Method for extracting acidic telopeptide-containing collagen and use thereof

Abstract

The application discloses an acid-containing telopeptide collagen extraction method and application thereof, relates to the technical field of biological materials, and comprises the following steps: removing fat from a bovine tissue, dividing the bovine tissue into uniform substances, sequentially soaking the uniform substances in an alkaline solution, a carbonate solution, an organic solvent solution and a salt solution, draining the uniform substances, soaking the uniform substances in an acid solution, removing impurities through centrifugation, neutralizing the acid solution and the alkaline solution, centrifuging, soaking the uniform substances in an acid solution again, ultrafiltrating, micropore filtering, and preparing the acid-containing telopeptide collagen through preparation means; the acid-containing telopeptide collagen with high purity and high telopeptide retention rate can be prepared; the telopeptide collagen of the acid-containing telopeptide collagen contains specific amino acids in a telopeptide region on a crosslinking site, is a key site for forming covalent crosslinking between collagen molecules and inside collagen fibers, has high mechanical strength, and is suitable for the fields of cartilage tissue repair and the like.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a method for extracting acidic telopeptide-containing collagen and its application. Background Technology

[0002] Type I collagen is an important component of soft tissue in mammals, occupying a crucial position in the animal body. Collagen plays a central role in tissue engineering, serving as the "gold standard material" for constructing biomimetic extracellular matrix (ECM). Its unique structure and biological functions make it a key foundational material for tissue repair and regeneration. As a tissue engineering scaffold material, its excellent natural ECM biomimetic properties, biocompatibility, and signal transduction functions are increasingly recognized by researchers.

[0003] Collagen in tissue engineering has shown unlimited potential in the transformation into medical devices. Collagen products already used in medicine include hemostatic sponges, hemostatic powder, mineralized bone powder, artificial blood vessel coatings, bioprosthetic valves, cartilage repair, artificial corneas, and collagen implants. However, most of these products are based on determinate collagen, and rarely use telopeptide-containing collagen. Perhaps due to the issue of immunogenicity, telopeptides are generally considered by scholars to be important substances that trigger immunogenicity. Zhang Ziqiang et al. (Zhang Ziqiang, Zhang Yihe, An Qi, et al. Immunogenicity study of determinate type I collagen[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(22):7.) described how determinate type I collagen prepared by enzymatic hydrolysis, dialysis, freeze drying, and sterilization can reduce adverse immune responses and inflammation by increasing collagen purity, reducing DNA residue, telopeptide residue, and α-Gal antigen. The latest research by Hu Zhangjie et al. (Hu Zhangjie, Zhang Baoguan, Zhang Zhiwu. Application of solid collagen-based materials in medical devices [J]. Chinese Journal of Tissue Engineering Research, 2025, 29(16):3503-3512.) points out the application of telopeptide-containing collagen in cartilage repair. How to obtain high-purity telopeptide-containing collagen has become an important bottleneck restricting the development of telopeptide-containing collagen.

[0004] Therefore, the preparation of high-purity and non-allergenic telopeptide collagen has become an important measure to broaden the application of collagen in the medical field. Summary of the Invention

[0005] This application provides a method for extracting acidic telopeptide-containing collagen and its application, which solves the problems of low purity and high sensitization of telopeptide collagen in the prior art, and achieves high purity and low sensitization of telopeptide-containing collagen.

[0006] This application provides a method for extracting acidic collagen containing terminal peptides, comprising the following steps:

[0007] The fat in the beef tissue was removed and cut into uniform tissue blocks. The blocks were then soaked in alkaline solution, carbonate solution, organic solvent solution and salt solution in sequence and then drained.

[0008] The drained tissue blocks were soaked in an acidic solution for extraction.

[0009] After centrifugation to remove impurities, acid-base neutralization, secondary acid dissolution, ultrafiltration, and microfiltration, collagen containing telopeptides was obtained.

