Bionic polypeptide for inducing mineralization in type I collagenous fibers and application thereof

Abstract

The invention discloses a bionic polypeptide for inducing mineralization in type I collagenous fibers and application of the bionic polypeptide. The amino acid sequence of the novel NCPs bionic polypeptide comprises a collagen binding motif, a calcium ion chelation motif and a cell adhesion sequence; the amino acid sequence of the collagen binding motif comprises sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 3. The novel NCPs bionic polypeptide disclosed by the invention can stabilize an amorphous calcium phosphate precursor and specifically bind to type I collagen, so that ordered intra-fiber mineralization is realized. The polypeptide disclosed by the invention breaks through the limitation of single function of a traditional non-collagen simulant, and provides a core technology for constructing a tissue engineering scaffold of which the structure and the performance are highly bionic to those of natural bones.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a biomimetic polypeptide that induces mineralization within type I collagen fibers and its applications. Background Technology

[0002] Bone defects refer to the interruption or loss of bone tissue continuity caused by trauma, infection, tumor resection, etc., and have become a significant medical problem worldwide, greatly impacting patients' quality of life and healthcare systems. For bone defects exceeding critical sizes, autologous bone grafting is the "gold standard," but its availability is limited and it is prone to secondary complications; while metal prostheses and existing artificial bone materials have shortcomings in terms of biodegradability, mechanical properties, and bioactivity.

[0003] Although collagen / apatite hybrid scaffolds have been developed using conventional mineralization methods, a major limitation of these methods is that apatite typically nucleates only on the surface of collagen fibers. This limitation results in insufficient strength, as the physical properties of natural bone depend on its layered, interwoven nanostructures that constantly adapt to changing mechanical demands. Among the multiple structural layers of bone, nanoscale mineralized collagen is the most fundamental structural unit. The formation of this structure hinges on an ordered mineralization process: hydroxyapatite crystals preferentially nucleate and grow within specific interstitial regions formed by the periodic interlacing of collagen molecules, subsequently achieving mineralization from within the fibers to between them. This process involves the formation and directional transport of metastable precursors such as amorphous calcium phosphate, primarily regulated by non-collagenous proteins (NCPs).

[0004] NCPs can inhibit spontaneous extrafibrillary mineral deposition and promote controlled mineral nucleation on collagen templates. This dual role is crucial for determining the hierarchical structure and mechanical properties of bone. However, natural NCPs are difficult to extract and prone to mutation. Therefore, in the past few decades, researchers have focused on developing NCP mimics to further study the complex interactions between NCPs and minerals and collagen.

[0005] Given the abundance of acidic groups in the NCP protein backbone, researchers have successively used acidic polyelectrolytes such as polyaspartic acid and polyacrylic acid to achieve intracellular mineralization of collagen fibers through a "polymer-induced liquid precursor" process. However, these mimics have significant limitations: they can only mimic the ion chelating function of NCPs, lacking the ability to specifically bind to collagen fibers, resulting in high sensitivity to conditions such as molecular weight and concentration, limited regulatory efficiency, and unstable effects. Therefore, researchers have turned to finding alternatives that can better mimic the dual functions of NCPs. For example, some studies have attempted to combine polyacrylic acid with polyphosphates that have an affinity for type I collagen under alkaline conditions to construct a dual biomimetic system. However, developing a novel biomimetic molecule with a well-defined structure, simple synthesis, and the ability to achieve dual functions in one integrated manner has become a goal pursued in this field. This will open up new directions for the design and clinical translation of bone regeneration materials and promote the further development of the field of biomimetic tissue engineering. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a biomimetic polypeptide that induces mineralization within type I collagen fibers and its applications. The biomimetic polypeptide effectively induces the orderly nucleation and growth of hydroxyapatite within type I collagen fibers, achieving precise intrafiber mineralization. Simultaneously, the invention provides the application of this biomimetic polypeptide in the preparation of high-performance bone repair materials. By constructing a mineralized collagen structure at the nanoscale that mimics natural bone, it provides a core technology for developing next-generation high-performance bone repair materials.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a novel NCPs biomimetic polypeptide that induces mineralization within type I collagen fibers, wherein the amino acid sequence of the novel NCPs biomimetic polypeptide includes a collagen-binding motif, a calcium ion chelating motif, and a cell adhesion sequence; the amino acid sequence of the collagen-binding motif includes the sequences shown in SEQ ID NO.1-SEQ ID NO.3.

