Hydrogel scaffold for promoting tendon-bone healing

Abstract

The invention discloses a hydrogel scaffold for promoting tendon-bone healing, and belongs to the field of biological medicines. The drug-loaded composite hydrogel scaffold for promoting tendon-bone healing is prepared on the basis of silk fibroin (SF), nano-hydroxyapatite (nHA) and icariin (ICA) by adopting a photocuring cross-linking technology, and the hydrogel scaffold prepared by the invention is beneficial to tendon-bone repair, has excellent biological properties, controllable drug release ability and good mechanical properties, and can be used for preparing a drug-loaded hydrogel scaffold for promoting tendon-bone healing. While the tendon-bone interface repair is promoted, a feasible method is provided for the repair of skeletal muscle disease injuries caused by senile diseases, sports injuries and occupational diseases, and the method has wide application prospects in clinic.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a hydrogel scaffold for promoting tendon-bone healing. Background Art

[0002] Tendon-bone injuries are a typical symptom of musculoskeletal disorders, widely present in conditions such as degenerative diseases of the elderly, sports injuries, and occupational injuries. Their high incidence and difficulty in treatment make them a significant health issue affecting social labor capacity and the allocation of medical resources, severely impacting patients' quality of life and placing a heavy burden on the healthcare system. Due to the complex anatomical structure of the bone-tendon-bone interface, efficient tendon-bone healing, improved repair outcomes, shortened recovery periods, and reduced surgical failure rates are key areas of current orthopedic and tissue engineering research, and also have significant public health implications. Summary of the Invention

[0003] To overcome the deficiencies of the prior art, the present invention provides a hydrogel scaffold for promoting tendon-bone healing.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] A first aspect of the present invention provides use of a hydrogel scaffold in preparing a drug for promoting tendon-bone healing, wherein the hydrogel scaffold is prepared from silk fibroin, nano-hydroxyapatite and icariin.

[0006] Furthermore, the silk fibroin is methacrylated silk fibroin.

[0007] Furthermore, the concentration of the icariin is 10-10000 μM.

[0008] Furthermore, the concentration of icariin is 10 μM.

[0009] Furthermore, the silk fibroin is methacrylated silk fibroin.

[0010] Furthermore, the ratio of methacryloylated silk fibroin to nano-hydroxyapatite is 1:(0.25-0.75).

[0011] Furthermore, the medicine also includes artificial ligament.

[0012] Furthermore, the artificial ligament is a silk fiber artificial ligament.

[0013] Furthermore, the silk fibroin artificial ligament is a silk fibroin artificial ligament coated with SF.

[0014] Furthermore, the concentration of SF is 10%-30%.

[0015] Furthermore, the concentration of SF is 10%.

[0016] Furthermore, the preparation method of the hydrogel scaffold includes: preparing a hydrogel using methacrylylated silk fibroin and hydroxyapatite, and mixing the hydrogel with icariin-loaded nanoparticles.

[0017] Furthermore, the methacrylated silk fibroin is prepared by mixing silk fibroin with glycidyl methacrylate.

[0018] Furthermore, the hydrogel was prepared using a photocrosslinking method.

[0019] Furthermore, the photoinitiator used in the photocrosslinking method is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0020] Furthermore, icariin-loaded nanoparticles were prepared using ultracentrifugation.

[0021] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0022] A second aspect of the present invention provides use of a hydrogel scaffold in preparing a product for repairing or treating soft tissue damage, wherein the hydrogel scaffold is prepared from silk fibroin, nano-hydroxyapatite and icariin.

[0023] Furthermore, the soft tissue includes tendons, ligaments, etc.

[0024] Furthermore, the products include but are not limited to medicines, dressings and implants for damaged tissues.

[0025] A third aspect of the present invention provides use of a hydrogel scaffold in promoting the growth of MC3T3 cells or in preparing a drug for promoting the growth of MC3T3 cells. The hydrogel scaffold is prepared from silk fibroin, nano-hydroxyapatite and icariin.

[0026] Furthermore, the promoting of MC3T3 cell growth is promoting MC3T3 cell growth in vitro for non-therapeutic purposes.

[0027] A fourth aspect of the present invention provides a method for promoting the growth of MC3T3 cells, comprising culturing the cells using a hydrogel scaffold, wherein the hydrogel scaffold is prepared from silk fibroin, nano-hydroxyapatite and icariin.

[0028] Furthermore, the method is an in vitro non-therapeutic method.

[0029] Advantages and beneficial effects of the present invention:

[0030] The drug-loaded composite hydrogel scaffold prepared in this application is beneficial to tendon-bone repair. The scaffold has excellent biological properties, controllable drug release capacity and good mechanical properties. While promoting the repair of the tendon-bone interface, it also provides a feasible method for repairing skeletal muscle diseases caused by geriatric diseases, sports injuries and occupational diseases, and has broad application prospects in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram of the synthetic mechanism of SilMA;

[0032] Figure 2 It is from SF and SilMA 1 H NMR spectrum;

[0033] Figure 3 is the infrared spectra of SF and SilMA;

[0034] Figure 4 4A is a graph of 5% SilMA hydrogel, 4B is a graph of 10% SilMA hydrogel, 4C is a graph of 15% SilMA hydrogel, and 4D is a graph of 20% SilMA hydrogel.

[0035] Figure 5 Figures 5A and 5B show the gelation of SilMA / nHA hydrogels with different concentrations, respectively. Figure 5A shows S-H1 (10% SilMA / 2.5% nHA), Figure 5B shows S-H2 (10% SilMA / 5% nHA), and Figure 5C shows S-H3 (10% SilMA / 7.5% nHA).

[0036] Figure 6 are SEM images of SilMA hydrogels, wherein 6A is a 5% SilMA image, 6B is a 10% SilMA image, 6C is a 15% SilMA image, 6D is a 20% SilMA image, 6E is a SilMA hydrogel pore size comparison image, and 6F is a schematic diagram of the freeze-dried SilMA hydrogel;

[0037] Figure 7 are SEM images of SilMA / nHA hydrogels with different concentrations, among which 7A is S-H1 (10% SilMA / 2.5% nHA), 7B is S-H2 (10% SilMA / 5% nHA), 7C is S-H3 (10% SilMA / 7.5% nHA), 7D is a comparison of SilMA hydrogel pore sizes, and 7E is a schematic diagram of SilMA hydrogel after freeze-drying;

[0038] Figure 8 is the EDS analysis diagram of the composite hydrogel system;

[0039] Figure 9Figure 9 is a graph showing the swelling performance of hydrogels, where 9A is a graph showing the swelling curves of SilMA hydrogels with different concentrations, and 9B is a graph showing the equilibrium swelling degree of SH hydrogels with different concentrations;

[0040] Figure 10 10A is a graph showing the degradation trend of composite hydrogels, wherein 10A is a graph showing the swelling curves of SilMA hydrogels with different concentrations, and 10B is a graph showing the swelling degrees of SH hydrogels with different concentrations;

[0041] Figure 11 11A is a graph showing the compressive properties of the composite hydrogels, wherein 11B is a graph showing the elastic modulus of S hydrogels at different concentrations under different strains, 11C is a graph showing the stress-strain curves of SH hydrogels at different concentrations, and 11D is a graph showing the elastic modulus of SH hydrogels at different concentrations under different strains.

