Preparation method and application of a biomimetic multi-level spherical mineralized collagen scaffold

Abstract

The present invention discloses a preparation method and application of a bionic multi-stage spherical mineralized collagen scaffold. The collagen mineralization area of ​​the prepared mineralized collagen scaffold presents a specific distribution, and a bionic multi-stage spherical structure appears, which can highly simulate the mineralization pattern of the mineralization front of the organism. The mineralized collagen scaffold is formed by inducing the mineralization of type I collagen within collagen fibers by a specific mineralization system prepared in vitro, and presents a spherical structural unit after multi-stage assembly. The above-mentioned specific mineralization system is a calcium-phosphate precursor system stabilized by egg yolk phosphoprotein. The bionic multi-stage spherical mineralized collagen scaffold material of the present invention has excellent bone induction properties and unique mechanical properties, and is non-cytotoxic, and can be used as a bone tissue engineering scaffold material to promote bone defect repair.

Description

Technical Field

[0001] The present invention belongs to the field of bone tissue engineering and biomaterials, and particularly relates to a preparation method and application of a bionic multi-stage spherical mineralized collagen scaffold. Background Art

[0002] Bone defects caused by trauma, tumors, and infection are common and pose significant risks. In clinical treatment, autologous bone transplantation is the gold standard for bone defect repair, but issues such as limited availability and donor site damage limit its clinical application. While synthetic bone graft materials are widely available, they still cannot match the superior performance of natural bone tissue. This is due to their poor biomimetic properties, failing to accurately simulate the hierarchical structure of natural bone tissue and even failing to achieve functional biomimetic results.

[0003] Early artificial bone defect repair scaffold materials were mostly made by mechanically mixing hydroxyapatite, calcium salt particles, and collagen composites through physical means. For example, patent CN100341588C discloses a method for preparing porous collagen-composite nanohydroxyapatite artificial bone. While the resulting material can form a porous structure and possess certain mechanical properties, it is far from comparable to the stiffness and toughness of natural bone tissue. On the one hand, the basic building block of natural bone tissue is intrafibrillated mineralized collagen. Its microstructure is formed by collagen fibers acting as organic molecular templates, inducing the orderly deposition of amorphous calcium and phosphate precursors on or within their surfaces. Based on this, some scholars have constructed intrafibrous mineralized collagen scaffolds to simulate the nanostructure of bone tissue and used them for bone repair. For example, patent CN103830775B discloses a method for preparing a high-strength collagen-based artificial bone repair material. By self-assembling nano-calcium phosphate and collagen molecules, a bone repair material close to the strength of human cortical bone is formed. Although the material has excellent mechanical properties, in actual use, it often faces problems such as increased brittleness, inability to perfectly fit the surgical area, poor operability, and even causing bone nonunion in the defect area. Therefore, there are studies on the use of flexible scaffold materials for bone defect repair. For example, patent CN101234216B discloses a biomimetic glycosylated mineralized collagen / glycosylated chitosan / PLGA composite bone tissue engineering scaffold and its preparation method. Although the material has a three-dimensional mesh structure that is conducive to cell adhesion, proliferation, and differentiation on the surface of the scaffold material, this type of implant material often faces problems such as insufficient mineralization rate, poor biomimetic structure, and slow osteogenesis.

[0004] The mineralization front of natural bone tissue contains a spherical mineralized structure, with both mineralized and unmineralized areas. This spherical mineralized structure facilitates the transition from soft collagen to hard mineralized collagen. It also facilitates local stress interruption and stress dispersion when the collagen is subjected to stress, giving the mineralization front area good mechanical properties and appropriate toughness. Furthermore, during bone tissue development, the mineralization front continuously advances across the collagen, with calcium salt deposits exhibiting a spherical mineralized structure that radiates outward until they fuse together to form mineralized collagen, further assembling into a precise bone tissue structure. Therefore, the mineralization front is a unique entity in the field of biomineralization. It would be of great significance for biomimetic bone repair materials to reproduce the multi-level structure of the mineralization front. However, no studies have yet directly simulated the biomimetic multi-level mineralization front structure for bone defect repair in vitro. Summary of the Invention

