbable bone wax that promotes bone healing includes the following steps: According to the formula mass ratio, the biodegradable copolymer and PTMC are mixed evenly at 60°C to form a base material. The temperature is then lowered to 45°C, and hydrogenated castor oil, magnesium-calcium silicate-biopeptide composite microspheres, and mesoporous silica-supported Panax notoginseng saponins are added sequentially and mixed evenly to form a paste. The paste is then injected into a mold at 45°C and cooled and shaped at 2°C to obtain bone wax.

[0042] The raw materials and their sources involved in the above embodiments are as follows: PTMC is polytrimethylene carbonate, sourced from Wuhan Pushida Biotechnology Co., Ltd., with a purity of 98%. Hydrogenated castor oil is sourced from Ruichengkang Pharmaceutical Technology (Shaanxi) Co., Ltd., with a purity of over 99%. PEG-400 is polyethylene glycol with a number average molecular weight of 400, sourced from Xi'an Taihua Pharmaceutical Technology Co., Ltd. PEG-1000 is polyethylene glycol with a number average molecular weight of 1000, sourced from Nanjing Bermuda Biotechnology Co., Ltd. Stannous octoate has a purity of over 99.5%. D,L-lactide has a purity of over 99%. ε-caprolactone has a purity of over 99%. Dichloromethane has a purity of over 99.9%. n-Hexane has a purity of over 99%. Monocyclic urticaria is a farmed product, sourced from Changyi Haoyuan Aquaculture Co., Ltd. Papain is sourced from Chongqing Tianrun Biological Products Co., Ltd., with an enzyme activity of 100,000 / g. Trypsin, sourced from Qinhe Health Industry (Shaanxi) Co., Ltd., has an enzyme activity of 4000 U / g and is extracted from porcine pancreas. The purity of Ca(NO3)2·4H2O is above 99%. The purity of Mg(NO3)2·6H2O is above 98%. The purity of dopamine is above 99%. CTAB is hexadecyltrimethylammonium bromide, with a purity above 99%. TEOS is tetraethyl orthosilicate, with a purity above 99%. Total saponins from Panax notoginseng are sourced from Wuhan Xinxin Jiali Biotechnology Co., Ltd., with a purity above 98%.

[0043] Comparative Example 1 The difference from Example 1 is that the bone wax includes a biodegradable copolymer in a mass ratio of 16:20:11:4:3, PTMC, magnesium-calcium silicate-biopeptide composite microspheres, mesoporous silica-supported Panax notoginseng saponins, and hydrogenated castor oil.

[0044] Comparative Example 2 The difference from Example 1 is that the bone wax includes a biodegradable copolymer in a mass ratio of 27:9:5:4:3, PTMC, magnesium-calcium silicate-biopeptide composite microspheres, mesoporous silica-supported Panax notoginseng saponins, and hydrogenated castor oil.

[0045] Comparative Example 3 The difference from Example 1 is that in the preparation of the biodegradable copolymer, PEG-1000 was used for all PEG.

[0046] Comparative Example 4 The difference from Example 1 is that D,L-lactide is not added in the preparation of the biodegradable copolymer, and ε-caprolactone monomer is used instead of D,L-lactide.

[0047] Comparative Example 5 The difference from Example 1 is that ε-caprolactone monomer is not added in the preparation of the biodegradable copolymer, and D,L-lactide is used instead of ε-caprolactone monomer.

[0048] Comparative Example 6 The difference from Example 1 is that trypsin is not used for enzymatic hydrolysis in the preparation of the biopeptides.

[0049] Comparative Example 7 The difference from Example 1 is that papain is not used for enzymatic hydrolysis in the preparation of the biopeptides.

[0050] Comparative Example 8 The difference from Example 1 is that in the preparation of the biopeptide, papain is replaced by bromelain (enzyme activity 100,000 / g).

[0051] I. Hemolysis rate test: Sample preparation: Bone wax was pulverized under sterile conditions. Particles between 150-200 mesh were taken and added to 0.9% sterile physiological saline preheated at 37℃ at a ratio of 0.5 g / mL. The mixture was aseptically extracted in a constant temperature shaker at 37℃ and 120 rpm for 72 h. The extract was filtered through a 0.22 μm sterile filter membrane to obtain the original bone wax extract.

[0052] Detection method: Prepare a 2% red blood cell suspension. Set up an experimental group, a positive control group, and a negative control group, with 3 replicates per group.

