A method for preparing mineralized collagen based on synchronous biomimetic calcification and silicification strategy
By preparing mineralized collagen raw materials through a simultaneous biomimetic calcification and silicification strategy, the problems of high preparation cost and numerous steps in existing technologies have been solved. This approach achieves uniform mineralization and enhanced bioactivity of collagen fibers, making it suitable for the preparation of bone repair materials.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- WEST ANHUI UNIV
- Filing Date
- 2026-03-13
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies for preparing mineralized adhesive raw materials suffer from high costs, numerous steps, long mineralization cycles, and difficulty in large-scale preparation. Furthermore, there is a lack of research on biomimetic silicification, which poses risks to bioactivity and safety.
By employing a simultaneous biomimetic calcification and silicification strategy, using low-cost silicates as the silicon source, and controlling pH value and constant temperature stirring conditions, rapid mineralization of collagen is achieved, thus preparing mineralized collagen raw materials that combine biomimetic calcification and silicification.
It achieves uniform mineralization of collagen fibers, enhances bioactivity and biocompatibility, promotes the preparation of bone repair materials, and has better biocompatibility and bone-promoting activity.
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Figure CN122141007A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of natural polymer compound processing and medical materials technology, and more specifically, to a method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy. Background Technology
[0002] Bone, as one of the most important organs in the human body, plays vital biological roles, such as supporting and protecting organs, participating in bodily movement, and storing minerals. Due to factors such as disease, natural disasters, traffic accidents, and population aging, humans frequently face the painful challenge of bone defects. Currently, the most widely used clinical treatments for bone defects are autologous bone and allogeneic bone transplantation. However, autologous bone transplantation faces challenges such as limited bone tissue sources and the risk of secondary trauma to patients, while allogeneic bone transplantation is prone to immune rejection, posing risks to the treatment of bone defects. Therefore, developing bone repair materials that mimic the structure and function of natural bone and possess good bioactivity is of significant research importance.
[0003] From a materials science perspective, bone is a composite material with a complex hierarchical structure, composed of both inorganic and organic components. The main inorganic component is hydroxyapatite (HAP), accounting for approximately 60-70%, while the main organic component is collagen, accounting for approximately 20-30%. In vivo, bone formation is closely related to the process of biomineralization, which is essentially the orderly deposition and crystallization of inorganic mineral ions on a collagen fiber template, assembling layer by layer to form mineralized collagen fibers. The mineralized collagen fibers formed during biomineralization are the basic building blocks of bone and accompany the entire bone formation process. Therefore, the in vitro synthesis of mineralized collagen using a biomimetic mineralization strategy as a bone repair material shows promising application prospects.
[0004] To date, inspired by this, researchers have extensively studied the mechanism of biomimetic collagen mineralization and the structure and properties of its building materials, laying the foundation for the in vitro synthesis of ideal bone repair materials. Early studies primarily focused on the compositional ratio of inorganic minerals and collagen in bone, preparing collagen / hydroxyapatite composites through simple blending. However, this approach failed to achieve effective integration of hydroxyapatite and collagen. In recent years, numerous researchers have prepared stable amorphous calcium phosphate precursor solutions using polymers or small-molecule organic acids. These solutions inhibit calcium phosphate nucleation and crystallization while inducing intracellular mineralization within collagen fibers, achieving a high degree of biomimicry in structure and composition to the mineralized material compared to natural bone. However, this technology suffers from drawbacks such as high cost of mineralization solutions, numerous mineralization steps, long mineralization cycles, and difficulty in large-scale production, thus limiting its practical application.
