Multi-modal response bone tissue engineering material, preparation method and application
By preparing multimodal responsive bone tissue engineering materials, the problem of poor bone healing in diabetic fractures was solved, mechanical fixation, ion release and photothermal therapy were achieved, and fracture repair and regeneration were significantly promoted.
Patent Information
- Application Number
- CN202510777027.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-11
- Publication Date
- 2025-09-05
AI Technical Summary
Existing technologies have the problems of poor bone healing, high trauma and high infection risk in treating diabetic fractures. Conventional bone cement and autologous allogeneic bone transplants are non-degradable, cytotoxic, donor scarcity and immune rejection.
The multimodal responsive bone tissue engineering material is made of methacrylated gelatin, methacrylated sodium hyaluronate, 2,4,6-trimethylbenzoyl lithium phosphate, michelene and nanohydroxyapatite through UV cross-linking and curing. The material has mechanical support, ion release and photothermal therapy capabilities, promoting fracture repair.
This material significantly accelerated bone regeneration in diabetic fractures, enhanced mechanical properties, promoted vascularization and osteogenic differentiation, achieved stable fixation of fractures and regulation of the microenvironment, and improved fracture repair effects.
Smart Images
Figure CN120586159A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical tissue engineering, and in particular to a multimodal response bone tissue engineering material, a preparation method and an application thereof. Background Art
[0002] Diabetic fractures are caused by microvascular lesions, oxidative stress, and chronic inflammation induced by long-term hyperglycemia, leading to local angiogenesis disorders and inhibition of osteoblast function, often manifesting as delayed healing or non-union. Conventional clinical treatment is often accompanied by problems such as poor bone healing, high trauma, and high risk of infection. Although the application of bone cement can provide immediate mechanical fixation, it has problems such as non-degradability, cytotoxicity, and lack of biological activity. Autologous and allogeneic bone transplantation are limited by donor scarcity and immune rejection. Therefore, there is an urgent need to develop a material that can stabilize the fracture site, restore bone integrity, and regulate the microenvironment to promote bone regeneration and repair. Summary of the Invention
[0003] To develop a material that can stabilize fracture sites, restore bone integrity, and regulate the microenvironment to promote bone regeneration and repair, the present invention provides a multimodal bone tissue engineering material, preparation method, and application. The multimodal bone tissue engineering material provided by the present invention can achieve mechanical fixation, ion release, and photothermal therapy, effectively promoting fracture repair.
[0004] The present invention provides a multimodal responsive bone tissue engineering material, wherein the multimodal responsive bone tissue engineering material is prepared by mixing methacrylated gelatin, methacrylated sodium hyaluronate, 2,4,6-trimethylbenzoyl lithium phosphate, michelene and nano-hydroxyapatite and then cross-linking and curing the mixture through ultraviolet light. The usage ratio of the methacrylated gelatin, methacrylated sodium hyaluronate, 2,4,6-trimethylbenzoyl lithium phosphate, michelene and nano-hydroxyapatite is 0.1 g~1 g: 0.01 g~0.1 g: 0.0025 g~0.025 g: 0.05 mL~0.5 mL: 0.01 g~0.1 g.
[0005] The multimodal bone tissue engineering material provided by the present invention features michalcone, which responds to near-infrared photothermal light. Nanohydroxyapatite releases multiple ions, including calcium, phosphorus, magnesium, and strontium, synergistically promoting vascularization and osteogenic differentiation. Methacrylamide gelatin, sodium methacrylated hyaluronate, and lithium 2,4,6-trimethylbenzoyl phosphate provide mechanical support. Validation in an animal model of diabetic fractures demonstrates that the multimodal bone tissue engineering material provided by the present invention can significantly accelerate bone regeneration.
[0006] Furthermore, the pore size of the multimodal response bone tissue engineering material is 100 μm to 300 μm.
[0007] The present invention also provides a method for preparing the multimodal responsive bone tissue engineering material, which specifically comprises the following steps: dissolving methacrylated gelatin, methacrylated sodium hyaluronate, and 2,4,6-trimethylbenzoyl lithium phosphate in deionized water to form a methacrylated gelatin / methacrylated sodium hyaluronate solution; Adding the michelene solution to the methacrylated gelatin / methacrylated sodium hyaluronate solution and mixing the mixture to obtain a michelene-methacrylated gelatin / methacrylated sodium hyaluronate solution; Adding nano-hydroxyapatite to the michelene-methacrylated gelatin / methacrylated hyaluronate sodium solution and mixing the mixture to obtain a nano-hydroxyapatite-michelene-methacrylated gelatin / methacrylated hyaluronate sodium solution; The michelene-methacrylated gelatin / methacrylated sodium hyaluronate solution is irradiated with ultraviolet light to complete cross-linking and curing, thereby obtaining the multimodal responsive bone tissue engineering material.
