Collagen-like protein driven hydroxyapatite assembled skeleton repair material and preparation method thereof

Abstract

The embodiment of the invention discloses a bone repair material assembled by hydroxyapatite driven by collagen-like protein and a preparation method of the bone repair material. The preparation method of the bone repair material comprises the following steps: preparing recombinant collagen HAP-CLP3-Hhc; the preparation method comprises the following steps: preparing a cholesterol modified recombinant collagen HAP-CLP3-Hhc-CHol; the preparation method comprises the following steps: preparing a coupling product HAP-CLP3-Hhc-beta-CD; and preparing the bone repair material HAP-CLP3 / beta-CD / HAP. The bone repair material HAP-CLP3 / beta-CD / HAP prepared by the invention has good cytocompatibility, biodegradability and mechanical properties, can promote the growth of new bones, and shows potential application value in bone repair. Furthermore, a new thought and a new direction are developed for the design of functional biological materials for tissue regeneration, so that the requirements of tissue regeneration such as bone repair and the like are met.

Description

Technical Field

[0001] Embodiments of the present invention relate to the technical field of bone regeneration biomaterials, and particularly to a bone repair material driven by collagen-like protein to assemble hydroxyapatite and a preparation method thereof. Background Art

[0002] Existing preparation technologies for bone repair materials include: (1) 3D printing technology. For example, a bone tissue engineering scaffold of a biodegradable polyester / bioceramic composite material is made by 3D printing technology using hydroxyapatite and polylactic acid (PLA) as raw materials. Its advantages are good biocompatibility and uniform structure, providing favorable conditions for cell growth. However, the mechanical properties of hydroxyapatite in the material are relatively poor, restricting the application scope of the material. (2) Plasma immersion ion implantation (PIII) technology. The advantage of this technology is that the preparation of the material is completed in one step, and the implantation dose can be accurately controlled, simplifying the preparation process, reducing tissue inflammation, and accelerating new bone formation. However, this technology has high requirements and requires special instrument equipment for operation, restricting its practical application. (3) Micro-nano structured biomaterials. This material has good cell compatibility and is beneficial to cell growth, but has defects such as poor mechanical properties and high production costs, restricting its application and development. Summary of the Invention

[0003] Therefore, embodiments of the present invention provide a bone repair material driven by collagen-like protein to assemble hydroxyapatite and a preparation method thereof. The present invention uses collagen-like protein (HAP-CLP3) as a substrate and prepares a hard bone repair biomaterial through a host-guest assembly strategy. Verified by in vitro tests, it has a promoting effect on cell proliferation and osteogenic differentiation, showing potential application value in bone repair. Further, it opens up new ideas and directions for the design of tissue regeneration functional biomaterials to meet the needs of tissue regeneration such as bone repair.

[0004] To achieve the above object, embodiments of the present invention provide the following technical solutions:

[0005] According to the first aspect of embodiments of the present invention, a preparation method of a bone repair material driven by collagen-like protein to assemble hydroxyapatite, the method comprising the following steps:

[0006] (1) Recombinant collagen HAP-CLP3-Hhc is obtained by recombination, expression and purification of collagen-like protein HAP-CLP3 containing a hydroxyapatite binding domain and hedgehog protein through genetic engineering technology;

[0007] (2) HAP-CLP3-Hhc reacts with cholesterol in the presence of a buffer to obtain cholesterol-modified recombinant collagen HAP-CLP3-Hhc-Chol;

[0008] (3) After the β-cyclodextrin is aminated with 2-(2-chlorophenyl)ethylamine, the obtained aminated β-cyclodextrin is subjected to a coupling reaction with HAP-CLP3-Hhc in the presence of EDC, NHS and a solvent to obtain a coupling product HAP-CLP3-Hhc-β-CD;

[0009] (4) HAP-CLP3-Hhc-Chol, HAP-CLP3-Hhc-β-CD and hydroxyapatite are mixed evenly in the presence of a solvent to obtain a bone repair material HAP-CLP3 / β-CD / HAP.