[0005] It is worth noting that the inorganic components of bone also contain other trace elements, such as magnesium, zinc, fluorine, and silicon, which are essential for bone formation and growth. Domestic and international research on the physicochemical properties and biological functions of silicon has confirmed that it is an essential trace element for the normal growth and development of tissues such as bone, cartilage, and tendons. However, current research on biomimetic collagen mineralization mainly focuses on the biomimetic calcification of collagen, with relatively little research on the biomimetic silicification of collagen. Researchers have used compounds such as choline chloride or polyacrylamide chloride to regulate the hydrolysis of tetraethyl orthosilicate to form a mineralizing solution, inducing biomimetic silicification or silicon-calcium hybridization of collagen. Biomimetic silicified or silicon-calcium hybrid collagen scaffolds prepared using this technique contain active silanol groups, which can promote osteogenic differentiation of mesenchymal stem cells, exhibiting good biological activity and showing excellent prospects for bone repair applications. However, there are also problems such as high precursor preparation costs, numerous mineralization steps, and potential risks arising from differences in composition compared to natural bone. Therefore, finding low-cost and safe silicon source alternatives and rapidly preparing mineralized adhesive raw materials using biomineralization is an urgent problem to be solved. Summary of the Invention
[0006] Therefore, this invention aims to propose a method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy. Using low-cost silicates as the silicon source, the method achieves rapid mineralization of collagen by simultaneously carrying out silicification and calcification, thereby overcoming the shortcomings of existing technologies.
[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy includes the following steps: Step 1: Dissolve collagen in buffer solution under ice-water bath conditions to prepare a neutral collagen solution with a collagen concentration of 0.01%-0.1%. Then add soluble calcium salt to the neutral collagen solution and mix thoroughly. Adjust the pH to 8-10 under ice-water bath conditions. Then stir under constant temperature conditions to prepare a calcium-containing collagen fiber suspension. Step 2: Under continuous constant temperature stirring conditions, the mixed solution of silicate and phosphate is added to the calcium-containing collagen fiber suspension to obtain the final solution. The pH is adjusted to 8-10, and the solution is continuously stirred at a constant temperature for 6-24 hours. Then the precipitate is separated and washed with water. After freeze drying, the mineralized gum raw material with both biomimetic calcification and silicification can be prepared. The constant temperature conditions are in the range of 28-37℃, the stirring time is 15-30 minutes, and the stirring speed is 300-1000 r / min. Furthermore, the collagen in step 1 is any one of fish skin type I collagen, bovine skin type I collagen, porcine skin type I collagen, and bovine Achilles tendon type I collagen.
[0008] Further, the buffer solution in step 1 is any one of tris(hydroxymethyl)aminomethane buffer, 2-morpholine ethanesulfonic acid buffer, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer, with a concentration of 0.01-0.05 mol / L and a pH value of 6-8.
[0009] Furthermore, the soluble calcium salt in step 1 is any one of anhydrous calcium chloride, calcium chloride dihydrate, and calcium nitrate.
[0010] Furthermore, in the calcium-containing collagen fiber suspension in step 1, the concentration of calcium ions is 0.005-0.015 mol / L.
[0011] Furthermore, the mixed solution of silicate and phosphate mentioned in step 2 is prepared by mixing silicate solution and phosphate solution at a volume ratio of 1:1.
[0012] Preferably, the silicate solution concentration is 0.05-0.1 mol / L, and it is prepared by dissolving either sodium silicate pentahydrate or sodium silicate nonahydrate in deionized water.
[0013] Preferably, the phosphate solution concentration is 0.05-0.2 mol / L, and it is prepared by dissolving any one of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate in deionized water.
[0014] Furthermore, before adding the silicate and phosphate mixed solution in step 2 to the collagen fiber suspension containing calcium ions, the pH value of the mixed solution needs to be adjusted to 9-11 using hydrochloric acid or sodium hydroxide with a concentration of 4-6 mol / L. Furthermore, the final solution in step 2 has a calcium ion to phosphate ion molar ratio of (1.5-2):1.
[0015] The beneficial effects of this invention are as follows: In the preparation method of this invention, soluble calcium salt is pre-mixed thoroughly with a low-concentration collagen solution, and the pH of the solution is adjusted to 8-10. This effectively promotes the ionization of carboxyl groups in the side chains of collagen molecules, thereby chelating with calcium ions and facilitating the adsorption of calcium ions. Then, under constant temperature stirring conditions, self-assembly is performed to prepare a dispersed calcium-containing collagen fiber suspension, which provides in-situ nucleation sites for calcification and silicification. Further addition of a silicate and phosphate mixed solution with a pH of 9-11 allows phosphate ions to bind with calcium ions on the collagen fibers and release H+. + This promotes the hydrolysis of silicate ions into orthosilicic acid and the formation of amorphous silicon dioxide. Simultaneously, due to H... +Consumption can promote further calcification, which is beneficial to the synthesis of hydroxyapatite. This allows biomimetic calcification and silicification to proceed in situ, synchronously, and rapidly, which is conducive to the uniform mineralization and mass production of collagen fibers.