[0005] To address the problems in the prior art, the present invention proposes a method for preparing and applying a biomimetic multi-level spherical mineralized collagen scaffold. The biomimetic multi-level spherical mineralized collagen scaffold is formed by inducing partial intra-collagen mineralization of type I collagen material using a phosvitin-calcium phosphate precursor system.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned biomimetic multi-stage spherical mineralized collagen scaffold, the specific technical solution comprising the following steps:

[0007] 1) Weigh the flaky crystalline phosvitin solid at room temperature;

[0008] 2) adding a calcium source to a buffer solution to prepare a calcium buffer solution, and adding a phosphorus source to a buffer solution to prepare a phosphorus buffer solution; the pH of the buffer solution is 7.2-7.5;

[0009] 3) adding a calcium buffer and a phosphate buffer sequentially to the phosvitin from step 1) to obtain a phosvitin-calcium-phosphate precursor system; the resulting phosvitin-calcium-phosphate precursor system has a calcium:phosphorus molar concentration ratio of 1.67:1 to 2.14:1; and the phosvitin concentration is 37.5-300 μg / mL;

[0010] 4) adding the type I collagen material to the aforementioned phosvitin-calcium-phosphate precursor system for in vitro mineralization; after the mineralization is completed, a scaffold material is obtained, and the scaffold material is fished out and dried to obtain a biomimetic multi-stage spherical mineralized collagen scaffold material.

[0011] As a preferred embodiment of the present invention, in step 2), the calcium source is one or more of calcium chloride, calcium hydroxide, and calcium carbonate; the phosphorus source is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate; and the buffer is a Tris-HCl buffer system or a Hepes buffer system.

[0012] As a preferred embodiment of the present invention, in step 2), the calcium ion concentration in the calcium buffer is 1.67mM-9mM, and the phosphate ion concentration in the phosphate buffer is 1mM-4.2mM;

[0013] As a preferred embodiment of the present invention, in step 3), the order of adding the calcium buffer and the phosphate buffer to the phosvitin is first adding the calcium buffer and then adding the phosphate buffer; wherein the added volumes of the calcium buffer and the phosphate buffer are equal.

[0014] As a preferred embodiment of the present invention, in step 4), the type I collagen material is derived from a multilayer rat tail collagen model, a recombinant rat tail collagen model, a turkey tendon collagen model, a recombinant bovine Achilles tendon-derived collagen model, or a demineralized dentin collagen model.

[0015] As a preferred embodiment of the present invention, in step 4), the in vitro mineralization time is 0.5 days to 7 days, and the mineralization temperature is 24° C. to 37° C.

[0016] The present invention also provides a biomimetic multi-stage spherical mineralized collagen scaffold material prepared by the above method.

[0017] As a preferred embodiment of the present invention, the mineralized structure of the scaffold material is characterized by a multi-level spherical shape. The scaffold material has both mineralized and unmineralized areas. The collagen in the mineralized area undergoes intrafiber mineralization, and the texture becomes hard after calcium and phosphorus deposition. The collagen fibers in the unmineralized area maintain the original rhythmic strip structure and have a soft texture.

[0018] The biomimetic multi-level spherical mineralized collagen scaffold material prepared according to the method of the present invention has excellent osteoinductive properties, good mechanical properties, and no cytotoxicity, and can be used directly or indirectly as a scaffold material for bone defect repair or bone tissue engineering.

[0019] Compared with the prior art, the present invention has the following advantages.

