Method for improving bone induction activity of bio-derived hydroxyapatite

Abstract

The invention discloses a method for improving bone induction activity of bio-derived hydroxyapatite, and belongs to the technical field of medical materials. The preparation method aims at the characteristics that bio-derived hydroxyapatite has a natural three-dimensional pore structure, but is lack of bone induction activity and poor in mechanical property; according to the present invention, the biological derived hydroxyapatite is compounded with the icariin solution, and then is subjected to dual compounding with the icariin solution and the bone matrix gelatin solution so as to achieve the purposes of bone induction promotion and mechanical property strengthening. The icariin is derived from a traditional Chinese medicine herba epimedii, is a traditional kidney-tonifying and bone-strengthening traditional Chinese medicine and has the effects of improving osteoblast activity and promoting osteoblast proliferation and differentiation, and after the icariin and the hydroxyapatite composite material are implanted into a body, the bone induction activity of the hydroxyapatite can be enhanced in a short time; the bone matrix gelatin is formed by carrying out mild physical and chemical treatment on bone tissues, is mainly a mixed component of coarsely extracted I-type collagen and non-collagen, has viscosity, contains BMPs, has good bone induction activity and slow release characteristics, plays a slow release role in icariin, and improves the compressive strength of hydroxyapatite at the same time. The prepared bio-derived hydroxyapatite for improving the bone induction activity retains a natural pore structure, is good in biological safety, has the bone induction activity, is high in compressive strength, wide in source, relatively low in production cost and stable in bone induction activity, and provides a better bone repair scheme for clinic.

Description

Technical Field

[0001] The invention relates to the technical field of medical materials, and in particular to a method for improving the bone induction activity of biologically derived hydroxyapatite. Background Art

[0002] In the process of clinical treatment of bone defects, it is usually necessary to fill the defect site with bone graft materials to promote bone healing. Commonly used materials include autologous bone, allogeneic bone, artificial bone and xenogeneic bone.

[0003] Autologous bone comes from one's own iliac bone and other locations. Autologous bone transplantation has good osteoinduction, osteoconduction and osteogenesis abilities, and has good biosafety. It is the gold standard for bone defect repair. However, the disadvantages of autologous bone transplantation are obvious. Not only is the quantity limited, but it may also cause donor site pain and local complications such as hematoma, fracture, infection, and postoperative scar hyperplasia. Therefore, its clinical application is limited.

[0004] Allogeneic bone is a bone tissue material derived from the human body. After decellularization and other treatments, it has low immunogenicity and has the advantages of good bone induction and bone conduction. However, allogeneic bone comes from healthy human bone tissue, has a limited source, and cannot fully meet clinical bone grafting needs.

[0005] Xenogeneic bone materials are abundant in sources, easy to obtain in large quantities and process and store, and are simple to process. Their cost is much lower than that of homologous bone or artificial synthetic materials, but they need to be processed to remove immunogenicity. Taking bioderived hydroxyapatite obtained by high-temperature calcination of bovine cancellous bone as an example, it is an ordered crystal of bone inorganic matter; high-temperature calcination effectively removes antigens while retaining the natural porous structure, and its main component is high-purity hydroxyapatite; it has a calcium-phosphorus ratio and pore structure similar to human bone, and has a large specific surface area, which is conducive to the recombination of cytokines and the growth of new bone tissue cells, and can provide a favorable microenvironment for the growth of new bone tissue and blood vessels. Bioderived hydroxyapatite has good biocompatibility and bone conduction ability, but it also has the problems of insufficient bone induction activity and low compressive strength. Solving this problem will help to better promote the repair of bone defects.

[0006] According to the 2020 edition of the Chinese Pharmacopoeia, epimedium is the dried leaves of Epimedium brevicornuMaxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim., or Epimedium koreanum Nakai, all of the Berberidaceae family. As a commonly used Chinese medicine, epimedium has the effects of strengthening kidney yang, strengthening tendons and bones, and removing rheumatism. It is often used to treat osteoporosis. Icariin is the main active ingredient of epimedium, a flavonoid compound with a wide range of pharmacological activities. Icariin can increase osteoblast activity, improve bone metabolism, repair damaged bone tissue, promote osteoblast proliferation and differentiation, and has certain bone induction activity.

