PDRN-crosslinked collagen gel and preparation method thereof

Abstract

The invention relates to the technical field of biological materials, and particularly discloses PDRN-crosslinked collagen gel and a preparation method thereof.According to the gel, through a TCFH-NMI system mediated amidation reaction, polydeoxyribonucleotide (PDRN) and a collagen molecular chain are subjected to covalent crosslinking, and a stable three-dimensional network structure is formed. The preparation method comprises the following steps: respectively dissolving collagen and PDRN, then mixing, adding NMI, adding TCFH in batches, carrying out a cross-linking reaction, and after the reaction is finished, carrying out dialysis purification to obtain the PDRN-cross-linked collagen gel. The prepared gel has good structural stability, mechanical performance and biocompatibility, can effectively delay PDRN release and improve the in-vivo stability of the material, and is suitable for the fields of wound repair, skin tissue engineering scaffolds, medical beauty filling materials and the like.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, specifically to a PDRN-crosslinked collagen gel and its preparation method. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) are a class of bioactive substances composed of deoxynucleotide fragments. They possess good biocompatibility and safety, and have clear effects in promoting cell proliferation, tissue repair, and anti-inflammatory regulation. They have already been applied in wound repair, soft tissue injury repair, and medical aesthetic regeneration. With the development of extraction and purification technologies, the application scope of high-purity PDRN products continues to expand.

[0003] Collagen is an important component of the human extracellular matrix, possessing excellent biocompatibility, biodegradability, and cell adhesion properties, and is widely used in tissue engineering scaffolds and medical repair materials. Recombinant collagen has even greater advantages in application due to its controllable source and low immune risk. However, collagen materials often suffer from problems such as low mechanical strength, insufficient in vivo stability, and susceptibility to enzymatic degradation. In practical applications, cross-linking modification is often necessary to improve its structural stability and lifespan.

[0004] Existing collagen cross-linking methods mostly employ glutaraldehyde, genipin, or carbodiimide systems. Some of these cross-linking agents pose cytotoxicity or residue risks, and some methods require harsh reaction conditions or organic solvent systems, resulting in insufficient environmental friendliness and safety controllability. Furthermore, current technologies often use physical mixing or encapsulation methods to combine PDRN and collagen, lacking a stable covalent bond structure, which can lead to problems such as rapid release of active ingredients, insufficient stability, and poor functional persistence. Based on the above, this invention provides a PDRN-cross-linked collagen gel and its preparation method. Summary of the Invention

[0005] To address the shortcomings of existing technologies, such as the rapid release of active ingredients and poor system stability caused by physical mixing of PDRN and collagen, as well as the high risk of toxic residues, harsh reaction conditions, and poor environmental friendliness of traditional collagen cross-linking methods, this invention provides a PDRN-cross-linked collagen gel and its preparation method.

[0006] In a first aspect, the present invention provides a PDRN-crosslinked collagen gel, employing the following technical solution: A PDRN-crosslinked collagen gel, wherein the PDRN-crosslinked collagen gel is formed by covalent crosslinking of PDRN and collagen through a TCFH-NMI-mediated amidation reaction, wherein PDRN forms amide bonds with carboxyl groups on the collagen molecular chain through amino groups in its molecular structure.

[0007] Preferably, the PDRN-crosslinked collagen gel has the following performance characteristics: pH 6.5-6.8, osmotic pressure 285-290 mOsmol / kg; swelling degree 28-36 in 0.9 wt% sodium chloride solution; storage modulus 300-350 Pa and loss modulus 200-250 Pa at a frequency of 5 Hz.

[0008] Preferably, the collagen is selected from one or more of type I collagen, type III collagen, type V collagen, and recombinant collagen.

[0009] Preferably, the collagen has a molecular weight of 50-300 kDa; the PDRN has a molecular weight of 1-50 kDa and a purity of ≥95%.

