Collagen biomimetic mineralization scaffold and preparation method thereof
A collagen-inspired mineralization scaffold was prepared by using a synergistic composite material of ColI, nHA, and nPB. This method addresses the shortcomings of existing bone repair scaffolds in terms of mechanical properties, bioactivity, and biocompatibility, and achieves excellent biomechanical support and osteogenic promotion during the bone defect repair process.
Patent Information
- Application Number
- CN202511074603.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-05-29
- Filing Date
- 2025-08-01
- Publication Date
- 2025-12-16
AI Technical Summary
Existing bone repair scaffolds suffer from insufficient mechanical properties, limited bioactivity regulation capabilities, poor biocompatibility, and limited biological functions, resulting in low osteogenic repair efficiency and making it difficult to meet the repair needs of large-area or complex bone defects.
A collagen-inspired biomimetic mineralization scaffold was prepared using a synergistic composite material of ColI, nHA, and nPB via a hanging drop crosslinking method to form an nHA coating. Combined with treatment with calcium chloride and sodium dihydrogen phosphate, the mechanical strength and biocompatibility of the scaffold were improved, osteoblast proliferation and differentiation were promoted, and immune rejection was reduced.
It provides excellent biomechanical support during the bone defect repair process, promotes new bone formation, reduces immune rejection, improves osteoblast activity and survival rate, and enhances repair quality and efficiency.
Smart Images
Figure CN121130176A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to bone tissue engineering and regenerative medicine, and in particular to a collagen biomimetic mineralization scaffold and its preparation method. Background Technology
[0002] Current clinical bone defect repair mainly relies on traditional methods such as autologous bone transplantation, allogeneic bone transplantation, and bone stimulation techniques. However, these strategies still have significant limitations: while autologous transplantation has the advantage of biocompatibility, it faces the risks of insufficient donor bone volume and secondary damage, making it difficult to meet the needs of repairing large bone defects; although allogeneic transplantation can expand the sources of bone, problems such as immune rejection and low bone integration efficiency still limit its application effectiveness; bone stimulation techniques, represented by minimally invasive drilling, can initiate the repair process by inducing stem cell migration, but the newly formed bone tissue often exhibits structural disorder and insufficient mechanical properties, making it difficult to achieve functional regeneration of load-bearing bone. In addition, existing technologies have poor adaptability to complex anatomical morphologies and lack the ability to synergistically regulate multiple factors in the bone regeneration microenvironment. Based on this, bone tissue engineering technology provides an innovative solution for bone defect repair by integrating biomimetic scaffold design and bioactive factor delivery systems. Modern manufacturing technology has achieved precise construction of multi-scale structural scaffolds, whose mechanical properties can be graded to simulate the biomechanical characteristics of natural bone tissue. However, existing bone repair scaffolds generally have the following drawbacks: (1) Insufficient mechanical properties make it difficult to effectively withstand the complex and variable stress environment of the osteochondral interface. After implantation, the scaffold is prone to deformation or breakage, resulting in unsatisfactory repair effects; (2) Most traditional bone repair scaffolds mainly provide physical support. Although they can fill the bone defect area to a certain extent, they lack effective bioactivity regulation capabilities and are difficult to actively promote the adhesion, proliferation and differentiation of osteoblasts. After implantation, the scaffold itself cannot improve the local microenvironment or effectively induce new bone formation, resulting in a slow osteogenic repair process and limited repair quality and efficiency, which seriously affects the regeneration effect of large-area or complex bone defects; (3) After implantation, traditional bone repair scaffold materials are prone to immune rejection due to limited biocompatibility, leading to local inflammation or other adverse reactions, which in turn affects the bone repair effect and the patient's recovery process; (4) Traditional bone repair scaffolds often have the problem of single biological function, mainly relying on the passive support of the material itself and lacking the active promotion effect on bone tissue regeneration, resulting in low osteogenic repair efficiency and ultimately affecting the healing quality and speed of bone defects. Summary of the Invention
[0003] Purpose of the invention: The purpose of this invention is to provide a collagen biomimetic mineralization scaffold with excellent mechanical properties and excellent cell proliferation-promoting properties.
[0004] Another object of the present invention is to provide a method for preparing the above-mentioned stent.
[0005] Technical solution: The collagen biomimetic mineralization scaffold of the present invention uses ColI and nPB as the framework and nHA as the surface coating. The mass ratio of ColI and nPB is 9-30 mg / ug. The framework is formed by the hanging drop crosslinking method, and the nHA is deposited on the surface of the framework by the alternating immersion method.