[0010] Furthermore, the bovine tissue is bovine Achilles tendon, bovine hide, or bovine cartilage tissue;

[0011] The cut tissue blocks are fibrous, sheet-like, or granular in shape. The dimensions of fibrous tissue blocks are 5cm-10cm in length, 1mm-1.5mm in width, and 1mm-1.5mm in height. The dimensions of sheet-like tissue blocks are 5cm-10cm in length, 5cm-10cm in width, and 1mm-1.5mm in height. The dimensions of granular tissue blocks are 1mm-1.5mm in length, 1mm-1.5mm in width, and 1mm-1.5mm in height.

[0012] Furthermore, the alkaline solution is a 0.05M-0.5M sodium hydroxide solution or a 0.05M-0.5M potassium hydroxide solution;

[0013] The carbonate solution is a 0.05M-1.0M sodium carbonate solution, a 0.05M-1.0M sodium bicarbonate solution, or a 0.08M-0.3M calcium hydroxide solution;

[0014] The organic solvent solution is a 20%-95% ethanol solution or a 20%-70% diethyl ether solution;

[0015] The salt solution is at least one of 0.9%-20% sodium chloride solution, potassium chloride solution, or EDTA solution;

[0016] The soaking time for each solution is 30 min to 12 h.

[0017] Furthermore, the acidic solution is one of 10mM-50mM hydrochloric acid, 0.03mM-0.2M phosphoric acid, 0.05mM-0.2M citric acid, 0.05M-0.5M acetic acid, 0.05M-0.5M formic acid, or 0.01M-0.1M nitric acid, and the soaking time is 3 to 25 days.

[0018] Furthermore, the centrifugal force for centrifugal impurity removal is 5000g-80000g;

[0019] The alkaline solution used for acid-base neutralization is one of 1M-10M sodium hydroxide solution, 1M-10M potassium hydroxide solution, or 0.05M-0.5M calcium hydroxide solution, adjusting the pH to 5-7; the secondary acid dissolution uses the acidic solution from step S2 to dissolve until transparent; ultrafiltration uses a hollow fiber column with a pore size of 100 kDa; microfiltration uses a 0.22 μm microporous membrane.

[0020] Furthermore, the terminal peptide-containing collagen is type I collagen, which is prepared into a solution with a protein content of 1 mg / ml to 20 mg / ml by vacuum concentration.

[0021] The above-mentioned application of acidic telopeptide-containing collagen in the preparation of soft tissue repair materials involves reconstituted vacuum-concentrated telopeptide-containing collagen with 0.1M acetic acid solution to a final concentration of 40mg / ml, adding heparan sulfate and oxidized konjac glucomannan, and then 3D printing to obtain the repair material.

[0022] The amount of heparan sulfate added is 1% of the mass of the telopeptide collagen, and the amount of oxidized konjac glucomannan added is 3% of the mass of the telopeptide collagen.

[0023] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages:

[0024] By employing an extraction process combining acidic solution soaking with centrifugation, acid-base neutralization, secondary acid dissolution, ultrafiltration, and microfiltration, the problems of low purity and high allergenicity of telopeptide-containing collagen in existing technologies were effectively solved, thus achieving the preparation of high-purity, low-allergenic telopeptide-containing collagen. Furthermore, by using a gradient soaking treatment of bovine tissue with alkaline solution, carbonate solution, organic solvent solution, and salt solution, non-collagenous components (such as cell membrane proteins, lipids, and nucleic acid fragments) in the tissue were effectively removed, achieving complete preservation of the collagen telopeptide structure and efficient removal of impurities.

[0025] By combining terminal peptide-containing collagen with heparan sulfate and oxidized konjac glucomannan, and using 3D printing technology to construct a ternary dynamic network structure, the problems of insufficient mechanical strength and lack of repair signals in traditional collagen raw materials are effectively solved, thereby realizing the structural strengthening and active repair functions of soft tissue repair materials.

[0026] By adding NaOH solution dropwise during the 3D printing process to adjust the pH to 7.2, and combining it with a low-temperature environment (4℃) and genipin cross-linking treatment, the ternary network structure of collagen-heparan sulfate-oxidized konjac glucomannan was effectively stabilized, thereby achieving mechanical stability and biocompatibility of the repair material after implantation. Attached Figure Description

[0027] Figure 1The image shows the SDS-PAGE results of the telopeptide-containing collagen. In the image, the "marker" lane represents the standard molecular weight sample, and the "sample" lane represents the telopeptide-containing collagen.