[0010] Secondly, the present invention provides a method for preparing PDRN-crosslinked collagen gel, employing the following technical solution: A method for preparing PDRN-crosslinked collagen gel includes the following steps: S1. Preparation of raw material solution: Dissolve collagen in buffer solution, adjust pH to prepare collagen solution; dissolve PDRN (polydeoxyribonucleic acid) in deionized water to prepare PDRN solution; mix collagen solution and PDRN solution evenly to obtain mixed solution; S2, Crosslinking reaction: Add NMI (N-methylimidazole) to the mixed solution, stir until homogeneous, then add TCFH (N,N,N′,N′-tetramethylchloromethanemidazone hexafluorophosphate) in three equal portions, stir to react, let stand, and obtain the reaction product solution; S3. Purification: The reaction product solution is dialyzed to obtain PDRN-crosslinked collagen gel.

[0011] Preferably, the buffer in step S1 is MES buffer, HEPES buffer, or PBS buffer, and the buffer concentration is 10-50 mM.

[0012] Preferably, in step S2, TCFH is added in three equal amounts, with an interval of 10-15 minutes between each addition.

[0013] Preferably, in step S2, the stirring reaction temperature is 20-25℃, the reaction time is 2-6h, and the standing time is 6-18h.

[0014] Preferably, in step S3, dialysis is performed using a dialysis bag with a molecular weight cutoff of 10-50 kDa, and the dialysis time is 24-72 hours, during which the dialysis fluid is replaced 3-5 times.

[0015] Preferably, the PDRN-crosslinked collagen gel obtained in step S3 can be further freeze-dried to obtain PDRN-crosslinked collagen powder, and the obtained powder can still restore the gel morphology after rehydration.

[0016] Preferably, the vacuum freeze-drying conditions are -50℃ to -80℃, vacuum degree ≤10Pa, and drying time is 12-48h.

[0017] Thirdly, the present invention provides an application of PDRN-crosslinked collagen gel in the preparation of wound repair materials, skin tissue engineering scaffolds, or medical aesthetic filler materials.

[0018] In this invention, PDRN is stably linked to collagen molecular chains via an amidation reaction mediated by the TCFH-NMI system, forming a uniform and stable cross-linked network structure. This structure not only improves the mechanical stability and anti-degradation properties of collagen materials but also effectively delays the diffusion and release of PDRN in bodily fluids, prolonging its biological activity. During release, PDRN promotes cell proliferation and angiogenesis, while collagen provides a good scaffold for cell adhesion and growth. The synergistic effect of both can further improve tissue repair efficiency, giving the resulting gel material excellent application potential in wound repair and cosmetic filling.

[0019] In summary, the present invention has the following beneficial effects: 1. Achieving stable covalent binding between PDRN and collagen to improve system stability: This invention uses an amidation reaction mediated by the TCFH-NMI system to form stable amide bonds between PDRN and collagen molecular chains, avoiding the problem of easy diffusion and loss of active ingredients in traditional physical mixing systems, thereby significantly improving the system stability and effective action time of the material.

[0020] 2. Significantly improves the mechanical properties and anti-degradation properties of gel materials: Through cross-linking reaction, a uniform and stable network structure is formed, which improves the mechanical stability of collagen materials and their ability to be degraded in antibody liquid environment, so that the resulting gel has better morphology maintenance ability during in vivo application.

[0021] 3. Achieve sustained release of PDRN and enhance the duration of bioactivity: The cross-linked network structure can slow down the release rate of PDRN in the body fluid environment, prolong its bioactivity duration, and thus enhance tissue repair and regeneration effects.

[0022] 4. It has dual functions of structural support and biological repair: Collagen provides cells with a good scaffold for adhesion and growth, while PDRN can promote cell proliferation and angiogenesis. The two work together to further improve tissue repair efficiency.

[0023] 5. The reaction system is green and safe, and suitable for the preparation of biomedical materials: The cross-linking reaction of this invention is completed in an aqueous or buffer system, avoiding the toxic residue problems that may be caused by traditional organic solvent systems, and improving the biosafety and environmental friendliness of the obtained gel material.

[0024] 6. Uniform and controllable crosslinking process, stable product performance: By adding the crosslinking agent in stages, the uniformity of the crosslinking reaction is further improved, avoiding excessive local crosslinking, making the gel structure more uniform and stable, thereby ensuring the repeatability of material performance.

