Cellulose-hydroxyapatite composite for bone repair and method for preparing the same

Abstract

The application provides a cellulose-hydroxyapatite composite material for bone repair and a preparation method thereof, and belongs to the technical field of biomedical materials. The preparation method comprises the following steps: uniformly mixing acetic acid bacteria liquid and nutrient liquid, and standing and culturing at normal temperature and pressure to form a bacterial cellulose gel with a hierarchical structure; placing the bacterial cellulose gel in an alkali solution for heating and soaking; after impurities and residual bacteria are removed, washing the bacterial cellulose gel with deionized water until neutral to obtain a purified bacterial cellulose gel; dissolving collagen in an acetic acid solution to obtain a collagen solution; soaking the purified bacterial cellulose gel in the collagen solution to obtain bacterial cellulose loaded with collagen; and placing the bacterial cellulose loaded with collagen in a mineralization solution for in-situ mineralization and drying treatment to obtain the cellulose-hydroxyapatite composite material. The composite material provided by the application has excellent mechanical properties and good biocompatibility and bone regeneration capacity.

Description

Technical Field

[0001] At least one embodiment of the present invention relates to a biomedical material, and more particularly to a cellulose-hydroxyapatite composite material for bone repair and a method for preparing the same. Background Technology

[0002] Fractures and bone injuries caused by traffic accidents and osteoporosis are common problems in the field of bone repair. While bone, as a load-bearing tissue, has a certain self-repairing ability, it still relies on bone grafting surgery when faced with large-area defects. Bone grafting surgery mainly includes autologous transplantation, allogeneic transplantation, and synthetic grafting. To date, autologous transplantation remains the "gold standard" in bone repair, but synthetic grafting has also become an important transplantation method. Summary of the Invention

[0003] In view of this, the present invention provides a cellulose-hydroxyapatite composite material for bone repair and a method for preparing the same. The cellulose-hydroxyapatite composite material has excellent mechanical properties, good biocompatibility and bone repair ability, and has good application prospects in the field of biomedical materials.

[0004] According to one aspect of the present invention, a method for preparing a cellulose-hydroxyapatite composite material is provided, comprising:

[0005] The bacterial culture of Acetobacter xylinus (ATCC 23767, Latin name Gluconacetobacter xylinus) was mixed evenly with the nutrient solution and then incubated statically at room temperature and pressure to form a bacterial cellulose gel with a hierarchical structure.

[0006] The bacterial cellulose gel was placed in an alkaline solution and heated to remove impurities and residual bacteria. Then, it was rinsed with deionized water until neutral to obtain the purified bacterial cellulose gel.

[0007] Collagen was dissolved in acetic acid solution to obtain a collagen solution; the purified bacterial cellulose gel was immersed in the collagen solution to obtain collagen-loaded bacterial cellulose; and

[0008] Bacterial cellulose loaded with collagen was placed in a mineralization solution, mineralized in situ, and dried to obtain a cellulose-hydroxyapatite composite material.

[0009] According to another aspect of the present invention, a cellulose-hydroxyapatite composite material obtained by the above preparation method is provided, comprising:

[0010] The organic phase has a hierarchical structure and includes collagen-loaded bacterial cellulose.

[0011] The inorganic phase, including hydroxyapatite particles, is uniformly loaded in the organic phase.

[0012] According to another aspect of the present invention, the above-described cellulose-hydroxyapatite composite material is provided for use in the preparation of biomedical materials for bone repair.

[0013] According to the preparation method of cellulose-hydroxyapatite composite material provided in the above embodiments of the present invention, bacterial cellulose loaded with collagen is used as a biomineralization template (i.e., bacterial cellulose loaded with collagen is used as a carrier for subsequent hydroxyapatite mineralization deposition), and cellulose-hydroxyapatite composite material is obtained through in-situ mineralization to simulate the structure and composition of bone tissue, thereby meeting clinical needs and realizing bone tissue replacement.