[0053] Experimental group: 1.0 mL of bone wax extract and 0.1 mL of erythrocyte suspension were added; Positive control group: Add 1.0 mL of distilled water and 0.1 mL of red blood cell suspension; Negative control group: 1.0 mL of physiological saline and 0.1 mL of red blood cell suspension were added; After gently mixing each group, incubate at 37℃ for 60 min, centrifuge at 3000 rpm for 5 min, collect the supernatant, and measure the absorbance (OD value) at 545 nm using an ELISA reader. Hemolysis rate (%) = (Average OD value of experimental group - Average OD value of negative control group) / (Average OD value of positive control group - Average OD value of negative control group) × 100%

[0054] II. Hemostatic performance test: Sample preparation: Bone wax was pressed into round discs with a diameter of 8 mm and a thickness of 2 mm under sterile conditions, and 5 parallel samples were set for each group.

[0055] Test method: Rabbit whole blood (anticoagulated with sodium heparin) and physiological saline were mixed at a 1:1 volume ratio to prepare simulated blood. A bone wax disc was placed at the bottom of a sterile test tube, 2.0 mL of simulated blood was quickly added, and the timing was started. The test tube was placed in a 37°C water bath, and the test tube was tilted every 10 seconds. The time required for the blood to stop flowing was observed, which was the clotting time.

[0056] III. Degradation performance testing: Sample preparation: Bone wax was pressed into round pieces with a diameter of 8 mm and a thickness of 2 mm, and the initial mass (m0) was weighed. Three parallel samples were set for each group.

[0057] Test method: Immerse bone wax discs in 20 mL of simulated body fluid (SBF, pH 7.4) and incubate in a constant temperature shaker at 37℃ and 120 rpm. Remove the samples on days 7, 14, 28, and 56, rinse gently with deionized water, and vacuum dry at 45℃ to constant weight. Weigh the remaining mass (m1); mass residue rate (%) = (m1 / m0) × 100%; record the number of days required for the bone wax discs to completely degrade (mass residue rate 0).

[0058] IV. Detection of osteoblast proliferation activity: Sample preparation: Bone wax extract stock solution was prepared using the same method as for hemolysis rate detection. It was diluted to a concentration of 50% (v / v) with DMEM complete medium containing 10% fetal bovine serum (hereinafter referred to as medium) to serve as the experimental group solution. A mixture of physiological saline and osteogenic induction medium in equal volumes was used as the negative control group solution.

[0059] Detection method: Mouse preosteoblasts MC3T3-E1 were subjected to a assay of 5 × 10⁻⁶ cells / mL. 3 Cells were seeded at a density of 1 / well in 96-well plates and cultured for 24 hours until adherence. The old medium was then discarded. Experimental and negative control solutions were added separately, with 6 replicates per group. Blank wells (containing only culture medium) were also included. Cells were cultured for another 7 days, and 10 μL of CCK-8 reagent was added to each well. After incubation at 37°C for 2 hours, the OD value was measured at 450 nm using a microplate reader. Relative cell proliferation rate (RGR, %) = (OD value of experimental group - OD value of blank well) / (OD value of negative control group - OD value of blank well) × 100%.

[0060] V. Detection of osteoblast differentiation activity: Sample preparation: Bone wax extract stock solution was prepared using the same method as for hemolysis rate detection. It was diluted to a concentration of 50% (v / v) with osteogenic induction medium containing 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate (hereinafter referred to as osteogenic induction medium) to serve as the experimental group solution. A mixture of physiological saline and osteogenic induction medium in equal volumes was used as the blank control group solution.

[0061] Detection method: MC3T3-E1 cells were cultured at a concentration of 2×10⁻⁶.5 Cells were seeded at a density of 100% per well in 6-well plates. After adhesion, the plates were replaced with experimental and blank control solutions. Each group had 3 replicates. Cells were collected after 14 days of culture, lysed, and ALP was measured at 405 nm according to the alkaline phosphatase (ALP) assay kit instructions. Total protein concentration in the same lysate was determined using the BCA method. ALP activity was expressed as U / mg prot (units of enzyme activity per milligram of protein).

[0062] VI. Bone matrix mineralization capacity testing: Sample preparation: The bone wax extract stock solution was prepared using the same method as for hemolysis rate detection. It was diluted to a concentration of 50% (v / v) with osteogenic induction medium containing 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate (hereinafter referred to as osteogenic induction medium). A mixture of physiological saline and osteogenic induction medium in equal volumes was used as the blank control solution.

