Parallel silk scaffold with anti-inflammatory activity, preparation method and application

Abstract

The present invention discloses a parallel silk scaffold with anti-inflammatory activity, a preparation method and an application thereof, belonging to the field of biomedicine technology. The method comprises the following steps: using mulberry silk as a raw material, degumming, dissolving, dialysis and centrifuging to obtain a silk fibroin solution; mixing polyvinyl alcohol and the silk fibroin solution, adding Klotho protein, then ultrasonicating, drying, water-dissolving and centrifuging the mixed solution to obtain silk fibroin nanospheres loaded with Klotho protein; mixing the Klotho protein-loaded silk fibroin nanospheres with the silk fibroin solution in proportion, and subjecting the mixture to directionally freezing, freeze-drying, cross-linking and re-freeze-drying to obtain a Klotho protein-loaded nanosphere-combined parallel silk sponge scaffold. The composite scaffold constructed by the present invention has a parallel topological structure and excellent mechanical strength; and has nanospheres with drug loading and continuous drug release properties, providing a drug sustained-release system for damaged tissue areas, and has a certain effect on alleviating inflammatory reactions and promoting the repair of tissues such as tendons and ligaments.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a parallel silk scaffold with anti-inflammatory activity, a preparation method and an application thereof. Background Art

[0002] Tendon injuries are common sports-related illnesses, affecting tens of millions of people. Currently, there is a lack of effective treatment options for these injuries. Tendons have poor autologous repair capabilities after injury, often forming pathological repairs such as fibrous scarring. This results in weaker tendon stress than native tendons and a high rate of secondary injury, severely impacting patients' quality of life and athletes' careers. Therefore, repairing tendon injuries is a significant challenge.

[0003] Currently, common clinical treatments for tendon injuries, such as surgical suturing, autologous transplantation, allografts, xenografts, and prosthetic devices, are designed to maintain function and alleviate symptoms, but are not effective in promoting tendon tissue repair and regeneration. Furthermore, these methods have significant limitations, such as donor site lesions, chronic inflammation, rejection, and functional impairment. Therefore, developing methods that can effectively promote tendon repair is of paramount clinical significance and application value.

[0004] Tissue engineering and regenerative medicine, one of the most progressive and challenging frontiers in medical life sciences in recent years, offer new hope for the repair and regeneration of these difficult-to-heal tissues. However, ideal tissue engineering scaffolds for the repair and regeneration of these difficult-to-heal tissues, such as tendons, are currently lacking.

[0005] Silk fibroin, a natural polymeric protein, is considered a promising material for tissue regeneration due to its widespread availability, low cost, good biocompatibility, excellent mechanical properties, and controllable degradation. Previous researchers have used silk fibroin to construct parallel scaffolds with tendon-inspired structures, achieving some therapeutic results, but the repair effects still fall short of the required tendon motor function.

[0006] The host's inflammatory response is a key factor in determining the success of tendon repair and regeneration following biomaterial implantation. Persistent inflammation can lead to tendon fibrosis and adhesive scar formation, impairing tissue function and increasing the risk of secondary injury. Therefore, regulating the inflammatory microenvironment is a crucial component of tendon tissue engineering. However, currently, there are no tissue engineering scaffolds that promote tendon repair through controlled drug release to modulate the inflammatory response. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a parallel silk scaffold with anti-inflammatory activity, a preparation method and application. The scaffold is made of Klotho protein-loaded nanoparticles (Klotho@NS) and silk fibroin through directional freezing and cross-linking reaction. It can regulate the local inflammatory microenvironment of the tissue and effectively promote high-quality repair and regeneration of damaged tissue.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] A method for preparing a parallel silk scaffold, characterized by comprising the following steps:

[0010] Mulberry silk is degummed, dissolved, dialyzed and centrifuged to obtain a silk fibroin solution;

[0011] After mixing the silk fibroin solution, polyvinyl alcohol and Klotho protein in proportion, the mixture is sonicated, dried, dissolved in water and centrifuged to obtain a Klotho protein-loaded nanoparticle solution;

[0012] The silk fibroin solution is mixed with a Klotho protein-loaded nano-microsphere solution, and after directionally freezing, freeze-drying, cross-linking, and freeze-drying again, a parallel silk scaffold with Klotho protein-loaded nano-microspheres is obtained.

[0013] Furthermore, in the mixed solution of silk fibroin, polyvinyl alcohol and Klotho protein, the concentration of the silk fibroin solution is 5% (w / v).