[0020] The biomimetic multi-stage spherical mineralized collagen scaffold material preparation method of the present invention is simple and has a concise process. The scaffold is formed by inducing partial intra-collagen mineralization of type I collagen using a phosvitin-calcium-phosphate precursor system as a mineralizing solution. The main reason for the generation of its mineralized structure is that the preparation technology of the calcium-phosphate precursor system stabilized by phosvitin has significant technical sensitivity. Only under specific phosvitin-calcium-phosphate ion concentration and ratio conditions can a unique mineralized structure be formed and an efficient mineralization rate be possessed. The fundamental reason is that the formation and regulation mechanism of the calcium-phosphate precursor system stabilized by phosvitin is innovative, and is significantly different from the previous mechanisms of polymer-stabilized calcium-phosphate precursors or phosphoprotein-stabilized calcium-phosphate precursors. The mechanism is primarily manifested in the fact that phosvitin undergoes conformational changes (e.g., changes in the proportion of its secondary β-sheet structure) in a specific mineralization system, thereby anchoring calcium and phosphate ions to form unique phosvitin-stabilized calcium-phosphate self-assembled aggregates. These aggregates rapidly adsorb to collagen fibrils and form unique spherical adsorption zones, ultimately inducing the formation of a unique spherical mineralized structure and achieving a highly efficient mineralization rate. This mechanism of phosvitin-calcium-phosphate precursor formation and its regulation of collagen mineralization have not been previously reported in the field of mineralization and represent a significant mechanistic innovation. The resulting mineralization product (a biomimetic multi-level spherical mineralized collagen scaffold) and mineralization rate (mineralization within a single collagen fiber is complete within 12 hours) differ from previous studies.

[0021] In addition, the scaffold is compared with other mineralized collagen scaffolds prepared based on calcium-phosphate minerals. The latter are mostly formed by physical means of mechanical mixing and forging and sintering. The collagen fibers show complete mineralization and are hard in texture. The scaffold collagen mineralized area and unmineralized area coexist in the present invention, which can achieve the simultaneous distribution of soft and hard areas. On the one hand, the collagen texture becomes hard after calcium and phosphate deposition in the mineralized area, and its mineralization pattern is consistent with the spherical mineralization pattern of the mineralization front area of ​​the organism in nature. At the same time, the collagen in the mineralized area has high hardness and good toughness. When it is used as a bone repair material or bone tissue engineering material for local bone defect, the collagen in the mineralized area can play a load-bearing and supporting role as a force-bearing part. On the other hand, the collagen fibers in the unmineralized area maintain the original rhythmic strip structure and are soft in texture. The collagen in the unmineralized area can be further mineralized under specific induction conditions to form a hard mineralized collagen. When it is used as a bone repair material or bone tissue engineering material for local bone defect, the unmineralized collagen can be used as a local stress dispersion area to interrupt the excessive stress stimulation of the defect. In addition, the repair process of tissue at the bone defect site is essentially a gradual reconstruction process from soft tissue to hard tissue. Existing mineralized collagen scaffolds are mostly fully mineralized, and they often have problems with bone non-union and difficulty in shaping during actual application. The unmineralized area of ​​the mineralized collagen scaffold described in the present invention is in contact with the bone defect. After induction, the collagen in the unmineralized area gradually hardens and can be embedded with the soft tissue in the bone defect area and promote its reconstruction, thereby achieving a transition from soft to hard texture at the interface between the material and the bone defect. This intermediate material with both soft and hard properties is more in line with the natural bone formation of organisms, and is of great significance for the research and development of artificial bone repair materials with bidirectional bionic structure and function. The bionic multi-stage spherical mineralized collagen scaffold described in the present invention has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Image of phosvitin-stabilized calcium-phosphate precursor under cryo-electron microscopy.

[0023] Figure 2 : Transmission electron microscopy images of spherical mineralized structures of multilayered rat tail collagen fibers after 3 days of in vitro mineralization induction.

[0024] Figure 3 : Scanning electron microscopy images of the spherical mineralized structure of the recombinant bovine Achilles tendon-derived collagen scaffold after 3 days of in vitro mineralization induction.

[0025] Figure 4 : Collagen mineralization images of different mineralization system concentrations and different mineralization times under scanning electron microscopy.