[0007] Bone matrix gelatin is made of bone tissue after mild physical and chemical treatment. It is mainly a mixture of type I collagen and non-collagenous proteins. It has a certain viscosity and contains BMPs. It not only has good osteoinductive activity, but also can be used as a sustained-release agent for drugs. After being double-compounded with bio-derived hydroxyapatite and icariin, it not only has osteoinductive activity when implanted in the body, but also has a sustained-release effect on icariin, prolonging the duration of the material's osteoinductive activity, and can also improve the compressive strength of bio-derived hydroxyapatite materials, achieving the purpose of promoting bone formation and bone defect repair.

[0008] Patent (CN 109985277 B) provides a porous hydroxyapatite / chitosan composite scaffold for bone tissue engineering and a preparation method thereof, wherein chitosan is used to enhance the mechanical properties of hydroxyapatite and increase the adhesion to bone cells; Patent (CN 109833513 B) discloses a degradable hydroxyapatite scaffold and a preparation method and application thereof, wherein low-crystallinity hydroxyapatite pellets are accumulated in a mold, and the hydroxyapatite pellets are coated by injecting a polymer solution, and after vacuum drying, molten polymer is added again to firmly combine the pellets together to obtain a porous scaffold, which does not require high-temperature sintering and can retain the original properties of hydroxyapatite. The obtained low-crystallinity hydroxyapatite scaffold has the advantages of high degradability, high strength, three-dimensional pore connectivity, controllable pore size, and good osteogenesis. However, the above-mentioned hydroxyapatite is artificially synthesized, does not have the natural pore structure and chemical composition of bio-derived materials, and lacks bone induction activity. The patent (CN 107485730 B) discloses a method for improving the cell compatibility of bio-derived hydroxyapatite, which uses supercritical carbon dioxide for defatting and deproteinization, leaving no organic matter residue, and can effectively improve the biocompatibility and biomechanical properties of the prepared material, but the mechanical properties are poor and lack bone induction activity. Current technology cannot solve the bone induction activity and mechanical properties of animal-derived bio-derived hydroxyapatite, which affects the large-scale application of bio-derived hydroxyapatite in bone repair. Summary of the invention

[0009] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a method for improving the osteoinductive activity of bio-derived hydroxyapatite materials. The icariin used has a good osteogenic effect, and the compound with bio-derived hydroxyapatite can effectively solve the problem of insufficient osteoinductive activity of the existing bio-derived hydroxyapatite. At the same time, in order to solve the problem of too fast release of icariin after implantation and the problem of insufficient compressive strength of bio-derived hydroxyapatite materials, bone matrix gelatin is used to attach to the surface of the material to play a role in osteoinduction, and to a certain extent, the release of icariin is delayed, and at the same time, the compressive strength of the material is increased.

[0010] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: the present invention provides a method for improving the osteoinductive activity of biologically derived hydroxyapatite materials, comprising the following steps: Step 1: The method for preparing the bio-derived hydroxyapatite material comprises: Cancellous bone tissue from qualified human donors or animals is obtained under sterile conditions. The soft tissue on the bone surface is completely removed with a scalpel. The bone is processed into blocks, slices or columns using a bone saw and rinsed. The rinsed bone material is ultrasonically cleaned with purified water at room temperature until there is no blood. 0.5mol / L The method comprises the following steps: soaking the material in a NaOH solution at room temperature for 2 to 4 hours; then ultrasonically cleaning the material with purified water at 70 to 80°C for at least 10 times, each time for at least 1 hour; drying the material in a blast drying oven at 50 to 60°C for at least 5 hours; processing the dried material to the required specifications, such as bone particles with a particle size of 0.25 to 2.00 mm, blocks of 2 to 6 mm*2 to 6 mm*2 to 6 mm or other required specifications; placing the processed material in a muffle furnace for calcination at a calcination temperature of 600 to 1000°C for a calcination time of 2 to 8 hours; washing the obtained material with purified water to a pH of 6.0 to 8.0; drying the material in a blast drying oven, packaging the material in a double-layer sterilization bag and irradiating the material for sterilization at a sterilization dose of 15 to 25 KGy, and obtaining a bio-derived hydroxyapatite material.