[0014] According to the cellulose-hydroxyapatite composite material provided in the above embodiments of the present invention, the cellulose-hydroxyapatite composite material has a hierarchical organic phase and an inorganic phase uniformly dispersed in the organic phase, which makes the composite material have excellent mechanical properties, good biocompatibility and bone regeneration ability, and can simulate the structural components of bone tissue as a tissue surgical implant material or bone repair material for application in the field of biomedical materials. Attached Figure Description

[0015] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings of the embodiments will be briefly introduced below. Obviously, the drawings described below only relate to some embodiments of the present invention, and are not intended to limit the present invention.

[0016] Figure 1 A flowchart illustrating the preparation method of the cellulose-hydroxyapatite composite material provided in this embodiment of the invention;

[0017] Figure 2 This is a schematic diagram illustrating the preparation process of the cellulose-hydroxyapatite composite material provided in an embodiment of the present invention;

[0018] Figure 3 The images show scanning electron microscope (SEM) images of the purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1.

[0019] Figure 4 The X-ray diffraction (XRD) patterns are of the purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1.

[0020] Figures 5A-5BThe following are the FTIR spectra of the purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1.

[0021] Figure 6A The stress-strain curves are for the purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1.

[0022] Figure 6B Young's modulus diagrams of purified bacterial cellulose gel (BC) of Comparative Example 1, collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1 are shown.

[0023] Figure 7 Comparison of maximum fracture strength and Young's modulus of cellulose-hydroxyapatite composite materials (BC / Col / HAp) obtained in Examples 1 to 5;

[0024] Figure 8 Thermogravimetric curves of purified bacterial cellulose gel (BC) of Comparative Example 1, collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1 are shown.

[0025] Figure 9 The images show the cell compatibility test results of collagen-loaded bacterial cellulose (BC / Col) in Comparative Example 2 and cellulose-hydroxyapatite composite material (BC / Col / HAp) in Example 1 of this invention.

[0026] Figure 10 Microscopic images of BC / Col / Hap cells without bone marrow stem cell inoculation on day 7, bacterial cellulose loaded with collagen (BC / Col) of Comparative Example 2 of the present invention inoculated with bone marrow stem cells on day 7, and cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1 inoculated with bone marrow stem cells on day 7, stained with alizarin red. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and the accompanying drawings. However, this invention can be implemented in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided to make the invention thorough and complete, and to fully convey the scope of the invention to those skilled in the art. In the accompanying drawings, for clarity, the dimensions and relative dimensions of layers and regions may be exaggerated, and the same reference numerals denote the same elements throughout.

[0028] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The terms “comprising,” “including,” etc., as used herein indicate the presence of the stated features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0029] In related technologies, bone tissue is an organic-inorganic composite material with a natural multi-level structure. Its inorganic phase is mainly composed of hydroxyapatite, which endows it with excellent mechanical properties and provides support. Therefore, this invention constructs a high-performance bone repair material by simulating the unique structure and composition of natural bone tissue and employing an ultra-low energy consumption biomimetic mineralization strategy.

[0030] Figure 1 A flowchart illustrating the preparation method of the cellulose-hydroxyapatite composite material provided in this embodiment of the invention.

[0031] Figure 2 This is a schematic diagram illustrating the preparation process of the cellulose-hydroxyapatite composite material provided in an embodiment of the present invention.

[0032] According to an exemplary embodiment of the present invention, the present invention provides a method for preparing a cellulose-hydroxyapatite composite material, referring to... Figure 1 , Figure 2 As shown, it includes: operations S1 to S5.

[0033] In step S1, the Acetobacter xylinum bacterial culture is mixed evenly with the nutrient solution and then incubated statically at room temperature and pressure to form a bacterial cellulose gel (BC) with a hierarchical structure.

[0034] In some embodiments, the concentration (OD) of Acetobacter xylinum bacterial culture 600 The value is between 0.8 and 1.2.