[0010] Further, in step (1), the amino acid sequence of the recombinant collagen HAP-CLP3-Hhc is SEQ ID No.1.

[0011] Further, in step (2), a HAP-CLP3-Hhc solution with a concentration of 1 mg / mL is mixed with TBS buffer in a volume ratio of 1:0.8 - 1.2, allowed to stand for 10 - 15 minutes, and cholesterol with a final concentration of 20 - 40 mM is added under stirring conditions, and stirred overnight at room temperature to obtain a solution containing cholesterol-modified recombinant collagen HAP-CLP3-Hhc-Chol, wherein the HAP-CLP3-Hhc solution uses an elution buffer as the solvent.

[0012] Further, in step (3), β-cyclodextrin is mixed with a 10 - 15% aqueous solution of 2-(2-chlorophenyl)ethylamine in a mass-to-volume ratio of 0.2:5 - 6, allowed to stand at room temperature for 30 - 40 min, heated to evaporate water at 65 - 70 °C to obtain a viscous solid, added with an aqueous NaOH solution, soaked, precipitated with methanol, and dried to obtain aminated β-cyclodextrin.

[0013] Further, in step (3), EDC, NHS, and aminated β-cyclodextrin are sequentially added to a HAP-CLP3-Hhc solution with a concentration of 0.1 mg / mL, mixed evenly, and allowed to stand at room temperature for 30 min to obtain a solution containing HAp-CLP3-Hhc-β-CD; wherein the HAP-CLP3-Hhc solution uses MES buffer as the solvent, and the volume-to-mass ratio of the HAP-CLP3-Hhc solution to EDC, NHS, and aminated β-cyclodextrin is 1:0.4:0.6:300.

[0014] Further, in step (4), HAP-CLP3-Hhc-Chol is dissolved in DMSO to obtain a HAP-CLP3-Hhc-Chol solution, and HAP-CLP3-Hhc-β-CD is dissolved in PBS buffer to obtain a HAP-CLP3-Hhc-β-CD solution;

[0015] The HAP-CLP3-Hhc-Chol solution and the HAP-CLP3-Hhc-β-CD solution are mixed evenly, and then hydroxyapatite is added. After mixing evenly, it is dried to form a bone repair material HAP-CLP3 / β-CD / HAP.

[0016] Furthermore, the mass ratio of HAP-CLP3-Hhc-Chol, HAP-CLP3-Hhc-β-CD to hydroxyapatite is 1:1.5 - 2:2.5 - 3.

[0017] According to the second aspect of the embodiments of the present invention, the present invention provides a bone repair material, which is made by the method described in any one of the above.

[0018] The embodiments of the present invention have the following advantages:

[0019] Based on the recombinant collagen-like protein (HAP-CLP3-Hhc) and β-cyclodextrin (β-CD), the present invention forms a bone repair material through host-guest coordination and incorporation of hydroxyapatite. Among them, collagen can promote growth, and hydroxyapatite can be non-covalently assembled. The formed bone repair material HAP-CLP3 / β-CD / HAP has good cell compatibility, biodegradability, and mechanical properties, and can promote new bone growth, showing potential application value in bone repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can be obtained according to the provided drawings.

[0021] Figure 1 It is a schematic assembly diagram of the bone repair material provided by the present invention;

[0022] Figure 2 It is a sequence design diagram of the recombinant collagen HAP-CLP3-Hhc provided by the present invention;

[0023] Figure 3 It is an identification diagram of Coomassie brilliant blue staining of the recombinant collagen HAP-CLP3-Hhc SDS-PAGE provided by the present invention;

[0024] Figure 4 It is the CCK-8 determination result of NIH-3T3 cells provided by the present invention;

[0025] Figure 5CCK-8 assay results of MC3T3-E1 subclone 14 cells provided by the present invention;