[0028] Figure 2 The image shows the SDS-PAGE results of determinated peptides. In the image, the "marker" lane represents the standard molecular weight sample, and the "original sample" lane represents determinated peptide collagen.

[0029] Figure 3 The left side shows a 3D hydrogel formed by determinate collagen and the right side shows a 3D hydrogel formed by terminate collagen.

[0030] Figure 4 Schematic diagram of collagen containing telopeptides;

[0031] Figure 5 Schematic diagram of determinate collagen;

[0032] Figure 6 Comparison of elastic modulus between collagen containing telopeptides and collagen without telopeptides;

[0033] Figure 7 The creep / recovery effect of hydrogels prepared with telopeptide collagen;

[0034] Figure 8 Stress relaxation of hydrogels prepared from telopeptide collagen. Detailed Implementation

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0036] Example 1: A method for extracting acidic telopeptide-containing collagen, specifically including: a method for preparing telopeptide-containing collagen and its application, comprising the following steps:

[0037] S1. Remove fat from bovine tissue and cut it into uniform tissue blocks. Soak the blocks in an alkaline solution, carbonate solution, organic solvent solution, and salt solution in sequence, then drain. The bovine tissue consists of bovine Achilles tendon, hide, and cartilage. The uniformly sized tissue blocks after cutting can be fibrous, sheet-like, or granular. If fibrous, the dimensions are between 5cm*1mm*1mm and 10cm*1.5mm*1.5mm; if sheet-like, the dimensions are between 5cm*5cm*1mm and 10cm*10cm*1.5mm; if granular, the dimensions are between 1mm*1mm*1mm and 1.5mm*1.5mm*1.5mm.

[0038] The alkaline solution is a 0.05M-0.5M sodium hydroxide solution or a 0.05M-0.5M potassium hydroxide solution;

[0039] The carbonate solution is a 0.05M-1.0M sodium carbonate solution, a 0.05M-1.0M sodium bicarbonate solution, or a 0.08M-0.3M calcium hydroxide solution;

[0040] The organic solvent solution is a 20%-95% ethanol solution or a 20%-70% diethyl ether solution;

[0041] The salt solution is at least one of 0.9%-20% sodium chloride solution, potassium chloride solution, or EDTA solution;

[0042] The soaking time for each solution is 30 min to 12 h.

[0043] S2. After soaking and draining, add the bovine tissue pieces to an acidic solution and soak them. The acidic solution is one of the following: 10mM-50mM hydrochloric acid, 0.03mM-0.2M phosphoric acid, 0.05mM-0.2M citric acid, 0.05M-0.5M acetic acid, 0.05M-0.5M formic acid, or 0.01-0.1M nitric acid. The soaking time is 3 to 25 days.

[0044] S3. Then, after centrifugation to remove impurities, acid-base neutralization, centrifugation, secondary acid dissolution, ultrafiltration, microfiltration, and preparation, high-purity collagen containing telopeptides can be obtained. The obtained collagen containing telopeptides is type I collagen, which is then concentrated under vacuum to prepare a solution with a protein content of 1-20 mg / ml.

[0045] Centrifugation is used to remove impurities that have not been acid-digested, with a centrifugal force of 5000g-80000g.

[0046] Acid-base neutralization involves using an alkaline solution to stop the acidolysis reaction. The alkaline solution used includes one of the following: 1M-10M sodium hydroxide solution, 1M-10M potassium hydroxide solution, or 0.05M-0.5M calcium hydroxide solution, adjusting the pH to 5-7. Secondary acid dissolution involves using the acidic solution from step S2 again to dissolve the solution until it becomes transparent.

[0047] The ultrafiltration is a hollow fiber column ultrafiltration with a pore size of 100 kDa;

[0048] Microfiltration is performed using a microporous membrane with a diameter of 0.22 μm.

[0049] Specific example 1: S1 Take 25g of cowhide, remove the fat, and then use a knife to cut the tissue into uniform granules of about 1mm in length, width and height. Soak the granules in 2L of 0.05M sodium hydroxide solution, 2L of 5% sodium carbonate solution, 2L of 50% ether solution and 2L of 0.9% / L sodium chloride solution for 30min each. Remove the residual fat in the tissue. Under high salt conditions, the cells in the tissue will be cleaved and extracted. Drain the water and set aside.

