Recombinant humanized type III collagen-hydroxyapatite composite bone repair material and its application

Abstract

The present invention relates to a recombinant humanized type III collagen-hydroxyapatite composite bone repair material and its preparation method and application. At present, the industry has always expected to achieve efficient production of macromolecular humanized collagen or whole human collagen, which is a technical difficulty that is difficult to completely break through. The molecular weight of the recombinant humanized type III collagen of the present invention is close to that of natural collagen; at the same time, its encoding gene is optimized to achieve efficient and secretory expression of recombinant collagen with high yield. The composite bone repair material prepared with the collagen of the present invention has a more uniform microstructure than the products prepared with commercially available natural collagen or type III human collagen, and achieves the combination of hydroxyapatite grains and collagen fibers at the nanoscale. At the same time, there is a multi-scale pore structure inside the material, which achieves the purpose of better biomimetic natural bone composite material preparation. Therefore, this product can be widely used in dental (jaw) bone defects, as well as dental or bone surgery such as filling and repair of bone defects, nonunion, delayed bone healing or nonunion caused by various reasons, so that more patients can benefit from it.

Description

Technical Field

[0001] The present invention belongs to the fields of bioengineering and biomaterials, and particularly relates to a recombinant humanized type III collagen-hydroxyapatite composite bone repair material, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen is the most abundant protein family in animals. Its biological activity enables it to participate in cell migration, differentiation, and reproduction, providing mechanical strength to connective tissue. It also promotes cell growth and possesses properties such as hemostasis, biocompatibility, and biodegradability. Based on these properties, collagen is widely used in medical fields such as burns, trauma, corneal diseases, wound hemostasis, drug delivery, and sustained-release technology.

[0003] To date, genes encoding over 30 types of collagen chains have been discovered, forming more than 16 types of collagen molecules, the most common of which are types I, II, III, V, and XI. Type I collagen is the most abundant in animals, accounting for 80-90% of all collagen. It has the strongest effect on bodily functions, and therefore is the most widely used in clinical practice. Type I collagen is primarily found in adult skin, tendons, and bone tissue, while type III collagen is primarily found in infant skin or the lining of blood vessels, the intestines, and in adult cartilage, vitreous, and intervertebral discs. Type III collagen (CLO3A1) has a strong synthesis capacity and can differentiate bone marrow-derived stem cells into repair-enhancing fibroblasts during wound repair. Therefore, in biomedical engineering, type III collagen is more widely used in repairing tissue wounds, supporting cell reorganization, improving skin condition, and enhancing skin elasticity and suppleness.

[0004] Traditional collagen production relies primarily on extraction from animal tissues via acid-base hydrolysis. However, this process produces a complex product with low purity. Furthermore, the product easily loses some of its biological activity during the extraction process, leading to clinical rejection and significant viral risks. Furthermore, the low content of type III collagen in animal tissues limits its potential for natural extraction.

[0005] In recent years, with the development of recombinant DNA technology, researchers have selected various host cells (such as E. coli, yeast, insects, mammals, etc.) to fully utilize the excellent properties of collagen, hoping to produce recombinant human collagen with good safety, high reproducibility, and stable quality. Although the E. coli expression system has the characteristics of high expression, it has disadvantages such as heat generation, difficulty in purification of inclusion body expression, no post-translational modification in prokaryotic systems, and low product biological activity. While expression systems such as mammalian cells and insect cells have post-translational modification functions, their applications are limited by the disadvantages of long production cycles, difficulty in cultivation, high cost, and low expression levels. As a microbial system, the Pichia pastoris expression system takes into account the high expression level of the product, easy purification, and post-translational modification functions, and has very outstanding advantages in expressing collagen. The molecular weight of collagen subunits is generally around 120kDa, which is much larger than that of general biologically active proteins, and its amino acid sequence contains a large number of Gly-XY (X and Y represent amino acids, where Y is mostly proline) repeats. Therefore, for recombinant collagen or analogues obtained by biological fermentation methods relying on genetic engineering technology, there are great difficulties in expressing the length and expression level of the product.

[0006] Therefore, the existing public reports on recombinant human (human-like) III collagen mainly use partial fragments of natural human type III collagen or partial human type III collagen fragments spliced ​​with other types of human collagen fragments for recombinant expression. For example, Chinese patent CN111363029A discloses a method for expressing recombinant human type III collagen mature peptide by Pichia pastoris, wherein the expressed collagen is only 498 amino acids. Another example is a recombinant human collagen disclosed in Chinese patent CN103122027B, wherein the expressed protein is a spliced ​​peptide segment of 257 to 501 amino acids composed of human collagen type III peptide segment and human collagen type II peptide segment. For example, Chinese patent CN103725623A discloses a Pichia pastoris engineered bacterium that secretes and expresses human type III collagen α chain protein, and its construction method and application. Although it is claimed that it can secrete and express human type III collagen mature peptide with 1069 amino acids, no detailed evidence of expression is provided. Moreover, it is well known that the commonly used Escherichia coli and yeast expression systems do not contain proline hydroxylase. Therefore, without the introduction of external proline hydroxylase (such as P4H) for co-expression, the collagen directly expressed by yeast cannot contain hydroxyproline. Therefore, it is impossible to calculate the collagen expression level by hydroxyproline content in the patent, and its expression level is only 1g / L, which is still low for industrial production. On the other hand, compared with small molecule humanized collagen or human-like collagen, humanized collagen with large molecular weight has the following advantages: (1) small molecule humanized collagen or human-like collagen has a small molecular weight and can provide fewer cross-linking reaction site groups. Therefore, under the same cross-linking conditions, large molecule humanized collagen is more easily cross-linked and fixed. The prepared biomaterial has a longer upper limit of degradation time (this time can be adjusted by cross-linking process). Especially for the application scenario of biomaterials for in vivo repair, a longer degradation time is likely to play a role along with the repair of the wound, while small molecule collagen biomaterials are likely to be completely degraded and absorbed before the wound is completely repaired, resulting in the material disintegration and wound repair being out of sync. (2) Small molecule humanized collagen or artificially designed human-like collagen is more like gelatin, a degradation product of large molecule collagen in terms of molecular weight. Since the integrity of the molecule is lost to a large extent, the collagen activity properties that rely on large molecule collagen are weakened or lost to a large extent in the body. Therefore, a larger molecular weight and a structure closer to natural collagen have greater advantages.

[0007] Currently, the industry has been hoping to achieve the production of large-molecule humanized collagen or fully human collagen, but due to the limitations of existing recombinant expression technology, it has always been a technical difficulty that is difficult to completely overcome.

[0008] The recombinant humanized collagen III of the present invention is based on the amino acid sequence of the human type III collagen α1 (COL3A1) chain, and the amino acid sequence is effectively screened. Not only does it retain the core functional sequence of collagen, but its molecular weight is closer to that of natural collagen. At the same time, its encoding gene is optimized to achieve efficient and secretory expression of the recombinant collagen and increase production. Summary of the Invention

[0009] The present invention aims to provide a recombinant humanized collagen protein expressed by Pichia pastoris, which has a molecular weight close to that of natural human protein, has a high expression level, and has excellent biological functions.

[0010] One of the objectives of the present invention is to provide a recombinant humanized type III collagen, which is secreted and expressed by Pichia pastoris, and the amino acid sequence is shown in SEQ ID NO.4.

[0011] Preferably, the encoding gene of the recombinant humanized type III collagen is shown as SEQ ID NO.5.

[0012] Another object of the present invention is to provide a method for preparing recombinant humanized type III collagen, which comprises the following steps:

[0013] (1) Cloning the above gene sequence into an expression vector, transforming Pichia pastoris into engineered bacteria, and screening to obtain engineered bacteria that can efficiently express recombinant humanized collagen;

[0014] (2) Fermenting and culturing the genetically engineered bacteria to induce expression of recombinant humanized collagen to obtain a fermentation broth containing the target protein;

[0015] (3) Purify the fermentation broth to obtain recombinant humanized type III collagen.

[0016] Preferably, the expression vector is pGAPZαA, and the Pichia pastoris engineered bacterium is Pichia pastoris GS115.

[0017] Another object of the present invention is to provide a use of recombinant humanized type III collagen in the preparation of hemostatic sponges, bone repair materials, medical or aesthetic filling materials, and cosmetics.

[0018] Another object of the present invention is to provide a recombinant humanized type III collagen hemostatic sponge, wherein the recombinant humanized type III collagen hemostatic sponge contains the above-mentioned recombinant humanized type III collagen.

[0019] Preferably, the recombinant humanized type III collagen hemostatic sponge is prepared according to the following method:

[0020] (1) adding recombinant humanized type III collagen to an acidic solution to prepare a collagen solution;

[0021] (2) The collagen solution is freeze-dried and then cross-linked to obtain the product.