[0042] Figure 12 This is a flow chart for the preparation of icariin-loaded nanoparticles;

[0043] Figure 13 are SEM images of drug-loaded hydrogels, where 13A is the SEM image of icariin-loaded nanoparticles (ICA nps), 13B is the SEM image of drug-loaded hydrogel (SHI), 13C is the size distribution diagram of icariin-loaded silk fibroin nanoparticles (ICA nps), and 13D is the size distribution diagram of drug-loaded hydrogel (SHI);

[0044] Figure 14 are FTIR spectra of drug-loaded nanoparticles, where 14A is the SF spectrum, 14B is the nHA spectrum, 14C is the ICA spectrum, and 14D is the FTIR spectrum of ICA, SF, and ICA nps;

[0045] Figure 15 is the in vitro cumulative release percentage curve of drug-loaded nanoparticles;

[0046] Figure 16 are the XRD patterns of the drug-loaded hydrogels, where 16A is the XRD pattern of S(SilMA) hydrogel, 16B is the XRD pattern of SH hydrogel, 16C is the XRD pattern of SHI hydrogel, and 16D is the XRD pattern of S, SH, and SHI hydrogels;

[0047] Figure 17 are XPS patterns of drug-loaded hydrogels, where 17A is the XRS pattern of S (SilMA) hydrogel, 17B is the XRS pattern of SH hydrogel, 17C is the XRS pattern of SHI hydrogel, and 17D is the XRS pattern of S, SH, and SHI hydrogels;

[0048] Figure 1818A is a SEM image of the silk fibroin fiber after coating with 10% RSF, 18B is a SEM image of the silk fibroin fiber after coating with 20% RSF, and 18C is a SEM image of the silk fibroin fiber after coating with 30% RSF;

[0049] Figure 19 19A is a water contact angle diagram of uncoated silk fibroin fiber ligament, 19B is a water contact angle diagram of silk fibroin fiber ligament after 10% SF coating, and 19C is a comparison diagram of the water contact angles of silk fibroin fiber ligament before and after coating;

[0050] Figure 20 20A is a diagram showing the swelling and degradation of the drug-loaded hydrogel, and 20B is a diagram showing the degradation of the drug-loaded hydrogel.

[0051] Figure 21 This is a diagram of the establishment of the rabbit anterior cruciate ligament defect model;

[0052] Figure 22 22A is a graph showing the OD values of CCK8 at different drug concentrations, and 22B is a graph showing the survival rates of CCK8 at different drug concentrations.

[0053] Figure 23 Figure 23A is a graph showing the proliferation of L929 cells in the ligament layer. Figure 23A is a graph showing the OD results of CCK8, Figure 23B is a graph showing the live and dead staining of cells in the 10% SF-ALs group, Figure 23C is a graph showing the live and dead staining of cells in the 20% SF-ALs group, and Figure 23D is a graph showing the live and dead staining of cells in the 30% SF-ALs group.

[0054] Figure 24 Figure 24A is a graph showing the proliferation of MC3T3-E1 cells in the bone layer, wherein 24A is a graph showing the OD results of CCK8, 24B is a graph showing the live-dead staining of S hydrogel cells, 24C is a graph showing the live-dead staining of SH hydrogel cells, and 24D is a graph showing the live-dead staining of SHI hydrogel cells;

[0055] Figure 25 Figure 25A shows the adhesion of L929 cells to the ligament layer, and Figure 25B shows the adhesion of MC3T3-E1 cells to the bone layer hydrogel.

[0056] Figure 26 Figure 26A shows the results of ALP staining and Figure 26B shows the results of ARS staining.

[0057] Figure 27 This is the Micro CT analysis result 4 weeks after surgery;

[0058] Figure 28 These are histological images of the tissue repair process in different experimental groups 4 weeks after surgery. 28A is the HE staining result, 28B is the Masson staining result, 28C is the toluidine blue staining result, and 28D is the CD31 staining result. DETAILED DESCRIPTION

[0059] The following provides definitions of some terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0060] The present invention provides an application of a hydrogel scaffold in preparing a medicine for promoting tendon-bone healing. The hydrogel scaffold is prepared from silk fibroin, nano-hydroxyapatite and icariin.

[0061] The preparation method of the hydrogel scaffold comprises: preparing a hydrogel by using methacrylylated silk fibroin and hydroxyapatite, and mixing the hydrogel with icariin-loaded nanoparticles.

[0062] In some embodiments, the shape of the hydrogel scaffold body includes one of a cylinder, a cube, a cuboid, and a sphere, and can be designed according to actual conditions without specific limitation.

[0063] In some embodiments, the hydrogel used in the hydrogel scaffold body includes one of a photocrosslinked hydrogel and a non-photocrosslinked hydrogel.

[0064] Among them, the photocrosslinked hydrogel includes one of glycidyl methacrylate (GMA), glycidyl methacrylate-modified hyaluronic acid (GMHA), gelatin methacrylate anhydride (GelMA), and hyaluronic acid methacrylate anhydride (MAHA).

[0065] In some embodiments, any hydrogel that is cross-linked by other methods (e.g., temperature, chemical initiator) other than photocross-linking can also be used. The non-photocross-linked hydrogel includes one of chitosan hydrogel, agarose hydrogel, chitosan / poly-N-acryloylglycine composite hydrogel, and cyclodextrin hydrogel modified with acryloyl chloride.

[0066] The drug also includes pharmaceutically acceptable excipients.

[0067] In some embodiments, "pharmaceutically acceptable" is intended to encompass any carrier that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host to which it is administered. The pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, preservatives, anti-adhesives, integrants, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0068] In some embodiments, the dosage form of the drug includes but is not limited to an injectable dosage form or a dressing.

[0069] The injectable dosage forms include, but are not limited to, injection solutions, injection solutions for intravenous drip, injection suspensions, sterile powders for injection, intravenous injections, water injections, injection emulsions, powder injections, injections, sterile powder injections, freeze-dried powder injections, etc.

[0070] The present invention will be further described below with reference to specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0071] Example 1 Preparation and characterization of photocrosslinkable silk fibroin hydrogel

[0072] 1 Experimental methods

[0073] 1.1 Extraction of silk fibroin (SF)

[0074] Silk fibroin was extracted using an alkaline degumming method. Silkworm cocoons were dried, cut evenly, and placed in a 0.05M Na₂CO₃ solution, boiled for 60 minutes. The silk was then repeatedly scrubbed with deionized water until it was smooth and free of bubbles, removing the sericin and sodium carbonate. The degummed silk was then placed in a 60°C drying oven and dried for at least 8 hours until completely dry. The dried silk was then dissolved in a 9.3M lithium bromide (LiBr) solution at 60°C for 1 hour, then transferred to a dialysis bag (MWCO 12-14 kDa) and dialyzed against deionized water at 4°C for 5-7 days, with the deionized water changed 4-5 times daily. The resulting SF aqueous solution was then centrifuged, frozen, and dried to obtain silk fibroin (SF), which was then stored at 4°C until ready for use.

[0075] 1.2 Synthesis of methacryloylated silk fibroin (SilMA)

[0076] 20 g of degummed, dried silk was placed in 100 ml of a 9.3 M LiBr solution at 60°C. After the silk was completely dissolved and the solution turned clear yellow, 4, 6, and 8 mL of glycidyl methacrylate (GMA) were evenly added dropwise at a rate of 0.5 mL / min. The mixture was stirred on a thermostatic magnetic stirrer at 60°C and 300 rpm for 3 h. After the reaction, the resulting mixture was transferred to a dialysis bag (MWCO 12-14 kDa) and dialyzed against deionized water at 4°C for 5-7 days, with the deionized water changed 4-5 times daily. After dialysis, the SilMA was centrifuged, frozen, and dried, and stored at 4°C until ready for use.