[0008] Furthermore, the ultraviolet light has a wavelength of 365 nm and an irradiation time of 30 s.
[0009] Furthermore, the usage ratio of the methacrylated gelatin, methacrylated sodium hyaluronate, 2,4,6-trimethylbenzoyl lithium phosphate, michelene and nano-hydroxyapatite is 0.1 g~1 g: 0.01 g~0.1 g: 0.0025 g~0.025 g: 0.05 mL~0.5 mL: 0.01 g~0.1 g.
[0010] The present invention also provides an application of the multimodal response bone tissue engineering material in repairing diabetic fractures.
[0011] Furthermore, the application subjects are db / db diabetic mice with bone defects.
[0012] Furthermore, the multimodal responsive bone tissue engineering material is used to promote bone regeneration and increase bone density.
[0013] The multimodal responsive bone tissue engineering material provided by the present invention can achieve mechanical fixation, ion release, and photothermal therapy, effectively promoting the repair of fractures.
[0014] Compared with the prior art, the present invention has the following beneficial effects: The multimodal response bone tissue engineering material prepared by the present invention adds the two-dimensional layer structure of MXene and nHAP, which enhances the mechanical properties of bone cement and can meet the requirements of load-bearing bone fixation.
[0015] In the multimodal responsive bone tissue engineering material prepared by the present invention, nHAP-mediated ion release is combined with near-infrared-triggered PTT. MXene and nHAP synergistically enhance mechanical properties and multi-ion release, combined with NIR-PTT to regulate the fracture microenvironment, thereby accelerating bone regeneration and repair.
[0016] The preparation method of the multimodal responsive bone tissue engineering material in the present invention is relatively simple and easy to operate. It does not require in vitro cell inoculation. By implanting the material at the tibial fracture site of db / db diabetic mice, it can significantly promote vascularization and bone regeneration, greatly improving the fracture repair effect. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0018] Figure 1 Preparation process, scanning electron microscopy, and energy spectrum of nHAP; In the figure, A is the nHAP preparation process; B is the scanning electron microscopy image of nHAP; C is an element contained in nHAP.
[0019] Figure 2 The appearance and scanning electron microscope images of different groups of materials are shown; In the figure, A shows the appearance of different groups of materials; B is the scanning electron microscope image of different groups of materials.
[0020] Figure 3 The mechanical properties, ion release and degradation diagrams of different groups of materials are shown; In the figure, A is the stress-strain diagram of different groups of materials; B is the cumulative ion release of different groups of materials; C is the degradation rate of different groups of materials.
[0021] Figure 4 These are the results of cell live-death tests on different groups of materials.
[0022] Figure 5 Shown are the results of cell scratch tests for different groups of materials.
[0023] Figure 6 These are micro-CT scan images of fractures repaired with different groups of materials 4 and 8 weeks later.
[0024] Figure 7These are H&E staining and Masson staining images of different groups of materials after fracture repair for 4 and 8 weeks.
[0025] Figure 8 Bone mineral density (BMD) and bone volume / tissue volume (BV / TV) were analyzed 8 weeks after fracture repair in different groups of materials; In the figure, A shows the bone mineral density (BMD) analysis of the different groups of materials after 8 weeks of fracture repair; B is the bone volume / tissue volume (BV / TV) analysis of different groups of materials after 8 weeks of fracture repair. DETAILED DESCRIPTION
[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.
[0027] Example 1: Preparation of bone tissue engineering materials with different groups of multimodal responses Nanohydroxyapatite (nHAP) was prepared by water bath method. The preparation process is shown in Figure 1 The specific preparation steps are as follows: first, 30 g of oyster shell powder and 23.7 g of diammonium hydrogen phosphate were dissolved in 500 mL of ddH2O, and the mixture was evenly dispersed in the solution by magnetic stirring. Then, the pH value of the solution was adjusted to 9 with NaOH, and the reaction was stirred continuously at a constant temperature of 90°C for 12 h to promote the formation of calcium ions (Ca 2+ ) and phosphate ions (PO4 3- ) undergoes precipitation reaction in the solution to generate hydroxyapatite (Ca 10 After the reaction, the product was centrifuged and washed three times with anhydrous ethanol and then ddH2O, respectively. Finally, it was dried in a 60°C vacuum oven for 16 hours to obtain nanohydroxyapatite (nHAP). Before use, nHAP was placed in a clean bench and sterilized with ultraviolet light for 12 hours.