[0035] In some embodiments, the nutrient solution comprises a mixture of white sugar and black tea.

[0036] In some embodiments, the volume ratio of Acetobacter xylinum bacterial culture to nutrient solution is (1~50):1, and the volume ratio can be, for example, 1:1, 10:1, 20:1, 40:1, 50:1, but is not limited to the values ​​mentioned above.

[0037] In step S2, the bacterial cellulose gel is placed in an alkaline solution and heated to remove impurities and residual bacteria. Then, it is rinsed with deionized water until neutral to obtain purified bacterial cellulose gel.

[0038] In some embodiments, the alkaline solution includes at least one of KOH, NaOH, and Na2CO3.

[0039] In some embodiments, the concentration of the alkali solution is 0.25 mol / L to 2.5 mol / L, for example, 0.25 mol / L, 0.5 mol / L, 1.0 mol / L, 1.5 mol / L, 2.0 mol / L, 2.5 mol / L, but is not limited to the values ​​listed.

[0040] In some embodiments, the heating temperature for immersing the bacterial cellulose gel in an alkaline solution is 60°C to 100°C, for example, 60°C, 80°C, or 100°C, but is not limited to the values ​​mentioned.

[0041] Control the alkali concentration and heating / soaking temperature within the aforementioned ranges to completely remove residual bacteria and sugars from the bacterial cellulose gel. The experimental phenomenon is that the bacterial cellulose gel changes from brownish-yellow to white; observing this color change confirms the treatment is complete. After alkali treatment, soak in water to remove any remaining alkali.

[0042] In step S3, collagen is dissolved in acetic acid solution to obtain collagen solution.

[0043] In some embodiments, the concentration of the acetic acid solution is 0.5 v / v% to 5.0 v / v%, for example, it can be 0.5 v / v%, 1.0 v / v%, 1.5 v / v%, 2.0 v / v%, 5.0 v / v, but is not limited to the values ​​mentioned.

[0044] In some embodiments, the mass percentage of collagen in the collagen solution is 0.1 wt% to 10.0 wt%, for example, 0.1 wt%, 0.5 wt%, 1.0 wt%, 5.0 wt%, or 10.0 wt%, but is not limited to the values ​​listed.

[0045] In step S4, the purified bacterial cellulose gel is immersed in a collagen solution to obtain collagen-loaded bacterial cellulose (BC / Col).

[0046] In some embodiments, the soaking time is 12h to 72h, for example, 12h, 20h, 24h, 48h, 72h, but is not limited to the values ​​mentioned.

[0047] In step S5, collagen-loaded bacterial cellulose is placed in a mineralization solution, mineralized in situ, and dried to obtain a cellulose-hydroxyapatite composite material.

[0048] In some embodiments, the mineralizing solution includes calcium chloride, 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), sodium chloride, potassium chloride, dipotassium hydrogen phosphate, and sodium silicate. The role of sodium chloride, potassium chloride, dipotassium hydrogen phosphate, and sodium silicate in the mineralizing solution is to balance the anion and cation ratio. 4-hydroxyethylpiperazine ethanesulfonic acid acts as a pH adjuster to regulate the pH value of the mineralizing solution.

[0049] In some embodiments, the concentration of calcium chloride is 1.0 mmol / L to 4.0 mmol / L, for example, 1.0 mmol / L, 2.0 mmol / L, 2.1 mmol / L, 2.2 mmol / L, 2.3 mmol / L, 2.4 mmol / L, 2.5 mmol / L, and 4.0 mmol / L, but not limited to these values. The concentration of calcium chloride is preferably 2.0 mmol / L, so that the Young's modulus of the cellulose-hydroxyapatite composite material formed by in-situ mineralization is greater than 15 GPa, to meet the clinical requirements of biomedical materials.

[0050] In some embodiments, the concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 5.0 mmol / L to 40.0 mmol / L, for example, 5.0 mmol / L, 10.0 mmol / L, 15.0 mmol / L, 20.0 mmol / L, or 40.0 mmol / L, but is not limited to the values ​​listed.