[0077] 1.3 Preparation of photocrosslinked silk fibroin hydrogel

[0078] The silk fibroin modified with glycidyl methacrylate can be irradiated with ultraviolet light in the presence of a photoinitiator to initiate free radical polymerization and undergo intermolecular cross-linking to form a gel. The specific preparation steps are as follows:

[0079] (1) At room temperature, freeze-dried SilMA was weighed and dissolved in deionized water to obtain SilMA solution (5 wt%, 10 wt%, 15 wt%, 20 wt%).

[0080] (2) At room temperature, nanohydroxyapatite (nHA) was added to the SilMA solution at a ratio of SilMA: nHA of 1:0.25, 1:0.5, and 1:0.75 to obtain a SilMA / nHA composite solution.

[0081] (3) Under light-shielding conditions, add 0.5 wt% of the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) to the solution obtained in (1) and (2) above and fully dissolve it.

[0082] (4) The composite solution was injected into a transparent glass bottle and cross-linked under 365 nm UV light at room temperature for 5 min to obtain the photocross-linked hydrogel (SH, SilMA / nHA).

[0083] (5) The numbers of composite materials with different solid contents are shown in Table 1.

[0084] Table 1 Composite material number

[0085]

[0086] (6) Observe the photocrosslinking properties of composite materials with different contents and record the changes before and after irradiation.

[0087] 1.4 H NMR spectroscopy ( 1 H NMR) determination

[0088] In order to determine the degree of substitution (DS) of the synthesized SilMA, a certain amount of SilMA was dissolved in 0.5 mL / 10 mg of D2O and the structure was analyzed using a 400 Hz nuclear magnetic resonance spectrometer (NMR-H). 1 H NMR spectroscopy. The DS of SilMA was determined based on the percentage of modified lysine. Each spectrum was normalized using the proton signals generated by the aromatic amino acids of SF and SilMA at 6.9 to 7.5 ppm. The DS was calculated based on the integrated area of the peak where the lysine methylene protons appear at 2.8 to 2.95 ppm. Therefore, the DS of SilMA was determined by the following formula:

[0089]

[0090] 1.5 Fourier transform infrared spectroscopy (FTIR) analysis

[0091] Fourier transform infrared spectroscopy was used to characterize the characteristic peaks of the amide bond of SilMA to determine the degree of grafting of the methacrylamide group. 1g of SF and SilMA were thoroughly mixed with KBr powder at room temperature and then pressed into pellets. Subsequently, the sample was scanned using an infrared spectrometer (FTIR) with a wavenumber range of 400–4000 cm -1 , and finally the infrared absorption spectrum of the sample is obtained.

[0092] 1.6 Observation of hydrogel structure and morphology

[0093] The composite solution obtained in 1.3 was injected into molds (D = 8 mm, H = 5 mm) and UV-cured. Hydrogels with different ratios were freeze-dried and then quenched with liquid nitrogen. The quenched surface was placed face-up on a stage and gold-sprayed. The cross-sectional morphology was observed using a scanning electron microscope (SEM). Energy dispersive spectroscopy (EDS) was used to analyze the elemental composition, content, and distribution of the surface micro-regions on the cross-sections.

[0094] 1.7 Mechanical testing of hydrogels

[0095] Use the mold in 1.6 to prepare several cylindrical hydrogels. During the preparation, the liquid is added slowly to avoid the generation of bubbles. After the hydrogels are prepared, the compression test of different hydrogels is carried out using a universal dynamic mechanical analyzer (Q800, America). -1 The compression test is performed at a rate of 100 nm. The test is completed when cracks appear in the hydrogel. The results are used to generate a compressive stress curve and calculate the elastic modulus.

[0096] 1.8 Swelling test of hydrogel

[0097] Prepare several cylindrical hydrogels using the silicone mold in 1.6, and completely immerse the prepared composite hydrogel (W0) in PBS solution. Incubate in a constant temperature shaker at 37°C and 40 rpm, and remove samples at different time points (1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, 96 h). Wipe the surface moisture and continue to weigh (mass W t The swelling ratio is calculated according to the following formula: Swelling ratio = (W t Three parallel controls were set for each sample for observation and data analysis.

[0098] 1.9 Hydrogel degradation test

[0099] After the freeze-dried hydrogel is fully expanded, record the weight W t The samples were immersed in PBS solution containing 0.5 U / L type II collagenase, shaken in a constant temperature shaker at 37°C and 40 rpm, and taken out and weighed at different time points (1h, 2h, 4h, 8h, 12h, 24h, 48h, 96h) (W d Finally, the degradation efficiency of the hydrogel was calculated as follows: degradation rate = (W t -W d ) / W t ×100%. Three parallel controls were selected for each sample for observation and data analysis.

[0100] 1.10 Statistical Analysis

[0101] All results are expressed as mean ± standard deviation (mean ± SD), and data were analyzed using SPSS. All experiments were repeated three times, and the statistical significance level was set at P < 0.05.

[0102] 2 Experimental results

[0103] 2.1 Synthesis mechanism and structure of SilMA

[0104] The synthetic mechanism of SilMA is as follows Figure 1 As shown in Figure 2, silk fibroin molecules contain multiple hydroxyl (-OH) and amino (-NH2) groups, which are active reaction sites. After adding acrylic acid (or methacrylic acid) and its esterifying agent, the hydroxyl (-OH) or amino (-NH2) groups of silk fibroin react with the carboxyl (-COOH) groups in acrylic acid (MA) through nucleophilic addition reaction, forming ester bonds and grafting the acrylate groups onto the silk fibroin molecules.

[0105] 2.2 1 H NMR analysis

[0106] SF and SilMA with different GMA concentrations (GMA concentrations of 4ml, 6ml, and 8ml) were subjected to H NMR spectroscopy. As the acrylate groups in GMA react with the amino or hydroxyl groups of silk fibroin, cross-linking forms new chemical bonds and chemical shifts occur, such as Figure 2 Compared to SF, SilMA exhibits significantly stronger signal intensities at δ = 5.72ppm / 5.77ppm and δ = 1.8ppm, representing the vinyl and methyl groups of methacrylate, respectively. Calculation of the grafting yields of the three synthesized SilMA concentrations showed that the grafting yields ranged from 20% to 40%, which is consistent with literature.

[0107] 2.3 FTIR analysis

[0108] SF and SilMA with different reaction concentrations (GMA concentrations are 4ml, 6ml, and 8ml) were subjected to FTIR testing. The infrared spectra of SF and SilMA are shown in Figure 2. Figure 3 The typical amide characteristic peaks of SF are at 1639 cm -1 、1512 cm -1 and 1234cm -1 In contrast, SilMA has a peak at 951 cm -1 The absorption peak at is significantly enhanced, indicating that the methacrylamide group has been successfully grafted, corresponding to the increase in the double bond content in the RR'C=CH2 characteristic peak.

[0109] 2.4 Hydrogel photocrosslinking effect

[0110] The gelation of SilMA hydrogels with different concentrations is shown in Figure 2. Figure 4 The uniformly mixed liquid was placed in a glass bottle for subsequent observation. At room temperature, all groups of samples were liquid and showed no gelation. As the SilMA concentration increased, the sample color gradually deepened and turned yellow. After UV irradiation, all samples gelled, and the color deepened relative to before gelation, further indicating that the SilMA photosensitive group was successfully grafted and the gelation effect was good.

