Recombinant human-derived collagen sponge and method for preparing the same

Abstract

This invention discloses a recombinant human collagen cross-linked sponge and its preparation method. The method involves first hydrolyzing a silane coupling agent under alkaline conditions to form a hydrolysate, then adding recombinant human collagen, mixing thoroughly, freeze-drying to form a sponge, and finally heating to cross-link and solidify it, thus obtaining the recombinant human collagen cross-linked sponge. This invention offers a simple preparation method, and the resulting recombinant human collagen cross-linked sponge exhibits excellent mechanical properties, stable biodegradability, and no cytotoxicity, making it a promising candidate for human repair materials.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology and relates to a recombinant human collagen cross-linked sponge and its preparation method. Background Technology

[0002] Recombinant human collagen is produced by artificially designing and synthesizing a human collagen gene monomer based on the repetitive characteristic sequence of the glycine-XY (Gly-XY) tripeptide in the collagen domain of the α1 chain of human type III collagen. This monomer is then linked by a series of somatic cleavage enzymes to construct an expression vector containing six identical human collagen gene monomers. The vector is then transformed into Pichia pastoris and subjected to high-density fermentation, separation, and purification (Chinese Patent ZL201110327865.5).

[0003] Recombinant human collagen, as a type of protein molecule, exhibits high water solubility, resulting in a lack of mechanical strength and structural stability in water, which greatly limits its further applications. To slow down the biodegradation of recombinant human collagen and improve its mechanical stability in aqueous solutions, it is typically cross-linked using chemical or physical methods. Physical cross-linking generally requires sophisticated and complex processes, and its cross-linking effect is limited. Chemical cross-linking is relatively simpler and has a better cross-linking effect; however, widely used chemical cross-linking agents (such as glutaraldehyde, hexamethylene isocyanate, and 1,4-butanediol diglycidyl ether) have significant drawbacks when used to cross-link collagen. Glutaraldehyde, a commonly used cross-linking agent, can easily cause local tissue calcification and cytotoxic reactions when its residues remain. Although 1,4-butanediol diglycidyl ether exhibits low cytotoxicity, it has been proven to pose a risk of cancer, and according to ISO 10993, its residue level is strictly controlled below 2 ppm. Therefore, there is a need to find novel chemical cross-linking agents that can significantly improve solution stability and mechanical strength while possessing good biocompatibility. There are currently no reports on using inorganic phases to crosslink recombinant human collagen through hybridization and to regulate its properties. Summary of the Invention

[0004] To address the problems of poor mechanical strength and structural stability, and high biotoxicity after chemical cross-linking of existing cross-linked recombinant human collagen, this invention provides a recombinant human collagen cross-linked sponge with excellent mechanical properties, stable biodegradation, and no cytotoxicity, as well as its preparation method.

[0005] The technical solution of the present invention is as follows:

[0006] A method for preparing recombinant human collagen cross-linked sponge includes the following steps:

[0007] Step 1: Add the silane coupling agent to water, adjust the pH to 9-10, stir to fully hydrolyze it into particles, and obtain the hydrolysate;

[0008] Step 2: Add recombinant human collagen to the hydrolysate and stir until evenly mixed. Pour the mixture into a mold and freeze-dry to form a recombinant human collagen sponge. Then, cross-link and cure the recombinant human collagen sponge at 40-80℃ for 2-10 hours to obtain a recombinant human collagen cross-linked sponge.

[0009] Preferably, in step 1, the silane coupling agent is selected from one or more of the following: γ-(2,3-epoxypropoxy)propyltrimethoxysilane (GPTMS) and γ-glycidoxypropyltriethoxysilane containing epoxy groups (GPTES), tetraethoxysilane (TEOS), vinyltriethoxysilane (VTEO), 3-aminopropyltrimethoxysilane (APTMS), and 3-aminopropyltriethoxysilane (APTES), more preferably γ-(2,3-epoxypropoxy)propyltrimethoxysilane or tetraethoxysilane.

[0010] Preferably, in step 1, a co-solvent is added to promote the hydrolysis of the silane coupling agent, for example, ethanol is used as the co-solvent.

