Hydrogel complex as well as preparation method and application thereof

Abstract

The invention relates to the field of biological materials and drug delivery, in particular to a hydrogel complex as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing PLGA-PFD microspheres: dissolving 200mg of polylactic acid-glycolic acid copolymer in dichloromethane to form a solution A, adding 2-20mg of pirfenidone into the solution A, sequentially adding 2% of gelatin solution, 0.5% of polyvinyl alcohol and 0.5% of gelatin solution, uniformly mixing, removing an organic solvent, and freeze-drying to fix the form to form the PLGA-PFD microspheres; preparation of the hydrogel complex: dispersing the PLGA-PFD microspheres in a hydrogel matrix, uniformly stirring to prepare a hydrogel solution, printing the hydrogel solution into printing hydrogel by adopting a 3D printing technology, modifying the printing hydrogel by using antibacterial peptide to obtain the hydrogel complex, and drying the hydrogel complex to obtain the PLGA-PFD hydrogel complex. Slow release and controlled release of the PFD and the same period of chronic inflammation in the bone healing period are realized.

Description

Technical Field

[0001] The present invention relates to the field of biomaterials and drug delivery, and in particular to a hydrogel complex and a preparation method and application thereof. Background Art

[0002] In modern warfare and military operations, blast injuries, gunshot wounds, and burns are complex and severe, posing a significant threat to military life and combat effectiveness. Among postoperative trauma repair materials, hydrogels are highly flexible and can be 3D printed. The development of hydrogel dressings with rapid hemostasis, antibacterial properties, and anti-inflammatory properties can address battlefield emergency care and improve treatment efficiency. Hydrogel materials that promote tissue regeneration have enormous potential for wound repair, particularly for complex injuries such as burns and blasts, and play a significant role at the intersection of national defense and biomedical sciences. In recent years, the rapid development of tissue engineering technology has provided new therapeutic strategies and technical support for bone repair. New regenerative medicine materials offer excellent safety, controlled degradation, a low risk of immune rejection, and flexible printing, enabling innovative applications of core bioregenerative medicine materials within the national defense and military industries. Traditional drug delivery systems (such as oral or parenteral) suffer from uneven drug distribution and insufficient local concentration, making them ineffective in treating localized infections and fibrotic diseases.

[0003] Among antimicrobial peptides (AMPs), lysine is a class of small peptides with broad-spectrum antimicrobial activity, effectively inhibiting the growth and reproduction of bacteria, fungi, viruses, and even some parasites. Their mechanism of action primarily involves disrupting the integrity of microbial cell membranes, leading to leakage of cellular contents and thus killing pathogens. Compared with traditional antibiotics, AMPs offer significant advantages such as reduced resistance to drug resistance, rapid action, and low host cell toxicity, thus demonstrating significant potential in the field of anti-infective therapy. However, the clinical application of AMPs still faces numerous challenges, the most prominent of which is their poor stability and susceptibility to protease degradation.

[0004] Pirfenidone (PFD) is a new pyridone compound with broad-spectrum antifibrotic properties that can prevent and reverse fibrosis and scar formation. Approved by the US Food and Drug Administration, it is the first drug to demonstrate efficacy in idiopathic pulmonary fibrosis (IPF) and scar inhibition through repeated, randomized, placebo-controlled Phase III clinical trials. The drug also demonstrates promising efficacy in fibrotic diseases such as renal interstitial fibrosis and hepatic fibrosis, with a modest inhibitory effect on fibroblasts. Despite its diverse medicinal applications, PFD suffers from difficult-to-control release rates in traditional administration methods (such as oral administration or local injection) for the treatment of grade 1 infected bone defects, resulting in fluctuating drug concentrations. Summary of the Invention

[0005] To solve the above problems, the present invention provides a hydrogel complex and a preparation method and application thereof.

[0006] A method for preparing a hydrogel composite comprises the following steps: Preparation of PLGA-PFD microspheres: Dissolve 200 mg of poly(lactic-co-glycolic acid) in dichloromethane to form solution A. Add 2-20 mg of pirfenidone to solution A. Then, add 2% gelatin solution, 0.5% polyvinyl alcohol, and 0.5% gelatin solution in that order. Mix thoroughly, remove the organic solvent, and freeze-dry to fix the morphology to form PLGA-PFD microspheres. Preparation of hydrogel complex: PLGA-PFD microspheres are dispersed in a hydrogel matrix and stirred evenly to prepare a hydrogel solution. The hydrogel solution is printed into a printed hydrogel using 3D printing technology, and the printed hydrogel is modified with antimicrobial peptides to obtain a hydrogel complex.

[0007] Preferably, the antimicrobial peptide is polylysine.

[0008] Preferably, the printed hydrogel is immersed in a 10% polylysine solution for 40 h to 50 h and freeze-dried to obtain a hydrogel complex.

[0009] Preferably, during the preparation of the hydrogel matrix, stirring is performed for 2 to 5 hours to achieve uniform dispersion and expel air bubbles.

