A polyphenol-modified composite hydrogel scaffold and preparation method and application thereof

Abstract

This invention relates to a tissue repair material, disclosing a polyphenol-modified composite hydrogel scaffold, its preparation method, and its applications. The method includes the following steps: preparing a polycaprolactone scaffold using electrospinning technology; modifying bioactive glass with polyphenols and copper ions to obtain a polyphenol-modified copper-containing bioactive glass suspension; mixing the polyphenol-modified copper-containing bioactive glass suspension with a hydrogel solution and uniformly coating it onto the surface of the polycaprolactone scaffold; and performing a photocrosslinking reaction on the coated polycaprolactone scaffold to obtain a polyphenol-modified copper-containing bioactive glass composite hydrogel scaffold. This invention provides physical support for cell adhesion, arrangement, and proliferation, while also possessing multiple biological functions such as promoting angiogenesis, anti-oxidation, and immune regulation.

Description

Technical Field

[0001] This invention relates to a tissue repair material, and more particularly to a composite hydrogel scaffold. Background Technology

[0002] Traumatic bone injury, especially complex trauma accompanied by combined bone and soft tissue defects, remains a critical clinical challenge in regenerative medicine. These injuries are often caused by high-energy trauma, tumor resection, or advanced musculoskeletal diseases. Repairing them requires not only restoring the mechanical load-bearing function of bone but also simultaneously restoring the morphology and physiological function of soft tissues such as muscles, tendons, and skin. However, bone tissue and soft tissues (such as skin) differ significantly in mechanical properties, extracellular matrix (ECM) composition, and regenerative microenvironment, making the integrated regeneration of the hard-soft tissue interface a significant technical bottleneck.

[0003] The emergence of guided bone regeneration (GBR) membrane technology provides crucial strategic support for addressing this challenge. An ideal GBR membrane should possess both physical barrier properties and bioactivity, mechanically preventing soft tissue ingrowth while actively regulating and promoting underlying bone regeneration. In recent years, research focus has gradually shifted to developing bifunctional barrier membranes that mimic the structure of the natural periosteum. The natural periosteum, as a highly vascularized connective tissue covering the bone surface, not only plays a physical protective role but also precisely regulates the coupling process of osteogenic and angiogenesis through its intrinsic cellular and cytokine network. Therefore, constructing repair materials that can mimic the structure and function of the natural periosteum has significant research value and clinical implications for achieving integrated repair of musculoskeletal tissues.

[0004] Advances in tissue engineering have offered new insights into solving this challenge. An ideal bone and soft tissue repair scaffold should be able to reproduce the multi-scale structural features of natural tissues and possess the biological activity to induce tissue-specific regeneration.

[0005] Among existing scaffold fabrication technologies, electrospinning is favored due to its ability to mimic the nanofiber topology of the extracellular matrix (ECM). Polycaprolactone (PCL) has gained widespread application due to its excellent mechanical properties and processability, but its hydrophobicity and lack of bioactive groups limit its further clinical translation. Photocrosslinked hydrogels typically possess good biocompatibility and cell adhesion capabilities, providing a hydrated three-dimensional microenvironment for cells.

[0006] Therefore, existing technologies have developed composite systems of electrospun PCL fibers and hydrogels. These systems can guide cell migration through the ordered topology of PCL fibers and promote cell behavior through the hydrophilic network of hydrogels, exhibiting a synergistic enhancement effect in tissue engineering such as skin wound repair. However, in practical applications, the effectiveness of this composite system in promoting angiogenesis, antibacterial activity, antioxidation, and immunomodulation still needs further improvement. Summary of the Invention