[0011] Preferably, in step 1, the hydrolysis time is 30–60 min.

[0012] Preferably, in step 2, the recombinant human collagen is produced by fermentation of Pichia pastoris with accession number CGMCC No. 5021, which has been fully disclosed in Chinese patent ZL201110327865.5.

[0013] Preferably, in step 2, the concentration of recombinant human collagen in the mixed solution is 10-15 wt.%, and the concentration of silane coupling agent is 5-15 wt.%, more preferably 5-10 wt.%.

[0014] Preferably, in step 2, the stirring time is 1 to 10 hours.

[0015] Preferably, in step 2, the mixed solution is poured into a mold, rapidly frozen at -70°C, and then freeze-dried.

[0016] Preferably, in step 2, the crosslinking curing temperature is 50–60°C and the crosslinking curing time is 4–6 h.

[0017] Furthermore, the present invention provides the application of the above-mentioned recombinant human collagen cross-linked sponge in the preparation of bone defect repair materials, oral mucosa repair materials, skin tissue repair materials, etc.

[0018] This invention utilizes the hydrolysis of silane under alkaline conditions to form silica-like particles with a large number of active groups. Using inorganic particles as crosslinking sites, the particles undergo covalent crosslinking reactions with amino and carboxyl groups present on recombinant human collagen segments under certain conditions to form a stable crosslinked structure. The large specific surface area porous recombinant human collagen crosslinked sponge is achieved by freeze-drying.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] (1) The recombinant human collagen cross-linked sponge of the present invention uses recombinant human collagen as raw material, which effectively avoids rejection and animal virus toxicity problems when used in humans.

[0021] (2) This invention uses inorganic particles formed after the hydrolysis of silane coupling agent as crosslinking sites, avoiding the use of toxic chemical reagents. At the same time, it endows the crosslinked recombinant human collagen sponge with the advantages of good structural stability, slowed degradation rate and significantly improved mechanical properties, and has potential application prospects in the field of human body repair materials.

[0022] (3) The preparation method of the present invention is simple, batch-stable, easy to operate, and can be mass-produced. Attached Figure Description

[0023] Figure 1 The images are of the hydrolysate of γ-(2,3-epoxypropoxy)propyltrimethoxysilane under an optical microscope. a) is the hydrolysate with a pH of 9-10, and b) is the hydrolysate with a pH of 7-8.

[0024] Figure 2 The images show physical and SEM images of recombinant human collagen cross-linked sponge samples: a) physical image of GPTMS-crosslinked recombinant human collagen cross-linked sponge, b) physical image of TEOS-crosslinked recombinant human collagen cross-linked sponge, c) SEM image of GPTMS-crosslinked recombinant human collagen cross-linked sponge, and d) SEM image of TEOS-crosslinked recombinant human collagen cross-linked sponge.

[0025] Figure 3 Tensile strength and elongation at break of recombinant human collagen crosslinked sponges with different GPTMS contents;

[0026] Figure 4 Figures show the swelling degree and degradation stability of recombinant human collagen cross-linked sponge samples. a) Swelling rate of recombinant human collagen cross-linked sponges with different GPTMS contents; b) Degradation rate of recombinant human collagen cross-linked sponges with different GPTMS contents after 3 days.

[0027] Figure 5Figure 1 shows the in vitro cytotoxicity test results of GPTMS recombinant human collagen cross-linked sponge (I-SCF) samples with different contents (0wt%, 1wt%, 5wt%, 15wt%). Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, all materials and reagents used in the following embodiments are commercially available. Recombinant humanized collagen was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.

[0029] Comparative Example 1

[0030] First, take 8.5g of deionized water and adjust the pH to 9-10, stirring for 30 minutes. Then, add 1.5g of recombinant humanized collagen to the solution and stir to dissolve for 2.5 hours. After that, pour the mixture into a mold and freeze it at -70℃ for 5 hours, then freeze-dry it for 24 hours to obtain a recombinant humanized collagen sponge. Finally, dry it at 60℃ for 6 hours to obtain a recombinant humanized collagen cross-linked sponge.