[0025] 7. The process is simple and suitable for large-scale production: The preparation method adopted in this invention has mild conditions, stable and reliable process steps, and does not require the use of complex equipment, making it suitable for large-scale industrial production and clinical application. Attached Figure Description

[0026] Figure 1 This is the structural formula of TCFH and NMI in this invention.

[0027] Figure 2 This is a schematic diagram of the reaction in which collagen and PDRN are covalently cross-linked through an amidation reaction mediated by TCFH-NMI to form a PDRN-crosslinked collagen gel in this invention. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0029] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0031] Type I collagen was purchased from Solarbio, product number: C8063; PDRN was purchased from Aladdin, product number: I1506387; PBS buffer was purchased from aladdin, product number: P743267; NMI purchased from MERCK, product number: 336092; TCFH was purchased from MERCK, product number: 09658.

[0032] To more clearly demonstrate the covalent cross-linking mechanism between PDRN and recombinant collagen in this invention, the structural formulas of TCFH and NMI are as follows: Figure 1 As shown, the reaction diagram is as follows: Figure 2 As shown.

[0033] Example 1 A method for preparing PDRN-crosslinked collagen gel includes the following steps: S1. Preparation of raw material solutions: Type I collagen was dissolved in 10 mM PBS buffer to prepare a collagen solution with a concentration of 10 mg / mL, and the pH of the collagen solution was adjusted to 6.0 with 1 mol / L sodium hydroxide solution; PDRN was dissolved in deionized water to prepare a PDRN solution with a concentration of 10 mg / mL; then the collagen solution and PDRN solution were mixed evenly at a mass ratio of 1:0.8 to obtain a mixed solution. S2, Crosslinking reaction: NMI was added to the mixed solution all at once, and the mixture was stirred at room temperature for 10 min to ensure uniform dispersion. Then, TCFH was added to the reaction system in three equal portions, with an interval of 10 min between each addition. The molar ratio of TCFH to carboxyl groups in collagen was controlled at 1:1, and the molar ratio of NMI to TCFH was controlled at 2:1. The reaction was stirred at 20℃ and 100 rpm for 2 h. After the reaction was completed, the system was transferred to a refrigerated environment at 2℃ and allowed to stand for 6 h to obtain the reaction product solution. S3. Purification: The reaction product solution was transferred into a dialysis bag with a molecular weight cutoff of 10 kDa. The dialysis bag was placed in a dialysis tank filled with deionized water and dialyzed in deionized water for 24 hours. The dialysate was changed every 6 hours during the process, and the dialysate was changed 3 times to remove unreacted substances and byproducts, thus obtaining PDRN-crosslinked collagen gel.

[0034] Example 2 A method for preparing PDRN-crosslinked collagen gel includes the following steps: S1. Preparation of raw material solutions: Type I collagen was dissolved in 30mM PBS buffer to prepare a collagen solution with a concentration of 20mg / mL, and the pH of the collagen solution was adjusted to 6.5 with 1mol / L sodium hydroxide solution; PDRN was dissolved in deionized water to prepare a PDRN solution with a concentration of 20mg / mL; then the collagen solution and PDRN solution were mixed evenly at a mass ratio of 1:1 to obtain a mixed solution. S2, Crosslinking reaction: NMI was added to the mixed solution all at once, and the mixture was stirred at room temperature for 10 min to disperse the system evenly. Then, TCFH was added to the reaction system in three equal portions, with an interval of 12 min between each addition. The molar ratio of TCFH to carboxyl groups in collagen was controlled at 2:1, and the molar ratio of NMI to TCFH was controlled at 3:1. The reaction was stirred at 22℃ and 200 rpm for 4 h. After the reaction was completed, the system was transferred to a refrigerated environment at 2℃ and allowed to stand for 12 h to obtain the reaction product solution. S3. Purification: The reaction product solution was transferred into a dialysis bag with a molecular weight cutoff of 30 kDa. The dialysis bag was placed in a dialysis tank filled with deionized water and dialyzed in deionized water for 48 hours. The dialysate was changed every 8 hours during the process, and the dialysate was changed 4 times to remove unreacted substances and byproducts, thus obtaining PDRN-crosslinked collagen gel.

