Recombinant collagen type iii, hydrogel, preparation method and application

Abstract

The application discloses recombinant collagen type III, a hydrogel, a preparation method and application, and belongs to the technical field of medical materials. The protein has an amino acid sequence as shown in SEQ ID No. 2 or 3, and has excellent biocompatibility and cell activity. The hydrogel based on the protein is formed by mixing a solution of the protein with a solution containing oxidized sodium alginate and hydroxypropyl methyl cellulose, adjusting pH, sequentially introducing an EDC / NHS crosslinking system and calcium ions, and standing. The process constructs a stable double-network structure of EDC / NHS catalyzed covalent crosslinking network and OSA-Ca 2+ ion crosslinking network synergistic effect. The obtained hydrogel has high mechanical strength, excellent toughness, long-lasting wet tissue adhesion and suitable swelling performance, can effectively adapt to the complex stress changes of dynamic wounds, and provides an active matrix for cells, and promotes tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to recombinant type III collagen, hydrogel, preparation method and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Dynamic wounds are common in highly mobile areas such as joints, where continuous movement causes the covering dressing to endure complex cyclic stresses over a long period. Currently used clinical dressings, due to insufficient mechanical fit, are prone to displacement or failure under repeated traction, which not only damages the wound barrier but may also interfere with the normal healing process.

[0004] Hydrogel materials are considered ideal candidate materials due to their biomimetic high water content and good biocompatibility. Among them, collagen has attracted much attention due to its inherent biological functions. However, traditional collagen-based hydrogels generally suffer from core defects such as weak mechanical properties and poor fatigue resistance. Most existing modification strategies can only improve one aspect of performance, making it difficult to achieve high strength, high elastic recovery, and durable adhesion synergistically while ensuring biocompatibility. This is precisely the comprehensive solution urgently needed for dynamic wound care. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide recombinant type III collagen, hydrogel, preparation method, and applications. Based on bioinformatics methods, this invention rationally designs and optimizes the coding sequence of human type III collagen, successfully developing a recombinant human collagen with a simple preparation process, high yield, strong stability, excellent bioactivity, and a triple-helix structure. This provides a new solution for the development of related biomedical materials and functional products.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a recombinant type III collagen, the amino acid sequence of which is shown in SEQ ID No. 2 or SEQ ID No. 3.

[0008] In a second aspect, the present invention provides a gene encoding the recombinant type III collagen described in the first aspect, wherein the nucleotide sequence is any one of SEQ ID No. 4-5.

[0009] A third aspect of the present invention provides a method for preparing recombinant type III collagen hydrogel, comprising:

[0010] Mix solution A with solution B, adjust the pH, add the cross-linking agent and calcium salt, and let stand to obtain the final product.

[0011] Solution A is the recombinant type III collagen solution described in the first aspect;

[0012] Solution B is a hydroxypropyl methylcellulose (HPMC) solution containing oxidized sodium alginate (OSA).

[0013] In some embodiments of the present invention, the degree of oxidation of the oxidized sodium alginate is 25% to 35%.

[0014] In some embodiments of the present invention, the concentration of the recombinant type III collagen solution is 8-12 mg / mL;

[0015] The concentration of sodium alginate in solution B is 15-25 mg / mL, and the concentration of hydroxypropyl methylcellulose is 1-3 mg / mL.

[0016] The volume ratio of solution A to solution B is (0.9~1.1):(0.9~1.1).

[0017] In some embodiments of the present invention, the solvent of solution A includes any one or more of acetic acid, hydrochloric acid, phosphoric acid, citric acid, and ascorbic acid.

[0018] In some embodiments of the present invention, the method for preparing the oxidized sodium alginate includes:

[0019] Sodium alginate aqueous solution and oxidant were mixed and oxidized under light-protected conditions to terminate the reaction. Alcohol was added to precipitate the precipitate, which was then collected by filtration. The precipitate was redissolved in water, dialyzed, and freeze-dried to obtain oxidized sodium alginate.

[0020] In some embodiments of the present invention, the concentration of the sodium alginate aqueous solution is 1-5 wt%, preferably 2-4 wt%.

[0021] In some embodiments of the present invention, the oxidant includes sodium periodate; the mass ratio of sodium alginate to sodium periodate is (1.8~2.2):1.

[0022] In some embodiments of the present invention, the oxidation time is 4-5 h;

[0023] In some embodiments of the present invention, ethylene glycol is added to terminate the reaction; after the addition of ethylene glycol, stirring is continued for 0.5 to 1 h in the dark; the amount of ethylene glycol used is 4 to 6 mL / g sodium alginate.

[0024] In some embodiments of the present invention, the alcohol includes ethanol, and the amount of the alcohol used is 70-90 mL / g sodium alginate.

[0025] In some embodiments of the present invention, solution A is mixed with solution B, the pH is adjusted to 5.4-5.6, stirred for 15-30 minutes, mixed evenly, and allowed to stand to obtain solution C; a crosslinking agent is added to solution C, stirred evenly, and calcium salt aqueous solution is added dropwise under stirring conditions. After the addition is complete, the mixture is allowed to stand and gel to obtain recombinant type III collagen hydrogel.

[0026] In some embodiments of the present invention, the crosslinking agent comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS); the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and recombinant type III collagen is 1:1:(4~8).