[0026] Figure 5 : The results of the effect of bionic multi-level spherical mineralized collagen scaffold prepared from rat tail collagen on the proliferation ability of mouse BMSC cells.

[0027] Figure 6: The results of Alizarin red staining were used to evaluate the osteogenic differentiation ability of mouse BMSC cells induced by different collagen scaffolds.

[0028] Figure 7 : Surface height map and elastic modulus curve of biomimetic multi-level spherical mineralized collagen scaffold prepared from rat tail collagen under atomic force microscope.

[0029] Figure 8 : Circular dichroism spectroscopy characterization of protein conformational changes in phosvitin-calcium phosphate precursor system. DETAILED DESCRIPTION

[0030] The present invention will be further described and illustrated below in conjunction with specific embodiments. The embodiments are merely illustrative of the present disclosure and do not limit its scope. The technical features of the various embodiments of the present invention may be combined accordingly, provided that there is no conflict between them.

[0031] Example 1

[0032] (1) Weighing of phosvitin

[0033] Weigh the crystalline phosvitin solid at room temperature and place it at the bottom of a 15 mL centrifuge tube. The weighing requirement is to control the final phosvitin concentration in the mineralization solution to 75 μg / mL.

[0034] (2) Selection of calcium-phosphorus system

[0035] A Tris-HCl buffer system (containing a molar concentration of 150 mM NaCl, a molar concentration of 42 mM Tris-HCl, and a molar concentration of 8 mM Tris-base) was prepared, and the pH of the system was titrated to 7.4. Anhydrous calcium chloride granules and potassium dihydrogen phosphate powder were weighed and dissolved in the Tris-HCl buffer system to obtain a calcium / Tris-HCl buffer and a phosphate / Tris-HCl buffer. The calcium ion concentration in the calcium / Tris-HCl buffer was 7.0 mM, and the phosphate ion concentration in the phosphate buffer was 4.2 mM.

[0036] (3) Preparation of phosvitin-calcium-phosphate precursor system

[0037] 5 mL of the calcium / Tris-HCl buffer described in Example (2) was measured and added to the centrifuge tube containing phosvitin described in (1), and then an equal volume of phosvitin / Tris-HCl buffer was slowly added. The mixture was gently pipetted and mixed for about 20 times. The resulting solution was clear and transparent, with no visible turbidity or precipitation. The phosvitin-calcium-phosphorus precursor system was obtained and used as a mineralization solution for inducing type I collagen mineralization. Figure 1Cryo-electron microscopy showed that in the mineralization system of this example, 75 μg / mL of phosvitin can stabilize supersaturated calcium and phosphate ions, and the formed phosvitin-calcium and phosphate precursor can undergo a self-assembly process to form a unique aggregate structure.

[0038] (4) Preparation of type I collagen model (multilayered rat tail collagen model)

[0039] Rat tail collagen fibers were exfoliated and diluted to 15 mg / mL with 0.3 M glacial acetic acid. The fibers were magnetically stirred at room temperature for 72 hours until completely dissolved. The dialysis bag was soaked in 75% ethanol for 25 minutes and rinsed with ultrapure water. The extracted collagen was then diluted with 0.3 M glacial acetic acid to a concentration of 5 mg / mL and placed into the dialysis bag. The bag was then infiltrated with 1× PBS buffer for 4 days, followed by reverse osmosis with pure water for 3 days to prepare a self-assembled multilayered rat tail collagen model.

[0040] (5) Preparation of biomimetic multi-level spherical mineralized scaffolds by mineralization of multilayered rat tail collagen model induced by phosvitin-calcium phosphate precursor

[0041] The self-assembled multilayered rat tail collagen was diluted and dripped onto a carbon-supported nickel mesh. Excess moisture was then absorbed with absorbent paper and then placed in the phosvitin-calcium-phosphate precursor system described in Section (3) of this Example. Mineralization was performed in a constant temperature incubator (28°C) for 6 hours, 12 hours, 1 day, and 2 days. At the corresponding mineralization time point, the carbon-supported nickel mesh was removed with tweezers and air-dried at room temperature (20-25°C). A biomimetic multi-level spherical mineralized collagen scaffold was then prepared on the nickel mesh. Figure 2 Transmission electron microscopy was used to show that rat tail collagen formed a large number of spherical mineralized structures after being induced by phosvitin-calcium phosphate precursor (the collagen in the mineralized area was a black opaque area, and the collagen in the non-mineralized area was a translucent area).