[0011] Step 2, the preparation method of the icariin solution comprises: Take commercially available icariin, dissolve it in water for injection, stir evenly, and prepare a suspension of 1.0-100 mg / L.

[0012] Step 3, compounding the bio-derived hydroxyapatite material with the icariin suspension comprises: The bio-derived hydroxyapatite material is placed in a mold, and the mold is placed in an oscillator with an oscillation speed of 100 to 120 r / min. The icariin suspension is added and soaked at room temperature for 1 to 24 hours. During this period, the orientation of the bio-derived hydroxyapatite material is manually adjusted 2 to 6 times, and then freeze-dried after adsorption saturation. The material is quickly pre-frozen in a refrigerator below -70°C, the freeze-drying chamber of the freeze dryer is pre-cooled to -40°C, and then the material is placed in the freeze-drying chamber to start freeze-drying; the temperature in the pre-freezing stage is not higher than -40°C, the time is 6 to 12 hours, the vacuum degree in the sublimation stage is 10 to 100 Pa, and the time is 10 to 24 hours, and the icariin / bio-derived hydroxyapatite composite material is obtained.

[0013] Step 4, the preparation method of the bone matrix gelatin comprises: Cortical bone from qualified human donors or animals is obtained, washed and crushed, and cortical bone particles with a particle size of 0.2-0.8 mm are sieved and washed with purified water; soaked in a chloroform / methanol mixture for 4-24 hours for degreasing; demineralized in a 0.1-0.6 mol / L hydrochloric acid solution for 4-24 hours; soaked in a 1-2 mol / L CaCl2 solution for 4-24 hours to remove low molecular weight proteoglycans; soaked in a 0.5 mol / L EDTA solution for at least 4 hours to remove non-apatite calcium and part of the proteoglycans; soaked in a 2-8 mol / L LiCl solution for 4-24 hours to shrink collagen fibers and remove part of the high molecular weight proteoglycans; washed with purified water, stirred and dissolved with 45-50°C purified water; centrifuged for 10-30 minutes, discarded the supernatant, and the obtained precipitate was added to purified water at a volume ratio of 1:1-5 and fully mixed to obtain a bone matrix gelatin solution. Icariin is added to the bone matrix gelatin solution and mixed to obtain a 1.0-100.0 mg / L icariin-bone matrix gelatin solution.

[0014] Step 5, the composite method of the icariin-bone matrix gelatin solution and the icariin / bioderived hydroxyapatite material is as follows: Negative pressure adsorption is used with a pressure value of 1 to 50 kPa to make the bone matrix gelatin fill the pores of the bio-derived hydroxyapatite material and infiltrate for 1 to 24 hours; the centrifugal radius is 10 cm and the speed is less than 100 r / min for 1 to 3 minutes; freeze-drying is carried out, and the freeze-drying method is as follows: the temperature in the pre-freezing stage is not higher than -50°C, the time is 6 to 10 hours, and the vacuum degree in the sublimation stage is 20 to 100 Pa, and the time is 10 to 36 hours.

[0015] Furthermore, the cancellous bone material in step 1 may be derived from animals or humans, and preferably bovine cancellous bone.

[0016] Furthermore, the compounding of the bio-derived hydroxyapatite material and the icariin solution in step 3 is characterized in that the volume ratio of the icariin solution to the bio-derived hydroxyapatite material is 1 to 10:1.

[0017] Furthermore, the human qualified donor or animal cortical bone mentioned in step 4 is preferably human qualified donor cortical bone.

[0018] Furthermore, the composite of the icariin-bone matrix gelatin solution and the icariin / bioderived hydroxyapatite material in step five is characterized in that the volume ratio of the icariin-bone matrix gelatin solution to the icariin / bioderived hydroxyapatite composite material is 1 to 2:1.

