Silk fibroin-hyaluronic acid composite hydrogel as well as preparation method and application thereof

Abstract

The invention discloses silk fibroin-hyaluronic acid composite hydrogel as well as a preparation method and application thereof, and belongs to the technical field of tissue engineering. Fibroin obtained through degumming is dissolved, a silk fibroin solution is obtained through dialysis and purification, the prepared silk fibroin and hyaluronic acid serve as raw materials, 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and 2-morpholinoethanesulfonic acid (MES) serve as cross-linking agents, and the composite hydrogel material is prepared. The degradable plastic has good mechanical property and swelling property, and is relatively slow to degrade. Meanwhile, the hydrogel has good biocompatibility, and cell proliferation can be promoted; an in-vitro hBMSC chondrogenic differentiation induction experiment shows that the HBMSC has chondrogenic induction capability. The silk fibroin-hyaluronic acid composite hydrogel disclosed by the invention can be used for repairing articular cartilage injury.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and in particular to a silk fibroin-hyaluronic acid composite hydrogel and a preparation method and application thereof. Background Art

[0002] Articular cartilage injury is a common orthopedic condition, with an incidence of up to 80% in people over 60 years old. It is primarily caused by trauma, degenerative changes, or inflammatory diseases such as osteoarthritis. Articular cartilage lacks vascularity and innervation, and its ability to repair itself after damage is very limited. Even minor injuries can trigger progressive degeneration, ultimately leading to loss of joint function and persistent pain. While traditional treatments such as microfracture, autologous cartilage transplantation, and joint replacement can alleviate symptoms to some extent, these approaches still have significant limitations. For example, donor sources for autologous cartilage transplantation are limited, and immunogenicity issues can lead to rejection. According to statistics, the 10-year failure rate of joint replacement is as high as 10%-15%. Therefore, the development of novel biomaterials to promote cartilage regeneration and repair has become a research hotspot in biomedical engineering. Currently, the treatment of cartilage injury remains a challenging task. Recent cartilage repair methods, including cartilage pericardium, scaphoid fracture, allograft, and cartilage pericardium xenograft transplantation, have failed to provide complete and durable healing. Therefore, cartilage regeneration remains a hurdle in tissue engineering, although research into potential artificial cartilage substitutes continues as regenerative strategies continue.

[0003] Hydrogels are biomaterials with three-dimensional network structures that have attracted attention for their ability to closely mimic the structure and composition of human tissue. Studies have shown that hydrogels can have water contents exceeding 90%, similar to the water content of natural cartilage (70%-80%). Furthermore, they possess excellent biocompatibility, tunable mechanical properties, and biomimetic extracellular matrix (ECM) and drug-loading properties, making them ideal cartilage replacement materials. In recent years, with the rapid development of biomaterials, tissue regeneration, and medical applications, research on the application of hydrogels in cartilage repair has made significant progress. Their functions have expanded from simple structural support to include active regulation of cellular behavior (such as promoting chondrocyte proliferation and differentiation), delivery of bioactive factors (such as TGF-β and BMP-2), and response to dynamic microenvironments (such as pH and temperature changes). Currently, hydrogel-based tissue engineering is a promising strategy for cartilage defect repair and has been extensively studied. The study of hydrogels as effective therapeutic materials for replacing or regenerating weak articular cartilage has increased the focus on synthetic polymers. To achieve more ideal therapeutic goals, it is necessary to prepare intelligent hybrid hydrogels with complex structures and strengthen in-depth research on their mechanical and biological behavior to ensure that tissue engineering implants possess strong tissue interactions, good absorbability, and a hierarchical structure. While some progress has been made in the application of hydrogels in cartilage repair, issues remain, such as high cost, complex hydrogel preparation processes, insufficient mechanical properties, and limited chondrogenic induction. Silk fibroin and hyaluronic acid have been used to prepare hydrogels in previous studies. Nicole et al. used horseradish peroxidase to crosslink tyrosine-modified hyaluronic acid with silk fibroin to prepare a hydrogel scaffold. Yan Shuqin et al. used freeze-drying to prepare porous silk / hyaluronic acid blend membranes. Compared to chemical crosslinking, enzyme catalytic activity is easily affected by the environment, resulting in low crosslinking efficiency. Furthermore, enzyme crosslinking requires strictly controlled reaction conditions, increasing process complexity. Furthermore, the resulting enzyme-crosslinked network may be weak, resulting in insufficient mechanical properties for the hydrogel. Furthermore, these materials have not been studied for biomedical applications or drug loading. At present, loading drugs into hydrogels and applying them to cartilage repair is a research hotspot in the biomedical field.