[0026] Figure 6 Results of dead / live staining images of NIH-3T3 cells provided by the present invention;

[0027] Figure 7 Results of dead / live staining image display of MC3T3-E1 subclone 14 cells provided by the present invention;

[0028] Figure 8 Degradation results of HAP-CLP3 / β-CD / HAP and HAP / β-CD provided by the present invention;

[0029] Figure 9 Experimental results of the migration rate of NIH-3T3 cells provided by the present invention;

[0030] Figure 10 Experimental results of the migration rate of MC3T3-E1 subclone 14 cells provided by the present invention. Detailed implementation manners

[0031] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0032] See Figure 1 , the present invention provides a bone repair material driven by collagen-like protein for hydroxyapatite assembly, and its construction process is as follows:

[0033] Recombinant collagen-like protein containing hydroxyapatite binding sites (HAP-CLP3) is recombined with Hedgehog to obtain recombinant collagen HAP-CLP3-Hhc.

[0034] Recombinant collagen HAP-CLP3-Hhc is modified by cholesterol (Chol) to obtain HAP-CLP3-Hhc-Chol ( Figure 1A). β - Cyclodextrin (β - CD) was conjugated with recombinant collagen (HAp - CLP3 - Hhc) through an amination reaction using EDC and NHS to obtain HAP - CLP3 - Hhc - β - CD. β - CD is a macrocyclic host molecule that can perform host - guest complexation with high affinity with the guest Chol molecule. Incorporating hydroxyapatite nanoparticles into the bone repair material, hydroxyapatite binds to the hydroxyapatite - binding site (HAP) in collagen (HAP - CLP3 - Hhc) to drive the assembly of the conjugate polymer into a supramolecular bone repair material HAP - CLP3 / β - CD / HAP with self - healing behavior ( Figure 1 B). The HAP - CLP3 / β - CD / HAP bone repair material has the ability to form new bone and can promote the regeneration and reconstruction of bone defects. ( Figure 1 C). It provides a new direction for the design of bone repair materials.

[0035] Example 1

[0036] This example provides a preparation method for a bone repair material driven by collagen - like protein to assemble hydroxyapatite:

[0037] (1) Preparation of recombinant collagen HAP - CLP3 - Hhc

[0038] The sequences of the hydroxyapatite - binding site (HAP), hedgehog protein (Hhc) sequence, and collagen sequence (CLP3, derived from the scl - 2 gene of Streptococcus pyogenes) were sequence - recombined. Specifically, see Figure 2 , the hedgehog protein (Hhc) sequence was inserted at the C - terminus of collagen (CLP3), a globular polypeptide (V - domain) was introduced at the N - terminus of CLP3 and then the HAP sequence was inserted. Meanwhile, the polypeptide structures CPPC and RGD were inserted between CLP3 and Hhc, and CPPC was inserted between v - domain and HAP. The overlap PCR technique was used to introduce the above sequences and obtain the gene sequence encoding recombinant collagen HAP - CLP3 - Hhc. Then, the HAP - CLP3 - Hhc gene sequence and the pET - 28a vector were digested with EcoR I and Hind III enzymes and ligated. Subsequently, transformation (into DH5α), plasmid extraction, digestion identification, and sequencing were carried out. Finally, the pET - 28a - HAP - CLP3 - Hhc recombinant plasmid was successfully constructed.