[0016] The mineralized adhesive raw material prepared by the present invention through simultaneous biomimetic calcification and silicification can introduce silica and hydroxyapatite into collagen fibers. Both of these materials have good bioactivity. Biocompatibility and alkaline phosphatase experiments show that this mineralized adhesive raw material has better biocompatibility and osteopromoting activity than biomimetic calcified adhesive raw material, and can provide technical support for the preparation of biomimetic bone repair materials. Attached Figure Description
[0017] Figure 1 The images are scanning electron microscope (SEM) images of the mineralized adhesive raw materials prepared in Example 5 and Comparative Example 1, and the unmineralized adhesive raw material prepared in Comparative Example 2. The scale bar is 1 μm.
[0018] Figure 2 The energy dispersive spectroscopy (EDS) spectra of the mineralized adhesive raw materials prepared in Example 5 and Comparative Example 1, and the unmineralized adhesive raw materials prepared in Comparative Example 2.
[0019] Figure 3 The infrared spectra of the mineralized adhesive raw materials prepared in Example 5 and Comparative Example 1, and the unmineralized adhesive raw materials prepared in Comparative Example 2 are shown.
[0020] Figure 4 The X-ray diffraction patterns are those of the mineralized adhesive raw materials prepared in Example 5 and Comparative Example 1, and the unmineralized adhesive raw materials prepared in Comparative Example 2.
[0021] Figure 5 Thermogravimetric analysis curves of the mineralized adhesive raw materials prepared in Example 5 and Comparative Example 1 are shown.
[0022] Figure 6 The images show transmission electron microscopy (TEM) images (left) and selected area electron diffraction (SED) patterns (right) of the mineralized adhesive raw materials prepared in Example 5 and Comparative Example 1, with a scale bar of 200 nm.
[0023] Figure 7 The effects of the mineralized gum raw material extracts prepared in Example 5 and Comparative Example 1 on the cell proliferation of BMSCs were investigated.
[0024] Figure 8 The alkaline phosphatase activity results of BMSCs induced by the mineralized gel raw materials prepared in Example 5 and Comparative Example 1 at 3, 7 and 14 days of osteogenic differentiation are shown. Detailed Implementation
[0025] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.
[0026] Example 1 This embodiment proposes a method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy, including the following steps: Step 1: Dissolve collagen in buffer solution under ice-water bath conditions to prepare a neutral collagen solution with a collagen concentration of 0.05%. Then add soluble calcium salt to the neutral collagen solution and mix thoroughly. Adjust the pH to 9 under ice-water bath conditions and stir for a certain period of time under constant temperature conditions to obtain a calcium-containing collagen fiber suspension. The collagen is type I collagen from fish skin.
[0027] The buffer solution was tris(hydroxymethyl)aminomethane buffer with a concentration of 0.03 mol / L and a pH of 8.
[0028] The soluble calcium salt is anhydrous calcium chloride.
[0029] The concentration of calcium ions in the calcium-containing collagen fiber suspension is 0.010 mol / L.
[0030] Step 2: Under continuous constant temperature stirring, the mixed solution of silicate and phosphate is added to the calcium-containing collagen fiber suspension to obtain the final solution. The pH is adjusted to 9, and the solution is continuously stirred at a constant temperature for 15 hours. Then the precipitate is separated and washed with water. After freeze drying, the mineralized gum raw material with both biomimetic calcification and silicification can be prepared. The mixed solution of silicate and phosphate mentioned in step 2 is prepared by mixing silicate solution and phosphate solution at a volume ratio of 1:1.
[0031] The silicate solution has a concentration of 0.08 mol / L and is prepared by dissolving sodium silicate pentahydrate in deionized water.
[0032] The phosphate solution concentration was 0.12 mol / L, prepared by dissolving disodium hydrogen phosphate in deionized water.