[0051] In some embodiments, the concentration of sodium chloride is 100.0 mmol / L to 300.0 mmol / L, for example, 100.0 mmol / L, 200.0 mmol / L, or 300.0 mmol / L, but is not limited to the values ​​mentioned.

[0052] In some embodiments, the concentration of potassium chloride is 0.5 mmol / L to 5.0 mmol / L, for example, 0.5 mmol / L, 1.0 mmol / L, 2.0 mmol / L, or 5.0 mmol / L, but is not limited to the values ​​listed.

[0053] In some embodiments, the concentration ratio of dipotassium hydrogen phosphate to calcium chloride is 1:2.

[0054] In some embodiments, the concentration of sodium silicate is 0.5 mmol / L to 20.0 mmol / L, for example, 0.5 mmol / L, 1.0 mmol / L, 5.0 mmol / L, 10.0 mmol / L, or 20.0 mmol / L, but is not limited to the values ​​listed.

[0055] In some embodiments, in-situ mineralization is carried out at 37°C for 12 to 72 hours. Drying treatment may be, for example, drying at room temperature for 24 hours.

[0056] According to the preparation method of cellulose-hydroxyapatite composite material provided in the above embodiments of the present invention, bacterial cellulose loaded with collagen is used as a biomineralization template (i.e., bacterial cellulose loaded with collagen is used as a carrier for subsequent hydroxyapatite mineralization deposition), and cellulose-hydroxyapatite composite material is obtained through in-situ mineralization to simulate the structure and composition of bone tissue, thereby meeting clinical needs and realizing bone tissue replacement.

[0057] According to an exemplary embodiment of the present invention, the present invention provides a cellulose-hydroxyapatite composite material, comprising:

[0058] An organic phase with a hierarchical structure, comprising bacterial cellulose loaded with collagen; and an inorganic phase comprising hydroxyapatite particles uniformly loaded in the organic phase.

[0059] In embodiments of the present invention, the Young's modulus of the cellulose-hydroxyapatite composite material is 9.2 GPa to 17.8 GPa. More preferably, by adjusting the preparation process parameters of the cellulose-hydroxyapatite composite material, the Young's modulus of the cellulose-hydroxyapatite composite material is controlled to be 15 GPa to 17.8 GPa, thereby meeting the clinical needs of biomaterials.

[0060] In embodiments of the present invention, the maximum tensile strength of the cellulose-hydroxyapatite composite material is 440.9 MPa to 852.8 MPa.

[0061] In the embodiments of the present invention, the cellulose-hydroxyapatite composite material has a hierarchical organic phase and an inorganic phase uniformly dispersed in the organic phase, which makes the composite material have excellent mechanical properties, good biocompatibility and bone regeneration ability. It can simulate the structural components of bone tissue and be used as a tissue surgical implant material or bone repair material in the field of biomedical materials.

[0062] According to an exemplary embodiment of the present invention, the present invention provides the application of the cellulose-hydroxyapatite composite material as described above in the preparation of biomedical materials for bone repair.

[0063] The following illustrative description illustrates a cellulose-hydroxyapatite composite material for bone repair and its preparation method. It should be noted that this illustration is merely a specific embodiment of the present invention and does not limit the scope of protection of the present invention.

[0064] Comparative Example 1

[0065] A purified bacterial cellulose gel (BC) was prepared using the following method:

[0066] Preparation of bacterial cellulose gel:

[0067] Add 200 mL of Acetobacter xylinum bacterial culture (ATCC 23767, OD) 600 A bacterial cellulose gel with a value of 1.2 (Latin name Gluconacetobacter xylinus) was placed in a container with 100 mL of nutrient solution (9.0 wt% white sugar + 0.9 wt% black tea) and incubated at room temperature and pressure. After 3 days, a bacterial cellulose gel with a hierarchical structure was formed.