[0004] Hydrogels based on natural biomaterials are used in bone tissue engineering and articular cartilage repair and treatment, demonstrating their adaptability to the articular cartilage microenvironment and safety. However, developing hydrogels that simultaneously meet both functional and biocompatible requirements is challenging. Therefore, developing hydrogels that are adaptable to the articular cartilage microenvironment and promote cartilage repair has significant research value and potential. Summary of the Invention

[0005] To address the above technical problems, the present invention provides a silk fibroin-hyaluronic acid composite hydrogel, its preparation method, and its application. The present invention utilizes the excellent mechanical properties and good biocompatibility of silk fibroin. Hyaluronic acid, a component of the extracellular matrix (ECM), has a high affinity for cells and can promote cell proliferation. Therefore, the composite hydrogel material of the present invention combines the advantages of both components and can induce the chondrogenic differentiation of hBMSCs for use in the repair and treatment of articular cartilage damage.

[0006] The first objective of the present invention is to provide a silk fibroin-hyaluronic acid composite hydrogel. This composite hydrogel material exhibits a three-dimensional porous network structure, exhibiting excellent mechanical properties and biocompatibility. This porous network promotes cell growth and proliferation and can also be loaded with drugs. When used for cartilage repair, the composite hydrogel can be injected into the joint cavity to release the drug for therapeutic purposes. The present invention provides a hydrogel material with excellent mechanical properties and biocompatibility.

[0007] Based on the above objectives, the present invention adopts the following technical solutions:

[0008] Step 1: Remove the silkworm cocoons, cut the silkworm pieces into pieces and clean them, and then obtain a regenerated silk fibroin solution with a concentration of 2-10% w / v after degumming, dissolving, dialysis and concentration;

[0009] Step 2: Dissolve a certain amount of hyaluronic acid in pure water by stirring to obtain a 1-10% w / v hyaluronic acid solution;

[0010] Step 3: Mixing the silk fibroin solution and the hyaluronic acid solution in a certain mass ratio, adding a crosslinker, stirring for 15 to 60 minutes, and standing at 4°C for 4 to 12 hours; preferably, the mass ratio of the silk fibroin solution to the hyaluronic acid solution is (12 to 2):3; the crosslinker is 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and 2-morpholinoethanesulfonic acid (MES), with a mass ratio of (3-1):1:1, and the crosslinker accounts for 5 to 50% of the total mass of the composite hydrogel;

[0011] Step 4: Place the mixed solution described in step 3 in a water bath at 37° C. for 5 to 40 minutes to obtain a silk fibroin-hyaluronic acid composite hydrogel.

[0012] The second object of the present invention is to provide a bone damage repair material, comprising a silk fibroin-hyaluronic acid composite hydrogel material.

[0013] The third object of the present invention is to provide the use of the silk fibroin-hyaluronic acid composite hydrogel and / or the cartilage damage repair material containing the silk fibroin-hyaluronic acid composite hydrogel material in the preparation of an injection for treating articular cartilage damage repair.

[0014] The present invention has the following beneficial effects:

[0015] (1) The material used in the present invention is a natural polymer material. By combining silk fibroin and hyaluronic acid, the mechanical properties of the hydrogel can be enhanced, the synergy of biological activity can be improved, and the degradation is controllable, breaking through the limitations of a single natural polymer material. It is particularly suitable for the field of tissue engineering that requires long-term mechanical support and biological activity.

[0016] (2) The raw materials used in the present invention are low in cost, the material preparation steps are simple, and the material is easy to industrially synthesize and produce, and has commercial application value.

[0017] (3) The composite hydrogel of the present invention has good mechanical properties and biocompatibility, and can promote cell growth and proliferation.