[0031] Example 1

[0032] First, 0.5 g of γ-(2,3-epoxypropoxy)propyltrimethoxysilane was added to 8.0 g of deionized water, and the pH was adjusted to 9-10. The mixture was stirred and hydrolyzed for 30 min. Then, 1.5 g of recombinant humanized collagen was added to the solution, and the mixture was stirred and dissolved for 2.5 h. After dissolution, the mixture was poured into a mold and rapidly frozen at -70℃ for 5 h. Following freeze-drying for 24 h, a recombinant humanized collagen sponge was obtained. This sponge was then post-cured at 60℃ for 6 h to obtain a cross-linked recombinant humanized collagen sponge.

[0033] Example 2

[0034] First, 0.5g of tetraethoxysilane was added to 7.0g of deionized water, along with 1g of ethanol as a co-solvent. The pH was adjusted to 9-10, and the mixture was stirred and hydrolyzed for 30 minutes. Then, 1.5g of recombinant humanized collagen was added to the solution, and the mixture was stirred and dissolved for 2.5 hours. After dissolution, the mixture was poured into a mold and rapidly frozen at -70℃ for 5 hours. Following freeze-drying for 24 hours, a recombinant humanized collagen sponge was obtained. This sponge was then post-cured at 60℃ for 6 hours to obtain a cross-linked recombinant humanized collagen sponge.

[0035] Example 3

[0036] First, 1.0 g of γ-(2,3-epoxypropoxy)propyltrimethoxysilane was added to 7.5 g of deionized water, and the pH was adjusted to 9-10. The mixture was stirred and hydrolyzed for 30 min. Then, 1.5 g of recombinant humanized collagen was added to the solution, and the mixture was stirred and dissolved for 2.5 h. After dissolution, the mixture was poured into a mold and rapidly frozen at -70℃ for 5 h. Following freeze-drying for 24 h, a recombinant humanized collagen sponge was obtained. This sponge was then post-cured at 60℃ for 6 h to obtain a cross-linked recombinant humanized collagen sponge.

[0037] Example 4

[0038] First, 1.5 g of γ-(2,3-epoxypropoxy)propyltrimethoxysilane was added to 7.0 g of deionized water, and the pH was adjusted to 9-10. The mixture was stirred and hydrolyzed for 30 min. Then, 1.5 g of recombinant humanized collagen was added to the solution, and the mixture was stirred and dissolved for 2.5 h. After dissolution, the mixture was poured into a mold and rapidly frozen at -70℃ for 5 h. Following freeze-drying for 24 h, a recombinant humanized collagen sponge was obtained. This sponge was then post-cured at 60℃ for 6 h to obtain a cross-linked recombinant humanized collagen sponge.

[0039] Comparative Example 2

[0040] First, 0.5 g of γ-(2,3-epoxypropoxy)propyltrimethoxysilane was added to 8.0 g of deionized water, and the pH was adjusted to 7-8. The mixture was stirred and hydrolyzed for 30 min. Then, 1.5 g of recombinant humanized collagen was added to the solution, and the mixture was stirred and dissolved for 2.5 h. After dissolution, the mixture was poured into a mold and rapidly frozen at -70℃ for 5 h. Following freeze-drying for 24 h, a recombinant humanized collagen sponge was obtained. This sponge was then post-cured at 60℃ for 6 h to obtain a cross-linked recombinant humanized collagen sponge.

[0041] Characterization example

[0042] 1. Tensile test

[0043] The mechanical properties of each recombinant human collagen cross-linked sponge were determined according to the test method of pharmaceutical industry standard YY / T 0471.4-2004. The specific steps are as follows: The recombinant human collagen cross-linked sponge samples were cut into 2cm × 8cm pieces and then fixed on a texture analyzer with a clamping distance of 5cm. Uniaxial tensile tests were conducted under constant temperature and humidity conditions of 25℃ and 50% to test the tensile strength of the recombinant human collagen cross-linked sponge.