[0022] Preferably, the acidic solution can be acetic acid or dilute hydrochloric acid. More preferably, the acidic solution is acetic acid,

[0023] Preferably, the crosslinking is vacuum thermal crosslinking or chemical crosslinking. More preferably, the crosslinking is vacuum thermal crosslinking.

[0024] Preferably, the recombinant humanized type III collagen hemostatic sponge is prepared according to the following method:

[0025] (1) adding 1% to 3% (wt / vol) of the above-mentioned recombinant humanized type III collagen to a 1% to 3% (vol / vol) acetic acid solution, and stirring with a magnetic stirrer at room temperature until the protein is completely dissolved to prepare a collagen solution;

[0026] (2) The collagen solution is injected into a suitable mold and freeze-dried. The freeze-dried sample is vacuum-heat cross-linked in a vacuum drying oven to obtain a collagen hemostatic sponge sample.

[0027] Another purpose of the present invention is to use the hemostatic sponge in hemostasis of wounds during surgeries such as surgery, obstetrics and gynecology, plastic surgery, and stomatology.

[0028] The above-mentioned hemostatic sponge is mainly obtained by direct freeze-drying of a collagen solution, to which no other raw materials are added. Therefore, the applicant also tried to add other raw materials (such as hydroxyapatite) to prepare a composite material on this basis. To the applicant's surprise, compared with natural collagen, the dispersion of hydroxyapatite powder in the collagen solution of the present invention was greatly improved, which is more conducive to simulating the structure of natural bone. In addition, the actual repair efficacy of the bone repair composite material prepared by this process is also much higher than that of natural collagen or other recombinant collagen. The collagen of the present invention is also more advantageous in the preparation of composite materials such as bone repair.

[0029] Therefore, another object of the present invention is to provide a humanized type III collagen-hydroxyapatite composite bone repair material, wherein the humanized type III collagen-hydroxyapatite composite bone repair material contains the above-mentioned recombinant humanized type III collagen.

[0030] Preferably, the bone repair material further contains hydroxyapatite.

[0031] Preferably, the bone repair material is prepared according to the following method:

[0032] (1) adding recombinant humanized type III collagen to an acidic solution to prepare a collagen solution;

[0033] (2) adding hydroxyapatite powder to the above collagen solution to prepare a slurry containing hydroxyapatite;

[0034] (3) freeze-drying the slurry and then cross-linking to obtain the product.

[0035] Preferably, the acidic solution can be acetic acid or dilute hydrochloric acid. More preferably, the acidic solution is acetic acid,

[0036] Preferably, the crosslinking is vacuum thermal crosslinking or chemical crosslinking. More preferably, the crosslinking is vacuum thermal crosslinking.

[0037] Preferably, the preparation method of the above-mentioned humanized collagen-hydroxyapatite composite bone repair material is as follows:

[0038] (1) adding 1% to 3% (wt / vol) of the above-mentioned recombinant humanized type III collagen to a 1% to 3% (vol / vol) acetic acid solution, and stirring with a magnetic stirrer at room temperature until the protein is completely dissolved to prepare a collagen solution;

[0039] (2) adding 1% to 5% (wt / vol) of 200 nm hydroxyapatite powder to the above collagen solution and stirring in an ice-water bath to prepare a slurry containing hydroxyapatite;

[0040] (3) The slurries are injected into appropriate molds and freeze-dried. The freeze-dried samples are vacuum-heat cross-linked in a vacuum drying oven to obtain collagen-hydroxyapatite composite bone repair material samples.

[0041] Another object of the present invention is to provide a recombinant humanized type III collagen-hydroxyapatite composite bone repair material for use in filling and repairing tooth (jaw) bone defects; and in filling and repairing bone defects, nonunion, delayed bone healing or nonunion caused by various reasons.

[0042] Compared with the prior art, the present invention has the following significant advantages: (1) The designed human-like collagen gene is based on the amino acid sequence of natural type III human collagen, and residues 154 to 1232 are screened to retain the core functional sequence of collagen. In this way, the molecular weight is closer to that of natural collagen, which is more conducive to increasing the yield while achieving secretory expression of recombinant collagen, is easy to purify, and has excellent biological function; (2) The use of the pGAPZαA vector can achieve high-level expression of the recombinant humanized collagen gene in Pichia pastoris, and no methanol induction is required during the fermentation process, the fermentation cycle is short, the cost is low, and the production process is relatively safe; (3) The secretory expression of the recombinant humanized collagen can be achieved by inducing the Pichia pastoris engineered bacteria, and the secretory expression is conducive to the separation and purification of the target protein, and can also shorten the production cycle and reduce the production cost; (4) The target protein with a purity of more than 97% can be obtained by only one-step salting-out crude purification and one-step hydrophobic chromatography purification, which is simple to operate, has a short production cycle, and has significant economic benefits. (5) The hemostatic sponge prepared using the recombinant humanized type III collagen of the present invention has a better hemostatic effect than the hemostatic sponge prepared using commercially available type III human collagen and commercially available hemostatic sponge. (6) The bone repair material prepared using the recombinant humanized type III collagen of the present invention has a better function of promoting osteoblast growth than the bone repair material prepared using commercially available type III human collagen or natural type I collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the electrophoresis diagram of the linearized nucleic acid of the recombinant humanized collagen A expression vector.

[0044] Figure 2 This is the electrophoresis diagram of the linearized nucleic acid of the recombinant humanized collagen B (before optimization) expression vector.

[0045] Figure 3 This is the electrophoresis diagram of the linearized nucleic acid of the recombinant humanized collagen B (optimized) expression vector.

[0046] Figure 4 This is the electrophoresis diagram of the linearized nucleic acid of the recombinant humanized collagen C expression vector.

[0047] Figure 5 This is an SDS-PAGE analysis of the induction expression of recombinant humanized collagen A. Lane 1 is the GS115 competent induced expression supernatant (negative control); lane 2 is recombinant humanized collagen A.

[0048] Figure 6This is an SDS-PAGE analysis of the induction expression of recombinant humanized collagen B (before optimization). Lane 1 is the GS115 competent induced expression supernatant (negative control); lane 2 is the recombinant humanized collagen B (before optimization).

[0049] Figure 7 This is an SDS-PAGE analysis of the inducible expression of recombinant humanized collagen B (after optimization). Lane 1 is recombinant humanized collagen B (before optimization); lane 2 is recombinant humanized collagen B (after optimization).

[0050] Figure 8 This is an SDS-PAGE analysis of the induction expression of recombinant humanized collagen C. Lane 1 is the GS115 competent induced expression supernatant (negative control); lane 2 is recombinant humanized collagen C.

[0051] Figure 9 This is the SDS-PAGE detection image of the recombinant humanized collagen fermentation broth.

[0052] Figure 10 This is an SDS-PAGE detection chart of crude purification of recombinant humanized collagen.

[0053] Figure 11 This is the chromatogram of recombinant humanized collagen purification.

[0054] Figure 12 This is the SDS-PAGE detection picture after purification of recombinant humanized collagen

[0055] Figure 13 This is an in vitro hemostasis model evaluating related devices and blood flow. The first photo on the left shows the evaluation device developed in accordance with industry standard YY1477.5-2020. The three photos on the right show the location where blood enters the syringe containing the test sample.

[0056] Figure 14 Schematic diagram of the hemostatic sponge implantation site

[0057] Figure 15 20G05 VS "Aiweiting" implant site muscle tissue microscopic examination results

[0058] Figure 16 Schematic diagram of the application sites of rabbit irritation or intradermal reaction test, in the figure 1 - head; 2 - test site; 3 - control site; 4 - hairless back area; 5 - tail

[0059] Figure 17 Results of acute toxicity experiment on collagen sponge in ICR mice

[0060] Figure 18Microstructure of 20G05-HA bone repair material

[0061] Figure 19 Microstructure of 19G291-HA bone repair material

[0062] Figure 20 Microstructure of COL-HA bone repair material

[0063] Figure 21 20G05-HA cells were inoculated and cultured for 2, 5, and 7 days

[0064] Figure 22 19G291-HA cells were inoculated and cultured for 2 days

[0065] Figure 23 COL-HA cells were inoculated and cultured for 2, 5, and 7 days

[0066] Figure 24 Schematic diagram of the application site of 20G05-HA bone repair material stimulation or intradermal reaction experiment

[0067] Figure 25 Results of acute toxicity test on ICR mice using 20G05-HA bone repair material

[0068] Figure 26 Cell growth on the surface and sides of the sample inoculated with MC3T3-E1 cells after 7 days of culture

[0069] Figure 27 Cell growth on the surface and side of the sample inoculated with MC3T3-E1 cells on day 7 of culture

[0070] Figure 28 Cell growth on the surface and side of the sample inoculated with MC3T3-E1 cells on day 7 of culture

[0071] Figure 29 Cell growth on the surface and side of the sample inoculated with MC3T3-E1 cells after 14 days of culture

[0072] Figure 30 Cell growth on the surface and side of the sample inoculated with MC3T3-E1 cells after culturing for 14 days with 20G05-HA-2

[0073] Figure 31 Cell growth on the surface and side of the sample inoculated with MC3T3-E1 cells on 20G05-HA-3 for 14 days DETAILED DESCRIPTION

[0074] The present invention will be described in further detail below with reference to the embodiments and accompanying drawings.