[0009] The novel NCPs-inspired peptides of this invention can stabilize amorphous calcium phosphate precursors and specifically bind to type I collagen, achieving ordered intrafibrous mineralization. These peptides overcome the limitations of traditional non-collagen mimics with their single function, providing a core technology for constructing tissue engineering scaffolds with highly biomimetic structures and properties of natural bone.

[0010] SEQ ID NO.1: FKYRPRYFL.

[0011] SEQ ID NO.2: GLRSKSKKF.

[0012] SEQ ID NO.3: QYPDATDED.

[0013] Preferably, the amino acid sequence of the novel NCPs biomimetic polypeptide includes the sequences shown in SEQ ID NO.4-SEQ ID NO.6.

[0014] SEQ ID NO.4: FKYRPRYFLTKREEVDRGD.

[0015] SEQ ID NO. 5: GLRSKSKKFTKREEVDRGD.

[0016] SEQ ID NO. 6: QYPDATDEDTKREEVDRGD.

[0017] Secondly, the present invention provides a method for preparing a novel NCPs biomimetic polypeptide that induces mineralization within type I collagen fibers as described in the first aspect. The preparation method includes: analyzing type I collagen binding motifs in osteopontin and osteosialin using three-dimensional structural visualization software, predicting concentrated amino acid regions that are within 3 angstroms of type I collagen or have hydrogen bonding interactions, screening sequences with affinity reaching the nanomolar level using molecular docking software as type I collagen binding motifs, and combining collagen binding motifs, calcium ion chelating motifs, and cell adhesion sequences to obtain the desired product.

[0018] Preferably, the method of combining the components includes using a solid-phase synthesis method to sequentially synthesize a cell adhesion sequence, a calcium ion chelating motif, and a collagen binding motif along the C-terminus to the N-terminus direction.

[0019] Thirdly, the present invention provides a method for inducing mineralization within type I collagen fibers, the method comprising: mixing type I collagen with a calcium source solution containing a novel NCPs biomimetic polypeptide for inducing mineralization within type I collagen fibers as described in the first aspect and co-incubating, followed by adding a phosphorus source solution.

[0020] Preferably, the concentration of the novel NCPs biomimetic peptide in the calcium source solution is 20-30 μg / mL (e.g., 20 μg / mL, 25 μg / mL or 30 μg / mL).

[0021] Preferably, the mineralization solution contains 0.05%-0.2% (w / v) of type I collagen, for example, 0.05%, 0.1% or 0.2%.

[0022] Preferably, the calcium source solution comprises Solution.

[0023] Preferably, the phosphorus source solution includes Solution.

[0024] Preferably, in the calcium source solution The final concentration is 4-4.5 mM, for example, 4 mM, 4.2 mM or 4.5 mM.

[0025] Preferably, in the phosphorus source solution The final concentration is 1.5-2.1 mM, for example 1.5 mM, 1.8 mM or 2.1 mM.

[0026] Preferably, the incubation time is 5-15 min, for example 5 min, 10 min or 15 min.

[0027] Fourthly, the present invention provides the application of the novel NCPs biomimetic peptides described in the first aspect, which induce mineralization within type I collagen fibers, in the preparation of high-performance bone repair materials.

[0028] Fifthly, the present invention provides the application of the novel NCPs biomimetic peptides described in the first aspect for inducing mineralization within type I collagen fibers in the induction of mineralization within type I collagen fibers.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The biomimetic peptide provided by this invention innovatively integrates three functional domains—collagen targeting, mineralization nucleation, and cell adhesion—into a single molecule, overcoming the limitations of existing mimics and enabling precise and efficient simulation of the core functions of natural NCPs.

[0031] (2) This invention has achieved two core breakthroughs in biomimetic polypeptide sequence design: First, it screens out a new collagen binding sequence through computational simulation; second, it focuses on the “RGD” integrin binding site contained in osteopontin (OPN) and osteosialin (BSP) for the first time and incorporates it into the design, whose function goes beyond the single category of existing mimics that only regulate mineralization.