[0014] Furthermore, in the mixed solution of silk fibroin, polyvinyl alcohol and Klotho protein, the concentration of the polyvinyl alcohol solution is 5% (w / v).

[0015] Furthermore, in the mixed solution of silk fibroin, polyvinyl alcohol and Klotho protein, the concentration of Klotho protein is 10 μg / ml.

[0016] Furthermore, in the mixed solution of silk fibroin, polyvinyl alcohol and Klotho protein, the volume ratio of the silk fibroin solution to the polyvinyl alcohol solution is 1:4.

[0017] Furthermore, in the mixed solution of silk fibroin and Klotho protein-loaded nanoparticles, the concentration of the silk fibroin solution is 5% (w / v).

[0018] Furthermore, in the mixed solution of silk fibroin and Klotho protein-loaded nanospheres, the volume ratio of the silk fibroin solution to the Klotho protein-loaded nanospheres solution is 3:1.

[0019] The directional freezing process is to place the mixed solution of the silk fibroin and Klotho protein-loaded nanospheres in a polystyrene cylindrical tube, place it on a pre-cooled aluminum plate, wrap it with a sponge, and place it in a -80°C refrigerator overnight. The cross-linking process is to solidify it by covalent cross-linking with 90% methanol.

[0020] Beneficial effects of the present invention:

[0021] The silk scaffold with anti-inflammatory activity, made from Klotho@NS and silk fibroin, is a sustained-release system for the continuous delivery of Klotho protein. It can significantly reduce the inflammatory response around damaged tissue and promote high-quality tissue repair and regeneration.

[0022] The invention prepares a sustained-release anti-inflammatory drug using silk fibroin as a carrier. The silk fibroin has good biocompatibility and can be degraded in the body.

[0023] The tissue repair scaffold with anti-inflammatory activity prepared by the present invention has a parallel tendon bionic structure after directional freezing and cross-linking reaction, which can better meet the biological performance requirements of tendon tissue repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] Figure 1 This is an electron microscopic photograph of the parallel silk scaffold with anti-inflammatory activity in Example 1 of the present invention;

[0026] Figure 2 This is a graph showing the degradation performance of the parallel silk scaffold with anti-inflammatory activity in Example 1 of the present invention;

[0027] Figure 3 These are H&E staining images of rat Achilles tendon injury repaired 8 weeks after injury in Example 2 of the present invention (Figures a and b are the control group and the Klotho@NS composite scaffold group, respectively);

[0028] Figure 4 These are immunohistochemical staining images of rat Achilles tendon injury repaired 8 weeks after Example 2 of the present invention (Figures a and b are TNF-α staining of the control group and the Klotho@NS composite scaffold group, respectively; Figures c and d are IL-1β staining of the control group and the Klotho@NS composite scaffold group, respectively);

[0029] Figure 5 This is a general morphological diagram of the parallel silk scaffold with anti-inflammatory activity in Example 3 of the present invention. DETAILED DESCRIPTION

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0031] A parallel silk scaffold with anti-inflammatory activity, preparation method and application,

[0032] Example 1

[0033] A parallel silk scaffold with a tendon-like bionic structure is prepared by directional freezing and cross-linking of silk fibroin. The preparation method comprises the following steps:

[0034] (1) Preparation of silk fibroin solution:

[0035] a) Degumming: 10 g of mulberry silk (grade 6A) was placed in 4 L of 0.02 M sodium carbonate aqueous solution, incubated at 100°C in a water bath for 30 min, and then washed with 2 L of pure water by shaking. This process was repeated three times to remove the sericin and leave the fibroin. The fibroin was then dried at 60°C to obtain 7 g of dried fibroin, which was set aside.

[0036] b) Dissolution: The dried silk fibroin was dissolved in 9.3M lithium bromide aqueous solution at 20% (w / v) and heated at 60°C for 1 hour until the silk fibroin was dissolved to obtain a mixture containing silk fibroin and a small amount of insoluble particles.

[0037] c) Dialysis: The mixed solution was dialyzed using a regenerated cellulose dialysis bag (molecular weight cut-off 12,000-14,000 Daltons) against sterile pure water (10 times the volume of the mixed solution) six times over three days to remove lithium bromide ions from the solution and obtain a retentate;

[0038] d) Centrifugation: The dialyzed silk fibroin solution was centrifuged twice at 9000 rpm for 20 min at 4°C to remove impurities in the solution;

[0039] e) freeze-drying the retentate at -45°C and 20 Pa for 48 hours and storing at -80°C for later use;

[0040] (2) Preparation of a parallel silk scaffold with a tendon-inspired bionic structure: The high-concentration silk fibroin solution prepared by the method (1) was diluted with sterile pure water to a final concentration of 6% (w / v) and placed in a polystyrene cylindrical tube (12 mm in diameter × 52 mm in height).