[0050] S2. Add the product obtained in step S1 to 10L of 0.5M citric acid solution and acid dissolve for 3 days to dissociate the collagen fiber network inside the tissue into monomeric or polymeric collagen.

[0051] S3. The substance obtained in step S2 is centrifuged at 5000g to remove un-acidified impurities. Then, 0.05M sodium hydroxide solution is added to adjust the pH of the overall solution to 5. The precipitate is then removed, and 10L of 0.5M citric acid solution is added again to dissolve it until clear. Small molecule impurities in the solution are then removed by ultrafiltration using a 100KD hollow fiber column, and polymers and impurities are removed by filtration through a 0.22μm microporous filter. The resulting sample is freeze-dried to a concentration of 1mg / ml to obtain a commercially viable 1mg / ml sample.

[0052] Specific example 2: S1. Take 25g of beef Achilles tendon, remove the fat, and then use a knife to cut the tissue into uniform granular materials with a length and width of about 1.5mm. Soak the granules in 2L of 0.5M sodium hydroxide solution, 2L of 1.0M sodium carbonate solution, 2L of 70% ether solution, and 2L of 20% sodium chloride solution for 12 hours each to remove the fat involved in the tissue and dissociate the substances inside the cells.

[0053] S2. Add the product obtained in step S1 to 10 L of 0.2 M citric acid solution and acid hydrolyze for 25 days;

[0054] S3. The substance obtained in step S2 is centrifuged at 80,000g to remove un-acidified impurities. Then, 0.05M sodium hydroxide solution is added to adjust the pH of the overall solution to 5. The precipitate is then removed, and 0.4L of 0.2M citric acid solution is added again to dissolve it until clear. Small molecule impurities in the solution are then removed by ultrafiltration using a 100KD hollow fiber column, and polymers and impurities are removed by microfiltration using a 0.22µm micropore filter. The resulting sample is freeze-dried to a concentration of 20mg / ml to obtain a commercially viable 20mg / ml sample.

[0055] Specific example 3: S1. Take 25g of cowhide, remove the fat, and then use a knife to cut the tissue into uniform granules with a length, width and height of about 1.5mm. Soak the granules in 2L of 0.5M sodium hydroxide solution, 2L of 50% sodium carbonate solution, 2L of 70% ether solution and 2L of 10% sodium chloride solution for 12 hours each. Each soaking requires draining.

[0056] S2. Add the product obtained in step S1 to 10 L of 0.5 M acetic acid solution and acid hydrolyze for 25 days.

[0057] S3. The substance obtained in step S2 is centrifuged at 10000g to remove un-acidified impurities. Then, 1M sodium hydroxide solution is added to adjust the pH of the overall solution to 7. The precipitate is then removed, and 1.0L of 0.5M acetic acid solution is added again to dissolve it until clear. Small molecule impurities in the solution are then removed by ultrafiltration using a 100KD hollow fiber column, and polymers and impurities are removed by filtration through a 0.22μm micropore. The resulting sample is diluted to a concentration of 10mg / ml to obtain a commercially viable 10mg / ml sample.

[0058] Specific example 4: S1. Take 25g of Achilles tendon, remove the fat, and then use a knife to cut the tissue into uniform slices about 5cm*5cm*1mm long. Soak the slices in 2L of 0.05M sodium hydroxide solution, 2L of 1M sodium carbonate solution, 2L of 50% ether solution, and 2L of 10% sodium chloride solution for 30 minutes each. Drain the slices after each soaking.

[0059] S2. Add the product obtained in step S1 to 10 L of 0.1 M formic acid solution and dissolve in acid for 3 days.

[0060] S3. The substance obtained in step S2 is centrifuged at 5000g to remove un-acidified impurities. Then, 0.05M sodium hydroxide solution is added to adjust the pH of the overall solution to 7. The precipitate is then removed, and 10L of 0.1M formic acid solution is added again to dissolve it until clear. Small molecule impurities in the solution are then removed by ultrafiltration using a 100KD hollow fiber column, and polymers and impurities are removed by microfiltration using a 0.22μm microporous filter. The resulting sample is diluted to a concentration of 2mg / ml to obtain a commercially viable 2mg / ml sample.

