A recombinant collagen wound dressing and its preparation method

Abstract

This invention relates to the field of dressing technology, specifically to a recombinant collagen wound dressing and its preparation method. The dressing has a membrane structure, and its raw materials include selectively methacrylamide-treated recombinant collagen concentrate, sodium oxyhyaluronate, and carboxymethyl chitosan. A permanent cross-linked collagen network is formed through visible light curing, and a multi-layered stable network is constructed by combining the cross-linking between sodium oxyhyaluronate and carboxymethyl chitosan, as well as calcium ion-induced ionic micro-cross-linking. The preparation method, through precise control of the degree of methacrylamide and optimization of the component addition sequence (especially the surface atomization and internal penetration of carboxymethyl chitosan), ensures good processability of the precursor while enabling the final product to maintain excellent wet mechanical properties, morphological stability, fluid management ability, and gentle dressing change compliance even after absorbing wound exudate.

Description

Technical Field

[0001] This invention relates to the field of dressing technology, and in particular to a recombinant collagen wound dressing and its preparation method. Background Technology

[0002] In the field of wound repair and care, collagen is widely used in the development of wound dressings due to its excellent biocompatibility and cell migration-promoting properties. The emergence of recombinant humanized collagen further avoids the potential immunogenicity and pathogen risks of animal-derived collagen, providing a safer raw material option for high-end dressings. Imparting photosensitive properties to collagen through chemical modifications such as methacrylation, followed by visible light-induced curing to form a hydrogel network, has become one of the mainstream technical routes for preparing collagen dressings that can be molded in situ and have good adhesion. However, the covalent cross-linked network formed by photocuring alone often suffers significant degradation in mechanical properties under moist conditions. It is difficult to maintain sufficient morphological stability and mechanical strength after absorbing a large amount of wound exudate, which can easily lead to swelling, deformation, or even structural disintegration of the dressing on the wound, affecting its continuous coverage and protective function.

[0003] To improve wet-state performance, existing technologies attempt to introduce other polymers or cross-linking mechanisms into the collagen network. For example, sodium hyaluronate, chitosan, or their derivatives are blended with modified collagen to enhance the dressing's absorbency due to their strong hydrophilicity. However, simple physical blending often leads to uneven distribution of components and weak interactions between different polymer chains. Under the impact of seepage, the network structure is prone to loosening, resulting in limited liquid retention and potential backflow, increasing the risk of maceration of surrounding healthy tissue. Another approach is to construct dynamic covalent bonds or physical cross-links to enhance network stability. However, such rapid gelation reactions cause a sharp increase in the viscosity of the precursor body fluid, severely impairing its fluidity and spreadability. This poses significant challenges to the preparation of uniform films through processes such as coating, making it difficult to achieve large-scale, controllable dressing formation.

[0004] Therefore, existing technologies face a dilemma: pursuing good processability of precursors (such as low viscosity and easy spreadability) often requires sacrificing the network stability and shape retention of the final dressing in a wet state; conversely, pre-constructing a strongly interacting network to improve wet performance can impair processability. Especially for wound dressings that require multiple clinical needs such as good adhesion, high absorbency, excellent wet strength, and easy peeling during dressing changes (with minimal damage), how to construct a multi-layered, synergistic, stable network structure in the final product through precise material design and process control, while ensuring processability, thereby comprehensively balancing wet mechanical properties, exudate management capabilities, and usability, remains a critical technological bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0005] In view of this, the purpose of this invention is to propose a recombinant collagen wound dressing and its preparation method, so as to solve the problem that the recombinant collagen photocurable dressing prepared by the prior art is difficult to achieve excellent wet mechanical stability, morphological retention after absorbing exudate and mild dressing change compliance at the same time, while ensuring good processability of the precursor and mild curing under visible light.

[0006] Based on the above objectives, the present invention provides a recombinant collagen wound dressing, wherein the recombinant collagen wound dressing is a film dressing comprising a collagen cross-linking network formed by curing a selectively methacrylated recombinant collagen concentrate under visible light and sodium oxyhyaluronate distributed in the collagen cross-linking network, wherein carboxymethyl chitosan is infiltrated on the surface of the film dressing and forms a cross-linking structure with the sodium oxyhyaluronate; The raw materials for preparing the film dressing, by weight, include: 29,000-32,000 parts of selectively methacrylamide recombinant collagen concentrate, 150-250 parts of sodium hyaluronate oxide, and 20-35 parts of carboxymethyl chitosan. The selectively methacrylated recombinant collagen concentrate is obtained by dialysis and concentration of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa and recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa after methacrylation, and the retention rate of free primary amines after methacrylation is 60%-75%; the degree of aldehyde conversion of the oxidized sodium hyaluronate is 12.8%-24.6%.

[0007] Preferably, the raw materials for preparing the selectively methacrylamide recombinant collagen concentrate, by weight, include: 42,000-47,000 parts of sterile water for injection, 1,500-1,900 parts of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa, 600-1,000 parts of recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa, and 315-450 parts of methacrylic anhydride.

[0008] Preferably, the oxidized sodium hyaluronate is prepared by oxidizing 350-500 parts of sodium hyaluronate with a molecular weight of 200-400 kDa with 35-60 parts of sodium periodate, and then sealing the remaining oxidant with 12-20 parts of ethylene glycol.

[0009] Preferably, the periphery of the film dressing has a sealing area with a width of 2-3 mm, and by weight, 14-20 parts of carboxymethyl chitosan are further infiltrated into the sealing area.

