A recombinant collagen wound dressing and a preparation method thereof
By modifying recombinant humanized type III collagen with chitosan and cross-linking it with polyvinyl alcohol, and combining it with nano-silver, a stable dressing network is formed, which solves the problem of insufficient mechanical properties of natural collagen in the wound healing process, and achieves effective protection and rapid healing of the wound.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XINJIANG AIYOUHE BIOPHARMACEUTICAL CO LTD
- Filing Date
- 2025-09-04
- Publication Date
- 2026-06-26
AI Technical Summary
Natural collagen loses its mechanical properties significantly after absorbing water, becomes easily broken, and cannot form a sustainable physical barrier. Furthermore, it lacks sufficient support in deep wounds, affecting wound healing.
Recombinant humanized type III collagen was used to modify chitosan and form a cross-linked network with polyvinyl alcohol and genipin. Nano-silver was dispersed in the gel and compounded on the surface of PEG-6000 modified nonwoven fabric to enhance the mechanical stability and anti-enzymatic ability of the dressing.
It significantly improves the mechanical stability and enzymatic resistance of the dressing, provides a suitable microenvironment to promote wound healing, reduces the risk of infection, and improves wound repair efficiency.
Smart Images
Figure CN121081708B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of dressing technology, and particularly relates to a recombinant collagen wound dressing and its preparation method. Background Technology
[0002] A wound refers to damage to human tissues or organs caused by physical, chemical, or other factors. Human skin serves as a barrier to maintain the stability of the internal environment and prevent microbial invasion. When the normal physiological functions of the skin are impaired, healing is a dynamic and complex process, including the coagulation phase, inflammation phase, cell proliferation phase, and new tissue remodeling phase. The healing rate of a wound is affected by factors such as the type of dressing, pathological conditions, and the degree of wound trauma. Dressings, as temporary skin substitutes, can play a role in hemostasis, wound protection, and infection prevention, providing a favorable microenvironment for the recovery or reconstruction of the skin barrier function.
[0003] Medical dressings are a class of medical materials used to cover the surface of wounds and provide temporary protection for damaged skin. They can control or prevent wound infection and provide an environment conducive to wound healing. Ideal wound dressings should have the characteristics of accelerating wound healing, preventing infection, and restoring skin structure and properties.
[0004] Collagen is a major structural protein in the extracellular matrix of animals, possessing a variety of biological functions. It is widely present in human tissues such as skin, bone, and muscle, participating in cell proliferation, differentiation, migration, and signal transduction, playing a supporting, repairing, and protective role, such as maintaining the structure of various tissues, promoting cell attachment and proliferation, and repairing tissues and organs. It was one of the earliest biomaterials approved by the FDA and CSFDA, and due to its unique bioactivity, it is widely used in tissue engineering, biomedicine, drug delivery, and other fields. Collagen is the main structural protein of the extracellular matrix, possessing multiple biological functions, such as maintaining the structure of various tissues, promoting cell attachment, proliferation, migration, and repairing tissues and organs. To mimic the skin barrier function, Batheja et al., under laboratory conditions, cultured only human fibroblasts and collagen, and obtained artificial skin tissue with physiological indicators similar to natural human skin.
[0005] The intermolecular forces of natural collagen are mainly hydrogen bonds, lacking a strong covalent cross-linking network. Collagen swells after absorbing water, significantly reducing its mechanical properties and making it prone to rupture during dressing changes or patient activity. It cannot form a continuous physical barrier, increasing the risk of wound infection. When used for deep wounds (such as subcutaneous tissue defects), the collagen scaffold is prone to collapse due to insufficient self-support, failing to maintain the "three-dimensional spatial structure" required for the wound, thus hindering the infiltration of fibroblasts and endothelial cells and tissue regeneration. Summary of the Invention
[0006] This invention aims to provide a recombinant collagen wound dressing and its preparation method. The recombinant collagen wound dressing uses recombinant humanized type III collagen as a matrix, modified with chitosan to ensure tight adhesion to the wound surface. Polyvinyl alcohol (PVA) is used as a mechanical reinforcing agent, and genipin is used as a natural cross-linking agent to form a recombinant collagen gel. Nano-silver is dispersed within the gel and then composited onto the surface of a PEG-6000 modified nonwoven fabric, making the nonwoven fabric easily peelable from the recombinant collagen gel. This significantly improves the mechanical stability, enzymatic resistance, and repair efficiency of the recombinant collagen dressing.