[0075] Example 1 Construction of a Pichia pastoris expression system containing type III collagen gene

[0076] (1) Construction of recombinant expression vector

[0077] 1) Genetic Design

[0078] Based on the amino acid sequence of human type III collagen α1 chain (P02461-1) published in the protein resource database UniProt (website: http: / / www.uniprot.org / ), the core functional amino acids of collagen were retained and the amino acid sequence was optimized according to the codon preference of Pichia pastoris. The main design schemes are as follows: (1) Residues 1 to 153 of the N-terminal propeptide and residues 1233 to 1466 of the C-terminal propeptide were removed, and amino acids 154 to 1232 were retained; (2) Residues 1 to 153 of the N-terminal propeptide were removed, and amino acids 154 to 1466 were retained; (3) Amino acids 154 to 615 were selected. Then, an EcoRI restriction site was introduced at the 5' end, a NotI restriction site was carried at the 3' end, and a stop codon was introduced before the 3' NotI restriction site. All genes were codon-optimized for the Pichia pastoris expression system, and then the relevant gene sequences were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The corresponding amino acid sequences and nucleotide sequences are shown in the following table:

[0079] Table 1 Sequence design of different human type III collagen

[0080]

[0081]

[0082] 2) Connection conversion

[0083] The pGAPZαA expression vector and each COL3A1 gene were double-digested with EcoRI and NotI, respectively, at 37°C for 4 hours. The pGAPZαA vector backbone and COL3A1 gene were then excised from gels and ligated using the following system: 2 μl of vector fragment, 6 μl of target gene, 1 μl of T4 DNA ligase, and 1 μl of T4 DNA ligase buffer, for 1 hour at 22°C. The ligation products were transformed into E. coli TOP10 cloning host cells and plated on LB plates containing 25 μg / ml bleomycin. Single colonies were picked and transferred to 2 ml sterile tubes containing 300 μl of LB liquid medium. After incubation at 37°C, 220 rpm for 4 hours, 1 μl of the culture medium was collected for PCR analysis. Positive single colonies were identified as recombinant E. coli harboring the target gene. Positive single clones were inoculated into 200 ml of liquid LB medium and cultured overnight at 37°C and 220 rpm. Plasmids were extracted using the Tiangen Endotoxin-Free Plasmid Extraction Kit to obtain a large number of recombinant expression plasmids that can be used to transform Pichia pastoris.

[0084] (2) Transformation of Pichia pastoris

[0085] The above recombinant expression plasmid was linearized using AvrII endonuclease ( Figure 1-4 The linearization system consisted of 3 μl of AvrII enzyme, 100 μg of recombinant expression plasmid, 100 μl of 10× Cutsmart Buffer, and ddH2O to 1 ml. Enzyme digestion was performed at 37°C for 8–12 hours. The linearization system was first inactivated at 65°C for 5 minutes. The enzyme was then inactivated by adding 900 μl of isopropanol, incubating at -20°C for 10 minutes, and centrifuging at 14,000 rpm at 4°C to collect the precipitate. The precipitate was washed twice with 70% ethanol, air-dried in a laminar flow hood, and then added with 80 μl of ddH2O to obtain the linearized product. The linearized product was then transformed into competent Pichia pastoris GS115 cells, and recombinant yeast strains were obtained by plating on YPD plates containing 0.1 mg / ml bleomycin. Single colonies were picked from the electroporation plates and replicated on YPD plates containing different concentrations of bleomycin to obtain multi-copy recombinant yeast strains resistant to 2 mg / ml bleomycin.

[0086] (3) Expression identification

[0087] The above multi-copy yeast strains and GS115 competent cells were inoculated into 5 ml of BMGY medium (GS115 competent cells were used as negative control and induced at the same time), cultured at 30°C, 200 rpm for 3 days, supplemented with 1% glycerol every 24 h, and the induced expression supernatant was collected for SDS-PAGE analysis ( Figure 5-8), according to the SDS-PAGE test results, the designed four fragments can effectively express recombinant humanized collagen, but the expression levels of each protein are quite different, as shown in the following table:

[0088] Table 2 Collagen expression in different designs

[0089]

[0090] Based on the molecular weight and expression level of the expressed protein, the strain expressing recombinant humanized collagen B (after optimization) was finally determined as the genetically engineered bacteria expressing human-like collagen. This was subsequently used as the engineering strain for the invention description. The expression of other fragments did not achieve ideal results.

[0091] Example 2 Induced expression of recombinant humanized collagen

[0092] The genetically engineered bacteria are fermented and cultured to obtain a fermentation liquid containing recombinant humanized collagen.

[0093] The fermentation medium used was:

[0094] (1) Seed medium (YPD): peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L;

[0095] (2) Fermentation medium (BSM): phosphoric acid 26.7 ml / L, CaSO4·2H2O 0.93 g / L, K2SO4 18.2 g / L, MgSO4·2H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40 g / L, PTM1 4.0 ml / L.

[0096] PTM1: CuSO4·5H2O 6.0g / L, KI 0.088g / L, MnSO4·2H2O 3.0g / L, Na2M O O4·2H2O0.2g / L, H3BO3 0.02g / L, CoCl2·6H2O 0.5g / L, ZnCl2 20.0g / L, FeSO4·7H2O 65.0g / L, Biotin 0.2g / L, concentrated H2SO4 5.0ml, filter sterilize, and store at 4℃.

[0097] (3) Feed medium: 50% glycerol (W / V), containing 12 ml of PTM1 trace elements per 1 L.

[0098] The fermentation and induction expression process of recombinant humanized collagen engineering bacteria is as follows:

[0099] First, the engineered bacteria were inoculated into YPD liquid medium for cultivation at 30°C.600 When the cell count reaches 4-8, it can be used as a fermentation seed. Set the fermentation tank parameters to 30°C, 200-700 rpm, 4 L / min of ventilation (automatically adjusted by the fermentation tank), maintain dissolved oxygen above 30%, and adjust the pH to 5.0 with ammonia water. Then, add the fermentation seed liquid to a sterile fermentation tank and begin fermentation. When the carbon source is exhausted, start adding feed medium. When the wet weight of the bacteria reaches 220 g / L, end the fermentation and collect the fermentation liquid by centrifugation.

[0100] The expression of target protein was detected by SDS-PAGE. Figure 9 As shown, the yield is approximately 10 g / L.

[0101] The present invention utilizes Pichia pastoris to produce recombinant humanized collagen, and does not require the addition of methanol during the fermentation process, thereby saving production costs, simplifying the operation process, shortening the fermentation cycle, and ensuring high-level expression of the recombinant humanized collagen.

[0102] Example 3 Purification of recombinant humanized collagen

[0103] The fermentation broth is purified to obtain recombinant humanized collagen. The purification steps are as follows:

[0104] (1) Add ammonium sulfate to the fermentation broth at a concentration of 160 g / L, then centrifuge at 8000 rpm to collect the salting-out precipitate, and redissolve the salting-out precipitate with purified water ( Figure 10 );

[0105] (2) Add 0.8 M ammonium sulfate to the above-mentioned salting-out precipitation and redissolved product, adjust the pH to 7.4, filter through a filter membrane, and perform chromatography purification using a hydrophobic chromatography column. The purification binding buffer is 10 mM sodium dihydrogen phosphate, 0.8 M ammonium sulfate, pH 7.4, and the elution buffer is 10 mM sodium dihydrogen phosphate, pH 7.4. During the purification process, the elution peak ( Figure 11 ), SDS-PAGE test results show that after this purification, the purity of recombinant humanized collagen can reach more than 97% ( Figure 12 ).

[0106] (3) After freeze-drying, pure recombinant humanized collagen was obtained.

[0107] The purification method of the present invention uses only one step of salting out and one step of hydrophobic chromatography purification. No liquid change is required during the purification process, the operation is simple, the production cycle is short, and the final purity can reach more than 97%, which can meet the requirements of biomaterial development.

[0108] Example 4 Safety test of collagen raw materials

[0109] In order to verify whether the recombinant humanized collagen 20G05 expressed in the present invention meets the requirements of biosafety standards, the following safety evaluation was performed, using natural bovine type I collagen COL (purchased from Hebei Kaolisen Biotechnology Co., Ltd.) as a control.