[0068] Preparation of purified bacterial cellulose gel:

[0069] The obtained bacterial cellulose gel block was taken out and placed in a 0.75 mol / L sodium hydroxide solution and soaked at 80°C. After removing impurities and residual bacteria, it was rinsed with deionized water until neutral to obtain purified bacterial cellulose gel (BC).

[0070] Comparative Example 2

[0071] A collagen-loaded bacterial cellulose (BC / Col) was prepared using the following method:

[0072] Preparation of collagen solution:

[0073] Acetic acid was added to deionized water to form an acetic acid solution of 1.5 v / v%, and then collagen (purchased from Shanghai Yuanye Biotechnology Co., Ltd., molecular weight 2000~3000) was dissolved in it until completely dissolved to obtain a collagen solution containing 2.0 wt% collagen.

[0074] The purified bacterial cellulose gel (BC) from Comparative Example 1 was immersed in the above collagen solution for 48 hours, and then washed with deionized water to obtain collagen-loaded bacterial cellulose (BC / Col).

[0075] Example 1

[0076] S1. Preparation of bacterial cellulose:

[0077] Add 200 mL of Acetobacter xylinum bacterial culture (ATCC 23767, OD) 600 A bacterial cellulose (BC) gel with a value of 1.2 (Latin name Gluconacetobacter xylinus) was placed in a container with 100 mL of nutrient solution (9.0 wt% white sugar + 0.9 wt% black tea) and incubated at room temperature and pressure. After 3 days, a bacterial cellulose (BC) gel with a hierarchical structure was formed.

[0078] S2, Bacterial cellulose purification:

[0079] The obtained bacterial cellulose gel block was taken out and placed in a 0.75 mol / L sodium hydroxide solution and soaked at 80°C. After removing impurities and residual bacteria, it was rinsed with deionized water until neutral to obtain purified bacterial cellulose gel.

[0080] S3. Preparation of collagen solution:

[0081] Acetic acid was added to deionized water to form a 1.5 v / v% acetic acid solution. Collagen was then dissolved in the solution until it was completely dissolved, resulting in a collagen solution containing 2.0 wt% collagen.

[0082] S4. The purified bacterial cellulose gel was soaked in the above collagen solution for 48 hours, and then washed with deionized water to obtain collagen-loaded bacterial cellulose (BC / Col).

[0083] S5. Preparation of cellulose-hydroxyapatite composite material:

[0084] Bacterial cellulose (BC / Col) loaded with collagen was placed in a mineralization solution and mineralized at 37°C for 18 h, and then dried at room temperature for 24 h. The mineralization solution was prepared by dissolving 4-hydroxyethylpiperazine ethanesulfonic acid, anhydrous calcium chloride, sodium chloride, potassium chloride, dipotassium hydrogen phosphate, and sodium silicate in deionized water, with concentrations of 5.0 mmol / L, 2.0 mmol / L, 100.0 mmol / L, 1.0 mmol / L, 1.0 mmol / L, and 3.0 mmol / L, respectively, to obtain a cellulose-hydroxyapatite composite material.

[0085] Example 2

[0086] Cellulose-hydroxyapatite composite material was prepared using the same method as in Example 1, except that the concentration of anhydrous calcium chloride was replaced with 1.0 mmol / L and the concentration of dipotassium hydrogen phosphate was replaced with 0.5 mmol / L.

[0087] Example 3

[0088] Cellulose-hydroxyapatite composite material was prepared using the same method as in Example 1, except that the concentration of anhydrous calcium chloride was replaced with 2.5 mmol / L and the concentration of dipotassium hydrogen phosphate was replaced with 1.25 mmol / L.

[0089] Example 4

[0090] Cellulose-hydroxyapatite composite material was prepared using the same method as in Example 1, except that the concentration of anhydrous calcium chloride was replaced with 3.0 mmol / L and the concentration of dipotassium hydrogen phosphate was replaced with 1.5 mmol / L.