[0044] 2. Swelling degree test

[0045] Accurately weigh the recombinant human collagen cross-linked sponge to a weight of Wd, then add it to PBS and let it stand at room temperature for 24 hours. Take out the fully expanded recombinant human collagen cross-linked sponge, remove the free water on the surface of the recombinant human collagen cross-linked sponge with absorbent paper, and weigh the recombinant human collagen cross-linked sponge to a weight of Wg. Calculate the degree of expansion (%) = (Wg-Wd / Wd)×100%.

[0046] The swelling degree measured using the method described above can indirectly determine the cross-linking status of the recombinant human collagen cross-linked sponge. A lower swelling degree indicates a higher degree of cross-linking and a more stable structure in the tested sponge.

[0047] 3. Degradation rate test

[0048] First, the recombinant human collagen cross-linked sponge was accurately weighed and the data was recorded.

[0049] The prepared sponges were placed in excess PBS aqueous solution and incubated at 37°C for 3 days. The recombinant human collagen cross-linked sponges were then removed, rinsed three times with distilled water, frozen overnight, freeze-dried, and weighed (Wt). The degradation rate in water was calculated using the formula (Wo-Wt) / Wo×100%.

[0050] The degradation rate measured using the above method can be used to determine the degradation stability of the recombinant human collagen cross-linked sponge. The lower the degree of degradation, the more stable the surface structure.

[0051] 4. In vitro cytotoxicity test

[0052] The in vitro cytotoxicity test was conducted according to EN ISO 10993-5:2009 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test", which tested the cell proliferation rate.

[0053] ① First, the recombinant human collagen cross-linked sponge was mixed with RPMI1640 culture medium at 0.001 g / ml and placed in a 37℃, 5% carbon dioxide incubator for 24 hours for extraction. The mixture was then filtered through a 0.22 μm microporous membrane to remove bacteria and obtain the extract.

[0054] ②Inoculate a 1×10⁵ cells / mL L929 cell suspension into a 96-well cell culture plate and incubate it in a 37℃ CO₂ incubator for 24 hours. After the cells adhere and grow, remove the supernatant and divide them into a control group and an experimental group.

[0055] ③ The control group was given RPMI1640 culture medium; the experimental group was given RPMI1640 culture medium containing 50% of the above extract; the control group and the experimental group were placed in a 37℃ carbon dioxide incubator for further culture. After 2 days, they were taken out, and 20μL of MTT solution (5mg / ml) was added to each well of the culture plate. The plates were then cultured at 37℃ for 4 hours and the culture was terminated.

[0056] ④ Carefully aspirate the culture supernatant from the wells, add 200 μL of DMSO to each well, shake for 10 minutes to mix, and then measure the absorbance value at 630 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0057] ⑤ Calculate the relative cell proliferation rate (RCR) according to the following formula: RCR (%) = (average absorbance value of experimental group / average absorbance value of control group) × 100%;

[0058] ⑥ The relationship between relative cell proliferation rate (RCR) and cytotoxicity grade is as follows:

[0059] RCR not less than 100%, cytotoxicity grade 0;

[0060] The RCR was 75-99%, and the cytotoxicity grade was 1.

[0061] The RCR was 50-74%, and the cytotoxicity grade was 2.

[0062] The RCR was 25-49%, and the cytotoxicity grade was 3.

[0063] The RCR ranged from 1% to 24%, and the cytotoxicity grade was 4.

[0064] The RCR was 0%, and the cytotoxicity grade was 5.

[0065] The biocompatibility of the recombinant human collagen cross-linked sponge was determined using the relative cell proliferation rate (RCR) measured as described above. A higher RCR indicates better biocompatibility of the recombinant human collagen cross-linked sponge.

[0066] Figure 1 Images of the hydrolyzed silane solution of γ-(2,3-epoxypropoxy)propyltrimethoxysilane under an optical microscope. a) shows the hydrolyzed solution from Example 1 with a pH of 9-10, where uniformly distributed particles are visible. b) shows the hydrolyzed solution from Comparative Example 2 with a pH of 7-8, where no obvious particles are formed. This demonstrates that solution pH affects the degree of hydrolysis of the silane coupling agent.