[0035] Example 3 A method for preparing PDRN-crosslinked collagen gel includes the following steps: S1. Preparation of raw material solutions: Type I collagen was dissolved in 50 mM PBS buffer to prepare a collagen solution with a concentration of 30 mg / mL, and the pH of the collagen solution was adjusted to 7.0 with 1 mol / L sodium hydroxide solution; PDRN was dissolved in deionized water to prepare a PDRN solution with a concentration of 30 mg / mL; then the collagen solution and PDRN solution were mixed evenly at a mass ratio of 1:1.2 to obtain a mixed solution. S2, Crosslinking reaction: NMI was added to the mixed solution all at once, and the mixture was stirred at room temperature for 10 min to disperse the system evenly. Then, TCFH was added to the reaction system in three equal portions, with an interval of 15 min between each addition. The molar ratio of carboxyl groups in TCFH to collagen was controlled at 3:1, and the molar ratio of NMI to TCFH was controlled at 5:1. The reaction was stirred at 25℃ and 300 rpm for 6 h. After the reaction was completed, the system was transferred to a refrigerated environment at 2℃ and allowed to stand for 18 h to obtain the reaction product solution. S3. Purification: The reaction product solution was transferred into a dialysis bag with a molecular weight cutoff of 50 kDa. The dialysis bag was placed in a dialysis tank filled with deionized water and dialyzed in deionized water for 72 hours. The dialysate was changed every 12 hours during the process, and the dialysate was changed 5 times to remove unreacted substances and byproducts, thus obtaining PDRN-crosslinked collagen gel.

[0036] Comparative Example 1 A method for preparing PDRN-crosslinked collagen gel includes the following steps: S1. Preparation of raw material solutions: Type I collagen was dissolved in 30mM PBS buffer to prepare a collagen solution with a concentration of 20mg / mL, and the pH of the collagen solution was adjusted to 6.5 with 1mol / L sodium hydroxide solution; PDRN was dissolved in deionized water to prepare a PDRN solution with a concentration of 20mg / mL; then the collagen solution and PDRN solution were mixed evenly at a mass ratio of 1:1 to obtain a mixed solution. S2, Crosslinking reaction: The mixed solution was stirred at 22℃ and 200rpm for 4 hours to make the system uniformly dispersed and obtain the reaction product solution; S3. Purification: The reaction product solution was transferred into a dialysis bag with a molecular weight cutoff of 30 kDa. The dialysis bag was placed in a dialysis tank filled with deionized water and dialyzed in deionized water for 48 hours. The dialysate was changed every 8 hours during the process, and the dialysate was changed 4 times to remove unreacted substances and byproducts, thus obtaining PDRN-crosslinked collagen gel.

[0037] Comparative Example 2 A method for preparing PDRN-crosslinked collagen gel includes the following steps: S1. Preparation of raw material solutions: Type I collagen was dissolved in 30mM PBS buffer to prepare a collagen solution with a concentration of 20mg / mL, and the pH of the collagen solution was adjusted to 6.5 with 1mol / L sodium hydroxide solution; PDRN was dissolved in deionized water to prepare a PDRN solution with a concentration of 20mg / mL; then the collagen solution and PDRN solution were mixed evenly at a mass ratio of 1:1 to obtain a mixed solution. S2, Cross-linking reaction: NHS was added to the mixed solution all at once, and the mixture was stirred at room temperature for 10 min to disperse the system evenly. Then, EDC was added to the reaction system in three equal portions, with an interval of 12 min between each addition. The molar ratio of carboxyl groups in EDC to collagen was controlled at 2:1, and the molar ratio of NHS to EDC was controlled at 3:1. The reaction was stirred at 22℃ and 200 rpm for 4 h. After the reaction was completed, the system was transferred to a refrigerated environment at 2℃ and allowed to stand for 12 h to obtain the reaction product solution. S3. Purification: The reaction product solution was transferred into a dialysis bag with a molecular weight cutoff of 30 kDa. The dialysis bag was placed in a dialysis tank filled with deionized water and dialyzed in deionized water for 48 hours. The dialysate was changed every 8 hours during the process, and the dialysate was changed 4 times to remove unreacted substances and byproducts, thus obtaining PDRN-crosslinked collagen gel.