[0010] Preferably, the raw materials for preparing the film dressing further include 30-50 parts of lithium phenyl-2,4,6-trimethylbenzoylphosphinate and 10-20 parts of calcium chloride dihydrate, by weight.

[0011] Preferably, the visible light curing process involves irradiation with visible light at a wavelength of 405 nm for 50-70 seconds, with a cumulative light dose of 500-700 mJ / cm². 2 .

[0012] Furthermore, the present invention also provides a method for preparing a recombinant collagen wound dressing, comprising the following steps: S1. Recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa and recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa were dissolved in sterile water for injection and subjected to methacrylation reaction. The reaction was terminated when the free primary amine retention rate reached 60%-75%. After dialysis and concentration, a selectively methacrylated recombinant collagen concentrate was obtained. S2. Oxidize sodium hyaluronate and block the remaining oxidant. After dialysis and freeze-drying, oxidized sodium hyaluronate is obtained. S3. The selectively methacrylated recombinant collagen concentrate obtained in step S1 is mixed with the sodium oxidized hyaluronic acid obtained in step S2, and then phenyl-2,4,6-trimethylbenzoyl lithium phosphine and calcium chloride dihydrate are added and coated into a film. S4. Atomize and spray carboxymethyl chitosan endosmosis solution onto the membrane surface obtained in step S3; S5. The film-forming body after step S4 is cured by visible light irradiation and then equilibrated with sterile sodium chloride solution to obtain recombinant collagen wound dressing.

[0013] The beneficial effects of this invention are: First, a network framework with a gradient structure was constructed by compounding recombinant humanized type III collagen with different molecular weights. Higher molecular weight collagen provides good mechanical support and network framework, while lower molecular weight collagen enhances the penetration and filling capacity of chain segments. The synergy between the two enables the formed precursor to have suitable spreading properties, while the three-dimensional network formed after curing has both toughness and load-bearing capacity in a wet state, effectively resisting swelling and deformation caused by leakage.

[0014] Secondly, by precisely controlling the degree of methacrylation, the retention rate of free primary amines in the collagen molecular chain is limited to a specific range. This design allows the collagen molecules to retain sufficient photosensitive groups to ensure the formation of a stable and permanent network under visible light curing, while also preserving ample active amino sites. These reserved amino groups lay the foundation for subsequent reactions with oxidized sodium hyaluronate and surface-infiltrated carboxymethyl chitosan, thereby constructing reversible dynamic cross-linking points within and on the surface of the photocured network. This significantly improves the dressing's shape recovery and tear resistance in humid environments.

[0015] Furthermore, by employing a process sequence that involves the delayed addition of oxidized sodium hyaluronate combined with the surface atomization and infiltration of carboxymethyl chitosan, the construction of the dynamic cross-linked network is cleverly divided into two steps. This method avoids the premature reaction of oxidized sodium hyaluronate with collagen amines, which would lead to a sharp increase in precursor viscosity, thus ensuring the uniformity and operability of the coating. After film formation, the surface-infiltrated carboxymethyl chitosan and the oxidized sodium hyaluronate in the system form a gradient cross-link from the surface inwards, further enhancing the stability of the dressing surface layer and its interfacial control ability for wound exudate, improving fluid retention and reducing backflow.

[0016] Finally, calcium chloride is introduced before the dressing is applied. Its calcium ions form transiently reversible ionic micro-crosslinks with the carboxyl groups in collagen, sodium oxyhyaluronate, and carboxymethyl chitosan. These temporary ionic bonds improve the precursor's anti-sagging and edge-forming properties during the initial coating process, ensuring the integrity and regularity of the dressing. After the dressing has cured and absorbed exudate, the dynamic reversible nature of the ionic micro-crosslinks prevents the material from becoming overly rigid, helping to maintain the overall soft and adherent properties of the dressing. This achieves good edge stability while ensuring smooth peeling during dressing changes and reducing the risk of secondary damage to newly formed tissue. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0018] Raw materials and sources: Recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa, catalog number rp212898, lyophilized powder, derived from microbial recombinant expression, animal-derived component negative, carrier-free, low endotoxin, sterile filtered, purity not less than 95%, Shanghai Aladdin Biochemical Technology Co., Ltd.; Recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa, catalog number rp212896, lyophilized powder, derived from microbial recombinant expression, animal-derived component negative, carrier-free, low endotoxin. Aseptic filtration, purity not less than 95%, Shanghai Aladdin Biochemical Technology Co., Ltd.; Sodium hyaluronate, catalog number IS9017, molecular weight approximately 200kDa-400kDa, Beijing Solarbio Science & Technology Co., Ltd.; Carboxymethyl chitosan, white to light yellow free-flowing powder, degree of carboxylation not less than 80.0%, viscosity not higher than 80mPa·s, Jiangsu Aoxin Bioengineering Co., Ltd.; Lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, catalog number 940704, purity not less than 95%, Merck Life Sciences. Methacrylamide, sodium periodate, ethylene glycol, calcium chloride dihydrate, sodium chloride, sodium hydroxide, hydrochloric acid, and sterile water for injection are all commercially available analytical grade or biological reagent grade.