[0010] Preferably, during the preparation of the PLGA-PFD microspheres, stirring is performed for 5 to 10 hours to mix the microspheres uniformly, thereby promoting the formation of a cross-linked network between the amino groups in the gelatin and the carboxyl groups in the sodium alginate.

[0011] Preferably, during the preparation of the PLGA-PFD microspheres, stirring is performed for 5 to 10 hours to remove the organic solvent.

[0012] Preferably, the hydrogel matrix is ​​prepared by mixing a 40% by mass gelatin solution and a 30% by mass sodium alginate solution in a volume ratio of 1:1, uniformly dispersing the mixture and removing bubbles to obtain a gelatin / sodium alginate premix, and adding 6% by mass lithium magnesium silicate and fully dispersing the mixture to form a hydrogel matrix. The hydrogel complex prepared by the preparation method.

[0013] Application of the hydrogel complex in the preparation of tissue repair products.

[0014] Preferably, the tissue repair refers to infectious wound repair, bone tissue engineering and anti-fibrosis treatment.

[0015] Preferably, the bone tissue engineering is infectious bone defect.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The hydrogel complex of the present invention can achieve sustained and controlled release of PFD through PLGA microspheres in the same cycle as chronic inflammation during bone healing. At the same time, the degradation time of PLGA is relatively long, about 5-6 weeks, which realizes the control of release rate.

[0017] The hydrogel complex of the present invention can deliver antimicrobial peptides and bifenidone in a dual-track manner, and can be applied to infectious wound repair, bone tissue engineering and anti-fibrosis treatment.

[0018] This invention innovatively applies PFD to orthopedic conditions, using PLGA (poly(lactic-co-glycolic acid)) microspheres encapsulated with PFD-antimicrobial peptide EPL for the treatment of grade 1 infected bone defects. Its low hydrophilicity enables it to maintain high mechanical strength during the early healing phase, making it suitable for fracture repair. In the early stages of fracture healing (e.g., within 8 weeks), the callus cannot withstand high loads, and PLGA 85:15 provides adequate support. As bone healing progresses, its mechanical properties gradually decline, matching the increased load-bearing capacity of the bone. The degradation rate of PLGA matches the time window for bone repair. The slower degradation rate of PLGA 85:15 allows for long-term support during the bone remodeling phase.

[0019] The high drug loading and targeting of the microspheres can reduce systemic exposure and lower side effects. The surface properties of the 10μm microspheres can optimize their retention time in bone tissue. The pore structure between the microspheres can promote cell migration and tissue regeneration. The specificity of the PLGA material can protect PFD from enzymatic hydrolysis and oxidation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the XRD of the hydrogel complex, where A is the XRD of the original hydrogel, B is the XRD of the modified hydrogel complex, PFD represents bifenadone, Lap represents hydroxyapatite, Mix represents the common original hydrogel complex, Ant represents antimicrobial peptide, Apa represents gelatin, and Alg represents sodium alginate.

[0021] Figure 2 For hydrogels only than pheniramine infrared detection.

[0022] Figure 3 IR detection of antimicrobial peptide EPL in hydrogels only.

[0023] Figure 4 Infrared detection of hydrogel complex.

[0024] Figure 5Coupling of hydrogel complex HPLC drug release.

[0025] Figure 6 The above is the release curve of hydrogel-loaded PFD, which shows the HPLC drug release at different times, A is 0h, 0.5h, 1h and 6h, and B is 24h, 72h, 96h and 4 weeks.

[0026] Figure 7 Schematic diagram of the overall function of hydrogel.

[0027] Figure 8 This is the design diagram of the hydrogel 3D printer, where AB is the original diagram for constructing the hydrogel three-dimensional structure, and CF is a panoramic and planar schematic diagram of a single-layer hydrogel.

[0028] Figure 9 This is the overall design drawing.

[0029] Figure 10 Scanning electron microscope image of PLGA-PFD microspheres.

[0030] Figure 11 The antibacterial experiment of the hydrogel complex is shown in Figure 2. A is the antibacterial ring. (1) in A is a hydrogel with a size of 10mm×10mm×3mm and co-cultured for 0h. (2) in A is a hydrogel with a size of 10mm×10mm×3mm and co-cultured for 48h. (3) in A is a hydrogel with a size of 15mm×5mm×3mm and co-cultured for 0h. (4) in A is a hydrogel with a size of 15mm×5mm×3mm and co-cultured for 48h. B is bacterial culture. (1) in B is 1×10 8 U, B (2) is 1×10 7 U, B (3) is 1×10 6 U.

[0031] Figure 12 Figure 3 is the biological toxicity and efficacy test of bifenidone on fibroblasts (CCK8). Compared with the group without drug addition, * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001.

[0032] Figure 13 Figure 3 is the biological toxicity test of bifenidone on BMCS (CCK8), * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001.

[0033] Figure 14 This is the DR result of the animal experiment, indicating that the sores on the experimental animals gradually became larger after modeling.

[0034] Figure 15Micro-CT results show confirmed infection. A is a three-dimensional scan of the radius, and B is a three-dimensional visualization image.