[0033] Before adding the silicate and phosphate mixture to the collagen fiber suspension containing calcium ions, the pH of the mixture needs to be adjusted to 10 using 5 mol / L hydrochloric acid or sodium hydroxide. The final solution has a calcium ion to phosphate ion molar ratio of 1.7:1.
[0034] The constant temperature conditions are defined as follows: temperature range of 32℃, stirring time of 22 minutes, and stirring speed of 650 r / min.
[0035] In this invention, the dissolving, mixing, stirring, freeze-drying, etc., all follow the conventional principles of chemical processes, and those skilled in the art can perform specific operations based on common knowledge.
[0036] Example 2 The difference between this embodiment and Embodiment 1 is that: Prepare a neutral collagen solution with a collagen concentration of 0.01%; Add the soluble calcium salt to the neutral collagen solution and mix thoroughly, then adjust the pH to 9 under an ice-water bath. The collagen is bovine type I collagen.
[0037] The buffer solution was 2-morpholine ethanesulfonic acid buffer with a concentration of 0.01 mol / L and a pH of 7.
[0038] The soluble calcium salt is calcium chloride dihydrate.
[0039] The concentration of calcium ions in the calcium-containing collagen fiber suspension is 0.005 mol / L.
[0040] Under continuous constant temperature stirring conditions, a mixed solution of silicate and phosphate was added to a calcium-containing collagen fiber suspension to obtain the final solution. The pH was adjusted to 8, and the solution was stirred at a constant temperature for 6 hours. The silicate solution has a concentration of 0.05 mol / L and is prepared by dissolving sodium silicate nonahydrate in deionized water.
[0041] The phosphate solution concentration was 0.05 mol / L, prepared by dissolving sodium dihydrogen phosphate in deionized water.
[0042] Before adding the silicate and phosphate mixture to the collagen fiber suspension containing calcium ions, the pH of the mixture needs to be adjusted to 9 using 4 mol / L hydrochloric acid or sodium hydroxide. The final solution has a calcium ion to phosphate ion molar ratio of 1.5:1.
[0043] The constant temperature conditions were maintained at 37°C for 15 minutes and at a stirring speed of 300 r / min.
[0044] Example 3 The difference between this embodiment and Embodiment 1 is that: Prepare a neutral collagen solution with a collagen concentration of 0.1%; Add the soluble calcium salt to the neutral collagen solution and mix thoroughly, then adjust the pH to 10 in an ice-water bath. The collagen is type I collagen from pigskin.
[0045] The buffer solution was 4-hydroxyethylpiperazine ethanesulfonic acid buffer with a concentration of 0.05 mol / L and a pH of 6.
[0046] The soluble calcium salt is calcium nitrate.
[0047] The concentration of calcium ions in the calcium-containing collagen fiber suspension is 0.015 mol / L.
[0048] Under continuous constant temperature stirring conditions, a mixed solution of silicate and phosphate was added to a calcium-containing collagen fiber suspension to obtain the final solution. The pH was adjusted to 10, and the solution was continuously stirred at a constant temperature for 24 hours. The silicate solution concentration is 0.1 mol / L.
[0049] The phosphate solution concentration was 0.2 mol / L, prepared by dissolving dipotassium hydrogen phosphate in deionized water.
[0050] Before adding the silicate and phosphate mixture to the collagen fiber suspension containing calcium ions, the pH of the mixture needs to be adjusted to 11 using 6 mol / L hydrochloric acid or sodium hydroxide. The final solution has a calcium ion to phosphate ion molar ratio of 2:1.
[0051] The constant temperature conditions were maintained at 35℃, with a stirring time of 30 minutes and a stirring speed of 1000 r / min.
[0052] Example 4 The phosphate solution is prepared by dissolving potassium dihydrogen phosphate in deionized water.
[0053] Example 5 This embodiment proposes a method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy, including the following steps: (1) Dissolve tris(hydroxymethyl)aminomethane in deionized water to a concentration of 0.05 mol / L and adjust the pH to 7.4. Then, under ice-water bath conditions, dissolve bovine type I collagen sponge in tris(hydroxymethyl)aminomethane buffer to prepare a neutral collagen solution with a collagen concentration of 0.1%. Then add anhydrous calcium chloride and stir evenly to prepare a neutral collagen solution containing calcium ions. Adjust the pH to 9 under ice-water bath conditions, with a calcium ion concentration of 0.008 mol / L. Then, under 37°C water bath conditions, stir at a constant temperature of 600 r / min for 30 min to prepare a collagen fiber suspension containing calcium ions.