[0007] This invention addresses the issue that the effects of existing electrospun PCL fiber and hydrogel composite systems on biological functions such as promoting angiogenesis, antibacterial activity, antioxidant activity, and immunomodulation need to be further enhanced. It provides a polyphenol-modified composite hydrogel scaffold, its preparation method, and its application.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A method for preparing a polyphenol-modified composite hydrogel scaffold includes the following steps: Polycaprolactone scaffolds were prepared using electrospinning technology. Modifying bioactive glass with polyphenols and copper ions to obtain a polyphenol-modified copper-containing bioactive glass suspension includes: adding bioactive glass to a polyphenol solution to obtain polyphenol-modified bioactive glass; mixing the polyphenol-modified bioactive glass with a copper chloride solution; and chelating the polyphenol with Cu²⁺ using the ortho-phenolic hydroxyl groups of the polyphenol to obtain the polyphenol-modified copper-containing bioactive glass suspension. A polyphenol-modified copper-containing bioactive glass suspension was mixed with a hydrogel solution and uniformly coated onto the surface of a polycaprolactone scaffold. After coating, the polycaprolactone scaffold was photocrosslinked to obtain a polyphenol-modified copper-containing bioactive glass composite hydrogel scaffold.

[0009] Preferably, the preparation of the polycaprolactone scaffold via electrospinning technology includes: Dichloromethane and N,N-dimethylformamide were mixed evenly in a volume ratio of 2:1 to 5:1 to obtain mixed solution A. Polycaprolactone was dissolved in mixed solution A to obtain a polycaprolactone solution with a concentration of 8-15% w / v; Polycaprolactone scaffolds were obtained by electrospinning a polycaprolactone solution.

[0010] Preferably, electrospinning is carried out at a flow rate of 3.0-6.0 mL / h under a high voltage of 16 kV.

[0011] Preferably, the modification of bioactive glass with polyphenols and copper ions to obtain a polyphenol-modified copper-containing bioactive glass suspension includes: Bioactive glass was added to a polyphenol solution to obtain polyphenol-modified bioactive glass with a final concentration of 10-30 mg / mL; Polyphenol-modified bioactive glass was mixed with a copper salt solution with a concentration of 0.2-2 mol / L at a volume ratio of 1:1-1:3 to obtain a polyphenol-modified copper-containing bioactive glass suspension.

[0012] Preferably, the polyphenol solution includes one or more of the following: a tannic acid solution with a concentration of 2-5 mg / mL, an anthocyanin solution with a concentration of 0.25-1 mg / mL, and a dopamine solution with a concentration of 1-4 mg / mL.

[0013] Preferably, the step of mixing the polyphenol-modified copper-containing bioactive glass suspension with a hydrogel solution and uniformly coating it onto the surface of a polycaprolactone scaffold includes: A hydrogel solution was prepared by containing 0.5% (w / v) lithium phenyl-2,4,6-trimethylbenzoyl phosphate, wherein the hydrogel solution is any one of the following: a 5-15% w / v methacrylamide gelatin hydrogel solution, a 1-2% w / v methacrylamide hyaluronic acid hydrogel solution, or a 7.5-20% w / v methacrylamide collagen hydrogel solution; The polyphenol-modified copper-containing bioactive glass suspension was mixed with the hydrogel solution to obtain mixed system B; Mixture system B is uniformly coated on the surface of the polycaprolactone scaffold.

[0014] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphate containing 0.5% w / v.

[0015] Preferably, the volume ratio of the polyphenol-modified copper-containing bioactive glass suspension to the methacrylamide gelatin hydrogel solution is 1:5-1:9, the volume ratio of the polyphenol-modified copper-containing bioactive glass suspension to the methacrylamide hyaluronic acid hydrogel solution is 1:1-1:3, and the volume ratio of the polyphenol-modified copper-containing bioactive glass suspension to the methacrylamide collagen hydrogel solution is 1:5-1:9.

[0016] A polyphenol-modified composite hydrogel scaffold is prepared using the aforementioned method for preparing a polyphenol-modified composite hydrogel scaffold.

[0017] Application of a polyphenol-modified composite hydrogel scaffold in the preparation of bone or soft tissue repair products.