[0041] a) Directional freezing: A pre-cooled aluminum plate (-80°C) was placed at the bottom of the cylindrical tube. The cylindrical tube was wrapped with a sponge and placed in a -80°C freezer overnight to control the growth direction of ice crystals in the mixed solution and form parallel functionalized silk scaffolds.

[0042] b) Freeze-drying, cross-linking, and re-freeze-drying: The frozen scaffold is moved to a freeze dryer, freeze-dried for 24 hours, and then cross-linked with 90% methanol; finally, the scaffold is freeze-dried again to obtain a simple parallel silk scaffold, that is, a parallel silk scaffold with a tendon-inspired structure.

[0043] Example 2

[0044] A nanoparticle (Klotho@NS) with anti-inflammatory activity is prepared by phase separation of Klotho protein, PVA solution, and silk fibroin solution. The preparation method comprises the following steps:

[0045] (1) Preparation of silk fibroin solution:

[0046] a) Degumming: 10 g of mulberry silk (grade 6A) was placed in 4 L of 0.02 M sodium carbonate aqueous solution, incubated at 100°C in a water bath for 30 min, and then washed with 2 L of pure water by shaking. This process was repeated three times to remove the sericin and leave the fibroin. The fibroin was then dried at 60°C to obtain 7 g of dried fibroin, which was set aside.

[0047] b) Dissolving: Dissolving the dried silk fibroin at 20% (w / v) in a 9.3 M lithium bromide aqueous solution, heating at 60° C. for 1 hour until the silk fibroin is dissolved, to obtain a mixture containing the silk fibroin and a small amount of insoluble particles;

[0048] c) Dialysis: The mixed solution was dialyzed using a regenerated cellulose dialysis bag (molecular weight cut-off 12,000-14,000 Daltons) against sterile pure water (10 times the volume of the mixed solution) six times over three days to remove lithium bromide ions from the solution and obtain a retentate;

[0049] d) Centrifugation: The dialyzed silk fibroin solution was centrifuged twice at 9000 rpm for 20 min at 4°C to remove impurities in the solution;

[0050] e) freeze-drying the retentate at -45°C and 20 Pa for 48 hours and storing at -80°C for later use;

[0051] (2) Preparation of nanoparticles (Klotho@NS) with anti-inflammatory activity:

[0052] a) Mixing: Dilute the high-concentration silk fibroin solution prepared in method (1) with sterile pure water to a final concentration of 5% (w / v). Add the silk fibroin solution and 4 times the volume of an equal-concentration PVA solution to a 15 ml centrifuge tube, mix thoroughly, and then add 10 μg / ml Klotho protein. Keep the mixture away from light throughout the preparation process.

[0053] b) Ultrasound: Place the mixed solution at least 1 cm below the ultrasonic probe, move the probe up and down, and sonicate at 25% amplitude for 30 seconds;

[0054] c) Drying: Pour the sonicated solution evenly into a culture dish, covering the entire bottom of the dish, and place in a fume hood to dry overnight.

[0055] d) Dissolution: Add 4 times the volume of pure water to the above culture dish, mix well with a pipette, and shake on a shaker at room temperature for 30 minutes;

[0056] e) Centrifugation: The dissolved liquid is centrifuged at 11,000 rpm and 4°C for 20 minutes. After discarding the supernatant, an equal volume of pure water to the original silk fibroin solution is added and the mixture is stored at 4°C until use. Klotho protein-loaded nanoparticles are obtained, i.e., nanoparticles with anti-inflammatory activity.

[0057] Example 3

[0058] A parallel silk scaffold with anti-inflammatory activity is prepared by directional freezing and cross-linking reaction of nano-microspheres loaded with Klotho protein and silk fibroin. The preparation method comprises the following steps:

[0059] (1) Preparation of silk fibroin solution:

[0060] a) Degumming: 10 g of mulberry silk (grade 6A) was placed in 4 L of 0.02 M sodium carbonate aqueous solution, incubated at 100°C in a water bath for 30 min, and then washed with 2 L of pure water by shaking. This process was repeated three times to remove the sericin and leave the fibroin. The fibroin was then dried at 60°C to obtain 7 g of dried fibroin, which was set aside.

[0061] b) Dissolution: The dried silk fibroin was dissolved in 9.3M lithium bromide aqueous solution at 20% (w / v) and heated at 60°C for 1 hour until the silk fibroin was dissolved to obtain a mixture containing silk fibroin and a small amount of insoluble particles.