[0110] <Cytotoxicity Assay>

[0111] Brief description of experimental method:

[0112] Table 3

[0113] Cell name L929 culture medium MEM + 10% FBS medium Number of seed plates 5000 pieces / hole Dosage Protein solution administration Co-culture conditions 24h，(37±1)℃ Detection method CCK8 method Determination method Survival rate>70%, determined to be non-cytotoxic

[0114] The two collagen raw materials, 20G05 and COL, were dissolved in culture medium at a concentration of 1.25 mg / mL. After filtration, L929 cell toxicity tests were performed to evaluate the cytotoxicity of the collagen raw materials themselves. The experimental results are as follows:

[0115] Table 4

[0116]

[0117] The above results indicate that the 20G05 and COL raw materials exhibit no significant cytotoxicity and are relatively safe for use in the preparation of collagen hemostatic sponges. Based on this, it can be inferred that the recombinant humanized type III collagen of the present invention can be used in the preparation of a variety of products, including bone repair materials, hemostatic sponges, implantable fillers for medical or aesthetic purposes, and cosmetics, without any safety concerns.

[0118] Example 5 Preparation of hemostatic sponge samples

[0119] 1% (wt / vol) of ① recombinant type III humanized collagen 20G05, ② recombinant type III humanized collagen 19G291 (purchased from Jiangshan Juyuan Biotechnology Co., Ltd., or prepared according to Chinese patent CN102443057B), and ③ natural bovine type I collagen COL (purchased from Hebei Kaolisen Biotechnology Co., Ltd.) were added to a 3% (vol / vol) acetic acid solution, respectively. The solutions were magnetically stirred at room temperature until the proteins were completely dissolved and filtered using a filter membrane to prepare three collagen solutions.

[0120] The three collagen solutions were injected into 100*100 mm molds respectively and freeze-dried in a freeze dryer according to the freeze-drying curve (keeping at -20°C for 5 hours and gradually heating to room temperature within 30 hours) to prepare collagen sponge samples.

[0121] The three freeze-dried collagen sponge samples were placed in a vacuum drying oven and vacuum-crosslinked at 150°C for 10 hours to obtain collagen hemostatic sponge samples, which were respectively designated as 20G05-DHT, 19G291-DHT, and COL-DHT.

[0122] This example is only used to illustrate the present invention, but is not intended to limit the scope of the present invention. The relevant preparation conditions are not limited to the above conditions. For example, the acetic acid solution can be further replaced by dilute hydrochloric acid, and the vacuum thermal crosslinking method can also be replaced by a chemical crosslinking method.

[0123] Example 6 Evaluation of water absorption of hemostatic sponge

[0124] Water absorption is an important physical and chemical performance indicator of hemostatic sponges. A high water absorption rate (the industry standard is >2000%) is the basic condition for achieving hemostasis. The water absorption rate of three collagen sponges was tested as follows:

[0125] Take a certain amount of sponge, weigh the dry weight as m1, immerse it in purified water, press lightly with tweezers until it is completely soaked, and leave it for 2 hours to ensure that all air is completely removed to ensure the integrity of the sample. After the sample absorbs enough water, use small tweezers to gently pinch a corner and remove it from the water. Be careful not to squeeze the sample. Hold the tweezers lightly on the water surface to drain for 1 minute, then weigh the wet weight of the sample again as m2. The water absorption rate is calculated according to the following formula. Repeat the test 5 times and take the average value. Calculation formula: Water absorption rate (%) = (m2-m1) / m1*100%. The water absorption rate of the three samples and the reference product "Aiweiting Microfiber Hemostatic Collagen (Sponge)" (purchased from Bard Medical Technology (Shanghai) Co., Ltd.) were tested separately. The results are shown in the following table.

[0126] Table 5 Water absorption of each sample

[0127]

[0128] The experimental results show that the water absorption rates of the three samples and the control are all higher than the 2000% water absorption rate requirement stipulated by the industry standard. Among them, the water absorption rates of the 20G05-DHT and COL-DHT samples are higher, which has certain advantages in achieving hemostatic function.

[0129] Example 7 Evaluation of the in vitro coagulation efficacy of hemostatic sponge

[0130] Hemostatic function is the main indicator for evaluating the efficacy of hemostatic sponges. In vitro coagulation experiments were used to compare the efficacy of collagen hemostatic sponges. The specific methods are as follows:

[0131] Experimental animals: rabbits

[0132] Test samples: 1) Negative control group: gauze (10 mg / sample); 2) Positive control: Aiweiting microfiber hemostatic collagen (sponge) (10 mg / sample); 3) Test sample: 2 types of recombinant collagen hemostatic sponges (10 mg / sample)

[0133] Operation steps: 1) Prepare anticoagulated rabbit blood: Mix 15 mg / mL EDTA anticoagulant with fresh rabbit blood at a ratio of 1:9; 2) Place the test sample (negative control, positive control, test sample) in a 50 mL centrifuge tube, and prepare a group of empty 50 mL centrifuge tubes as a blank control group; 3) Mix anticoagulated rabbit blood and 0.2 M calcium chloride solution at a ratio of 9:1, and add 0.5 mL of mixed rabbit blood to each centrifuge tube; 4) Place the centrifuge tube containing rabbit blood in a 37°C water bath for 5 minutes, during which time ensure that the sponge is in full contact with the blood; 5) Add 40 mL of distilled water to each centrifuge tube and shake at 37°C and 120 rpm / min for 20 minutes (to rupture free red blood cells); 6) Take the supernatant in the centrifuge tube and detect the absorbance at 540 nm.

[0134] Data processing method: BCI is an indicator of coagulation effect and is processed as follows:

[0135] BCI (%) = sample absorbance / blank control group absorbance * 100% (the smaller the BCI value, the better the hemostatic effect of the hemostatic material)

[0136] In vitro coagulation function tests were performed on the two samples and the reference substance "Aiweiting Microfibrillar Hemostatic Collagen (Sponge)". The results are shown in the following table.

[0137] Table 6

[0138]

[0139] The results of in vitro coagulation experiments show that compared with the commercially available reference product "Aiweiting Microfiber Hemostatic Collagen (Sponge)" and other hemostatic sponges prepared with recombinant type III collagen, 20G05-DHT has obvious efficacy advantages (the higher the BCI value, the worse the coagulation effect). Therefore, the hemostatic sponge prepared with 20G05 collagen can achieve better hemostatic function.

[0140] Example 8 In vitro hemostasis model evaluation

[0141] Experimental animals: rabbits

[0142] Test products: 1) Sample: 20G05 recombinant type III humanized collagen sponge; 2) Control: Aiweiting microfibrillar hemostatic collagen (sponge)

[0143] Operation steps (developed in accordance with industry standard YY1477.5-2020):

[0144] 1) Experimental device construction:

[0145] The experimental device for constructing an in vitro model of hemostasis performance mainly consists of a sample clamping device, a negative pressure system, and connecting pipes, such as Figure 13 As shown:

[0146] Use a 1mL syringe barrel and a 0.3mm flat needle. Cut the sample into 5mm diameter, 2mm thick discs. Use the syringe's core rod to insert each sample into the bottom of the syringe. Then, push the rubber fixture (cut off the blind end of the syringe's rubber piston to make a fixture with a through hole, and the uncut smooth end will contact the sample) over the sample. Connect the tail end of the syringe to the negative pressure line to complete the device.

[0147] 2) Prepare anticoagulated rabbit blood: Mix 15 mg / mL EDTA anticoagulant with fresh rabbit blood at a ratio of 1:9.

[0148] 3) Cut the test sample and reference substance according to the requirements of step 1 to prepare three replicates. Load each of the cut samples into a 1 mL syringe according to the method of step 1 for later use.

[0149] 4) Perform the experiment sequentially: 0.9 mL of anticoagulated rabbit blood and 0.1 mL of 0.2 M calcium chloride solution are mixed in a 2 mL centrifuge tube. The centrifuge tube is placed at the bottom of a clamping device. The syringe containing the test sample prepared in step 3 is mounted on the clamping device. The negative pressure line is connected and a constant negative pressure is activated. The syringe is lowered and the needle is inserted into the rabbit blood. The rabbit blood is allowed to rise at a uniform rate and pass through the test sample.

[0150] 5) Start timing from the moment the blood contacts the bottom surface of the test sample, and observe the rise of the blood within 60 seconds. After contact with the test sample, the hemostatic effect of the sample will slow the rate of blood rise to varying degrees. By comparing the rise of the test sample and the control, the difference in hemostatic effect can be analyzed.