[0063] Detection method: MC3T3-E1 cells were cultured at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1 / well in 6-well plates. After adhesion, the solution was replaced with the experimental solution and the blank control solution. Each group had 3 replicates. The solution was changed every 3 days and cultured for 28 days. The old solution was discarded, the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, stained with 1% Alizarin Red S solution (pH 4.2) for 20 min, and rinsed thoroughly with deionized water. The mineralized nodules were dissolved with 10% cetylpyridinium chloride solution, and the OD value was measured at 562 nm. The mineralization capacity was expressed as the multiple of the OD value of the experimental group to the OD value of the blank control group.

[0064] VII. Anti-inflammatory performance test: Sample preparation: Bone wax extract stock solution was prepared using the same method as for hemolysis rate detection. It was diluted to a concentration of 50% (v / v) with DMEM medium containing 10% fetal bovine serum (hereinafter referred to as medium) as a pretreatment solution.

[0065] Detection method: Mouse monocytes / macrophages RAW264.7 were processed at a concentration of 1×10⁻⁶. 6 Cells / well density seeded in 6-well plates, with the following three groups, each group having 3 replicates: Experimental group: Cells were incubated with the pretreatment solution for 2 hours; LPS model group: cells were incubated for 2 hours with a mixture of physiological saline and culture medium in equal volume ratio; Normal control group: Cells were incubated for 2 hours with a mixture of physiological saline and culture medium in equal volume ratio; After incubation, both the experimental group and the LPS model group were stimulated with LPS at a final concentration of 100 ng / mL for 24 h, while the normal control group was not stimulated with LPS. Cell supernatant was collected, and factor concentrations were detected according to the instructions of the TNF-α and IL-10 ELISA kits. The rate of change in factor concentration was calculated as follows: Rate of change in factor concentration = (Experimental group - Normal control group) / (LPS model group - Normal control group) × 100%.

[0066] 8. Angiogenesis Potential Detection: Sample preparation: Bone wax extract stock solution was prepared using the same method as for hemolysis rate detection. It was diluted to a concentration of 50% (v / v) with DMEM medium containing 10% fetal bovine serum (hereinafter referred to as medium) to serve as the experimental group solution. A mixture of physiological saline and medium in equal volumes was used as the blank control group solution.

[0067] Detection method: Human umbilical vein endothelial cells (HUVECs) were prepared at a concentration of 5 × 10⁻⁶. 5 Cells were seeded at a density of 1 / well in 6-well plates. After adhesion, the cells were replaced with experimental and blank control solutions. Each group had 3 replicates. After 24 hours of culture, the cell supernatant was collected, and the VEGF concentration was measured according to the VEGF ELISA kit instructions. The VEGF concentration change rate was calculated as: VEGF concentration change rate = (experimental group - blank control group) / blank control group × 100%.

[0068] IX. Materials-Cell Compatibility Testing: Sample preparation: Bone wax was made into round discs with a diameter of 8 mm and a thickness of 2 mm, sterilized by 15 kGy γ-rays, and then placed at the bottom of a 24-well plate.

[0069] Detection method: MC3T3-E1 cell suspension was prepared at 5×10⁻⁶. 4 The concentration of cells / well was directly added to the material surface, with 3 replicates per group. After 4 hours of culture, the cells were gently rinsed 3 times with PBS, fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and the number of adherent cells was counted in 5 random fields of view under a microscope (200x) and the average value was taken.

[0070] 10. Detection of key osteogenic factor secretion: Sample preparation: Bone wax extract stock solution was prepared using the same method as for hemolysis rate detection. It was diluted to a concentration of 50% (v / v) with osteogenic induction medium containing 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate (hereinafter referred to as osteogenic induction medium) to serve as the experimental group solution. A mixture of physiological saline and osteogenic induction medium in equal volumes was used as the blank control group solution.

[0071] Detection method: MC3T3-E1 cells were cultured at 5×10⁻⁶ cells / year. 5Cells were seeded at a density per well in 6-well plates. After adhesion, the cells were replaced with experimental and blank control solutions, respectively. Each group had 3 replicates. After 14 days of culture, the cell supernatant was collected, and the concentration of BMP-2 in the supernatant was measured according to the BMP-2 ELISA kit instructions. The rate of change in BMP-2 concentration was calculated as follows: BMP-2 concentration change rate = (experimental group - blank control group) / blank control group × 100%.