[0027] In some embodiments of the present invention, the concentration of the calcium salt aqueous solution is 0.1~0.5 M, and 0.5~1.5 mL of calcium salt aqueous solution is added for every 10 mL of mixed solution.

[0028] In a fourth aspect, the present invention provides a recombinant type III collagen hydrogel, which is prepared by the preparation method described in the third aspect.

[0029] A fifth aspect of the present invention provides the application of the recombinant type III collagen described in the first aspect or the recombinant type III collagen hydrogel described in the fourth aspect in biomedical materials.

[0030] In some embodiments of the present invention, the biomedical material includes any one or more of collagen hemostatic cotton, surgical sutures, and wound dressings.

[0031] The beneficial effects of this invention are as follows:

[0032] This invention provides a recombinant type III collagen. Through molecular design of its amino acid sequence (such as SEQ ID No. 2 or 3), it not only fully retains the core bioactive regions of natural type III collagen that promote cell adhesion, migration, and proliferation, but also achieves high purity and good solubility in recombinant expression. This design effectively avoids the immune risks associated with animal-derived collagen and lays the foundation for maintaining structural homogeneity and functional stability in solution (such as solution A), thus providing a core biomaterial with well-defined activity and controllable quality for the subsequent construction of high-performance hydrogels.

[0033] This invention, based on the aforementioned recombinant type III collagen, innovatively combines it with OSA and HPMC, and further covalently crosslinks it with Ca through EDC / NHS. 2+The synergistic effect of ionic cross-linking constructs a stable dual-network hydrogel. This structure ingeniously solves the fundamental problem of weak mechanical properties in traditional collagen gels, while achieving high strength, high elastic recovery, and durable tissue adhesion, enabling it to closely adhere to and adapt to the complex stress changes of dynamic wounds. Furthermore, this hydrogel possesses suitable swelling properties, good biocompatibility, and active repair guidance capabilities derived from recombinant collagen, thus providing an integrated, high-performance active repair solution for dynamic wounds. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 This is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image of the purified recombinant type III collagen obtained in Example 1 of this invention. Lane A represents the sample before anion purification, lane B represents the flow-through after anion purification, and lane C represents the elution after anion purification.

[0036] Figure 2 The sponge is the recombinant type III collagen obtained in Example 1 of this invention after freeze-drying.

[0037] Figure 3 This is an SDS-PAGE image of the purified recombinant type III collagen obtained in Example 2 of this invention. Lane A represents the sample before anion purification, lane B represents the flow-through after anion purification, and lane C represents the elution after anion purification.

[0038] Figure 4 The sponge obtained by freeze-drying recombinant type III collagen in Example 2 of this invention.

[0039] Figure 5 The cell adhesion effects of recombinant type III collagen at different concentrations obtained in Examples 1 and 2 of this invention are shown.

[0040] Figure 6 This is a photograph of the recombinant type III collagen hydrogel prepared in Example 5 of the present invention.

[0041] Figure 7 This is a scanning electron microscope (SEM) image of the recombinant type III collagen hydrogel prepared in Example 5 of the present invention.

[0042] Figure 8 The tensile stress-strain curves are shown for the recombinant type III collagen hydrogels prepared in Examples 3 to 8 of this invention.

[0043] Figure 9The tensile Young's modulus diagrams are for the recombinant type III collagen hydrogels prepared in Examples 3 to 8 of this invention.

[0044] Figure 10 The image shows the cyclic compression stress-strain curve of the recombinant type III collagen hydrogel prepared in Example 5 of this invention.

[0045] Figure 11 The adhesion strength of the recombinant type III collagen hydrogels prepared in Examples 3 to 8 of this invention.

[0046] Figure 12 The swelling diagrams are of the recombinant type III collagen hydrogels prepared in Examples 3 to 8 of this invention.

[0047] Figure 13 Cell viability of the recombinant type III collagen hydrogels prepared in Examples 3 to 8 of this invention. Detailed Implementation

[0048] This invention provides recombinant type III collagen, hydrogel, preparation method, and applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0049] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0050] This invention is based on the complete amino acid sequence of human type III collagen, systematically screening and identifying its core functional sequences with key biological activities. Based on these sequence characteristics, through rational design and targeted repetitive construction, a series of recombinant type III collagens with well-defined structures and enhanced functions were obtained. This invention uses recombinant type III collagen as the core active ingredient, constructing a covalently cross-linked network of EDC / NHS and OSA-Ca... 2+ A stable dual-network structure with synergistic effects of ionic cross-linking networks has been developed to create a hydrogel with both excellent mechanical properties and bioactivity, suitable as a dressing for wounds subjected to repeated stretching. The covalently cross-linked network provides a high-strength, stable framework for the hydrogel, while the ionic cross-linked network provides good toughness and energy dissipation. Together, these components endow the material with fatigue resistance, high adhesion, and suitable swelling stability, providing durable mechanical support and a suitable moist microenvironment for tissue repair. Simultaneously, based on the inherent bioactivity of recombinant type III collagen, it actively guides and accelerates tissue repair, thereby achieving safe and efficient wound healing.

[0051] The present invention provides a recombinant type III collagen, the amino acid sequence of which is shown in SEQ ID No. 2 or SEQ ID No. 3.