[0054] (2) Take appropriate amounts of sodium silicate pentahydrate and disodium hydrogen phosphate and dissolve them in deionized water to make the concentrations of sodium silicate and disodium hydrogen phosphate 0.08 mol / L and 0.096 mol / L, respectively. Stir the sodium silicate solution and disodium hydrogen phosphate solution at a volume ratio of 1:1 to obtain a mixed solution of silicate and phosphate.
[0055] (3) Adjust the pH of the mixed solution prepared in step (2) to 10.5 using 4 mol / L hydrochloric acid. Then, according to the molar ratio of calcium ions to phosphate ions of 1.67, add the silicate and phosphate mixed solution to the collagen fiber suspension containing calcium ions prepared in step (1). Stir at 600 r / min for 24 h in a water bath at 37°C. Separate the precipitate and wash it with water. Repeat this process 3 times. Freeze the precipitate in a freezer at -20°C and freeze dry it using a freeze dryer to obtain the mineralized gum raw material prepared based on the strategy of simultaneous biomimetic calcification and silicification.
[0056] (4) Sterilize the synchronous biomimetic calcification / silicification adhesive raw material obtained in step (3) using 10 kGy γ rays.
[0057] Comparative Example 1 A method for preparing mineralized collagen based on a biomimetic calcification strategy (1) Dissolve tris(hydroxymethyl)aminomethane in deionized water to a concentration of 0.05 mol / L and adjust the pH to 7.4. Then, under ice-water bath conditions, dissolve bovine type I collagen sponge in tris(hydroxymethyl)aminomethane buffer to prepare a neutral collagen solution with a collagen concentration of 0.1%. Then add anhydrous calcium chloride and stir evenly to prepare a neutral collagen solution containing calcium ions. Adjust the pH to 9 under ice-water bath conditions, with a calcium ion concentration of 0.008 mol / L. Then, under 37°C water bath conditions, stir at a constant temperature of 600 r / min for 30 min to prepare a collagen fiber suspension containing calcium ions.
[0058] (2) Dissolve an appropriate amount of disodium hydrogen phosphate in deionized water to make the concentration of disodium hydrogen phosphate 0.048 mol / L. Adjust the pH of the disodium hydrogen phosphate solution to 10.5 using 4 mol / L hydrochloric acid. Then, according to the molar ratio of calcium ions to phosphate ions of 1.67, add the disodium hydrogen phosphate solution to the collagen fiber suspension containing calcium ions prepared in step (1). Stir at a constant temperature of 600 r / min for 24 h in a water bath at 37℃. Separate the precipitate and wash it with water. Repeat this process 3 times. Freeze the precipitate in a freezer at -20℃ and freeze dry it using a freeze dryer to obtain the mineralized collagen raw material prepared based on the biomimetic calcification strategy.
[0059] (3) Sterilize the biomimetic calcification adhesive raw material obtained in step (2) using 10 kGy γ rays.
[0060] Comparative Example 2 A method for preparing unmineralized collagen (1) Dissolve tris(hydroxymethyl)aminomethane in deionized water to a concentration of 0.05 mol / L and adjust the pH to 7.4. Then, under ice-water bath conditions, dissolve bovine type I collagen sponge in tris(hydroxymethyl)aminomethane buffer to prepare a neutral collagen solution with a collagen concentration of 0.1%. Then add anhydrous calcium chloride and stir evenly to prepare a neutral collagen solution containing calcium ions. Adjust the pH to 9 under ice-water bath conditions, with a calcium ion concentration of 0.008 mol / L. Then, under 37°C water bath conditions, stir at a constant temperature of 600 r / min for 30 min to prepare a collagen fiber suspension containing calcium ions.
[0061] (2) The calcium-containing collagen fiber suspension prepared in step (1) was placed in a water bath at 37°C and stirred at a constant speed of 600 r / min for 24 h. The precipitate was separated and washed with water. This process was repeated 3 times. The precipitate was frozen in a freezer at -20°C and then freeze-dried using a freeze dryer to obtain unmineralized collagen raw material.
