Functionalized janus extracellular matrix hydrogel scaffolds, methods of making and using the same

Abstract

The application provides a functionalized Janus extracellular matrix hydrogel scaffold, a preparation method and application thereof. The scaffold comprises a decellularized duck small intestinal mucosa layer which is co-functionalized by a polymeric flavonoid substance with a benzopyran ring as a basic structural unit and trivalent gallium ions, and a hydrogel layer with in-situ gelation and self-repairing properties formed on one side of the mucosa layer. In use, the functionalized side without gel is oriented towards the repaired tendon, and the side with gel is oriented towards the surrounding soft tissue, so as to form an inner tissue integration interface and an outer anti-adhesion barrier interface. The scaffold retains the collagen fiber structure of the decellularized matrix, has antioxidant, microenvironment-responsive release, antibacterial and good blood compatibility, can reduce peritendinous fibrous adhesion in an animal model of tendon rupture, improve tendon sliding and improve the mechanical strength of the repaired part.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials, decellularized extracellular matrix, and tendon repair technology, and particularly to a Janus scaffold composed of a functionalized decellularized duck small intestinal mucosa layer and an oxidized hyaluronic acid-carboxymethyl chitosan hydrogel layer, its preparation method, and its application in reducing peritendinous adhesions and promoting tendon function repair. Background Technology

[0002] Repairing flexor tendon injuries requires not only the reconnection of the ruptured tissue and restoration of its load-bearing capacity, but also the ability of the repaired tendon to maintain low-resistance gliding within the tendon sheath. Tendons have low blood supply and cell density, limiting their intrinsic repair capacity; surgical injuries can also cause inflammation and fibrosis in surrounding tissues, and exogenous fibrous tissue can form scar bridging across the gliding interface between the tendon and tendon sheath, thus restricting joint movement.

[0003] Existing membranes, gels, electrospun materials, and polysaccharide coatings primarily utilize physical isolation or local drug delivery mechanisms. Homogeneous materials often struggle to simultaneously meet opposing interfacial requirements: the side facing the tendon should retain natural microstructures suitable for tissue integration and matrix reconstruction, while the side facing surrounding tissues should form a moist, flexible, and low-adhesion barrier. Simply enhancing barrier properties may hinder tissue repair and integration, while simply increasing tissue adhesion can easily exacerbate peritendinous scarring. Summary of the Invention

[0004] The purpose of this invention is to provide a functionalized Janus extracellular matrix / hydrogel scaffold, which has different and synergistic interface functions on both sides of the scaffold, thereby reducing peritendinous adhesions while maintaining or improving the quality of tendon repair.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a functionalized Janus extracellular matrix / hydrogel scaffold, comprising a first functional layer and a second functional layer disposed on one side of the first functional layer. The first functional layer is a decellularized duck small intestinal mucosal scaffold PCGa@dDIS functionalized with polymeric flavonoids with benzopyran rings as basic structural units and trivalent gallium ions; the second functional layer is a hydrogel layer formed by oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS) through dynamic Schiff base bonds. The exposed surface of PCGa@dDIS serves as a tendon contact surface, and the OHA / CMCS hydrogel surface serves as an anti-adhesion barrier surface facing the surrounding soft tissue.

[0006] A second aspect of the present invention provides a method for preparing the Janus scaffold, comprising: preparing a decellularized duck small intestinal mucosal scaffold; impregnating and functionalizing it with a polymeric flavonoid / trivalent gallium composite solution; obtaining OHA by oxidizing hyaluronic acid with sodium periodate; mixing OHA and CMCS solution and coating it on one side of PCGa@dDIS, and forming a gel in situ using the dynamic Schiff base reaction of aldehyde and amino groups.

[0007] Preferably, the duck small intestinal mucosa is sonicated in 75% ethanol for 20 min, then shaken in a solution containing 0.5% sodium dodecyl sulfate and 1% Triton X-100 for 24 h, and then treated with DNase I, thoroughly washed, frozen at -80 ℃ and freeze-dried.

[0008] Preferably, the concentration of the polymeric flavonoid is 0.5 mg / mL, the trivalent gallium ions are provided by gallium nitrate nonahydrate, and the molar ratio of the polymeric flavonoid to the trivalent gallium ions is 1:1, 1:2 or 1:4, more preferably 1:2.

[0009] Preferably, OHA and CMCS are prepared into 5% solutions by mass using phosphate buffer, and after mixing, they are immediately coated onto one side of PCGa@dDIS so that the hydrogel layer and the matrix layer form a continuous interface, while the other side retains the exposed natural fibrous structure.