[0018] This invention, by adopting the above technical solutions, has significant technical effects: This invention integrates polyphenol-modified copper-containing bioactive glass into a polycaprolactone / photocurable hydrogel electrospinning scaffold system, which can simulate the extracellular matrix structure of natural musculoskeletal tissue, providing physical support for cell adhesion, arrangement and proliferation. At the same time, it can achieve the continuous release of copper ions and polyphenol molecules, thereby promoting multiple biological functions such as angiogenesis, anti-oxidation and immune regulation, and ultimately achieving comprehensive regulation from macroscopic structure to microscopic regenerative microenvironment. Attached Figure Description

[0019] Figure 1 Macroscopic images of the electrospinning supports prepared according to Examples 1-5.

[0020] Figure 2 Transmission electron microscope (TEM) images of the electrospinning scaffolds prepared according to Examples 1-5.

[0021] Figure 3 Images showing the live and dead dyeing effects of the electrospinning scaffold extract prepared according to Examples 1-5.

[0022] Figure 4 Figure 1 shows the results of CCK-8 cell proliferation experiments using the electrospinning scaffold extract prepared according to Examples 1 and 6-7.

[0023] Figure 5 The antibacterial effect diagrams of the electrospinning scaffold extract prepared according to Examples 1-5 are shown, where A is an actual image of the antibacterial effect of the scaffold against Staphylococcus aureus; and B is an actual image of the antibacterial effect of the scaffold against Escherichia coli.

[0024] Figure 6 The effect of the electrospun scaffold extract prepared according to Examples 1-5 on angiogenesis is shown in the figure.

[0025] Figure 7 In vitro ALP staining images of the electrospinning scaffold extract prepared according to Examples 1-5.

[0026] Figure 8 Actual images showing the recovery effect of electrospun scaffolds prepared according to Examples 1-5 on infected wounds.

[0027] Figure 9 HE staining images of electrospun scaffolds prepared according to Examples 1-5 after infection of wounds.

[0028] Figure 10 Masson staining images of the electrospun scaffolds prepared according to Examples 1-5 after infection of the wound.

[0029] Figure 11 The electrospun scaffolds prepared according to Examples 1-5 were used for laser Doppler perfusion imaging of infected wounds.

[0030] Figure 12 CT images of skull injuries using electrospun scaffolds prepared according to Examples 1-5.

[0031] Figure 13 Histochemical staining images of the electrospun scaffolds prepared according to Examples 1-5 for use in skull injuries. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0033] Example 1 This embodiment presents a polyphenol-modified composite hydrogel scaffold that integrates polyphenol-modified copper-containing bioactive glass (poly-CuBG) into a polycaprolactone / photocurable hydrogel electrospun scaffold system. The polycaprolactone hydrogel composite fiber scaffold mimics the extracellular matrix structure of natural musculoskeletal tissue, providing physical support for cell adhesion, alignment, and proliferation. Simultaneously, the polyphenol-modified copper-containing bioactive glass (poly-CuBG) is used to achieve the continuous release of Cu²⁺ ions and polyphenol molecules, thereby promoting multiple biological functions such as angiogenesis, antioxidation, and immune regulation, ultimately achieving comprehensive regulation from macroscopic structure to the microscopic regenerative microenvironment.

[0034] Specifically, it is prepared by the following method, including the following steps: Step S1: Mix dichloromethane and N,N-dimethylformamide at a volume ratio of 4:1 to obtain mixed solution A; Step S2: Dissolve polycaprolactone (PCL) in the above mixed solution A to obtain a polycaprolactone solution with a concentration of 10% w / v, i.e., PCL solution; Step S3: Electrospinning is performed at a flow rate of 5.0 mL / h under a high voltage of 16 kV, with a collector spacing of 15 cm, to obtain a polycaprolactone scaffold, i.e., a PCL scaffold. Step S4: Prepare a polyphenol mixed solution by adding dopamine, tannic acid and anthocyanin to deionized water to obtain a polyphenol mixed solution. The polyphenol mixed solution includes a tannic acid solution with a final concentration of 2-5 mg / mL, an anthocyanin solution with a final concentration of 0.25-1 mg / mL and a dopamine solution with a final concentration of 1-4 mg / mL.