[0019] The beneficial effects of the present invention are: 1. The present invention uses icariin with osteoinductive activity as an osteoinductive factor to improve the osteoinductive activity of biologically derived hydroxyapatite. Compared with conventional growth factors, it has no teratogenicity, stable activity and low price.

[0020] 2. The present invention uses bone matrix gelatin as a sustained-release carrier of icariin, which is closer to the natural carrier of growth factors, can further improve the bone induction activity of the material, and enhance the compressive strength of the bio-derived hydroxyapatite material, thereby expanding the application range of the material.

[0021] 3. The method of first negative pressure compounding and then centrifugation of the present invention effectively retains the natural pore structure, which is conducive to the compounding of cytokines and the growth of new bone tissue cells.

[0022] 4. The double-compound method of the present invention can ensure the full and effective compounding of icariin, prolong the sustained-release time, optimize the sustained-release attempt, and continuously promote bone formation and bone defect repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the drawings are briefly introduced. Obviously, the drawings described below are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0024] Attached Figure 1 Photo of the appearance of bio-derived hydroxyapatite material without bone matrix gelatin Attached Figure 2 This is a photo of the composite material prepared by the present invention, which is white or slightly yellow, porous. Attached Figure 3 This is an electron micrograph of the material without centrifugation during the composite process with bone matrix gelatin. The interior of hydroxyapatite is filled with bone matrix gelatin and the pores are sealed. Attached Figure 4 This is a scanning electron microscope photo of the composite bone of the present invention. It can be seen that the microstructure is porous, uniform bone matrix gelatin can be seen on the surface of the material, and the pores are connected. Attached Figure 5 , 6 The compressive strength test process and results of the material of the present invention show that the compressive strength of the composite material is significantly improved compared with that of the uncomposite material. Attached Figure 7 This is the release curve of icariin of the material of the present invention. The material prepared by the present invention can achieve a good sustained release effect. Attached Figure 8 The results of the bone induction activity scoring of the material of the present invention in the nude mouse implantation experiment show that the bone induction activity of the composite material is significantly improved compared with that of the uncomposite material. Attached Fig. 9HE-stained sections of the uncomposite bone-inducing activity animal experiment, no new bone-forming elements were observed Attached Fig.10 HE staining section of the bone induction activity animal experiment of the composite material prepared by the present invention shows a large amount of cartilage Implementation

[0025] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Example

[0026] Embodiment 1 A method for improving the osteoinductive activity of a bioderived hydroxyapatite material, characterized in that the bioderived hydroxyapatite material is first compounded with an icariin solution, and then compounded with an icariin-bone matrix gelatin solution; 1. Preparation of bio-derived hydroxyapatite materials: The femurs of healthy adult cattle were obtained under sterile conditions, and the soft tissue on the bone surface was completely removed with a scalpel; a bone saw was used to process the bones into thin slices with a thickness of 5 mm and rinsed with purified water for 10 minutes; the rinsed bones were ultrasonically cleaned with purified water at room temperature until there was no blood; they were soaked in 0.5 mol / L NaOH solution at room temperature for 3 hours; then ultrasonically cleaned with purified water at 80°C for 10 times, each time for 1 hour; dried at 60°C in a blast drying oven for 5 hours; the dried materials were processed into blocks with specifications of 5mm*5mm*5mm and cylinders with a diameter of 2mm and a height of 6mm; the formed materials were placed in a muffle furnace for calcination at a calcination temperature of 800°C and a calcination time of 4 hours; the obtained materials were oscillated and washed with purified water until the pH was 6.0-8.0; after drying in a blast drying oven, they were packaged in double-layer sterilization bags and irradiated for sterilization, with a sterilization dose of 20KGy.

[0027] 2. Preparation of icariin solution: Take the icariin powder purchased from Yuanye Biotechnology, dissolve it in water for injection and stir evenly to make a 50 mg / L suspension.