[0010] A third aspect of the invention provides the use of the Janus scaffold in the preparation of medical implant materials for tendon rupture repair and reduction of peritendinous adhesions. In use, the PCGa@dDIS side faces the tendon, and the OHA / CMCS hydrogel side faces the tendon sheath or surrounding soft tissue.

[0011] Compared with the prior art, the present invention has at least the following beneficial effects: First, the decellularized duck small intestinal mucosa retains a continuous collagen fiber framework and major extracellular matrix components, providing a tissue-derived structural basis for the tendon contact surface; Second, the coordination of polymeric flavonoids with gallium ions and their non-covalent interaction with the matrix endows significant free radical scavenging and antibacterial properties without destroying the main collagen structure; Third, the OHA / CMCS dynamic hydrogel can gel in situ on one side and has self-healing properties, forming a flexible lateral anti-adhesion barrier; Fourth, the scaffold exhibits faster release of polymeric flavonoids and gallium ions in acidic and reactive oxygen species environments, and has a low hemolysis rate; Fifth, in a rat flexor tendon repair model, the Janus scaffold reduces peritendinous fiber connections, improves the plantar flexion angle under load, and increases the maximum failure load. Attached Figure Description

[0012] Figure 1The results of the preparation and validation of decellularized duck small intestinal mucosal scaffolds are shown in the following figures: A is a schematic diagram of the dDIS preparation process; B is a physical image of duck small intestine, duck small intestinal mucosal scaffold DIS, decellularized scaffold dDIS, and lyophilized dDIS; C is the residual DNA content; D is DAPI, H&E, Masson, and Sirius Red staining; E is genomic DNA agarose gel electrophoresis; and F and G are immunohistochemical staining and quantification of type I collagen, type III collagen, fibronectin, and laminin. Figure 2 The preparation, structural chemical characterization, and antioxidant results of PC / Ga functionalized dDIS are shown in Figure 1. A is a schematic diagram of PCGa@dDIS preparation, B is a scanning electron microscope image, C is the fiber size distribution, D is the three-dimensional morphology under atomic force microscopy, E is the average surface roughness, F is the X-ray photoelectron spectroscopy, G is the Fourier transform infrared spectrum, H is the physical image of the DPPH reaction solution, and I is the corresponding UV-Vis absorption spectrum. Figure 3 This document presents the preparation and physicochemical characterization of the PCGa@dDIS / OHA-CMCS Janus scaffold. A shows the Janus structure and dynamic Schiff base cross-linking schematic diagram; B shows the infrared spectra of HA and OHA; C shows the proton nuclear magnetic resonance spectra of HA and OHA; D shows the gelation results in the inverted bottle; E shows the hydrogel cleavage and self-healing results; F shows the scanning electron microscope image of the bilayer interface cross-section; G shows the actual images of the scaffold bending and torsion; H shows the in vitro degradation results; I shows the swelling results; J and K show the DPPH free radical scavenging results; and L and M represent Ga³ under different microenvironments. + Results of the release of polymeric flavonoids; Figure 4 The results show the antibacterial effects of PC / Ga functionalized scaffolds against Staphylococcus aureus and Escherichia coli. A represents DMAO / PI staining of live and dead bacteria, B represents plate colony images, C represents the relative survival rate of bacteria, and D represents scanning electron micrographs of bacterial attachment and morphology. Figure 5 The results show the in vivo evaluation of the Janus scaffold for repairing ruptured flexor tendons in rats. A represents the animal model and evaluation timeline, B and F represent plantar flexion images at 0, 5, 10, 15, and 20 g loads at 2 and 3 weeks post-operation, C and G represent gross images of the repaired tendon at 2 and 3 weeks post-operation, D and H represent the maximum failure load at 2 and 3 weeks post-operation, E and I represent the changes in plantar flexion angles at different load ranges at 2 and 3 weeks post-operation, J represents H&E staining, and K represents Masson staining. Figure 6 Images of hemolysis experiments and quantitative data on hemolysis rates of dDIS, PCGa@dDIS, and Janus stents; Figure 7The images show H&E staining of the heart, liver, spleen, lung, and kidney of rats in the blank group, dDIS group, PCGa@dDIS group, and Janus group at the experimental endpoint.