[0019] Example 1: Step 1: Add 45000 mg of sterile water for injection, 1750 mg of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa and 750 mg of recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa to a sterile reactor, stir at 300 r / min for 12 h at 4 °C to completely dissolve it, and then let it stand for 30 min to remove bubbles to obtain a recombinant collagen compound solution; Step 2: Prepare 2000 mg of 1 mol / L sodium hydroxide solution and 2000 mg of 1 mol / L hydrochloric acid solution as adjusting solutions. Under ice bath conditions, add 375 mg of methacrylic anhydride dropwise to the recombinant collagen compound solution obtained in Step 1 over 20 min, and maintain the pH of the system at 8.0 ± 0.2. After the addition is completed, continue the reaction for 20 min. Take samples every 10 min and use the ninhydrin colorimetric method to detect the free primary amine retention rate. When the free primary amine retention rate reaches 70%, immediately adjust the pH of the system to 6 with 1 mol / L hydrochloric acid solution to terminate the reaction. Then, put the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it in 8000 g of sterile deionized water at 4 °C for 48 h. Replace the 8000 g of sterile deionized water once every 6 h. After dialysis, concentrate it to 30000 mg using a 10000 Da ultrafiltration membrane at 4 °C to obtain a methacrylamide recombinant collagen concentrate. Step 3: Add 39600 mg of sterile deionized water and 400 mg of sodium hyaluronate to another sterile reactor, stir at room temperature for 12 h to completely dissolve, then add 45 mg of sodium periodate, and react under light-protected conditions for 6 h; subsequently add 15 mg of ethylene glycol and continue the reaction for 30 min to block the remaining oxidant; load the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze in 6000 g of sterile deionized water for 48 h, changing the 6000 g of sterile deionized water every 6 h, and freeze-dry after dialysis to obtain oxidized sodium hyaluronate; the degree of aldehyde was determined by hydroxylamine hydrochloride titration, and the degree of aldehyde was 18.5%; Step 4: Add 30,000 mg of selectively methacrylated recombinant collagen concentrate, 3,800 mg of sterile deionized water, and 200 mg of oxidized sodium hyaluronate to a sterile mixing tank in sequence, and stir at 4°C for 15 min; then adjust the pH of the system to 7; add 40 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphine dissolved in 1,600 mg of sterile deionized water, and stir in the dark for 5 min; 60 s before the film coating, add 15 mg of calcium chloride dihydrate dissolved in 485 mg of sterile deionized water, mix for 30 s, and immediately coat the film onto the release polyester film, controlling the wet film thickness to 1000 μm; Step 5: Prepare a separate carboxymethyl chitosan endosmosis solution, consisting of 25 mg carboxymethyl chitosan and 4975 mg sterile deionized water. Within 30 seconds after the coating in Step 4, spray 5000 mg of the endosmosis solution evenly over the entire membrane surface using an atomization method. The spraying time should be controlled within 15 seconds. After spraying, let it stand for 90 seconds. Step 6: Irradiate the film-forming material treated in Step 5 with 405nm visible light for 60s to achieve a cumulative light dose of 600mJ / cm². 2 After curing, the film is trimmed under sterile conditions and cut into 60mm×80mm sheets. Then, each sheet is placed in two sterile sodium chloride solutions of 30000mg each with a mass concentration of 9g / L for 10 minutes to equilibrate. Each wet film is sandwiched between two release films and gently pressed to remove the surface free liquid. The mass of a single sheet is controlled to be 3400mg, thus obtaining the recombinant collagen wound dressing.

[0020] Example 2: Step 1: Add 46,000 mg of sterile water for injection, 1,900 mg of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa and 600 mg of recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa to a sterile reactor, stir at 320 r / min at 4°C for 11 h to completely dissolve it, and then let it stand for 25 min to remove bubbles to obtain a recombinant collagen compound solution; Step 2: Prepare 1900 mg of 1 mol / L sodium hydroxide solution and 1900 mg of 1 mol / L hydrochloric acid solution as adjusting solutions. Under ice bath conditions, add 315 mg of methacrylic anhydride dropwise to the recombinant collagen compound solution obtained in Step 1 over 18 min, and maintain the pH of the system at 7.9 ± 0.2. After the addition is completed, continue the reaction for 18 min. Take samples every 10 min and use the ninhydrin colorimetric method to detect the free primary amine retention rate. When the free primary amine retention rate reaches 75%, immediately adjust the pH of the system to 6.1 with 1 mol / L hydrochloric acid solution to terminate the reaction. Then, put the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it in 8000 g of sterile deionized water at 4 °C for 48 h. Replace the 8000 g of sterile deionized water once every 6 h. After dialysis, concentrate it to 29000 mg using a 10000 Da ultrafiltration membrane at 4 °C to obtain a concentrated solution of methacrylamide recombinant collagen. Step 3: Add 40400 mg of sterile deionized water and 350 mg of sodium hyaluronate to another sterile reactor, stir at room temperature for 12 h to completely dissolve, then add 35 mg of sodium periodate, and react under light-protected conditions for 5 h; subsequently add 12 mg of ethylene glycol and continue the reaction for 30 min to block the remaining oxidant; load the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze in 6000 g of sterile deionized water for 48 h, changing the 6000 g of sterile deionized water every 6 h, and freeze-dry after dialysis to obtain oxidized sodium hyaluronate; the degree of aldehyde was determined by hydroxylamine hydrochloride titration, and the degree of aldehyde was 12.8%; Step 4: Add 29000 mg of selectively methacrylated recombinant collagen concentrate, 4300 mg of sterile deionized water, and 150 mg of oxidized sodium hyaluronate to a sterile mixing tank in sequence, and stir at 4°C for 15 min; then adjust the pH of the system to 6.9; add 30 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphine dissolved in 1500 mg of sterile deionized water, and stir in the dark for 4 min; 45 s before the film coating, add 10 mg of calcium chloride dihydrate dissolved in 500 mg of sterile deionized water, mix for 20 s, and immediately coat the film onto the release polyester film, controlling the wet film thickness to 850 μm; Step 5: Prepare a separate carboxymethyl chitosan endosmosis solution, consisting of 20 mg carboxymethyl chitosan and 4980 mg sterile deionized water. Within 30 seconds after the coating in Step 4, spray 5000 mg of the endosmosis solution evenly over the entire membrane surface using an atomization method. The spraying time should be controlled within 12 seconds. After spraying, let it stand for 80 seconds. Step 6: Irradiate the film-forming material treated in Step 5 with 405nm visible light for 50s to achieve a cumulative light dose of 500mJ / cm². 2After curing, the film is trimmed under sterile conditions and cut into 60mm×80mm sheets. Then, each sheet is placed in two sterile sodium chloride solutions of 30000mg each with a mass concentration of 9g / L for 10min to equilibrate. Each wet film is sandwiched between two release films and gently pressed to remove the surface free liquid. The mass of a single sheet is controlled to be 3200mg to obtain the recombinant collagen wound dressing.