[0067] Figure 2Images of the recombinant human collagen cross-linked sponge samples are shown below: a) GPTMS-crosslinked recombinant human collagen cross-linked sponge; b) TEOS-crosslinked recombinant human collagen cross-linked sponge; c) SEM image of GPTMS-crosslinked recombinant human collagen cross-linked sponge; d) TEOS-crosslinked recombinant human collagen cross-linked sponge. It can be seen that the cross-linked sponges all possess a large specific surface area and a porous microstructure.

[0068] Figure 3 The figures show the tensile strength and elongation at break of recombinant human collagen cross-linked sponge samples with different GPTMS contents. It can be seen that the tensile strength of the recombinant human collagen cross-linked sponge gradually increases with increasing GPTMS content, indicating that the addition of GPTMS significantly enhances the mechanical strength. The elongation at break initially increases and then decreases, indicating that excessively high levels of inorganic particles are detrimental to the elongation at break.

[0069] Figure 4 The figures show the swelling degree and degradation stability of the recombinant human collagen cross-linked sponge samples. a) The swelling rate of the recombinant human collagen cross-linked sponge with different GPTMS contents is shown. The swelling rate decreases with increasing GPTMS content, indicating that GPTMS and recombinant collagen have undergone covalent cross-linking and the cross-linking density increases with increasing GPTMS content. b) The degradation rate of the recombinant human collagen cross-linked sponge with different GPTMS contents after 3 days is shown. The degradation rate decreases with increasing GPTMS content, indicating that GPTMS effectively improves the degradation stability of the collagen sponge through covalent cross-linking.

[0070] Figure 5 The in vitro cytotoxicity test results of GPTMS recombinant human collagen cross-linked sponge (I-SCF) samples with different contents (0wt%, 1wt%, 5wt%, 15wt%) are shown in the figure. According to EN ISO 10993-5:2009 "Biological evaluation of medical devices - Part 5: In vitro cytotoxicity test", the cell proliferation rate is higher with the increase of GPTMS content, indicating that the recombinant human collagen cross-linked sponge has better biocompatibility.

Claims

1. A method for preparing recombinant human collagen cross-linked sponge, characterized in that, Includes the following steps: Step 1: Add the silane coupling agent to water, add the co-solvent ethanol to promote the hydrolysis of the silane coupling agent, adjust the pH to 9~10, stir to allow it to fully hydrolyze and form particles, and obtain the hydrolysate; Step 2: Add recombinant human collagen to the hydrolysate and stir until evenly mixed. Pour the mixture into a mold, freeze it rapidly at -70℃, and then freeze-dry it to form a recombinant human collagen sponge. Then, cross-link and solidify the recombinant human collagen sponge at 40~80℃ for 2~10 hours to obtain a recombinant human collagen cross-linked sponge.

2. The preparation method according to claim 1, characterized in that, In step 1, the silane coupling agent is selected from γ-(2,3-epoxypropoxy)propyltrimethoxysilane, γ-glycidoxypropyltriethoxysilane, tetraethoxysilane, vinyltriethoxysilane, 3-aminopropyltrimethoxysilane, or 3-aminopropyltriethoxysilane.

3. The preparation method according to claim 1, characterized in that, In step 1, the hydrolysis time is 30-60 minutes; in step 2, the recombinant human collagen is obtained from Pichia pastoris with accession number CGMCC No. 5021. Pichia pastoris Produced by fermentation.

4. The preparation method according to claim 1, characterized in that, In step 2, the concentration of recombinant human collagen in the mixed solution is 10-15 wt.%, and the concentration of silane coupling agent is 5-15 wt.%.

5. The preparation method according to claim 1, characterized in that, In step 2, the concentration of the silane coupling agent in the mixed solution is 5~10 wt.%.

6. The preparation method according to claim 1, characterized in that, In step 2, the stirring time is 1 to 10 hours.

7. The preparation method according to claim 1, characterized in that, In step 2, the cross-linking curing temperature is 50~60℃ and the cross-linking curing time is 4~6h.

8. The recombinant human collagen cross-linked sponge prepared by any one of the preparation methods according to claims 1 to 7.

9. The application of the recombinant human collagen cross-linked sponge according to claim 8 in the preparation of bone defect repair materials, oral mucosa repair materials or skin tissue repair materials.

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