Injectable bone repair material based on superfine eggshell powder and preparation method of injectable bone repair material

Abstract

The invention discloses an injectable bone repair material based on superfine eggshell powder and a preparation method of the injectable bone repair material, and belongs to the field of biomedical materials. According to the material, waste eggshells serve as raw materials, superfine eggshell powder is obtained through alkaline cleaning and ball milling, the superfine eggshell powder is converted into an apatite matrix through treatment of a dipotassium phosphate (K2HPO4) solution, and silver hybrid bacterial cellulose nanofibers are compounded to enhance the mechanical property and the antibacterial function. The obtained material has excellent injectability, the mechanical strength can be matched with the human bone tissue, the degradation period is synchronous with the bone regeneration rate, and the material has excellent antibacterial performance. According to the invention, waste resources are utilized to reduce the cost, and a low-temperature treatment process is combined, so that the material has mechanical adaptability, osteoinductivity and broad-spectrum antibacterial property, and an efficient, safe and environment-friendly solution is provided for osteoporotic fracture and bone defect repair.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to an injectable bone repair material prepared based on ultrafine eggshell powder and a preparation method thereof. Background Art

[0002] Recent national population surveys have shown that my country's population is aging rapidly. One of the common diseases associated with aging is osteoporosis, a systemic skeletal disease primarily caused by increased bone resorption by osteoclasts and a relative decrease in bone formation by osteoblasts, resulting in an imbalance between bone formation and resorption. Osteoporosis is characterized by decreased bone density and strength, an increased risk of fractures, and associated pain and functional limitations. Osteoporotic fractures are associated with high mortality and disability rates, making them a leading cause of disability and mortality in elderly patients and placing a significant social and economic burden on the healthcare system.

[0003] Current osteoporotic fracture treatment faces dual challenges. Traditional fixation relies on autogenous bone or metal implants, which can lead to donor-site complications and stress shielding. Furthermore, artificial bone repair materials must balance mechanical compatibility, bioactivity, and controllable degradation. Existing bone repair materials are primarily classified into three categories: organic bone cements, inorganic bone cements, and composite bone repair materials. Commonly used organic bone cements, such as polymethyl methacrylate (PMMA) bone cements, are non-degradable, and the exothermic curing process and residual monomers can damage surrounding tissue. Inorganic bone cements generally exhibit good tissue compatibility and osteoinductivity, but they also have their own limitations. For example, calcium sulfate cements degrade rapidly in vivo; calcium phosphate cements exhibit low mechanical properties; and magnesium phosphate cements exhibit poor water resistance. Composite materials, such as hydroxyapatite composites, are expensive, and their traditional preparation processes rely on high-temperature calcination, which consumes significant energy. Furthermore, given the irregular morphology of most bone defects in clinical practice, shaped materials struggle to achieve a close fit with tissue, thus compromising repair effectiveness. In contrast, injectable bone repair materials that integrate both integrated and repair functions are attracting considerable attention. However, existing injectable materials generally have the problem of single function: mechanical enhancers are non-degradable, antibacterial additives are prone to aggregation and cytotoxicity is uncontrollable, etc. Therefore, the development of a new green and low-cost injectable bone repair material is of great significance.

[0004] Eggshells, a waste resource produced in the millions of tons globally each year, are primarily composed of calcium carbonate, a substance highly similar to the inorganic components of bone. Previous studies have shown that eggshells have enormous potential for bone tissue engineering applications: eggshell powder, when treated with a phosphate solution, can be converted into bone-like apatite, with a calcium-to-phosphorus ratio close to that of natural bone, possessing both osteoconductive and inductive potential. Patent CN202310085516.X proposes the use of eggshell membranes to prepare intelligent membranes for guided bone tissue regeneration. A team at the University of Massachusetts has demonstrated excellent bone regeneration capabilities in skull defect repair using composite hydrogel scaffolds prepared from eggshell powder. In summary, waste eggshells can be used to develop injectable bone repair materials, providing an efficient, safe, and low-cost solution for bone defects. Summary of the Invention

[0005] This invention provides an injectable bone repair material based on ultrafine eggshell powder. The ultrafine eggshell powder is prepared from discarded eggshells and treated with a K2HPO4 solution to enhance its osteoinductivity. Silver-hybridized bacterial cellulose nanofibers are also added to provide antibacterial properties and enhance the material's mechanical strength. The resulting material can be injected into bone defects or bone loss sites. After solidification, it effectively enhances the mechanical strength of bone tissue, guides the migration and proliferation of host bone cells, and stimulates the differentiation of stem cells into osteoblasts. The material's degradation rate synchronizes with bone regeneration, and it exhibits excellent and stable antibacterial properties, effectively preventing postoperative infection.