[0028] 3. Composite of bio-derived hydroxyapatite material and icariin solution: The bio-derived hydroxyapatite material was placed in a cylindrical mold with a diameter of 20 mm and a height of 50 mm. The icariin suspension was added according to the volume ratio of icariin suspension to bio-derived hydroxyapatite of 2:1, and soaked at room temperature for 4 hours, with an oscillation speed of 100 r / min. The orientation of the bio-derived hydroxyapatite material was manually adjusted every hour for a total of 3 times, and then freeze-dried after adsorption saturation. The material was pre-frozen in a -70°C refrigerator for 4 hours, the freeze-drying chamber of the freeze dryer was pre-cooled to -40°C, and then the material was placed in the freeze-drying chamber to start freeze-drying; the pre-freezing stage temperature was -40°C, the time was 6 hours, and the vacuum degree of the sublimation stage was less than 100 Pa, the time was 24 hours, and the icariin / bio-derived hydroxyapatite composite material was obtained.

[0029] 4. Preparation of Icariin-Bone Matrix Gelatin Solution: The cortical bone was obtained from the middle part of the femur of a qualified human donor, washed with purified water and crushed, and the cortical bone particles of 0.2-0.5 mm were screened with a stainless steel mesh; soaked in a chloroform / methanol mixture for 4 hours for degreasing; demineralized in a 0.6 mol / L hydrochloric acid solution for 24 hours, and the acid was changed every 6 hours; soaked in a 2 mol / L CaCl2 solution for 4 hours to remove low molecular weight proteoglycans; soaked in a 0.5 mol / L EDTA solution for 4 hours to remove non-hydroxyapatite calcium and part of the proteoglycans; soaked in an 8 mol / L LiCl solution for 24 hours to shrink the collagen fibers and remove part of the high molecular weight proteoglycans; washed with purified water, stirred and dissolved with 50°C purified water; centrifuged at a centrifugal radius of 10 cm and a speed of 8000 r / min for 10 min, the supernatant was discarded, and the obtained precipitate was added to the injection water at a volume ratio of 1:1 and mixed thoroughly to obtain a bone matrix gelatin solution. Icariin was added into the bone matrix gelatin solution to prepare a 50 mg / L icariin-bone matrix gelatin solution.

[0030] 5. Composite of icariin-bone matrix gelatin solution and icariin / bioderived hydroxyapatite material: Negative pressure adsorption was used with a pressure of 30 kPa to fill the pores of the bio-derived hydroxyapatite material with bone matrix gelatin and infiltrate for 1 hour; the centrifugal radius was 10 cm and the speed was 80 r / min for 3 minutes; and freeze-drying was performed. The freeze-drying method was as follows: the pre-freezing stage temperature was -50°C for 6 hours, and the sublimation stage vacuum was less than 100 Pa for 36 hours, thus obtaining a bone repair material with improved bone induction activity.

[0031] Embodiment 2 A method for improving the osteoinductive activity of a bioderived hydroxyapatite material, characterized in that the bioderived hydroxyapatite material is first compounded with an icariin solution, and then compounded with icariin-bone matrix gelatin; 1. Preparation of bio-derived hydroxyapatite materials: Bilateral femurs of healthy adult pigs were obtained under sterile conditions, and the soft tissue on the bone surface was completely removed with a scalpel; a bone saw was used to process it into thin slices with a thickness of 5 mm and rinsed with purified water for 10 minutes; the rinsed bovine bones were ultrasonically cleaned with purified water at room temperature until there was no blood; they were soaked in 0.5 mol / L NaOH solution at room temperature for 3 hours; then ultrasonically cleaned with purified water at 80°C for 20 times, each time for 1 hour; dried at 60°C in a blast drying oven for 5 hours; the dried material was processed into blocks with a specification of 5mm*5mm*5mm; the formed material was placed in a muffle furnace for calcination at a calcination temperature of 700°C and a calcination time of 6 hours; the obtained material was oscillated and washed with purified water until the pH was 6.0-8.0; after drying in a blast drying oven, it was packaged in double-layer sterilization bags and irradiated for sterilization, with a sterilization dose of 20KGy.