[0151] Experimental results:

[0152] 1) The rise of blood pressure within 60 seconds is as follows Figure 13 As shown;

[0153] 2) In the 20G05 recombinant type III humanized collagen sponge group, blood no longer rose after passing through the test sample. Analysis of the test sample revealed internal coagulation, which was the primary cause of the blood's continued rise. In the control group, blood rose to a significantly higher level within 60 seconds and continued to rise, indicating that the control's ability to slow the rate of blood rise was limited. Therefore, a comparison of the blood rise in the test sample and control indicates that the 20G05 recombinant type III humanized collagen sponge demonstrated significantly superior hemostatic function to the control in the in vitro hemostasis model, demonstrating a clear advantage in achieving hemostasis.

[0154] Example 9 Hemostatic sponge liver hemostasis test

[0155] Test products: 1) Sample: 20G05 recombinant type III humanized collagen sponge; 2) Control: Aiweiting microfiber hemostatic collagen (sponge), Bonase medical collagen sponge (purchased from Bonage Technology (Beijing) Co., Ltd.).

[0156] Test method:

[0157] 1) Cut the hemostatic sponge and sterile gauze into several squares with a side length of 1.5 cm and similar thickness. Weigh them and place them on the surgical tray for later use.

[0158] 2) Weigh the rats. Inject 10% chloral hydrate anesthetic into the abdomen according to the body weight to give the rats general anesthesia. Fix the rats (back facing down). Remove the 4×4 cm area around the chest. 2 The hair of the area was disinfected with 75% medical alcohol and covered with a sterile towel after disinfection. The entire experimental process was performed under sterile conditions.

[0159] 3) Make a 3-cm vertical incision along the midline of the upper abdomen, below the costal arch. Open the abdomen layer by layer, cutting through the muscle layer to expose the liver. Place sterile gauze beneath the incision and free the middle lobe of the liver from the abdominal cavity onto the gauze.

[0160] 4) Use gauze to clean the surface of the liver and surrounding fluid. Use a scalpel to partially resect the liver. When bleeding is evident, quickly wipe away the blood with gauze. Immediately apply hemostatic material to the bleeding incision and apply gentle pressure.

[0161] 5) Cover the wound with hemostatic material for 1 minute, then gently remove it and observe the wound for bleeding. If active bleeding occurs, continue covering and applying pressure. If the material is completely soaked with blood, replace it with a new, identical material. Repeat the application of pressure until the wound stops bleeding. The hemostatic standard is the absence of active bleeding within 30 seconds after removing the sponge. Record the amount of bleeding from the liver wound.

[0162] Result evaluation

[0163] Bleeding amount (g) = after hemostasis (weight of gauze and hemostatic material) - before hemostasis (weight of gauze and hemostatic material)

[0164] Table 7

[0165] Weight of gauze and hemostatic materials before hemostasis (g) Weight of gauze and hemostatic materials after hemostasis (g) Amount of bleeding (g) Ai Weiting 19.2 20.7 1.5 20G05 19.5 19.7 0.2 Bonasse 126.4 127 0.6

[0166] Aiweiting: Blood coagulation was not completed at the end of the trial, and active bleeding was still occurring in the wound.

[0167] 20G05: The wound size is similar to that of Aiweiting. Coagulation is completed at the end of the test, and there is no need to press during the hemostasis process. It can stick to the wound by itself. After there is no active bleeding, it can be easily removed without adhesion.

[0168] Bonase: The size of the wound is similar to that of Aiweiting. At the end of the trial, there is still slight active bleeding in the wound. The wound cannot be closed by itself and needs to be pressed.

[0169] Example 10 Safety Evaluation of Hemostatic Sponge

[0170] In order to verify that the hemostatic sponge prepared with 20G05 recombinant humanized collagen meets the biosafety standards, the following safety evaluation was conducted, using the commercially available "Aiweiting microfiber hemostatic collagen (sponge)" as a control.

[0171] <Cytotoxicity Evaluation>

[0172] Extraction method: Extraction ratio: 3cm 2 / mL, extraction solution: MEM medium + 10% FBS, extraction time: (24±2)h, extraction temperature: (37±1)℃. Brief description of experimental method:

[0173] Table 8

[0174] Cell name L929 Number of seed plates 5000 pieces / hole Dosage Extract administration Co-culture time 24h Detection method CCK8 method Determination method Survival rate>70%, determined to be non-cytotoxic

[0175] The cytotoxicity evaluation tests of the 20G05 sponge sample and the reference substance "Aiweiting microfibrillar hemostatic collagen (sponge)" were performed respectively, and the results are shown in the following table.

[0176] Table 9

[0177]

[0178] From the cytotoxicity results, it can be seen that compared with the commercially available reference product "Aiweiting Microfiber Hemostatic Collagen (Sponge)", the hemostatic sponge prepared by 20G05 collagen also has no obvious cytotoxicity.

[0179] <Hemolysis test>

[0180] Extraction method: Extraction ratio: 3cm 2 / mL, extraction solution: normal saline, extraction time: (72±2)h, extraction temperature: (37±1)℃.

[0181] The specific method is as follows:

[0182] 1) Test sample preparation: ① Sample extract preparation: Prepare according to the above extraction method; ② Positive control: Purified water, no preparation required; ③ Negative control: 0.9% sodium chloride injection, no preparation required.

[0183] 2) Preparation of Red Blood Cell Suspension: Take sterile defibrinated rabbit blood, add 10-fold the volume of 0.9% sodium chloride injection, and mix thoroughly. Centrifuge at 300g for 15 minutes, remove the pellet, and discard the supernatant. Continue centrifuging until the supernatant no longer appears red. Dilute the resulting red blood cells with 0.9% sodium chloride injection to a 2% red blood cell suspension for later use.

[0184] 3) Sample addition and incubation:

[0185] Table 10

[0186] Sample extract / mL 0.9% sodium chloride injection / mL Purified water / mL 2% red blood cell suspension / mL Sample Group 1 0 0 1 Positive control 0 0 1 1 Negative control 0 1 0 1

[0187] Add samples according to the above table, mix well, and immediately place in a 37°C water bath and incubate for 3 hours.

[0188] 4) Observation and Evaluation of Results: ① Incubate each tube in the aqueous solution for 15 min, 30 min, 45 min, 1 h, 2 h, and 3 h. Visually observe the results. Pour the contents of the tube and centrifuge at 800g for 5 min. Transfer 200 μL of the supernatant to a 96-well plate. Place the 96-well plate in a microplate reader and measure the absorbance at 540 nm. ② A hemolysis rate of less than 5% meets the test requirements.

[0189] HR=(AB) / (CB)×100%

[0190] Where: HR: hemolysis rate of the test sample (%); A: absorbance of the test sample group; B: absorbance of the negative control group; C: absorbance of the positive control group

[0191] The prepared 20G05 collagen sponge was evaluated by in vitro hemolysis test with the negative control saline and the positive control purified water. The experimental results are shown in the following table:

[0192] Table 11

[0193]

[0194] The in vitro hemolysis experiment showed that the results of the in vitro hemolysis evaluation of the 20G05 recombinant type III humanized collagen sponge met the requirements of the national standard (hemolysis rate <5%) and was safe and reliable in terms of hemolysis.

[0195] Example 11 Hemostatic sponge muscle implantation experiment

[0196] The specific method is as follows:

[0197] Experimental animals: rabbits (New Zealand rabbits, 2.0-3.0 kg, male)

[0198] Procedure: 1) Using the trocar implantation method, place the implant parallel to the long axis of the muscle fiber. 2) When using the paraspinal muscles of rabbits, implant four test material samples into the muscle on one side of the spine of each rabbit, parallel to the spine, 25 to 50 mm from the midline, with each implant spaced approximately 25 mm apart. Implant four control material samples on the other side of the spine using the same method. Implantation site: Figure 14 shown.

[0199] Implantation specifications: Refer to the requirements for block materials in GB16886.6-2015 Biological Evaluation of Medical Devices Part 6: Local Reaction Test after Implantation, and make test samples with a diameter of 1-3 mm, a length of 10 mm, and rounded ends.

[0200] Clinical Observation: Each implant site should be inspected for changes in normal tissue architecture, preferably including assessment of local draining lymph nodes. Low-power magnification is recommended. The nature and extent of any observed tissue reactions, such as hematoma, edema, cysts, and / or other gross findings, should be recorded. The presence, morphology, and location of the implant, including any possible residue of resolvable material, should also be documented. Gross photographs should be documented.

[0201] In addition to inspection of the implant site, a gross necropsy should be performed if appropriate if the animal shows signs of distress or reaction to the implant.

[0202] Implant sample collection and evaluation: The tissue at the implant site was collected, fixed, dehydrated, and embedded to make a wax block. The wax block was sectioned, stained with HE, and the sample was evaluated histologically under an optical microscope.