[0039] Express the pET-28a-HAP-CLP3-Hhc recombinant plasmid using BL21 Escherichia coli. The expression process is as follows: First, transfer the pET-28a-HAP-CLP3-Hhc recombinant plasmid into BL21 expression-competent cells. Pick a single clone into a 5 mL kanamycin antibiotic (50 mg / ml) medium, and then transfer it into a 1.5 L medium containing kanamycin antibiotic (50 mg / ml) for expansion culture. When cultured at 37 °C until the OD 600 value reaches 0.6 - 0.8, add IPTG with a concentration of 0.3 mM to induce protein expression, and place the bacteria at 16 °C for expression for 24 - 30 hours. Then, centrifuge the bacteria at 8000 g for 25 minutes at 4 °C to collect the bacteria. Use a low-temperature high-pressure homogenizer to break the bacteria, purify the recombinant protein through a nickel column, and finally elute the target protein with an elution buffer (The preparation method of 1 L elution buffer: Mix 50 mL of 1 M Tris-HCl (pH = 8.0), 10 mL of 5 M NaCl aqueous solution, 300 mL of 1 M imidazole aqueous solution, and 640 mL of deionized water). Collect the eluate containing the target protein, and identify the target protein by Coomassie brilliant blue staining using SDS-PAGE ( Figure 3 ).

[0040] The amino acid sequence of recombinant collagen HAP-CLP3-Hhc is as follows:

[0041] MADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDGGPCPPCNPYHPTIPQSVHGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPAGPKGEQGPQGLPGKDGEAGAQGPAGPMGPAGEQGEKGEPGTQGAKEDRGETGPKGPKGERGEAGPAGKDGEPGPVGPARGDGKGGSGGSTVHGCFTPESTALLESGVRKPLGELSIGDRVLSMTANGQAVYSEVILFMDRNLEQMQNFVQLHTDGGAVLTVTPAHLVSVWQPESQKLTFVFADRIEEKNQVLVRDVETGELRPQRVVKVGSVRSKGVVAPLTREGTIVVNSVAASCYAVINSQSLAHWGLAPMRLLSTLEAWLPAKEQLHSSPKVVSSAQQQNGIHWYANALYKVKDYVLPQSWRHDGHHHHHHHHG* (SEQ ID No.1).

[0042] (2) Preparation of cholesterol-modified recombinant collagen HAP-CLP3-Hhc-Chol

[0043] Using an ultrafiltration tube (Millipore UFC910096, USA), the eluate containing the target protein obtained in step (1) was concentrated to a HAP-CLP3-Hhc solution with a concentration of 1 mg / mL at 3500 rpm. The concentrated HAP-CLP3-Hhc solution with a concentration of 1 mg / mL was mixed with TBS buffer (containing 100 mM TCEP, 0.5 mM EDTA2NA, 0.2% Triton X-100, pH = 7.4) at a volume ratio of 1:1, allowed to stand for 10 min, and cholesterol with a final concentration of 30 mM was added under stirring conditions (while mixing), and stirred overnight (12 h) at room temperature to obtain a solution containing cholesterol-modified recombinant collagen HAP-CLP3-Hhc-Chol.

[0044] (3) Preparation of HAP-CLP3-Hhc-β-CD

[0045] Weigh 0.2 g of β-cyclodextrin (β-CD) into a 50 mL centrifuge tube, add 5 mL of a 10% aqueous solution of 2-(2-chlorophenyl)ethylamine, mix well, let stand at room temperature for 30 min, heat to evaporate the water at 65 °C until a viscous solid is finally formed. Add an equal volume (about 5 mL) of a 15% aqueous NaOH solution to the viscous solid, soak at 25 °C for 1 h, then add an equal volume (about 10 mL) of methanol to precipitate, and dry at 65 °C for 10 min to obtain aminated β-cyclodextrin.

[0046] Use MES buffer (0.1 M MES, 0.5 M NaCl, pH = 6) to replace the HAP-CLP3-Hhc solution with a concentration of 1 mg / mL in step (2) with a HAP-CLP3-Hhc solution of the same concentration. Add 0.4 mg of EDC powder to 1 mL of the HAP-CLP3-Hhc solution replaced with MES above, mix well and let stand for 30 min, then add 0.6 mg of NHS powder, mix well and let stand for 30 min, and then add 0.3 g of aminated β-cyclodextrin, mix well, and let stand at room temperature for 30 min to obtain a solution containing HAP-CLP3-Hhc-β-CD.

