Bone material as well as preparation method and application thereof
By fermenting Bacillus licheniformis and compounding it with polyvinyl alcohol and polylactic acid, a new type of bone material was prepared, which solved the problem of insufficient compatibility and mechanical properties of existing materials, and achieved improvements in biocompatibility, degradability and safety.
Patent Information
- Application Number
- CN202411741727.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-29
- Publication Date
- 2025-02-28
AI Technical Summary
The existing artificial bone materials have limited compatibility with the human body, which poses safety risks, and the mechanical properties of natural materials are poor, prone to brittle cracking and deforming, and some of them have heavy metal residues that lead to cytotoxicity.
The flounder fish bones were fermented by Bacillus licheniformis ACCC 10236, combined with polyvinyl alcohol and polylactic acid, and prepared new bone materials through high-pressure homogenization and high-temperature curing.
It improves the mechanical properties of halibut fish bones, enhances biocompatibility, reduces the toxicity of heavy metals, and has no inflammation or thrombosis reactions in the material, which can automatically degrade in the human body. It is suitable for a variety of medical scenarios.
Smart Images
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Abstract
Description
Technical Field
[0001] The present application relates to the field of medical technology, and in particular to a bone material and a preparation method and application thereof. Background Art
[0002] The demand for treatment and prosthetics for bone diseases such as bone injury, osteoporosis, and arthritis has always been high, but traditional treatment methods often have certain limitations. Therefore, research on artificial bones can provide humans with a new treatment method. However, most of the current artificial bone materials have limited compatibility with the human body and have certain safety risks.
[0003] Natural materials have excellent biological activity and high safety. Currently, the most commonly used natural materials in bones are cow bones, shark bones, etc. However, natural materials have poor mechanical properties, are prone to brittle cracking, deformation and damage, and some have heavy metal residues that cause them to be cytotoxic.
[0004] Therefore, there is an urgent need for new bone materials that are close to natural bones, have good biocompatibility, are non-toxic, and are degradable. Summary of the invention
[0005] The purpose of the present invention is to provide a new bone material which is close to natural bone, has good biocompatibility, is non-toxic, degradable and highly safe.
[0006] In one aspect, the present application provides a method for preparing a bone material, the method comprising:
[0007] Step 1, drying and crushing the flounder bones to obtain fish bone powder;
[0008] Step 2: mixing the fish bone powder with water, inoculating Bacillus licheniformis ACCC 10236, fermenting, sterilizing and drying to obtain fish bone fermented powder;
[0009] Step 3: Mix the fish bone fermentation powder with polyvinyl alcohol and polylactic acid to obtain a mixed powder, homogenize under high pressure and then solidify at high temperature to obtain an artificial bone material.
[0010] Preferably, the mesh size of the fish bone powder is 80-100 mesh, more preferably, 100 mesh.
[0011] Furthermore, the OD of the Bacillus licheniformis ACCC 10236 is 600 Is 3-4.
[0012] Preferably, OD 600 is 4.
[0013] Furthermore, the inoculation amount of the Bacillus licheniformis ACCC 10236 is 1%-5%.
[0014] Preferably, the inoculation amount is 1%.
[0015] Furthermore, the fermentation treatment conditions are 25°C-37°C, 200-300rpm, and the fermentation treatment time is 1-2h.
[0016] Preferably, the fermentation treatment conditions are 37° C., 220 rpm, and enzymatic treatment for 2 h.
[0017] Furthermore, the fish bone powder is mixed with water at a solid-liquid ratio of 1:(5-10).
[0018] Preferably, the fish bone powder is mixed with water at a solid-liquid ratio of 1:10.
[0019] Preferably, the sterilization condition is 121° C. for 60 min.
[0020] Those skilled in the art can understand that the sterilization here is intended to kill all bacteria, so the sterilization conditions and sterilization methods can be selected according to the actual situation, and no excessive restrictions are imposed here. Similarly, those skilled in the art can select different drying methods to dry the above materials, as long as the materials can be completely dried.
[0021] In this application, Bacillus licheniformis ACCC 10236 is used for the first time to ferment flounder bones. It has been verified that the fermentation process can effectively improve the mechanical properties of flounder bones and improve their safety, solve the problem of low biocompatibility of flounder bones, and the fermented material also has the effect of promoting bone cell proliferation. This is because Bacillus licheniformis ACCC 10236 can effectively transform harmful substances such as heavy metals in fish bones into non-toxic components, thereby improving the multiple properties of the material.
[0022] Furthermore, the mass ratio of the fish bone fermentation powder, polyvinyl alcohol and polylactic acid is (10-20): (10-20): (1-5).
[0023] Preferably, the mass ratio of the fish bone fermentation powder, polyvinyl alcohol and polylactic acid is 20:10:5.
[0024] Furthermore, the mass ratio of the mixed powder to water is (1-5):1.
[0025] Preferably, the mass ratio of the mixed powder to water is 2:1.