[0032] (3) This invention is the first to use the biomimetic peptide as an ideal model tool for in-depth research on mineralization mechanism, clarifying the precise influence of key parameters such as sample addition order, peptide concentration and mineral concentration on mineralization process, verifying the key role of non-classical crystallization path and the core regulatory function of NCPs in mineral formation. This understanding provides direct theoretical guidance for the active design and optimization of new generation bone repair materials.

[0033] (4) The biomimetic polypeptide-guided intracellular mineralization method constructed in this invention fundamentally overcomes the limitations of traditional mineralization methods. As a key technology for guiding the development of bone tissue scaffolds that are highly biomimetic to natural bone in terms of structure and performance, this method shows broad application prospects. Attached Figure Description

[0034] Figure 1 This is a chemical structure diagram of a biomimetic polypeptide;

[0035] Figure 2 This is the mass spectrum of a biomimetic polypeptide;

[0036] Figure 3 This is a high-performance liquid chromatogram of a biomimetic polypeptide;

[0037] Figure 4 Figure 1 shows the characterization of the ability of biomimetic peptides to stabilize mineral precursors. Figure 2a is the particle size analysis diagram of calcium phosphate solution containing biomimetic peptides, and Figure 3b is the SEM image of mineralized collagen in high-concentration mineralized solution containing biomimetic peptides.

[0038] Figure 5 Figure 1 shows the characterization of the binding ability of biomimetic peptides and type I collagen. Figure 2a is a circular dichroism chromatogram of a mixture of biomimetic peptides and type I collagen at different concentration ratios, and Figure 3b is a confocal microscope image of the two.

[0039] Figure 6 The image shows the detection results of the effect of biomimetic peptides on cell proliferation.

[0040] Figure 7 TEM image of biomimetic polypeptide-induced mineralization within type I collagen fibers;

[0041] Figure 8 Macroscopic morphology of bone tissue scaffolds prepared from biomimetic peptides;

[0042] Figure 9 SEM comparison images of bone tissue scaffolds prepared with and without the added biomimetic peptides;

[0043] Figure 10 A comparison of the mechanical properties of bone tissue scaffolds prepared with and without the added biomimetic peptides. Detailed Implementation

[0044] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0045] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0046] Example 1

[0047] Design and synthesis of biomimetic peptides.

[0048] (1) Sequence design of biomimetic peptides

[0049] The proteins OPN and BSP, which are highly expressed in bone tissue and share common functional characteristics, were selected as the design basis. First, Pymol was used to analyze the collagen-binding motifs of BSP and OPN, predicting the concentrated amino acid regions within 3 Å of collagen or with hydrogen bonding, namely “FKYRPRYFL” from BSP, and “GLRSKSKKF” and “QYPDATDED” from OPN. Further affinity calculations using GNINA showed that the peptide “FKYRPRYFL” had the best affinity constant for type I collagen, reaching 5.78 nM, significantly higher than “GLRSKSKKF” (5.05 μM) and “QYPDATDED” (10.7 μM), thus it was selected as the collagen-binding motif. Simultaneously, the hydrophilic C-terminal sequence “TKREEVD” of amelogenin was selected as the calcium ion chelation and mineralization nucleation motif, and the classic cell adhesion sequence “RGD” was introduced. Finally, the three functional domains were combined to construct the biomimetic polypeptide (SEQ ID NO.1).

[0050] (2) Solid-phase synthesis of biomimetic peptides

[0051] A 50 mg / mL solution was prepared by dissolving 0.5 g of ninhydrin in 10 mL of anhydrous ethanol and used as the detection reagent.

[0052] Mix 490 mL DMF and 10 mL DBU, add 25 g anhydrous piperazine, and sonicate to dissolve to obtain the deprotectant.

[0053] The coupling agent was obtained by mixing 475 mL of DMF and 25 mL of N-methylmorpholine evenly.

[0054] The pyrolysis solution was prepared by mixing TFA, triisopropylsilane and water in a volume ratio of 95:2.5:2.5.