[0062] (3) Sterilize the unmineralized rubber raw material obtained in step (2) using 10 kGy γ rays.
[0063] Experiments were conducted to investigate the morphology, structure, composition, biocompatibility, and in vitro osteogenic differentiation-promoting capacity of the scaffold materials prepared in Example 5, Comparative Example 1, and Comparative Example 2. (1) Characterization experiments of morphology, structure and composition of scaffold materials The scaffold materials prepared in Example 5, Comparative Example 1, and Comparative Example 2 were cut into 1×1cm blocks, placed on a stage coated with conductive adhesive, and sputtered with gold. The surface microstructure of the scaffold materials was characterized using field emission scanning electron microscopy, and the elemental composition of the scaffold materials was analyzed using energy dispersive spectroscopy. The composition and crystal structure of the scaffold materials prepared in Example 5, Comparative Example 1, and Comparative Example 2 were analyzed using infrared spectroscopy and X-ray crystal diffraction. The content of organic and inorganic matter in the mineralized adhesive raw materials was analyzed by thermogravimetric analysis to determine the degree of mineralization. A small amount of the un-lyophilized mineralized adhesive raw materials prepared in Example 5 and Comparative Example 1 was dispersed in deionized water, and 3 μL was dropped onto an ultrathin copper mesh. The mineralization of the collagen fibers was analyzed by transmission electron microscopy and selected electron diffraction.
[0064] (2) Biocompatibility test of mineralized adhesive raw materials The mineralized gel raw materials prepared in Example 5 and Comparative Example 1 were added to DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin, and extracted for 24 h at 37°C and 5% CO2 to obtain scaffold extract. Bone marrow mesenchymal stem cell (BMSC) suspension in logarithmic growth phase was seeded into 96-well plates (20,000 cells / well) and cultured at 37°C and 5% CO2 for 24 h, after which the original culture medium was discarded. Then, 200 μL of scaffold extract was added, and the plates were incubated at 37°C and 5% CO2 for 1, 3, 5, and 7 days. For the blank control group, 200 μL of DMEM medium was used instead of the scaffold extract, and the remaining procedures were the same. Next, the well plates were removed at the specified time, and 10 μL of CCK-8 standard reagent was added under light-protected conditions. The plates were then incubated in a 5% CO2, 37°C incubator for 4 h, and the absorbance at 450 nm was measured using a microplate reader to analyze the cytotoxicity of the materials.
[0065] (3) In vitro osteogenic differentiation-promoting experiment of mineralized gel raw materials The mineralized gel raw materials prepared in Example 5 and Comparative Example 1 were used to prepare osteogenic induction medium containing the materials at a ratio of mineralized gel raw material mass to osteogenic induction medium volume of 1:2, serving as the experimental group; the blank control group used sterile PBS instead of mineralized gel raw materials. Logarithmic growth phase BMSCs were digested with trypsin and then added to DMEM medium, and evenly seeded into 6-well plates at a density of 1 million cells / well. The plates were incubated for 24 hours in a 5% CO2, 37°C incubator. Then, the original medium was removed, and 1 mL of osteogenic induction medium from the experimental group and blank control group was added. The plates were cultured for 3, 7, and 14 days, respectively, with the medium changed every 24 hours. At the set time points, the plates were removed, washed three times with sterile PBS, digested with 0.25% trypsin, centrifuged, and the precipitate was collected, mixed with sterile PBS, and used as the test sample. Then, 50 μL of alkaline phosphatase (ALP) kit buffer and 50 μL of matrix solution were added to a 96-well plate, followed by 5 μL of the sample to be tested. After incubation at 37°C for 15 min, 150 μL of chromogenic reagent was added, and the absorbance at 520 nm was measured using a microplate reader to analyze the in vitro osteogenic ability of the mineralized gel raw material.