[0032] 2. Preparation of icariin solution: Take the icariin powder purchased from Yuanye Biotechnology, dissolve it in water for injection and stir evenly to make a 40 mg / L suspension.

[0033] 3. Composite of bio-derived hydroxyapatite material and icariin solution: The bio-derived hydroxyapatite material was placed in a cylindrical mold with a diameter of 40 mm and a height of 50 mm. The icariin suspension was added according to the volume ratio of icariin solution to bio-derived hydroxyapatite of 3:1, and the material was soaked at room temperature for 4 hours, with an oscillation speed of 120 r / min. The orientation of the bio-derived hydroxyapatite material was manually adjusted every hour for a total of 3 times, and then freeze-dried after adsorption saturation. The material was pre-frozen in a -70°C refrigerator for 4 hours, the freeze-drying chamber of the freeze dryer was pre-cooled to -40°C, and then the material was placed in the freeze-drying chamber to start freeze-drying; the pre-freezing stage temperature was -40°C, the time was 6 hours, and the vacuum degree of the sublimation stage was less than 100 Pa, the time was 24 hours, and the icariin / bio-derived hydroxyapatite composite material was obtained.

[0034] 4. Preparation of Icariin-Bone Matrix Gelatin Solution: Cortical bones of healthy cattle were taken, washed with purified water and crushed, and the stainless steel mesh was used to sieve the cortical bone particles of 0.2-0.5 mm; soaked in chloroform / methanol mixture for 4 hours to degrease; demineralized in 0.6 mol / L hydrochloric acid solution for 24 hours, and the acid was changed every 6 hours; soaked in 2 mol / L CaCl2 solution for 6 hours to remove low molecular weight proteoglycans; soaked in 0.5 mol / L EDTA solution for 6 hours to remove non-hydroxyapatite calcium and some proteoglycans; soaked in 8 mol / L LiCl solution for 48 hours to shrink collagen fibers and remove some high molecular weight proteoglycans; washed with purified water, stirred and dissolved with 45℃ purified water; centrifuged at a centrifugal radius of 10 cm and a speed of 8000 r / min for 10 min, discarded the supernatant, and the obtained precipitate was added to injection water at a volume ratio of 1:1 and mixed thoroughly to obtain bone matrix gelatin solution. Icariin was added to the bone matrix gelatin solution to prepare a 30 mg / L icariin-bone matrix gelatin solution.

[0035] 5. Composite of icariin-bone matrix gelatin solution and icariin / bioderived hydroxyapatite material: Negative pressure adsorption was used with a pressure value of 10 kPa to allow the bone matrix gelatin to fill the pores of the bio-derived hydroxyapatite material and infiltrate for 1 hour; the centrifugal radius was 10 cm and the speed was 100 r / min for 3 minutes; freeze-drying was performed, and the freeze-drying method was as follows: the pre-freezing stage temperature was -50°C for 6 hours, and the sublimation stage vacuum was less than 100 Pa for 24 hours, and the bone repair material with improved bone induction activity was obtained ( Figure 2 , 4 ).

[0036] Comparative Example 1 The control group in this comparative example is bio-derived hydroxyapatite, and its preparation method is the same as that described in Example 1. The compressive strength of bio-derived hydroxyapatite and composite bone repair material were tested respectively. Specifically, bio-derived hydroxyapatite and composite bone repair material with specifications of 20mm*10mm*10mm were placed on a universal mechanical machine for compressive strength testing. The mechanical machine speed was 5mm / min, and the maximum force was recorded to calculate the compressive strength. Figure 5 , 6 As shown in the figure, the average compressive strength of bio-derived hydroxyapatite was (0.785 ± 0.18) mPa, and the average compressive strength of composite bone was (2.22 ± 0.23) mPa. The compressive strength of composite bone was significantly higher than that of the untreated group. This indicates that the compressive strength of bio-derived hydroxyapatite was improved after composite treatment.