[0006] The method for preparing the stent according to the present invention includes the following steps:
[0007] (1) Add anhydrous ethanol to the mixture of EDC and NHS, and after dissolution, a crosslinking agent is obtained;
[0008] (2) Add the crosslinking agent to the mixed powder of Col I and nPB, shake it to form a suspension, then add water dropwise until Col I powder and nPB powder are initially crosslinked, let it stand to complete the crosslinking, and obtain Col I-nPB scaffold.
[0009] (3) Add calcium chloride to Tris-HCl buffer to obtain calcium chloride working solution; wash the ColI-nPB scaffold obtained in step (2) and place it in calcium chloride working solution for incubation to obtain ColI-nPB-calcium chloride scaffold.
[0010] (4) After absorbing the surface moisture of the Col I-nPB-calcium chloride scaffold, place it in a sodium dihydrogen phosphate solution and incubate.
[0011] (5) Repeat steps (3)-(4) at least twice, wash, and obtain a collagen biomimetic mineralized scaffold.
[0012] Preferably, the mass ratio of EDC to NHS in step (1) is 0.2-1:1, and more preferably 0.5:1.
[0013] Preferably, the ratio of EDC to anhydrous ethanol added in step (1) is 4-20 g / L, and more preferably 10 g / L.
[0014] Preferably, in step (2), the ratio of the amount of Col I powder to the crosslinking agent added is 2-6 g / L, and more preferably 6 g / L.
[0015] Preferably, in step (3), the concentration of the Tris-HCl buffer solution is 0.03-0.06M, and more preferably 0.05M.
[0016] Preferably, in step (3), the ratio of calcium chloride to Tris-HCl buffer added is 20-25 g / L, and more preferably 22 g / L.
[0017] Preferably, in step (4), the concentration of the sodium dihydrogen phosphate solution is 15-19 g / L, more preferably 17.14 g / L.
[0018] Preferably, in steps (3) and (4), the incubation temperature is 30-40°C, and more preferably 37°C.
[0019] Preferably, in steps (3) and (4), the incubation time is 0.8-1.5h, and more preferably 1h.
[0020] Invention Principle: This invention utilizes a synergistic composite of three materials—collagen I (Col I), nano-hydroxyapatite (nHA), and nano-Prussian blue (nPB)—combined with a hanging drop crosslinking method to prepare a collagen-inspired biomimetic mineralization scaffold. This is the first time a hanging drop crosslinking method has been employed. During the crosslinking process, ultrapure water is added dropwise to prevent uneven crosslinking. This dropwise addition effectively controls the degree of crosslinking, which is judged by the amount of flocculent material produced, ensuring incomplete or even impossible crosslinking. During the mineralization process of the Col I-nPB scaffold, an nHA coating forms on its surface. The introduction of nHA enhances the mechanical strength and osteoconductivity of the biomimetic mineralization scaffold, and helps promote osteogenic differentiation and new bone formation of bone marrow mesenchymal stem cells (BMSCs). Col I, an important component of the natural bone matrix, effectively promotes osteoblast attachment and proliferation while gradually degrading during tissue repair. nHA, an inorganic mineral component of bone tissue, possesses excellent osteoinduction and mineralization-promoting functions, accelerating new bone formation. nPB, a functional component, plays a crucial immunomodulatory role. nPB exhibits excellent antioxidant properties, effectively scavenging local oxygen free radicals, reducing oxidative stress in the implantation area, thereby inhibiting the release of pro-inflammatory cytokines and lowering the immune system's rejection response to the implanted scaffold. The innovative pendant drop cross-linking molding technology allows for precise control of the scaffold's three-dimensional structure and pore morphology under mild conditions, mimicking the complex microenvironment of natural bone tissue and enhancing cell penetration, angiogenesis, and nutrient exchange, thus achieving superior biomechanical support and bioactivity guidance during bone defect repair.