[0035] Figure 16 Schematic diagram of animal surgery and CRP confirmation of infection model: The CRP test performed after the operation showed that the experimental animals had entered the infection state and the model was successful.

[0036] Figure 17 Cytoskeleton and cell nucleus analysis after co-culture of hydrogel with fibroblasts and bone marrow mesenchymal stem cells.

[0037] Figure 18 HE staining of the radius of the control group.

[0038] Figure 19 HE staining of radius in the experimental group. DETAILED DESCRIPTION

[0039] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0040] The PLGA-PFD microspheres in this invention maintain the effective concentration of the drug for a long time and achieve a sustained-release effect. As an advanced drug carrier, poly(lactic-co-glycolic acid) (PLGA) microspheres can significantly prolong the duration of effective drug concentration in the body and achieve a sustained-release effect. This property gives PLGA microspheres significant advantages in drug delivery systems, effectively enhancing the therapeutic efficacy of drugs while reducing dosing frequency and lowering drug toxicity and side effects.

[0041] The sustained-release mechanism of PLGA-PFD microspheres is primarily based on their biodegradability. In the body, the PLGA microspheres gradually degrade, resulting in sustained drug release, maintaining drug concentrations at therapeutically effective levels for extended periods. This sustained-release property not only improves drug bioavailability but also reduces peak drug concentrations in the body, minimizing potential toxicity to normal tissues.

[0042] Furthermore, the physicochemical properties of PLGA-PFD microspheres, such as particle size, composition, and surface properties, can be flexibly adjusted to meet the needs of different drugs and treatments. By optimizing these parameters, PLGA microspheres can further improve drug stability and delivery efficiency, providing strong support for personalized medicine.

[0043] In summary, PLGA-PFD microspheres, as a highly efficient drug delivery carrier, significantly improve the therapeutic effect and safety of drugs through their sustained-release properties, showing broad application prospects.

[0044] The antimicrobial peptide EPL, as the active drug, exhibits broad-spectrum antimicrobial activity. It was physically immobilized on the surface via impregnation. Bifenidone, as the active drug, exhibits anti-fibrotic and anti-inflammatory effects. PLGA microspheres, with a particle size of 10 μm, encapsulate bifenidone to achieve sustained release, consistent with the bone infection cycle and enhancing efficacy. 8% calcium gluconate enhances the stability and mechanical properties of the hydrogel.

[0045] Preparation of hydrogel complexes, such as Figure 9 As shown: (1) Preparation of hydrogel matrix: 1) Spray the surface of the magnetic stirrer with 75% alcohol and wipe it with cotton balls for disinfection, place it in a clean bench, turn on the ultraviolet lamp, and irradiate it in the bench for 30 minutes; 2) Use a magnetic stirrer to prepare a 40% gelatin solution at 35°C and a 30% sodium alginate solution at room temperature, then mix the gelatin solution and the sodium alginate solution in a ratio of 1:1, continue stirring at 35°C for 2 hours, and obtain a gelatin / sodium alginate premix; 3) Add 6% lithium magnesium silicate by weight and ultrasonicate for 30 minutes to form a hydrogel matrix. The prepared hydrogel matrix makes the hydrogel complex have good printing performance.

[0046] (2) Preparation of PLGA-PFD microspheres: 200 mg of PLGA 85:15 was dissolved in 15 mL of dichloromethane by the double emulsion solvent evaporation method to form solution A. Pirfenidone was added to solution A at different doses (2, 5, 10, 15, and 20 mg). Solution A was used as the oil phase. After fully dissolved, it was added to 10 mL of 2% gelatin solution to form a colostrum solution. The colostrum solution was quickly poured into 100 mL of 0.5% PVA (polyvinyl alcohol) and stirred at 10,000 r / min for 10 s in an ice bath to form a double emulsion solution. The double emulsion solution was poured into 500 mL of 0.5% gelatin solution and magnetically stirred at room temperature for 6 h to remove the residual organic solvent. The solution was mechanically stirred at 2,000 rad at 40 °C for 6 h, and then washed three times with anhydrous ethanol to remove the organic solvent. The solution was freeze-dried for 24 h to fix the morphology to form PLGA-PFD microspheres. The prepared PLGA-PFD microspheres were unidirectionally distributed with a size of 20 μm.

[0047] (3) PLGA-PFD microspheres were collected and freeze-dried and stored at -80°C. The drug loading of PLGA-PFD microspheres was observed under transmission electron microscopy and stored in a -20°C refrigerator.

[0048] (4) Dispersing the PLGA-PFD microspheres prepared in step (2) in the hydrogel matrix prepared in step (1) and stirring evenly to prepare a hydrogel solution.

[0049] (5) The hydrogel solution prepared in (4) was printed into a printed hydrogel using 3D printing technology at 40°C. During printing, the hydrogel solution was printed layer by layer into the desired shape using additive manufacturing equipment, and 8% calcium gluconate crosslinking agent was added to achieve the final shape. Figure 8 shown.