[0038] Comparative Example 3 A method for preparing PDRN-crosslinked collagen gel includes the following steps: S1. Preparation of raw material solutions: Type I collagen was dissolved in 30mM PBS buffer to prepare a collagen solution with a concentration of 20mg / mL, and the pH of the collagen solution was adjusted to 6.5 with 1mol / L sodium hydroxide solution; PDRN was dissolved in deionized water to prepare a PDRN solution with a concentration of 20mg / mL; then the collagen solution and PDRN solution were mixed evenly at a mass ratio of 1:1 to obtain a mixed solution. S2, Cross-linking reaction: Genipin is added to the mixed solution at one time, so that the mass fraction of genipin in the system is 0.1%. The mixture is stirred at 30°C for 12 hours to induce a cross-linking reaction and obtain the reaction product solution. S3. Purification: The reaction product solution was transferred into a dialysis bag with a molecular weight cutoff of 30 kDa. The dialysis bag was placed in a dialysis tank filled with deionized water and dialyzed in deionized water for 48 hours. The dialysate was changed every 8 hours during the process, and the dialysate was changed 4 times to remove unreacted substances and byproducts, thus obtaining PDRN-crosslinked collagen gel.

[0039] Comparative Example 4 A method for preparing PDRN-crosslinked collagen gel includes the following steps: S1. Preparation of raw material solutions: Type I collagen was dissolved in 30mM PBS buffer to prepare a collagen solution with a concentration of 20mg / mL, and the pH of the collagen solution was adjusted to 6.5 with 1mol / L sodium hydroxide solution; PDRN was dissolved in deionized water to prepare a PDRN solution with a concentration of 20mg / mL; then the collagen solution and PDRN solution were mixed evenly at a mass ratio of 1:1 to obtain a mixed solution. S2, Cross-linking reaction: Add glutaraldehyde solution to the mixed solution at one time, so that the mass fraction of glutaraldehyde in the system is 0.1%, stir the reaction at 22°C for 2 hours to allow the system to undergo a cross-linking reaction, and obtain the reaction product solution; S3. Purification: The reaction product solution was transferred into a dialysis bag with a molecular weight cutoff of 30 kDa. The dialysis bag was placed in a dialysis tank filled with deionized water and dialyzed in deionized water for 48 hours. The dialysate was changed every 8 hours during the process, and the dialysate was changed 4 times to remove unreacted substances and byproducts, thus obtaining PDRN-crosslinked collagen gel.

[0040] Comparative Example 5 A method for preparing PDRN-crosslinked collagen gel includes the following steps: S1. Preparation of raw material solutions: Type I collagen was dissolved in 30mM PBS buffer to prepare a collagen solution with a concentration of 20mg / mL, and the pH of the collagen solution was adjusted to 6.5 with 1mol / L sodium hydroxide solution; PDRN was dissolved in deionized water to prepare a PDRN solution with a concentration of 20mg / mL; then the collagen solution and PDRN solution were mixed evenly at a mass ratio of 1:1 to obtain a mixed solution. S2, Crosslinking reaction: NMI was added to the mixed solution all at once, and the mixture was stirred at room temperature for 10 min to disperse the system evenly. Then, TCFH was added to the reaction system all at once. The molar ratio of TCFH to carboxyl groups in collagen was controlled at 2:1, and the molar ratio of NMI to TCFH was controlled at 3:1. The reaction was stirred at 22℃ and 200 rpm for 4 h. After the reaction was completed, the system was transferred to a 2℃ refrigerated environment and allowed to stand for 12 h to obtain the reaction product solution. S3. Purification: The reaction product solution was transferred into a dialysis bag with a molecular weight cutoff of 30 kDa. The dialysis bag was placed in a dialysis tank filled with deionized water and dialyzed in deionized water for 48 hours. The dialysate was changed every 8 hours during the process, and the dialysate was changed 4 times to remove unreacted substances and byproducts, thus obtaining PDRN-crosslinked collagen gel.