[0052] In the full-length triple helix region of the Homo sapiens COL3A1 (NP_000081) protein, gapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeg (SEQ ID No. 1) was selected as a repeating unit and copied 8 or 9 times to obtain recombinant type III collagen with an amino acid sequence as shown in SEQ ID No. 2 or SEQ ID No. 3.

[0053] SEQ ID No. 2:

[0054] Gapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeggapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeg gapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgegGapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeg GapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgegGapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeg gapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeggapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeg.

[0055] SEQ ID No. 3:

[0056] Gapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeggapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeggapgergrpglpg aagapgergppglagapgppgppgdkgepgergapgekgegGapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgegGapgergrpglpgaagapgergppgla gapgppgppgdkgepgergapgekgegGapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeggapgergrpglpgaagapgergppglagapgppgppgdkge pgergapgekgeggapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgegGapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeg.

[0057] The present invention also provides a gene encoding the above-mentioned recombinant type III collagen, wherein the nucleotide sequence is any one of SEQ ID No. 4-5.

[0058] In this invention, the sequence encoding the aforementioned recombinant type III collagen was codon-optimized, and the entire gene was synthesized and ligated into a plasmid to construct an expression vector. After the above optimization, its nucleotide sequence is shown in SEQ ID No. 4 or SEQ ID No. 5.

[0059] SEQ ID No.4:

[0060]

[0061] SEQ ID No.5:

[0062]

[0063] This invention provides a method for preparing the above-mentioned recombinant type III collagen, comprising:

[0064] (1) Gene design and synthesis

[0065] Based on the recombinant humanized type III collagen nucleotide sequence shown in SEQ ID No. 2 or SEQ ID No. 3, the whole gene was synthesized, and the gene fragment was cloned into an expression vector to construct a recombinant expression plasmid.

[0066] (2) Construction of expression strains and protein expression

[0067] The resulting recombinant expression plasmid was transformed into *E. coli* BL21(DE3) competent cells and plated on LB agar containing kanamycin, incubated overnight at 37°C. Single colonies were picked and inoculated onto LB broth containing kanamycin, and cultured at 37°C with shaking until OD (out of cells). 600 The initial concentration was 0.4–0.6, and IPTG was added to a final concentration of 0.1–1.0 mM. Expression was induced at 37°C for 4–6 h. The bacterial cells were collected by centrifugation.

[0068] (3) Crude protein purification

[0069] The bacterial cell pellet was resuspended in Buffer A (50 mM Tris, 250 mM NaCl, pH 8.0), incubated on ice for 1 hour, and then sonicated. The supernatant was collected by centrifugation. The supernatant was initially purified by one or more of isoelectric point precipitation and ammonium sulfate precipitation to obtain crude purified protein.

[0070] (4) Protein purification

[0071] The crude purified protein was dissolved in 50 mM Tris buffer (pH 8.0) and further purified by anion exchange chromatography to obtain high-purity recombinant humanized type III collagen.

[0072] (5) Protein exchange and lyophilization

[0073] After purification using anion exchange chromatography, the protein solution was directly dialyzed into distilled water. Finally, the dialyzed sample was freeze-dried to obtain the recombinant humanized type III collagen product.

[0074] This invention also provides a method for preparing recombinant type III collagen hydrogel, comprising:

[0075] Mix solution A with solution B, adjust the pH, add the cross-linking agent and calcium salt, and let stand to obtain the final product.

[0076] Solution A is the aforementioned recombinant type III collagen solution;

[0077] Solution B is a hydroxypropyl methylcellulose solution containing sodium alginate.

[0078] This invention utilizes a covalent cross-linking network mediated by a cross-linking agent (EDC / NHS) and oxidized sodium alginate-Ca 2+ The synergistic construction of the ionic cross-linking network forms a stable and uniform dual-network structure. This structure is visually represented by a porous morphology with uniform size distribution and complete shape. The covalent cross-linking network provides a high-strength and stable framework for the hydrogel, while the ionic cross-linking network contributes good toughness and energy dissipation capacity. The synergistic effect of the two enables the hydrogel to simultaneously possess high mechanical strength, high toughness, and outstanding fatigue resistance, meeting the mechanical support requirements of repeated stretching or wounds in active areas.

[0079] In one specific embodiment, the concentration of the recombinant type III collagen solution is 8-12 mg / mL, preferably 10 mg / mL;

[0080] In solution B, the concentration of sodium alginate is 15-25 mg / mL, preferably 20 mg / mL; and the concentration of hydroxypropyl methylcellulose is 1-3 mg / mL, preferably 2 mg / mL.

[0081] The volume ratio of solution A to solution B is (0.9~1.1):(0.9~1.1), preferably 1:1.

[0082] By precisely optimizing the concentration and ratio of each key component, an ideal dual-network structure was achieved. Collagen concentration ensured sufficient bioactivity and structural basis; sodium alginate concentration directly regulated the density of covalent and ionic dual crosslinking; HPMC concentration affected the rheology of the prepolymer and network uniformity; and a 1:1 volume ratio ensured that the precursors of the two networks could be fully and uniformly mixed and reacted. The synergistic effect of these parameters resulted in a hydrogel possessing high mechanical strength, excellent toughness, a stable porous structure, and good biocompatibility.