[0050] (6) Modify the printed hydrogel to obtain a hydrogel complex: dissolve 10 g of antimicrobial peptide polylysine (EPL) in 90 mL of deionized water to form a 10% polylysine solution. Soak the 15 mm × 15 mm × 3 mm printed hydrogel in 15 mL of 10% polylysine solution for 48 h and freeze-dry to obtain a hydrogel complex.

[0051] To enhance the biocompatibility and cell adhesion of the hydrogel, the printed hydrogel was completely immersed in a 10% pure water solution of polylysine to ensure uniform adsorption of polylysine molecules on its surface. Immersion treatments were performed for varying durations (e.g., 12, 24, and 48 hours). The distribution of polylysine on the hydrogel surface was observed using scanning electron microscopy (SEM). Cell experiments verified its effect on promoting cell adhesion and growth. It was found that a 48-hour immersion time achieved an ideal adsorption and uniform distribution of polylysine on the hydrogel surface, significantly enhancing the hydrogel's biocompatibility and cell adhesion.

[0052] The overall schematic diagram of hydrogel function is as follows Figure 7 As shown, a. Preparation of 3D designed hydrogel: According to the specific conditions of the bone infected defect wound, personalized hydrogel is designed and prepared using three-dimensional (3D) printing technology. This design ensures that the hydrogel can accurately match the geometry of the defect site, thereby achieving the best therapeutic effect. b. Biocompatibility and drug delivery of hydrogel complexes: The resulting hydrogel complex has excellent biocompatibility, can establish close contact with the host tissue, and reduce immune rejection. In addition, drugs or cells can be loaded in the hydrogel to achieve local delivery of drugs or cells and enhance the therapeutic effect. c. Biological effects of hydrogels: Hydrogels exhibit stable drug release characteristics in the body, have high biological activity, and can effectively promote tissue repair. Its metabolic rate is controllable and the immune response is small, which helps to reduce side effects during treatment.

[0053] Verification experiment 1. Physical characterization X-ray diffraction was used to analyze the 3D printing raw materials gelatin, sodium alginate, lithium magnesium silicate, and the hydrogel composite after printing.

[0054] See the results Figure 1 The X-ray diffraction peaks of gelatin, sodium alginate, lithium magnesium silicate, and the hydrogel composite are each uniquely representative. The peaks of lithium magnesium silicate are primarily located at 2θ values ​​of 20°, 35°, and 61°, those of gelatin are primarily located at 2θ values ​​of 22°, and those of sodium alginate are primarily located at 2θ values ​​of 13°, indicating that the quality of these raw materials meets the requirements. The diffraction peaks of the hydrogel composite are primarily located at 2θ values ​​of 22°, 35°, and 61°, reflecting the presence of all the raw materials in the composite material.

[0055] The broad 2θ diffraction peak at 12-33° for gelatin indicates moderate crystallinity. The loss of the 13° peak for sodium alginate is likely due to the strong interaction between sodium alginate and gelatin, and the formation of a more regular structure in the composite hydrogel after crosslinking with calcium ions. The decrease in the 35° and 61° peaks in the hydrogel composite is likely due to the nanostructured structure of lithium magnesium silicate, which has a very small particle size and is tightly bound to the composite hydrogel due to charge adsorption, resulting in lower crystallinity in the hydrogel composite. This suggests that the hydrogel composite forms a regular, stable structure.

[0056] The infrared spectrum results are as follows Figures 2 to 4 As shown, the biocomposite of the hydrogel composite has obvious peaks at 2943 cm', 1418 cm1, 1640 cm·, and 1013 cm", which are characteristic absorption peaks of gelatin sodium alginate and lithium magnesium silicate, respectively, indicating that the main components of the raw materials in the sample have not changed after mixed printing. The characteristic absorption peaks of lithium magnesium silicate all appear in the hydrogel composite, indicating that the layered crystal structure of lithium magnesium silicate in the hydrogel composite has not changed and functions in the mixture in the form of nanosheets.