[0041] Performance verification I. Testing Items and Methods To comprehensively evaluate the overall performance of the PDRN-crosslinked collagen gel of this invention, and considering the application requirements of biomaterials in wound repair, skin tissue engineering, and cosmetic fillers, the PDRN-crosslinked collagen gels prepared in Examples 1-3 and Comparative Examples 1-5 were subjected to the following core performance tests. All test methods were optimized and formulated according to relevant standards or industry standard methods: (a) Basic physicochemical properties testing pH value detection: Referring to YY / T 0962-2021 "Cross-linked sodium hyaluronate gel for plastic surgery", a precision pH meter was used to directly measure the pH value of the gel sample at room temperature of 25℃. The measurement was performed in parallel for 3 times and the average value was taken.

[0042] A freezing point osmolality analyzer was used. A gel sample was taken, and purified water was added at a mass ratio of 1:5. The mixture was thoroughly mixed, allowed to stand for equilibrium, and then centrifuged to collect the supernatant as the test solution. Further dilution was performed if necessary to ensure the measured value fell within the instrument's linear range. Calibration and determination were performed according to Chapter 0632 of the Chinese Pharmacopoeia, Part IV, "Determination of Osmolality". The unit is mOsmol / kg. Three parallel determinations were performed, and the average value was taken.

[0043] Swelling degree test: Take a 500-mesh sieve (8cm×8cm square, folded into a 4cm×4cm×2cm square groove) and place it in a drying oven. Dry it at 80℃ to constant weight and record it as m0. Weigh 0.3g (accurate to 0.001g) of gel sample and place it on the sieve. Place the sieve in an evaporating dish and add 30mL of 0.9wt% sodium chloride solution (simulating the human body fluid environment) at once. Let it stand at 25℃ for 30min to allow the gel to fully swell. Remove the sieve and sample, and use filter paper to absorb the liquid at the bottom and around until the filter paper is dry. Weigh it and record it as m1. Then place the sieve in a drying oven and dry it at 80℃ to constant weight and record it as m2. Calculate the swelling degree according to the formula: Q=(m1-m2) / (m2-m0). Perform three parallel measurements and take the average value.

[0044] (ii) Stability testing The PDRN-crosslinked collagen gel samples prepared in Examples 1-3 and Comparative Examples 1-5 were sealed in sterile vials and stored in a refrigerated environment at 5°C and a room temperature environment at 25°C for 30 days in the dark. The appearance of the samples (water separation, collapse, softening, structural rupture, discoloration, etc.) was observed at 0, 5, 10 and 30 days, and the swelling degree change rate was measured. Swelling degree change rate (%) = (swelling degree after storage - initial swelling degree) / initial swelling degree × 100%.

[0045] (III) Mechanical property testing (viscoelasticity) Using a rotational rheometer, the viscoelasticity of the gel sample was tested at 25℃ and 5Hz using a flat plate clamp (40mm in diameter and 1mm in gap). The storage modulus (G', reflecting the gel's elasticity and mechanical support capacity) and loss modulus (G'', reflecting the gel's viscosity) were recorded. The tests were performed in parallel three times, and the average value at each frequency was taken.

[0046] (iv) Biocompatibility testing Cytotoxicity assay (CCK-8 assay): Human skin fibroblasts (HSF) were harvested and seeded in 96-well plates (1×10⁻⁶ cells / wells). 4 Cells / well) were cultured for 24 h. Then, an equal volume of medium containing different PDRN-crosslinked collagen gel sample extracts (gel sample to medium volume ratio 1:10, extracted at 37℃ for 24 h) was added to the original medium, and cultured for another 48 h. CCK-8 reagent (10 μL per well) was added, and after incubation for 4 h, the absorbance value (OD value) at 450 nm was measured using an ELISA reader. Cell viability was calculated as follows: Cell viability (%) = (OD value of sample group / OD value of blank control group) × 100%. The blank control group was fed medium without PDRN-crosslinked collagen gel sample extracts. Five groups were measured in parallel, and the average value was taken.