[0035] Step S5: Add the bioactive glass to the above polyphenol mixed solution, stir at room temperature for 6 hours to fully disperse, and obtain polyphenol-modified bioactive glass with a final concentration of 10-30 mg / mL; Step S6: Prepare a copper chloride solution to obtain a copper chloride solution with a concentration of 1 mol / L; Step S7: Mix the polyphenol-modified bioactive glass obtained in step S5 with an equal volume of copper chloride solution, and use the ortho-phenolic hydroxyl groups of polyphenols to chelate with Cu²⁺ to obtain a polyphenol-modified copper-containing bioactive glass suspension. Step S8: Prepare a hydrogel solution using an aqueous solution containing 0.5% w / v lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP) as a solvent. The prepared hydrogel solution is a 10% w / v methacrylamide gelatin hydrogel solution, i.e., GelMA hydrogel solution. Step S9: Mix the polyphenol-modified copper-containing bioactive glass suspension obtained in step S7 with the GelMA hydrogel solution and then uniformly coat it on the surface of the PCL scaffold. The volume ratio of the two is 1:5. Step S10, photocrosslinking for 60s yields a polyphenol-modified copper-containing bioactive glass composite hydrogel scaffold, namely PCL / GelMA@Poly-CuBG.

[0036] Example 2 Similar to Example 1, except that this example does not include steps S4-S10, and directly prepares PCL without hydrogel coating.

[0037] Example 3 Similar to Example 1, the difference is that this example does not include steps S4-S7, but instead directly coats the 10% (w / v) GelMA hydrogel solution obtained in step S8 onto the surface of the PCL scaffold in step S3, and finally obtains PCL / GelMA.

[0038] Example 4 Similar to Example 1, the difference is that steps S5-S7 are not included in this example. Instead, the polyphenol mixed solution obtained by mixing dopamine, tannic acid and anthocyanin at final concentrations of 3 mg / mL, 0.75 mg / mL and 3 mg / mL respectively in step S4 is mixed with the GelMA hydrogel solution obtained in step S8 at a volume ratio of 1:5 and then coated on the surface of the PCL scaffold to finally obtain PCL / GelMA@Poly.

[0039] Example 5 Similar to Example 1, the difference is that steps S6 and S7 are not included in this example, that is, the added polyphenol-modified bioactive glass is no longer chelated with copper ions, and finally PCL / GelMA@Poly-BG is obtained.

[0040] Example 6 Similar to Example 1, the difference is that in this example, step S8 prepares a 2% w / v methacrylamide hyaluronic acid hydrogel solution, i.e., HAMA hydrogel solution, and finally obtains PCL / HAMA@Poly-CuBG.

[0041] Example 7 Similar to Example 1, the difference is that in this example, step S8 prepares a 15% w / v methacrylamide collagen hydrogel solution, namely ColMA hydrogel solution, and finally obtains PCL / ColMA@Poly-CuBG.

[0042] Example 8 In vitro biocompatibility assessment I. Cell Culture BMSCs and RAW 264.7 cells were cultured in Durbeco modified Eagle medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. HUVECs were cultured in endothelial cell medium containing 5% FBS and 1% endothelial cell growth supplement. All cells were cultured in a humidified environment at 37°C with 5% carbon dioxide.

[0043] II. Preparation of scaffold leachate The composite hydrogel scaffolds (1 cm × 1 cm) prepared according to Examples 1-6 were immersed in serum-free culture medium and incubated at 37°C at a ratio of 1 cm² / mL for 24 hours. The supernatant was collected, filtered through a 0.22 μm filter membrane, and stored at -20°C until use.

[0044] 3. Live / dead staining method Bone marrow mesenchymal stem cells (BMSCs) were used at a rate of 2 × 10⁻⁶. 4 Cells were seeded at a density of 10 cells / well on different scaffold materials prepared according to Examples 1-5 in 24-well plates (with blank controls in a group of cells cultured normally without the addition of extract). After 24 and 48 hours of culture, fluorescence images were acquired by microscope after staining with calcein AM (2 μM) and propidium iodide (4 μM) at 37°C for 30 minutes.