[0111] When nHA (nanohydroxyapatite) was added to a 10% SilMA solution, the properties of the solution (such as color, viscosity, transparency, etc.) changed significantly with the increase of nHA concentration. These changes were mainly related to the dispersion of nHA particles, surface reaction, and interaction with the SilMA matrix. Figure 5As shown, the hydrogel exhibits increased viscosity, decreased transparency, and a more milky or off-white color. Especially at high nHA concentrations, the color turbidity increases significantly. In particular, as nHA concentration increases, the efficiency of the cross-linking reaction may decrease, and the curing time gradually increases, because nHA particles hinder the penetration of UV light. However, the stiffness of the cross-linked material also increases, and the osteoinductive and mechanical properties of the material are enhanced.

[0112] 2.5 Scanning electron microscopy (SEM) characterization

[0113] The internal structure morphology of the brittle fracture of pure SilMA hydrogel after freeze drying is as follows: Figure 6 As shown in the figure, all samples exhibited a similar, loose, porous network structure with regular shapes and interconnected pores. The pores had clear boundaries, were neatly arranged, and had a relatively flat surface, which was conducive to cell growth and provided space for information transmission and nutrient transport. Furthermore, with increasing SilMA (Sil) concentration, the pore size of the gel first increased and then decreased. The average pore size of the gel with 20% SilMA was the smallest, at 14.264±6.14323 μm. The average pore size of the gel with 10% SilMA was the largest, at 35.87752±10.48112 μm.

[0114] like Figure 7 As shown, spherical nHA particles were uniformly embedded in the hydrogel. At 2.5 wt% nHA, the pore structure was relatively uniform, with small and dispersed nHA particles and a smooth hydrogel surface. The average pore size was 9.8727±1.81326 μm. At 5 wt% nHA, HA particles began to aggregate, the surface roughness increased, and small, lumpy particles were present. The average pore size was 17.04553±3.66018 μm. At 7.5 wt% nHA, the pores became tighter, with significant aggregation of nHA particles. The hydrogel surface was irregularly distributed, with distinct particle clusters. The pore structure may be partially filled with nHA particles. The average pore size was 13.9446±3.13557 μm. High-magnification images revealed the presence of pores between the spherical nHA particles, which facilitated cell adsorption and growth factor accumulation, thereby accelerating new bone formation and vascularization at the tendon-bone interface.

[0115] 2.6 EDS analysis

[0116] The distribution of C, Ca, O, P and N elements in the cross section of the material was analyzed by EDS. Figure 8The characteristic elements of nHA, Ca (blue) and P (green), are visually reflected in the composite hydrogel, showing uniform dispersion and complete overlap, further demonstrating the uniform embedding and dispersion of nHA particles within the carrier. Other elements, derived from other components of the hydrogel, provide a stable support environment for the nHA particles.

[0117] 2.7 Hydrogel swelling properties

[0118] The swelling curve of photocurable hydrogel is shown in Figure 9 As shown in the figure, the swelling of the hydrogel is fast at first and then slows down. In the initial water absorption stage, the swelling rate is the fastest. After 24 hours, it gradually becomes flat and the swelling rate slows down until the equilibrium water absorption is reached.

[0119] SilMA hydrogels also exhibit variability in swelling. Low-concentration hydrogels absorb water faster, swell more, and ultimately absorb a higher amount of water. As concentration increases, the hydrogel absorbs water more slowly, swells less, and absorbs less water. By adjusting the concentration of SilMA hydrogels, their swelling characteristics can be tailored to meet specific performance requirements in different application scenarios.

[0120] The swelling degree of the SH composite hydrogel decreased with increasing nHA concentration. Its swelling performance was maximum at an nHA concentration of 2.5% (Q = 2.37 ± 0.12). When the nHA concentration increased to 5% and 7.5%, the swelling degrees reached 2.54 ± 0.10 and 2.75 ± 0.17, respectively. This may be due to the low water retention capacity of nHA. Low concentrations of nHA may be more evenly dispersed in the hydrogel, which does not significantly affect the hydrogel's swelling properties. Furthermore, with higher nHA concentrations, the average pore size of the composite hydrogel decreased, and the binding became tighter. This decreased water permeability may increase the resistance of water molecules to entering the hydrogel, resulting in a decrease in the hydrogel's swelling degree.

[0121] 2.8 Degradation properties of hydrogels

[0122] The degradation performance of materials is one of the important indicators for evaluating tissue engineering scaffolds. The degradation process is usually a complex process involving factors such as chemical bond breakage, molecular chain breakage, and water molecule penetration within the hydrogel. Degradable scaffolds can avoid secondary surgery and thus reduce the infection rate. Figure 10 As shown in the figure, the degradation rates of all sample groups showed a trend of first fast and then slow.

[0123] As SilMA concentration increased, the degradation rate slowed. After 4 days, the mass loss in the 5 wt% SilMA group was 34.59%, the 10 wt% SilMA group was 35.38%, the 15 wt% SilMA group was 23.61%, and the 20 wt% SilMA group was 21.28%. Low-concentration SilMA hydrogels typically have a looser network structure, allowing water to easily penetrate the hydrogel's network, resulting in a faster degradation rate.

[0124] The degradation rate of SH hydrogels in each group slowed down with the increase of nHA concentration. After 48 h, the remaining masses of 2.5% nHA, 5% nHA, and 7.5% nHA groups were 56.47%, 59.36%, and 71.31%, respectively.

[0125] 2.9 Mechanical properties of hydrogels

[0126] from Figure 11 As can be seen from A, with the increase of S concentration, the stress value of S (SilMA) hydrogel also increases, showing that the compressive performance is gradually enhanced. This shows that the increase of SilMA makes the internal structure of the hydrogel more compact and improves its mechanical strength. Elastic modulus statistics ( Figure 11 B) shows that the elastic modulus of the 20% SilMA group was significantly higher than that of the 5% SilMA group. The elastic modulus of the 5% SilMA group at 20% strain was 0.92±0.08 kPa, and at 40% strain was 5.92±0.27 kPa. The elastic modulus of the 20% SilMA group at 20% strain was 7.46±0.82 kPa, and at 40% strain was 55.73±2.87 kPa. This suggests that a higher concentration of SilMA imparts superior deformation resistance to the hydrogel.

[0127] As the nHA content increases, the compressive strength of the hydrogel is further improved, especially in a larger strain range, which indicates that the addition of HAp strengthens the rigidity and stability of the hydrogel ( Figure 11 C). Elastic modulus statistics chart ( Figure 11 D) shows that the elastic modulus of the S-H3 group with a higher HAp content is significantly higher than that of the S-H1 group. The elastic modulus of the S-H1 group at 20% strain is 0.46±0.06 kPa, and the elastic modulus at 40% strain is 2.50±0.28 kPa; the elastic modulus of the S-H3 group at 20% strain is 0.80±0.16 kPa, and the elastic modulus at 40% strain is 7.97±2.24 kPa, indicating that the incorporation of HAp significantly enhances the mechanical properties of the hydrogel.