[0083] In one specific embodiment, the solvent of solution A includes, but is not limited to, any one or more of acetic acid, hydrochloric acid, phosphoric acid, citric acid, and ascorbic acid, preferably acetic acid. The acidic solution, as a collagen solvent, provides an acidic environment, causing the collagen carboxyl groups to protonate, the amino groups to become positively charged, and the intermolecular repulsion to increase, thus dissolving the collagen.

[0084] In one specific embodiment, the oxidation degree of the oxidized sodium alginate is 25% to 35%.

[0085] In one specific embodiment, the method for preparing the oxidized sodium alginate includes:

[0086] Sodium alginate aqueous solution and oxidant were mixed and oxidized under light-protected conditions to terminate the reaction. Alcohol was added to precipitate the precipitate, which was then collected by filtration. The precipitate was redissolved in water, dialyzed, and freeze-dried to obtain oxidized sodium alginate.

[0087] This invention chemically modifies natural sodium alginate through a controllable oxidation process (such as controlling the oxidant, time, and temperature), precisely introducing aldehyde groups that can react with the amino groups of collagen, while partially retaining the guluronic acid sequence that interacts with calcium ions. This ensures that the obtained oxidized sodium alginate has the stable and reproducible chemical structure necessary to achieve dual cross-linking function.

[0088] In one specific embodiment, the concentration of the sodium alginate aqueous solution is 1-5 wt%, preferably 2-4 wt%, and more preferably 3 wt%.

[0089] In one specific embodiment, the oxidant includes sodium periodate; the mass ratio of sodium alginate to sodium periodate is (1.8~2.2):1.

[0090] In one specific embodiment, the oxidation time is 4 to 5 hours.

[0091] In one specific embodiment, ethylene glycol is added to terminate the reaction; after the addition of ethylene glycol, stirring is continued for 0.5 to 1 h in the dark; the amount of ethylene glycol used is 4 to 6 mL / g sodium alginate.

[0092] It is understandable that the dosage of 4~6 mL / g sodium alginate refers to the ratio of ethylene glycol to sodium alginate in this invention being 4~6 mL:1g, that is, 4~6 mL of ethylene glycol is added for every 1 g of sodium alginate.

[0093] In one specific embodiment, the alcohol includes ethanol, and the amount of alcohol used is 70~90 mL / g sodium alginate.

[0094] It is understandable that the dosage of 4~6 mL / g sodium alginate refers to the ratio of alcohol to sodium alginate in this invention being 70~90 mL:1g, that is, 70~90 mL of alcohol is added for every 1 g of sodium alginate.

[0095] The alcohol is preferably ethanol.

[0096] In one specific embodiment, solution A is mixed with solution B, the pH is adjusted to 5.4-5.6, stirred for 15-30 minutes, mixed evenly, and allowed to stand to obtain solution C; cross-linking agent is added to solution C, stirred evenly, and calcium salt aqueous solution is added dropwise under stirring conditions. After the addition is complete, the mixture is allowed to stand and gel to obtain recombinant type III collagen hydrogel.

[0097] In one specific embodiment, the crosslinking agent comprises 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS); the mass ratio of EDC, NHS, and recombinant type III collagen is 1:1:(4~8). This ratio ensures that the EDC / NHS system is sufficient to efficiently catalyze the formation of amide bonds between collagen molecules and between collagen and oxidized sodium alginate, constructing a stable covalently crosslinked main network. Simultaneously, this ratio range avoids over-crosslinking (which would impair gel elasticity and biocompatibility) or under-crosslinking (resulting in insufficient mechanical strength) that may result from excessive crosslinking agent, thus precisely achieving the synergy between the covalent network and the ionic network, enabling the hydrogel to possess both excellent mechanical properties and suitable biological functions.

[0098] In one specific embodiment, the concentration of the calcium salt aqueous solution is 0.1~0.5 M, and 0.5~1.5 mL of calcium salt aqueous solution is added for every 10 mL of mixed solution.

[0099] The present invention also provides a recombinant type III collagen hydrogel, which is prepared by the above preparation method.

[0100] Thanks to its stable dual-network structure, the hydrogel adheres firmly to the wound surface and is not easily detached during movement. Simultaneously, it possesses suitable swelling properties, effectively absorbing wound exudate and maintaining a moist healing environment while avoiding excessive expansion that could reduce adhesion or compress the wound. This achieves an ideal balance between moisturization and fixation in dynamic wound care.

[0101] This invention uses recombinant type III collagen as its core, ensuring not only good biocompatibility but also endowing it with inherent bioactivity. This hydrogel provides a favorable microenvironment for cell growth, exhibiting a certain cell proliferation-promoting effect, thereby actively guiding and accelerating the tissue repair process.

[0102] This invention allows for effective control of the mechanical strength, toughness, and swelling rate of hydrogels by adjusting conditions such as the ratio of dual-network components and crosslinking density. The hydrogel ultimately prepared by this invention possesses excellent mechanical adaptability, stable physicochemical properties, and positive biological functions, making it particularly suitable for nursing scenarios such as chronic wounds and wounds in active areas where high mechanical performance is required. This provides an innovative dressing solution for safe and efficient tissue repair.

[0103] Therefore, the present invention also provides the application of the above-mentioned recombinant type III collagen or the above-mentioned recombinant type III collagen hydrogel in biomedical materials.

[0104] In one specific embodiment, the biomedical material includes, but is not limited to, any one or more of collagen hemostatic cotton, surgical sutures, and wound dressings. This invention is particularly suitable for preparing high-performance dynamic wound dressings.