[0047] Animal experiment: Twenty-four five-week-old female nude mice were randomly divided into eight groups (corresponding to groups 1-3 of Examples and groups 1-5 of Comparative Examples), with three mice in each group. 0.2 mL of gel sample was injected subcutaneously into the back of each nude mouse. The injection site was observed for adverse reactions such as redness, swelling, inflammation, and necrosis at 0, 1, 3, 7, and 14 days after injection. The gel filling effect (whether it was palpable and whether the shape was stable) was also recorded.

[0048] II. Test Results and Analysis (a) The results of the basic physical and chemical properties test are shown in Table 1.

[0049] Table 1. Results of Basic Physicochemical Properties Tests Results analysis: pH and osmotic pressure: The pH of all samples was in the range of 6.65-6.80, and the osmotic pressure was 284-289 mOsmol / kg, which is highly matched with the physiological environment of the human body, indicating that the samples have the basic conditions to be compatible with human tissues.

[0050] Swelling degree: The swelling degree of Examples 1-3 was 28.62-35.39, which is within a suitable range. This ensures that the gel can fully expand in the body fluid environment to fill defects or provide support, without causing structural rupture due to excessive swelling. Comparative Example 1 (physical mixing) had no stable swelling value because it did not form a covalent cross-linked network and could not maintain the gel morphology. It was easily dispersed and dissolved in sodium chloride solution. The swelling degree of Comparative Examples 2-4 was lower than that of the Examples. Among them, Comparative Example 3 (genipin cross-linking) had the lowest swelling degree because of its low cross-linking efficiency and dense network structure, which limited its water absorption capacity. The swelling degree of Comparative Example 5 (TCFH added at once) (29.84) was slightly lower than that of the Examples, indicating that adding TCFH in three stages is more conducive to forming a uniform cross-linked network and improving water absorption and swelling performance.

[0051] (ii) The results of the storage stability test are shown in Table 2.

[0052] Table 2 Storage stability test results (30 days) Results Analysis: After 30 days of storage at 5℃ and 25℃, Examples 1-3 maintained a uniform and transparent gel morphology without any abnormal appearance changes. The swelling degree change rate ranged only 1.8-2.7%, indicating that the gel network structure was stable and could meet the transportation and storage requirements in practical applications. Comparative Example 1 (physical mixing) had the worst stability due to the lack of covalent cross-linking and completely dissolved at room temperature. Comparative Examples 3 (genipin cross-linking) and 4 (glutaraldehyde cross-linking) showed degradation and collapse within 30 days due to low cross-linking efficiency or uneven cross-linking, and the swelling degree change rate was significantly higher than that of Examples. Comparative Example 2 (EDC / NHS cross-linking) caused discoloration and shrinkage due to residual organic solvent. Comparative Example 5 (TCFH added at once) had better stability than Comparative Examples 2-4, but slightly worse than Examples. This was because the one-time addition of TCFH caused violent local reactions and insufficient uniformity of the cross-linked network, which affected long-term stability.

[0053] (III) The test results of mechanical properties (viscoelasticity) are shown in Table 3.

[0054] Table 3. Viscoelasticity test results (average value at 5Hz) Results Analysis: Storage modulus (G') is the core indicator for evaluating the mechanical support performance of gels. The higher the G', the stronger the gel elasticity and the better the support capacity. The G' of Examples 1-3 ranged from 316.54 to 324.78 Pa, and the loss factor ranged from 0272 to 0.312, indicating that the gels are mainly elastic and have excellent mechanical support performance, which can meet the needs of tissue support in wound repair and shape maintenance in medical aesthetic filling. Comparative Example 1 (physical mixing) had no cross-linked network, so no elasticity could be detected, and there was no mechanical support capacity. Comparative Example 3 (genipin cross-linking) had the lowest G' due to low cross-linking efficiency, loose network structure, and the worst mechanical performance. Comparative Example 4 (glutaraldehyde cross-linking) had a lower G' than the examples and a higher loss factor, indicating that the gel viscosity ratio increased, elasticity was insufficient, and structural uniformity was poor. Comparative Example 2 (EDC / NHS cross-linking) had a G' of 185.62 Pa, and the mechanical performance was average. Comparative Example 5 (TCFH added at once) had a lower G' than the examples due to excessive local cross-linking and uneven overall network, resulting in slightly inferior elasticity and support capacity compared to the examples.

