Application of ordered regenerative mechanics biomimetic silk fibroin hydrogel in preparation of dressing for promoting scarless wound repair
By preparing ordered regenerative biomimetic silk fibroin hydrogels that mimic the mechanical properties of salamander skin, the biomimetic problem of the mechanical microenvironment in scarless wound regeneration was solved, achieving ordered regeneration of scarless wound repair and tissue regeneration.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTHERN UNIVERSITY OF SCIENCE AND TECHNOLOGY
- Filing Date
- 2025-09-02
- Publication Date
- 2026-07-03
AI Technical Summary
How to achieve biomimetic design of the mechanical microenvironment to promote orderly regeneration and repair of wounds with both morphology and function, especially in scarless wound regeneration, and realize the biomimetic design of the orderly regenerative mechanical microenvironment.
By preparing ordered regenerative biomimetic silk fibroin hydrogels to simulate the low mechanical properties of salamander skin, and utilizing the cross-linking reaction of methacryloyl fibroin (SFMA) and silk fibroin nanofibers (BSNF), a hydrogel material with a Young's modulus of 0.9–2.7 kPa is formed, which promotes scarless wound repair.
It significantly promotes scarless wound repair, hair follicle and sebaceous gland regeneration, and the extracellular matrix of the newly formed tissue is reconstructed in a state similar to that of normal skin tissue. Its simple composition makes it easy to translate into clinical practice.
Smart Images
Figure CN121102562B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of tissue repair materials technology, and provides the application of ordered regenerative mechanical biomimetic silk fibroin hydrogel in the preparation of dressings that promote scarless wound repair. Background Technology
[0002] Wound regeneration and repair require the synergistic effects of various wound repair-related cells, such as macrophages, fibroblasts, and epidermal cells, as well as extracellular matrix proteins and bioactive factors, both spatially and temporally to ensure orderly repair and complete skin tissue regeneration and functional reconstruction. However, orderly wound regeneration and repair depend on a suitable microenvironment, including appropriate mechanical and signaling molecules. Numerous studies suggest that the mechanical microenvironment influences orderly wound regeneration by altering the signaling molecules of mechanically sensitive cells related to wound repair. It is a crucial factor affecting the regeneration of skin appendages such as hair follicles, sebaceous glands, and sweat glands, and the regeneration of these appendages is a key indicator of scarless wound regeneration. Therefore, how to biomimeticize the mechanical microenvironment to promote orderly regeneration and repair that combines both morphological and functional aspects is a critical scientific problem that urgently needs to be solved in the field of wound regeneration and repair.
[0003] The resting tension and stiffness of the skin of the newt (Ambystomamexicanum), which possesses fully ordered regenerative repair capabilities, are significantly lower than those of mouse and human skin, which exhibit incomplete ordered regeneration. These low mechanical properties of newt skin are a crucial basis for its unique ordered regenerative capacity; these lower skin mechanical properties are defined as the "ordered regenerative mechanical microenvironment." The ordered regenerative mechanical microenvironment is a vital factor in promoting the orderly repair of skin tissue, and how to achieve biomimetic application of this microenvironment to wound regeneration and repair remains a challenging problem in this field. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide the application of ordered regenerative biomimetic silk fibroin hydrogel in the preparation of dressings that promote scarless wound repair.
[0005] To achieve the above objectives, the present invention provides the following technical solution:
[0006] This invention provides the application of ordered regenerative biomimetic silk fibroin hydrogel in the preparation of dressings that promote scarless wound repair. The silk fibroin hydrogel has similar mechanical properties to salamander skin and is obtained through a cross-linking reaction of methacryloyl fibroin SFMA and silk fibroin nanofibers BSNF.
[0007] Preferably, the effective Young's modulus of the silk fibroin hydrogel is 0.9 to 2.7 kPa.