[0105] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0106] Example 1

[0107] This embodiment provides a recombinant type III collagen, the preparation method of which is as follows:

[0108] In the full-length triple helix region of the Homo sapiens COL3A1 (NP_000081) protein, gapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeg (SEQ ID No. 1) was selected as a repeating unit and repeated 8 times to obtain recombinant humanized type III collagen with the amino acid sequence shown in SEQ ID No. 2, with a theoretical molecular weight of approximately 38.8 kDa.

[0109] The sequence encoding this protein was codon-optimized (nucleotide sequence SEQ ID No. 4), and the whole gene was synthesized and ligated into the pET28a plasmid to construct the expression plasmid. The expression plasmid was transformed into *E. coli* BL21(DE3) competent cells, plated on LB solid medium containing kanamycin, and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing kanamycin, and cultured at 37°C with shaking until OD (out of control). 600 The concentration was 0.4–0.6, and isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1–1.0 mM. Expression was induced at 37°C for 4–6 h, and the cells were collected by high-speed centrifugation. After resuspending the cells in phosphate buffer, they were homogenized by high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation.

[0110] The supernatant from the above-mentioned crushed and centrifuged mixture was adjusted to pH 5.2 with concentrated hydrochloric acid and allowed to stand for 1 h for acid precipitation. After precipitation, the precipitate was collected by high-speed centrifugation and dissolved in 50 mM Tris buffer (pH=8). Further purification was performed by anion exchange chromatography: the chromatography column was equilibrated under 50 mM Tris conditions, and contaminating proteins were washed with 50 mM Tris, 50-100 mM NaCl, and pH=8. The target protein was then eluted with 50 mM Tris, 300-500 mM NaCl, and pH=8.

[0111] The apparent molecular weight of recombinant collagen was analyzed using SDS-PAGE technology. The results are as follows: Figure 1 As shown, the purified sample did not exhibit a single main band, but rather two protein bands with strong signals, clear morphology, and similar widths appeared at approximately 43 kDa and 39 kDa. This dual-main-band phenomenon suggests that the target protein may exist in different higher-order structures or aggregate states in the sample, leading to differences in its migration behavior in the denaturing gel, thus causing a shift in apparent molecular weight. Furthermore, several weakly colored extraneous protein bands were observed outside the target region, indicating that the purity of the product under the current purification process has room for improvement, and further optimization of chromatographic conditions can remove trace impurities.

[0112] Example 2

[0113] This embodiment provides a recombinant type III collagen, the preparation method of which is as follows:

[0114] In the full-length triple helix region of the Homo sapiens COL3A1 (NP_000081) protein, gapgergrpglpgaagapgergppglagapgppgppgdkgepgergapgekgeg (SEQ ID No. 1) was selected as a repeating unit and repeated 9 times to obtain recombinant humanized type III collagen with the amino acid sequence shown in SEQ ID No. 3, with a theoretical molecular weight of approximately 43.6 kDa.

[0115] The sequence encoding this protein was codon-optimized (nucleotide sequence SEQ ID No. 5), and the whole gene was synthesized and ligated into the pET28a plasmid to construct the expression plasmid. The expression plasmid was transformed into *E. coli* BL21(DE3) competent cells, plated on LB solid medium containing kanamycin, and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing kanamycin, and cultured at 37°C with shaking until OD (out of control). 600 The concentration was 0.4–0.6, and IPTG was added to a final concentration of 0.1–1.0 mM. Expression was induced at 37°C for 4–6 h, and the cells were collected by high-speed centrifugation. After resuspending the cells in phosphate buffer, they were homogenized by high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation.

[0116] The supernatant from the above-mentioned crushed and centrifuged mixture was adjusted to pH 5.2 with concentrated hydrochloric acid and allowed to stand for 1 h for acid precipitation. After precipitation, the precipitate was collected by high-speed centrifugation and dissolved in 50 mM Tris buffer (pH=8). Further purification was performed by anion exchange chromatography: the chromatography column was equilibrated under 50 mM Tris conditions, and contaminating proteins were washed with 50 mM Tris, 50-100 mM NaCl, and pH=8. The target protein was then eluted with 50 mM Tris, 300-500 mM NaCl, and pH=8.

[0117] The apparent molecular weight of recombinant collagen was analyzed using SDS-PAGE. The results are as follows: Figure 3 As shown, the purified sample exhibits a strong, deeply stained main band at approximately 44 kDa, with its migration position consistent with the theoretical molecular weight, indicating good expression levels of the target protein. The main band is intact and has clear edges, confirming its good enrichment. Simultaneously, a small number of lighter-colored non-target bands can be observed below the main band, indicating the presence of trace amounts of low-molecular-weight impurities or protein degradation fragments. However, these do not affect the preliminary assessment of the purity and properties of the main product, and the purification results meet the basic requirements of most downstream applications.