[0061] Comparative Example 1: The only difference from Specific Example 3 is that 0.75g of pepsin was added when adding the acid solution in step S2;

[0062] Comparative Example 2: The only difference from Specific Example 3 is that it was soaked in 2L of 50% sodium carbonate solution, 2L of 10% sodium chloride solution, 2L of 70% ether solution, and 2L of 0.5M sodium hydroxide solution for 12 hours each.

[0063] Comparative Example 3: The only difference from Specific Example 3 is that it was soaked in 2L of 70% ether solution, 2L of 50% sodium carbonate solution, 2L of 0.5M sodium hydroxide solution, and 2L of 10% sodium chloride solution for 12 hours each.

[0064] Comparative Example 4: The only difference from Specific Example 3 is that it was soaked in 2L of 10% sodium chloride solution, 2L of 70% ether solution, 2L of 50% sodium carbonate solution, and 2L of 0.5M sodium hydroxide solution for 12 hours each.

[0065] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0066] Acids break down the ionic bonds, hydrogen bonds, and some weaker covalent bonds (such as Schiff bases formed by aldehyde-amine condensation) between collagen fibers, causing the tightly cross-linked collagen fiber network to dissociate into monomeric collagen or smaller polymers. Acid treatment itself does not sever the peptide bonds of the collagen molecule backbone; it affects the intermolecular interactions and solvation rather than the intramolecular covalent structure. Therefore, the complete structure of the collagen molecule, including the crucial telopeptide region, is preserved intact.

[0067] The subsequent transition from alkali to salt solution gradually removes residual fat, lyses cell membranes, and dissolves non-collagenous proteins and glycosaminoglycans. Osmotic pressure is adjusted during the gradual pH reduction to salt solution transition, opening up the tissue. Lipid-soluble substances are extracted using organic solutions. Finally, high salt concentrations precipitate some impurities, reducing metal ions that may participate in cross-linking or act as enzyme cofactors, preventing non-specific reactions during subsequent acidolysis, and stabilizing collagen. This process is conducted at relatively mild concentrations and times (alkali concentration 0.05-0.5M, time 30 min-12 h), aiming to remove non-collagenous components from the tissue without significantly damaging the triple helix structure and telomeres of collagen itself.

[0068] The pH is adjusted to 5-7 using an alkaline solution (close to the isoelectric point of collagen), causing telopeptide-containing collagen molecules to precipitate due to their lowest solubility near neutral pH. Many other proteins (with different isoelectric points) or small molecule impurities remain in the supernatant or are removed by centrifugation. The neutralized precipitated collagen is then redissolved in an acidic solution, where the acid restores the collagen's solubility (making it positively charged). Only collagen that can be completely dissolved at this specific acid concentration (usually structurally intact collagen that has depolymerized into monomers) can proceed to the next step. Undissolved collagen may be overly cross-linked or denatured aggregates and will be discarded, providing a clear and homogeneous starting solution for subsequent ultrafiltration and filtration steps.

[0069] The telopeptide-containing collagen prepared in this embodiment exhibits low allergenicity, primarily due to its highly efficient impurity removal and preservation of natural structure. The most prevalent immunogenic substances in animal tissues are non-collagenous proteins (such as cell membrane proteins, cytoplasmic proteins, serum proteins, and glycoproteins). The powerful impurity removal combination of the transition solution (alkali, salt, and organic solvents), along with subsequent centrifugation, neutralization precipitation, ultrafiltration, and filtration, systematically and extremely effectively removes these non-collagenous protein components. Organic solvents (ether / ethanol) effectively remove lipids (including potential lipid-related antigens), while alkali treatment and EDTA also contribute to the removal of nucleic acid fragments. Alkali treatment, high-salt treatment, and organic solvent treatment have a certain effect on removing or inactivating bacterial endotoxins (LPS). The final 0.22 μm sterile filtration further removes microorganisms and endotoxin aggregates.

