Collagen-like protein-driven hydroxyapatite-assembled bone repair material and its preparation method

Abstract

The embodiment of the present invention discloses a bone repair material that is assembled by collagen-like proteins and driven by hydroxyapatite, and a preparation method thereof. The preparation method of the bone repair material comprises: preparing recombinant collagen HAP-CLP3-Hhc; preparing cholesterol-modified recombinant collagen HAP-CLP3-Hhc-Chol; preparing a coupling product HAP-CLP3-Hhc-β-CD; and preparing a bone repair material HAP-CLP3 / β-CD / HAP. The bone repair material HAP-CLP3 / β-CD / HAP prepared by the present invention has good cell compatibility, biodegradability, and mechanical properties, can promote new bone growth, and shows potential application value in bone repair. Furthermore, it opens up new ideas and directions for the design of functional biomaterials for tissue regeneration to meet the needs of tissue regeneration such as bone repair.

Description

Technical Field

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

[0002] Existing bone repair material preparation technologies include: (1) 3D printing technology. For example, a biodegradable polyester / bioceramic composite bone tissue engineering scaffold is produced using hydroxyapatite and polylactic acid (PLA) as raw materials through 3D printing technology. Its advantages are good biocompatibility and uniform structure, which provide favorable conditions for cell growth. However, the mechanical properties of hydroxyapatite in the material are relatively poor, which limits the application range of this material. (2) Plasma immersion ion implantation (PIII) technology. The advantage of this technology is that the material preparation is completed in one step, and the injection dose can be accurately controlled, which simplifies the preparation process, can reduce tissue inflammation, and accelerate the formation of new bones. However, this technology has high requirements and requires special instruments and equipment to operate, which limits its application in practice. (3) Micro-nanostructured biomaterials. This material has good cell compatibility and is conducive to cell growth, but it has the disadvantages of poor mechanical properties and high production costs, which limit its application development. Summary of the Invention

[0003] To this end, embodiments of the present invention provide a bone repair material featuring collagen-like protein-driven hydroxyapatite assembly and a method for preparing the material. Using a collagen-like protein (HAP-CLP3) as a substrate and employing a host-guest assembly strategy, the present invention prepares a hard bone repair biomaterial. In vitro experiments have demonstrated its ability to promote cell proliferation and osteogenic differentiation, demonstrating potential application in bone repair. Furthermore, the material opens new avenues for the design of functional biomaterials for tissue regeneration, addressing the needs of bone repair and other tissue regeneration applications.

[0004] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0005] According to a first aspect of an embodiment of the present invention, a method for preparing a bone repair material driven by collagen-like protein to assemble hydroxyapatite comprises the following steps:

[0006] (1) The collagen-like protein HAP-CLP3 containing the hydroxyapatite binding domain and the hedgehog protein were recombined, expressed and purified through genetic engineering technology to obtain the recombinant collagen protein HAP-CLP3-Hhc;

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

[0008] (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 the coupling product HAP-CLP3-Hhc-β-CD;

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

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

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

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

[0013] Furthermore, in step (3), EDC, NHS, and aminated β-cyclodextrin were sequentially added to a HAP-CLP3-Hhc solution with a concentration of 0.1 mg / mL, mixed, 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 used MES buffer as a solvent, and the volume mass ratio of the HAP-CLP3-Hhc solution to EDC, NHS, and aminated β-cyclodextrin was 1:0.4:0.6:300.

[0014] Furthermore, 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 were mixed, and hydroxyapatite was added. After mixing, the mixture was dried to form the 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 a second aspect of the embodiments of the present invention, the present invention provides a bone repair material, which is made by any of the methods described above.