[0118] Performance validation: Cell adhesion activity assay

[0119] To verify whether the recombinant type III collagen obtained in the examples possesses biological activity as a cell adhesion and migration matrix, a relative cell adhesion experiment was conducted. The specific steps are as follows:

[0120] Add EDC / NHS mixtures containing 100 μg / mL, 50 μg / mL, and 10 μg / mL recombinant human type III collagen to 48-well plates, respectively, with four replicates for each concentration. Wells containing an equal volume of PBS serve as negative controls. The coating process lasts for 1 hour to allow the material to fully gel and form a matrix. After coating, seed each well with 5 × 10⁶ cells / well. 4 L929 cells were cultured at 37°C, 5% CO2, and 95% humidity for 2 hours to allow cell adhesion. After culture, the culture medium in each well was aspirated, and the cells were gently washed three times with PBS to remove any unadhered cells. Cell adhesion rate was then detected using the CCK-8 assay: absorbance was measured at 450 nm using a microplate reader. In this assay, OD... 450 The value is positively correlated with the number and activity of cells adhering to the pores; the higher the value, the greater the amount of cell adhesion and the better the activity.

[0121] Figure 5It is clearly demonstrated that the recombinant type III collagen prepared in Examples 1 and 2 possesses significant cell adhesion bioactivity. For example... Figure 5 As shown, at all test concentrations (10, 50, 100 μg / mL), whether in Example 1 or Example 2, the corresponding OD values ​​were... 450 The values ​​were all significantly higher than those of the control group (commercially available collagen). This directly proves that the collagen obtained by the method of this invention can effectively promote the adhesion of L929 cells, and the adherent cells maintain good metabolic activity. Further analysis shows that the relative adhesion activity of cells exhibits a clear concentration-dependent relationship. In both examples, OD 450 The values ​​all increased stepwise with increasing collagen coating concentration (from 10 μg / mL to 100 μg / mL). This trend confirms that the active function of the collagen in this invention is controllable and predictable, and the interaction strength between cells and the material substrate can be effectively regulated by adjusting its application concentration.

[0122] The experimental results strongly validate that the hydrogel constructed with the recombinant type III collagen of this invention as its core can provide a favorable adhesion microenvironment for cells. This characteristic is crucial for its application as a wound dressing, because good cell adhesion is a key initial step in guiding cell migration, proliferation, and ultimately achieving active tissue repair. This biologically supports the beneficial effect of the material of this invention in "actively guiding and accelerating tissue repair."

[0123] Because the recombinant type III collagen obtained in Example 2 has the same OD at the same concentration 450 The value is higher, so it was chosen to prepare hydrogels and the following experiments were conducted.

[0124] Example 3

[0125] This embodiment provides a recombinant type III collagen hydrogel, the preparation method of which includes the following steps:

[0126] Recombinant type III collagen solid was dissolved in 0.3 M acetic acid solution and stirred at 200 rpm until completely dissolved to obtain solution A. The concentration of recombinant type III collagen in solution A was 10 mg / mL.

[0127] A 3% aqueous solution of sodium alginate (SA) was reacted with sodium periodate under light-protected conditions for 4-5 hours, with a mass ratio of sodium alginate to sodium periodate of 2:1. Ethylene glycol (5 mL / g SA) was added to terminate the reaction, and the mixture was stirred under light-protected conditions for 0.5-1 hours. Ethanol (80 mL / g SA) was then added to the mixture to oxidize sodium alginate (OSA) and precipitate it. The precipitate was collected by filtration. The precipitate was redissolved in deionized water and dialyzed with deionized water for 2 days. Finally, the dialysate was freeze-dried to obtain a white solid OSA.

[0128] HPMC solution (concentration 2 mg / mL, HPMC selected is USP2910-E3) was prepared using a hot / cold water method: First, approximately 1 / 3 of the required amount of deionized water was heated to 85-90°C. While stirring, the HPMC powder was slowly and evenly dispersed in the hot water to form a suspension. Then, the remaining 2 / 3 of the cold water or ice water was rapidly added, and stirring was continued until the HPMC was completely dissolved to form a clear solution. OSA (final concentration 20 mg / mL) solid was added to the HPMC solution, and after stirring evenly, solution B was obtained.

[0129] Mix solution A and solution B at a volume ratio of 1:1 and stir at 400 rpm for 15-30 minutes at room temperature to achieve initial homogenization. Adjust the pH of the mixture to 5.5 with dilute NaOH solution (0.1 M), stir at room temperature for 15 minutes, sonicate at 40 kHz for 5 minutes, and let stand at 4°C for 2 hours. Add EDC solution (20 mg / mL) and NHS solution (20 mg / mL), with a mass ratio of EDC:NHS:recombinant type III collagen of 1:1:8, and stir until homogenized. Under gentle stirring at 150 rpm, slowly add 0.1 M CaCl2 solution at a volume ratio of 10:1 to the above system at a rate of approximately 1 drop per second. After stirring, pour the mixture into a mold and let it stand at room temperature to obtain collagen hydrogel W1C1.

[0130] Example 4

[0131] This embodiment provides a recombinant type III collagen hydrogel, the preparation method of which differs from that of Example 3 in that: 0.15 M CaCl2 solution is slowly added dropwise to the above system at a volume ratio of 10:1, stirred well, poured into a mold, and allowed to stand at room temperature to obtain collagen hydrogel W1C2. The remaining steps are completely consistent with those of Example 3.

[0132] Example 5

[0133] This embodiment provides a recombinant type III collagen hydrogel, the preparation method of which differs from that of Example 3 in that: EDC:NHS:recombinant type III collagen = 1:1:6 by mass, to obtain collagen hydrogel W2C1. The remaining steps are completely consistent with those of Example 3.