[0055] Weigh 300 mg of Fmoc-(Boc)-Wang resin and place it in a peptide synthesis tube. Add activated DMF to swell the resin for 6 h, then dry it under vacuum. Add a deprotecting agent to the resin, react on a shaker for 15 min, and wash three times alternately with DCM and DMF, then dry it under vacuum. Take a small amount of resin and place it in a boiling water bath with the test reagent for 1 min. If the resin changes color, the deprotection is complete.

[0056] Dissolve 10 molar amounts of Fmoc-Val-OH and HBTU in 10 mL of coupling agent and react on a shaker for 10 min for pre-activation. Mix the pre-activated amino acid with the successfully deprotected resin and react on a shaker for 1 h. After the reaction, use a circulating water vacuum pump to remove the liquid from the peptide synthesis tube. Wash three times alternately, first with DCM and then with DMF, and remove the liquid again. Take a small amount of resin and add the detection reagent to a boiling water bath for 1 min. If the resin color does not change, the coupling is complete. Repeat the deprotection-coupling process until the last amino acid is deprotected.

[0057] The resin was transferred to a magnetic bottle, placed in a rotor, and stirred at 200 rpm for 3 hours in an ice-water bath. After lysis, the resin was removed by filtration, and the filtrate was purged with nitrogen until it became a slurry. Ice-crystal methyl tert-butyl ether was added to precipitate a white solid. The solid was centrifuged, resuspended, and washed three times, then air-dried overnight to obtain a dry white powder, which is the biomimetic polypeptide. The chemical structure of the biomimetic polypeptide is shown below. Figure 1 As shown.

[0058] Example 2

[0059] Mass spectrometry analysis of biomimetic peptides.

[0060] The biomimetic peptide prepared in Example 1 was dissolved in acetonitrile by ultrasonication and then characterized by molecular weight using a time-of-flight mass spectrometer (Autoflex_MAX). Figure 2 As shown, the actual measured mass-to-charge ratio (m / z) of the biomimetic peptide in the mass spectrum is 2475.86, while the theoretical mass-to-charge ratio (m / z) of the molecular ion peak is 2475.29. The two are in high agreement, indicating the successful synthesis of the peptide.

[0061] Example 3

[0062] Purity verification of biomimetic peptides.

[0063] The purity of the biomimetic peptide prepared in Example 1 was analyzed by high performance liquid chromatography (HPLC). A YMC-Triart C18 column was used as the chromatographic column, and gradient elution was performed using a water-acetonitrile system containing 0.1% TFA as the mobile phase. The detection results are as follows: Figure 3 As shown, the biomimetic peptide has a purity of 97.35%, which meets the requirements for subsequent use.

[0064] Example 4

[0065] Characterization of the ability of biomimetic peptides to stabilize mineral precursors.

[0066] The particle size distribution of the calcium phosphate mineralization solution containing the biomimetic polypeptide was determined at multiple time points (immediate, 6 h, 18 h, 42 h) using a Malvern nanoparticle size analyzer (Nano-ZS), with each group repeated three times. The composition of the mineralization solution was 5 mL 9 mM. 5 mL 4.2 mM And 20 µg / mL biomimetic peptides.

[0067] The results are as follows Figure 4 As shown in Figure a, the experimental group solution initially exhibited three discrete peak regions. Over time, the particle size gradually concentrated at approximately 500 nm, and no peaks larger than this size appeared. This indicates that the polypeptide can effectively chelate calcium ions, inhibit the nucleation of apatite crystals, and has a stabilizing effect on amorphous calcium phosphate precursors.

[0068] The microstructure of collagen mineralized in a high-concentration calcium-phosphorus mineralization solution was observed using a cold field emission scanning electron microscope (S-4800); the high-concentration calcium-phosphorus mineralization solution consisted of 2.5 mL of 90 mM... 2.5 mL 42 mM And 20 µg / mL biomimetic peptides.

[0069] Figure 4 Figure b shows that the minerals are arranged in an orderly manner along the collagen fibers, with a dense and smooth morphology. This indicates that the biomimetic polypeptide effectively regulates crystal nucleation and growth direction by stabilizing the amorphous mineral precursor, thereby significantly improving the microstructure and orderliness of mineralized collagen.

[0070] Example 5

[0071] Characterization of the binding ability of biomimetic peptides to type I collagen.