[0091] Example 5

[0092] Cellulose-hydroxyapatite composite material was prepared using the same method as in Example 1, except that the concentration of anhydrous calcium chloride was replaced with 4.0 mmol / L and the concentration of dipotassium hydrogen phosphate was replaced with 2.0 mmol / L.

[0093] The purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1 were subjected to scanning electron microscopy (SEM) analysis. The results are shown in [link to SEM]. Figure 3 As shown.

[0094] like Figure 3 As shown, no inorganic phase was observed in the purified bacterial cellulose gel (BC) of Comparative Example 1 and the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2. The cellulose-hydroxyapatite composite material of Example 1 contained inorganic phase particles inside the hierarchical organic phase (as shown in the rectangular box). This indicates that mineralization was achieved inside the collagen-loaded bacterial cellulose (BC / Col) in Example 1, and the preparation of the cellulose-hydroxyapatite composite material (BC / Col / HAp) was successfully realized.

[0095] The purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) prepared in Comparative Example 2, and the cellulose-hydroxyapatite composite material (BC / Col / HAp) prepared in Example 1 were subjected to XRD tests. The test results are shown in [link to XRD analysis]. Figure 4 As shown.

[0096] like Figure 4As shown, the diffraction patterns of the purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1 show peaks at 14.1° (100), 16.4° (010), and 22.3° (110) with diffraction angles of 2θ, corresponding to the characteristic peaks of bacterial cellulose gel (BC), which is a typical type I cellulose crystal structure. The diffraction pattern of the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1 shows peaks at 25.8° (002), 31.7° (211), and 32.2° (300) with diffraction angles of 2θ, corresponding to the characteristic peaks of hydroxyapatite (HAp). This indicates that the cellulose-hydroxyapatite composite material (BC / Col / HAp) was successfully prepared in Example 1.

[0097] Infrared spectroscopy was performed on the purified bacterial cellulose (BC) gel of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite material of Example 1. The test results are shown in [reference missing]. Figures 5A-5B As shown, where Figure 5A The scanning range is 4000~400cm. -1 Infrared spectrum, Figure 5B for Figure 5A Enlarged view of a section (1200~600cm) -1 ).

[0098] like Figure 5A , Figure 5B As shown, the infrared spectra of the cellulose-hydroxyapatite composite material in Example 1 are at 1164 cm⁻¹. -1 1108cm -1 1058cm -1 Characteristic peaks of bacterial cellulose (BC) appear; at 1547 cm⁻¹ -1 The presence of a characteristic peak of amide II (NH bending), corresponding to the characteristic peak of collagen, indicates successful collagen loading; at 610 cm⁻¹ -1 The corresponding PO4 appears 3- The V4 characteristic peak indicates that the cellulose-hydroxyapatite composite material was successfully prepared in Example 1.

[0099] Stress-strain tests and Young's modulus tests were performed on the purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite materials (BC / Col / HAp) obtained after drying for 24 hours in Examples 1-5, respectively. The test results are shown in [reference missing]. Figures 6A-6B , Figure 7As shown. The stress-strain test was conducted using a Mark-10 EAM303 tensile testing machine. The test humidity was 25%, the temperature was 30℃, the tensile speed was 0.5 mm / min, the sample size was 4 mm, and the composite material was subjected to axial tension in a dry state (after drying at room temperature for 24 hours). The Young's modulus was obtained based on the stress-strain ratio within 1% of the strain.

[0100] Figure 6A The stress-strain curves are for the purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1.

[0101] Figure 6B The Young's modulus diagrams are for the purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1.

[0102] like Figure 6A , Figure 6B As shown, the purified bacterial cellulose gel (BC) of Comparative Example 1 has a Young's modulus of 9.4 GPa and a maximum tensile strength of 157.7 MPa. The collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2 has a Young's modulus of 8.9 GPa and a maximum tensile strength of 370.9 MPa.