[0134] Example 6

[0135] This embodiment provides a recombinant type III collagen hydrogel, the preparation method of which differs from that of Example 3 in that: EDC:NHS:recombinant type III collagen = 1:1:6, by mass; 0.15 M CaCl2 solution is slowly added dropwise to the above system at a volume ratio of 10:1, stirred well, poured into a mold, and allowed to stand at room temperature to obtain collagen hydrogel W2C2. The remaining steps are completely consistent with those of Example 3.

[0136] Example 7

[0137] This embodiment provides a recombinant type III collagen hydrogel, the preparation method of which differs from that of Example 3 in that the mass ratio of EDC:NHS:collagen protein is 1:1:4, resulting in collagen hydrogel W3C1. The remaining steps are completely consistent with those of Example 3.

[0138] Example 8

[0139] This embodiment provides a recombinant type III collagen hydrogel, the preparation method of which differs from that of Example 3 in that: the mass ratio of EDC:NHS:collagen protein is 1:1:4; 0.15 M CaCl2 solution is slowly added dropwise to the above system at a volume ratio of 10:1, stirred well, poured into a mold, and allowed to stand at room temperature to obtain collagen hydrogel W3C2. The remaining steps are completely consistent with those of Example 3.

[0140] Performance testing

[0141] The properties of the recombinant type III collagen hydrogels obtained in Examples 3 to 8 were tested. The test content and results are as follows:

[0142] 1. Scanning electron microscopy (SEM) analysis

[0143] The internal structure of the hydrogel was examined using a GeminiSEM360 scanning electron microscope (Carl Zeiss AG, Germany). Prior to examination, the hydrogel was freeze-dried and coated with gold. SEM images of Example 5 (W2C1) are shown below. Figure 7 As shown, the hydrogel exhibits a porous morphology with uniform pore size distribution and intact shape, intuitively reflecting the EDC / NHS covalent cross-linking network and OSA-Ca 2+ The ion cross-linked network constitutes a stable and uniform network structure.

[0144] 2. Mechanical properties

[0145] Tensile tests were performed on the recombinant type III collagen hydrogels obtained in Examples 3-8, and cyclic compression tests were performed on the recombinant type III collagen hydrogel obtained in Example 5. The test speed for all tests was 10 mm / min. Compression / tensile strength was defined as the stress at which the collagen hydrogel fractured. The fatigue resistance of the hydrogels was characterized by 10 cycles of cyclic compression tests at 75% strain, with no interval between cycles. Three samples were tested in each cycle.

[0146] The stress-strain and corresponding Young's modulus experimental results of the tensile test are as follows: Figure 8 , Figure 9 As shown, the mechanical behavior of the hydrogels obtained in different embodiments clearly reflects the synergistic mechanism of the dual-network structure. Among them, Examples 7 (W3C1 = 308 kPa) and 8 (W3C2 = 330.2 kPa), with high chemical crosslinking, exhibit higher moduli, indicating that the high-density covalent crosslinking network effectively constructs a strong and rigid skeleton; however, their tensile strain is lower (W3C1 = 169.7%, W3C2 = 148.9%), indicating that a single reinforcing covalent network limits the dynamic energy dissipation of the ionic crosslinking network, leading to a relative decrease in the material's ductility. Examples 3 (W1C1 = 225.9%) and 4 (W1C2 = 219.3%), with low chemical crosslinking, exhibit higher tensile strain, but lower moduli (W1C1 = 80.1 kPa, W1C2 = 115.6 kPa), reflecting that when the covalent network is too weak, it cannot form effective support, and even with ionic crosslinking, it is difficult to improve the overall load-bearing capacity. Examples 5 (W2C1) and 6 (W2C2) exhibit a more synergistic dual-network characteristic: based on a covalent network framework with a suitable crosslinking density, W2C2 (0.15 M Ca) 2+ The modulus (180.2 kPa) is higher than that of W2C1 (0.1 M Ca). 2+ The 150 kPa pressure demonstrates how ionic crosslinking supplements stiffness, effectively dissipating energy through reversible dissociation / recombination, thus imparting superior toughness to the material while maintaining strength. The cyclic compression test results of Example 5 (W2C1) are as follows: Figure 10 As shown, the W2C1 hydrogel exhibits a similar load-unload curve after 10 cycles under high strain conditions of 75%, indicating its excellent elasticity and fatigue resistance. This confirms that the present invention, through specific dual-network construction and control of component conditions, can obtain collagen hydrogels with high mechanical strength and high toughness.

[0147] 3. Adhesion properties

[0148] To evaluate the adhesion properties of the hydrogel of this invention to skin tissue, a shear strength test was conducted on pigskin in the examples. After the hydrogel was bonded to the surface of pigskin, a shear force parallel to the bonding surface was applied at a test speed of 10 mm / min. Three samples were tested in each instance. The adhesion strength is as follows: Figure 11 As shown, at lower levels of chemical crosslinking (Examples 3 and 4), the Ca2+ level is increased. 2+ The concentration (W1C2, 22.1 kPa) showed higher adhesion strength than the low concentration sample (W1C1, 18.0 kPa); while at moderate levels of chemical crosslinking (Examples 5 and 6), lower Ca... 2+ The concentration (W2C1, 32.5 kPa) was actually superior to the higher concentration sample (W2C2, 28.5 kPa). This trend indicates that the improvement in adhesion strength does not depend on simply enhancing one type of crosslinking, but rather requires the two to achieve an optimal ratio. The adhesive properties of this dual-network hydrogel ensure that the present invention can firmly adhere to the wound surface and is not easily detached in moving areas, fully meeting the core requirements of a highly mechanically adaptable wound care hydrogel.