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

[0019] This invention utilizes a recombinant collagen-like protein (HAP-CLP3-Hhc) and β-cyclodextrin (β-CD) through host-guest coordination, and incorporates hydroxyapatite to form a bone repair material. Collagen promotes growth, and hydroxyapatite allows for non-covalent assembly. The resulting bone repair material, HAP-CLP3 / β-CD / HAP, exhibits excellent cytocompatibility, biodegradability, and mechanical properties, promoting new bone growth and demonstrating potential application in bone repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

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

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

[0023] Figure 3 This is a Coomassie Brilliant Blue staining identification diagram of the recombinant collagen HAP-CLP3-Hhc SDS-PAGE provided by the present invention;

[0024] Figure 4 The CCK-8 assay results of NIH-3T3 cells provided by the present invention;

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

[0026] Figure 6 The NIH-3T3 cell dead / alive staining image results provided by the present invention;

[0027] Figure 7 The dead / alive staining image of MC3T3-E1 subclone 14 cells provided by the present invention is displayed;

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

[0029] Figure 9 The experimental results of NIH-3T3 cell migration rate provided by the present invention;

[0030] Figure 10 The experimental results of the migration rate of MC3T3-E1 subclone 14 cells provided by the present invention are as follows: DETAILED DESCRIPTION

[0031] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

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

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

[0034] The recombinant collagen HAP-CLP3-Hhc was modified with cholesterol (Chol) to obtain HAP-CLP3-Hhc-Chol ( Figure 1A). β-cyclodextrin (β-CD) was coupled to recombinant collagen (HAp-CLP3-Hhc) via EDC and NHS via an amination reaction to obtain HAP-CLP3-Hhc-β-CD. β-CD is a macrocyclic host molecule that can undergo high-affinity host-guest complexation with the guest Chol molecule. Hydroxyapatite nanoparticles were incorporated into the bone repair material. Hydroxyapatite bound to the hydroxyapatite binding site (HAP) in collagen (HAP-CLP3-Hhc) to drive the assembly of the conjugated polymer into a supramolecular bone repair material with self-healing behavior, HAP-CLP3 / β-CD / HAP ( Figure 1 B). 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) provides a new direction for the design of bone repair materials.

[0035] Example 1

[0036] This embodiment provides a method for preparing a bone repair material that is assembled by collagen-like protein-driven hydroxyapatite:

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

[0038] The hydroxyapatite binding site (HAP) sequence, the hedgehog protein (Hhc) sequence and the collagen sequence (CLP3, derived from the scl-2 gene of Streptococcus pyogenes) were recombined. Figure 2 The Hedgehog protein (Hhc) sequence was inserted into the C-terminus of collagen (CLP3), and the globular polypeptide (V-domain) was introduced into the N-terminus of CLP3, followed by the HAP sequence. Furthermore, the polypeptide structures CPPC and RGD were inserted between CLP3 and Hhc, and CPPC was inserted between the V-domain and HAP. Overlap PCR was used to introduce these sequences and generate the gene encoding the recombinant collagen HAP-CLP3-Hhc. The HAP-CLP3-Hhc gene sequence was then digested with EcoRI and HindIII enzymes and ligated into the pET-28a vector. Subsequently, the plasmid was transformed into DH5α, and the plasmid was extracted, identified by enzyme digestion, and sequenced. Finally, the pET-28a-HAP-CLP3-Hhc recombinant plasmid was successfully constructed.

[0039] The pET-28a-HAP-CLP3-Hhc recombinant plasmid was expressed in BL21 Escherichia coli. The expression process was as follows: the pET-28a-HAP-CLP3-Hhc recombinant plasmid was first transferred into the BL21 expression competent cell, a single clone was picked and placed in 5 mL of kanamycin (50 mg / ml) culture medium, and then transferred to 1.5 L of culture medium containing kanamycin (50 mg / ml) for expansion and cultured at 37 °C until the OD 600 When the value was 0.6-0.8, 0.3 mM IPTG was added to induce protein expression, and the cells were placed at 16°C for expression for 24-30 hours. The cells were then collected by centrifugation at 8000g for 25 minutes at 4°C. The cells were disrupted using a low-temperature high-pressure homogenizer, and the recombinant protein was purified using a nickel column. Finally, the target protein was eluted with elution buffer (1L elution buffer was prepared by mixing 50 mL of 1M Tris-HCl (pH=8.0), 10 mL of 5M NaCl aqueous solution, 300 mL of 1M imidazole aqueous solution, and 640 mL of deionized water). The eluate containing the target protein was collected and the target protein was identified 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] * (SEQ ID No. 1).