[0007] To achieve the above objectives, the present invention provides a method for preparing a recombinant collagen wound dressing, comprising the following steps:
[0008] S1. A recombinant collagen solution is mixed with a chitosan solution to obtain a recombinant collagen mixture. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is added to the recombinant collagen mixture to form a stable composite system. The modified recombinant collagen solution is obtained by dialysis using a dialysis bag.
[0009] S2. Take the modified recombinant collagen solution, add polyvinyl alcohol solution and nano silver solution, sonicate, add genipin solution, stir in the dark to obtain recombinant collagen gel solution;
[0010] S3. Soak the nonwoven fabric in an ethanol solution, rinse it with distilled water, immerse it in a polyethylene glycol solution, treat it with constant temperature shaking, rinse it with distilled water, and vacuum dry it to obtain the modified nonwoven fabric.
[0011] S4. Lay the modified nonwoven fabric flat on a horizontal glass plate, take the recombinant collagen gel solution, coat it evenly on the surface of the nonwoven fabric with a coater, incubate it in a constant temperature incubator, and allow it to crosslink in the dark to obtain the nonwoven dressing; immerse the nonwoven dressing in PBS solution, take it out and freeze dry it under vacuum, and sterilize it with γ-rays to obtain the recombinant collagen dressing.
[0012] Preferably, in step S1, the recombinant collagen is recombinant humanized type III collagen, and the recombinant collagen solution comprises recombinant collagen and PBS solution, wherein the mass-to-volume ratio of the recombinant collagen to the PBS solution is (0.8-1.2) g: 50 mL; the chitosan solution comprises chitosan and a 1% (v / v) aqueous acetic acid solution, wherein the mass-to-volume ratio of the chitosan to the 1% (v / v) aqueous acetic acid solution is (0.8-1.2) g: 50 mL, and the pH of the chitosan solution is adjusted to 6.7-6.9 with NaOH.
[0013] Preferably, in step S1, the volume ratio of the recombinant collagen solution to the chitosan solution is (9-22):4; the mass-to-volume ratio of the mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and recombinant collagen is (0.08-0.12) g:130 mL; the dialysis bag has a specification of 10 kDa; and the dialysis time is 40-48 h.
[0014] Preferably, in step S2, the polyvinyl alcohol solution is an aqueous solution with a mass concentration of 0.8% to 1.2%, and the volume ratio of the polyvinyl alcohol solution to the modified recombinant collagen solution is (0.9 to 1.1):1.
[0015] Preferably, in step S2, the concentration of the nano-silver solution is 1 mg / mL, and the volume ratio of the nano-silver solution to the modified recombinant collagen solution is (0.8–1.2):10; the ultrasonic treatment power is 150–250 W, and the time is 4–6 min; the genipin solution is a DMSO solution containing 2% genipin by mass, and the volume ratio of the genipin solution to the modified recombinant collagen solution is (0.8–1.2):20; the light-protected stirring speed is 80–120 rpm, and the time is 20–40 min.
[0016] Preferably, in step S3, the ethanol solution is an aqueous solution with a volume concentration of 70%–80%; the soaking time is 20–40 min; the polyethylene glycol solution is an aqueous solution of polyethylene glycol 6000 with a mass concentration of 1.5%–2.5%; the constant temperature oscillation temperature is 35–40°C, the rotation speed is 80–120 rpm, and the time is 1–2 h; the vacuum drying temperature is 50–70°C, and the time is 2–4 h.
[0017] Preferably, in step S4, the coating thickness is 0.1–0.5 mm; the incubation temperature is 35–40°C for 20–28 h; the immersion time in PBS solution is 25–35 min; the vacuum freeze-drying temperature is -55°C to -70°C for 20–28 h; and the gamma ray sterilization dose is 20–30 kGy.