[0047] (4) Preparation of bone repair material HAP-CLP3 / β-CD / HAP

[0048] Place the solution containing HAP-CLP3-Hhc-Chol obtained in step (2) and the solution containing HAP-CLP3-Hhc-β-CD obtained in step (3) in a -80 °C refrigerator and freeze for 3 h, then place them in a freeze dryer to freeze-dry to obtain HAP-CLP3-Hhc-Chol powder and HAP-CLP3-Hhc-β-CD powder.

[0049] Dissolve 0.102 g of HAP-CLP3-Hhc-Chol powder in 200 μL of DMSO to obtain a HAP-CLP3-Hhc-Chol solution, dissolve 0.202 g of HAP-CLP3-Hhc-β-CD powder in 200 μL of PBS buffer (pH = 7.4) to obtain a HAP-CLP3-Hhc-β-CD solution, mix the HAP-CLP3-Hhc-Chol solution and the HAP-CLP3-Hhc-β-CD solution, add 0.3 g of hydroxyapatite during the mixing process, and dry at 60 °C to form the bone repair material HAP-CLP3 / β-CD / HAP.

[0050] Test Example 1

[0051] NIH-3T3 cells and MC3T3-E1 subclone 14 cells for laboratory use were commercially purchased and used for in vitro experiments. They were cultured in DMEM (Gibco, 11885084 and 10566016, US) medium containing 10% FBS (fetal bovine serum, Gibco, 10100154, US) and 1% double antibody (penicillin-streptomycin solution, HyClone, SV30010, US) in a constant temperature incubator at 37°C with CO₂.

[0052] Cell compatibility test

[0053] To evaluate cell viability, NIH-3T3 and MC3T3-E1 subclone 14 cells were cultured in DMEM medium containing 10% FBS and 1% double antibody. 0.25%, 0.375%, and 0.5% of the dried bone repair material were immersed in the medium, and the cells were cultured with the leachate. The cytotoxic effect of the hydrogel on the cells was detected by dead / live staining. Green fluorescence represents live cells, and red fluorescence represents dead cells. In addition, the viability of NIH-3T3 and MC3T3-E1 subclone 14 cells was detected by the CCK8 experiment. 1×10 4 cells were seeded in 96-well plates and cultured with the hydrogel leachate for 20 h. The old medium was discarded, new medium was added, CCK8 reagent was added, and it was placed in the incubator for 30 min. The optical density (OD) at 450 nm was measured and normalized to the medium control. All tests were repeated three times, and the cytotoxicity of the hydrogel was evaluated.

[0054] Through the CCK-8 assay results ( Figure 4 and Figure 5 ), as well as the dead / live staining data of NIH-3T3 ( Figure 6 ) and MC3T3-E1 subclone 14 cells ( Figure 7 ), it was shown that the bone repair material had good cell compatibility.

[0055] Test Example 2

[0056] The bone repair material (HAP-CLP3 / β-CD / HAP) prepared in Example 1 and the control group (HAP / β-CD) were placed in PBS (0.01M, pH = 7.4) respectively to measure the degradation property.

[0057] Among them, the preparation method of HAP / β-CD is as follows: Take 0.02 g of freeze-dried HAP-CLP3-Hhc-β-CD powder, redissolve it with 200 μL of 0.01 M PBS, then add 0.3 g of hydroxyapatite, and dry it at 60 °C to form the control group (HAP / β-CD) of the bone repair material.

[0058] The degradation of the bone repair material (HAP-CLP3 / β-CD / HAP) and the control group (HAP / β-CD) was evaluated by OD measurement.

[0059] Weigh 0.2 g of the bone repair material (HAP-CLP3 / β-CD / HAP) and the control group (HAP / β-CD) with the same mass respectively, place them in 5 mL of PBS (0.01 M, pH = 7.4), add 10 μL of proteinase K (30 U mL -1 ) respectively, mix well, and place at 37 °C. Measure the protein concentration at different time intervals.