[0042] Example 2:

[0043] (1) Weighing of phosvitin

[0044] Weigh the crystalline phosvitin solid at room temperature and place it at the bottom of a 50 mL centrifuge tube. The weighing requirement is to control the final phosvitin concentration in the mineralization solution to 150 μg / mL.

[0045] (2) Selection of calcium-phosphorus system

[0046] Prepare a Tris-HCl buffer system (containing 150 mM NaCl, 42 mM Tris-HCl, and 8 mM Tris-base) and titrate the system to pH 7.4. Weigh anhydrous calcium chloride and potassium dihydrogen phosphate to maintain a calcium ion concentration of 7.0 mM and a phosphate ion concentration of 4.2 mM in the calcium / Tris-HCl buffer. Dissolve the weighed anhydrous calcium chloride and potassium dihydrogen phosphate in the Tris-HCl buffer system to obtain a calcium / Tris-HCl buffer and a phosphate / Tris-HCl buffer, respectively.

[0047] (3) Preparation of phosvitin-calcium-phosphate precursor system

[0048] 10 mL of the calcium / Tris-HCl solution described in Example (2) was measured and added to the centrifuge tube containing phosvitin described in (1), and then an equal volume of phosvitin / Tris-HCl solution was slowly added. The mixture was gently blown about 20 times to mix. The resulting solution was clear and transparent, with no visible turbidity or precipitation. The phosvitin-calcium-phosphorus precursor system was obtained and used as a mineralization solution to induce type I collagen mineralization.

[0049] (4) 50 g of fresh bovine Achilles tendon was removed of attached muscle, ground in a mortar, and thoroughly washed with physiological saline. Rinse with pure water and place in a 200 mL beaker. Digest with 100 mL of 2% acetic acid solution containing pepsin under magnetic stirring for 72 h to obtain a collagen solution. The solution was then placed in glass bottles and subjected to a gradient freeze-drying method (stored at 4°C for 24 h, -20°C for 24 h, and -80°C for 24 h, followed by freeze-drying in a vacuum freeze dryer for 48 h) to obtain a recombinant bovine Achilles tendon-derived collagen model.

[0050] (5) Preparation of biomimetic multi-level spherical mineralized collagen scaffolds by mineralization of recombinant bovine Achilles tendon-derived collagen model induced by phosvitin-calcium phosphate precursor

[0051] The recombinant bovine Achilles tendon-derived collagen model was cut into small pieces, preferably 0.5 cm × 0.5 cm × 0.5 cm in size, and placed in the mineralization solution prepared with the phosvitin-calcium phosphate precursor system described in Section (3) of this Example. The pieces were mineralized in a constant temperature incubator (30°C) for 3 days. After the mineralization was completed, the recombinant bovine Achilles tendon-derived collagen model pieces were removed with tweezers, gently washed with ultrapure water, and dehydrated with ethanol gradients (30%, 50%, 70%, 90%, and 100%) for 10 minutes each gradient, followed by dehydration twice with 100% ethanol. This produced a biomimetic multi-stage spherical mineralized collagen scaffold. Figure 3 Scanning electron microscopy showed that the phosvitin-calcium phosphate precursor described in this example induced intrafiber mineralization of recombinant bovine Achilles tendon-derived collagen, and the mineralized area was a protruding spherical structure.