[0070] The acid hydrolysis process does not denature collagen, and the collagen molecules retain their natural triple helix structure (renaturation). Without the use of proteases that break peptide bonds, the primary structure (amino acid sequence) of collagen is intact, providing a basis for proper renaturation. The immunogenicity of collagen with a natural triple helix structure is much lower than that of denatured gelatin (completely denatured collagen). The intact natural conformation may hide some potential linear antigenic epitopes.

[0071] The telopeptide region itself contains some antigenic epitopes; however, in the intact natural triple-helix collagen molecule, these epitopes are spatially shielded or have low conformation dependence. More importantly, this approach completely removes highly immunogenic non-collagenous impurities, making the residual and weak immunogenicity from the telopeptide negligible overall.

[0072] Terminal peptide regions (especially those rich in lysine (Lys) and hydroxylysine (Hyl) residues) are key sites for the formation of covalent cross-links between collagen molecules. Lysine oxidase (LOX) catalyzes the oxidation and deamination of Lys / Hyl residues in the terminal peptide region to generate aldehyde groups (allysine / hydroxyallysine). These aldehyde groups can spontaneously react with the ε-amino group of Lys / Hyl in another molecule's helical region or terminal peptide region to form aldehyde-amine condensation products (Schiff base) or condense with another aldehyde group to form aldol condensation products (Aldol condensation). These products further rearrange and reduce to form stable mature covalent cross-links (such as hydroxylysylpyridinium (HP), lysylpyridinium (LP), histidine-hydroxyopen-chain lysine (HHMD), etc.). Retaining intact terminal peptides means that collagen molecules retain the ability to form these natural and mature cross-links.

[0073] Because telopeptide-free collagen has lost key telopeptide cross-linking sites, its molecules can only rely on weak non-covalent interactions (such as hydrogen bonds, electrostatic interactions, and hydrophobic interactions) or require external cross-linking agents (such as glutaraldehyde and carbodiimide EDC) to form cross-links. Such cross-linked networks are usually weak, have poor stability, and are also affected in terms of biocompatibility.

[0074] Using Specific Example 3 and Comparative Example 1 as experiments, the final product was subjected to electrophoretic testing according to YYT 1453-2016, the method for characterizing type I collagen in tissue-engineered medical device products. The electrophoresis results are as follows: Figure 1 , Figure 2 As shown, the SDS-PAGE electrophoresis bands are intact, and the sample clearly consists of two α1 bands and one α2 band. To more intuitively demonstrate the differences in collagen containing telopeptides, Figure 4 A schematic diagram of collagen containing telopeptides is provided. Figure 5 A schematic diagram of determinate collagen is provided, showing that terminate collagen has a larger molecular weight compared to determinate collagen.

[0075]

[0076]

[0077] The obtained pure collagen was subjected to shear modulus testing, and the elastic modulus was measured using a dynamic mechanical analyzer (DMA) at 25°C. The difference in elastic modulus between the end-containing and end-removed collagen was compared. The results are as follows: Figure 6 And analyze the creep / recovery of collagen containing telopeptides (see [reference]). Figure 7 Stress relaxation is seen Figure 8 .

[0078] Purity and immunogenicity were verified by detecting residual impurities. Nucleic acid (ultraviolet spectrophotometry (A260 / A280)), lipid residue (sulfur-phosphorus-iron colorimetric method), endotoxin (Limulus amebocyte lysate (LAL) reagent dynamic turbidimetric method), and ash / inorganic matter (residue on ignition method (ash)) were detected. The results are shown in Table 1.

[0079] Table 1 Experimental Results

[0080]

[0081] Example 2: The above example prepared an extraction scheme for collagen containing telopeptides. This example provides an application of collagen containing telopeptides, which is applied to soft tissue repair.

[0082] Take the vacuum-concentrated collagen containing telopeptides, redissolve it with 0.1M acetic acid solution (pH 3.0) to a final concentration of 40mg / ml, then add heparan sulfate at a rate of 1% of the mass of the collagen containing telopeptides, stir at low speed at 4℃ for 12h to dissolve, and obtain a transparent viscous collagen solution.