[0018] (4) The composite hydrogel of the present invention can induce hBMSC to differentiate into cartilage and has cartilage induction ability, and can be used in bone tissue repair engineering and for the repair and treatment of articular cartilage damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 The scanning electron micrographs 100X (left) and 200X (right) of the silk fibroin-hyaluronic acid composite hydrogel obtained in Example 1 of the present invention are shown;

[0021] Figure 2 is the stress-strain curve of the silk fibroin-hyaluronic acid composite hydrogel obtained in Example 1 of the present invention;

[0022] Figure 3 is the swelling curve of the silk fibroin-hyaluronic acid composite hydrogel obtained in Example 1 of the present invention;

[0023] Figure 4 is the degradation curve of the silk fibroin-hyaluronic acid composite hydrogel obtained in Example 1 of the present invention;

[0024] Figure 5 The toxic effects of the silk fibroin-hyaluronic acid composite hydrogel obtained in Example 1 of the present invention on L929 and hBMSCs;

[0025] Figure 6 The effect of the silk fibroin-hyaluronic acid composite hydrogel obtained in Example 1 of the present invention on the proliferation of L929 and hBMSCs;

[0026] Figure 7 The effect of the silk fibroin-hyaluronic acid composite hydrogel obtained in Example 1 of the present invention on the activity of hBMSCs;

[0027] Figure 8 This is a graph showing the results of Alcian blue staining of the silk fibroin-hyaluronic acid composite hydrogel obtained in Example 1 of the present invention for inducing hBMSC into chondrogenic differentiation in vitro for 7 and 14 days;

[0028] Figure 9 This is a graph showing the results of real-time fluorescence quantitative PCR of the silk fibroin-hyaluronic acid composite hydrogel obtained in Example 1 of the present invention inducing hBMSCs to differentiate into chondrogenic cartilage for 7 and 14 days in vitro;

[0029] Figure 10 The immunoblotting results (top) and band gray value statistics (bottom) of the silk fibroin-hyaluronic acid composite hydrogel obtained in Example 1 of the present invention for inducing hBMSC into chondrogenic differentiation in vitro for 7 and 14 days are shown. DETAILED DESCRIPTION

[0030] The following are specific examples of the present invention, which further describe the technical solutions of the present invention. The described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0031] Example 1.

[0032] Preparation of silk fibroin-hyaluronic acid composite hydrogel.

[0033] Remove the cocoons from the silkworm cocoons, cut them into pieces, and clean them. Degumming is performed by boiling the silkworm flakes in a Na2CO3 solution for 20-60 minutes. After degumming, they are rinsed with pure water and dried at 60°C to obtain degummed silk. Cut the degummed silk into small pieces and place them in a beaker. Add 9.3M LiBr and dissolve at 60°C for 1-6 hours. The dissolved silk fibroin is then dialyzed and purified to obtain a 6% w / v silk fibroin solution. Dissolve a certain amount of hyaluronic acid in pure water with stirring to obtain a 3% w / v hyaluronic acid solution.

[0034] The silk fibroin solution and the hyaluronic acid solution were mixed in a ratio of 8:2 to prepare a mixed solution with a concentration of 5.4% w / v; the crosslinking agents EDC, NHS and MES were added in a ratio of 3:1:1, accounting for 20% of the total mass of the composite hydrogel, and stirred to allow the crosslinker and the material to react for 30 minutes, and then placed in a 4°C refrigerator for 8 hours; then, the gel solution was converted into a gel in a 37°C water bath for 25 minutes to obtain a silk fibroin-hyaluronic acid composite hydrogel.

[0035] Example 2

[0036] Different from Example 1, when the dissolved silk fibroin was dialyzed and purified and a certain mass of hyaluronic acid was dissolved by stirring with pure water, an 8% w / v silk fibroin solution and a 6% w / v hyaluronic acid solution were obtained, respectively. The silk fibroin solution and the hyaluronic acid solution were mixed in a ratio of 2:1 to prepare a mixed solution with a concentration of 7.3% w / v.