[0026] Preferably, the method further comprises the steps of irradiating and sterilizing the bone material and packaging it.
[0027] In a preferred embodiment, a method for preparing a bone material comprises:
[0028] Step 1, drying and crushing the flounder bones to 80-100 mesh to obtain fish bone powder;
[0029] Step 2: Mix the fish bone powder with water at a solid-liquid ratio of 1: (5-10), and inoculate Bacillus licheniformis ACCC 10236 (OD 600 =3-4), 25°C-37°C, 200-300rpm, fermentation treatment for 1-2h, sterilization at 121°C for 30-60min, drying until the mass no longer changes, to obtain fish bone fermentation powder;
[0030] Step 3: Take the fish bone fermentation powder, polyvinyl alcohol, and polylactic acid in a mass ratio of (10-20): (10-20): (1-5) and mix them by ball milling to obtain a mixed powder. Then, mix the mixed powder with water in a mass ratio of (1-5): 1, and after high-pressure homogenization (60-80MPa, 10-30min), put it into a mold and cure it at 160℃-200℃ for 60-120min to obtain an artificial bone material.
[0031] Step 4: irradiate and sterilize the bone material and package it.
[0032] The mold can be selected according to actual conditions and is not limited here.
[0033] On the other hand, the present application also provides a bone material prepared by the method.
[0034] Furthermore, the bone material can promote the proliferation of bone cells.
[0035] Furthermore, the tensile strength of the bone material can reach 399.25 MPa and the hardness can reach 258.62 MPa.
[0036] Furthermore, the bone material has no inflammatory or thrombotic reaction.
[0037] Furthermore, the bone material is degradable.
[0038] All components of the bone material of the present application are degradable materials and can be automatically degraded in the human body.
[0039] On the other hand, the present application also provides a product comprising the bone material.
[0040] Preferably, the products include various bone material products such as bone defect repair products, joint replacement products, and dental implant products.
[0041] The present invention has the following beneficial effects:
[0042] 1. This application first uses Bacillus licheniformis ACCC 10236 to ferment flounder bones to obtain a new bone material. The material has no potential cytotoxicity and has a certain promoting effect on cell proliferation, and has the potential to promote bone cell growth. In addition, the fermentation process also improves the mechanical properties of flounder bones, making them closer to the data of real human bones.
[0043] 2. This application also uses polyvinyl alcohol, polylactic acid and fish bone fermentation powder to prepare a new bone material. The material has mechanical properties close to those of the human body, has no inflammatory and thrombotic reactions and can be degraded in the human body, and is widely applicable to a variety of medical scenarios.
[0044] 3. This application also provides a method for preparing a new type of bone material, providing technical support for the industrial production of bone materials. DETAILED DESCRIPTION
[0045] In order to more clearly illustrate the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.
[0046] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.
[0047] Among them, Bacillus licheniformis ACCC 10236 and Bacillus subtilis ACCC11089 were purchased from China Agricultural Microbiological Culture Collection Center, and Bacillus licheniformis CCTCC AB 92069 was purchased from China Center for Type Culture Collection. Polyvinyl alcohol (PVA) CAS No.: 9002-89-5, polylactic acid CAS No.: 26100-51-6.
[0048] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.
[0049] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in microbiology, biochemistry, analytical chemistry, cell culture, and related fields conventional in the technical field.
[0050] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and purified water.
[0051] In the following examples, unless otherwise specified, % means wt %, i.e. weight percentage.
[0052] Example 1
[0053] In this embodiment, flounder bones are used as the raw material of artificial bone material for the first time. Flounder bones are also rich in calcium and phosphorus, have a high density, and have great potential as a bone filling material.
[0054] The fish bones and fish meat of flounder were separated by using a bone-meat separator until there was no large piece of fish meat left on the fish bones, and the fish bones were washed, dried and crushed to 100 mesh to obtain fish bone powder. The fish bone powder was mixed with water at a solid-liquid ratio of 1:10, and different inoculants were inoculated with different inoculum amounts (all inoculants were kept at OD 600 4), fermentation treatment, sterilization at 121° C. for 60 min, and drying to obtain fish bone fermentation powder. The specific conditions are shown in Table 1.
[0055] Human osteosarcoma MG-63 cells in logarithmic growth phase were cultured in MEM medium, supplemented with 10% fetal bovine serum and 1% double antibody. The inoculated cells were placed in a carbon dioxide incubator at 37°C and 5% CO2 for routine culture overnight. After adding 1g of fish bone fermentation powder and continuing to culture for 24h, the relative cell proliferation rate was detected by CCK-8 method. The blank control group was added with an equal amount of serum-free medium, and the negative control group was added with 1g of fish bone powder instead of fish bone fermentation powder.
[0056] Table 1 Results of relative cell proliferation rate
[0057]
[0058]
[0059] From the results in Table 1, it can be seen that the use of Bacillus licheniformis ACCC 10236 to treat flounder bones can obtain safe bone materials. This material has no potential cytotoxicity and has a certain degree of promoting effect on cell proliferation, and has the potential to promote bone cell growth.