[0057] Figure 2 Medium, 3446.82cm -1 :This absorption peak is usually associated with OH stretching vibration and may come from the water or hydroxyl groups contained in the hydrogel; 2925.90cm -1 and 2853.20cm -1 :These two absorption peaks are usually associated with CH stretching vibration, indicating that the hydrogel contains alkyl chains; 2011.99cm -1 :This peak may be related to C≡N (cyano) stretching vibration, suggesting that the hydrogel may contain nitrogen-containing compounds; 1665.15cm -1 : This absorption peak is usually associated with C=O stretching vibration and may indicate the presence of an amide bond. It is a characteristic absorption peak of gelatin; 1631.16 cm -1 and 1492.13cm -1:These two peaks may be related to NH bending vibration, further supporting the hypothesis that the hydrogel contains protein components; 1459.86cm -1 :This peak may be related to CH bending vibration, which once again emphasizes the presence of alkyl chains in the hydrogel; 1387.87cm -1 and 1254.57cm -1 :These absorption peaks may be related to CN stretching vibration, indicating that the hydrogel contains carbamate bonds; 1156.33cm -1 and 953.25cm -1 : These peaks may be related to CO stretching vibrations, commonly observed in polysaccharides such as sodium alginate; 526.23 cm -1 : This absorption peak may be related to C-Cl stretching vibration, suggesting that the hydrogel may contain chlorine-containing compounds. Figure 3 Medium, 3441.08cm -1 : This absorption peak is usually associated with OH stretching vibration, indicating the presence of hydroxyl groups in the hydrogel; 3062.18 cm -1 and 2923.17cm -1 :These absorption peaks are usually associated with CH stretching vibration, indicating that the hydrogel contains alkyl chains; 2852.46cm -1 :This absorption peak is also related to CH stretching vibration, further supporting the conclusion that alkyl chains exist in the hydrogel; 1874.64cm -1 and 1811.72cm -1 :These absorption peaks may be related to C=O stretching vibration, indicating that the hydrogel contains carbonyl groups; 1585.96cm -1 and 1530.89cm -1 :These absorption peaks are usually associated with NH bending vibration, indicating that the hydrogel contains amino groups; 1490.27cm -1 :This absorption peak may be related to CH bending vibration, further supporting the conclusion that there are alkyl chains in the hydrogel; 1455.71cm -1 :This absorption peak is usually associated with CH bending vibration, indicating that the hydrogel contains alkyl chains; 1428.03cm -1 :This absorption peak may be related to the CN stretching vibration, indicating that the hydrogel contains carbamate bonds; 1381.59cm -1 and 1320.77cm -1 :These absorption peaks may be related to CN stretching vibration, further supporting the conclusion that the hydrogel contains urethane bonds; 1275.69cm -1 and 1211.45cm -1 :These absorption peaks may be related to CO stretching vibration, indicating that the hydrogel contains ether bonds; 1184.31cm -1and 1136.39cm -1 :These absorption peaks may be related to CO stretching vibration, further supporting the conclusion that the hydrogel contains ether bonds; 1031.65cm -1 :This absorption peak may be related to COC stretching vibration, indicating that the hydrogel contains ether bonds; 1041.48cm -1 and 991.36cm -1 :These absorption peaks may be related to CO stretching vibration, further supporting the conclusion that the hydrogel contains ether bonds; 889.24cm -1 and 826.88cm -1 :These absorption peaks may be related to CH bending vibration, indicating that the hydrogel contains alkyl chains; 701.20cm -1 :This absorption peak may be related to C-Cl stretching vibration, suggesting that the hydrogel may contain chlorine-containing compounds; 611.31cm -1 :This absorption peak may be related to C-Br stretching vibration, suggesting that the hydrogel may contain bromine-containing compounds; 529.45cm -1 、504.17cm -1 and 478.46cm -1 : These absorption peaks may be related to skeleton vibration, indicating that the hydrogel contains a specific skeleton structure. Figure 4 Medium, 3277.27cm -1 : This absorption peak is usually associated with OH or NH stretching vibration, indicating that the hydrogel contains hydroxyl or amino groups; 2937.68cm -1 and 2875.70cm -1 :These two absorption peaks are usually associated with CH stretching vibration, indicating that the hydrogel contains alkyl chains; 1656.82cm -1 : This absorption peak is usually associated with C=O stretching vibration and may indicate the presence of an amide bond. It is a characteristic absorption peak of many biological macromolecules such as proteins and polysaccharides; 1533.01 cm -1 :This absorption peak may be related to NH bending vibration, further supporting the hypothesis that the hydrogel contains amino groups; 1455.40cm -1 and 1407.29cm -1 :These absorption peaks may be related to CH bending vibration, which once again emphasizes the presence of alkyl chains in the hydrogel; 1245.78cm -1 : This absorption peak may be related to CO stretching vibration and is usually observed in polysaccharides such as sodium alginate; 1021.69cm -1 :This absorption peak may be related to COC stretching vibration, indicating that the hydrogel contains ether bonds; 448.22cm -1 : This absorption peak may be related to skeleton vibration, indicating that the hydrogel contains a specific skeleton structure.

[0058] Scanning electron microscope images of PLGA-PFD microspheres are shown in Figure 2. Figure 10 As shown, the microspheres are unidirectionally distributed with a diameter of 20 μm.

[0059] To observe the growth status of the third generation fibroblasts and BMCS cells after 7 days of culture, phalloidin staining was used to show the overall cell skeleton. After counterstaining the nucleus, the morphology of individual cells was observed under a high-power microscope. As shown in the figure, the cells can show spindle-shaped or polygonal extension in the hydrogel complex, cell proliferation and division, and cell contact, which can indicate that the cells have a good growth state in the hydrogel complex. The results are shown in the figure. Figure 17 As shown, the first row shows a fibroblast control group. The second row shows fibroblasts co-cultured with different concentrations of hydrogel complexes, at loading levels of 2, 5, 10, and 20 mg / ml PFD. Cell morphology changed significantly and cells gradually died. The third row shows BMCS co-cultured with different concentrations of hydrogel complexes, at loading levels of 2, 5, 10, and 20 mg / ml EPL, with no significant effect.

[0060] 2. Drug release experiment The prepared hydrogel complex was placed in artificial bone marrow fluid and an in vitro release experiment was carried out at 37°C.