[0072] Table 1. Test Results (Average Values)

[0073] Note: "-" indicates that the item was not tested.

[0074] The bone waxes in Examples 1 to 3 utilize a hydrophilic-hydrophobic interwoven three-dimensional network constructed from a biodegradable copolymer and PTMC. A specific ratio of PEG-400 and PEG-1000 optimizes hydrophilicity and swelling properties, while PTMC degrades slowly via surface erosion. The three components synergistically achieve a precise match between the degradation rate and the bone regeneration cycle, preventing early collapse or prolonged retention. Hydrogenated castor oil optimizes the ointment's adhesion and plasticity, ensuring immediate hemostasis, while the degradation products are all biocompatible, maintaining an extremely low hemolysis rate. Magnesium-calcium silicate-biopeptide composite microspheres serve as the core signal source, with a porous structure loading biopeptides and slowly releasing magnesium. 2+ With silicate ions, bioactive peptides drive cell proliferation and differentiation by activating osteogenic signaling pathways, Mg 2+ The silicate ions provide continuous active stimulation for bone healing; dopamine modification strengthens the interfacial binding between the microspheres and the substrate and bone tissue, ensuring stable release of bioactive peptides. Mesoporous silica provides physical protection and controlled sustained release of Panax notoginseng saponins, exerting anti-inflammatory effects to improve the healing microenvironment and promoting angiogenesis to ensure nutrient supply. It forms an orderly synergy with bioactive peptides and inorganic ions in anti-inflammatory, angiogenic, and osteogenic effects. Each component plays a precise role at different stages of bone healing: the hemostatic component quickly seals bleeding, the anti-inflammatory component relieves inflammation in a timely manner, and the osteogenic and angiogenic components continuously drive bone regeneration, while ensuring excellent biocompatibility and cell adaptability.

[0075] In Comparative Example 1, the amount of biodegradable copolymer decreased while the amount of PTMC increased. Biodegradable copolymers are the main carriers of active ingredients. The increased proportion of PTMC weakens the swelling effect and microenvironment regulation ability of the biodegradable copolymer system, leading to an imbalance in the release kinetics of active ingredients. At the same time, the high flexibility and dense structure of PTMC reduces the hydrophilicity of the material, slows down its interaction with blood, prolongs hemostasis time, and alters the degradation rate due to the slow degradation characteristics of PTMC, resulting in poorer overall compatibility with the bone regeneration cycle.

[0076] In Comparative Example 2, the amount of magnesium-calcium silicate-biopeptide composite microspheres was reduced: the composite microspheres are the core signal source for bone induction, and their reduction directly led to a decrease in magnesium content. 2+ The total release of silicate ions and bioactive peptides was insufficient, weakening the specific signals driving osteogenic differentiation. Although the anti-inflammatory and pro-angiogenic effects of Panax notoginseng total saponins were still present, the lack of synergistic osteogenic signals led to a decrease in bone regeneration performance. At the same time, due to the weakened auxiliary effect of bioactive peptides in regulating inflammatory factors, anti-inflammatory and pro-angiogenic indicators were also correspondingly reduced.

[0077] In Comparative Example 3, the PEG component was changed to consist entirely of PEG-1000: the short chains of PEG-400 can increase the hydrophilic sites on the material surface, forming an optimal distribution of hydrophilic segments with PEG-1000, thus optimizing interfacial interactions. Using only PEG-1000 would result in tightly wrapped hydrophilic segments, reduced accessibility to hydrophilicity, affecting the initial interactions between the material and body fluids and cells, leading to decreased cell adhesion. The longer PEG-1000 chains create steric hindrance on the polymer backbone, slightly slowing down the degradation rate.

[0078] In Comparative Example 4, no D,L-lactide was added, but excess ε-caprolactone was used. The amorphous polylactic acid segments formed by the polymerization of D,L-lactide are crucial for ensuring the copolymer's degradation rate and structural openness. Their absence significantly increases the copolymer's crystallinity, resulting in a denser molecular chain arrangement that hinders water molecule penetration and ester bond hydrolysis, thus slowing down degradation. The dense material surface also impedes cell adhesion and the release of active ingredients. Furthermore, its excessive hardness and brittleness affect its adhesion to bone surfaces, leading to decreased hemostatic performance.