[0006] The specific steps include:

[0007] Step 1: clean the eggshells, soak them in an alkaline solution or detergent to remove the protein, wash and dry them, and then ball-mill them until D50 is less than 10 μm to obtain ultrafine eggshell powder.

[0008] Step 2: centrifuging the bacterial cellulose homogenate, mixing the homogenate mixture with sulfuric acid, taking the precipitate after centrifugation, washing it with deionized water, and centrifuging and dehydrating it to obtain bacterial cellulose nanofibers.

[0009] Step 3: Add the bacterial cellulose nanofibers obtained in step 2 to the AgNO3 solution, stir and disperse, cool to room temperature, add hydrazine hydrate solution to carry out silver hybridization reaction, and wash to obtain silver hybridized bacterial cellulose nanofibers.

[0010] Step 4: Stir and mix the ultrafine eggshell powder obtained in step 1 and the silver hybrid bacterial cellulose nanofibers obtained in step 3 in a K2HPO4 solution to form a uniform slurry to obtain a primary setting bone repair material.

[0011] Step 5, solidifying the initially set bone repair material obtained in step 4 to obtain a final set bone repair material.

[0012] Preferably, in step 1, the concentration of NaOH is 1-10%, the soaking time is 2-12 hours, the ball-to-material mass ratio of the ball mill is (5-10):1, and the ball mill speed is 200-400 rpm.

[0013] Preferably, in step 2, the speed of the homogenizer is 8000-10000 rpm, and the volume concentration of sulfuric acid is 40-90%.

[0014] Preferably, in step 3, the AgNO3 concentration is 0.05-0.2 mol / L, the stirring and heating temperature is 30-60°C, the concentration of the hydrazine hydrate solution is 0.2%-1%, the silver hybridization reaction temperature is 30-60°C, and the time is 4-8h.

[0015] Preferably, in step 4, the mass ratio of ultrafine eggshell powder to silver hybrid bacterial cellulose nanofibers is 1:1 to 7:1, the concentration of K2HPO4 is 0.5-2 mol / L, and the stirring temperature is 40-80°C.

[0016] Preferably, the curing conditions in step 5 are 35-38° C., 80-100% humidity, and the curing time is 3-10 days.

[0017] Compared with the prior art, the present invention has the following significant advantages:

[0018] (1) The present invention can efficiently utilize waste eggshell resources and convert eggshell powder into apatite through low-temperature treatment with a K2HPO4 solution, thereby reducing raw material costs and shortening the process cycle.

[0019] (2) The present invention improves the compressive strength of the material by introducing silver hybrid bacterial cellulose nanofibers. + Sustained release achieves broad-spectrum antibacterial effect, effectively preventing postoperative infection without the need for additional antibiotics.

[0020] (3) The present invention uses non-toxic natural materials (eggshell and bacterial cellulose) for bone repair, which has high clinical adaptability and excellent safety.

[0021] (4) The present invention uses K2HPO4 as a curing agent to promote the hydration of ultrafine eggshell powder, optimize injectability and degradation controllability, and match the bone regeneration process. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below with reference to the embodiments.

[0023] Example 1

[0024] Step 1: Take 100g of fresh eggshells, wash them with clean water, crush them into small pieces, soak them in 200mL of 5% NaOH solution, shake them at a constant temperature of 37°C for 4h to remove residual protein, rinse them with deionized water until neutral (pH≈7), dry them at 60°C for 24h, and ball mill the dried eggshells in a ball mill with a ball-to-material ratio of 10:1 and a rotation speed of 300rpm for 6h to obtain ultrafine eggshell powder with D50<10μm.

[0025] Step 2: Cut the bacterial cellulose into small pieces, centrifuge the mixture at 10,000 rpm for 30 minutes using a tissue homogenizer, transfer the homogenized mixture into a three-necked flask containing 80% (V / V) sulfuric acid, vigorously stir in a constant temperature water bath, centrifuge at 8,000 rpm for 15 minutes, take the precipitate, wash it three times with deionized water, and centrifuge the purified bacterial cellulose nanofiber suspension at 10,000 rpm for 20 minutes, dehydrate it, and obtain bacterial cellulose nanofibers.