[0021] Example 3: Step 1: Add 44,000 mg of sterile water for injection, 1,600 mg of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa and 900 mg of recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa to a sterile reactor, stir at 280 r / min for 13 h at 4 °C to completely dissolve it, and then let it stand for 30 min to remove bubbles to obtain a recombinant collagen compound solution; Step 2: Prepare 1950 mg of 1 mol / L sodium hydroxide solution and 1950 mg of 1 mol / L hydrochloric acid solution as adjusting solutions. Under ice bath conditions, add 360 mg of methacrylic anhydride dropwise to the recombinant collagen compound solution obtained in Step 1 over 20 min, and maintain the pH of the system at 8.0 ± 0.2. After the addition is completed, continue the reaction for 20 min. Take samples every 10 min and use the ninhydrin colorimetric method to detect the free primary amine retention rate. When the free primary amine retention rate reaches 65%, immediately adjust the pH of the system to 6.0 with 1 mol / L hydrochloric acid solution to terminate the reaction. Then, put the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it in 8200 g of sterile deionized water at 4 °C for 48 h. Change the 8200 g of sterile deionized water once every 6 h. After dialysis, concentrate it to 30000 mg using a 10000 Da ultrafiltration membrane at 4 °C to obtain a methacrylamide recombinant collagen concentrate. Step 3: Add 39800 mg of sterile deionized water and 420 mg of sodium hyaluronate to another sterile reactor, stir at room temperature for 12 h to completely dissolve, then add 42 mg of sodium periodate, and react under light-protected conditions for 6 h; subsequently add 14 mg of ethylene glycol and continue the reaction for 30 min to block the remaining oxidant; load the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze in 6000 g of sterile deionized water for 48 h, changing the 6000 g of sterile deionized water every 6 h, and freeze-dry after dialysis to obtain oxidized sodium hyaluronate; the degree of aldehyde was determined by hydroxylamine hydrochloride titration, and the degree of aldehyde was 15.6%; Step 4: Add 30,000 mg of selectively methacrylated recombinant collagen concentrate, 3,600 mg of sterile deionized water, and 180 mg of oxidized sodium hyaluronate to a sterile mixing tank in sequence, and stir at 4°C for 15 min; then adjust the pH of the system to 7.0; add 38 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphine dissolved in 1,600 mg of sterile deionized water, and stir in the dark for 5 min; 55 s before the film coating, add 12 mg of calcium chloride dihydrate dissolved in 500 mg of sterile deionized water, mix for 30 s, and immediately coat the film onto the release polyester film, controlling the wet film thickness to 950 μm; Step 5: Prepare a separate carboxymethyl chitosan endosmosis solution, consisting of 24 mg carboxymethyl chitosan and 4976 mg sterile deionized water. Within 30 seconds after the coating in Step 4, spray 5000 mg of the endosmosis solution evenly over the entire membrane surface using an atomization method. The spraying time should be controlled within 15 seconds. After spraying, let it stand for 90 seconds. Step 6: Irradiate the film-forming material treated in Step 5 with 405nm visible light for 55s to achieve a cumulative light dose of 550mJ / cm². 2 After curing, the film is trimmed under sterile conditions and cut into 60mm×80mm sheets. Then, each sheet is placed in two sterile sodium chloride solutions of 30000mg each with a mass concentration of 9g / L for equilibration for 10min. Each wet film is sandwiched between two release films and the surface free liquid is gently pressed out. The mass of a single sheet is controlled to be 3300mg, thus obtaining the recombinant collagen wound dressing.