[0008] Preferably, the silk fibroin hydrogel is prepared by the following method:
[0009] (1) Degumming raw silk to obtain degummed silk fibroin;
[0010] (2) Preparation of methacrylamide silk fibroin SFMA: Degummed silk fibroin was dissolved in lithium bromide solution, glycidyl methacrylate (GMA) was added, and the reaction was carried out to obtain SFMA;
[0011] (3) Preparation of silk fibroin nanofiber solution, i.e. BSNF solution: Degummed silk fibroin was dissolved in lithium bromide solution, dialyzed, centrifuged to obtain silk fibroin aqueous solution, concentrated, self-assembled into metastable silk fibroin particles, diluted with deionized water, and incubated to obtain BSNF solution.
[0012] (4) Prepare SFMA solution using deionized water, mix SFMA solution, BSNF solution and photoinitiator aqueous solution evenly to obtain a mixed solution, adjust the pH of the mixed solution to 10.5, pour it into a mold, perform photocrosslinking reaction, and perform post-treatment to obtain the silk fibroin hydrogel material.
[0013] Preferably, the specific method of step (1) is as follows: add 2.5g of raw silkworm silk to 1L of boiling 2.12g / L sodium carbonate solution, continue to boil and stir for 30-60 minutes, take it out and wash it with deionized water 3-5 times, and dry it in an oven at 60℃.
[0014] Preferably, in step (2), the 9.3 mol / L lithium bromide solution is heated to 60°C, degummed silk fibroin is added, and stirred to dissolve it to a concentration of 200 g / L. GMA is then added to make the concentration 424 mmol / L.
[0015] Preferably, in step (2), the reaction conditions are: 60℃ for 3 to 6 hours.
[0016] Preferably, in step (2), after the reaction is completed, the mixture is dialyzed in deionized water for 3 to 8 days using a 12-14 kDa dialysis membrane to remove lithium bromide and unreacted GMA. The mixture is then centrifuged 1 to 2 times to remove impurities and freeze-dried to obtain SFMA.
[0017] Further preferred centrifugation conditions are: 4℃, 9000rpm, 20 minutes.
[0018] Preferably, in step (3), the 9.3 mol / L lithium bromide solution is heated to 60°C, degummed silk fibroin is added, and the solution is stirred to dissolve it. The solution is then dialyzed in deionized water for 3 to 8 days using a 12-14 kDa dialysis membrane to remove lithium bromide. The solution is then centrifuged 1 to 2 times to remove impurities, resulting in a silk fibroin aqueous solution with a mass concentration of 4-6%.
[0019] Preferably, in step (3), the solution is concentrated to 20% of its original volume at 60°C to obtain a concentrated solution, which is then self-assembled into metastable silk fibroin particles. The concentrated solution is then diluted to 2% with deionized water and incubated to obtain a BSNF solution.
[0020] Preferably, in step (3), the incubation conditions are: 60°C sealed incubation until a gel is formed.
[0021] Preferably, in step (4), the photoinitiator aqueous solution is obtained by dissolving the photoinitiator LAP in water; when preparing the mixture, the mass concentration of the SFMA solution used is 10%, the mass concentration of the BSNF solution is 2%, and the mass concentration of the photoinitiator aqueous solution is 0.5%; the volume ratio of SFMA solution, BSNF solution, and photoinitiator aqueous solution is 1:1:1.
[0022] Preferably, in step (4), the pH is adjusted using a 5% sodium hydroxide solution or a 99% triethanolamine solution.
[0023] Preferably, in step (4), the photocrosslinking conditions are: 405nm light source, 10–60 mw / cm². 2 The time is 0.5 to 4 minutes.
[0024] Preferably, in step (4), the post-treatment method is to soak in phosphate buffer (2.0mM KH2PO4, 137mM NaCl, 10.0mM Na2HPO4, 2.7mM KCl, pH 7.4) for 12 to 24 hours.
[0025] The beneficial effects of this invention are:
[0026] This invention provides the application of ordered regenerative biomimetic silk fibroin hydrogel in the preparation of dressings that promote scarless wound repair. First, degummed silk fibroin is used as raw material to prepare methacrylamide silk fibroin and silk fibroin nanofibers, and then SFMA solution and BSNF solution are obtained. Finally, the SFMA solution, BSNF solution and photoinitiator aqueous solution are mixed evenly to obtain a mixed solution. The pH of the mixed solution is adjusted to 10.5, and photocrosslinking is performed.