[0066] Experimental results Figure 1The images show scanning electron microscope (SEM) images of the materials prepared in Example 5, Comparative Example 1, and Comparative Example 2. As can be seen from the images, the collagen fiber surface of Comparative Example 2 is smooth, without granular mineral accumulation, indicating no mineralization. Example 5 and Comparative Example 1 both exhibit typical microstructures of mineralized collagen fibers, with granular inorganic mineralization dispersed within and on the surface of the crisscrossing collagen fibers, indicating that mineralization has occurred in both. Furthermore, the inorganic mineral content on the surface of the material prepared in Example 5 is significantly higher than that in Comparative Example 1, demonstrating that the strategy of simultaneous biomimetic calcification and silicification can effectively improve the mineralization degree of mineralized collagen.
[0067] Figure 2 The figures show the energy dispersive spectroscopy (EDS) spectra of the materials prepared in Example 5, Comparative Example 1, and Comparative Example 2. In the figures, the EDS of the synchronous biomimetic calcified / silicified collagen prepared in Example 5 shows significant characteristic peaks for C, N, O, Ca, P, and Si elements, indicating that biomimetic calcification and biomimetic silicification occurred in Example 5. The biomimetic calcified collagen prepared in Comparative Example 1 shows significant characteristic peaks for C, N, O, Ca, and P elements, but no characteristic peak for Si, indicating that biomimetic calcification occurred. In contrast, only C, N, and O elements were detected in the collagen prepared in Comparative Example 2, indicating that no mineralization occurred.
[0068] Figure 3 The figures show the infrared spectra of the materials prepared in Example 5, Comparative Example 1, and Comparative Example 2. As can be seen from the figures, the materials prepared in Example 5, Comparative Example 1, and Comparative Example 2 exhibit characteristic absorption peaks of the typical amide A, B, I, II, and III bands of type I collagen near 3390–3400, 2940, 1648, 1542, and 1239 cm⁻¹. Furthermore, Example 5 and Comparative Example 1 show characteristic absorption peaks at 1029, 603, and 562 cm⁻¹ at phosphate groups, confirming the presence of hydroxyapatite and the occurrence of biomimetic calcification. Example 5 shows characteristic absorption peaks at 793 and 469 cm⁻¹ at silicate groups, indicating the occurrence of biomimetic silanization.
[0069] Figure 4 The X-ray diffraction patterns of the materials prepared in Example 5, Comparative Example 1, and Comparative Example 2 are shown in the figures. As can be seen from the figures, the synchronous biomimetic calcified / silicified collagen prepared in Example 5 and the biomimetic calcified collagen prepared in Comparative Example 1 exhibit peaks near 26°, 32.2°, 39.9°, 47°, and 50.8° (2θ). After comparison with the standard card of hydroxyapatite, these peaks are confirmed to be characteristic absorption peaks of the (002), (211), (112), (300), (310), and (222) crystal planes of hydroxyapatite. The broad peak near 32.2° (2θ) indicates a low-crystallinity hydroxyapatite component. The collagen prepared in Comparative Example 2 only exhibits a broad peak near 21° (2θ), which is attributed to the amorphous carbon absorption characteristic peak of collagen, indicating that no mineralization has occurred in Comparative Example 2.
[0070] Figure 5 The thermogravimetric analysis (TGA) diagrams of the mineralized collagen prepared in Example 5 and Comparative Example 1 are shown. As can be seen from the diagrams, the synchronous biomimetic calcified / silicified collagen prepared in Example 5 has a higher degree of mineralization compared to the biomimetic calcified collagen prepared in Example 2. At 800°C, the residual mass of inorganic minerals reaches 63.22%, which is close to the 60-70% inorganic content in natural bone tissue.
[0071] Figure 6 The images show transmission electron microscopy (TEM) images (left) and selected area electron diffraction (SED) patterns (right) of the mineralized collagen prepared in Example 5 and Comparative Example 1. As can be seen from the images, the black needle-like minerals in Example 5 and Comparative Example 2 grow along the collagen fiber axis and exhibit the characteristic diffraction rings of hydroxyapatite, indicating that biomimetic calcification occurred in both cases. Furthermore, in Example 5, in addition to the needle-like hydroxyapatite, spherical silica is uniformly dispersed on the fiber surface, coexisting with the hydroxyapatite. This indicates that simultaneous biomimetic calcification / silicification occurred in Example 5, and the simultaneous occurrence of biomimetic silicification does not significantly affect the formation of hydroxyapatite.