[0149] 4. Swelling test

[0150] The weight of the hydrogel in the embodiment at different time points was recorded by weighing. The swelling ratio (SR) reflects the degree of swelling of the gel in different solutions, and the calculation formula is as follows:

[0151]

[0152] Where W0 represents the original weight before soaking, W t This represents the weight of the hydrogel at time point t. The swelling time points are: 0 h, 1 h, 5 h, 12 h, 24 h, and 48 h.

[0153] Swelling test results are as follows Figure 12 As shown, Examples 3 and 4 (W1C1, W1C2) exhibited higher swelling rates due to their low covalent crosslinking density and network defects; Examples 5 to 8 (W2C1, W2C2, W3C1, W3C2) all showed suitable swelling rates. This swelling characteristic allows the hydrogel to effectively absorb wound exudate while avoiding excessive expansion, thus achieving an ideal moist environment and fit in wound care.

[0154] 5. Cell viability

[0155] L929 cells were distributed at a rate of 1 × 10⁶ cells per well. 4Each sample was inoculated into a 96-well plate and cultured in complete culture medium for 24 h. Then, the extract of the hydrogel from the previous example was added to each well. After further culturing for 72 h, 10% (v / v) of CCK-8 solution was added to each well, and after incubation for 2 h, the OD was measured using a microplate reader. 450 Cell viability is calculated. The cell viability results are as follows: Figure 13 As shown, all examples exhibited excellent cell viability, with Examples 3 to 6 (W1C1, W1C2, W2C1, W2C2) demonstrating good biocompatibility and a certain proliferative effect. This indicates that the present invention can provide a favorable microenvironment for cell growth.

[0156] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant type III collagen, characterized in that, Its amino acid sequence is shown in SEQ ID No. 2 or SEQ ID No.

3.

2. A gene encoding the recombinant type III collagen of claim 1, characterized in that, The nucleotide sequence is any one of SEQ ID No. 4-5.

3. A method for preparing recombinant type III collagen hydrogel, characterized in that, include: Mix solution A with solution B, adjust the pH, add the cross-linking agent and calcium salt, and let stand to obtain the final product. Solution A is the recombinant type III collagen solution according to claim 1; Solution B is a hydroxypropyl methylcellulose solution containing sodium alginate; The crosslinking agent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide; the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide and recombinant type III collagen is 1:1:(4~8); The calcium salt is an aqueous solution of calcium salt, and the concentration of the aqueous solution of calcium salt is 0.1~0.5 M. 0.5~1.5 mL of the aqueous solution of calcium salt is added for every 10 mL of mixed solution.

4. The preparation method according to claim 3, characterized in that, The oxidation degree of the oxidized sodium alginate is 25%~35%; The concentration of the recombinant type III collagen solution is 8-12 mg / mL; The concentration of sodium alginate in solution B is 15-25 mg / mL, and the concentration of hydroxypropyl methylcellulose is 1-3 mg / mL. The volume ratio of solution A to solution B is (0.9~1.1):(0.9~1.1).

5. The preparation method according to claim 3, characterized in that, The solvent of solution A includes any one or more of acetic acid, hydrochloric acid, phosphoric acid, citric acid, and ascorbic acid.

6. The preparation method according to claim 3, characterized in that, The method for preparing the oxidized sodium alginate includes: Sodium alginate aqueous solution and oxidant were mixed and oxidized under light-protected conditions to terminate the reaction. Alcohol was added to precipitate the precipitate, which was then collected by filtration. The precipitate was redissolved in water, dialyzed, and freeze-dried to obtain oxidized sodium alginate.

7. The preparation method according to claim 6, characterized in that, The concentration of the sodium alginate aqueous solution is 1-5 wt%; The oxidant includes sodium periodate; the mass ratio of sodium alginate to sodium periodate is (1.8~2.2):1; The oxidation time is 4-5 hours; The reaction was terminated by adding ethylene glycol; after adding ethylene glycol, stirring was continued in the dark for 0.5-1 h; the amount of ethylene glycol used was 4-6 mL / g sodium alginate; The alcohol includes ethanol, and the amount of alcohol used is 70~90 mL / g sodium alginate.

8. The preparation method according to claim 3, characterized in that, Mix solution A with solution B, adjust the pH to 5.4-5.6, stir for 15-30 min, mix well, and let stand to obtain solution C; add cross-linking agent to solution C, stir well, and add calcium salt aqueous solution dropwise under stirring conditions. After the addition is complete, let stand to gel, and obtain recombinant type III collagen hydrogel.

9. A recombinant type III collagen hydrogel, characterized in that, It is prepared by the preparation method described in any one of claims 3-8.

10. The application of the recombinant type III collagen of claim 1 or the recombinant type III collagen hydrogel of claim 9 in the preparation of biomedical materials; The biomedical materials include any one or more of the following: collagen hemostatic cotton, surgical sutures, and wound dressings.

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