[0037] Example 3

[0038] Different from Example 1, when the dissolved silk fibroin was dialyzed and purified and a certain mass of hyaluronic acid was dissolved by stirring with pure water, a 2% w / v silk fibroin solution and a 4% w / v hyaluronic acid solution were obtained, respectively. The silk fibroin solution and the hyaluronic acid solution were mixed in a ratio of 1:1 to prepare a mixed solution with a concentration of 3% w / v.

[0039] Example 4

[0040] Different from Example 2, the mass ratio of the cross-linking agents EDC, NHS and MES is 1:1:1.

[0041] Example 5

[0042] Different from Example 3, the mass ratio of the cross-linking agents EDC, NHS and MES is 2:1:1.

[0043] Taking Example 1 as an example, tests were conducted on micromorphology, mechanical properties, swelling properties, degradation properties, biocompatibility, and in vitro chondrogenic induction ability.

[0044] (1) Microscopic morphology observation of hydrogel

[0045] The composite hydrogel obtained in Example 1 was frozen at -80°C overnight, freeze-dried using a vacuum freeze dryer, and fractured with liquid nitrogen. The cut surface was sprayed with gold to enhance conductivity and observation effect. The sample was attached to the sample stage with conductive glue and observed and photographed using a Czech VAGE-3-SBH scanning electron microscope. Figure 1 Scanning electron micrographs at 100X (left) and 200X (right). The results show that the hydrogel exhibits a three-dimensional porous network structure. This porous network facilitates the exchange of water molecules, drug molecules, nutrients, and metabolites.

[0046] (2) Determination of mechanical properties

[0047] The composite hydrogel obtained in Example 1 was prepared in the size of 10 mm (diameter) × 15 mm (height). The compression mechanical test was carried out at room temperature at a compression deformation rate of 1 mm / min on a CMT-100 electronic universal testing machine. Three replicates were set for each group (tested in a wet state). The results are shown in Figure 2 ,The results showed that the mechanical properties of the composite hydrogel exhibited a compressive strength of about 87KPa at a strain of 60%, ,and had good mechanical properties.

[0048] (3) Swelling properties

[0049] The initial mass of the composite hydrogel after freeze-drying is recorded as W i Subsequently, the cells were placed in PBS (pH = 7.4) at 37°C, taken out at fixed time points (5, 10, 30, 60, 90, 120, 180 min), weighed, and recorded as W. t , until the mass does not change. The swelling ratio SR is calculated according to the following formula:

[0050]

[0051] Three parallel samples were taken for each sample to be measured and statistically analyzed and compared. The experimental results are shown in Figure 3 The results showed that the mass swelling rate of the composite hydrogel when it reached dynamic equilibrium was 569.98±15.12%, which showed good swelling properties, was beneficial to the exchange and transportation of nutrients, and was of great significance to the growth and migration of chondrocytes and the regeneration and repair of articular cartilage damage.

[0052] (4) Degradation performance

[0053] Hyaluronidase was prepared into a 50% concentration solution using a PBS solution with a pH value of 7.4, and sterilized by filtering with a biological filter membrane. The weight of the dried hydrogel was recorded as W0, and sterilized by irradiation under ultraviolet conditions. The dried composite hydrogel was placed in the same volume of PBS and enzyme solution respectively. In a shaker at 37°C and 100rpm, samples were taken at 1, 2, 3, 4, 5, 6, 7 and 8 weeks (fresh PBS and enzyme solution were replaced every 2 days), and the composite hydrogel soaked in the enzyme solution was washed 3 times with pure water, then dried with a vacuum freeze dryer, and the weight after drying (W1) was recorded. The degradation rate W was calculated according to the following formula L :

[0054]

[0055] Three parallel samples were taken for each sample to be measured and statistically analyzed and compared. The experimental results are shown in Figure 4The results showed that the hydrogel had good biodegradability, with the degradation rates in PBS and enzyme solution reaching 51.93±2.040% and 57.60±1.623% on day 56, respectively.