[0018] Compared with the prior art, the beneficial effects of the present invention are reflected in:
[0019] (1) Genipin masks protease recognition sites by forming pyrrolidone bonds with the lysine amino groups of recombinant collagen. It can also react with the hydroxyl groups of polyvinyl alcohol to form a ternary cross-linked network of "recombinant collagen-genipin-polyvinyl alcohol," reducing the degradation of recombinant collagen by proteases on the wound surface. The degradation products are glycine, proline, and other amino acids naturally present in the body, which can participate in wound repair. Utilizing the positive charge properties of chitosan, it forms strong electrostatic adsorption with wound tissue. PEG-6000 is used to modify the surface of the nonwoven fabric, reducing its surface energy through hydrogen bonding and decreasing physical adhesion to the gel layer. This makes the cohesive force of the gel layer greater than the interfacial adhesion force with the nonwoven fabric, facilitating the separation of the nonwoven fabric from the wound.
[0020] (2) Polyvinyl alcohol (PVA) molecular chains form an interpenetrating network with collagen through hydrogen bonds, which not only enhances the wet tensile strength of the dressing but also improves water absorption through the microcrystalline structure, avoiding the defects of excessive swelling or drying and crusting of pure collagen. The cross-linking of PVA and recombinant collagen forms a porous gel structure, providing spatial support for epithelial cell migration and granulation tissue formation, thus aiding wound healing. Nano-silver is uniformly embedded in the gel network through ultrasonic dispersion, achieving long-lasting antibacterial effects and preventing wound infection that hinders healing. Attached Figure Description
[0021] Figure 1 Comparative images showing the healing of full-thickness skin defects in mice. Detailed Implementation
[0022] The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.
[0023] The main compounds used in the examples and comparative examples were all commercially available products and were not subjected to any further purification treatment.
[0024] Example 1
[0025] A method for preparing a recombinant collagen wound dressing includes the following steps:
[0026] S1. Dissolve 10g of recombinant humanized type III collagen in 500mL of PBS solution to obtain a recombinant collagen solution. Dissolve 3g of chitosan in 150mL of 1% acetic acid solution and adjust the pH to 6.8 with NaOH to obtain a chitosan solution. Mix 500mL of recombinant collagen solution and 150mL of chitosan solution to obtain a recombinant collagen mixture. Add 0.5g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the recombinant collagen mixture and stir magnetically at 500rpm for 2h to form a stable complex system. Dialyze the mixture using a 10kDa dialysis bag for 44h to obtain a modified recombinant collagen solution.
[0027] S2. Take 100 mL of modified recombinant collagen solution, add 100 mL of 0.8% polyvinyl alcohol aqueous solution, stir at 100 rpm for 1 h, then add 10 mL of 1 mg / mL nano silver solution, sonicate at 200 W for 5 min, then add 5 mL of 2% genipin DMSO solution, stir at 100 rpm for 30 min in the dark to obtain recombinant collagen gel solution.
[0028] S3. Soak a 5cm×5cm nonwoven fabric in a 75% ethanol solution for 30 minutes, rinse it three times with distilled water, then immerse it in a 2% polyethylene glycol 6000 solution, shake it at 37℃ and 100rpm for 1.5 hours, rinse it three times with distilled water, and vacuum dry it at 60℃ for 3 hours to obtain the modified nonwoven fabric.
[0029] S4. The modified nonwoven fabric is laid flat on a horizontal glass plate. The recombinant collagen gel solution is taken and evenly coated onto the surface of the nonwoven fabric using a coater. The coating thickness is 0.2 mm. The nonwoven fabric is placed in a constant temperature incubator at 37℃ and kept in the dark for 24 hours for cross-linking incubation to obtain the nonwoven fabric dressing. The nonwoven fabric dressing is immersed in PBS solution for 30 minutes, then removed and freeze-dried at -60℃ for 24 hours. It is then sterilized with 25 kGy dose γ-rays to obtain the recombinant collagen dressing.
[0030] Example 2
[0031] A method for preparing a recombinant collagen wound dressing includes the following steps:
[0032] S1. Dissolve 8g of recombinant humanized type III collagen in 500mL of PBS to obtain a recombinant collagen solution. Dissolve 3.2g of chitosan in 200mL of 1% acetic acid solution and adjust the pH to 6.7 with NaOH to obtain a chitosan solution. Mix 450mL of the recombinant collagen solution and 200mL of the chitosan solution to obtain a recombinant collagen mixture. Add 0.4g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the recombinant collagen mixture and stir magnetically at 500rpm for 2h to form a stable complex system. Dialyze the mixture using a 10kDa dialysis bag for 40h to obtain a modified recombinant collagen solution.