[0028] Preparation of methacrylated gelatin / sodium methacrylated hyaluronate solution: In a clean hood, add 1 g of methacrylated gelatin (GelMA), 100 mg of sodium methacrylated hyaluronate (HAMA), and 25 mg of lithium 2,4,6-trimethylbenzoyl phosphate (LAP) to 9.5 mL of sterile deionized water. Stir continuously in a water bath (200 rpm / min, 37°C) to fully dissolve the mixture to obtain the methacrylated gelatin / sodium methacrylated hyaluronate (GelMA / HAMA) solution.
[0029] Preparation of michelene-methacrylated gelatin / sodium methacrylated hyaluronate solution: Dilute a 10 mg / mL michelene (MXene) stock solution with deionized water to a 1 mg / mL MXene solution. Add 500 μL of the MXene solution to the prepared GelMA / HAMA solution to obtain a michelene-methacrylated gelatin / sodium methacrylated hyaluronate (MXene-GelMA / HAMA) solution.
[0030] Preparation of 0.5% nanohydroxyapatite-michene-methacrylated gelatin / sodium methacrylated hyaluronate solution: 0.05 g of nHAP was added to the prepared MXene-GelMA / HAMA solution and mixed to obtain a 0.5% nanohydroxyapatite-michene-methacrylated gelatin / sodium methacrylated hyaluronate (0.5% nHAP-MXene-GelMA / HAMA) solution.
[0031] Preparation of 1% nanohydroxyapatite-michene-methacryloyl gelatin / sodium methacrylated hyaluronate solution: 0.1 g of nHAP was added to the prepared MXene-GelMA / HAMA solution and mixed to obtain a 1% nanohydroxyapatite-michene-methacryloyl gelatin / sodium methacrylated hyaluronate (1% nHAP-MXene-GelMA / HAMA) solution.
[0032] Preparation of 2% nanohydroxyapatite-michene-methacrylated gelatin / sodium methacrylated hyaluronate solution: 0.2 g of nHAP was added to the prepared MXene-GelMA / HAMA solution and mixed to obtain a 2% nanohydroxyapatite-michene-methacrylated gelatin / sodium methacrylated hyaluronate (2% nHAP-MXene-GelMA / HAMA) solution.
[0033] Scanning electron microscopy analysis ( Figure 1 B) shows that the surface of nHAP material has a petal-like structure, and the energy spectrum analysis ( Figure 1C) shows that elements such as magnesium and strontium are distributed in the nHAP material.
[0034] The obtained GelMA / HAMA solution, MXene-GelMA / HAMA solution, 0.5% nHAP-MXene-GelMA / HAMA solution, 1% nHAP-MXene-GelMA / HAMA solution, and 2% nHAP-MXene-GelMA / HAMA solution were irradiated under 365 nm ultraviolet light for 30 s. The photoinitiator MXene generated active free radicals under ultraviolet excitation, which triggered the covalent cross-linking of the double bonds of GelMA / HAMA to form a three-dimensional network structure. After cross-linking and curing, the materials were freeze-dried to obtain different groups of multimodal response bone tissue engineering materials ( Figure 2 A). The pore sizes of different groups of materials were analyzed using scanning electron microscopy. The results showed that the pore sizes of the bone tissue engineering materials with different multimodal responses were approximately 100 μm to 300 μm ( Figure 2 B), where 1% nHAP-MXene-GelMA / HAMA material exhibited better mechanical properties ( Figure 3 The results of cell viability tests showed that the biocompatibility of each material group was good, and the cell proliferation phenomenon of the 1% nHAP-MXene-GelMA / HAMA material was more obvious ( Figure 4 ); The results of the cell scratch test showed that the 1% nHAP-MXene-GelMA / HAMA material exhibited good ability to promote endothelial cell migration and tube formation ( Figure 5 ).
[0035] Example 2: Application of multimodal responsive bone tissue engineering materials in promoting diabetic fracture repair Eight-week-old, SPF-grade db / db diabetic male mice weighing approximately 38 g and with blood glucose levels between 17 and 21 mmol / L were selected. After isoflurane anesthesia, the surgical area was depilated and prepared, disinfected three times with iodine tincture. A sterile surgical drape was then placed. Using an autoclaved scalpel and forceps, a longitudinal incision was made along the medial side of the tibia, along the skin and subcutaneous tissue. The muscle was bluntly dissected to expose the tibial shaft. A 1 mm bone defect was created in the mid-tibial region using an electric oscillating saw to simulate a fracture. The defect was then flushed with saline. The resulting GelMA / HAMA solution, MXene-GelMA / HAMA solution, and 1% nHAP-MXene-GelMA / HAMA solution were injected into the fracture site, respectively. After curing by irradiation with 365 nm UV light for 30 seconds, the incisions were sutured and externally fixed with a custom splint. This resulted in the treatment groups with different multimodal bone tissue engineering materials. The control group did not receive injections and was sutured directly after surgery. PTT was performed using an 808 nm near-infrared laser (NIR) at an irradiance of 500 mW / cm² for 10 minutes each on days 3, 7, 14, 21, 28, 35, 42, 49, and 56 after surgery. Four and eight weeks after implantation, whole tibiae were harvested from mice treated with the multimodal responsive bone tissue engineering materials in the different groups and fixed in 4% paraformaldehyde.