[0061] Samples were taken regularly and the release of antimicrobial peptides and bifenidone was determined by high performance liquid chromatography (HPLC).

[0062] Experimental procedures HPLC drug release profile (1) Sample preparation Cut the PLGA-PFD microspheres into samples of appropriate size to ensure consistent drug loading in each sample. Place the samples individually into dialysis bags with a molecular weight cutoff (MWCO) of 8,000 to 14,000.

[0063] (2) Dialysis conditions The dialysis bag was placed in 100 mL of PBS buffer solution at pH 7.4 and the temperature was set to 37°C.

[0064] Samples were taken at the following time points: 0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, and 12 h.

[0065] 2 mL of dialysate was sampled each time for HPLC analysis, and an equal amount of fresh dialysate was added in time.

[0066] (3) HPLC analysis Chromatographic column: C18 reverse phase column (such as Agilent ZorbaxSB-C18, 4.6 mm × 150 mm, 5 μm).

[0067] Mobile phase: water-acetonitrile-trifluoroacetic acid (TFA)-triethylamine (72:28:0.1:0.15).

[0068] Flow rate: 1.0 mL / min.

[0069] Column temperature: 30℃.

[0070] Detection wavelength: 314nm~310nm.

[0071] Injection volume: 20 μL.

[0072] (4) Data processing A standard curve of bifenidone was drawn to ensure a linear relationship between drug concentration and peak area.

[0073] The drug concentration at each time point was calculated based on the standard curve.

[0074] The cumulative drug release rate was calculated as the ratio of the total mass of released drug to the drug loading amount.

[0075] The release curve is drawn with time as the horizontal axis and the cumulative drug release rate as the vertical axis.

[0076] The results are as follows Figure 5 As shown in Table 1, 0 hours (blue dashed line): represents the PFD content of the hydrogel complex before the start of the release experiment; 0.5 hours to 12 hours (pink to yellow dotted line): shows the rapid release of PFD in the initial stage, and the peak area increases rapidly, indicating that the initial release rate of the drug in the hydrogel is high; 24 hours to 96 hours (green solid line to dark green dotted line): shows that the release rate of PFD begins to slow down, and the increase in the peak area decreases, indicating that the drug release enters a slower stage; 4 weeks to 8 weeks (brown to purple dotted line): shows the long-term release behavior of PFD, and the peak area continues to increase slowly, indicating that the hydrogel complex can achieve sustained release of PFD. The hydrogel complex released 40.8% of antimicrobial peptides within 72 hours and 60% of bifenidone in 4 weeks, and the release rate was consistent with the Higuchi kinetic model.

[0077] The release profile of PFD is as follows Figure 6As shown, in the initial state of 0 hours (blue dotted line), the release amount of PFD is low; in the time period of 0.5 hours to 12 hours (pink to yellow dotted line), the release amount of PFD increases significantly, showing the rapid release of the drug in the initial stage; over time, from 24 hours to 96 hours (green solid line to dark green dotted line), the release rate of PFD gradually slows down, indicating that the drug release enters a slower stage; in the long-term observation of 4 weeks to 8 weeks (brown to purple dotted line), the release amount of PFD continues to increase slowly, showing that the hydrogel complex can achieve sustained release of PFD.

[0078] Release of EPL from the hydrogel: Initially, at 0 hours (blue dashed line), the amount of EPL released was also low. From 24 to 72 hours (red solid line to brown dashed-dotted line), the amount of EPL released increased significantly, demonstrating rapid initial drug release. Over time, from 96 hours to 4 weeks (green solid line to gray dashed-dotted line), the release rate of EPL gradually slowed, indicating that drug release entered a slower phase. During long-term observation at 8 weeks (white dashed-dotted line), the amount of EPL released continued to slowly increase, demonstrating that the hydrogel complex was capable of achieving sustained EPL release.

[0079] Table 1 Release data 3. Antibacterial performance test The hydrogel complexes with the sizes of 15mm×15mm×3mm and 10mm×10mm×3mm were mixed with 1×10 Staphylococcus aureus. 8 The cells were co-cultured in a U CO2 bacterial incubator for 0 h and 48 h, and the antibacterial activity was detected by inhibition zone test, with 3 parallel experiments in each group.

[0080] The results are as follows Figure 11 As shown in the figure, the hydrogel complex has a significant inhibitory effect on bacteria, with the diameter of the inhibition zone reaching 8 mm. A high concentration of Staphylococcus aureus was detected in the bone tissue samples of the infection group, with a colony count of 1.2×10 6 ±0.3×10 6 CFU / g bone tissue indicated that infection was successfully established. No bacterial growth was detected in the bone tissue samples of the control group, confirming the specificity of the infection.