[0203] Evaluation indicators: 1) degree of inflammation; 2) material parameters, such as rupture and / or fragmentation, shape and location of degradable material residues.

[0204] The 20G05 collagen sponge and the positive control substance "Aiweiting microfiber hemostatic collagen (sponge)" were used to test the local reaction after intramuscular implantation in rabbits. The experimental results are as follows:

[0205] Clinical Observation and Evaluation: One rabbit died one day after implantation (due to postoperative infection). The remaining rabbits showed no bleeding, swelling, or sample expulsion at the implantation site on days 1, 3, and 5. During the implantation period, the animals were observed to be in good general condition and showed no abnormalities.

[0206] Anatomical observation and evaluation: No inflammatory reaction or other abnormalities were observed in the muscle tissue at the implantation site 1, 2, 4, and 8 weeks after implantation.

[0207] Microscopic evaluation: Microscopic examination results are as follows: Figure 15 shown.

[0208] Both the 20G05 sponge sample and the positive control substance "Aiweiting" showed normal degradation status in rabbit muscles. It was preliminarily judged that the degradation period of the 20G05 sample in rabbit muscles was 4-8 weeks.

[0209] The results of the local reaction test after intramuscular implantation in rabbits showed that the 20G05 collagen sponge met the requirements of national standards.

[0210] Example 12 Hemostatic sponge stimulation or intradermal reaction experiment

[0211] Extraction method: Extraction ratio: 3cm 2 / mL, extraction solution: normal saline, extraction time: (72±2)h, extraction temperature: (37±1)℃.

[0212] Fixation method: Use breathable tape for fixation (tape and gauze may fall off after fixation, so frequent observation is required).

[0213] Animals administered: Rabbits

[0214] The specific method is as follows:

[0215] 1) Test sample preparation: ① Test sample: sample extract. ② Positive control: 20% SDS saline solution (prepare immediately for use). ③ Negative control: saline solution.

[0216] 2) Dosage method: ① Cut the absorbent gauze into 2.5cm×2.5cm size and stack 5-6 layers. ② Drop the corresponding extract onto the gauze. The amount of extract should be enough to soak the gauze. Generally, drop 0.5mL on each gauze. Figure 16 Apply the patch to the indicated areas on both sides of the animal's back. ③ Secure the patch with a bandage (semi-occlusive or occlusive) for at least 4 hours. ④ After the contact period, remove the patch and mark the contact area with permanent ink. ⑤ Remove any remaining test material using appropriate methods, such as washing with warm water or other suitable non-irritating solvents and wiping dry. ⑥ Route of administration: Skin contact. ⑦ Dosage frequency and rate: Single contact test, secure the patch for at least 4 hours.

[0217] 3) Observation and Scoring: ① Observation Frequency and Duration: For single-contact testing, record the contact site condition at (1±0.1) h, (24±2) h, (48±2) h, and (72±2) h after patch removal. ② Observation Content: Observe and score the erythema and edema induced by the test material on the animal's skin over the specified time period. ③ Animal Reaction Observation and Assessment: Record the score for each test site according to the table below.

[0218] Table 12 Skin reaction scoring system

[0219]

[0220] 4) Result evaluation:

[0221] ①Calculate the primary irritation index (PII)

[0222] After 72 hours of scoring, the primary irritation scores of all erythema and edema caused by the test materials at (24 ± 2) hours, (48 ± 2) hours, and (72 ± 2) hours for each animal were added together, and then the sum of all scores was divided by 6 (two test / observation sites, three time points) to obtain the PII.

[0223] Note: Only the observation data of (24±2)h, (48±2)h and (72±2)h were used for calculation.

[0224] ②Report the corresponding reaction type

[0225] According to the primary irritation index, the reaction type of each group of test samples is reported in the table below to evaluate the potential of the hemostatic sponge samples to produce skin irritation under the test conditions.

[0226] Table 13 Rabbit primary or cumulative irritation index type

[0227] Average score Reaction type 0～0.4 Very slight 0.5～1.9 Mild 2～4.9 poisoning 5～8 severe

[0228] Sample 20G05 collagen sponge, negative control saline, and positive control 20% SDS were used to conduct rabbit irritation or intradermal reaction tests. The experimental results are shown in the following table:

[0229] Table 14 Rabbit irritation or intradermal reaction test results 1

[0230]

[0231] Table 15 Rabbit irritation or intradermal reaction test results 2

[0232]

[0233] The results of the rabbit irritation or intradermal reaction test showed that the irritation index of the 20G05-DHT sample extract on rabbit skin was consistent with that of the negative control saline. Therefore, the results of the 20G05 collagen sponge rabbit irritation or intradermal reaction test evaluation met the requirements of the national standard.

[0234] Example 13 Acute toxicity test of hemostatic sponge

[0235] Extraction method: Extraction ratio: 3cm 2 / mL, extraction solution: physiological saline / vegetable oil, extraction time: (72±2)h, extraction temperature: (37±1)℃.

[0236] Experimental animals: ① Species / Strain: ICR mice; ② Gender: Male; Animal grade: ③ SPF grade; ④ Animal size: 17-23g

[0237] The specific method is as follows: 1) Dosing frequency and rate: Dosing is based on the animal's body weight, single administration, and the injection speed is constant and does not exceed 0.1 mL / s.

[0238] Table 16 Grouping and dosing information

[0239]

[0240] 2) Clinical Observation: ① Test Animals: 1-4 groups of animals; ② Observation Frequency and Duration: After the extract injection, observe the animal's immediate reaction. Observe and record the general condition, toxicity signs, and number of deaths in each group 4, 24, 48, and 72 hours later. ③ Observation Details: Includes, but is not limited to, animal death or near-death, as well as mental state, behavioral activity, feeding habits, stool characteristics, respiratory status, and eyelid condition.

[0241] 3) Weight: ① Animals to be tested: 1-4 groups of animals; ② Testing period: Day 1-Day 4, with body weight measured at a fixed time daily. Moribund animals are not weighed; ③ Testing duration: Planned to end on Day 4. Plans developed under unknown circumstances may be adjusted based on actual conditions, and changes to the plan must be recorded.

[0242] 4) Result Evaluation: ① If the reaction of the test group animals is no greater than that of the solvent control group animals during the 72-hour observation period, the test sample is judged to have no acute systemic toxic reaction. ② If two or more test group animals develop moderate toxic symptoms or die, or three or more test group animals experience a weight loss exceeding 10%, the test sample is judged to have an acute systemic toxic reaction. ③ If the test animals develop mild toxic symptoms, or no more than one animal develops moderate toxic symptoms or dies, or if there are no toxic symptoms but the animals in the group generally experience a weight loss, 10 additional mice will be retested.

[0243] Sample 20G05 collagen sponge was subjected to acute toxicity test in ICR mice. The experimental results are shown in the following table and Figure 17 As shown:

[0244] Table 17 Results of acute toxicity test in mice

[0245]

[0246]

[0247] Table 18

[0248]

[0249] The results of the acute toxicity study on ICR mice showed no significant differences in body weight between the 20G05 saline / vegetable oil extract group and the solvent control group. Furthermore, behavioral observations revealed that all mice in each group were normal. The results of the acute toxicity study on the 20G05 collagen sponge in ICR mice met the requirements of national standards.

[0250] Example 14 Preparation of hemostatic sponges with different collagen concentrations and evaluation of their water absorption

[0251] <Sample preparation>

[0252] 3%, 5%, and 10% (wt / vol) recombinant type III humanized collagen 20G05 were added to 1% (vol / vol) acetic acid solution, respectively. The solution was magnetically stirred at room temperature until the protein was completely dissolved, and then filtered using a filter membrane to prepare three collagen solutions.

[0253] The three collagen solutions were injected into 100*100 mm molds respectively and freeze-dried in a freeze dryer according to the freeze-drying curve (keeping at -20°C for 5 hours and gradually heating to room temperature within 30 hours) to prepare collagen sponge samples.

[0254] The three freeze-dried recombinant type III humanized collagen sponge samples were placed in a vacuum drying oven and vacuum-crosslinked at 150°C for 10 hours to obtain recombinant type III humanized collagen sponge samples, which were designated as 20G05-1, 20G05-2, and 20G05-3, respectively.

[0255] <Water Absorption Evaluation>

[0256] The specific experimental method is carried out with reference to Example 6.

[0257] The water absorption rate of the three samples was tested and compared with the control. The results are shown in the following table.

[0258] Table 19

[0259]

[0260] The experimental results show that the water absorption rates of the three samples are all higher than the 2000% water absorption rate requirement stipulated by the industry standard. Among them, 20G05-1 has the highest water absorption rate. The other two samples also have high water absorption rates, which are higher than the control, and have certain advantages in achieving hemostatic function.