[0056] (5) Biocompatibility

[0057] The composite hydrogel obtained in Example 1 was soaked in PBS and placed in a shaker at 37°C and 100 rpm for 24 hours to obtain extracts of different hydrogel experimental groups. The prepared extract was sterilized by filtering with a 0.22 μm biological filter membrane. Cells were cultured with complete culture medium containing the extract, and the CCK-8 method was used to detect the toxicity, vitality and proliferation effects of the composite hydrogel on L929 and hBMSC, and the AO / PI method was used to detect the effect on the viability of hBMSC. The results showed that there was no significant difference in the toxicity and vitality of the composite hydrogel on cells compared with the blank control ( Figure 5 ), indicating that the composite hydrogel had no cytotoxic effect; in the 3-day proliferation assay, the composite hydrogel had a significant proliferation-promoting effect compared with the blank control on the 3rd day ( Figure 6 ); however, AO / PI staining showed that the composite hydrogel had no effect on cell viability ( Figure 7 ); In summary, the composite hydrogel has good biocompatibility and can promote cell proliferation.

[0058] (6) Study on in vitro chondrogenic induction ability

[0059] The composite hydrogel obtained in Example 1 was soaked in PBS and placed in a shaker at 37°C and 100rpm for 24 hours to obtain extracts of different hydrogel experimental groups. The prepared extract was filtered and sterilized with a 0.22μm biological filter membrane. The hBMSCs were cultured with the induction complete medium containing the extract for 7 days and 14 days, respectively. The uninduced group was used as a negative control, and the induction complete medium containing TGF-β3 was used as a positive control (the uninduced group was cultured for too long, the cells were in poor condition or even died, so there was only a positive control group at 14 days); its chondrogenic induction ability was evaluated macroscopically by Alcian blue staining, its chondrogenic induction ability was studied at the gene level by real-time fluorescence quantitative PCR, and its chondrogenic induction ability was studied at the protein level by immunoblotting. The results showed that the Alcian blue staining area of the composite hydrogel group was deeper than that of the negative control and the positive control ( Figure 8 ), and by RT-qPCR ( Figure 9 ) and Western blot( Figure 10 ) The results showed that the composite hydrogel had obvious chondrogenic induction ability and could promote hBMSC chondrogenic differentiation in a shorter time compared with the positive control.

[0060] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A silk fibroin-hyaluronic acid composite hydrogel material, characterized in that: A composite hydrogel is prepared with silk fibroin and hyaluronic acid as raw materials and EDC, NHS and MES as cross-linking agents. The composite hydrogel has a three-dimensional porous network structure.

2. A method for preparing the silk fibroin-hyaluronic acid composite hydrogel material as claimed in claim 1, characterized in that: The following steps are involved: Step 1: Remove the silkworm cocoons, cut the silkworm pieces into pieces and clean them, and then obtain a regenerated silk fibroin solution with a concentration of 2-10% w / v after degumming, dissolving, dialysis and concentration; Step 2: Dissolve a certain amount of hyaluronic acid in pure water by stirring to obtain a 1-10% w / v hyaluronic acid solution; Step 3: Mix the silk fibroin solution and the hyaluronic acid solution in a ratio of (12-2):3, add the cross-linking agent, stir, and let stand at 4°C for 4-12 hours; Step 4: Place the mixed solution in step 3 in a water bath at 37° C. for 5 to 40 minutes to obtain a silk fibroin-hyaluronic acid composite hydrogel.

3. The silk fibroin-hyaluronic acid composite hydrogel material according to claim 2, characterized in that: The cross-linking agents are EDC, NHS and MES.

4. The preparation method according to claim 3, characterized in that The mass ratio of the cross-linking agent is (3-1):1:1, and the cross-linking agent accounts for 5-50% of the total mass of the composite hydrogel.

5. The preparation method according to claim 2, characterized in that After adding the cross-linking agent, stir for 15 to 60 minutes and then let it stand at 4°C.

6. A cartilage damage repair material, characterized in that: The invention comprises the silk fibroin-hyaluronic acid composite hydrogel material as claimed in claim 1.

7. Use of the silk fibroin-hyaluronic acid composite hydrogel material according to claim 1 in the preparation of an injection for treating articular cartilage damage and repair.

8. Use of the cartilage damage repair material according to claim 6 in the preparation of an injection for treating articular cartilage damage repair.

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