[0081] 4. Anti-fibrosis performance experiment Fibroblasts were seeded at a density of 5000-10000 cells / well in a 96-well plate with 100 μL complete medium per well. 5 ng / ml TGF-β1 was added to stimulate the fibroblasts. After the cells attached (approximately 24 hours), different concentrations of pirfenidone (0, 0.01, 0.1, 0.5, 1.0, 1.5 and 2.0 mg / ml) were added and treated for 48 hours. A blank control group (without drug) and a DMSO control group (containing only 0.1% DMSO) were set up, with 5 replicate wells in each group, and the cells were cultured in an incubator.

[0082] Fibroblasts were seeded at a density of 5,000–10,000 cells / well in a 96-well plate with 100 μL of complete culture medium per well. After the cells had adhered (approximately 24 hours), the culture medium was replaced with medium containing various concentrations of bifenidone. A blank control group (drug-free) and a DMSO control group (containing only 0.1% DMSO) were established.

[0083] Five replicate wells were set up for each group and cultured in an incubator.

[0084] The cells were then transferred to a 15mm×15mm×3mm hydrogel complex and co-cultured with fibroblasts in a cell incubator with a stable temperature (37°C), a stable CO2 level (5%), a constant pH (pH: 7.2-7.4), and a high relative saturation humidity (95%) for 48 hours. The cell viability of the TGF-β1 stimulation group was significantly improved, with 5ng / ml being the optimal stimulation concentration of TGF-β1.

[0085] Subsequently, fibroblasts were treated with 5 ng / ml TGF-β1 and co-treated with different concentrations of pirfenidone (0, 0.01, 0.1, 0.5, 1.0, 1.5, and 2.0 mg / ml) for 48 h.

[0086] The experimental results are as follows Figure 12 As shown in the results, 0.1 mg / ml pirfenidone significantly inhibited TGF-β1-induced fibroblast proliferation (P < 0.05), with the inhibitory effect being even more pronounced at 1.5 mg / ml. However, increasing the pirfenidone concentration did not further enhance the inhibitory effect. Furthermore, the pirfenidone dose also significantly promoted the proliferation of bone marrow mesenchymal stem cells.

[0087] The anti-fibrosis effect was evaluated by detecting collagen secretion and cell proliferation. Figure 13 As shown, 1.5 mg / ml is the optimal concentration.

[0088] The results are as follows Figure 12-13 As shown in the results, the hydrogel complex can significantly inhibit the secretion of collagen and reduce the degree of fibrosis.

[0089] 5. Biocompatibility test Bone marrow mesenchymal stem cells were seeded at a density of 5,000-10,000 cells / well in a 96-well plate with 100 μL of complete culture medium per well. After the cells adhered (approximately 24 hours), the culture medium was replaced with various concentrations of bifenidone. A blank control group (drug-free) and a DMSO control group (containing only 0.1% DMSO) were set up.

[0090] Five replicate wells were set up for each group and cultured in an incubator.

[0091] The cells were then transferred to a 15 mm × 15 mm × 3 mm hydrogel complex and co-cultured therewith, and the cell survival rate was detected by CCK8 assay using the same method.

[0092] The results showed that the hydrogel complex had good biocompatibility. The cell differences before and after co-culture suspension were calculated using a cell counting plate, and the cell survival rate exceeded 85%.

[0093] 6. In vivo experiments of hydrogel complexes (1) Establishment of rabbit radius infected bone defect model 2% sodium pentobarbital (2.1 ml / kg) was injected into the ear vein. After anesthesia took effect, the hair on the right forelimb was trimmed. The rabbit was fixed in the supine position on a rabbit stand. After routine disinfection and draping, the skin and subcutaneous tissue were longitudinally incised along the radius in the middle of the limb. After thorough hemostasis, the radius was fully exposed. A bone defect of approximately 10 mm × 10 mm in length and width was created at the distal end of the radius with a bone drill. A 1.5 × 10 mm 8 CFU·ml -1 1 ml of Staphylococcus aureus (ATCC25923) was inoculated into the medullary cavity, and the incision was closed. After two weeks of continued feeding, bacterial culture was positive, establishing a rabbit bone infection model (Photo 3-1). Before implantation, the same surgical technique was used to thoroughly debride the distal radius, leaving a 10 mm long wound.

[0094] (2) Testing indicators 1. General observation After surgery, observe the animals' incision healing, whether there is redness, swelling, sinus formation, and whether the animals die.

[0095] 2. Determination of serum C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR).

[0096] Six animals were randomly selected from each group at six time points: before implantation and at 1, 3, 7, 2, and 4 weeks after surgery. 3.6 ml of venous blood was drawn from the marginal ear vein. Two ml of blood was used to quantitatively measure serum CRP using immunoturbidimetry with a special protein analyzer, and the remaining 1.6 ml was used to measure ESR using a Monitor-J automated erythrocyte sedimentation rate analyzer.

[0097] Serological test results such as Figure 16 As shown in Table 2, the white blood cell count and C-reactive protein (CRP) level in the infection group were significantly increased, which were 18.5×10 3 ±2.3×10 3 cells / μL and 12.4±1.8mg / L, indicating the presence of systemic inflammatory response.