[0045] IV. Cell Proliferation Experiment Cell proliferation of the electrospun scaffolds prepared according to Examples 1 and 6-7 was detected using a CellCounting Kit-8 (the blank control group consisted of normally cultured cells without the addition of extraction medium). Bone marrow mesenchymal stem cells (BMSCs) were seeded at a density of 5 × 10³ cells / well on 96-well plates. After 24 and 48 hours of culture, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance was measured at 450 nm using a microplate reader.

[0046] like Figure 1 As shown, the electrospun composite hydrogel membranes prepared according to Examples 1-5 all exhibit a uniform and defect-free structure on the surface of each group of membranes; Figure 2 As shown, transmission electron microscopy revealed that the electrospun fibers successfully formed a bilayer structure.

[0047] like Figure 3 , Figure 4 As shown, in vitro biocompatibility experiments indicate that this composite form of electrospun hydrogel scaffold does not exhibit significant cytotoxicity, and the cell proliferation trend is more pronounced in the copper-containing group. These results demonstrate that the prepared composite membrane (especially the copper-doped variant) possesses excellent biocompatibility and bioactivity, making it suitable for potential tissue engineering applications.

[0048] V. In vitro antibacterial activity assay The antibacterial activity of the scaffold material against Gram-positive Staphylococcus aureus (ATCC 25923) and Gram-negative Escherichia coli (ATCC 25922) was evaluated using the diffusion plate method. Bacterial suspensions (10... 6 CFU / mL was prepared in Luria-Bertani (LB) broth. Scaffold membranes (1 cm × 1 cm) prepared according to Examples 1-5 were placed in 24-well plates and incubated with 1 mL of bacterial suspension at 37°C for 24 hours and 48 hours, respectively (the blank control consisted of normally cultured bacteria without scaffold extract treatment). After incubation, the bacterial suspension was diluted and spread onto LB agar plates. After overnight incubation at 37°C, bacterial colonies were counted and photographed.

[0049] like Figure 5 As shown in Figure A, this experiment found that the PCL / GelMA@Poly-CuBG group exhibited the most significant antibacterial effect against Staphylococcus aureus at both time points, with a significantly reduced bacterial colony count compared to the control group and other groups. The PCL / GelMA@Poly-BG group and the PCL / GelMA@Poly group also showed moderate antibacterial activity, while the PCL / GelMA group and the PCL group showed limited inhibitory effects, similar to the Ctrl group.

[0050] like Figure 5 As shown in Figure B, the PCL / GelMA@Poly-CuBG group maintained the highest antibacterial efficacy against Escherichia coli at both 24 and 48 hours, indicating its broad-spectrum antibacterial potential. The PCL / GelMA@Poly-BG and PCL / GelMA@Poly groups showed moderate inhibitory effects, while the PCL / GelMA and PCL groups showed almost no antibacterial activity.

[0051] These results indicate that incorporating Poly-CuBG into the PCL / GelMA scaffold significantly enhances its antibacterial properties against both Gram-positive and Gram-negative bacteria.

[0052] Example 9: In vitro angiogenesis experiment 50 μL of Matrigel was coated onto each well of a 96-well plate and polymerized at 37°C for 30 minutes. Human umbilical vein endothelial cells (HUVECs) were suspended in different scaffold leachates prepared according to Examples 1-5 (the blank control group consisted of normally cultured cells without leachate), at a concentration of 2 × 10⁻⁶. 4 Cells were seeded at a density per well on Matrigel substrate. After 6 hours of incubation, the formation of tubular structures was observed and images were acquired using an inverted microscope.

[0053] like Figure 6 As shown, the results of this embodiment demonstrate that the PCL / GelMA@Poly-CuBG group exhibits the most complete and complex capillary-like network structure, characterized by a high-density network structure and clearly defined tubular morphology. Compared with all control groups, the total tube length of the PCL / GelMA@Poly-CuBG group showed a statistically significant increase, indicating its stronger angiogenesis-promoting ability in in vitro experiments.