[0062] c) Dialysis: The mixed solution was dialyzed using a regenerated cellulose dialysis bag (molecular weight cut-off 12,000-14,000 Daltons) against sterile pure water (10 times the volume of the mixed solution) six times over three days to remove lithium bromide ions from the solution and obtain a retentate;

[0063] d) Centrifugation: The dialyzed silk fibroin solution was centrifuged twice at 9000 rpm for 20 min at 4°C to remove impurities in the solution;

[0064] e) freeze-drying the retentate at -45°C and 20 Pa for 48 hours and storing at -80°C for later use;

[0065] (2) Preparation of Klotho protein-loaded nanoparticles (Klotho@NS):

[0066] a) Mixing: Dilute the high-concentration silk fibroin solution prepared in method (1) with sterile pure water to a final concentration of 5% (w / v). Add the silk fibroin solution and 4 times the volume of an equal-concentration PVA solution to a 15 ml centrifuge tube, mix thoroughly, and then add 10 μg / ml Klotho protein. Keep the mixture away from light throughout the preparation process.

[0067] b) Ultrasound: Place the mixed solution at least 1 cm below the ultrasonic probe, move the probe up and down, and sonicate at 25% amplitude for 30 seconds;

[0068] c) Drying: Pour the sonicated solution evenly into a culture dish, covering the bottom of the dish, and place in a fume hood to dry overnight;

[0069] d) Dissolution: Add 4 times the volume of pure water to the above culture dish, mix well with a pipette, and shake on a shaker at room temperature for 30 minutes;

[0070] e) Centrifugation: Centrifuge the above-mentioned dissolved liquid at 11000 rpm and 4°C for 20 min, discard the supernatant, add pure water in an amount equal to the volume of the original silk fibroin solution, and store at 4°C for later use.

[0071] (3) Preparation of a parallel silk scaffold with anti-inflammatory activity: The high-concentration silk fibroin solution prepared by method (1) was diluted with sterile purified water to a final concentration of 5% (w / v). The silk fibroin solution was mixed with the Klotho@NS prepared in method (2) at a volume ratio of 3:1 and placed in a polystyrene cylindrical tube (12 mm diameter × 52 mm height).

[0072] a) Directional freezing: A pre-cooled aluminum plate (-80°C) was placed at the bottom of the cylindrical tube. The cylindrical tube was wrapped with a sponge and placed in a -80°C freezer overnight to control the growth direction of ice crystals in the mixed solution and form parallel functionalized silk scaffolds.

[0073] b) Freeze-drying, cross-linking, and re-freeze-drying: The frozen scaffold was moved to a freeze dryer, freeze-dried for 24 hours, and then cross-linked with 90% methanol; finally, the scaffold was freeze-dried again to obtain a parallel silk scaffold composited with Klotho@NS, a tissue repair scaffold with anti-inflammatory activity ( Figure 5The freeze-dried scaffold showed a parallel arrangement of biomimetic structures under an electron microscope ( Figure 1 ) and showed excellent controllable degradation performance ( Figure 2 Compared with traditional parallel silk fibroin scaffolds, the silk scaffold with anti-inflammatory activity prepared by the present invention, after assembling NS loaded with Klotho protein, forms a sustained-release system for the continuous delivery of Klotho protein, significantly reducing the inflammatory response around damaged tissue and promoting high-quality tissue repair and regeneration.

[0074] Example 4

[0075] A parallel silk scaffold with anti-inflammatory activity is prepared by directional freezing and cross-linking of Klotho protein-loaded nanoparticles and silk fibroin. The method for using the parallel silk scaffold in Achilles tendon injury includes the following steps:

[0076] (1) Rat Achilles tendon injury model: Nine adult female SD rats (weighing 200-220 g) were anesthetized with sodium pentobarbital by intraperitoneal injection. A 1-cm longitudinal incision was made at the heel, and the skin and fascia were cut layer by layer to expose the Achilles tendon. The Achilles tendon was cut in the middle to create a 6-mm long defect window.

[0077] (2) Application of the parallel silk scaffold of composite Klotho@NS: The parallel silk scaffold of composite Klotho@NS in Example 3 (6 mm in length x 2 mm in width x 1 mm in height) was sutured together with suture thread at the defect window created in (1); the control group used the parallel silk scaffold of Example 1 (6 mm in length x 2 mm in width x 1 mm in height) and suture thread at the defect window created in (1); then the muscles and skin were sutured layer by layer. After the operation, the rats were fed with standard animal feed and moved freely.