[0098] Table 2 ESR results at various time points before and after surgery (mm·h -1 ) 3. Radiological observation After confirming successful infection model establishment by leukocyte detection, we used digital radiography (DR) to track defect healing at 2, 4, and 8 weeks. The entire right forelimb of the rabbit was dissected and perfused with 4% paraformaldehyde for X-ray imaging. The specimen was then gently separated from the muscle separator. The specimen was scanned using a microcomputed tomography system (YXLON, Germany), and three-dimensional images were reconstructed using VG Studio MAX software (Volume Graphics, Heidelberg, Germany) to analyze bone formation. Scanning parameters were set at 80 kV and 55.6 μA, with an image pixel size of 17 μm. The ratio of bone volume to total volume (BV / TV) was measured. Anteroposterior and lateral radiographs of the transplanted limb were obtained at each time point in each group according to the experimental design to observe the radiographic appearance of the graft.

[0099] To evaluate the effect of the hydrogel composite in the treatment of infected bone defects, we established a rat femoral infected bone defect model, e.g. Figure 16 This model involved surgically creating a critical bone defect in the mid-femoral region and implanting a gelatin sponge containing Staphylococcus aureus at the defect site to simulate common clinical bone infection. Four weeks after surgery, histological analysis, bacterial culture, and imaging examinations confirmed the success of the model. Figure 14 and Figure 15 shown.

[0100] Histological analysis Figure 18The infiltration of inflammatory cells (such as neutrophils and macrophages) and the distribution of inflammatory cells: Inflammatory cells are mainly distributed in the damaged area of ​​bone tissue, indicating that the bone defect site of the infection group has a significant inflammatory response, manifested as a large number of inflammatory cell infiltrations, including neutrophils and macrophages. The HE staining results are as follows Figure 19 The results showed that after hydrogel implantation, the bone tissue structure generally showed good continuity and integrity. Specifically, the following manifestations were observed: trabecular formation: New trabeculae gradually formed, revealing a clear trabecular structure. The trabeculae gradually became orderly and well-connected with the surrounding tissue. Bone marrow cavity recovery: The cellular composition within the bone marrow cavity gradually recovered, showing abundant bone marrow cells and vascular structure. The cells within the bone marrow cavity were neatly arranged, demonstrating a good tissue structure. This indicates that the bone tissue structure at the infection site was disordered, with broken trabeculae and the bone marrow cavity filled with inflammatory cells and necrotic tissue. In contrast, the bone tissue structure of the control group (uninfected) was intact, with orderly trabeculae and normal cellular composition within the bone marrow cavity.

[0101] Imaging tests (DR and CT scans) Figure 14 The infected group showed significant bone resorption and periosteal reaction at the bone defect site. X-rays revealed decreased bone density and thickened periosteum in the defect area, typical imaging features of infected bone defects. CT scans further revealed three-dimensional structural changes in the bone defect area, including trabecular disruption and expansion of the bone marrow cavity.

[0102] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0103] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0104] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing a hydrogel composite, characterized in that: The following steps are involved: Preparation of PLGA-PFD microspheres: Dissolve 200 mg of poly(lactic-co-glycolic acid) in dichloromethane to form solution A. Add 2-20 mg of pirfenidone to solution A. Then, add 2% gelatin solution, 0.5% polyvinyl alcohol, and 0.5% gelatin solution in that order. Mix thoroughly, remove the organic solvent, and freeze-dry to fix the morphology to form PLGA-PFD microspheres. Preparation of hydrogel complex: PLGA-PFD microspheres are dispersed in a hydrogel matrix and stirred evenly to prepare a hydrogel solution. The hydrogel solution is printed into a printed hydrogel using 3D printing technology, and the printed hydrogel is modified with antimicrobial peptides to obtain a hydrogel complex.

2. The preparation method according to claim 1, characterized in that The antimicrobial peptide is polylysine.

3. The preparation method according to claim 2, characterized in that The printed hydrogel was soaked in a 10% polylysine solution for 40 h to 50 h and freeze-dried to obtain a hydrogel complex.

4. The preparation method according to claim 1, characterized in that During the preparation of the hydrogel matrix, the mixture is stirred for 2 to 5 hours to achieve uniform dispersion and expel air bubbles.

5. The preparation method according to claim 1, characterized in that During the preparation of the PLGA-PFD microspheres, the mixture was stirred for 5 to 10 hours to mix thoroughly.

6. The preparation method according to claim 1, characterized in that During the preparation of the PLGA-PFD microspheres, stirring is performed for 5 to 10 hours to remove the organic solvent.

7. The preparation method according to claim 1, characterized in that Preparation of the hydrogel matrix: 40% by mass gelatin solution and 30% by mass sodium alginate solution were mixed in a volume ratio of 1:1, uniformly dispersed, and bubbles were removed to obtain a gelatin / sodium alginate premix. 6% by mass lithium magnesium silicate was added and fully dispersed to form a hydrogel matrix. The hydrogel complex prepared by the preparation method according to claim 1 .

9. Use of the hydrogel complex according to claim 8 in preparing tissue repair products.

10. The use according to claim 9, characterized in that The tissue repair refers to infected wound repair, bone tissue engineering and anti-fibrosis treatment.

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