[0036] After sampling, micro-computed tomography was used to reconstruct the method to evaluate the regeneration of new bone tissue after treatment with bone tissue engineering materials of different groups with multimodal responses. The specific implementation method was as follows: all tibial samples were scanned by micro-CT instrument, and the results are shown in Figure 6 After micro-CT scanning, H&E staining and Masson staining were used to evaluate the regeneration of new bone tissue after treatment with bone tissue engineering materials in different groups with multimodal responses. The results are shown in Figure 7 The bone mineral density (BMD) and bone volume / tissue volume (BV / TV) values in the micro-CT scan results 8 weeks after implantation were analyzed by software. The analysis results are shown in Figure 8 . Combined Figure 6 、 Figure 7 、 Figure 8 It can be seen that the BMD and BV / TV values of the 1% nHAP-MXene-GelMA / HAMA-NIR group were significantly higher than those of the other groups, and a satisfactory bone regeneration effect was achieved compared with the other groups, which indicates that the multimodal responsive bone tissue engineering material provided by the present invention can significantly enhance bone regeneration.
[0037] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.
[0038] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.
Claims
1. A multimodal responsive bone tissue engineering material, characterized in that: The multimodal responsive bone tissue engineering material is prepared by mixing methacrylated gelatin, methacrylated sodium hyaluronate, 2,4,6-trimethylbenzoyl lithium phosphate, michelene and nano-hydroxyapatite and then cross-linking and curing them with ultraviolet light. The usage ratio of the methacrylated gelatin, methacrylated sodium hyaluronate, 2,4,6-trimethylbenzoyl lithium phosphate, michelene and nano-hydroxyapatite is 0.1 g~1 g: 0.01 g~0.1 g: 0.0025 g~0.025 g: 0.05 mL~0.5 mL: 0.01 g~0.1 g.
2. The multimodal responsive bone tissue engineering material according to claim 1, characterized in that: The pore size of the multimodal responsive bone tissue engineering material is 100 μm to 300 μm.
3. A method for preparing the multimodal response bone tissue engineering material according to claim 1, characterized in that: The specific steps include: dissolving methacrylated gelatin, methacrylated sodium hyaluronate, and 2,4,6-trimethylbenzoyl lithium phosphate in deionized water to form a methacrylated gelatin / methacrylated sodium hyaluronate solution; Adding the michelene solution to the methacrylated gelatin / methacrylated sodium hyaluronate solution and mixing the mixture to obtain a michelene-methacrylated gelatin / methacrylated sodium hyaluronate solution; Adding nano-hydroxyapatite to the michelene-methacrylated gelatin / methacrylated hyaluronate sodium solution and mixing the mixture to obtain a nano-hydroxyapatite-michelene-methacrylated gelatin / methacrylated hyaluronate sodium solution; The nano-hydroxyapatite-michaelene-methacrylated gelatin / methacrylated sodium hyaluronate solution is irradiated with ultraviolet light to complete cross-linking and curing, thereby obtaining the multimodal responsive bone tissue engineering material.
4. The method for preparing a multimodal response bone tissue engineering material according to claim 3, characterized in that: The wavelength of the ultraviolet light is 365 nm, and the irradiation time is 30 s.
5. The method for preparing a multimodal response bone tissue engineering material according to claim 3, characterized in that: The usage ratio of the methacrylated gelatin, methacrylated sodium hyaluronate, 2,4,6-trimethylbenzoyl lithium phosphate, michelene and nano-hydroxyapatite is 0.1 g~1 g: 0.01 g~0.1 g: 0.0025 g~0.025 g: 0.05 mL~0.5 mL: 0.01 g~0.1 g.
6. Use of the multimodal responsive bone tissue engineering material according to claim 1 in repairing diabetic fractures.
7. Use of the multimodal responsive bone tissue engineering material in diabetic fracture repair according to claim 6, characterized in that: The application subjects are db / db diabetic mice with bone defects.
8. Use of the multimodal responsive bone tissue engineering material in diabetic fracture repair according to claim 6, characterized in that: The multimodal responsive bone tissue engineering material is used to promote bone regeneration and increase bone density.