[0078] Evaluation of repair effect: The animals were sacrificed 8 weeks after surgery and samples were collected for histological evaluation. Figure 3 Histological staining showed that compared with the control group ( Figure 3 a) In the Klotho@NS composite scaffold group, the proportion of immune cells was lower and the proportion of fibroblast-like cells was higher. The cell morphology and matrix secretion were closer to natural tendon tissue, and the tendon injury site had a better repair effect ( Figure 3 b) Immunohistochemical staining Figure 4 The results showed that compared with the control group ( Figure 4 a, c), the expression of pro-inflammatory factors TNF-α and IL-1β in the Klotho@NS composite scaffold group was significantly reduced, indicating that the Klotho composite scaffold can effectively inhibit tendon inflammation and promote tendon injury repair ( Figure 4 b, d).

[0079] The present invention selects membrane-bound Klotho as an anti-inflammatory factor and optimizes the bionic parallel silk scaffold of the tendon. Using silk materials, while maintaining the topological structure of the tendon, combined with Klotho protein, a composite scaffold material with the function of reducing tendon inflammatory response is constructed to promote high-quality regeneration and repair of tendons, which has extremely important clinical significance and application value.

[0080] The composite scaffold constructed by the present invention has a parallel topological structure and excellent mechanical strength; it also has nano-microspheres with drug loading and continuous drug release properties, providing a drug sustained-release system for the damaged tissue area, which can effectively reduce the inflammatory response and promote the repair of tissues such as tendons and ligaments. The developed composite scaffold uses silk fibroin as a carrier, has good biocompatibility, strong mechanical properties, and good drug sustained-release properties, providing a new type of cell-free tissue engineering scaffold for the clinical treatment of tissue damage, and has broad clinical application prospects.

[0081] Throughout this specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0082] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention, and such changes and modifications fall within the scope of the invention as claimed.

Claims

1. A method for preparing a parallel silk scaffold, characterized in that: The parallel silk scaffold is used to prepare a tendon injury repair material, and the method comprises the following steps: Mulberry silk is degummed, dissolved, dialyzed and centrifuged to obtain a silk fibroin solution; After mixing the silk fibroin solution, polyvinyl alcohol solution and Klotho protein in proportion, the mixture is sonicated, dried, dissolved in water and centrifuged to obtain a Klotho protein-loaded nanoparticle solution; The silk fibroin solution is mixed with the Klotho protein-loaded nanoparticle solution, and after directionally freezing, freeze-drying, cross-linking, and freeze-drying again, a parallel silk scaffold with Klotho protein-loaded nanoparticles is obtained.

2. The method for preparing a parallel silk scaffold according to claim 1, characterized in that: In the mixed solution of silk fibroin solution, polyvinyl alcohol solution and Klotho protein, the concentration of the silk fibroin solution is 5%, w / v.

3. The method for preparing a parallel silk scaffold according to claim 1, wherein: In the mixed solution of silk fibroin solution, polyvinyl alcohol solution and Klotho protein, the concentration of polyvinyl alcohol solution is 5%, w / v.

4. The method for preparing a parallel silk scaffold according to claim 1, wherein: In the mixed solution of silk fibroin solution, polyvinyl alcohol solution and Klotho protein, the concentration of Klotho protein is 10 μg / ml.

5. The method for preparing a parallel silk scaffold according to claim 1, wherein: In the mixed solution of silk fibroin solution, polyvinyl alcohol solution and Klotho protein, the volume ratio of the silk fibroin solution to the polyvinyl alcohol solution is 1:

4.

6. The method for preparing a parallel silk scaffold according to claim 1, characterized in that: In the mixed solution of the silk fibroin solution and the Klotho protein-loaded nanoparticles, the concentration of the silk fibroin solution is 5%, w / v.

7. The method for preparing a parallel silk scaffold according to claim 1, characterized in that: In the mixed solution of the silk fibroin solution and the Klotho protein-loaded nanospheres, the volume ratio of the silk fibroin solution to the Klotho protein-loaded nanospheres solution is 3:

1.

8. The method for preparing a parallel silk scaffold according to claim 1, characterized in that: The directional freezing process is to place the silk fibroin solution and the mixed solution of Klotho protein nanoparticles in a polystyrene cylindrical tube, place it on a pre-cooled aluminum plate, wrap it with a sponge, and place it in a -80°C refrigerator overnight; The cross-linking process is to solidify 90% methanol through covalent cross-linking.

9. The silk scaffold prepared by the preparation method according to any one of claims 1 to 8.

Images