[0033] S2. Take 100 mL of modified recombinant collagen solution, add 90 mL of 1.2% polyvinyl alcohol aqueous solution, stir at 100 rpm for 1 h, then add 8 mL of 1 mg / mL nano silver solution, sonicate at 150 W for 6 min, then add 4 mL of 2% genipin DMSO solution, stir at 80 rpm for 40 min in the dark to obtain recombinant collagen gel solution.
[0034] S3. Soak a 5cm×5cm nonwoven fabric in a 70% ethanol solution for 40 minutes, rinse it three times with distilled water, then immerse it in a 1.5% polyethylene glycol 6000 solution, shake it at 35℃ and 120rpm for 2 hours, rinse it three times with distilled water, and vacuum dry it at 50℃ for 4 hours to obtain the modified nonwoven fabric.
[0035] S4. The modified nonwoven fabric is laid flat on a horizontal glass plate. The recombinant collagen gel solution is taken and evenly coated onto the surface of the nonwoven fabric using a coater. The coating thickness is 0.1 mm. The nonwoven fabric is placed in a constant temperature incubator at 35℃ and kept in the dark for 28 hours for cross-linking incubation to obtain the nonwoven fabric dressing. The nonwoven fabric dressing is immersed in PBS solution for 25 minutes, taken out and freeze-dried at -55℃ for 28 hours. It is then sterilized with 20 kGy dose γ-ray to obtain the recombinant collagen dressing.
[0036] Example 3
[0037] A method for preparing a recombinant collagen wound dressing includes the following steps:
[0038] S1. 13.2 g of recombinant humanized type III collagen was dissolved in 550 mL of PBS solution to obtain a recombinant collagen solution. 2.4 g of chitosan was dissolved in 100 mL of 1% acetic acid solution, and the pH was adjusted to 6.9 with NaOH to obtain a chitosan solution. 550 mL of the recombinant collagen solution and 100 mL of the chitosan solution were mixed to obtain a recombinant collagen mixture. 0.6 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was added to the recombinant collagen mixture, and the mixture was magnetically stirred at 500 rpm for 2 h to form a stable composite system. The mixture was dialyzed through a 10 kDa dialysis bag for 48 h to obtain a modified recombinant collagen solution.
[0039] S2. Take 100 mL of modified recombinant collagen solution, add 110 mL of 1% polyvinyl alcohol aqueous solution, stir at 100 rpm for 1 h, then add 12 mL of 1 mg / mL nano silver solution, sonicate at 250 W for 4 min, then add 6 mL of 2% genipin DMSO solution, stir at 120 rpm for 20 min in the dark to obtain recombinant collagen gel solution.
[0040] S3. Soak a 5cm×5cm nonwoven fabric in an 80% ethanol solution for 20 minutes, rinse it three times with distilled water, then immerse it in a 2.5% polyethylene glycol 6000 solution, shake it at 40℃ and 80rpm for 1 hour, rinse it three times with distilled water, and vacuum dry it at 70℃ for 2 hours to obtain the modified nonwoven fabric.
[0041] S4. The modified nonwoven fabric is laid flat on a horizontal glass plate. The recombinant collagen gel solution is taken and evenly coated onto the surface of the nonwoven fabric using a coater. The coating thickness is 0.5 mm. The nonwoven fabric is placed in a constant temperature incubator at 40℃ and kept in the dark for 20 h for cross-linking incubation to obtain the nonwoven dressing. The nonwoven dressing is immersed in PBS solution for 35 min, taken out and freeze-dried at -70℃ for 20 h. It is then sterilized with 30 kGy dose γ-ray to obtain the recombinant collagen dressing.
[0042] Comparative Example 1
[0043] A method for preparing unmodified chitosan recombinant collagen wound dressings includes the following steps:
[0044] S1. Dissolve 10g of recombinant humanized type III collagen in 500mL of PBS to obtain a recombinant collagen solution. Add 0.5g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the recombinant collagen solution and stir magnetically at 500rpm for 2h to form a stable complex system. Dialyze the solution using a 10kDa dialysis bag for 44h to obtain a modified recombinant collagen solution.
[0045] S2. Take 100 mL of modified recombinant collagen solution, add 100 mL of 0.8% polyvinyl alcohol aqueous solution, stir at 100 rpm for 1 h, then add 10 mL of 1 mg / mL nano silver solution, sonicate at 200 W for 5 min, then add 5 mL of 2% genipin DMSO solution, stir at 100 rpm for 30 min in the dark to obtain recombinant collagen gel solution.