[0052] In addition, in order to evaluate the biological and mechanical properties of the biomimetic multi-level spherical mineralized collagen scaffold material of the present invention, relevant experiments were conducted. The specific experimental examples are as follows:

[0053] Experimental Example 1: Characterization of the rate and effect of different concentrations of phosvitin-calcium phosphate precursor system inducing collagen fiber mineralization

[0054] Different concentrations of phosvitin-calcium-phosphate precursor systems were prepared according to the method described in Example 1 (the system had a calcium-phosphorus molar ratio of 1.67:1, a calcium ion concentration of 3.5 mM, a phosphate ion concentration of 2.1 mM, and phosvitin concentrations of 75 μg / mL, 150 μg / mL, and 350 μg / mL, respectively). Rat tail collagen was extracted and self-assembled according to the method described in Example 1, and then the nickel mesh was added. The collagen-loaded nickel mesh was then placed in each of the aforementioned phosvitin-calcium-phosphate precursor systems and mineralized in an incubator at 30°C for 6, 24, and 48 hours. At the corresponding mineralization time point, the nickel mesh was collected, dried, and the collagen mineralization in each group was observed under a transmission electron microscope.

[0055] Figure 4 Transmission electron microscopy results showed that the phosvitin-precursor system at concentrations of 75 μg / mL and 150 μg / mL could induce collagen fiber mineralization ( Figure 4 As shown in A and 4B, the collagen in the mineralized area turned darker), while the phosvitin-precursor system at a concentration of 350 μg / mL could not induce the mineralization of collagen fibers ( Figure 4 C, unmineralized collagen is light in color). The occurrence of collagen mineralization and the formation of globular mineralized structures described in the present invention are closely related to the concentration ratio of the phosvitin-calcium-phosphate precursor system. Collagen globular mineralization can only be induced within a specific range of conditions. For details, please refer to Table 1 for the recommended ratios of the phosvitin-stabilized calcium-phosphate precursor system.

[0056] Table 1

[0057]

[0058] At the same time, the phosvitin-calcium phosphate precursor system at a concentration of 75 μg / mL can induce partial mineralization of collagen fibers in 6 hours, which is faster than the previous mineralization system (which requires more than 3 days). Transmission electron microscopy shows that the phosvitin-calcium phosphate precursor self-assembled aggregates are adsorbed on the collagen fibers ( Figure 4 D) and induced mineralization, calcium and phosphorus deposition in the mineralized area, and collagen turned black ( Figure 4 D) The collagen in the unmineralized area shows a typical periodic strip structure.

[0059] Experimental Example 2: Evaluation of the initial adhesion and cell proliferation of biomimetic multi-level spherical mineralized collagen scaffolds prepared from rat tail collagen

[0060] According to the method described in Example 1, rat tail collagen was extracted, diluted, and self-assembled, and dropped on the surface of a glass slide. After drying at room temperature, a simple collagen scaffold derived from rat tail was obtained. The rat tail collagen scaffold was induced to mineralize by the yolk phosphoprotein-calcium phosphate precursor system in Example 1 to obtain a biomimetic multi-stage spherical mineralized collagen scaffold. The biomimetic spherical mineralized scaffold and the simple collagen scaffold were sterilized in advance and placed in a 96-well plate, with 6 wells per group. Mouse BMSC cells were inoculated in the well plate and co-cultured in a DMEM high-glucose medium environment (containing 15% fetal bovine serum and 1% double antibody) for 6 hours, 12 hours, 1 day, 4 days, and 7 days. The initial adhesion and proliferation of the two groups of cells were detected by CCK8 to evaluate the cytotoxicity of the biomimetic multi-stage spherical mineralized collagen scaffold material to mouse BMSC cells.

[0061] Depend on Figure 5 The results showed that after 6 and 12 hours of co-culture with the scaffold materials, there was no statistically significant difference in early cell adhesion (P>0.05). However, after 1, 4, and 7 days of co-culture, the proliferation of mouse BMSCs in the biomimetic spherical mineralized collagen scaffold group was significantly enhanced compared to the collagen scaffold group (P<0.05). These results demonstrate that the biomimetic multi-level spherical mineralized collagen scaffold material of the present invention is non-cytotoxic and has good biocompatibility.