[0261] Example 15 Preparation of hemostatic sponges with different cross-linking conditions and evaluation of water absorption

[0262] 1% (wt / vol) recombinant type III humanized collagen 20G05 was added to 3% (vol / vol) acetic acid solution, and the mixture was magnetically stirred at room temperature until the protein was completely dissolved. The mixture was then filtered using a filter membrane to prepare a collagen solution.

[0263] The collagen solutions were injected into 100*100 mm molds, and freeze-dried in a freeze dryer according to the freeze-drying curve (keeping at -20°C for 5 hours and gradually heating to room temperature within 30 hours) to prepare collagen sponge samples.

[0264] The freeze-dried collagen sponge sample was placed in a vacuum drying oven and vacuum thermally cross-linked at 140°C, 150°C, and 160°C for 10 hours to obtain three recombinant type III humanized collagen sponge samples, which were respectively designated as 20G05-140, 20G05-150, and 20G05-160.

[0265] Water absorption is an important physical and chemical performance indicator of hemostatic sponges. A high water absorption rate (the industry standard is >2000%) is the basic condition for achieving hemostasis. The water absorption rate of three collagen sponges was tested as follows:

[0266] Take a certain amount of sponge, weigh the dry weight as m1, immerse it in purified water, and gently press with tweezers until it is completely soaked. Let it sit for 2 hours to ensure that all air is completely removed to ensure the integrity of the sample. After the sample has absorbed enough water, use small tweezers to gently pinch a corner and remove it from the water, being careful not to squeeze the sample. Gently hold the tweezers on the water surface to drain for 1 minute, then weigh the wet weight of the sample again as m2. The water absorption rate is calculated as follows. Repeat the test 5 times and take the average value. Calculation formula: Water absorption rate (%) = (m2-m1) / m1*100%. The water absorption rate of the three samples was tested separately and compared with the control. The results are shown in the following table.

[0267] Table 20

[0268]

[0269] The experimental results show that the water absorption rates of the three samples are all higher than the 2000% water absorption rate requirement stipulated by the industry standard. Among them, 20G05-150 has the highest water absorption rate. The other two samples also have high water absorption rates, which are higher than the control, and have certain advantages in achieving hemostatic function.

[0270] Example 16 Preparation of bone repair materials

[0271] The hemostatic sponges related to the above embodiments are mainly obtained by direct freeze-drying of collagen solutions, without adding other raw materials. Therefore, the applicant also tried to add other raw materials (such as hydroxyapatite) to prepare composite materials. To the applicant's surprise, compared with natural collagen, the hydroxyapatite powder in the collagen solution of the present invention is better dispersed, which is more conducive to simulating the structure of natural bone. The bone repair composite material prepared by this process also has better actual repair efficacy. The collagen of the present invention has more advantages in the preparation of composite materials such as bone repair.

[0272] The specific sample preparation and evaluation are as follows:

[0273] 1% (wt / vol) of ① recombinant type III humanized collagen 20G05, ② recombinant type III humanized collagen 19G291 (purchased from Jiangshan Juyuan Biotechnology Co., Ltd., or prepared according to Chinese patent CN102443057B), and ③ natural bovine type I collagen COL (purchased from Hebei Kaolisen Biotechnology Co., Ltd.) were added to a 3% (vol / vol) acetic acid solution, respectively. The solutions were magnetically stirred at room temperature until the proteins were completely dissolved and filtered using a filter membrane to prepare three collagen solutions.

[0274] 1% (wt / vol) of 200 nm hydroxyapatite powder (Macklin, product number: H875580, batch number: C10963596) was added to each of the three collagen solutions, and the mixture was stirred in an ice-water bath for 30 minutes to prepare three slurries containing hydroxyapatite.

[0275] The three slurries were poured into 100 x 100 mm molds and freeze-dried in a freeze dryer according to the freeze-drying curve (-20°C for 5 hours, gradually warming to room temperature over 30 hours). The freeze-dried samples were then cross-linked in a vacuum oven at 150°C for 10 hours to produce collagen-hydroxyapatite composite bone repair material samples. The three bone repair materials prepared were designated 20G05-HA, 19G291-HA, and COL-HA.

[0276] This example is only used to illustrate the present invention, but is not intended to limit the scope of the present invention. The relevant preparation conditions are not limited to the above conditions. For example, the acetic acid solution can be further replaced by dilute hydrochloric acid, and the vacuum thermal crosslinking method can also be replaced by a chemical crosslinking method.

[0277] Example 17 Observation of the Microscopic Morphology of Bone Repair Materials

[0278] Autologous bone repair is the gold standard for bone repair technology, but it is subject to numerous limitations due to limited sources. Therefore, developing ideal bone repair materials is key to achieving better bone repair technology. Ideal bone biomaterials closely mimic the composition and structure of natural bone. The bone repair materials prepared by the above process are compositionally consistent with natural bone, so further evaluation of the microstructure of these samples is required.

[0279] Natural bone is a natural composite material composed of an extracellular matrix and embedded cells. The extracellular matrix is ​​composed of mineralized collagen fibers. The densely packed nanostructures and precise three-dimensional spatial organization of hydroxyapatite crystals within the collagen fibers provide strong support for the body. The high tensile strength of the collagen fibers and the compressive resistance of the nanocrystals combine ingeniously to determine the unique load-bearing properties of bone. Hydroxyapatite crystals, formed by overlapping collagen layers, form the framework of the bone matrix. This oriented structure provides strong bonding and is less susceptible to dislocations and stacking faults, which improves stability and performance. Analysis of bone structure reveals a complex structure at two distinct levels: hydroxyapatite reinforces collagen fibers to form a coaxial layered ring structure measuring 3-7 μm, and canaliculi reinforce interstitial bone at the millimeter to micrometer scale. This delicate structure and arrangement of the bone matrix ensures both structural stability and normal biological absorption and exchange.

[0280] Scanning electron microscopy was used to observe the micromorphology of the three bone repair materials and to compare and analyze the advantages and disadvantages of their microstructures in simulating natural bone. Figures 18 to 20 As shown:

[0281] Natural collagen has poor solubility and high solution viscosity, so the hydroxyapatite powder is poorly dispersed, the sample is uneven inside, and it is difficult to simulate the structure of natural bone. Figure 20 Both 20G05 and 19G291 recombinant collagens have good solubility and can be combined more evenly. Figure 18 、 19 Comparing the micromorphology of the two bone repair materials, 20G05-HA and 19G291-HA, the microstructure of 20G05-HA is more uniform, and the combination of hydroxyapatite grains and collagen fibers at the nanoscale is achieved. At the same time, there is a multi-scale pore structure inside the material, achieving the purpose of better biomimetic preparation of natural bone composite materials. It realizes multi-level structural bionics and further imitates the natural bone structure. The hydroxyapatite grains are embedded in the collagen fibers and are tightly combined. The hydroxyapatite crystals are closely arranged in the collagen fibers, forming a nanostructure and a precise three-dimensional spatial structure. This is mainly due to the molecular design of 20G05 collagen, which enables it to better composite with hydroxyapatite to simulate the composition and structure of natural bone.

[0282] Example 18 Evaluation of the biological functionality of bone repair materials

[0283] In order to verify the effectiveness of the materials for repair, a cell seeding test of MC3T3 cells was conducted, and the cell attachment and growth were observed using a scanning electron microscope. The three bone repair material samples were cut into 6mm*6mm*3mm small pieces for cell seeding test and comparative analysis of biological functionality. The specific steps are as follows:

[0284] Inoculating cells on the material: ① Observation of cell status: Take a bottle of cells and observe the cell status under a microscope. If the cells are growing well and have grown to more than 85% fusion, they can be digested; ② Digestion: Aspirate the culture medium, wash once with an appropriate amount of PBS according to the size of the culture bottle used, aspirate the PBS, and digest with trypsin (the amount of trypsin added should be sufficient to spread the bottom of the culture bottle after shaking, and do not use too much). Incubate at 37°C and observe under a microscope. If the cells begin to fall off, immediately add twice the amount of trypsin to stop the digestion. Repeatedly pipette to remove the cells in the bottle. Transfer the liquid in the culture bottle to a centrifuge tube and centrifuge at 250g for 5 minutes (pay attention to balance). Discard the supernatant, aspirate the culture medium into the centrifuge tube, and mix thoroughly to form a cell suspension; ③ Cell counting: Take 10μl of the cell suspension and add 10μl of trypan blue, mix well, and count under a microscope; ④ Prepare cell suspension: Take the cell suspension in step ② and add it to the culture medium. ⑤ Inoculation of cells: Before inoculation, moisten the sample with culture medium until the sample spreads evenly in the culture medium. After wetting, use sterile tweezers to add the cells to the well plate and aspirate the culture medium. Pipette the cell suspension into the thoroughly mixed solution and drop it onto the sample membrane surface. Be careful to spread the cell suspension evenly across the membrane surface to avoid excessive concentration, which can make it difficult to locate the inoculation point when photographing. ⑥ After the cells have fully adhered to the material, add culture medium and incubate for 2, 5, and 7 days.