[0046] S3. Soak a 5cm×5cm nonwoven fabric in a 75% ethanol solution for 30 minutes, rinse it three times with distilled water, then immerse it in a 2% polyethylene glycol 6000 solution, shake it at 37℃ and 100rpm for 1.5 hours, rinse it three times with distilled water, and vacuum dry it at 60℃ for 3 hours to obtain the modified nonwoven fabric.
[0047] S4. The modified nonwoven fabric is laid flat on a horizontal glass plate. The recombinant collagen gel solution is taken and evenly coated onto the surface of the nonwoven fabric using a coater. The coating thickness is 0.2 mm. The nonwoven fabric is placed in a constant temperature incubator at 37℃ and kept in the dark for 24 hours for cross-linking incubation to obtain the nonwoven fabric dressing. The nonwoven fabric dressing is immersed in PBS solution for 30 minutes, then removed and freeze-dried at -60℃ for 24 hours. It is then sterilized with 25 kGy dose γ-rays to obtain the recombinant collagen dressing.
[0048] Comparative Example 2
[0049] A method for preparing an uncrosslinked recombinant collagen gel wound dressing includes the following steps:
[0050] S1. Dissolve 10g of recombinant humanized type III collagen in 500mL of PBS solution to obtain a recombinant collagen solution. Dissolve 3g of chitosan in 150mL of 1% acetic acid solution and adjust the pH to 6.8 with NaOH to obtain a chitosan solution. Mix 500mL of recombinant collagen solution and 150mL of chitosan solution to obtain a recombinant collagen mixture. Add 0.5g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the recombinant collagen mixture and stir magnetically at 500rpm for 2h to form a stable complex system. Dialyze the mixture using a 10kDa dialysis bag for 44h to obtain a modified recombinant collagen solution.
[0051] S2. Take 100 mL of modified recombinant collagen solution, add 100 mL of 0.8% polyvinyl alcohol aqueous solution, stir at 100 rpm for 1 h, then add 10 mL of 1 mg / mL nano silver solution, sonicate at 200 W for 5 min, and stir at 100 rpm for 30 min in the dark to obtain recombinant collagen gel solution.
[0052] S3. Soak a 5cm×5cm nonwoven fabric in a 75% ethanol solution for 30 minutes, rinse it three times with distilled water, then immerse it in a 2% polyethylene glycol 6000 solution, shake it at 37℃ and 100rpm for 1.5 hours, rinse it three times with distilled water, and vacuum dry it at 60℃ for 3 hours to obtain the modified nonwoven fabric.
[0053] S4. The modified nonwoven fabric is laid flat on a horizontal glass plate. The recombinant collagen gel solution is taken and evenly coated onto the surface of the nonwoven fabric using a coater. The coating thickness is 0.2 mm. The nonwoven fabric is placed in a constant temperature incubator at 37℃ and kept in the dark for 24 hours for cross-linking incubation to obtain the nonwoven fabric dressing. The nonwoven fabric dressing is immersed in PBS solution for 30 minutes, then removed and freeze-dried at -60℃ for 24 hours. It is then sterilized with 25 kGy dose γ-rays to obtain the recombinant collagen dressing.
[0054] Comparative Example 3
[0055] A method for preparing a recombinant collagen wound dressing without nano-silver includes the following steps:
[0056] S1. Dissolve 10g of recombinant humanized type III collagen in 500mL of PBS solution to obtain a recombinant collagen solution. Dissolve 3g of chitosan in 150mL of 1% acetic acid solution and adjust the pH to 6.8 with NaOH to obtain a chitosan solution. Mix 500mL of recombinant collagen solution and 150mL of chitosan solution to obtain a recombinant collagen mixture. Add 0.5g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the recombinant collagen mixture and stir magnetically at 500rpm for 2h to form a stable complex system. Dialyze the mixture using a 10kDa dialysis bag for 44h to obtain a modified recombinant collagen solution.
[0057] S2. Take 100 mL of modified recombinant collagen solution, add 100 mL of 0.8% polyvinyl alcohol aqueous solution, stir at 100 rpm for 1 h, sonicate at 200 W for 5 min, then add 5 mL of 2% genipin DMSO solution, stir at 100 rpm for 30 min in the dark to obtain recombinant collagen gel solution.