[0062] Experimental Example 3: Alizarin red staining to evaluate the ability of different collagen scaffolds to induce osteogenic differentiation of mouse BMSCs

[0063] Simple rat tail collagen scaffolds were prepared according to the process described in Experimental Example 2. Mineralized collagen scaffolds were prepared using different mineralization systems, including biomimetic spherical mineralized collagen scaffolds induced by phosvitin-calcium phosphate precursor (75 μg / mL) and mineralized collagen scaffolds induced by polyelectrolyte biomimetic analog polyacrylic acid (75 μg / mL). The mineralization temperature was 30°C and the mineralization time was 3 days.

[0064] Mouse BMSCs were seeded in 12-well plates, with triplicate wells per group. Alizarin red staining was performed. The specific groups were as follows: phosvitin group: biomimetic spherical mineralized collagen scaffolds supplemented with phosvitin-calcium phosphate precursor (75 μg / mL); polyacrylic acid group: mineralized collagen scaffolds supplemented with polyacrylic acid-calcium phosphate precursor (75 μg / mL); osteogenic induction group: unmineralized rat tail collagen scaffolds supplemented with osteogenic induction medium (in addition to standard α medium, containing 10 mM sodium β-glycerophosphate, 100 nM dexamethasone, and 0.2 mM vitamin C); and control group: unmineralized rat tail collagen scaffolds supplemented with standard α medium. The culture medium for each group was supplemented with 10% fetal bovine serum and 1% double-stranded antibody. The scaffolds and cells were co-cultured for 7 days, and the culture medium was replaced every two days. The calcium salt deposition of cells in each group was detected by Alizarin red staining, and the ability of different mineralized collagen scaffold materials to promote the osteogenic differentiation of mouse BMSC cells was quantitatively evaluated.

[0065] Depend on Figure 6 The results showed that compared with other groups, the biomimetic spherical mineralized collagen scaffold induced by phosvitin-calcium phosphate precursor promoted the osteogenic differentiation of mouse BMCS cells significantly. After 7 days of co-culture, the intercellular calcium salt deposition was obvious (the calcium salt deposition was manifested as Figure 6 At the same time, the quantitative analysis results of Alizarin Red showed that the biomimetic spherical mineralized collagen scaffold of the present invention induced more calcium salt deposition in the cell matrix than the mineralized collagen scaffold induced by polyacrylic acid, and the differences among the groups were statistically significant (P<0.05), indicating that the scaffold of the present invention has good osteoinductive properties.

[0066] Experimental Example 4: Micromechanical properties of biomimetic multi-level spherical mineralized collagen scaffolds prepared from rat tail collagen:

[0067] Following the method and mineralization system described in Example 2, rat tail collagen was extracted, self-assembled, and deposited onto a glass slide. After three days of in vitro mineralization, a rat tail-derived biomimetic multi-level spherical mineralized collagen scaffold was obtained. The surface morphology and elastic modulus of the scaffold were examined using atomic force microscopy to evaluate its micromechanical properties.

[0068] Combine Figure 7 A and 7C show that compared with the unmineralized collagen scaffold (surface height: 119.3nm-336.2nm), the collagen fibers of the mineralized collagen scaffold (surface height: 157.3nm-516.3nm) are significantly taller and thicker. Figure 7 B and 7D show that the linear elastic modulus of the mineralized collagen scaffold along the longitudinal and transverse directions of the collagen long axis (about 50-350 GPa) is higher than that of the unmineralized collagen scaffold (about 25-75 GPa). The positions of the longitudinal and transverse linear modulus graphs correspond to Figure 7The lengths of the longitudinal and transverse regions at the cross arrows in A and 7C are both 400 nm. The results show that the micromechanical properties of the biomimetic spherical mineralized collagen scaffolds induced by the phosvitin-calcium phosphate precursor system are significantly improved.