[0022] Example 4: Step 1: Add 43,000 mg of sterile water for injection, 1,500 mg of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa and 1,000 mg of recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa to a sterile reactor, stir at 300 r / min for 13 h at 4 °C to completely dissolve it, and then let it stand for 30 min to remove bubbles to obtain a recombinant collagen compound solution; Step 2: Prepare 2050 mg of 1 mol / L sodium hydroxide solution and 2050 mg of 1 mol / L hydrochloric acid solution as adjusting solutions. Under ice bath conditions, add 345 mg of methacrylic anhydride dropwise to the recombinant collagen compound solution obtained in Step 1 within 20 min, and maintain the pH of the system at 8.0 ± 0.2. After the addition is completed, continue the reaction for 22 min. Take samples every 10 min and use the ninhydrin colorimetric method to detect the free primary amine retention rate. When the free primary amine retention rate reaches 68%, immediately adjust the pH of the system to 6.0 with 1 mol / L hydrochloric acid solution to terminate the reaction. Then, put the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it in 8500 g of sterile deionized water at 4 °C for 50 h. Change the 8500 g of sterile deionized water once every 6 h. After dialysis, concentrate it to 30000 mg using a 10000 Da ultrafiltration membrane at 4 °C to obtain a methacrylamide recombinant collagen concentrate. Step 3: Add 39200 mg of sterile deionized water and 450 mg of sodium hyaluronate to another sterile reactor, stir at room temperature for 12 h to completely dissolve, then add 50 mg of sodium periodate, and react under light-protected conditions for 6 h; subsequently add 16 mg of ethylene glycol and continue the reaction for 30 min to block the remaining oxidant; load the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze in 6000 g of sterile deionized water for 48 h, changing the 6000 g of sterile deionized water every 6 h, and freeze-dry after dialysis to obtain oxidized sodium hyaluronate; the degree of aldehyde was determined by hydroxylamine hydrochloride titration, and the degree of aldehyde was 21.7%; Step 4: Add 30,000 mg of selectively methacrylated recombinant collagen concentrate, 3,400 mg of sterile deionized water, and 220 mg of oxidized sodium hyaluronate to a sterile mixing tank in sequence, and stir at 4°C for 15 min; then adjust the pH of the system to 7.0; add 42 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphine dissolved in 1,600 mg of sterile deionized water, and stir in the dark for 5 min; 60 s before the film coating, add 15 mg of calcium chloride dihydrate dissolved in 500 mg of sterile deionized water, mix for 30 s, and immediately coat the film onto the release polyester film, controlling the wet film thickness to 1100 μm; Step 5: Prepare a separate carboxymethyl chitosan endosmosis solution, consisting of 30 mg carboxymethyl chitosan and 4970 mg sterile deionized water. Within 30 seconds after the coating in Step 4, spray 5000 mg of the endosmosis solution evenly over the entire membrane surface using an atomization method. The spraying time should be controlled within 16 seconds. After spraying, let it stand for 100 seconds. Step 6: Irradiate the film-forming material treated in Step 5 with 405nm visible light for 65s to achieve a cumulative light dose of 650mJ / cm². 2After curing, the film is trimmed under sterile conditions and cut into 60mm×80mm sheets. Then, each sheet is placed in two sterile sodium chloride solutions of 30000mg each with a mass concentration of 9g / L for equilibration for 10min. Each wet film is sandwiched between two release films and gently pressed to remove the surface free liquid. The mass of a single sheet is controlled to be 3450mg, thus obtaining the recombinant collagen wound dressing.

[0023] Example 5: Step 1: Add 47,000 mg of sterile water for injection, 1,800 mg of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa and 700 mg of recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa to a sterile reactor, stir at 300 r / min at 4°C for 11 h to completely dissolve it, and then let it stand for 25 min to remove bubbles to obtain a recombinant collagen compound solution; Step 2: Prepare 2100 mg of 1 mol / L sodium hydroxide solution and 2100 mg of 1 mol / L hydrochloric acid solution as adjusting solutions. Under ice bath conditions, add 390 mg of methacrylic anhydride dropwise to the recombinant collagen compound solution obtained in Step 1 over 22 min, and maintain the pH of the system at 8.1 ± 0.2. After the addition is completed, continue the reaction for 20 min. Take samples every 10 min and use the ninhydrin colorimetric method to detect the free primary amine retention rate. When the free primary amine retention rate reaches 72%, immediately adjust the pH of the system to 6.0 with 1 mol / L hydrochloric acid solution to terminate the reaction. Then, put the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it in 8800 g of sterile deionized water at 4 °C for 50 h. Replace the 8800 g of sterile deionized water once every 6 h. After dialysis, concentrate it to 31000 mg using a 10000 Da ultrafiltration membrane at 4 °C to obtain a concentrated solution of methacrylamide recombinant collagen. Step 3: Add 38800 mg of sterile deionized water and 380 mg of sodium hyaluronate to another sterile reactor, stir at room temperature for 12 h to completely dissolve, then add 40 mg of sodium periodate, and react under light-protected conditions for 5 h; subsequently add 13 mg of ethylene glycol and continue the reaction for 30 min to block the remaining oxidant; load the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze in 6000 g of sterile deionized water for 48 h, changing the 6000 g of sterile deionized water every 6 h, and freeze-dry after dialysis to obtain oxidized sodium hyaluronate; the degree of aldehyde was determined by hydroxylamine hydrochloride titration, and the degree of aldehyde was 17.0%; Step 4: Add 31000 mg of selectively methacrylated recombinant collagen concentrate, 3700 mg of sterile deionized water, and 200 mg of oxidized sodium hyaluronate to a sterile mixing tank in sequence, and stir at 4°C for 15 min; then adjust the pH of the system to 7.1; add 45 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphine dissolved in 1700 mg of sterile deionized water, and stir in the dark for 5 min; 50 s before the film coating, add 18 mg of calcium chloride dihydrate dissolved in 500 mg of sterile deionized water, mix for 25 s, and immediately coat the film onto the release polyester film, controlling the wet film thickness to 1050 μm; Step 5: Prepare a separate carboxymethyl chitosan endosmosis solution, consisting of 28 mg carboxymethyl chitosan and 4972 mg sterile deionized water. Within 30 seconds after the coating in Step 4, spray 5000 mg of the endosmosis solution evenly over the entire membrane surface using an atomization method. The spraying time should be controlled within 14 seconds. After spraying, let it stand for 90 seconds. Step 6: Irradiate the film-forming material treated in Step 5 with 405nm visible light for 60s to achieve a cumulative light dose of 600mJ / cm². 2 After curing, the film is trimmed under sterile conditions and cut into 60mm×80mm sheets. Then, each sheet is placed in two sterile sodium chloride solutions of 30000mg each with a mass concentration of 9g / L for equilibration for 10min. Each wet film is sandwiched between two release films and gently pressed to remove the surface free liquid. The mass of a single sheet is controlled to be 3500mg, thus obtaining the recombinant collagen wound dressing.