[0103] The cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1 achieved a Young's modulus of 17.8 GPa and a maximum tensile strength of 852.8 MPa. Compared to the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1 exhibited a 100% increase in Young's modulus, demonstrating superior mechanical properties, and its Young's modulus met the clinical requirements for cortical bone (15-20 GPa).

[0104] Figure 7 The graph shows a comparison of the maximum fracture strength and Young's modulus of the cellulose-hydroxyapatite composite materials (BC / Col / HAp) obtained in Examples 1 to 5.

[0105] Among the tests, the Young's modulus of the cellulose-hydroxyapatite composite material in Example 2 was 9.2 GPa, and the maximum tensile strength was 463.9 MPa. The Young's modulus of the cellulose-hydroxyapatite composite material in Example 3 was 13.8 GPa, and the maximum tensile strength was 638 MPa. The Young's modulus of the cellulose-hydroxyapatite composite material in Example 4 was 11.6 GPa, and the maximum tensile strength was 578.1 MPa. The Young's modulus of the cellulose-hydroxyapatite composite material in Example 5 was 10.6 GPa, and the maximum tensile strength was 440.9 MPa.

[0106] See Figure 7 As shown, when the concentration of calcium ions in the mineralization solution is 2.0 mmol / L, the prepared cellulose-hydroxyapatite composite material (BC / Col / HAp) has a Young's modulus greater than 15 GPa, which meets the clinical requirements for biomedical materials.

[0107] The thermogravimetric analysis (TG) curves of the purified bacterial cellulose gel (BC) of Comparative Example 1, the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2, and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1 at 30–800 °C are shown below. Figure 8 As shown.

[0108] refer to Figure 8 As shown, the remaining masses of BC and BC / Col after combustion were 20.4% and 14.76%, respectively, which also proves the successful introduction of collagen, leading to a further reduction in residual mass after high-temperature treatment. Since the decomposition temperature of HAp is above 1200℃, the final residual mass of BC / Col / HAp is as high as 31.08%, indicating that the content of the inorganic phase HAp in BC / Col / HAp is 16.32%.

[0109] Biocompatibility tests were conducted on the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2 and the cellulose-hydroxyapatite composite material of Example 1. Bone marrow stem cells were seeded onto the surface of the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2 and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1, respectively, and then cultured in a cell culture incubator. The growth status of cells on the membrane surface was observed by laser confocal microscopy (CLSM).

[0110] Figure 9 The images show the cell compatibility test results of collagen-loaded bacterial cellulose (BC / Col) in Comparative Example 2 and cellulose-hydroxyapatite composite material (BC / Col / HAp) in Example 1 of this invention.

[0111] refer to Figure 9As shown, the results indicate that after 7 days of cell culture, the surface of the collagen-loaded bacterial cellulose (BC / Col) in Comparative Example 2 had only a small number of cells, while the surface of the cellulose-hydroxyapatite composite material (BC / Col / HAp) in Example 1 had a large number of cells. Compared to BC / Col, BC / Col / Hap is more conducive to the adhesion and survival of bone marrow mesenchymal stem cells. This demonstrates that the cellulose-hydroxyapatite composite material formed after mineralization has good biocompatibility.

[0112] Bone repair capacity assessment tests were conducted on the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2 and the cellulose-hydroxyapatite composite material of Example 1. Bone marrow stem cells were seeded on the surface of the collagen-loaded bacterial cellulose (BC / Col) of Comparative Example 2 and the cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1, respectively, and then cultured in a cell culture incubator. The degree of differentiation of bone marrow stem cells on the material surface was observed under a microscope.

[0113] Figure 10 Microscopic images of BC / Col / Hap cells without bone marrow stem cell inoculation on day 7, bacterial cellulose loaded with collagen (BC / Col) of Comparative Example 2 of the present invention inoculated with bone marrow stem cells on day 7, and cellulose-hydroxyapatite composite material (BC / Col / HAp) of Example 1 inoculated with bone marrow stem cells on day 7, stained with alizarin red.