[0058] S3. Soak a 5cm×5cm nonwoven fabric in a 75% ethanol solution for 30 minutes, rinse it three times with distilled water, then immerse it in a 2% polyethylene glycol 6000 solution, shake it at 37℃ and 100rpm for 1.5 hours, rinse it three times with distilled water, and vacuum dry it at 60℃ for 3 hours to obtain the modified nonwoven fabric.
[0059] S4. The modified nonwoven fabric is laid flat on a horizontal glass plate. The recombinant collagen gel solution is taken and evenly coated onto the surface of the nonwoven fabric using a coater. The coating thickness is 0.2 mm. The nonwoven fabric is placed in a constant temperature incubator at 37℃ and kept in the dark for 24 hours for cross-linking incubation to obtain the nonwoven fabric dressing. The nonwoven fabric dressing is immersed in PBS solution for 30 minutes, then removed and freeze-dried at -60℃ for 24 hours. It is then sterilized with 25 kGy dose γ-rays to obtain the recombinant collagen dressing.
[0060] Comparative Example 4
[0061] A method for preparing unmodified nonwoven recombinant collagen wound dressing includes the following steps:
[0062] S1. Dissolve 10g of recombinant humanized type III collagen in 500mL of PBS solution to obtain a recombinant collagen solution. Dissolve 3g of chitosan in 150mL of 1% acetic acid solution. Adjust the pH to 6.8 with NaOH to obtain a chitosan solution. Mix 500mL of the recombinant collagen solution and 150mL of the chitosan solution to obtain a collagen mixture. Add 0.5g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the recombinant collagen mixture and stir magnetically at 500rpm for 2h to form a stable complex system. Dialyze the mixture using a 10kDa dialysis bag for 44h to obtain a modified recombinant collagen solution.
[0063] S2. Take 100 mL of modified recombinant collagen solution, add 100 mL of 0.8% polyvinyl alcohol aqueous solution, stir at 100 rpm for 1 h, then add 10 mL of 1 mg / mL nano silver solution, sonicate at 200 W for 5 min, then add 5 mL of 2% genipin DMSO solution, stir at 100 rpm for 30 min in the dark to obtain recombinant collagen gel solution.
[0064] S3. Soak a 5cm×5cm nonwoven fabric in a 75% ethanol solution for 30 minutes, rinse three times with distilled water, and then vacuum dry at 60℃ for 3 hours to obtain a dried nonwoven fabric. Lay the dried nonwoven fabric flat on a horizontal glass plate, take a recombinant collagen gel solution, and coat it evenly on the surface of the nonwoven fabric with a coating agent. The coating thickness is 0.2mm. Place it in a 37℃ constant temperature incubator and incubate for cross-linking in the dark for 24 hours to obtain a nonwoven dressing. Soak the nonwoven dressing in PBS solution for 30 minutes, take it out, and then freeze-dry it at -60℃ for 24 hours. Sterilize it with 25kGy dose γ-rays to obtain a recombinant collagen dressing.
[0065] Experimental Example 1
[0066] In vitro enzymatic digestion experiment:
[0067] Prepare a Tris-HCl mixed enzyme solution containing 100 U / mL metalloproteinase 9. Cut the above dressings into 1cm×1cm squares, accurately weigh the initial dry weight and record it as W0. Soak the dressings in PBS buffer (pH 7.4) for 1 hour to fully hydrate them. Add one pretreated dressing to each well of a 24-well plate, add 1 mL of the mixed enzyme solution, seal and incubate at 37℃ (50 rpm) for 2 hours. Remove the incubated dressings, rinse them three times with distilled water, freeze-dry them at -60℃ for 24 hours, weigh the residual dry weight and record it as W1. Calculate the loss rate (results are shown in Table 1). The formula for calculating the loss rate is: weight loss rate (%) = (W0 - W1) / W0 × 100%.