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

[0043] 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 using an ultrafiltration tube (Millipore UFC910096, USA) at 3500 rpm. The concentrated HAP-CLP3-Hhc solution with a concentration of 1 mg / mL was mixed with TBS buffer (containing a final concentration of 100 mM TCEP, 0.5 mM EDTA2NA, 0.2% Triton X-100, pH = 7.4) at a volume ratio of 1:1, and allowed to stand for 10 minutes. Cholesterol was added to a final concentration of 30 mM under stirring (mixing while adding), and stirred at room temperature overnight (12 hours) 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 2-(2-chlorophenyl)ethylamine solution, mix thoroughly, let stand at room temperature for 30 minutes, and heat at 65°C until the water evaporates, eventually forming a viscous solid. Add an equal volume of 15% aqueous NaOH solution (approximately 5 mL) to the viscous solid and soak at 25°C for 1 hour. Then, add an equal volume of methanol (approximately 10 mL) for precipitation, and dry at 65°C for 10 minutes to obtain aminated β-cyclodextrin.

[0046] The HAP-CLP3-Hhc solution with a concentration of 1 mg / mL in step (2) was replaced with a HAP-CLP3-Hhc solution of the same concentration using MES buffer (0.1 M MES, 0.5 M NaCl, pH = 6). 0.4 mg of EDC powder was added to 1 mL of the HAP-CLP3-Hhc solution replaced with MES, mixed and allowed to stand for 30 min, and then 0.6 mg of NHS powder was added, mixed and allowed to stand for 30 min, and then 0.3 g of aminated β-cyclodextrin was added, mixed, and allowed to 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] The HAP-CLP3-Hhc-Chol solution obtained in step (2) and the HAP-CLP3-Hhc-β-CD solution obtained in step (3) were respectively placed in a -80°C refrigerator for 3 h, and then placed in a freeze dryer for freeze drying to obtain HAP-CLP3-Hhc-Chol powder and HAP-CLP3-Hhc-β-CD powder.

[0049] 0.102 g of HAP-CLP3-Hhc-Chol powder was dissolved in 200 μL of DMSO to obtain a HAP-CLP3-Hhc-Chol solution. 0.202 g of HAP-CLP3-Hhc-β-CD powder was dissolved in 200 μL of PBS buffer (pH = 7.4) to obtain a HAP-CLP3-Hhc-β-CD solution. The HAP-CLP3-Hhc-Chol solution and the HAP-CLP3-Hhc-β-CD solution were mixed, and 0.3 g of hydroxyapatite was added during the mixing process. The mixture was dried 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 were purchased from commercial sources and used for in vitro experiments. They were cultured in DMEM (Gibco, 11 885 084 and 10 566 016, US) containing 10% FBS (fetal bovine serum (Gibco, 10 100 154, US)) and 1% double-antibody (penicillin-streptomycin solution (HyClone, SV30010, US)) in a 37°C, CO2 incubator.

[0052] Cytocompatibility testing

[0053] To evaluate cell viability, NIH-3T3 and MC3T3-E1 subclone 14 cells were cultured in DMEM containing 10% FBS and 1% double-antibody. 0.25%, 0.375%, and 0.5% dry bone repair materials were immersed in the culture medium. The cells were cultured with the leaching solution, and the effect of the hydrogel on cytotoxicity was detected by dead / live staining. Green fluorescence represents live cells, and red fluorescence represents dead cells. In addition, the CCK8 assay was used to detect the viability of NIH-3T3 and MC3T3-E1 subclone 14 cells with the hydrogel. 1×10 4 Cells were seeded in 96-well plates and cultured with hydrogel extract for 20 hours. The old culture medium was discarded, fresh culture medium was added, and CCK8 reagent was added. The plates were placed in an incubator for 30 minutes. 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 hydrogels was evaluated.

[0054] The results of CCK-8 assay ( Figure 4 and Figure 5 ), and NIH-3T3 ( Figure 6 ) and MC3T3-E1 subclone 14 cells ( Figure 7 ) The live and dead staining data showed 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 (0.01 M pH=7.4) to measure their degradation.