[0027] The applicant measured the mechanical properties of skin from various animals and determined that the mechanical properties of the skin of the salamander, a species known for its complete and orderly regeneration and repair, constitute the mechanical microenvironment for orderly tissue regeneration. Guided by the mechanical properties of salamander skin, the applicant designed and synthesized a biomimetic silk fibroin hydrogel material for orderly regeneration, which is used to promote scarless wound repair.
[0028] This invention, based on the skin mechanical properties of the salamander, a species known for its fully ordered regeneration, clarifies the scope of the tissue's ordered regenerative mechanical microenvironment and utilizes a hybrid SFMA / BSNF hydrogel to biomimeticize the skin mechanical properties of the salamander, thus preparing an ordered regenerative mechanical biomimetic silk fibroin hydrogel material. In mouse wound healing experiments, compared to the untreated blank control group, the existing SFMA hydrogel group alone, and the mouse skin mechanical biomimetic SFMA / BSNF silk fibroin hydrogel group, the salamander skin mechanical biomimetic SFMA / BSNF silk fibroin hydrogel (i.e., the ordered regenerative mechanical biomimetic silk fibroin hydrogel of this invention) significantly promoted scarless wound repair and significant regeneration of hair follicles and sebaceous glands. Furthermore, the materials used in this invention are of a single composition; both SFMA and BSNF are silk fibroin proteins, making them easier to translate into clinical applications and of great significance for the design and clinical application of wound repair materials.
[0029] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description
[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:
[0031] Figure 1 This diagram illustrates the preparation of ordered regenerative biomimetic silk fibroin hydrogel, comprising five parts: salamander skin mechanical property testing, raw silk degumming process, SFMA preparation process, BSNF preparation process, and ordered regenerative biomimetic silk fibroin hydrogel preparation process.
[0032] Figure 2 To compare the effects of different silk fibroin hydrogels on the repair of dorsal wounds in C57BL / 6J mice. The comparison included: (A) a blank control group, the existing SFMA hydrogel group, mouse skin biomechanical biomimetic SFMA / BSNF silk fibroin hydrogel, and salamander skin biomechanical biomimetic SFMA / BSNF silk fibroin hydrogel on the wound healing rate in mice; (B) H&E staining results; and (C) Masson staining results. Detailed Implementation
[0033] The present invention will be further described below with reference to specific embodiments.
[0034] Example:
[0035] like Figure 1 As shown, the preparation of the ordered regenerative biomimetic silk fibroin hydrogel involves the following specific steps:
[0036] S1. Mechanical Property Testing: Normal skin and scar tissue from salamanders were selected, and the mechanical properties of the skin were tested using a bio-nanoplasty instrument. The Young's modulus of the skin at different stages of wound repair and that of normal skin were compared to provide mechanical guidance for the preparation of ordered regenerative biomimetic silk fibroin hydrogels.
[0037] S2. Raw silk degumming: Add 8.48g of sodium carbonate to 4L of deionized water and boil, stirring until fully dissolved to obtain a sodium carbonate solution with a concentration of 2.12g / L. Add 10g of raw silkworm silk and continue to boil and stir for 30 minutes. Wash with deionized water 5 times to remove sericin from the surface of the raw silk. Dry in an oven at 60℃ to obtain degummed fibroin.
[0038] S3. SFMA Preparation: 20.19 g of lithium bromide was dissolved in deionized water until fully dissolved to obtain a lithium bromide solution with a concentration of 9.3 mol / L. 5 g of degummed silk fibroin was added and dissolved at 60 °C for 1 hour to make the concentration of degummed silk fibroin 200 g / L. 1.5 ml of GMA was added to make the concentration 424 mmol / L. The reaction was carried out at 60 °C for 6 hours. The solution was dialyzed with a 12 kDa dialysis membrane in deionized water for 7 days to remove lithium bromide and unreacted GMA, thus obtaining methacrylamide silk fibroin SFMA.