[0068] Table 1: Dressing weight loss rate
[0069] Group <![CDATA[Initial dry weight W0 / mg]]> <![CDATA[Residual dry weight W1 / mg]]> Weight loss rate (%) Example 1 43.2 42.8 0.93％ Example 2 43.6 42.9 1.61％ Example 3 42.9 42.3 1.40％ Comparative Example 1 43.5 40.8 6.21％ Comparative Example 2 43.7 29.6 32.27％ Comparative Example 3 43.1 41.7 3.25％ Comparative Example 4 43.4 42.5 2.07％
[0070] As shown in Table 1, the dressings prepared by the methods in Examples 1-3 had low weight loss rates, less than 2%, while Comparative Example 2, which did not involve cross-linking the recombinant collagen, had a loss rate exceeding 30%. This indicates that cross-linking recombinant collagen with polyvinyl alcohol can effectively protect the recombinant collagen from protease degradation. The slightly higher weight loss rate of Comparative Example 1 compared to the examples is because the recombinant collagen was not modified with chitosan, indicating that chitosan modification has a protective effect on the recombinant collagen to some extent. The slightly higher weight loss rate of Comparative Example 3 compared to the examples is because silver nanoparticles were not added, indicating that silver nanoparticles may inhibit the activity of metalloproteinases to some extent.
[0071] Experimental Example 2
[0072] In vitro antibacterial test:
[0073] Cut the above dressings into 1cm × 1cm squares and place them in a 24-well plate. Mix the dressings with 1mL of Staphylococcus aureus bacterial suspension (10... 6The bacterial culture (CFU / mL) was co-cultured for 12 h. After serial dilution, the culture was plated, with the untreated bacterial culture serving as the control group. The inhibition rate was calculated (results are shown in Table 2). The inhibition rate was calculated using the formula: Inhibition rate (%) = (Number of bacteria in the control group - Number of bacteria in the experimental group) / Number of bacteria in the control group × 100%.
[0074] Table 2: Antibacterial rate of dressings
[0075] Group Bacterial count / CFU Antibacterial rate (%) control group <![CDATA[8.2×10 5 ]]> 0 Example 1 <![CDATA[1.8×10 4 ]]> 97.8 Example 2 <![CDATA[2.5×10 4 ]]> 97.0 Example 3 <![CDATA[3.1×10 4 ]]> 96.2 Comparative Example 1 <![CDATA[8.4×10 4 ]]> 89.8 Comparative Example 2 <![CDATA[7.5×10 4 ]]> 90.9 Comparative Example 3 <![CDATA[2.5×10 5 ]]> 69.5 Comparative Example 4 <![CDATA[8.1×10 4 ]]> 90.1
[0076] As shown in Table 2, the antibacterial rates of the dressings prepared by the methods in Examples 1-3 are all above 95%, while the antibacterial rates of the dressings in Comparative Examples 1-4 are all lower than those in Examples 1-3. Among them, the antibacterial rate of Comparative Example 3 is less than 70%, which may be because the dressing does not contain nano-silver, indicating that nano-silver plays an important role in the antibacterial effect of the dressing.
[0077] Experimental Example 3
[0078] Mouse model of full-thickness skin defect:
[0079] Seventy Kunming mice were divided into seven groups, labeled and grouped according to Examples 1-3 and Comparative Examples 1-4. All seventy mice were anesthetized by intraperitoneal injection of 2% sodium pentobarbital (45 mg / kg). The backs were shaved and disinfected. After fixation, a 1 cm square incision was marked on the back, parallel to the spine. Full-thickness skin was excised along the marked line, down to the fascia layer, to form a wound. The dressings from Examples 1-3 and Comparative Examples 1-4 were aseptically cut into 1 cm squares. 900 μL of sterile saline was dripped onto the surface of each dressing, incubated at 37°C for 3 minutes, and then fixed to the mouse skin incision. The wound area of each mouse was measured on day 21 (e.g., ...). Figure 1 (As shown in the figure). The statistical results are shown in Table 3.
[0080] Table 3: Statistics on wound healing rate in mice
[0081] Group Wound healing rate on day 21 / % Example 1 99.63±0.22 Example 2 99.14±0.18 Example 3 99.27±0.24 Comparative Example 1 91.32±0.32 Comparative Example 2 90.15±0.27 Comparative Example 3 93.36±0.38 Comparative Example 4 94.23±0.29
[0082] like Figure 1 As shown in Table 3, the wound healing rate of mice using dressings from Examples 1-3 exceeded 99% after 21 days, while the healing rate of mice using dressings from Comparative Examples 1-3 was less than 95%, which was much lower than that of Examples 1-3, especially Comparative Example 2, which had the lowest rate of only 90%. This indicates that the dressing obtained by cross-linking recombinant collagen and polyvinyl alcohol has a better wound healing effect.