[0079] In Comparative Example 5, no ε-caprolactone was added, but excess D,L-lactide was used. ε-caprolactone provides flexible segments that regulate the mechanical properties and degradation characteristics of the copolymer. Without these segments, the copolymer is dominated by polylactic acid segments, leading to increased rigidity and decreased hydrophilicity, resulting in a slower and more uneven degradation rate. Simultaneously, insufficient material flexibility affects operability and tissue adhesion. Furthermore, the lactic acid produced from the decomposition of excess polylactic acid segments lowers the pH, and long-term residues can easily trigger inflammatory responses.

[0080] In Comparative Examples 6 to 8, a certain protease was omitted or the protease was changed: the enzymatic hydrolysis of monocyclic caustic solanine protein broke the specific cleavage synergy between papain and trypsin, resulting in changes in peptides rich in cell adhesion and osteogenic active sequences. The types, abundance and conformation of the optimal active sequences were altered, the proportion of specific active peptides in the biopeptide library decreased, and the decline in related indicators was directly weakened.

Claims

1. An absorbable bone wax that promotes bone healing, characterized in that, It includes a biodegradable copolymer with a mass ratio of (25-30):(8-10):(9-13):(3-5):(2-4), PTMC, magnesium-calcium silicate-biopeptide composite microspheres, mesoporous silica-supported Panax notoginseng saponins and hydrogenated castor oil; The biodegradable copolymer is prepared by reacting PEG, stannous octoate, D,L-lactide, and ε-caprolactone at a mass ratio of 100:(0.3-0.6):(115-135):(115-135) at 125℃-135℃ and precipitating in n-hexane; the PEG comprises PEG-400 and PEG-1000 at a mass ratio of (2-4):(5-7); The magnesium-calcium silicate-biopeptide composite microspheres are prepared by porous magnesium-calcium silicate microspheres and biopeptides adsorbed in phosphate buffer at a mass ratio of (10-15):(0.8-1.2), centrifuged to obtain solid, dispersed in Tris-HCl buffer containing dopamine at pH 8.2-8.5, stirred to prepare polydopamine coating layer, and the solid is obtained. The bioactive peptides are obtained by hydrolyzing a pulverized monocyclic urchin in deionized water with 1.5%–2% papain by mass of the pulverized material at pH 6.2–6.5 and 50–55°C for 2–2.5 hours, followed by hydrolysis with 1%–2% trypsin by mass of the pulverized material at pH 7.8–8.2 and 35–40°C for 1.5–2 hours, yielding peptide fractions between 1 kDa and 5 kDa. The porous magnesium-calcium silicate microspheres were prepared by sol-gel of Ca(NO3)2·4H2O, Mg(NO3)2·6H2O and TEOS in a mass ratio of (46-52):(7-9):(20-25), spray-dried, calcined at 650℃-750℃ for 3.5-4.5 hours, and ground through a 150-200 mesh sieve.

2. The absorbable bone wax for promoting bone healing according to claim 1, characterized in that, The preparation method of the biodegradable copolymer includes the following steps: PEG:stannous octoate:D,L-lactide:ε-caprolactone = 100:(0.3-0.6):(115-135):(115-135) by mass ratio; under argon protection, PEG, stannous octoate, D,L-lactide and ε-caprolactone monomers are stirred and reacted to obtain a reaction solution; Add anhydrous dichloromethane and stir to obtain a diluted solution; With stirring, the diluted solution was added dropwise to n-hexane to precipitate, centrifuged, the solid was collected, washed with n-hexane, and dried under vacuum to obtain the biodegradable copolymer.

3. The absorbable bone wax for promoting bone healing according to claim 2, characterized in that, The PEG comprises PEG-400 and PEG-1000 in a mass ratio of (2-4):(5-7); the stirring reaction is carried out at 125℃-135℃ for 22-26 hours; the amount of anhydrous dichloromethane is 8-12 times the mass of the reaction solution; the amount of n-hexane is 8-12 times the volume of the diluent; and all n-hexane is pre-cooled at 2℃-6℃.

4. The absorbable bone wax for promoting bone healing according to claim 1, characterized in that, The preparation method of the magnesium-calcium silicate-biopeptide composite microspheres includes the following steps: The porous magnesium-calcium silicate microspheres are dispersed in phosphate buffer at a mass ratio of (10-15):(120-180):(0.8-1.2). Biopeptides are added, the mixture is shaken to adsorb, centrifuged, and the solid is collected. The solid is then dispersed in Tris-HCl buffer (pH 8.2-8.5) containing 1 mg / mL-1.5 mg / mL dopamine. The mixture is stirred and centrifuged at room temperature in the dark. The solid is then washed with deionized water and freeze-dried to obtain the magnesium-calcium silicate-biopeptide composite microspheres.