[0024] Example 6: Step 1: Add 42000mg of sterile water for injection, 1850mg of recombinant humanized type III collagen with a relative molecular mass of 55.9kDa and 650mg of recombinant humanized type III collagen with a relative molecular mass of 28.6kDa to a sterile reactor, stir at 340r / min at 4℃ for 12h to completely dissolve it, and then let it stand for 35min to remove bubbles to obtain the recombinant collagen compound solution; Step 2: Prepare 2200 mg of 1 mol / L sodium hydroxide solution and 2200 mg of 1 mol / L hydrochloric acid solution as adjusting solutions. Under ice bath conditions, add 450 mg of methacrylic anhydride dropwise to the recombinant collagen compound solution obtained in Step 1 over 25 min, and maintain the pH of the system at 8.1 ± 0.2. After the addition is completed, continue the reaction for 25 min. Take samples every 10 min and use the ninhydrin colorimetric method to detect the free primary amine retention rate. When the free primary amine retention rate reaches 60%, immediately adjust the pH of the system to 5.9 with 1 mol / L hydrochloric acid solution to terminate the reaction. Then, put the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it in 9000 g of sterile deionized water at 4 °C for 54 h. Change the 9000 g of sterile deionized water once every 6 h. After dialysis, concentrate it to 32000 mg using a 10000 Da ultrafiltration membrane at 4 °C to obtain a concentrated solution of methacrylamide recombinant collagen. Step 3: Add 38400 mg of sterile deionized water and 500 mg of sodium hyaluronate to another sterile reactor, stir at room temperature for 12 h to completely dissolve, then add 60 mg of sodium periodate, and react under light-protected conditions for 8 h; subsequently add 20 mg of ethylene glycol and continue the reaction for 30 min to block the remaining oxidant; load the reaction solution into a dialysis bag with a molecular weight cutoff of 3500 Da, dialyze in 6000 g of sterile deionized water for 48 h, changing the 6000 g of sterile deionized water every 6 h, and freeze-dry after dialysis to obtain oxidized sodium hyaluronate; the degree of aldehyde was determined by hydroxylamine hydrochloride titration, and the degree of aldehyde was 24.6%; Step 4: Add 32000 mg of selectively methacrylated recombinant collagen concentrate, 3000 mg of sterile deionized water, and 250 mg of oxidized sodium hyaluronate to a sterile mixing tank in sequence, and stir at 4°C for 15 min; then adjust the pH of the system to 7.1; add 50 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphine dissolved in 1800 mg of sterile deionized water, and stir in the dark for 6 min; 75 s before the film coating, add 20 mg of calcium chloride dihydrate dissolved in 500 mg of sterile deionized water, mix for 40 s, and immediately coat the film onto the release polyester film, controlling the wet film thickness to 1200 μm; Step 5: Prepare a separate carboxymethyl chitosan endosmosis solution, consisting of 35 mg carboxymethyl chitosan and 4965 mg sterile deionized water. Within 30 seconds after the coating in Step 4, spray 5000 mg of the endosmosis solution evenly over the entire membrane surface using an atomization method. The spraying time should be controlled within 18 seconds. After spraying, let it stand for 110 seconds. Step 6: Irradiate the film-forming material treated in Step 5 with 405nm visible light for 70s to achieve a cumulative light dose of 700mJ / cm². 2After curing, the film is trimmed under sterile conditions and cut into 60mm×80mm sheets. Then, each sheet is placed in two sterile sodium chloride solutions of 30000mg each with a mass concentration of 9g / L for 10min to equilibrate. Each wet film is sandwiched between two release films and gently pressed to remove the surface free liquid. The mass of a single sheet is controlled to be 3600mg to obtain the recombinant collagen wound dressing.

[0025] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that in step 1, instead of adding 750 mg of recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa, 2500 mg of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa was added; the other conditions were the same as in Example 1.

[0026] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that in step 1, instead of adding 1750 mg of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa, 2500 mg of recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa was added; the other conditions were the same as in Example 1.

[0027] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that the amount of methacrylic anhydride used in step 2 was adjusted to 285 mg, and the retention rate of free primary amine was controlled to be 80% by the ninhydrin colorimetric method; the other conditions were the same as in Example 1.

[0028] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that the amount of methacrylic anhydride used in step 2 was adjusted to 520 mg, and the retention rate of free primary amine was controlled to be 45% by the ninhydrin colorimetric method; the other conditions were the same as in Example 1.

[0029] Comparative Example 5: The difference between Comparative Example 5 and Example 1 is that 200 mg of sodium oxyhyaluronate obtained in step 3 was added to the recombinant collagen complex solution obtained in step 1 before the start of the methacrylation reaction in step 2, and it was present in the same reaction system as methacrylic anhydride, instead of being added after step 2 was completed and concentrated; the other conditions were the same as in Example 1.

[0030] Comparative Example 6: The difference between Comparative Example 6 and Example 1 is that in step 4, 200 mg of sodium hyaluronate was used to replace 200 mg of oxidized sodium hyaluronate; the other conditions were the same as in Example 1.