[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The present invention provides a new process for preparing collagen biomimetic mineralization scaffolds, and innovatively adopts the hanging drop cross-linking molding technology to achieve better biomechanical support in the process of bone defect repair; (2) In the mineralization process of ColI-nPB scaffold, repeated soaking with calcium chloride and sodium dihydrogen phosphate forms an nHA coating on its surface. The introduction of nHA is beneficial to enhance the mechanical strength and osteoconductivity of the biomimetic mineralization scaffold; (3) Compared with traditional allogeneic scaffold materials, the biomimetic mineralization scaffold obtained by the present invention shows obvious advantages in biocompatibility, significantly reduces the immune rejection reaction after implantation, and promotes excellent cell proliferation performance; (4) Through the microenvironment regulation enabled by nPB, the scaffold can effectively reduce local inflammatory response after implantation, improve the activity and survival rate of osteoblasts, improve the implantation success rate and patient acceptance, reduce the incidence of postoperative complications, and further ensure the safety, stability and long-term efficacy of the osteogenic repair process. Attached Figure Description
[0022] Figure 1 Mechanical property test curves of the biomimetic mineralization scaffolds prepared in Examples 1-3 and Comparative Example 2;
[0023] Figure 2 The images show the cell proliferation test results of the biomimetic mineralized scaffolds prepared in Examples 1-3 and Comparative Examples 1-2. Detailed Implementation
[0024] The technical solution of the present invention will be further described below with reference to the embodiments.
[0025] Example 1
[0026] The collagen biomimetic mineralization scaffold of the present invention uses ColI and nPB as the framework and nHA as the surface coating. The mass ratio of ColI to nPB is 28.57 mg / ug. The framework is formed by a hanging drop crosslinking method, and the nHA is deposited on the surface of the framework by an alternating immersion method.
[0027] The preparation method of the collagen biomimetic mineralization scaffold of the present invention includes the following steps:
[0028] (1) Preparation of crosslinking agent: Weigh 0.5g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1g of N-hydroxysuccinimide (NHS), then add 50mL of anhydrous ethanol, and shake thoroughly at room temperature with the aid of a shaker until completely dissolved to obtain the crosslinking agent.
[0029] (2) Crosslinking: The prepared crosslinking agent was added to a mixture of 0.3g Col I powder and 10.5ug nPB powder and shaken thoroughly in a shaker until it became a suspension. Then, ultrapure water was added dropwise as soon as possible until Col I powder and nPB powder began to crosslink into flocculents. After standing at 37°C for 36 hours, the crosslinking was completed and Col I-nPB scaffold was obtained.
[0030] (3) nHA coating treatment:
[0031] Accurately weigh 0.394 g Tris-HCl and place it in a beaker. Add 50 mL of ultrapure water and stir with a magnetic stirrer until fully dissolved. Measure the pH with a pH meter and adjust the pH of the solution to 7.4 with 1 mol / L HCl and NaOH to obtain a 0.05 M Tris-HCl buffer solution. Add 1.1 g of anhydrous calcium chloride granules and stir until completely dissolved to obtain a calcium chloride working solution.
[0032] The Col I-nPB scaffold obtained in step (2) was fully immersed in purified water to remove surface residues. Then, it was carefully picked up and its surface excess moisture was gently absorbed with sterile absorbent paper. It was then placed in the calcium chloride working solution, sealed, and incubated at 37°C for 1 hour to obtain the Col I-nPB-calcium chloride scaffold.
[0033] (4) After incubation in calcium chloride solution, remove the Col I-nPB-calcium chloride scaffold, gently blot dry its surface moisture with sterile absorbent paper, and then place it in 17.14 g / L sodium dihydrogen phosphate solution, seal it, and incubate it at 37°C for 1 hour.
[0034] (5) Repeat steps (3)-(4) three times. After multiple treatments with calcium chloride and sodium dihydrogen phosphate, it is beneficial to form a stable nHA deposition coating. After the treatment is completed, rinse repeatedly with a large amount of purified water to remove residual ions and obtain a collagen biomimetic mineralization scaffold.
[0035] After the surface of the stent is blotted dry, it should be dried and stored at room temperature. Before use, the dried stent can be immersed in 75% ethanol for sterilization, then rinsed 5 times with ultrapure water and dried at room temperature for later use.
[0036] Example 2
[0037] The similarities between this embodiment and Embodiment 1 will not be repeated here. The difference is that the amount of ColI powder added in step (2) is 0.1g.
[0038] Example 3
[0039] The similarities between this embodiment and Embodiment 1 will not be repeated here. The difference is that the amount of ColI powder added in step (2) is 0.5g.
[0040] Comparative Example 1
[0041] The similarities between this comparative example and Example 1 will not be repeated here. The difference is that the amount of ColI powder added in step (2) is 0g.