[0060] Take 1 μL of the sample from the mixture and use a NanoDrop spectrophotometer (Thermo Fisher Scientific) to measure the protein concentration to quantify the accumulated free protein.

[0061] The degradation rate of the protein was calculated using the following formula:

[0062] Degradation rate (%) = [1 - (W 总 - W 游离 ) / W 游离 × 100%, where W 总 represents the total weight of the hydrogel, and W 游离 represents the weight of the free protein in the solution. The results are as Figure 8 shown. Compared with the control group, the bone repair material (HAP-CLP3 / β-CD / HAP) has good degradation performance.

[0063] Test Example 3

[0064] Measure the hardness to evaluate the mechanical properties of the bone repair material. Use an automatic Vickers microhardness tester (FM-800, Future-Tech, Kanagawa, Japan) to perform Vickers hardness testing. Set the loading force to 100 g, place the bone repair material (Example 1) with a size of 1 cm * 1 cm into the instrument, keep it for 10 s, and then remove the loading force. It is expressed by the Vickers hardness value (HV), and the results are averaged from 3 regions.

[0065] Use the Vickers hardness formula to calculate the hardness value at each position:

[0066] HV = 0.1891 × F / d2

[0067] Wherein: d is the average value of the diagonal length, and F is the test force (usually in N).

[0068] After testing, the Vickers hardness of the HAP-CLP3 / β-CD / HAP prepared in Example 1 was 22.03 HV.

[0069] The Vickers hardness of the control group (HAP / β-CD) was 19.18 HV.

[0070] The results showed that the Vickers hardness of the HAP-CLP3 / β-CD / HAP in Example 1 was significantly higher than that of the control group, indicating that the bone repair material provided by the present invention has good mechanical properties.

[0071] Test Example 4

[0072] NIH-3T3 and MC3T3-E1 subclone 14 cells were cultured separately in DMEM medium containing 10% FBS and 1% double antibody. When the confluence of the two types of cells reached about 80% or more, the cells were plated separately. The same volume of cell suspension was added to six-well plates for cell culture. When the cell confluence reached about 80% or more, scratches were made on the two types of cells.

[0073] The scratch areas were respectively added with 0.5% of the bone repair material (HAP-CLP3 / β-CD / HAP) and the leaching solution medium of the control group (HAP / β-CD) for cell culture. The blank control group (Control) was also used for cell culture. NIH-3T3 cells were photographed at 0 h, 12 h, and 24 h, and MC3T3-E1 subclone 14 cells were photographed at 0 h, 12 h, 24 h, and 36 h. And they were normalized to the medium control. All tests were repeated three times, and the effect of the hydrogel on cell migration was evaluated. The results are as Figure 9 (NIH-3T3), Figure 10 (MC3T3-E1 subclone 14) shown.

[0074] Through the scratch data of NIH-3T3 ( Figure 9 ) and MC3T3-E1 subclone 14 cells ( Figure 10 ), it was shown that the bone repair material (HAP-CLP3 / β-CD / HAP) promoted cell migration compared with the control group.

[0075] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made based on the present invention, which will be obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

[0076] Sequence Listing

[0077] <110> Zhongke Zhicao (Zhejiang) Technology Co., Ltd.

[0078] <120> Collagen-like Protein-driven Hydroxyapatite Assembly-based Bone Repair Material and Its Preparation Method

[0079] <130> GG241443212A

[0080] <160> 1

[0081] <170> PatentIn version 3.5

[0082] <210> 1

[0083] <211> 567

[0084] <212> PRT

[0085] <213> Artificial Sequence

[0086] <400> 1

[0087] Met Ala Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Glu Leu Ile 1 5 10 15

[0089] Gln Glu Leu Ala Gln Gly Leu Gly Gly Ile Glu Lys Lys Asn Phe Pro 20 25 30 35

[0091] Thr Leu Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu 40 45 50 55

[0093] Thr Tyr Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg 60 65 70 75

[0095] Leu Leu Lys Gly Ile Gln Asp His Ala Leu Asp Gly Gly Pro Cys Pro 80 85 90 95