[0054] Example 10 In vitro osteogenic differentiation experiment I. Alkaline phosphatase (ALP) staining BMSCs were divided into 5 × 10 4 Cells were seeded at a density in 24-well plates and cultured in osteogenic induction medium (OIM) containing 50 μg / mL ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone, with different scaffold extracts prepared according to Example 1 added (the blank control was a group of normally cultured cells without extracts). After 7 days of induction culture, cells were fixed with 4% paraformaldehyde and stained using an ALP staining kit (Beyotime) according to the manufacturer's instructions. The stained cells were observed and photographed under a microscope.

[0055] like Figure 7 As shown, the results indicate that BMSCs treated with PCL / GelMA@Poly-CuBG leachate exhibited the strongest ALP staining intensity across all groups, suggesting enhanced early osteogenic differentiation capacity. In contrast, the ALP staining intensity in the control group and the PCL group was relatively weak.

[0056] Example 11 In vivo animal experiments I. Rat model of infected wound healing (soft tissue) Eight-week-old male Sprague-Dawley rats were used in the experiment. A full-thickness skin defect model with bacterial infection was established: after anesthesia with sodium pentobarbital (40 mg / kg, intraperitoneal injection), a circular wound with a diameter of 15 mm was made on the skin of the back of each rat. 100 μL of Staphylococcus aureus suspension (10...) was inoculated. 8 An infection model was established using CFU / mL. Rats were randomly divided into six groups (n=5 per group): Defect group (untreated defect group), PCL group (based on Example 2), PCL / GelMA group (based on Example 3), PCL / GelMA@Poly group (based on Example 4), PCL / GelMA@Poly-BG group (based on Example 5), and PCL / GelMA@Poly-CuBG group (based on Example 1). After the scaffold material was placed in the wound, it was fixed with a transparent dressing, and wound photographs were taken at 0, 3, 7, and 14 days postoperatively.

[0057] On day 14, rats were sacrificed and wound tissue was collected. Tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for histological analysis. Hematoxylin-eosin (H&E) staining and Masson's trichrome staining were performed according to standard operating procedures. Figure 8-10 As shown.

[0058] The results showed that all wounds appeared similar on postoperative day 0. Wound contraction was observed in all groups over time, but the closure rate differed significantly. The PCL / GelMA@PolyCuBG group exhibited the fastest healing process, with the wound almost completely closed by day 14; while the Defect, PCL, and PCL / GelMA groups still showed noticeable unhealed areas.

[0059] H&E staining showed that the PCL / GelMA@PolyCuBG group exhibited well-structured new epidermis and abundant granulation tissue formation, with minimal inflammatory cell infiltration. In contrast, the Defect group and the PCL group showed incomplete epithelial regeneration and persistent inflammatory response. Masson trichrome staining revealed that the PCL / GelMA@PolyCuBG group had a wider and more orderly collagen deposition area. Quantitative analysis of collagen volume fraction confirmed that the collagen content in this group was significantly higher than that in all other groups, indicating superior extracellular matrix remodeling capacity.

[0060] 1. Laser Doppler perfusion imaging Blood perfusion at the wound site was assessed on day 14 using laser Doppler perfusion imaging. The wound area was scanned after the rats were anesthetized.

[0061] like Figure 11As shown, the results indicated that on day 14, the PCL / GelMA@Poly-CuBG group exhibited the highest blood flow signal intensity in the wound area, indicating significant recovery of local microcirculation. In contrast, the perfusion signals in the Defect group and the PCL group were relatively weak, suggesting a delay in the angiogenesis process. Quantitative analysis of the perfusion unit confirmed that the mean blood flow in the PCL / GelMA@Poly-CuBG group was significantly higher than that in all other groups, while the PCL / GelMA@Poly and PCL / GelMA@Poly-BG groups also showed moderate improvement compared to the unmodified control group.

[0062] II. Rat model of skull defect (bone tissue) To assess bone regeneration capacity, a rat model of skull defects was established. After anesthesia, a midline incision was made in the scalp, and the periosteum was inverted to expose the skull. Under continuous saline irrigation, a critical-sized defect (5 mm in diameter) was created on each parietal bone using a trephine. The defect site was treated with the same scaffolding group as described above (n=5 per group). After suturing, rats were sacrificed at 4 and 8 weeks postoperatively, and skull samples were collected.