[0083] In an ice bath, the collagen solution was mixed with PBS solution, and then oxidized konjac glucomannan was added. The amount of oxidized konjac glucomannan added was 3% of the mass of the collagen containing telopeptides, and the volume of PBS solution was 10% of the volume of the collagen solution. Then, 0.1M NaOH solution was added dropwise until the pH reached 7.2. The solution was then transferred to a syringe at 4°C. The printer was a basic 3D printer with the following parameters: needle: 22-27G conical needle (diameter 200μm), temperature: printing platform 15°C (delayed gel), ambient temperature 4°C; pressure: 100kPa, layer height: 150% of needle diameter, speed: 7mm / s.

[0084] After printing, incubate at 37°C for 1 hour, then immerse in 0.1% (w / v) genipin PBS solution (24 hours, 37°C), and then rinse with PBS; the desired result can be obtained.

[0085] The technical solutions described in the embodiments of this application have at least the following technical effects or advantages:

[0086] The resulting 3D hydrogel is as follows Figure 3 , Figure 3The left side shows a 3D hydrogel formed by determinate collagen and the right side shows a 3D hydrogel formed by terminate collagen. The 3D hydrogel formed by determinate collagen was prepared by Comparative Example 1, and the 3D hydrogel formed by terminate collagen was prepared by Specific Example 3.

[0087] Heparan sulfate carries a strong negative charge (sulfonic acid group, carboxyl group), which electrostatically binds to the telopeptide region of collagen containing telopeptides to form a "charge protection layer", physically shielding the exposure of aldehyde groups and reducing intermolecular Schiff base cross-linking. At the same time, heparan sulfate acts as a cell signaling molecule (binding growth factors such as FGF-2), activating cell migration pathways and promoting the directional migration of soft tissue repair cells (such as fibroblasts) into the gel.

[0088] The oxidized aldehyde group (-CHO) of oxidized konjac glucomannan competitively consumes the ε-amino group of collagen telopeptide to form a "collagen-oxidized konjac glucomannan" cross-link (replacing part of the collagen-collagen cross-link), breaking the original dense network. The long chain structure of oxidized konjac glucomannan is interspersed between collagen fibers, and the network toughness is enhanced by hydrogen bonds and van der Waals forces.

[0089] The aldehyde group of oxidized konjac glucomannan forms a new Schiff base bond with the amino group (N-acetylglucosamine) of heparan sulfate (heparan sulfate -NH2+ O=CH-oxidized konjac glucomannan → heparan sulfate -N=CH-oxidized konjac glucomannan), constructing a ternary dynamic network of "collagen-heparan sulfate-oxidized konjac glucomannan". This makes it stable in a neutral printing environment (pH 7.2). After implantation, some bonds are reversibly broken in a slightly acidic environment (pH 6.5-7.0), gradually releasing heparan sulfate signaling molecules. Under external force, the ternary network consumes energy through bond breaking and recombination, avoiding brittle fracture of collagen fibers.

[0090] Heparan sulfate releases FGF-2 signals, guiding cells to migrate directionally along the ternary network; the hydrophilic chains of oxidized konjac glucomannan increase porosity and enhance nutrient permeability (pore size increases from <10 μm to 20-50 μm), solving the problem of excessively rigid networks hindering cell migration and nutrient diffusion.

[0091] Heparan sulfate activates the Wnt / β-catenin pathway, promoting extracellular matrix (ECM) synthesis; oxidized konjac glucomannan degradation products (low molecular weight polysaccharides) have anti-inflammatory and antioxidant properties, reducing fibrosis in the repair zone and addressing the problem that pure collagen only provides structural support and lacks active repair signals.

[0092] To verify the signal-guided and anti-inflammatory effects, human dermal fibroblasts (HDFs, third-generation cells) were used as a cell model. Based on Example 3, heparan sulfate and oxidized konjac glucomannan were added, along with single-factor and comparative Example 1 (control group) for cross-compartment migration experiments. In a 2 mm aperture migration chamber, the upper chamber contained cell suspension (5 × 10⁵ cells / ml, 100 μl), and the lower chamber contained a 3D-printed gel. After 24 h, the cells were fixed and stained, the number of migrated cells was counted, and the cell penetration depth was measured by confocal microscopy. The experimental results are shown in Table 2.