[0097] Pro Cys Asn Pro Tyr His Pro Thr Ile Pro Gln Ser Val His Gly Pro 100 105 110 115

[0099] Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly Glu Ala 120 125 130 135

[0101] Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly Glu Gln Gly 140 145 150 155

[0103] Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Glu Asp Arg Gly Glu 160 165 170 175

[0105] Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly Pro Ala 180 185 190 195

[0107] Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Gly Pro Lys Gly 200 205 210 215

[0109] Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly Glu Ala Gly Ala 220 225 230 235

[0111] Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly Glu Gln Gly Glu Lys 240 245 250 255

[0113] Gly Glu Pro Gly Thr Gln Gly Ala Lys Glu Asp Arg Gly Glu Thr Gly 260 265 270 275

[0115] Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys 280 285 290 295

[0117] Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Gly Pro Lys Gly Glu Gln 300 305 310 315

[0119] Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly 320 325 330 335

[0121] Pro Ala Gly Pro Met Gly Pro Ala Gly Glu Gln Gly Glu Lys Gly Glu 340 345 350 355

[0123] Pro Gly Thr Gln Gly Ala Lys Glu Asp Arg Gly Glu Thr Gly Pro Lys 360 365 370 375

[0125] Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly 380 385 390 395

[0127] Glu Pro Gly Pro Val Gly Pro Ala Arg Gly Asp Gly Lys Gly Gly Ser 400 405 410 415

[0129] Gly Gly Ser Thr Val His Gly Cys Phe Thr Pro Glu Ser Thr Ala Leu 420 425 430 435

[0131] Leu Glu Ser Gly Val Arg Lys Pro Leu Gly Glu Leu Ser Ile Gly Asp 440 445 450 455

[0133] Arg Val Leu Ser Met Thr Ala Asn Gly Gln Ala Val Tyr Ser Glu Val 460 465 470 475

[0135] Ile Leu Phe Met Asp Arg Asn Leu Glu Gln Met Gln Asn Phe Val Gln 480 485 490 495

[0137] Leu His Thr Asp Gly Gly Ala Val Leu Thr Val Thr Pro Ala His Leu 500 505 510 515

[0139] Val Ser Val Trp Gln Pro Glu Ser Gln Lys Leu Thr Phe Val Phe Ala 520 525 530 535

[0141] Asp Arg Ile Glu Glu Lys Asn Gln Val Leu Val Arg Asp Val Glu Thr 540 545 550 555

[0143] Gly Glu Leu Arg Pro Gln Arg Val Val Lys Val Gly Ser Val Arg Ser 560 565 570 575

[0145] Lys Gly Val Val Ala Pro Leu Thr Arg Glu Gly Thr Ile Val Val Asn 580 585 590 595

[0147] Ser Val Ala Ala Ser Cys Tyr Ala Val Ile Asn Ser Gln Ser Leu Ala 560 565 570 575

[0149] His Trp Gly Leu Ala Pro Met Arg Leu Leu Ser Thr Leu Glu Ala Trp 580 585 590 595

[0151] Leu Pro Ala Lys Glu Gln Leu His Ser Ser Pro Lys Val Val Ser Ser 600 605 610 615

[0153] Ala Gln Gln Gln Asn Gly Ile His Trp Tyr Ala Asn Ala Leu Tyr Lys 620 625 630 635

[0155] Val Lys Asp Tyr Val Leu Pro Gln Ser Trp Arg His Asp Gly His His 640 645 650 655