[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing a recombinant collagen wound dressing, characterized in that, Includes the following steps: S1. A recombinant collagen solution is mixed with a chitosan solution to obtain a recombinant collagen mixture. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is added to the recombinant collagen mixture to form a stable composite system. The modified recombinant collagen solution is obtained by dialysis using a dialysis bag. S2. Take the modified recombinant collagen solution, add polyvinyl alcohol solution and nano silver solution, sonicate, add genipin solution, stir in the dark to obtain recombinant collagen gel solution; S3. Soak the nonwoven fabric in an ethanol solution, rinse it with distilled water, immerse it in a polyethylene glycol solution, treat it with constant temperature shaking, rinse it with distilled water, and vacuum dry it to obtain the modified nonwoven fabric. S4. Lay the modified nonwoven fabric flat on a horizontal glass plate, take the recombinant collagen gel solution, coat it evenly on the surface of the modified nonwoven fabric with a coater, place it in a constant temperature incubator for incubation, and allow it to crosslink in the dark to obtain the modified nonwoven dressing. The modified nonwoven dressing was immersed in PBS solution, then removed and freeze-dried under vacuum, and sterilized with gamma rays to obtain recombinant collagen wound dressing. The recombinant collagen is recombinant humanized type III collagen, and the recombinant collagen solution includes recombinant collagen and PBS solution. In S1, the volume ratio of the recombinant collagen solution to the chitosan solution is (9~22):4; the mass-to-volume ratio of the mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and recombinant collagen is (0.08~0.12) g:130 mL; the dialysis bag specification is 10 kDa; and the dialysis time is 40~48 h.
2. The method for preparing a recombinant collagen wound dressing according to claim 1, characterized in that, In S1, the mass-to-volume ratio of the recombinant collagen to the PBS solution is (0.8~1.2) g:50 mL; the chitosan solution comprises chitosan and a 1% (v / v) aqueous acetic acid solution, the mass-to-volume ratio of the chitosan to the 1% (v / v) aqueous acetic acid solution is (0.8~1.2) g:50 mL, and the pH of the chitosan solution is adjusted to 6.7~6.9 with NaOH.
3. The method for preparing a recombinant collagen wound dressing according to claim 1, characterized in that, In S2, the polyvinyl alcohol solution is an aqueous solution with a mass concentration of 0.8% to 1.2%, and the volume ratio of the polyvinyl alcohol solution to the modified recombinant collagen solution is (0.9 to 1.1):
1.
4. The method for preparing a recombinant collagen wound dressing according to claim 1, characterized in that, In S2, the concentration of the nano-silver solution is 1 mg / mL, and the volume ratio of the nano-silver solution to the modified recombinant collagen solution is (0.8~1.2):10; the ultrasonic treatment power is 150~250W, and the time is 4~6 min; the genipin solution is a DMSO solution containing 2% genipin by mass, and the volume ratio of the genipin solution to the modified recombinant collagen solution is (0.8~1.2):20; the light-protected stirring speed is 80~120 rpm, and the time is 20~40 min.
5. The method for preparing a recombinant collagen wound dressing according to claim 1, characterized in that, In step S3, the ethanol solution is an aqueous solution with a volume concentration of 70%~80%; the soaking time is 20~40 min; the polyethylene glycol solution is an aqueous solution of polyethylene glycol 6000 with a mass concentration of 1.5%~2.5%; the constant temperature oscillation temperature is 35~40℃, the rotation speed is 80~120 rpm, and the time is 1~2 h; the vacuum drying temperature is 50~70℃, and the time is 2~4 h.
6. The method for preparing a recombinant collagen wound dressing according to claim 1, characterized in that, In S4, the coating thickness is 0.1~0.5mm; the incubation temperature is 35~40℃ and the time is 20~28h; the immersion time in PBS solution is 25~35min; the vacuum freeze-drying temperature is -55℃~-70℃ and the time is 20~28h; and the γ-ray sterilization dose is 20~30kGy.
7. A recombinant collagen wound dressing, characterized in that, The preparation method according to any one of claims 1 to 6 is used.