[0069] Experiment 5: Characterization of the Secondary Conformation of the Phosvitin-Calcium Phosphate Precursor System by Circular Dichroism (CD)

[0070] The mineralization system concentrations in Example 1 were configured as follows: a pure phosvitin solution and a phosvitin-calcium phosphate precursor solution. Spectral data in the far-UV region of 190-260 nm was measured using a JASCO J-1700 circular dichroism spectrometer with a cell diameter of 1 cm, a collection interval of 1 nm, a scan speed of 200 nm / min, and three accumulations. Young's algorithm was used to calculate the secondary structure proportion of phosvitin in each system to analyze changes in the secondary structure of the protein in the system.

[0071] Figure 8 CD results showed that the secondary structure of phosvitin was mainly composed of γ-random coil structure. After the addition of calcium and phosphate ions, the proportion of β-turns in the γ-random coil structure in the secondary structure of phosvitin decreased, and the proportion of β-folds increased significantly, indicating that the interaction between calcium and phosphate ions and phosvitin changed the secondary structure of the protein, making its structure more compact. The change in the conformation of the protein is closely related to its function of inducing globular mineralization.

[0072] The above-described embodiments and experimental examples merely illustrate several embodiments and experimental methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. Persons skilled in the art will readily appreciate that variations and modifications may be made without departing from the scope of the present invention, and all such variations and modifications fall within the scope of protection of the present invention.

Claims

1. A method for preparing a biomimetic multi-stage spherical mineralized collagen scaffold material, characterized in that: The steps include: 1) Weigh the flaky crystalline phosvitin solid at room temperature; 2) adding a calcium source to a buffer solution to prepare a calcium buffer solution, and adding a phosphorus source to a buffer solution to prepare a phosphorus buffer solution; the pH of the buffer solution is 7.2-7.5; the buffer solution is a Tris-HCl buffer system or a Hepes buffer system; 3) adding a calcium buffer and a phosphate buffer sequentially to the phosvitin from step 1) to obtain a phosvitin-calcium-phosphate precursor system; the order of adding the calcium buffer and the phosphate buffer to the phosvitin is first adding the calcium buffer and then adding the phosphate buffer; wherein the added volumes of the calcium buffer and the phosphate buffer are equal; the resulting phosvitin-calcium-phosphate precursor system has a calcium:phosphate molar concentration ratio of 1.67:1 to 2.14:1; the concentration of the phosvitin is 37.5-300 μg / mL; the calcium ion concentration in the calcium buffer is 1.67 mM-9 mM, and the phosphate ion concentration in the phosphate buffer is 1 mM-4.2 mM; 4) adding the type I collagen material to the aforementioned phosvitin-calcium-phosphate precursor system for in vitro mineralization; the in vitro mineralization time is 0.5 days to 7 days, and the mineralization temperature is 24°C to 37°C; after the mineralization is completed, a scaffold material is obtained, and the scaffold material is removed and dried to obtain a biomimetic multi-level spherical mineralized collagen scaffold material.

2. The method for preparing the biomimetic multi-stage spherical mineralized collagen scaffold material according to claim 1, characterized in that: In step 2), the calcium source is one or more of calcium chloride, calcium hydroxide, and calcium carbonate, and the phosphorus source is one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.

3. The method for preparing the biomimetic multi-stage spherical mineralized collagen scaffold material according to claim 1, characterized in that: In step 4), the type I collagen material is derived from a multilayer rat tail collagen model, a recombinant rat tail collagen model, a turkey tendon collagen model, a recombinant bovine Achilles tendon-derived collagen model, or a demineralized dentin collagen model.

4. A biomimetic multi-level spherical mineralized collagen scaffold material prepared by the method according to any one of claims 1 to 3.

5. The biomimetic multi-stage spherical mineralized collagen scaffold material according to claim 4, characterized in that: The scaffold material has both mineralized and unmineralized areas. The collagen in the mineralized area undergoes intrafiber mineralization, and the texture becomes hard after calcium and phosphorus deposition. The collagen fibers in the unmineralized area maintain the original rhythmic strip structure and are soft in texture.

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