[0057] The preparation method of HAP / β-CD is as follows: 0.02 g of freeze-dried HAP-CLP3-Hhc-β-CD powder is taken and re-dissolved with 200uL 0.01M PBS. Then, 0.3g of hydroxyapatite is added and dried at 60°C to form the control group of bone repair material (HAP / β-CD).

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

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

[0060] A 1 μL sample was taken from the mixture and the protein concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific) to quantify the accumulated free protein.

[0061] The protein degradation rate 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, W 游离 It represents the weight of free protein in the solution. Figure 8 As shown, the bone repair material (HAP-CLP3 / β-CD / HAP) had good degradation performance compared with the control group.

[0063] Test Example 3

[0064] Hardness was measured to evaluate the mechanical properties of bone repair materials. Vickers hardness testing was performed using an automated Vickers microhardness tester (FM-800, Future-Tech, Kanagawa, Japan). A 1 cm x 1 cm area of bone repair material (Example 1) was placed in the instrument under a load of 100 g. After 10 seconds, the load was removed. The Vickers hardness value (HV) was expressed as the average of three areas.

[0065] Calculate the hardness value at each location using the Vickers hardness formula:

[0066] HV=0.1891×F / d2

[0067] Where: d is the average value of the diagonal length, 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 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-E1subclone 14 cells were cultured in DMEM containing 10% FBS and 1% double antibody. When the coverage of the two cells reached about 80% or more, the cells were plated separately and the same volume of cells was added to six-well plates for culture. When the cell coverage reached about 80% or more, the two cells were scratched.

[0073] 0.5% of the bone repair material (HAP-CLP3 / β-CD / HAP) and the control group (HAP / β-CD) extract medium were added to the scratches to culture cells. The blank control group (Control) was used to culture cells. NIH-3T3 cells were photographed at 0h, 12h, and 24h, and MC3T3-E1 subclone 14 cells were photographed at 0h, 12h, 24h, and 36h. They were standardized to the medium control. All tests were repeated three times, and the effect of the hydrogel on cell migration was evaluated. The results are shown in Figure 2. Figure 9 (NIH-3T3), Figure 10 (MC3T3-E1 subclone 14) as shown.

[0074] Through NIH-3T3 ( Figure 9 ) and MC3T3-E1 subclone 14 cells ( Figure 10 ) Scratch wound data showed 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 using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

[0076] Sequence Listing

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

[0078] <120> Bone repair material driven by collagen-like protein and hydroxyapatite assembly and preparation method thereof

[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

[0091] Thr Leu Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu 35 40 45

[0093] Thr Tyr Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg 50 55 60

[0095] Leu Leu Lys Gly Ile Gln Asp His Ala Leu Asp Gly Gly Pro Cys Pro 65 70 75 80

[0097] Pro Cys Asn Pro Tyr His Pro Thr Ile Pro Gln Ser Val His Gly Pro 85 90 95

[0099] Lys Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly Glu Ala 100 105 110

[0101] Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly Glu Gln Gly 115 120 125

[0103] Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Glu Asp Arg Gly Glu 130 135 140

[0105] Thr Gly Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly Pro Ala 145 150 155 160

[0107] Gly Lys Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Gly Pro Lys Gly 165 170 175

[0109] Glu Gln Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly Glu Ala Gly Ala 180 185 190

[0111] Gln Gly Pro Ala Gly Pro Met Gly Pro Ala Gly Glu Gln Gly Glu Lys 195 200 205

[0113] Gly Glu Pro Gly Thr Gln Gly Ala Lys Glu Asp Arg Gly Glu Thr Gly 210 215 220

[0115] Pro Lys Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys 225 230 235 240

[0117] Asp Gly Glu Pro Gly Pro Val Gly Pro Ala Gly Pro Lys Gly Glu Gln 245 250 255

[0119] Gly Pro Gln Gly Leu Pro Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly 260 265 270

[0121] Pro Ala Gly Pro Met Gly Pro Ala Gly Glu Gln Gly Glu Lys Gly Glu 275 280 285

[0123] Pro Gly Thr Gln Gly Ala Lys Glu Asp Arg Gly Glu Thr Gly Pro Lys 290 295 300