[0060] In addition, in this embodiment, the mechanical properties of the bone material were tested, including tensile strength and hardness. The tensile strength was tested using a bone compressive strength tester, and the hardness was tested using a microhardness tester. The load size was 0.98N and the loading time was 15s. The test results are shown in Table 2.
[0061] Table 2 Mechanical properties test results
[0062]
[0063]
[0064] From the results in Table 2, it can be seen that the use of Bacillus licheniformis ACCC 10236 to treat flounder bones can effectively improve the mechanical properties of flounder bones, making them closer to the data of real human bones.
[0065] Example 2
[0066] In order to make the bone material closer to the physical and chemical properties of human bones and facilitate molding and use, in this embodiment, the material is compounded with other components and its properties are appropriately adjusted.
[0067] The specific method includes: taking the fish bone fermentation powder obtained by optimizing Example 1 and mixing it with different degradable components, putting it into a ball mill for ball milling, then mixing the mixed powder with water in different mass ratios, and after high-pressure homogenization (60MPa, 10min), putting it into a mold and curing it at 180℃ for 60min to obtain an artificial bone material. The negative control group uses hydroxyapatite to replace the fish bone fermentation powder. The mechanical property test is the same as Example 1, and the molding rate is determined as the ratio of the number of qualified products (i.e., no cracks on the surface) to the total number. The results and specific conditions are shown in Table 3.
[0068] Table 3 Mechanical properties test results
[0069]
[0070] It can be seen from the results in Table 3 that the bone material prepared by polyvinyl alcohol, polylactic acid and fish bone fermentation powder has mechanical properties close to those of the human body, and compared with fish bone fermentation powder, the composition has better molding ability and can be widely used in various medical scenarios.
[0071] In summary, this embodiment provides an optimized novel bone material and a specific preparation method thereof, which specifically comprises the following steps:
[0072] Step 1: Use a meat-bone separator to separate the fish bones and fish meat of the flounder until there is no large piece of fish meat left on the fish bones, wash and dry the fish bones and crush them into 100 mesh to obtain fish bone powder.
[0073] Step 2: Mix the fish bone powder and water at a solid-liquid ratio of 1:10, and add Bacillus licheniformis ACCC 10236 (OD 600 =4), 37°C, 220rpm, fermentation treatment for 2h, sterilization at 121°C for 60min, drying until the mass no longer changes, to obtain fish bone fermentation powder.
[0074] Step 3: Take 20g of fish bone fermentation powder, 10g of polyvinyl alcohol (PVA) and 5g of polylactic acid and put them into a ball mill for ball milling. Then, mix the mixed powder with water in a mass ratio of 2:1. After high-pressure homogenization (60MPa, 10min), put it into a mold and cure it at 180℃ for 60min to obtain artificial bone material.
[0075] Step 4: irradiate and sterilize the bone material and package it.
[0076] Test Case
[0077] This test example is conducted on the bone material preferably obtained in Example 2. The inflammatory reaction and thrombosis experiments are tested by in vitro experiments, and the test standards are carried out in accordance with GB / T 16886.4-2022. The test results are shown in Table 4.
[0078] Table 4
[0079]
[0080] As shown in the results of Table 4, the bone material provided in Example 2 has excellent anti-inflammatory effects and does not cause thrombosis, and has the potential to be actually applied in a variety of medical scenarios.
[0081] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.
Claims
1. A method for preparing a bone material, characterized in that: The method comprises: Step 1, drying and crushing the flounder bones to obtain fish bone powder; Step 2: mixing the fish bone powder with water, inoculating Bacillus licheniformis ACCC10236, fermenting, sterilizing and drying to obtain fish bone fermented powder; Step 3: Mix the fish bone fermentation powder with polyvinyl alcohol and polylactic acid to obtain a mixed powder, homogenize under high pressure and then solidify at high temperature to obtain an artificial bone material.
2. The method according to claim 1, characterized in that The OD of the Bacillus licheniformis ACCC 10236 600 Is 3-4.
3. The method according to claim 2, characterized in that The inoculation amount of the Bacillus licheniformis ACCC 10236 is 1%-5%.
4. The method according to claim 1, characterized in that: The fermentation treatment conditions are 25°C-37°C, 200-300rpm, and the fermentation treatment is 1-2h.
5. The method according to claim 1, characterized in that The fish bone powder is mixed with water at a solid-liquid ratio of 1:(5-10).
6. The method according to claim 1, characterized in that The mass ratio of the fish bone fermentation powder, polyvinyl alcohol and polylactic acid is (10-20): (10-20): (1-5).
7. The method according to claim 1, characterized in that The mass ratio of the mixed powder to water is (1-5):
1.
8. The bone material prepared by the method according to any one of claims 1 to 7.
9. A product comprising the bone material according to claim 8.