[0157] His His His His His His Gly

[0158] 660 665。

Claims

1. A preparation method of a bone repair material driven by collagen-like protein to assemble hydroxyapatite, characterized in that, The method includes the following steps: (1) Recombinant collagen HAP-CLP3-Hhc is obtained by recombining, expressing, and purifying collagen-like protein HAP-CLP3 containing a hydroxyapatite-binding domain and hedgehog protein through genetic engineering techniques; (2) HAP-CLP3-Hhc reacts with cholesterol in the presence of a buffer to obtain cholesterol-modified recombinant collagen HAP-CLP3-Hhc-Chol; (3) After β-cyclodextrin is aminated with 2-(2-chlorophenyl)ethylamine, the resulting aminated β-cyclodextrin is coupled with HAP-CLP3-Hhc in the presence of EDC, NHS, and a solvent to obtain a coupling product HAP-CLP3-Hhc-β-CD; (4) HAP-CLP3-Hhc-Chol, HAP-CLP3-Hhc-β-CD, and hydroxyapatite are mixed in the presence of a solvent to obtain a bone repair material HAP-CLP3 / β-CD / HAP.

2. The preparation method of the bone repair material driven by collagen-like protein to assemble hydroxyapatite according to claim 1, characterized in that In step (1), the amino acid sequence of the recombinant collagen HAP-CLP3-Hhc is SEQ ID No.

1.

3. The preparation method of the bone repair material driven by collagen-like protein to assemble hydroxyapatite according to claim 1, characterized in that, In step (2), A solution of HAP-CLP3-Hhc with a concentration of 1 mg / mL is mixed with TBS buffer at a volume ratio of 1:0.8 - 1.2, allowed to stand for 10 - 15 minutes, and cholesterol with a final concentration of 20 - 40 mM is added under stirring conditions, and stirred overnight at room temperature to obtain a solution containing cholesterol-modified recombinant collagen HAP-CLP3-Hhc-Chol, wherein the HAP-CLP3-Hhc solution uses an elution buffer as the solvent.

4. The preparation method of the bone repair material driven by collagen-like protein to assemble hydroxyapatite according to claim 1, characterized in that In step (3), β-cyclodextrin is mixed with a 10 - 15% aqueous solution of 2-(2-chlorophenyl)ethylamine at a mass-volume ratio of 0.2:5 - 6, allowed to stand at room temperature for 30 - 40 min, heated to evaporate water at 65 - 70 °C to obtain a viscous solid, added with an aqueous NaOH solution, soaked, precipitated with methanol, and dried to obtain aminated β-cyclodextrin.

5. The preparation method of the bone repair material driven by collagen-like protein to assemble hydroxyapatite according to claim 1, characterized in that In step (3), EDC, NHS, and aminated β-cyclodextrin are sequentially added to a HAP-CLP3-Hhc solution with a concentration of 0.1 mg / mL, mixed evenly, and allowed to stand at room temperature for 30 min to obtain a solution containing HAP-CLP3-Hhc-β-CD; wherein the HAP-CLP3-Hhc solution uses MES buffer as the solvent, and the volume-mass ratio of the HAP-CLP3-Hhc solution to EDC, NHS, and aminated β-cyclodextrin is 1:0.4:0.6:

300.

6. The preparation method of the bone repair material driven by collagen-like protein to assemble hydroxyapatite according to claim 1, characterized in that In step (4), Dissolve HAP-CLP3-Hhc-Chol in DMSO to obtain a HAP-CLP3-Hhc-Chol solution, and dissolve HAP-CLP3-Hhc-β-CD in PBS buffer to obtain a HAP-CLP3-Hhc-β-CD solution; The HAP-CLP3-Hhc-Chol solution is mixed with the HAP-CLP3-Hhc-β-CD solution, and then hydroxyapatite is added. After mixing, it is dried to form the bone repair material HAP-CLP3 / β-CD / HAP.

7. The preparation method of the collagen-like protein-driven hydroxyapatite-assembled bone repair material according to claim 6, wherein The mass ratio of HAP-CLP3-Hhc-Chol, HAP-CLP3-Hhc-β-CD to hydroxyapatite is 1:1.5 - 2:2.5 - 3.

8. A bone repair material, characterized in that, It is made by the method described in any one of claims 1 - 7.

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