[0037] Comparative Example 2 The control group in this comparative example is the hydroxyapatite group derived from composite icariin-bone matrix gelatin (single gelatin composite group) and the icariin hydroxyapatite group soaked (single immersion group), and the experimental group is the double composite bone repair material prepared by this patent (double composite group), and its preparation method is the same as that described in Example 1. Take three groups of materials respectively, weigh about 2.0g and put them into a centrifuge tube containing 10mL PBS, place them in an oscillator and oscillate at 120 r / min at 37°C, aspirate the supernatant every 24 hours, and use high performance liquid chromatography to detect the icariin concentration in the supernatant, and add fresh PBS to continue oscillating, draw a concentration curve, and analyze the drug release. The single immersion group released completely after 4 days, and the icariin release of the single gelatin composite group and the double composite group can last for more than 12 days. The sustained-release concentration of icariin in the double composite group is higher than that of the single gelatin composite group, see Figure 7 shown.

[0038] Comparative Example 3 In this comparative example, the control group is uncompounded bio-derived hydroxyapatite, and its preparation method is the same as that described in Example 1, and the experimental group is the composite bone repair material prepared in this patent. The osteoinductive activity of the bio-derived hydroxyapatite and the composite bone repair material was tested respectively. Specifically, Eight healthy male athymic nude mice aged 6-9 weeks, weighing 21-24 g, were selected. The intermuscular space between the gluteus medius and gluteus maximus was selected as the implantation site; bioderived hydroxyapatite was implanted on the left side, and the composite bone repair material group was implanted on the right side. The health status and wound healing of the animals were observed and recorded after surgery; the eight animals were killed by vertebral dislocation on the 4th week after surgery, and the muscle tissue including the implanted samples was cut and fixed in 4% formaldehyde solution for 72 hours. The fixed tissue blocks were decalcified with 50% formic acid, dehydrated with conventional ethanol, and paraffin-embedded to prepare tissue sections and HE staining. The osteoinductive properties of the two implants were evaluated according to the methods recommended in YYT 1680-2020 "Evaluation of the in vivo osteoinductive properties of allogeneic repair materials and demineralized bone materials".

[0039] At least three sections were prepared for each sample, and the sections with the least wrinkles, the most uniform staining, and the largest amount of implanted material were used for evaluation of osteoinductive capacity. A 5×5 (24 mm2) grid was inserted into the eyepiece of the microscope. The 10× objective lens was used for observation of the athymic mouse implant sample sections, and the total magnification was 100×. When placing the specimen on the stage, the position of the sample section was adjusted so that the objective lens was above one of the four corners of the sample section. Each field of view was observed until the entire sample section was observed. A field of view is the area that can be observed within the 5×5 grid at 100× magnification. If it is not certain whether bone formation exists in a specific divided area, a higher magnification objective lens was used for observation. After confirmation, the 10× objective lens was changed back to observe other divided areas.

[0040] The observation results were counted and scored. The specific method is: count the total number of small squares containing ≥50% implanted bone and / or bone formation in the grid of all photos of each implantation site (A). Among them, if there is >50% blank area, fibrous connective tissue, skeletal muscle, etc. in the small square, regardless of whether there is new bone in it, it will not be counted. If the small square meets the above conditions and contains at least one new bone forming element, it is recorded as a positive square. New bone forming elements include: chondrocytes / chondrocytes, osteoblasts / osteocytes, cartilage, bone, new bone, reticular bone and bone marrow. Count the number of positive squares at each implantation site (B). Calculate the proportion of new bone forming elements β according to the following formula.

[0041] New bone forming element ratio β = B / A × 100% The results of the bone induction activity score of nude mice were as follows Figure 8 As shown in Figure 2, the bone induction activity score of the experimental group was significantly better than that of the control group; the HE staining results were as follows Fig. 9 , 10 As shown, chondrocytes were observed in the HE staining of the sections of the experimental group, while no new bone-forming elements were found in the control group, indicating that the bone-inducing activity of bio-derived hydroxyapatite was improved after the composite treatment.

[0042] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0043] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for improving the osteoinductive activity of a bioderived hydroxyapatite material, characterized in that: The bio-derived hydroxyapatite material is first compounded with an icariin suspension and then double compounded with an icariin-bone matrix gelatin solution.