[0031] Performance testing: General Instructions for Sample Preparation and Testing: The samples used for performance testing are Examples 1-6 and Comparative Examples 1-6. To ensure a fair comparison, unless otherwise specified, all samples were prepared using the same batch of raw materials, the same type of scraper, the same release polyester film, the same 405nm light source, and the same equilibration process. All finished products were cut to 60mm × 80mm; specimens for wet tensile testing were further cut to 15mm × 80mm; specimens for liquid absorbency, liquid control performance, and potential adhesion testing were cut to the dimensions specified for each test item. Unless otherwise specified, all finished products were equilibrated for 24 hours at (23±2)℃ and (50±5)% relative humidity before testing; wet-related items were pre-equilibrated in 9g / L sodium chloride solution for 30 minutes before testing.

[0032] Wet tensile properties: The test was conducted according to GB / T 1040.3-2006. Before testing, the specimens were immersed in a 9 g / L sodium chloride solution at (23±2)℃ for 30 min to simulate the service conditions. Five 15 mm × 80 mm strip specimens with a gauge length of 50 mm were taken from each group. After removal, the free liquid on both sides was removed by gently touching both sides with a dust-free filter paper for 10 s. The specimens were then immediately clamped in an electronic universal testing machine at a tensile speed of 50 mm / min. The maximum load and elongation at break were recorded, and the wet tensile strength was calculated based on the original cross-sectional area.

[0033] Liquid absorbency and liquid retention: The liquid absorbency method was performed according to YY / T 0471.1-2004. Three 50mm×50mm samples were taken for each group, and the initial mass m0 was recorded. The samples were immersed in a 9g / L sodium chloride simulated percolation solution at (37±1)℃ for 30min. After being taken out and suspended vertically for 30s, the mass m1 was weighed, and the liquid absorption rate was calculated as (m1-m0) / m0×100%. Then, the liquid-absorbed samples were placed between two layers of quantitative filter paper, and after standing under a 500g plate load for 60s, the mass m2 was weighed again, and the liquid retention rate was calculated as (m2-m0) / m0×100%.

[0034] Fluid control performance and post-exudation morphology retention: An in vitro wound model was established according to YY / T 1477.3-2016. The sample was covered with an effective area of ​​36 cm². 2 The surface of an in vitro wound model was continuously infused with simulated exudate, which was a 9 g / L sodium chloride aqueous solution containing 0.2 wt% bovine serum albumin. The infusion rate was set to 0.20 mL / min, the test temperature was (37±1) ℃, and the test was conducted continuously for 4 hours. The leakage volume, reabsorption volume, area retention rate after sample absorption, and edge curling height of the model were recorded. The area retention rate was calculated as the ratio of the projected area after absorption to the initial area, and the edge curling height was the maximum value of the four sides.

[0035] Potential adhesion: An in vitro model for evaluating the potential adhesion of wound dressings was established according to YY / T 1477.4-2017. A 40mm×70mm sample was placed on the surface of a simulated wound substrate containing 10wt% gelatin and 0.9wt% sodium chloride. After contact for 4 hours at (37±1)℃ and relative humidity not less than 90%, the peel force was measured on an electronic universal testing machine by peeling at 180° with a peeling speed of 100mm / min. The maximum peel force was recorded.

[0036] In vitro cytotoxicity: The sample extract was prepared according to GB / T 16886.12-2023. The final dressing, after all curing and equilibration processes were completed, was used, with a surface area equal to the extract volume of 6 cm². 2 The extract was added to MEM medium containing 10% fetal bovine serum at a ratio of / mL and extracted at (37±1)℃ for 24h; subsequently, L929 cells were analyzed by MTT assay according to GB / T 16886.5-2017, with a seeding density of 1×10⁶ cells / mL. 4 Cells were cultured in wells for 24 hours. After the cells adhered, the solution was replaced with sample extract. After another 24 hours of incubation, MTT working solution was added and the cells were cultured for another 4 hours. The absorbance was measured at 570 nm using a microplate reader, and the cell viability was calculated.

[0037] Skin irritation test: After the polar and non-polar extracts of the sample were prepared according to GB / T 16886.12-2023, the closed dressing test of rabbits was carried out according to GB / T 16886.23-2023. The dressing area was 25mm×25mm and the dressing time was 24h. Erythema and edema scores were recorded at 1h, 24h, 48h and 72h after the sample was removed, and the primary irritation index was calculated.

[0038] Skin sensitization test: Evaluation was conducted using the guinea pig maximization test according to GB / T 16886.10-2024. The sample extract was prepared according to GB / T 16886.12-2023. After completing the induction, withdrawal, and challenge periods as specified in the standard, erythema and edema reactions were recorded at 24h and 48h after challenge. Sensitization was determined according to the standard grading. The test results are shown in Table 1.

[0039] Table 1 Performance Test Results

[0040] Data Analysis: As can be seen from the data in Table 1, the recombinant collagen wound dressing prepared by this invention achieves a good overall balance between wet-state load-bearing, liquid absorption and retention, morphological stability after exudation, and low potential adhesion. It is more suitable for superficial wounds, abrasions, and postoperative incisions, which require continuous moist coverage and good dressing compliance. The reason may be that two recombinant humanized type III collagens of different molecular weights together form a network framework that combines film-forming properties and flexibility. The moderately retained free primary amines ensure the basic strength after visible light curing and retain reaction sites for the cross-linking of sodium hyaluronate and carboxymethyl chitosan. The carboxymethyl chitosan infiltrating into the film surface and the subsequently added sodium hyaluronate form a progressive dynamic network in the thickness direction. Combined with the reversible ionic microbridges provided by the calcium chloride dihydrate introduced briefly before scraping, the dressing can maintain edge integrity and is not easily torn under stress after absorbing exudate, and maintains a relatively mild interfacial contact state on the wound surface.