5. The absorbable bone wax for promoting bone healing according to claim 4, characterized in that, The preparation method of the bioactive peptides includes: pulverizing *Ulva monocyclicis* into a slurry, adding deionized water, adjusting the pH to 6.2–6.5, adding 1.5%–2% papain by weight of the slurry, enzymatically hydrolyzing at 50℃–55℃ for 2–2.5 h, inactivating the enzyme, centrifuging at 4000 rpm–5000 rpm for 15 min–20 min, collecting the supernatant, adjusting the pH to 7.8–8.2, adding 1%–2% trypsin by weight of the slurry, enzymatically hydrolyzing at 35℃–40℃ for 1.5 h–2 h, inactivating the enzyme, centrifuging at 8000 rpm–8500 rpm for 10 min–15 min, collecting the supernatant, separating the peptide fractions with molecular weights between 1 kDa and 5 kDa using an ultrafiltration membrane, and freeze-drying to obtain the bioactive peptides.

6. The absorbable bone wax for promoting bone healing according to claim 4, characterized in that, The preparation method of the porous magnesium-calcium silicate microspheres includes: dissolving Ca(NO3)2·4H2O:Mg(NO3)2·6H2O:TEOS in a mass ratio of (46-52):(7-9):(20-25); dissolving Ca(NO3)2·4H2O and Mg(NO3)2·6H2O in deionized water to obtain solution A; dissolving TEOS in anhydrous ethanol to obtain solution B; adding solution B to solution A under stirring to form a sol, spray drying to obtain precursor powder; heating the precursor powder to 650℃-750℃, calcining for 3.5h-4.5h, cooling, and grinding through a 150-200 mesh sieve to obtain porous magnesium-calcium silicate microspheres.

7. The absorbable bone wax for promoting bone healing according to claim 4, characterized in that, The oscillation adsorption is performed at 35℃~40℃ for 22h~26h; the stirring is performed at 150rpm~200rpm for 24h~28h.

8. The absorbable bone wax for promoting bone healing according to claim 1, characterized in that, The preparation method of mesoporous silica-loaded total saponins of Panax notoginseng includes the following steps: CTAB:deionized water:sodium hydroxide aqueous solution:TEOS in a mass ratio of (1-1.2):(500-550):(3.5-4.5):(4-5); CTAB is dissolved in deionized water, sodium hydroxide aqueous solution is added, TEOS is added under stirring, the mixture is stirred and reacted, centrifuged, the solid is collected, washed, and calcined to obtain mesoporous silica nanoparticles; the mesoporous silica nanoparticles are dispersed in phosphate buffer, total saponins of Panax notoginseng are added, the mixture is loaded by shaking in the dark, centrifuged, the solid is collected, rinsed, and freeze-dried to obtain mesoporous silica-loaded total saponins of Panax notoginseng.

9. The absorbable bone wax for promoting bone healing according to claim 8, characterized in that, The concentration of the sodium hydroxide aqueous solution is 1.8M to 2.0M; the TEOS is added and the reaction is stirred for 1.5h to 2.5h; the washing is performed by alternating between anhydrous ethanol and deionized water 2 to 3 times each; the calcination is performed by heating to 550℃ to 600℃ at a rate of 1℃ / min and calcining for 5h to 6h; the total saponins of Panax notoginseng are added at a ratio of 100μg to 120μg of total saponins of Panax notoginseng per milligram of mesoporous silica nanoparticles; the light-protected shaking loading is performed at 25℃ to 30℃ and 80rpm to 100rpm for 12h to 16h; the rinsing is performed with phosphate buffer solution pre-cooled to 4℃ to 8℃ with a pH of 7.0 to 7.

4.

10. The method for preparing an absorbable bone wax that promotes bone healing as described in claim 1, characterized in that, Includes the following steps: According to the formula mass ratio, the biodegradable copolymer and PTMC are mixed evenly at 60℃~65℃ to form a base material. The temperature is then lowered to 45℃~50℃, and hydrogenated castor oil, magnesium-calcium silicate-biopeptide composite microspheres, and mesoporous silica-supported Panax notoginseng saponins are added in sequence and mixed evenly to form a paste. The paste is then injected into a mold at 45℃~50℃ and cooled and shaped at 2℃~6℃ to obtain bone wax.

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