2. A method for improving the osteoinductive activity of bioderived hydroxyapatite material according to claim 1, characterized in that: The bio-derived hydroxyapatite material is derived from human or animal bone tissue and is an inorganic bone material obtained after a series of antigen removal treatments. The main component of the inorganic bone material is hydroxyapatite and the inorganic bone material has a natural porous structure.

3. A method for improving the osteoinductive activity of bioderived hydroxyapatite material according to claim 1, characterized in that: The compounding method of the bio-derived hydroxyapatite and icariin suspension is as follows: The bio-derived hydroxyapatite material is placed in a mold containing icariin suspension with a stirring function, and the mold is placed in an oscillator with an oscillation speed of 100-120 r / min for 1 to 24 hours, during which the orientation of the bio-derived hydroxyapatite material is adjusted 2-6 times; the material is taken out and placed in an environment below -70°C to complete pre-freezing for 10-30 minutes, and then the material is placed in a freeze drying chamber of a freeze dryer precooled to -40°C, the temperature in the pre-freezing stage is not higher than -40°C, the time is 6-12 hours, and the vacuum degree in the sublimation stage is 10-100 Pa for 10-24 hours, so as to obtain icariin / bio-derived hydroxyapatite material.

4. A method for improving the osteoinductive activity of bioderived hydroxyapatite material according to claim 1, characterized in that: The concentration of the icariin suspension is 1.0-100.0 mg / L.

5. A method for improving the osteoinductive activity of bioderived hydroxyapatite material according to claim 1, characterized in that: The volume ratio of the icariin suspension to the biologically derived hydroxyapatite is 1-10:

1.

6. The method for improving the osteoinductive activity of bioderived hydroxyapatite material according to claim 1, characterized in that: The preparation method of the icariin-bone matrix gelatin solution is as follows: Bone tissue from qualified human donors or animals is washed and crushed, and bone particles with a particle size of 0.2-0.8mm are taken and washed with purified water; soaked in a chloroform / methanol mixture for 4-24 hours for degreasing; demineralized in a 0.1-0.6 mol / L hydrochloric acid solution for 4-24 hours; soaked in a 1-2 mol / L CaCl2 solution for 4-24 hours to remove low molecular weight proteoglycans; soaked in a 0.1-0.5 mol / L EDTA solution for 4-24 hours to remove non-hydroxyapatite calcium and part of proteoglycans; soaked in a 2-8 mol / L LiCl solution for at least 24 hours to shrink collagen fibers and remove part of high molecular weight proteoglycans; washed with purified water, stirred and dissolved with 45-50℃ purified water; centrifuged for 10-30 minutes, discarded the supernatant, and the obtained precipitate was added to purified water at a volume ratio of 1:1-5 and mixed thoroughly to obtain a bone matrix gelatin solution. Icariin is added to the bone matrix gelatin solution and mixed to obtain an icariin-bone matrix gelatin solution.

7. A method for improving the osteoinductive activity of bioderived hydroxyapatite material according to claim 1, characterized in that: The concentration of the icariin-bone matrix gelatin solution is 1.0-100.0 mg / L.

8. A method for improving the osteoinductive activity of bioderived hydroxyapatite material according to claim 1, characterized in that: The composite method of the icariin-bone matrix gelatin solution and the icariin / bioderived hydroxyapatite composite material is as follows: (1) Negative pressure adsorption: negative pressure adsorption is used with a pressure of 1-50 kPa to allow bone matrix gelatin to fully fill the pores of the bio-derived hydroxyapatite material and infiltrate for 1 to 24 hours; (2) Expose pores: centrifuge radius 10 cm, speed less than 100 r / min, centrifuge for less than 1-3 minutes, and after centrifugation, the sample is quickly placed in a refrigerator not higher than -50°C for freezing; (3) Freeze drying: the temperature in the pre-freezing stage is not higher than -50°C, the time is 6-10 hours, and the vacuum degree in the sublimation stage is 20-100 Pa, the time is 10-36 hours.

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