[0039] Preparation of S4.BSNF: 20.19 g of lithium bromide was dissolved in deionized water until fully dissolved to obtain a lithium bromide solution with a concentration of 9.3 mol / L. 5 g of degummed silk fibroin was added and dissolved at 60 °C for 1 hour. The solution was dialyzed with a 12 kDa dialysis membrane for 3 days to remove lithium bromide and centrifuged twice (4 °C, 9000 rpm, 20 min) to remove impurities, resulting in a silk fibroin aqueous solution with a concentration of 6 wt%. The solution was then slowly concentrated at 60 °C to a mass concentration of 20%. The concentrated solution was diluted with deionized water to 2% and incubated in a sealed oven at 60 °C to obtain a BSNF solution with a mass concentration of 2%.
[0040] S5. Prepare an SFMA solution using deionized water. Mix a 10% (w / w) SFMA solution, a 2% (w / w) BSNF solution, and a 0.5% (w / w) photoinitiator aqueous solution (obtained by dissolving lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in water, or an LAP aqueous solution) at a volume ratio of 1:1:1 to obtain a homogeneous mixture. Pour the mixture into an 8mm mold and adjust the pH of the mixture to 10.5 using a 99% (w / w) triethanolamine solution. Perform a photocrosslinking reaction (405nm light source, 60mw / cm²). 2The sample was soaked in phosphate buffer (2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, 2.7 mM KCl, pH 7.4) for 12 hours to obtain a silk fibroin hydrogel with mechanical properties similar to those of the salamander skin measured in step S1, and an effective Young's modulus of 1.79 ± 0.94 kPa.
[0041] Comparative Example 1
[0042] Steps S1 and S4 are omitted. In step S5, a simple SFMA hydrogel is obtained by photocrosslinking after mixing the SFMA solution with the photoinitiator aqueous solution.
[0043] The rest is the same as in the embodiment.
[0044] Comparative Example 2
[0045] Step S1 selects mouse skin mechanical properties to provide guidance on the mechanical range of silk fibroin hydrogel.
[0046] In step S5, the pH of the mixture is adjusted to 9.2 using a 99% triethanolamine solution.
[0047] The rest is the same as in the example, and a mouse skin biomimetic SFMA / BSNF silk fibroin hydrogel with similar mechanical properties to mouse skin is obtained, with an effective Young's modulus of 14.20±8.99kPa.
[0048] After thoroughly soaking and washing the silk fibroin hydrogels obtained in Examples 1 and 2 with physiological saline, they were applied to the wound and covered with a waterproof film to keep the hydrogel and the wound moist. No dressing changes were required during this period until the excess hydrogel fell off on its own.
[0049] Figure 2This study compared the effects of different silk fibroin hydrogels on the repair of dorsal wounds in C57BL / 6J mice. (A) Compared to the blank control group and the existing SFMA hydrogel-only group (Comparative Example 1), the biomechanical biomimetic SFMA / BSNF silk fibroin hydrogel accelerated the wound healing rate in mice. The salamander skin biomechanical SFMA / BSNF biomimetic silk fibroin hydrogel (i.e., the ordered regenerative biomechanical biomimetic silk fibroin hydrogel obtained in the examples) was superior to the mouse skin biomechanical biomimetic SFMA / BSNF silk fibroin hydrogel (Comparative Example 2). (B) H&E staining results showed that the biomechanical biomimetic SFMA / BSNF silk fibroin hydrogel promoted the regeneration of hair follicles and sebaceous glands in the healed tissue, and the number of regenerated hair follicles and sebaceous glands in the salamander skin biomechanical biomimetic SFMA / BSNF silk fibroin hydrogel group was significantly greater than that in the mouse skin biomechanical biomimetic SFMA / BSNF silk fibroin hydrogel group. (C) Masson staining results showed that, compared with the dense parallel lamellar scar structure of the control group and the SFMA hydrogel group alone, the salamander skin biomechanical biomimetic SFMA / BSNF silk fibroin hydrogel promoted the remodeling of extracellular matrix proteins in the newly formed tissue after healing, forming a loose mesh-like structure similar to the structure of normal skin tissue.