[0072] The following samples were measured using a circular dichroism chromatograph (J-1500): a 1 mg / mL type I collagen solution, a 100 μg / mL biomimetic peptide solution, and a solution in which the biomimetic peptide and type I collagen were mixed in equal volumes at different mass concentration ratios (1:5, 1:2, 1:1).

[0073] like Figure 5 As shown in Figure a, the biomimetic polypeptide exhibits a random coil conformation in aqueous solution; type I collagen displays typical triple helix structure characteristics; the CD spectra of all mixed solutions show significant changes compared to the single components, and this change becomes more significant with the increase of the relative concentration of the polypeptide, which preliminarily proves that there is an interaction between the biomimetic polypeptide and type I collagen.

[0074] 1 mL of 0.0157 mg / mL FITC solution was mixed with 1 mL of 1 mg / mL biomimetic peptide solution, and 1 mL of 0.002 mg / mL Cy5 solution was mixed with 1 mL of 1 mg / mL type I collagen solution. Both groups were reacted overnight in a shaker under dark conditions. After the reaction, they were dialyzed for 24 h to remove free dye. 50 μL of FITC-labeled peptide solution was mixed with 50 μL of Cy5-labeled type I collagen solution and incubated for 30 min. After washing three times with PBS, the mixture was observed under a fluorescence microscope.

[0075] Figure 5 The results in Figure b show that a distinct yellow fluorescent signal is visible in the Merge image, indicating that the FITC-labeled biomimetic peptide (green) and Cy5-labeled type I collagen (red) have significant spatial co-localization, further confirming that they have a specific binding interaction.

[0076] Example 6

[0077] Evaluation of the cell proliferation activity of biomimetic peptides.

[0078] Cells were seeded at a density of 5000 cells / 100 µL of medium in 96-well plates and incubated at 37°C with 5% [presumably referring to a specific temperature range]. The culture was carried out under the same conditions for 24 h. Then the original culture medium was removed, and 90 µL of fresh culture medium and 10 µL of CCK-8 solution were added to each well. The culture was continued under the same conditions for 4 h. The absorbance of each well at 450 nm was measured using a microplate reader. The wells with cell-free culture medium were used as blank controls for background subtraction to obtain the corrected absorbance.

[0079] Experimental results are as follows Figure 6 As shown, compared with the group without added peptides, the biomimetic peptides did not exhibit cytotoxicity within the measured concentration range, and the cell survival rate increased with increasing concentration, indicating that the peptides have good biocompatibility and cell proliferation-promoting activity.

[0080] Example 7

[0081] Biomimetic peptides induce mineralization within type I collagen fibers.

[0082] (1) Comparison of sample addition order

[0083] Simultaneous sample addition: Add 9 mM... 4.2 mM A mixed solution of 20 μg / mL biomimetic peptides was added to a type I collagen solution (1 mg / mL), and the pH was adjusted to 7.4.

[0084] Stepwise sample addition: First, add the sample containing 9 mM... The solution of 20 μg / mL biomimetic peptides was pre-incubated with collagen solution for 10 min, and then 4.2 mM was added. Adjust the pH of the solution to 7.4.

[0085] At different mineralization time points (1 h, 12 h, 36 h, 72 h), 10 μL of mineralization solution was added dropwise to the carbon support membrane copper mesh. After standing for 30 s, it was rinsed three times with deionized water, excess liquid was absorbed by filter paper, and after drying at room temperature, it was observed by transmission electron microscopy (HT-7700).

[0086] TEM results as follows Figure 7 As shown, the stepwise addition strategy is superior to the simultaneous addition strategy. In the stepwise addition system, typical non-classical crystallization path characteristics can be observed: large amorphous precursor clusters form in the early stages of mineralization, gradually dissociating into nanoscale particles as the reaction progresses, and finally assembling into well-organized mineralized fiber bundles. In contrast, in the simultaneous addition system, extrafiber mineralization occurs, leading to disordered fiber bundle arrangement in the final product.