[0041] As can be seen from the data in Table 1 for Example 1, Comparative Example 1, and Comparative Example 2, when only a single molecular weight recombinant humanized type III collagen is used, the dressing performance deviates in one direction. When only a higher molecular weight component is used, the precursor viscosity is too high, resulting in insufficient uniformity in coating, spreading, and subsequent infiltration, thus limiting wet extensibility, edge smoothness, and liquid control ability. When only a lower molecular weight component is used, although the hydrophilic absorption tendency is enhanced, the network support skeleton is insufficient, making it more prone to relaxation and deformation after infiltration. Therefore, different molecular weight components are not simply additive, but rather form a significant synergistic effect in film formation, force transmission, and infiltration buffering, exhibiting a comprehensive effect greater than the sum of its parts.

[0042] As can be seen from the data in Table 1 for Examples 1, 3, and 4, both excessively high and low levels of free primary amine retention make it difficult to achieve balanced performance for wound applications. When the free primary amine retention is too high, the degree of methacrylylation is low, resulting in insufficient permanent network support. While the material is softer and absorbs liquid more effectively, its wet strength, rewetting control, and shape retention decrease, and the interface is more prone to viscous contact. When the free primary amine retention is too low, although the visible light-cured network is enhanced, the dynamic interaction sites with sodium oxyhyaluronate and carboxymethyl chitosan are reduced, making the dressing more prone to brittleness, curling, and tight adhesion.

[0043] As can be seen from the data in Table 1 for Example 1 and Comparative Example 5, even with basically the same types and amounts of raw materials, different timing of the addition of sodium oxyhyaluronate leads to different final results. Adding sodium oxyhyaluronate too early to the methacrylation reaction system prematurely complicates the local reaction environment around collagen molecules. This affects the uniform introduction of methacryl groups onto the collagen backbone and also easily leads to increased local viscosity and micro-association before film formation, weakening the open channels required for subsequent carboxymethyl chitosan infiltration. Therefore, although Comparative Example 5 did not lack key components, it was inferior to Example 1 in terms of wet mechanics, liquid control, and edge stability.

[0044] As can be seen from the data in Example 1 and Comparative Example 6 in Table 1, after replacing oxidized sodium hyaluronate with an equal amount of sodium hyaluronate, the material still maintains a certain degree of hydrophilicity and coverage, but its retention capacity, area stability, and low adhesion performance after liquid absorption are significantly weakened. The main reason for this is that unoxidized sodium hyaluronate can only provide thickening and water retention effects, and cannot further form a cross-linked network with recombinant humanized type III collagen and carboxymethyl chitosan. Therefore, it is difficult to continuously disperse stress and inhibit local flow after liquid absorption.

[0045] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A recombinant collagen wound dressing, characterized in that, The recombinant collagen wound dressing is a film dressing. The film dressing includes a collagen cross-linking network formed by curing a selectively methacrylated recombinant collagen concentrate with visible light and sodium oxyhyaluronate distributed in the collagen cross-linking network. Carboxymethyl chitosan is infiltrated into the surface of the film dressing and forms a cross-linking structure with the sodium oxyhyaluronate. The raw materials for preparing the film dressing, by weight, include: 29,000-32,000 parts of selectively methacrylated recombinant collagen concentrate, 150-250 parts of oxidized sodium hyaluronate, 20-35 parts of carboxymethyl chitosan, 30-50 parts of phenyl-2,4,6-trimethylbenzoyl lithium phosphinate and 10-20 parts of calcium chloride dihydrate; The selectively methacrylamide recombinant collagen concentrate was obtained by dialysis and concentration of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa and recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa after methacrylamide reaction, with a free primary amine retention rate of 60%-75%; the degree of aldehyde conversion of the oxidized sodium hyaluronate was 12.8%-24.6%. The raw materials for preparing the selectively methacrylated recombinant collagen concentrate, by weight, include: 42,000-47,000 parts of sterile water for injection, 1,500-1,900 parts of recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa, 600-1,000 parts of recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa, and 315-450 parts of methacrylic anhydride; The preparation method of the recombinant collagen wound dressing includes the following steps: S1. Recombinant humanized type III collagen with a relative molecular mass of 55.9 kDa and recombinant humanized type III collagen with a relative molecular mass of 28.6 kDa were dissolved in sterile water for injection and subjected to methacrylation reaction. The reaction was terminated when the free primary amine retention rate reached 60%-75%. After dialysis and concentration, a selectively methacrylated recombinant collagen concentrate was obtained. S2. Oxidize sodium hyaluronate and block the remaining oxidant. After dialysis and freeze-drying, oxidized sodium hyaluronate is obtained. S3. The selectively methacrylated recombinant collagen concentrate obtained in step S1 is mixed with the sodium oxidized hyaluronic acid obtained in step S2, and then phenyl-2,4,6-trimethylbenzoyl lithium phosphine and calcium chloride dihydrate are added and coated into a film. S4. Atomize and spray carboxymethyl chitosan endosmosis solution onto the membrane surface obtained in step S3; S5. The film-forming body after step S4 is cured by visible light irradiation and then equilibrated with sterile sodium chloride solution to obtain recombinant collagen wound dressing.

2. The recombinant collagen wound dressing according to claim 1, characterized in that, The oxidized sodium hyaluronate is prepared by oxidizing 350-500 parts of sodium hyaluronate with a molecular weight of 200-400 kDa with 35-60 parts of sodium periodate, and then sealing the remaining oxidant with 12-20 parts of ethylene glycol, by weight.

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