[0026] Step 3, the bacterial cellulose nanofibers obtained in step 2 were mixed with 100 mL of 0.1 mol / L AgNO3 solution, stirred at a constant temperature of 40°C for 12 hours, cooled to room temperature, and then 0.6% hydrazine hydrate solution was added dropwise to the mixture under stirring until the solution turned gray-green, and then heated to 45°C for 6 hours, centrifuged at 10,000 rpm for 10 minutes, and the precipitate was collected and washed three times with distilled water and anhydrous ethanol in sequence. After vacuum drying, silver hybrid bacterial cellulose nanofibers were obtained.

[0027] Step 4: Add the ultrafine eggshell powder obtained in step 1 and the silver-hybridized bacterial cellulose nanofibers obtained in step 3 to a 1 mol / L K2HPO4 solution at a mass ratio of 7:1 and stir until a uniform slurry is formed to obtain a primary bone repair material for later use.

[0028] Step 5, curing the initial bone repair material at 37 ° C and 100% humidity for 7 days to obtain the final bone repair material.

[0029] Example 2

[0030] Step 1: Take 100g of fresh eggshells, wash them with clean water, crush them into small pieces, soak them in 200mL of 5% NaOH solution, shake them at a constant temperature of 37°C for 4h to remove residual protein, rinse them with deionized water until neutral (pH≈7), dry them at 60°C for 24h, and ball mill the dried eggshells in a ball mill with a ball-to-material ratio of 10:1 and a rotation speed of 300rpm for 6h to obtain ultrafine eggshell powder with D50<10μm.

[0031] Step 2: Cut the bacterial cellulose into small pieces, centrifuge the mixture at 10,000 rpm for 30 minutes using a tissue homogenizer, transfer the homogenized mixture into a three-necked flask containing 80% (V / V) sulfuric acid, vigorously stir in a constant temperature water bath, centrifuge at 8,000 rpm for 15 minutes, take the precipitate, wash it three times with deionized water, and centrifuge the purified bacterial cellulose nanofiber suspension at 10,000 rpm for 20 minutes, dehydrate it, and obtain bacterial cellulose nanofibers.

[0032] Step 3, the bacterial cellulose nanofibers obtained in step 2 were mixed with 100 mL of 0.1 mol / L AgNO3 solution, stirred at a constant temperature of 40°C for 12 hours, cooled to room temperature, and then 0.6% hydrazine hydrate solution was added dropwise to the mixture under stirring until the solution turned gray-green, and then heated to 45°C for 6 hours, centrifuged at 10,000 rpm for 10 minutes, and the precipitate was collected and washed three times with distilled water and anhydrous ethanol in sequence. After vacuum drying, silver hybrid bacterial cellulose nanofibers were obtained.

[0033] Step 4: add the ultrafine eggshell powder obtained in step 1 and the silver hybrid bacterial cellulose nanofibers obtained in step 3 to a 1 mol / L K2HPO4 solution in a mass ratio of 3:1, and stir until a uniform slurry is formed to obtain a primary setting bone repair material for later use.

[0034] Step 5: Curing the initially set bone repair material at 37° C. and 100% humidity for 7 days to obtain the final set bone repair material.

[0035] Example 3

[0036] Step 1: Take 100g of fresh eggshells, wash them with clean water, crush them into small pieces, soak them in 200mL of 5% NaOH solution, shake them at a constant temperature of 37°C for 4h to remove residual protein, rinse them with deionized water until neutral (pH≈7), dry them at 60°C for 24h, and ball mill the dried eggshells in a ball mill with a ball-to-material ratio of 10:1 and a rotation speed of 300rpm for 6h to obtain ultrafine eggshell powder with D50<10μm.

[0037] Step 2: Cut the bacterial cellulose into small pieces, centrifuge the mixture at 10,000 rpm for 30 minutes using a tissue homogenizer, transfer the homogenized mixture into a three-necked flask containing 80% (V / V) sulfuric acid, vigorously stir in a constant temperature water bath, centrifuge at 8,000 rpm for 15 minutes, take the precipitate, wash it three times with deionized water, and centrifuge the purified bacterial cellulose nanofiber suspension at 10,000 rpm for 20 minutes, dehydrate it, and obtain bacterial cellulose nanofibers.