[0285] Fixation: Take out the cultured material. ①2.5% glutaraldehyde fixation: Aspirate the original culture medium, add PBS, blow and wash the material, aspirate PBS 3 times, add 2.5% glutaraldehyde and fix overnight (room temperature). ②Alcohol gradient elution: Aspirate the glutaraldehyde used to fix the cells, and use gradient alcohol to dehydrate the sample. Soak in 30%, 50%, 70%, 80%, 90%, and 95% alcohol for 10 to 15 minutes each, and soak in 100% alcohol 3 times, 10 minutes each time. ③Sample drying: Vacuum drying at 60°C, and store the dried sample in a desiccator.

[0286] Scanning electron microscopy observation: Surface scanning electron microscopy was used to observe the cell attachment, growth and proliferation on the samples.

[0287] The experimental results are as follows Figures 21 to 23 shown.

[0288] After culturing the 19G291-HA sample for 2 days, almost no cell attachment and growth were observed on the surface, and the sample disintegrated severely, making it difficult to maintain a good structure. The 5-day and 7-day experiments could not be continued, and its biological functionality was poor.

[0289] Comparing 20G05-HA with COL-HA, 20G05-HA significantly outperformed COL-HA in terms of cell number and proliferation rate. The 20G05-HA sample was able to better adhere to MC3T3 cells, with improved cell morphology and growth, demonstrating its enhanced osteoblast growth-promoting capabilities. The 20G05 sample, due to its molecular design, achieved a more biomimetic preparation and demonstrated significant advantages in biological functionality.

[0290] Example 19 Biosafety Evaluation of Bone Repair Materials

[0291] In order to verify whether the 20G05-HA recombinant III humanized collagen-hydroxyapatite composite bone repair material meets the requirements of biosafety standards, the following safety evaluation was conducted, using COL-HA as a control.

[0292] <Cytotoxicity Evaluation>

[0293] The specific experimental method is shown in Example 10.

[0294] The cytotoxicity evaluation tests of 20G05-HA and COL-HA samples were performed respectively, and the results are shown in the following table.

[0295] Table 21

[0296]

[0297] The cytotoxicity results showed that the cytotoxicity of 20G05-HA and COL-HA met the requirements of national standards, and there were no obvious safety issues in terms of cytotoxicity.

[0298] <Hemolysis test>

[0299] The specific experimental method is shown in Example 10.

[0300] The two samples 20G05-HA and COL-HA were evaluated by in vitro hemolysis test with the negative control saline and the positive control purified water. The experimental results are shown in the following table:

[0301] Table 22

[0302]

[0303] It can be seen from the in vitro hemolysis experiment that the results of the in vitro hemolysis evaluation of 20G05-HA and COL-HA samples meet the requirements of the national standard (hemolysis rate <5%), and are safe and reliable in terms of hemolysis.

[0304] Example 20 Bone repair material stimulation or intradermal reaction experiment

[0305] Fixed position button Figure 24 Other experimental methods were performed with reference to Example 12.

[0306] The sample 20G05-HA bone repair material, the negative control saline, and the positive control 20% SDS were subjected to rabbit irritation or intradermal reaction tests. The experimental results are shown in the following table:

[0307] Table 23 Rabbit irritation or intradermal reaction test results 1

[0308]

[0309] Table 24 Rabbit irritation or intradermal reaction test results 2

[0310]

[0311] The results of the rabbit irritation or intracutaneous reaction test showed that the irritation index of the 20G05-HA sample extract on rabbit skin was consistent with that of the negative control saline. Therefore, the results of the rabbit irritation or intracutaneous reaction test evaluation of the 20G05-HA bone repair material met the requirements of the national standard.

[0312] Example 21 Acute toxicity test of bone repair materials

[0313] Refer to the table below for grouping and dosing information, and refer to Example 13 for other experimental methods.

[0314] Table 25 Grouping and dosing information

[0315]

[0316] The acute toxicity test of sample 20G05-HA bone repair material in ICR mice was carried out. The experimental results are shown in the following table and Figure 25 As shown:

[0317] Table 26 Results of acute toxicity test in mice

[0318]

[0319]

[0320] Table 27

[0321]

[0322] The results of the acute toxicity study in ICR mice showed no significant differences in body weight between the saline / plant oil extract group and the solvent control group. Furthermore, behavioral observations revealed that all mice in each group were normal. The results of the acute toxicity study in ICR mice for the sample bone repair material met the requirements of national standards.

[0323] Example 22 Preparation of bone repair material samples with different collagen concentrations and evaluation of their biofunctionality

[0324] <Sample preparation>

[0325] 1%, 2%, and 3% (wt / vol) recombinant type III humanized collagen 20G05 were added to 1% (vol / vol) acetic acid solution, respectively. The solution was magnetically stirred at room temperature until the protein was completely dissolved, and then filtered using a filter membrane to prepare three collagen solutions.

[0326] 5% (wt / vol) of 200 nm hydroxyapatite powder (Macklin, product number: H875580, batch number: C10963596) was added to the three collagen solutions respectively, and stirred in an ice-water bath for 30 minutes to prepare three slurries containing hydroxyapatite.

[0327] The three slurries were poured into 100 x 100 mm molds and freeze-dried in a freeze dryer according to the freeze-drying curve (-20°C for 5 hours, gradually warming to room temperature over 30 hours). The freeze-dried samples were then heat-crosslinked in a vacuum oven at 150°C for 10 hours to produce collagen-hydroxyapatite composite bone repair material samples. The three prepared bone repair materials were designated 20G05-HA-1, 20G05-HA-2, and 20G05-HA-3.

[0328] <Biofunctionality Evaluation>

[0329] To verify the effectiveness of the materials for repair, a cell seeding experiment with MC3T3 cells was conducted, and cell attachment and growth were observed using a scanning electron microscope. The three bone repair material samples were cut into 6mm*6mm*3mm pieces for cell seeding experiments and comparative analysis of their biofunctionality. For detailed steps, see Example 18.

[0330] The experimental results are as follows Figures 26-31 shown.

[0331] From the cell attachment, growth and proliferation on the surface and sides, it can be seen that the 20G05-HA-1 / 2 / 3 samples can attach MC3T3 cells very well, and the cell morphology, growth and proliferation are all very good. It can be seen that the samples have better functionality in promoting osteoblast growth. Among them, 20G05-HA-2 is the best and can be used as the preferred process.

Claims

1. A recombinant humanized type III collagen-hydroxyapatite composite bone repair material, characterized in that: It contains recombinant humanized type III collagen, the amino acid sequence of which is shown in SEQ ID NO.4; The recombinant humanized type III collagen is prepared by the following method: (1) The gene sequence shown in SEQ ID NO.5 was cloned into an expression vector, and the Pichia pastoris engineering bacteria were transformed to obtain engineering bacteria that can efficiently express recombinant humanized collagen, wherein the expression vector is pGAPZαA and the Pichia pastoris engineering bacteria is Pichia pastoris GS115; (2) Fermenting and culturing the genetically engineered bacteria to induce expression of recombinant humanized collagen to obtain a fermentation broth containing the target protein; (3) Purify the fermentation broth to obtain recombinant humanized collagen.

2. A method for preparing a bone repair material, comprising the following steps: (1) adding the recombinant humanized type III collagen as described in claim 1 to an acidic solution to prepare a collagen solution; (2) adding hydroxyapatite powder to the above collagen solution to prepare a slurry containing hydroxyapatite; (3) freeze-drying the slurry and then cross-linking to obtain the product. 3 . The method for preparing a bone repair material according to claim 2 , wherein the acidic solution is acetic acid or dilute hydrochloric acid. 4 . The method for preparing a bone repair material according to claim 2 , wherein the crosslinking is vacuum thermal crosslinking or chemical crosslinking.

5. The method for preparing the bone repair material according to claim 2, comprising the following steps: (1) adding 1% to 3% (wt / vol) of the recombinant humanized type III collagen as described in claim 1 to a 1% to 3% (vol / vol) acetic acid solution, and stirring the mixture magnetically at room temperature until the protein is completely dissolved to prepare a collagen solution; (2) adding 1% to 5% (wt / vol) of 200 nm hydroxyapatite powder to the above collagen solution and stirring in an ice-water bath to prepare a slurry containing hydroxyapatite; (3) The slurry is injected into a suitable mold and freeze-dried. The freeze-dried sample is vacuum-heat-crosslinked in a vacuum drying oven at 140°C to 160°C to obtain a collagen-hydroxyapatite composite bone repair material sample.

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