[0128] Example 2 Preparation and Characterization of Icariin-Loaded Nanoparticle Hydrogel Scaffold

[0129] 1. Experimental Methods

[0130] 1.1 Preparation of drug-loaded nanoparticles

[0131] Icariin-loaded nanoparticles were prepared by ultracentrifugation. Figure 12 As shown. In a fume hood, a 10 mg / mL SF solution was slowly added to an acetone solution at a 1:5 v / v ratio. An appropriate amount of icariin (100 mg / mL) was then added. The mixture was incubated at 500 rpm overnight at room temperature until the acetone completely evaporated, resulting in a stable mixed solution. The suspension was then ultracentrifuged at 10,000 rpm for 20 minutes. After centrifugation, the supernatant was aspirated and washed three times with an equal volume of deionized water. Finally, the resulting precipitate was freeze-dried to obtain icariin-loaded nanoparticles (ICA NPs) and stored at -20°C until further use.

[0132] 1.2 Structural morphology observation of drug-loaded nanoparticles and drug-loaded composite hydrogels

[0133] Prepare drug-loaded composite hydrogels (SHIs) by referring to the procedure described in 1.3. Spray-coat the freeze-dried drug-loaded nanoparticles and composite hydrogels with gold. After presetting the electron microscopy parameters and selecting an appropriate magnification, take SEM images of the samples. Select three parallel controls for each sample for observation and data analysis.

[0134] 1.3 Fourier transform infrared spectroscopy (FTIR) detection

[0135] The ICA, SF, and ICA NPs samples were analyzed by Fourier transform infrared spectrometer (FTIR, INVENIO-R, Germany) in the range of 4000–400 cm -1 .

[0136] 1.4 Preparation of silk fibroin artificial ligament

[0137] In this study, the first step was to weave a silk artificial ligament. This artificial ligament was then degummed by boiling it with a 0.5% Na₂CO₃ solution at a bath ratio of 1:50 at 100°C for 30 minutes. The artificial ligament was then removed and thoroughly rinsed with deionized water. This degumming process was repeated three times to obtain degummed silk fibroin fiber ligaments (SF-ALs). Finally, the degummed SF-ALs were dried in a 60°C oven to complete the preparation of the artificial ligament material.

[0138] 1.5 Structural morphology observation Scanning electron microscopy (SEM)

[0139] The surface morphology of 10%, 20%, and 30% SF-ALs was observed using an S-4800 scanning electron microscope. Before the experiment, the samples were fixed to the microscope stage as required, adhered with conductive adhesive, and then gold-sprayed. SEM images were acquired using appropriate microscope parameters and magnification. Three replicates of each sample were selected for observation and data analysis.

[0140] 1.6 Contact angle test

[0141] The surface wettability of 10%, 20%, and 30% SF-ALs was evaluated using a contact angle meter (Theta, Sweden). The samples were fixed on a test platform, and deionized water was slowly dripped onto the sample surface using a microinjector. The static contact angle of the water droplet on the sample surface was recorded using measurement software. The measurement was repeated three times for each sample group, and the average value was calculated to analyze differences in surface hydrophilicity.

[0142] 1.7 Preparation and characterization of icariin-loaded nanoparticle hydrogel scaffolds

[0143] S, SH, and SHI sample powders were evenly spread on a sample stage for crystal phase analysis using an X-ray diffractometer (EMPYREAN, PANalytical, the Netherlands). The surface elemental composition and chemical bonding information of the S, SH, and SHI hydrogels were analyzed using X-ray photoelectron spectroscopy (XPS, AXIS Ultra DLD, Japan). Freeze-dried hydrogel samples were fixed to the sample stage and tested under ultrahigh vacuum conditions. The data were analyzed to determine the chemical state of the elements on the sample surfaces.

[0144] 1.8 Compression test of drug-loaded hydrogel

[0145] The composite solution with different drug contents was injected into the mold and solidified, and then transferred to the universal chemical analyzer. -1 The compression test is performed at a rate of 100 nm. The test is complete when cracks appear in the hydrogel. The results are used to generate a compressive stress curve, which is then used to calculate the compression modulus.

[0146] 1.9 Drug-loaded hydrogel swelling test

[0147] The freeze-dried composite hydrogel sample (initial mass W0) was immersed in PBS and shaken at 40 rpm in a 37°C constant temperature shaker. The sample was taken out at the specified time points (1 h, 2 h, 4 h, 12 h, 24 h, 48 h), the surface moisture was wiped off, and the sample was weighed (mass W0). t ). The swelling rate calculation formula is: Swelling rate = (W t-W0) / W0×100%. Three parallel controls were selected for each sample for observation and data analysis.

[0148] 1.10 Degradation test of drug-loaded hydrogel

[0149] After the freeze-dried hydrogel is fully expanded, record the weight W t The samples were immersed in PBS containing 0.5U / L type II collagenase and shaken in a constant temperature shaker at 40 rpm and 37°C. The samples were taken out at different time points (1h, 2h, 4h, 12h, 24h, 48h) and weighed. d Calculate the degradation efficiency of hydrogel: Degradation rate = (W t -W d ) / W t ×100%. Three parallel controls were selected for each sample for observation and data analysis.

[0150] 1.11 Drug Release Test of Drug-Loaded Hydrogel

[0151] Hydrogel samples with varying drug loading concentrations were placed in a PBS solution, ensuring complete immersion. The release medium was stirred at 300 rpm using a magnetic stirrer. Samples were taken at various time points (1 hour, 4 hours, 12 hours, 24 hours, 48 hours, 4 days, and 7 days). For each sampling, 1 mL of the test solution was removed and immediately replaced with the same volume of fresh release medium to maintain a constant total volume. The absorbance of the removed samples at a specific wavelength was measured using a UV spectrophotometer. The drug concentration in the sample was calculated, and the cumulative release amount was further estimated.

[0152] 1.12 Statistical Analysis

[0153] All results are expressed as mean ± standard deviation (mean ± SD) and analyzed using SPSS. All experiments were repeated three times, and differences were considered statistically significant at P < 0.05.

[0154] 2 Experimental results

[0155] 2.1 Micromorphology and particle size distribution of drug-loaded nanoparticles

[0156] Microscopic morphology of drug-loaded nanoparticles Figure 13 As shown. ICA NPs (ICA nps) particles have a uniform hexahedral structure with an average size of 456.23±112.04 nm, of which the smallest is 21 nm and the largest is 794 nm. The size is relatively uniform. Figure 13As shown in Figure 3, the size distribution approximates a normal distribution. After adding the drug to the SH composite solution, the average particle size of the SHI group particles was 11.33 ± 3.32 μm, with the smallest particle size being 4.76 μm and the largest being 17.08 μm. This is smaller than the particle size of the particles in the SH hydrogel alone, and the overall distribution is relatively uniform, demonstrating good dispersibility and stability. This may be because the presence of the drug may affect the cross-linking degree of the composite material, leading to changes in the spatial structure of the composite material.

[0157] 2.2 FTIR spectrum of drug-loaded nanoparticles

[0158] Figure 14 The following is a FTIR spectrum of the drug-loaded nanoparticles. The FTIR spectra of the samples in each group exhibited similar peak shapes, indicating that the basic structures of the materials remained stable. In the FTIR spectra, peaks at 1620 cm⁻¹ and 3273 cm⁻¹ correspond to carbonyl (C=O) and hydroxyl (OH), respectively. The characteristic peaks of ICA are primarily distributed at ~1700 cm⁻¹ (C=O stretching vibration), ~1600-1650 cm⁻¹ (aromatic ring backbone vibration), and ~1200 cm⁻¹ (CO-C stretching vibration). After drug loading, the peak at 1620-1630 cm⁻¹ intensified, indicating that the SF molecules favored a β-sheet structure during nanoparticle formation. Conversely, the characteristic peaks of ICA shifted and weakened, suggesting possible chemical or physical interactions between the protein and SF. Overall, the molecular structures remained relatively stable, indicating that the drug loading process was relatively gentle and did not cause significant damage to the material.