[0038] Step 3, the bacterial cellulose nanofibers obtained in step 2 were mixed with 100 mL of 0.1 mol / L AgNO3 solution, stirred at a constant temperature of 40°C for 12 hours, cooled to room temperature, and then 0.6% hydrazine hydrate solution was added dropwise to the mixture under stirring until the solution turned gray-green, and then heated to 45°C for 6 hours, centrifuged at 10,000 rpm for 10 minutes, and the precipitate was collected and washed three times with distilled water and anhydrous ethanol in sequence. After vacuum drying, silver hybrid bacterial cellulose nanofibers were obtained.

[0039] Step 4: add the ultrafine eggshell powder obtained in step 1 and the silver hybrid bacterial cellulose nanofibers obtained in step 3 to a 1 mol / L K2HPO4 solution in a mass ratio of 1:1, and stir until a uniform slurry is formed to obtain a primary setting bone repair material for later use.

[0040] Step 5: Curing the initially set bone repair material at 37° C. and 100% humidity for 7 days to obtain the final set bone repair material.

[0041] Comparative Example 1

[0042] This comparative example is basically the same as Example 1, except that in step 4, the silver-hybridized bacterial cellulose nanofibers obtained in step 3 are not added to the solution.

[0043] The results showed that the bone repair material prepared in this comparative example had inferior mechanical strength and antibacterial ability compared with the bone repair material prepared in Example 1.

Claims

1. A method for preparing an injectable bone repair material based on ultrafine eggshell powder, characterized in that: The following steps are involved: Step 1: After washing the eggshells, soaking them in an alkaline solution to remove residual protein, drying them, and ball-milling them to a particle size D50 < 10 μm to obtain ultrafine eggshell powder; Step 2, homogenizing the bacterial cellulose and purifying it with sulfuric acid to obtain bacterial cellulose nanofibers; Step 3, adding the bacterial cellulose nanofibers obtained in step 2 to an AgNO3 solution, stirring and dispersing, adding a hydrazine hydrate solution to carry out a silver hybridization reaction, and washing to obtain silver-hybridized bacterial cellulose nanofibers; Step 4, mixing the ultrafine eggshell powder obtained in step 1 with the silver-hybridized bacterial cellulose nanofibers obtained in step 3, adding K2HPO4 solution and stirring to form a uniform slurry to obtain a primary setting bone repair material; Step 5: solidify the initially set bone repair material to obtain the final set bone repair material.

2. The preparation method according to claim 1, characterized in that Preferably, the alkaline solution in step 1 is a NaOH solution with a mass concentration of 1% to 10%, the soaking time is 2 to 12 hours, the ball-to-material mass ratio is (5 to 10):1, and the ball milling speed is 200 to 400 rpm.

3. The preparation method according to claim 1, characterized in that The volume concentration of sulfuric acid in step 2 is 40% to 90%.

4. The preparation method according to claim 1, characterized in that The concentration of the AgNO3 solution in step 3 is 0.05-0.2 mol / L; the concentration of the hydrazine hydrate solution is 0.2%-1%, the reaction temperature is 30-60°C, and the reaction time is 4-8h.

5. The preparation method according to claim 1, characterized in that The mass ratio of the ultrafine eggshell powder to the silver hybrid bacterial cellulose nanofibers in step 4 is 1:1 to 7:1, the K2HPO4 concentration is 0.5-2 mol / L, and the stirring temperature is 40-80°C.

6. The preparation method according to claim 1, characterized in that In step 5, the curing conditions are 35-38° C., 80-100% humidity, and the curing time is 3-10 days.

7. A perfusable bone repair material based on ultrafine eggshell powder, characterized in that: Prepared by the preparation method according to any one of claims 1 to 6, the material comprises: (1) Apatite matrix formed by conversion of ultrafine eggshell powder into K2HPO4 solution; (2) silver hybrid bacterial cellulose nanofibers, uniformly dispersed in the matrix; (3) After solidification, the material has controllable degradability that matches the bone regeneration rate, a compressive strength of ≥15 MPa, and broad-spectrum antibacterial properties.