[0042] Comparative Example 2
[0043] The similarities between this comparative example and Example 1 will not be repeated here. The difference is that the amount of ColI powder added in step (2) is 0.05g.
[0044] The mechanical properties of the biomimetic mineralized scaffolds obtained in each experiment were tested using a Shanghai Hengyi M231PRO testing machine, and their stress-strain curves for compressive strength were obtained, as shown below. Figure 1 As shown. The specific test method is as follows: The sample is placed in the center of the compression plate and compressed at a constant strain rate of 1 mm / min. The stress-strain diagram is plotted using the force and deformation data, and the compressive modulus (E) is calculated, which is the slope of the stress-strain curve segment.
[0045] As shown in the figure, the compressive strength of the biomimetic mineralization scaffold increases with the increase of strain. When the strain is greater than 0.4%, the compressive strength of the biomimetic mineralization scaffold increases within a certain range with the increase of ColI addition (Examples 1-3, Comparative Example 2) when the amount of ColI addition increases to a certain extent (Example 3), the compressive strength of the scaffold decreases.
[0046] The cell proliferation-promoting properties of the biomimetic mineralized scaffolds obtained in each experiment were tested, using BMSCs as the cells. The structures are as follows: Figure 2 As shown. The Thermo SkyHigh multilayer microplate was used, and the specific testing method is as follows: CCK-8 experiment: Cells were seeded at a density of 10*3 / well in a 96-well plate. After 24 hours, the culture medium was changed, and 100 μL of the corresponding group gel extraction solution was added to each well for treatment. At a specific time point, the culture medium was removed and aspirated, and low-glucose complete culture medium containing 10% CCK-8 was added. Cell-free wells were set up as the control group. After incubation in the dark for 1 hour, the cells were removed, and the absorbance value was detected at 450 nm using a microplate reader. The higher the absorbance value, the more viable cells there are.
[0047] As shown in the figure, within a certain range, the cell survival rate first increases and then decreases with the increase of Col I addition. When the amount of Col I addition increases to a certain extent (Example 3), the cell survival rate begins to decline.
Claims
1. A collagen biomimetic mineralization scaffold, characterized in that, The collagen biomimetic mineralization scaffold uses ColI and nPB as the framework and nHA as the surface coating. The mass ratio of ColI to nPB is 9-30 mg / ug. The framework is formed by a hanging drop crosslinking method, and the nHA is deposited on the surface of the framework by an alternating immersion method.
2. A method for preparing a collagen biomimetic mineralization scaffold as described in claim 1, characterized in that, Includes the following steps: (1) Add anhydrous ethanol to the mixture of EDC and NHS, and after dissolution, a crosslinking agent is obtained; (2) Add the crosslinking agent to the mixed powder of Col I and nPB, shake it to form a suspension, then add water dropwise until Col I powder and nPB powder are initially crosslinked, let it stand to complete the crosslinking, and obtain Col I-nPB scaffold. (3) Add calcium chloride to Tris-HCl buffer to obtain calcium chloride working solution; wash the Col I-nPB scaffold obtained in step (2) and place it in calcium chloride working solution for incubation to obtain Col I-nPB-calcium chloride scaffold. (4) After absorbing the surface moisture of the Col I-nPB-calcium chloride scaffold, place it in a sodium dihydrogen phosphate solution and incubate. (5) Repeat steps (3)-(4) at least twice, wash, and obtain a collagen biomimetic mineralized scaffold.
3. The preparation method according to claim 1, characterized in that, The mass ratio of EDC to NHS in step (1) is 0.2-1:
1.
4. The preparation method according to claim 1, characterized in that, The ratio of EDC to anhydrous ethanol added in step (1) is 4-20 g / L.
5. The preparation method according to claim 1, characterized in that, In step (2), the ratio of the amount of Col I powder to the crosslinking agent added is 2-6 g / L.
6. The preparation method according to claim 1, characterized in that, In step (3), the concentration of the Tris-HCl buffer solution is 0.03-0.06M.
7. The preparation method according to claim 1, characterized in that, In step (3), the ratio of calcium chloride to Tris-HCl buffer added is 20-25 g / L.
8. The preparation method according to claim 1, characterized in that, In step (4), the concentration of the sodium dihydrogen phosphate solution is 15-19 g / L.
9. The preparation method according to claim 1, characterized in that, In steps (3) and (4), the incubation temperature is 30-40℃.
10. The preparation method according to claim 1, characterized in that, In steps (3) and (4), the incubation time is 0.8-1.5h.