[0159] 2.3 Drug release test of drug-loaded nanoparticles

[0160] from Figure 15 As can be seen from the figure, the encapsulation of icariin in the nanoparticles is relatively stable. It is not fully released in a short period of time, and a relatively slow release process can be maintained later. The nanoparticles show a relatively obvious "burst release" phase in the early release period, with the release amount rapidly increasing from nearly 0% to about 20%. This is a typical early release characteristic of nano drug delivery systems, usually due to the dissolution of some drug molecules distributed on the surface or in the shallower layers of the particles within a short period of time. Subsequently, the release rate slowed significantly, and the curve gradually flattened. At 48 hours, 23.77% was released. This process represents the slow diffusion of the drug from the interior of the particle to the outside or the release through material degradation.

[0161] 2.4 Characterization results of drug-loaded hydrogel

[0162] Figure 16The following is the X-ray diffraction (XRD) pattern of the drug-loaded hydrogel. The XRD pattern of SilMA mainly shows clear crystalline peaks in the 2θ range of 20-30, and the diffraction peaks become slightly broader, proving that it has a β-pleated structure. nHA is a highly crystalline material, showing clear and sharp diffraction peaks at positions such as 31.77°, 32.19°, 34.05°, and 46.71°, reflecting its hexagonal crystal structure. These diffraction peaks indicate that HAp has a high degree of crystallinity. The XRD pattern of SHI is a superposition of the diffraction peaks of SilMA and nHA. However, because the diffraction peak of nHA is very strong, icariin is an amorphous or low-crystallinity substance and usually has no significant diffraction peaks in the XRD pattern. Therefore, the addition of icariin usually does not show obvious diffraction peaks in the XRD pattern.

[0163] Figure 17 This is the X-ray photoelectron spectroscopy (XPS) of the drug-loaded hydrogel. The XPS spectrum of SilMA displays the typical elemental characteristics of silk fibroin and is relatively concise, consisting primarily of signals for the elements C, N, and O. At 284.6 eV, a C 1s peak, representing carbon-carbon (C-C) or carbon-hydrogen (C-H) bonds, can be observed. N 1s peaks can be observed around 399.5 eV, and O 1s peaks can be observed around 532-533 eV. After adding nHA to SilMA, the XPS spectrum displays new characteristic peaks associated with HAp, indicating the introduction of an inorganic component. As an inorganic material, HAp's representative peaks are Ca 2p and P 2p. The XPS spectrum of SHI is more complex than that of SH, particularly the changes in the C 1s and O 1s peaks, which reflect the presence of ICA and its interactions with other components.

[0164] 2.5 Surface morphology of silk fiber ligament

[0165] Figure 18 SEM micrographs of modified silk fibroin materials. The 10% RSF (A) sample exhibits a relatively smooth surface with distinct fibers, a thin coating structure, and a small amount of particles deposited on the surface, arranged neatly and tightly. The 20% RSF (B) coating exhibits a less smooth surface, with particles deposited on the surface of the silk fibroin fibers, forming a relatively uniform and stable film. The 30% RSF (C) coating exhibits excessive thickness, with particle accumulation and film cracking. While the coating is relatively stable, it may affect the mechanical properties and flexibility of the ligament.

[0166] 2.6 Water contact angle test results

[0167] The water contact angle changes before and after material modification were tested by the bubble method. Figure 19The results of water contact angle tests are presented. The water contact angle reflects the wettability of a material; the smaller the contact angle, the more hydrophilic it is. SF-ALs coated with 10% SF (RSF-ALs) were selected for contact angle testing. The tests showed that the contact angle of the uncoated SF-ALs was 126.08±1.46°. After coating, the contact angle dropped to 105.35±5.95°. Combined with the above SEM results, the coated material forms a groove structure on the surface, increasing surface roughness and thus reducing the contact angle and improving hydrophilicity.

[0168] 2.7 Swelling and degradation properties of drug-loaded hydrogels

[0169] The swelling of drug-loaded hydrogels Figure 20 As shown in Figure A, the swelling rates of all sample groups showed a trend of initially increasing rapidly and then decreasing. After 48 hours, the S group showed the greatest swelling change, increasing by 3.79 times the original value; the SHI group showed the smallest swelling change, increasing by 2.38 times the original value. The addition of ICA tightened the internal structure of the hydrogel, reduced its water absorption capacity, and reduced the influx of free water into the hydrogel.

[0170] The degradation trend of drug-loaded composite hydrogels is as follows Figure 20 As shown in Figure B, the degradation rates of all sample groups showed a trend of initially rapid and then slowing. After 48 hours, the S group showed the most significant mass loss, reaching 14.81%, while the SH group showed the least mass loss, at only 6.43%. The S group hydrogel degraded most rapidly, likely due to its loose porous structure and interstitial spaces. The addition of nHA improved structural stability and reduced the degradation rate. The degradation of the drug-loaded SHI hydrogel may be accompanied by the release of ICA nps, which may increase the micropores of the hydrogel and accelerate degradation. Therefore, the degradation rate of SHI is greater than that of SH.

[0171] Example 3 In vitro and in vivo biological evaluation of drug-loaded hydrogel scaffolds

[0172] 1 Experimental methods

[0173] 1.1 Culture of fibroblasts (L929)

[0174] Mouse fibroblast L929 cells were obtained from a previous cryopreservation in the laboratory. Sterile DMEM complete medium supplemented with 1% double-streptomycin (penicillin / streptomycin) and 10% fetal bovine serum (FBS) was prepared, sealed, and refrigerated at 4°C until ready for use. After thawing, cells were cultured in fresh DMEM complete medium, with the medium replaced every two days. Cells were passaged when they reached 80–90% confluence and cultured until P3, when they were in good condition, for later use.

[0175] 1.2 Cultivation of osteoblasts (MC3T3-E1)

[0176] MC3T3-E1 osteoblasts were purchased from the Cell Bank of the Chinese Academy of Sciences (CCTCC). Sterile α-MEM medium containing 10% FBS and 1% double-streptomycin (penicillin / streptomycin) was prepared, sealed, and stored in a refrigerator at 4°C until use. After thawing, cells were placed in a cell culture incubator. When cells reached 80–90% confluence, they were passaged and cultured for further use.

[0177] 1.3 Drug concentration screening

[0178] The CCK8 method was used for drug concentration screening. SHI hydrogels with different drug concentrations (10, 100, 500, 1000, 10000 μM) and blank controls (no drug) were set up. The hydrogels were sterile treated and placed in 24-well plates. Sterile DMEM complete medium was prepared for use. MC3T3-E1 cells were cultured to 70-80% confluency and the cell concentration was adjusted to 10 5 cells / mL, 100 μL of the cell suspension was aspirated and added to a 24-well plate and cultured for 1, 4, and 7 days. At different time points, serum-free culture medium and CCK8 reagent were mixed in a 10:1 ratio in the dark and incubated in an incubator for another 4 h. After incubation, 100 μL was aspirated from each well in a 96-well plate and the absorbance of each group was measured at 450 nm using a microplate reader. Three replicate wells were prepared for each sample.