[0087] (2) Optimization of peptide and mineral concentrations

[0088] Prepare the following mineralization systems respectively:

[0089] ①1 mL 9 mM Add 5 μg / mL biomimetic peptide and 1 mL of 1 mg / mL type I collagen solution, pre-incubate for 10 min, then add 1 mL of 4.2 mM... ;

[0090] ②1 mL 9 mM Add 25 μg / mL biomimetic peptide and 1 mL of 1 mg / mL type I collagen solution, pre-incubate for 10 min, then add 1 mL of 4.2 mM... ;

[0091] ③1 mL 9 mM Add 50 μg / mL biomimetic peptide and 1 mL of 1 mg / mL type I collagen solution, pre-incubate for 10 min, then add 1 mL of 4.2 mM... .

[0092] ④1 mL 9 mM Add 1 mL of 1 mg / mL type I collagen solution, pre-incubate for 10 min, then add 1 mL of 4.2 mM collagen solution. ;

[0093] ⑤ 5 mL 9 mM Add 25 μg / mL biomimetic peptide and 1 mL of 1 mg / mL type I collagen solution, pre-incubate for 10 min, then add 5 mL of 4.2 mM [protein / protein]. ;

[0094] ⑥ 0.5 mL 18 mM Add 25 μg / mL biomimetic peptide and 1 mL of 1 mg / mL type I collagen solution, pre-incubate for 10 min, then add 0.5 mL of 18 mM... .

[0095] All systems were adjusted to pH 7.4 to initiate the mineralization reaction. At 12 h and 72 h of mineralization, 10 μL of sample solution was added dropwise to the carbon-supported copper mesh. After standing for 30 s, the solution was rinsed three times with deionized water, excess liquid was absorbed with filter paper, and the solution was dried at room temperature before observation using a transmission electron microscope (HT-7700). TEM results showed that both peptide and mineral concentrations significantly affected the intrafiber / extrafiber mineralization rate and the mineralization rate of collagen fibers. Through systematic comparison of the mineralization effects under different conditions, the optimal process parameters were determined to be: a biomimetic peptide concentration of 25 μg / mL, and a mineralization system composition of 1 mg type I collagen and 5 mL of 9 mM... 25 μg / mL biomimetic peptide and 5 mL 4.2 mM By using a step-by-step sampling method, the fastest and most orderly intrafiber mineralization can be achieved.

[0096] Example 8

[0097] Bone tissue scaffolds were prepared by adding biomimetic peptides.

[0098] Add 50 mL of 9 mM The solution containing 25 μg / mL of the biomimetic peptide described in Example 1 was added to 10 mL of 1 mg / mL type I collagen solution, mixed, and allowed to stand for 10 min for pre-incubation; then 50 mL of 4.2 mM [protein / protein] was added. The solution was adjusted to pH 7.4 to initiate the mineralization reaction. After 72 h of mineralization, the resulting product was centrifuged and washed three times with deionized water. The final suspension was injected into a 5 mm × 5 mm cylindrical mold and freeze-dried to obtain a product with the following properties: Figure 8 The bone tissue framework shown.

[0099] Comparative Example 1

[0100] Preparation of bone tissue scaffold without the addition of the biomimetic polypeptide described in Example 1 of this invention.

[0101] Add 50 mL of 9 mM Add the solution to 10 mL of 1 mg / mL type I collagen solution, pre-incubate for 10 minutes, then add 50 mL of 4.2 mM collagen. The solution was adjusted to pH 7.4 to initiate the mineralization reaction. After 72 h of mineralization, the resulting product was centrifuged and washed three times with deionized water. The final suspension was injected into a 5 mm × 5 mm cylindrical mold and freeze-dried to obtain a bone tissue scaffold without the added biomimetic peptide.

[0102] Experimental Example 1

[0103] Physicochemical properties characterization of bone tissue scaffolds prepared with and without the added biomimetic peptides.

[0104] The microstructure of the mineralized collagen samples obtained in Example 8 and Comparative Example 1 was characterized using a cold field emission scanning electron microscope (S-4800). The results are as follows: Figure 9 As shown, in the sample of Example 8, the minerals are uniformly covered on the surface of the collagen fibers, forming a dense and complete coating layer, and the plate-like hydroxyapatite crystals are arranged in an orderly manner along the fiber axis; while in the sample of Comparative Example 1, the minerals are granular and randomly attached to the surface of the collagen fibers, with obvious local aggregation and structural defects.

[0105] Experimental Example 2

[0106] Mechanical properties characterization of bone tissue scaffolds prepared with and without the added biomimetic peptide.