[0055] (iv) Biocompatibility test results 1. The results of the cytotoxicity test are shown in Table 4.

[0056] Table 4. Cytotoxicity test results (48h cell survival rate) 2. The results of the animal experiments are shown in Table 5.

[0057] Table 5. Animal experiment results (14 days after injection) Results analysis: Cytotoxicity: The cell survival rates of Examples 1-3 were high, meeting the standard of non-cytotoxicity of biomaterials; the cell survival rate of Comparative Example 4 (glutaraldehyde crosslinking) was only 58.7%, due to the significant toxicity of glutaraldehyde residue; the cell survival rates of Comparative Examples 2-3 (EDC / NHS, genipin crosslinking) were reduced, indicating slight toxicity; Comparative Examples 1 and 5 showed no obvious toxicity, similar to the Examples.

[0058] Animal experiments: Examples 1-3 maintained stable filling effect within 14 days without any adverse reactions, demonstrating excellent biocompatibility; Comparative Example 4 caused redness and necrosis due to toxicity; Comparative Example 2 showed slight redness due to residual organic solvents; Comparative Examples 1 and 3 had poor filling effect due to lack of cross-linking or unstable cross-linking; Comparative Example 5 had good filling effect and biocompatibility, but was slightly inferior to the examples.

[0059] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A PDRN-crosslinked collagen gel, characterized in that, The PDRN-crosslinked collagen gel is formed by covalent crosslinking of PDRN and collagen through an amidation reaction mediated by TCFH-NMI. PDRN forms amide bonds with the carboxyl groups on the collagen molecular chain through the amino groups in its molecular structure.

2. The PDRN-crosslinked collagen gel according to claim 1, characterized in that, The PDRN-crosslinked collagen gel has the following performance characteristics: pH 6.5-6.8, osmotic pressure 285-290 mOsmol / kg; swelling degree 28-36 in 0.9 wt% sodium chloride solution; storage modulus 280-1050 Pa and loss modulus 80-285 Pa at a frequency of 5 Hz.

3. The PDRN-crosslinked collagen gel according to claim 1, characterized in that, The collagen is selected from one or more of type I collagen, type III collagen, type V collagen, and recombinant collagen.

4. The PDRN-crosslinked collagen gel according to claim 1, characterized in that, The collagen has a molecular weight of 50-300 kDa; the PDRN has a molecular weight of 1-50 kDa and a purity of ≥95%.

5. A method for preparing the PDRN-crosslinked collagen gel according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Preparation of raw material solution: Dissolve collagen in buffer solution, adjust pH to prepare collagen solution; dissolve PDRN in deionized water to prepare PDRN solution; mix collagen solution and PDRN solution evenly to obtain mixed solution; S2, Crosslinking reaction: Add NMI to the mixed solution, stir evenly, then add TCFH in three equal portions, stir the reaction, let stand, and obtain the reaction product solution; S3. Purification: The reaction product solution is dialyzed to obtain PDRN-crosslinked collagen gel.

6. The method for preparing PDRN-crosslinked collagen gel according to claim 5, characterized in that, In step S1, the buffer solution is MES buffer, HEPES buffer, or PBS buffer, and the buffer concentration is 10-50 mM.

7. The method for preparing PDRN-crosslinked collagen gel according to claim 5, characterized in that, In step S2, TCFH is added in three equal amounts, with an interval of 10-15 minutes between each addition.

8. The method for preparing PDRN-crosslinked collagen gel according to claim 5, characterized in that, In step S2, the stirring temperature is 20-25℃, the reaction time is 2-6h, and the settling time is 6-18h.

9. The method for preparing PDRN-crosslinked collagen gel according to claim 5, characterized in that, In step S3, dialysis is performed using a dialysis bag with a molecular weight cutoff of 10-50 kDa, and the dialysis time is 24-72 hours, during which the dialysis fluid is changed 3-5 times.

10. The use of the PDRN-crosslinked collagen gel according to any one of claims 1-4 in the preparation of wound repair materials, skin tissue engineering scaffolds or medical aesthetic filler materials.

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