[0050] In summary, ordered regenerative biomimetic silk fibroin hydrogels, which possess mechanical properties similar to those of salamander skin, are more conducive to promoting scarless wound repair. This biomimetic hydrogel can effectively accelerate wound repair in mice. Figure 2 (A) and promotes the regeneration of hair follicles and sebaceous glands in new tissues, whose extracellular matrix remodeling state is similar to that of normal skin tissue. Figure 2 (B)
[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. The use of an ordered regenerative mechanics biomimetic silk fibroin hydrogel in the preparation of a dressing for promoting scarless repair of a wound, characterized in that, The silk fibroin hydrogel, which has similar mechanical properties to salamander skin, was prepared by the following method: (1) Degumming of raw silk to obtain degummed silk fibroin; (2) Preparation of methacrylamide silk fibroin SFMA: Degummed silk fibroin was dissolved in lithium bromide solution, glycidyl methacrylate was added, and the reaction was carried out to obtain SFMA; (3) Preparation of silk fibroin nanofiber solution, i.e. BSNF solution: Degummed silk fibroin was dissolved in lithium bromide solution, dialyzed, centrifuged to obtain silk fibroin aqueous solution, concentrated, self-assembled into metastable silk fibroin particles, diluted with deionized water, and incubated to obtain BSNF solution. (4) Prepare SFMA solution using deionized water, mix SFMA solution, BSNF solution and photoinitiator aqueous solution evenly to obtain a mixed solution, adjust the pH of the mixed solution to 10.5, pour it into a mold, perform photocrosslinking reaction, and then perform post-treatment to obtain the silk fibroin hydrogel material.
2. Use according to claim 1, characterized in that, The effective Young's modulus of the silk fibroin hydrogel is 0.9–2.7 kPa.
3. Use according to claim 1, characterized in that, The specific method for step (1) is as follows: Add 2.5g of raw silkworm silk to 1L of boiling 2.12g / L sodium carbonate solution, continue to boil and stir for 30-60 minutes, take it out and wash it with deionized water 3-5 times, and dry it in a 60℃ oven.
4. Use according to claim 1, characterized in that, In step (2), the 9.3 mol / L lithium bromide solution is heated to 60°C, degummed silk fibroin is added, and it is stirred to dissolve it to a concentration of 200 g / L. GMA is then added to make the concentration 424 mmol / L.
5. The use according to claim 1, characterized in that, In step (2), the reaction conditions are: 60℃ for 3 to 6 hours.
6. Use according to claim 1, characterized in that, In step (2), after the reaction is complete, the mixture is dialyzed in deionized water for 3 to 8 days using a 12-14 kDa dialysis membrane to remove lithium bromide and unreacted GMA. After centrifugation 1 to 2 times to remove impurities, the mixture is freeze-dried to obtain SFMA.
7. The application according to claim 1, characterized in that, In step (3), the 9.3 mol / L lithium bromide solution is heated to 60°C, degummed silk fibroin is added, and the solution is stirred to dissolve it. The solution is then dialyzed in deionized water for 3 to 8 days using a 12-14 kDa dialysis membrane to remove lithium bromide. The solution is then centrifuged 1 to 2 times to remove impurities, resulting in a silk fibroin aqueous solution with a mass concentration of 4-6%.
8. The application according to claim 1, characterized in that, In step (3), the solution is concentrated to 20% of its original volume at 60°C to obtain a concentrated solution, which self-assembles into metastable silk fibroin particles. The concentrated solution is then diluted to 2% with deionized water and incubated to obtain a BSNF solution. The incubation conditions are: 60°C in a sealed environment until a gel is formed.
9. The application according to claim 1, characterized in that, In step (4), the photoinitiator aqueous solution is obtained by dissolving the photoinitiator LAP in water; when preparing the mixture, the mass concentration of the SFMA solution used is 10%, the mass concentration of the BSNF solution is 2%, and the mass concentration of the photoinitiator aqueous solution is 0.5%; the volume ratio of SFMA solution, BSNF solution, and photoinitiator aqueous solution is 1:1:1; Adjust the pH using a 5% sodium hydroxide solution or a 99% triethanolamine solution; The photo-crosslinking conditions are: 405 nm light source, 10-60 mw / cm 2 , time 0.5-4 minutes; The post-treatment method is to soak in phosphate buffer for 12–24 hours.