[0093] Table 2. Cell compatibility and migration verification

[0094]

[0095] Then, soft tissue repair was verified by squeezing the gel into a rabbit cartilage defect model (5×5×5mm hole for modeling). Two months later, the rabbit was euthanized and removed by gross dissection. The cartilage repair model did not show any serious inflammation, and there was obvious repair in the cartilage defect area.

[0096] The thickness of newly formed cartilage was measured using a microindenter to simulate deformation resistance under joint load and to detect the compressive modulus. Joint fluid was collected and the concentration of tumor necrosis factor α was determined using enzyme-linked immunosorbent assay (ELISA). The results are shown in Table 3.

[0097] Table 3. Results of cartilage repair experiments

[0098]

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. 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 method for extracting acid telopeptide-containing collagen, characterized by, The method comprises the following steps: The bovine tissue is deboned and cut into uniform tissue pieces, and then sequentially soaked in an alkaline solution, a carbonate solution, an organic solvent solution and a salt solution, and then drained; The alkaline solution is a 0.05M-0.5M sodium hydroxide solution or a 0.05M-0.5M potassium hydroxide solution; The carbonate solution is a 0.05M-1.0M sodium carbonate solution or a 0.05M-1.0M sodium bicarbonate solution; The organic solvent solution is a 20%-70% diethyl ether solution; The salt solution is at least one of a 0.9%-20% sodium chloride solution or a potassium chloride solution; The soaking time is 30min-12h; The drained tissue pieces are soaked in an acid solution for extraction; The acid solution is one of a 10mM-50mM hydrochloric acid solution, a 0.03mM-0.2M phosphoric acid solution, a 0.05mM-0.2M citric acid solution, a 0.05M-0.5M acetic acid solution, a 0.05M-0.5M formic acid solution or a 0.01M-0.1M nitric acid solution, and the soaking time is 3-25 days; After centrifugal impurity removal, acid-base neutralization, secondary acid dissolution, ultrafiltration and micropore filtration, a telopeptide-containing collagen is prepared; The centrifugal impurity removal is performed at a centrifugal force of 5000g-80000g; The acid-base neutralization uses one of a 1M-10M sodium hydroxide solution, a 1M-10M potassium hydroxide solution or a 0.05M-0.5M calcium hydroxide solution to adjust the pH to 5-7; the secondary acid dissolution uses an acid solution to dissolve until transparent; the ultrafiltration uses a hollow fiber column with a pore size of 100KDa; and the micropore filtration uses a 0.22μm micropore filter membrane.

2. The method of claim 1, wherein the acid endopeptidase-containing collagen extract is characterized by, The bovine tissue is bovine Achilles tendon, bovine skin or bovine cartilage tissue; The cut tissue pieces are in the shape of filaments, sheets or granules, wherein the filament-shaped tissue pieces have a size of 5cm-10cm in length x 1mm-1.5mm in width x 1mm-1.5mm in height, the sheet-shaped tissue pieces have a size of 5cm-10cm in length x 5cm-10cm in width x 1mm-1.5mm in height, and the granule-shaped tissue pieces have a size of 1mm-1.5mm in length x 1mm-1.5mm in width x 1mm-1.5mm in height.

3. The method for extracting acidic terminal peptide-containing collagen as described in claim 1, characterized in that, The telopeptide-containing collagen is type I collagen, and a solution with a protein content of 1mg / ml-20mg / ml is prepared by vacuum concentration.

4. Use of the acid, endopeptidase-containing collagen according to any one of claims 1 to 3 for the preparation of a cartilage repair material, characterized in that, The vacuum-concentrated telopeptide-containing collagen is redissolved in a 0.1M acetic acid solution to a final concentration of 40mg / ml, and then a heparan sulfate and oxidized konjac glucomannan are added to prepare a repair material by 3D printing; The heparan sulfate is added in an amount of 1% of the mass of the telopeptide-containing collagen, and the oxidized konjac glucomannan is added in an amount of 3% of the mass of the telopeptide-containing collagen.

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