[0179] 1.4 Cell seeding and cytotoxicity testing

[0180] Sterile SF-ALs (ligament layer) and drug-loaded hydrogel (SHI, bone layer) scaffold samples were prepared according to the above method and placed in 24-well culture plates and irradiated overnight with UV light. Second- and third-passage L929 cells and MC3T3 E1 cells were selected as seed cells for the ligament layer and bone layer, respectively. After trypsinization, the cell suspension was dripped onto the surface of each sample. After incubation in an incubator for 15 minutes, the medium was added to 1 mL. Experimental groups (containing materials and cells), control groups (containing cells but not materials), and blank groups (containing neither) were set up. Cultures were maintained in a cell culture incubator, with the cell medium replaced every two days.

[0181] Prepare CCK-8 working solution in the dark. Remove samples on days 1, 4, and 7, discard the culture medium, and replace with serum-free culture medium mixed with CCK-8 reagent at a volume ratio of 10:1. Protect cells from light and incubate in a humidified incubator for 4 hours. After incubation, transfer 100 μL per well to a 96-well plate and measure the absorbance of the solution at 450 nm using a microplate reader. Three replicates were used for each sample. Cell viability was analyzed and calculated as follows: cell viability (Viab. %) = [(As - Ab) / (Ac - Ab)] × 100% (As: experimental group, Ac: control group, Ab: absorbance of blank group).

[0182] 1.5 Cell viability analysis

[0183] After 1, 4, and 7 days of culture, live and dead cells in the samples were stained with FDA / PI dye to observe the effect of the scaffold on cell proliferation. At the predetermined time points, the cells were removed and rinsed 1-2 times with PBS to remove the culture medium. 200 μL of FDA / PI working solution was added to each well and incubated in the dark for 10 minutes. The samples were then removed and placed on glass slides, and cell growth was observed using confocal laser scanning microscopy (CLSM).

[0184] 1.6 Cell morphology observation

[0185] Prepare the phalloidin working solution according to the instructions. Remove the sample holders on days 1, 4, and 7 after cell inoculation. Wash with PBS once or twice, then fix the cells with 4% paraformaldehyde at room temperature. After washing with PBS, permeabilize with 0.1% Triton X-100 solution. Then, stain with the working solution and DAPI solution in the dark for 30 minutes and 30 seconds, respectively. Observe and record using a laser confocal scanning microscope. Prepare three replicates for each sample group.

[0186] 1.7 Osteogenesis Induction Observation

[0187] Alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining are important indicators for assessing osteoblast differentiation, and their activity levels reflect the degree of osteoblast differentiation. ALP and Alizarin Red S staining can visually reflect the manifestation of bone mineralization. After MC3T3-E1 cells adhere, osteogenic induction medium (containing sodium β-glycerophosphate, ascorbic acid, and DEX) is incubated every 2-3 days. After 14 days of culture, the plates are removed, the medium is aspirated, and the cells are rinsed twice with PBS. The cells are then fixed with 4% paraformaldehyde for 10-15 minutes and washed twice with PBS. Finally, appropriate amounts of ALP and ARS working solutions are added, respectively, and the cells are incubated in the dark for 30 minutes (37°C). Cell color changes are observed (cells with high ALP activity appear bluish-purple / dark blue, and calcified nodules appear red). The cells are washed three times with PBS and observed under a microscope and photographed.

[0188] 1.8 Establishment of rabbit anterior cruciate ligament defect model

[0189] The experiment used 2-3 month old male New Zealand white rabbits weighing approximately 2.5 kg, which were purchased from Chengdu Dashuo Technology Co., Ltd. and fed adaptively for 7 days. Before the experiment, the anterior drawer test and Lachman test of the knee joint were negative. Figure 21As shown. After weighing, sodium pentobarbital and Su Mian Xin II were used for anesthesia. 3% sodium pentobarbital was injected intravenously at 1 mL / kg, and Su Mian Xin II was injected intramuscularly at 0.1 mL / kg. The surgical area was prepared with a hair pusher and disinfected with iodine 3 times. The skin and deep fascia were cut open along the patellar ligament, the patella was lifted and the joint cavity was exposed, and the native ACL was removed along the femoral and tibial ends. The composite material was completely filled along the bone tunnel, and sutured after compression and hemostasis. After the graft was implanted, the filling effect was as shown Figure 21 As shown. Immediately after surgery, gentamicin injections were given, and penicillin injections were administered for three consecutive days to prevent infection. Following surgery, the animals were maintained for four and eight weeks, respectively, and samples were removed at the corresponding time points to observe the repair effects.

[0190] 1.9 Micro-CT analysis

[0191] Four and eight weeks after scaffold implantation, knee joint specimens were removed after an overdose of sodium pentobarbital (100 mg / kg) and immediately fixed in 4% paraformaldehyde. The apparent repair process was first documented, followed by micro-CT scanning and three-dimensional reconstruction of the modeled area to assess osteochondral repair. Three replicates were prepared for each group.

[0192] 1.10 Histological Staining of Postoperative ACL Reconstruction Sections

[0193] Tissue blocks from the repair site (ACL graft and bone tunnel) were fixed with 4% paraformaldehyde and decalcified with 10% EDTA (pH 7.4). After decalcification, the tissue was embedded in paraffin. Sections were then stained with HE, Masson's, toluidine blue, and COL1 to further investigate tendon-bone interface healing.

[0194] 2 Experimental results

[0195] 2.1 Drug concentration screening

[0196] The bioactivity of hydrogels with different drug loading contents is as follows Figure 22 The concentrations used in all experiments were based on the drug concentration. Figure 22 As shown in A, all OD values decreased with increasing concentration, and cell activity increased with increasing culture time; Figure 22 As shown in Figure 2, cell survival rates in all groups were around 100% on the first day, indicating a safe concentration range. On the fourth day, cell survival rates decreased to varying degrees. By the seventh day, cell survival rates in all groups increased significantly, reaching around 200%, with the 10 μM concentration showing the most significant improvement.

[0197] 2.2 Cytotoxicity and proliferation assays

[0198] In this study, L929 cells and MC3T3 E1 cells were selected as seed cells for the ligament layer and bone layer, respectively. Cell proliferation was assessed using the CCK-8 assay, and cell growth and morphology were observed using live-dead staining.

[0199] In the ligament layer, such as Figure 23 As shown in the figure, L929 cells were seeded on 10% SF (10% SF-ALs), 20% SF (20% SF-ALs), and 30% SF (30% SF-ALs) materials and cultured for 1, 4, and 7 days. With increasing culture time, the number of cells on each material surface showed a gradual increase. Comparison of OD values across the different groups revealed that the 10% SF-coated material exhibited higher cell proliferation, indicating that SF treatment improves cytocompatibility. Furthermore, analysis of cell proliferation rates revealed an overall favorable growth trend for L929 cells, with some differences in proliferation observed across the different materials. While differences between the groups were relatively small at 1 and 4 days of culture, OD values increased significantly by 7 days, indicating a significant increase in proliferation advantage, indicating that SF modification enhances the adhesion and proliferation of L929 cells on the material surfaces. In addition, in the 10% SF-ALs group, the OD value of L929 cells was the highest at 7 days, reaching 2.8 times that of the 30% SF-ALs group, which was significantly better than other groups, further verifying the advantages of this material in cell compatibility.