[0063] 1. Microscopic CT analysis The collected skull samples were fixed with 4% paraformaldehyde and then scanned using a micro-CT system (SkyScan1176, Bruker) with the following parameters: voltage 50kV, current 500 μA, and resolution 9 μm. Three-dimensional (3D) reconstruction was performed using the manufacturer's proprietary software.

[0064] 2. Histological and immunohistochemical analysis Skull samples were decalcified with 10% EDTA solution for 4 weeks, then embedded in paraffin and sectioned (5 μm thick). Histological evaluation was performed using hematoxylin-eosin (H&E) staining and Masson's trichrome staining. For immunohistochemical staining, sections were incubated overnight at 4°C with anti-COL1 antibody (1:200, abcam) and anti-Runx2 antibody (1:200, abcam), followed by HRP-labeled secondary antibody. Staining results were developed using DAB substrate and counterstained with hematoxylin. Image acquisition was performed using an optical microscope.

[0065] At 4 weeks post-surgery, only limited new bone formation was observed at the defect margins in the Defect, PCL, and PCL / GelMA groups. In contrast, the PCL / GelMA@Poly and PCL / GelMA@Poly-BG groups showed moderate bone ingrowth, while the PCL / GelMA@Poly-CuBG group exhibited the most extensive new bone formation, almost covering the entire defect area. By 8 weeks, the skull defect in the PCL / GelMA@Poly-CuBG group was almost completely closed, with new bone density comparable to the surrounding natural bone. Significant unhealed areas remained in the other groups (especially the Defect and PCL groups), indicating delayed or insufficient bone regeneration. Figure 12 As shown.

[0066] Histological staining results showed that by week 4, the PCL / GelMA@Poly-CuBG group exhibited abundant newly formed trabecular bone structures with dense collagen fibers (blue in Masson's trichrome staining). In contrast, other groups showed only sparse bone matrix and predominantly fibrous tissue infiltration. By week 8, the PCL / GelMA@Poly-CuBG group had formed structurally ordered layered bone tissue with a mature medullary cavity, its morphology similar to natural bone. The Defect and PCL groups were still mainly filled with fibrous connective tissue, with only trace bone formation visible at the defect edges, such as... Figure 13 As shown.

[0067] Based on the above experimental results, it was confirmed that this polyphenol-modified copper-containing bioactive glass composite hydrogel scaffold possesses excellent physicochemical properties, continuous copper ion release capability, outstanding cell compatibility, and potent antibacterial activity. The incorporation of Cu²⁺ can enhance the antibacterial performance of the bioactive glass. By inducing the generation of reactive oxygen species (ROS), it exhibits a broad-spectrum inhibitory effect on Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, as well as Gram-positive bacteria such as Staphylococcus aureus. It is of great significance for preventing infection after musculoskeletal injury and creating a sterile microenvironment conducive to regeneration.

[0068] Furthermore, this scaffold exhibits significant abilities to promote angiogenesis, accelerate infected wound healing, enhance osteogenic differentiation, and regulate the regenerative microenvironment through antioxidant effects and M2 macrophage polarization. In vivo evaluation in a rat model of skull defects confirmed that PCL / GelMA@Poly-CuBG significantly promotes bone regeneration, and no systemic toxicity was found in biocompatibility assessments. These findings collectively support the potential application of this composite scaffold in integrated repair of hard and soft tissues.

[0069] It is readily understood that those skilled in the art can combine, split, or reorganize the embodiments provided in this application to obtain other embodiments, none of which exceed the protection scope of this application.

[0070] In summary, the above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be covered by the present invention.