[0072] Figure 7 The results show the effect of mineralized collagen extracts prepared in Example 5 and Comparative Example 1 on BMSC cell proliferation. Clearly, the extract of synchronously biomimetic calcified / silicified collagen prepared in Example 5 promotes BMSC proliferation more effectively than the blank control group and the extract of biomimetic calcified collagen prepared in Comparative Example 1, exhibiting superior biocompatibility.
[0073] Figure 8 The figures show the alkaline phosphatase activity of BMSCs induced by mineralized collagen prepared in Example 5 and Comparative Example 1 at 3, 5, and 7 days. As can be seen from the figures, the synchronous biomimetic calcification / silicification collagen prepared in Example 5 significantly enhanced the alkaline phosphatase activity of BMSCs compared to the blank control group and the biomimetic calcified collagen prepared in Comparative Example 1. Furthermore, the promoting effect increased with time, demonstrating a superior ability to promote bone differentiation.
[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any modifications or improvements made without departing from the spirit and principle of the present invention are within the scope of protection claimed by the present invention.
Claims
1. A method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy, characterized in that: Includes the following steps: Step 1: Dissolve collagen in buffer solution under ice-water bath conditions to prepare a neutral collagen solution with a collagen concentration of 0.01%-0.1%. Then add soluble calcium salt to the neutral collagen solution and mix thoroughly. Adjust the pH to 8-10 under ice-water bath conditions. Then stir under constant temperature conditions to prepare a calcium-containing collagen fiber suspension. Step 2: Under continuous constant temperature stirring conditions, the mixed solution of silicate and phosphate is added to the calcium-containing collagen fiber suspension to obtain the final solution. The pH is adjusted to 8-10, and the mixture is continuously stirred at a constant temperature for 6-24 hours. Then the precipitate is separated and washed with water. After freeze drying, the mineralized gum raw material with both biomimetic calcification and silicification can be prepared. The constant temperature conditions are in the range of 28-37℃, the stirring time is 15-30 minutes, and the stirring speed is 300-1000 r / min.
2. The method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy according to claim 1, characterized in that: The collagen mentioned in step 1 is any one of fish skin type I collagen, bovine skin type I collagen, porcine skin type I collagen, and bovine Achilles tendon type I collagen.
3. The method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy according to claim 1, characterized in that: Step 1: The buffer solution is any one of tris(hydroxymethyl)aminomethane buffer, 2-morpholine ethanesulfonic acid buffer, or 4-hydroxyethylpiperazine ethanesulfonic acid buffer, with a concentration of 0.01-0.05 mol / L and a pH value of 6-8.
4. The method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy according to claim 1, characterized in that: The soluble calcium salt mentioned in step 1 is any one of anhydrous calcium chloride, calcium chloride dihydrate, or calcium nitrate.
5. The method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy according to claim 1, characterized in that: In the calcium-containing collagen fiber suspension described in step 1, the concentration of calcium ions is 0.005-0.015 mol / L.
6. The method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy according to claim 1, characterized in that: The mixed solution of silicate and phosphate mentioned in step 2 is prepared by mixing silicate solution and phosphate solution at a volume ratio of 1:
1.
7. The method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy according to claim 6, characterized in that: The silicate solution has a concentration of 0.05-0.1 mol / L and is prepared by dissolving either sodium silicate pentahydrate or sodium silicate nonahydrate in deionized water.
8. The method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy according to claim 6, characterized in that: The phosphate solution has a concentration of 0.05-0.2 mol / L and is prepared by dissolving any one of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, or potassium dihydrogen phosphate in deionized water.
9. The method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy according to claim 1, characterized in that: Before adding the silicate and phosphate mixture in step 2 to the collagen fiber suspension containing calcium ions, the pH of the mixture needs to be adjusted to 9-11 using hydrochloric acid or sodium hydroxide at a concentration of 4-6 mol / L.
10. The method for preparing mineralized collagen based on a simultaneous biomimetic calcification and silicification strategy according to claim 1, characterized in that: The molar ratio of calcium ions to phosphate ions in the final solution described in step 2 is (1.5-2):1.