[0200] In the osteoblast layer, the cells in each group grew with time. On the first day, the OD values between the groups were not much different. As time went by, the MC3T3-E1 cells in each group showed a growth trend, and the growth rate of the SHI group was the fastest. The live-dead staining results corresponded to the CCK-8 results. Figure 24 It can be seen that the cells adhered and stretched well in the scaffold, with few dead cells. The cell growth trend in each group was obvious, which further demonstrated that SHI had good biocompatibility in the osteogenic layer.

[0201] 2.3 Cytoskeleton staining

[0202] To observe the adhesion and spreading of L929 and MC3T3-E1 cells on the scaffolds, the cell nuclei and cytoskeleton were stained red and blue with DAPI and phalloidin, respectively, on day 7 of culture.

[0203] Figure 25Figure A shows a high number of L929 cells on the scaffold, with dense distribution and high cell aggregation in some areas. The 10% SF-ALs group exhibited the best cell spreading, with clear F-actin filaments, indicating that these conditions were suitable for cell growth. While the other groups also exhibited some spreading, F-actin staining revealed a relatively compact cytoskeletal structure in some areas, likely due to the high cell density, which limited the spreading of individual cells. This phenomenon may affect their interaction with the scaffold and potentially impact the biocompatibility of the material.

[0204] MC3T3 cells showed good spreading ability on the scaffold. Figure 25 Figure B shows that cells failed to fully spread on the S-group hydrogel, likely due to the material's surface properties, resulting in insufficient cell adhesion. Cells partially spread on the SH-group hydrogel, with a clear skeletal structure, indicating that the material supports cell growth, but some cells still did not fully spread. The SHI-group hydrogel had the highest cell density and well-spread cells, with intact and clear F-actin structures and interconnected cells, indicating that this scaffold material may be most suitable for the growth of MC3T3 cells.

[0205] 2.4 Osteogenesis Induction Results

[0206] ALP is an early marker of osteoblasts. Increased ALP activity usually indicates the initiation of osteogenic differentiation. ALP staining results are used to evaluate the early differentiation of osteoblasts. Figure 26 As shown in A, the surface of each group showed different degrees of blue-purple staining, and the staining depth and distribution were also different, indicating that MC3T3 cells underwent osteogenic differentiation on the surface of the scaffold. There were also differences in the osteogenic differentiation of materials in different groups. Among them, the SHI group showed denser and deeper blue-purple deposition, indicating stronger osteogenic ability. ARS staining is used to detect cell mineralization, especially in osteogenic differentiation studies, to observe calcium deposition. Figure 26 As shown in Figure B, all groups showed red staining, indicating that MC3T3 cells underwent a certain degree of mineralization on the scaffold. However, differences in osteogenic induction or scaffold material between groups affected the level of calcium deposition. The SHI group showed denser and darker red deposits, indicating greater calcium deposition and stronger osteogenic capacity.

[0207] 2.5 Rabbit ACL defect model

[0208] This study used New Zealand rabbits as an animal model for in vivo validation. Three experimental groups were set up: a blank group underwent only tendon bone defect modeling, a control group underwent only scaffold implantation, and an experimental group underwent drug-loaded hydrogel scaffold implantation. The animals gradually gained weight in each group, with no significant differences between the groups. Postoperatively, the subjects maintained normal diet, exercise, and mental well-being, with no significant abnormalities observed. Samples were taken four and eight weeks after implantation, revealing no inflammation, suppuration, or sample breakage or dislocation. Figure 27 Micro CT evaluation of defect repair four weeks after surgery is presented. Three-dimensional reconstructed and pseudo-color images show a significant increase in new bone tissue and visible trabecular bone structure in the SHI group, while the blank control group exhibited a significant cavity in the defect area, indicating limited bone tissue self-repair capacity.

[0209] Figure 28 The histological manifestations of different experimental groups during the tissue repair process were displayed, and the tissue morphology, collagen deposition and repair of new tissue were evaluated. Figure 28 Figure A shows the overall morphology of the tissue defect area observed through HE staining. There were some differences in tissue filling and repair between experimental groups. Some groups showed relatively uniform tissue regeneration, while others still showed fibrous tissue hyperplasia or inflammatory reactions.

[0210] Masson staining dyes collagen fibers blue and cell matrix red, and is used to evaluate collagen deposition. Figure 28 As shown in Figure B, the amount of collagen deposition varied between groups. Some experimental groups exhibited more pronounced blue areas, indicating greater collagen deposition. The dense and orderly arrangement of blue collagen fibers indicates good ECM formation, which aids tissue repair. In contrast, some groups exhibited less collagen deposition or a looser arrangement, potentially indicating poorer repair outcomes.

[0211] Figure 28 C: Different groups showed varying degrees of blue-purple staining in the repaired area. The SHI group had more uniform blue-purple areas, indicating better cartilage ECM formation and abundant GAG deposition, which facilitates cartilage repair. The other groups had lighter blue-purple staining, indicating less cartilage ECM formation and possibly weaker repair.

[0212] CD31 can be used to assess the formation of new blood vessels. The extent of CD31 staining in the repair area varied between experimental groups. Further observation of vascular structure revealed a widespread distribution of CD31-positive signals in the SHI group, suggesting a well-developed vascular network in the repaired tissue. However, weaker CD31 staining in the other groups indicated less angiogenesis.

[0213] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. Application of a hydrogel scaffold in the preparation of a drug for promoting tendon-bone healing, wherein the hydrogel scaffold is prepared from silk fibroin, nanohydroxyapatite, and icariin; Preferably, the silk fibroin is methacrylated silk fibroin.

2. The use according to claim 1, characterized in that The concentration of icariin is 10-10000 μM; Preferably, the concentration of icariin is 10 μ m ; Preferably, the silk fibroin is methacrylated silk fibroin.

3. The use according to claim 1, characterized in that The ratio of silk fibroin to nanohydroxyapatite is 1:(0.25-0.75).

4. The use according to claim 1, characterized in that The medicament also includes artificial ligaments.

5. The use according to claim 4, characterized in that The artificial ligament is a silk fiber artificial ligament; Preferably, the silk fibroin artificial ligament is a silk fibroin artificial ligament coated with SF; Preferably, the concentration of SF is 10%-30%; Preferably, the concentration of SF is 10%.

6. The use according to claim 1, characterized in that The preparation method of the hydrogel scaffold comprises: preparing a hydrogel using methacrylylated silk fibroin and hydroxyapatite, and mixing the hydrogel with icariin-loaded nanoparticles; Preferably, the methacrylylated silk fibroin is prepared by mixing silk fibroin with glycidyl methacrylate; Preferably, the hydrogel is prepared using a photocrosslinking method; Preferably, the photoinitiator used in the photocrosslinking method is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate; Preferably, the icariin-loaded nanoparticles are prepared by ultracentrifugation.

7. The use according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

8. Use of a hydrogel scaffold in preparing a product for repairing or treating soft tissue damage, wherein the hydrogel scaffold is prepared from silk fibroin, nanohydroxyapatite and icariin.

9. Use of a hydrogel scaffold for promoting MC3T3 cell growth or in the preparation of a drug for promoting MC3T3 cell growth, wherein the hydrogel scaffold is prepared from silk fibroin, nanohydroxyapatite, and icariin; Preferably, the promoting the growth of MC3T3 cells is promoting the growth of MC3T3 cells in vitro for non-therapeutic purposes.

10. A method for promoting the growth of MC3T3 cells, characterized in that: The method comprises the steps of using a hydrogel scaffold for culturing, wherein the hydrogel scaffold is prepared from silk fibroin, nano-hydroxyapatite and icariin.

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