Claims

1. A method for preparing a polyphenol-modified composite hydrogel scaffold, characterized in that, Includes the following steps: Polycaprolactone scaffolds were prepared using electrospinning technology. Modifying bioactive glass with polyphenols and copper ions to obtain a polyphenol-modified copper-containing bioactive glass suspension includes: adding bioactive glass to a polyphenol solution to obtain polyphenol-modified bioactive glass; mixing the polyphenol-modified bioactive glass with a copper chloride solution; and chelating the polyphenol with Cu²⁺ using the ortho-phenolic hydroxyl groups of the polyphenol to obtain the polyphenol-modified copper-containing bioactive glass suspension. A polyphenol-modified copper-containing bioactive glass suspension was mixed with a hydrogel solution and uniformly coated onto the surface of a polycaprolactone scaffold. After coating, the polycaprolactone scaffold was photocrosslinked to obtain a polyphenol-modified copper-containing bioactive glass composite hydrogel scaffold.

2. The method for preparing a polyphenol-modified composite hydrogel scaffold according to claim 1, characterized in that, The preparation of polycaprolactone scaffolds via electrospinning technology includes: Dichloromethane and N,N-dimethylformamide were mixed evenly in a volume ratio of 2:1 to 5:1 to obtain mixed solution A. Polycaprolactone was dissolved in mixed solution A to obtain a polycaprolactone solution with a concentration of 8-15% w / v; Polycaprolactone scaffolds were obtained by electrospinning a polycaprolactone solution.

3. The method for preparing a polyphenol-modified composite hydrogel scaffold according to claim 2, characterized in that, Electrospinning was carried out at a flow rate of 3.0-6.0 mL / h under a high voltage of 16 kV.

4. The method for preparing a polyphenol-modified composite hydrogel scaffold according to claim 1, characterized in that, The modification of bioactive glass with polyphenols and copper ions to obtain a polyphenol-modified copper-containing bioactive glass suspension includes: Bioactive glass was added to a polyphenol solution to obtain polyphenol-modified bioactive glass with a final concentration of 10-30 mg / mL; Polyphenol-modified bioactive glass was mixed with a copper salt solution with a concentration of 0.2-2 mol / L at a volume ratio of 1:1-1:3 to obtain a polyphenol-modified copper-containing bioactive glass suspension.

5. The method for preparing a polyphenol-modified composite hydrogel scaffold according to claim 4, characterized in that, The polyphenol solution includes one or more of the following: a tannic acid solution with a concentration of 2-5 mg / mL, an anthocyanin solution with a concentration of 0.25-1 mg / mL, and a dopamine solution with a concentration of 1-4 mg / mL.

6. A method for preparing a polyphenol-modified composite hydrogel scaffold according to claim 1 or 4, characterized in that, The process of mixing a polyphenol-modified copper-containing bioactive glass suspension with a hydrogel solution and uniformly coating it onto the surface of a polycaprolactone scaffold includes: A hydrogel solution was prepared by containing 0.5% (w / v) lithium phenyl-2,4,6-trimethylbenzoyl phosphate, wherein the hydrogel solution is any one of the following: a 5-15% w / v methacrylamide gelatin hydrogel solution, a 1-2% w / v methacrylamide hyaluronic acid hydrogel solution, or a 7.5-20% w / v methacrylamide collagen hydrogel solution; The polyphenol-modified copper-containing bioactive glass suspension was mixed with the hydrogel solution to obtain mixed system B; Mixture system B is uniformly coated on the surface of the polycaprolactone scaffold.

7. The method for preparing a polyphenol-modified composite hydrogel scaffold according to claim 6, characterized in that, The volume ratio of polyphenol-modified copper-containing bioactive glass suspension to methacrylamide gelatin hydrogel solution is 1:5-1:9; the volume ratio of polyphenol-modified copper-containing bioactive glass suspension to methacrylamide hyaluronic acid hydrogel solution is 1:1-1:3; and the volume ratio of polyphenol-modified copper-containing bioactive glass suspension to methacrylamide collagen hydrogel solution is 1:5-1:

9.

8. A polyphenol-modified composite hydrogel scaffold, characterized in that, It is prepared using the method described in any one of claims 1-7 for preparing a polyphenol-modified composite hydrogel scaffold.

9. The application of the polyphenol-modified composite hydrogel scaffold according to claim 8 in the preparation of bone tissue or soft tissue repair products.

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