[0107] Uniaxial compression tests were performed on the samples obtained in Example 8 and Comparative Example 1 using a universal testing machine (INSTRON 34SC-1). The displacement velocity was set to 2 mm / min, and the ultimate load of the load cell was set to 1 kN. The stress-strain curves of the samples were recorded within the strain range of 0–90%. Based on these curves, the tangent modulus was calculated as the elastic modulus of the material within the linear response interval; simultaneously, using 70% strain as a specified point, the area under the stress-strain curve within this strain range was calculated by integration to characterize the toughness modulus of the material.

[0108] like Figure 10 As shown, the bone scaffold prepared by Comparative Example 1 had a Young's modulus of 0.987 ± 0.04 MPa and a toughness modulus of 0.588 ± 0.06 MPa; while the bone scaffold prepared by Example 8 had a Young's modulus of 3.649 ± 0.27 MPa and a toughness modulus of 2.098 ± 0.34 MPa. Compared with the control group without the addition of biomimetic peptides, the macroscopic mechanical properties of the scaffold obtained in Example 8 were significantly enhanced.

[0109] In summary, the novel NCPs-inspired peptides of this invention can stabilize amorphous calcium phosphate precursors and specifically bind to type I collagen, achieving ordered intrafibrous mineralization. These peptides overcome the limitations of traditional non-collagen mimics with their singular function, providing a core technology for constructing tissue engineering scaffolds with highly biomimetic structures and properties of natural bone.

[0110] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A novel NCPs biomimetic polypeptide that induces mineralization within type I collagen fibers, characterized in that, The amino acid sequence of the novel NCPs biomimetic polypeptide includes a collagen-binding motif, a calcium ion chelating motif, and a cell adhesion sequence; the amino acid sequence of the collagen-binding motif includes the sequences shown in SEQ ID NO.1-SEQ ID NO.

3.

2. The novel NCPs biomimetic polypeptide according to claim 1, characterized in that, The amino acid sequence of the novel NCPs biomimetic polypeptide includes the sequences shown in SEQ ID NO.4-SEQ ID NO.

6.

3. A method for preparing a novel NCPs biomimetic polypeptide that induces mineralization within type I collagen fibers as described in claim 1 or 2, characterized in that, The preparation method includes: using three-dimensional structural visualization software to analyze the type I collagen binding motifs in osteopontin and osteosialin, predicting the concentrated amino acid regions that are within 3 angstroms of type I collagen or have hydrogen bonding interactions, using molecular docking software to screen sequences with affinity reaching the nanomolar level as type I collagen binding motifs, and combining the collagen binding motif, calcium ion chelating motif, and cell adhesion sequence to obtain the final product.

4. The preparation method according to claim 3, characterized in that, The method of combining the components includes using a solid-phase synthesis method to sequentially synthesize a cell adhesion sequence, a calcium ion chelating motif, and a collagen binding motif along the C-terminus to the N-terminus.

5. A method for inducing intracellular mineralization of type I collagen fibers, characterized in that, The method includes: mixing type I collagen with a calcium source solution containing a novel NCPs biomimetic polypeptide that induces mineralization within type I collagen fibers as described in claim 1 or 2, co-incubating, and then adding a phosphorus source solution.

6. The method according to claim 5, characterized in that, The concentration of the novel NCPs biomimetic peptides in the mineralization solution is 20-30 μg / mL. Preferably, the mass percentage of type I collagen in the calcium source solution is 0.05%-0.2% (w / v).

7. The method according to claim 5 or 6, characterized in that, The calcium source solution includes Solution; Preferably, the phosphorus source solution includes Solution; Preferably, in the calcium source solution The final concentration is 4-4.5 mM; Preferably, in the phosphorus source solution The final concentration is 1.5-2.1 mM.

8. The method according to any one of claims 5-7, characterized in that, The incubation time is 5-15 minutes.

9. The application of the novel NCPs biomimetic peptides that induce mineralization within type I collagen fibers as described in claim 1 or 2 in the preparation of high-performance bone repair materials.

10. The application of the novel NCPs biomimetic polypeptides described in claim 1 or 2 that induce mineralization within type I collagen fibers in the induction of mineralization within type I collagen fibers.

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