[0114] The results showed that, compared to the staining micrographs of collagen-loaded bacterial cellulose (BC / Col) in Comparative Example 2, bone marrow stem cells formed a large number of red complexes (calcium nodules) on the surface of the cellulose-hydroxyapatite composite material (BC / Col / HAp) in Example 1. Compared to the staining micrographs of BC / Col / Hap without bone marrow stem cell inoculation (i.e., the control group), the staining pattern of the staining micrographs in Example 1 changed, verifying that the cellulose-hydroxyapatite composite material (BC / Col / HAp) in Example 1 has a certain inducing effect on osteogenic differentiation, exhibiting excellent bone regeneration capacity and showing good application prospects in the field of bone repair.

[0115] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a cellulose-hydroxyapatite composite material, characterized in that, include: The Acetobacter xylinum bacterial culture was mixed evenly with the nutrient solution and then incubated statically at room temperature and pressure to form a bacterial cellulose gel with a hierarchical structure. The bacterial cellulose gel was placed in an alkaline solution and heated to remove impurities and residual bacteria. Then, it was rinsed with deionized water until neutral to obtain the purified bacterial cellulose gel. Collagen is dissolved in acetic acid solution to obtain collagen solution; The purified bacterial cellulose gel was immersed in the collagen solution to obtain collagen-loaded bacterial cellulose. as well as The collagen-loaded bacterial cellulose was placed in a mineralization solution, mineralized in situ, and dried to obtain the cellulose-hydroxyapatite composite material.

2. The preparation method according to claim 1, characterized in that, The volume ratio of the Acetobacter xylinum bacterial solution to the nutrient solution is (1~50):

1.

3. The preparation method according to claim 1, characterized in that, The alkaline solution includes at least one of KOH, NaOH, and Na2CO3; The concentration of the alkaline solution is 0.25 mol / L to 2.5 mol / L; The heating temperature for soaking in alkaline solution is 60℃~100℃.

4. The preparation method according to claim 1, characterized in that, The concentration of the acetic acid solution is 0.5 v / v% to 5.0 v / v%. The collagen content in the collagen solution is 0.1 wt% to 10.0 wt% by mass. The purified bacterial cellulose gel was soaked in the collagen solution for 12 to 72 hours.

5. The preparation method according to claim 1, characterized in that, The mineralizing solution includes calcium chloride, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium chloride, potassium chloride, dipotassium hydrogen phosphate, and sodium silicate.

6. The preparation method according to claim 5, characterized in that, The concentration of calcium chloride is 1.0 mmol / L to 4.0 mmol / L; The ratio of the concentration of dipotassium hydrogen phosphate to the concentration of calcium chloride is 1:2; The concentration of 4-hydroxyethylpiperazine ethanesulfonic acid is 5.0 mmol / L to 40.0 mmol / L; The concentration of sodium chloride is 100.0 mmol / L to 300.0 mmol / L; The concentration of potassium chloride is 0.5 mmol / L to 5.0 mmol / L; The concentration of sodium silicate ranges from 0.5 mmol / L to 20.0 mmol / L; The in-situ mineralization was carried out at 37°C for 12-72 hours.

7. A cellulose-hydroxyapatite composite material obtained by the preparation method according to any one of claims 1 to 6, characterized in that, include: The organic phase has a hierarchical structure and includes collagen-loaded bacterial cellulose. An inorganic phase, including hydroxyapatite particles, is uniformly loaded in the organic phase.

8. The cellulose-hydroxyapatite composite material according to claim 7, characterized in that, The Young's modulus of the cellulose-hydroxyapatite composite material is 9.2 GPa to 17.8 GPa.

9. The cellulose-hydroxyapatite composite material according to claim 7, characterized in that, The maximum tensile strength of the cellulose-hydroxyapatite composite material is 440.9 MPa to 852.8 MPa.

10. The use of a cellulose-hydroxyapatite composite material as described in any one of claims 7 to 9 in the preparation of biomedical materials for bone repair.

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