[0125] Gly Pro Lys Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly 305 310 315 320

[0127] Glu Pro Gly Pro Val Gly Pro Ala Arg Gly Asp Gly Lys Gly Gly Ser 325 330 335

[0129] Gly Gly Ser Thr Val His Gly Cys Phe Thr Pro Glu Ser Thr Ala Leu 340 345 350

[0131] Leu Glu Ser Gly Val Arg Lys Pro Leu Gly Glu Leu Ser Ile Gly Asp 355 360 365

[0133] Arg Val Leu Ser Met Thr Ala Asn Gly Gln Ala Val Tyr Ser Glu Val 370 375 380

[0135] Ile Leu Phe Met Asp Arg Asn Leu Glu Gln Met Gln Asn Phe Val Gln 385 390 395 400

[0137] Leu His Thr Asp Gly Gly Ala Val Leu Thr Val Thr Pro Ala His Leu 405 410 415

[0139] Val Ser Val Trp Gln Pro Glu Ser Gln Lys Leu Thr Phe Val Phe Ala 420 425 430

[0141] Asp Arg Ile Glu Glu Lys Asn Gln Val Leu Val Arg Asp Val Glu Thr 435 440 445

[0143] Gly Glu Leu Arg Pro Gln Arg Val Val Lys Val Gly Ser Val Arg Ser 450 455 460

[0145] Lys Gly Val Val Ala Pro Leu Thr Arg Glu Gly Thr Ile Val Val Asn 465 470 475 480

[0147] Ser Val Ala Ala Ser Cys Tyr Ala Val Ile Asn Ser Gln Ser Leu Ala 485 490 495

[0149] His Trp Gly Leu Ala Pro Met Arg Leu Leu Ser Thr Leu Glu Ala Trp 500 505 510

[0151] Leu Pro Ala Lys Glu Gln Leu His Ser Ser Pro Lys Val Val Ser Ser 515 520 525

[0153] Ala Gln Gln Gln Asn Gly Ile His Trp Tyr Ala Asn Ala Leu Tyr Lys 530 535 540

[0155] Val Lys Asp Tyr Val Leu Pro Gln Ser Trp Arg His Asp Gly His His 545 550 555 560

[0157] His His His His His His Gly

[0158] 565。

Claims

1. A method for preparing a bone repair material driven by collagen-like protein and hydroxyapatite assembly, characterized in that: The method comprises the following steps: (1) The collagen-like protein HAP-CLP3 containing the hydroxyapatite binding domain and the hedgehog protein were recombined, expressed and purified through genetic engineering technology to obtain the recombinant collagen protein HAP-CLP3-Hhc; (2) HAP-CLP3-Hhc reacts with cholesterol in the presence of 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 the 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 the bone repair material HAP-CLP3 / β-CD / HAP.

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

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

5. The method for preparing a bone repair material driven by collagen-like protein assembly of hydroxyapatite according to claim 1, characterized in that: In step (3), EDC, NHS, and aminated β-cyclodextrin were sequentially added to a 0.1 mg / mL HAP-CLP3-Hhc solution, mixed, 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 used MES buffer as a solvent, and the volume mass ratio of the HAP-CLP3-Hhc solution to EDC, NHS, and aminated β-cyclodextrin was 1:0.4:0.6:

300.

6. The method for preparing a bone repair material driven by collagen-like protein assembly of hydroxyapatite according to claim 1, characterized in that: In step (4), HAP-CLP3-Hhc-Chol was dissolved in DMSO to obtain a HAP-CLP3-Hhc-Chol solution, and HAP-CLP3-Hhc-β-CD was dissolved in PBS buffer to obtain a HAP-CLP3-Hhc-β-CD solution; The HAP-CLP3-Hhc-Chol solution and the HAP-CLP3-Hhc-β-CD solution were mixed, and hydroxyapatite was added. After mixing, the mixture was dried to form the bone repair material HAP-CLP3 / β-CD / HAP.

7. The method for preparing a bone repair material driven by collagen-like protein assembly of hydroxyapatite according to claim 6, characterized in that: 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 according to any one of claims 1 to 7.

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