[0013] Figure 8 A schematic diagram illustrating the mechanism of action of the Janus scaffold in the repair of flexor tendon injuries. Detailed Implementation

[0014] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0015] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0016] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0017] Functionalized Janus extracellular matrix hydrogel scaffold Decellularized extracellular matrix (ECM) retains natural matrix components and multi-scale structures such as collagen fibers, fibronectin, and laminin, making it suitable as a tendon contact interface. Animal mucosa can be used as a source of ECM; however, this study found that commonly used materials (such as pig intestines) still have a certain thickness after decellularization, resulting in a foreign body sensation when used for flexor tendon repair. Duck intestine mucosa, on the other hand, can be processed into thin, continuous, and compliant sheet materials, facilitating tendon wrapping and unilateral modification. However, unfunctionalized ECM has limited ability to regulate postoperative oxidative stress, bacterial contamination, and interfacial adhesion. Therefore, it is necessary to construct a spatially asymmetric Janus scaffold, forming an inward-facing ECM functional surface and an outward-facing hydrogel barrier surface on the same material, and endowing it with antioxidant, antibacterial, and responsive release properties through polyphenol-metal coordination to coordinate tendon tissue repair and peritendinous anti-adhesion. This invention is based on at least this principle.

[0018] One aspect of the present invention provides a functionalized Janus extracellular matrix hydrogel scaffold. In this document, the terms "Janus hydrogel," "Janus scaffold," "Janus extracellular matrix hydrogel," or similar terms refer to a bifacial or two-dimensional network structure material exhibiting typical spatial asymmetry in structure and function. The scaffold of the present invention comprises a first functional layer and a second functional layer bonded together. The first functional layer comprises a decellularized duck small intestinal mucosal scaffold functionalized with polymeric flavonoids based on benzopyran rings as basic structural units and trivalent gallium ions. The second functional layer is an oxidized hyaluronic acid and carboxymethyl chitosan hydrogel layer disposed only on one side of the first functional layer, wherein the oxidized hyaluronic acid and the carboxymethyl chitosan are linked by dynamic Schiff base bonds. The side of the first functional layer without hydrogel is used to form a tendon contact surface, and the second functional layer constitutes an anti-adhesion barrier surface facing the surrounding tissue.

[0019] In a preferred embodiment, the second functional layer of the present invention is a hydrogel layer disposed only on the smooth surface of the first functional layer (acellular duck small intestinal mucosal scaffold), thereby enabling the surface of the first functional layer without hydrogel (rough surface) to contact the tendon. In this invention, the smooth surface refers to the surface of the acellular duck small intestinal mucosal scaffold with a dense structure (exhibiting a relatively smooth morphology, i.e., the matrix layer), and the rough surface refers to the surface of the acellular duck small intestinal mucosal scaffold with a fibrous porous structure (exhibiting a rough morphology). This structural design comprehensively considers multiple factors. On the one hand, the exposed rough surface provides a tissue-derived structural basis for the tendon contact surface; on the other hand, the coordination of polymeric flavonoids with gallium ions and their non-covalent interaction with the matrix layer impart significant free radical scavenging and antibacterial properties without damaging the main collagen structure; furthermore, this structure can significantly improve the plantar flexion angle under load and increase the maximum failure load.

[0020] To obtain decellularized duck small intestinal mucosal scaffolds with both rough and smooth morphologies, the present invention prepares decellularized duck small intestinal mucosal scaffolds through the following steps: duck small intestine is pre-cleaned, treated with ethanol, decellularized with a combination of ionic and nonionic surfactants, treated with nucleases, washed, and freeze-dried.

[0021] In a preferred embodiment, the ethanol treatment comprises sonicating in 50%-90% (preferably 70-80%) ethanol for 5-40 min (preferably 15-25 min); the combined decellularization comprises shaking in a solution containing 0.1-5% (preferably 0.1-1%) sodium dodecyl sulfate and 0.1-5% (preferably 0.5-2%) Triton X-100 for 5-48 h (preferably 15-30 h). In one specific implementation, the ethanol treatment includes sonication in 75% ethanol for 20 min; the combined decellularization includes shaking treatment with a solution containing 0.5% sodium dodecyl sulfate and 1% Triton X-100 for 24 h.

[0022] In this invention, the polymerizable flavonoid is a polyhydroxy polymer composed of flavan-3-ol units (such as catechin, epicatechin, or their derivatives) linked by carbon-carbon bonds. Preferably, the polymerizable flavonoid is a natural polymer composed of benzopyran rings as basic structural units linked by repeating 4→8 or 4→6 bonds. In a preferred embodiment, the polymerizable flavonoid is a dimer or polymer formed from catechin and / or epicatechin. Depending on the degree of polymerization, dimers to tetramers are generally referred to as oligomers, and pentamers or higher are referred to as polymers. In a preferred embodiment, the polymerizable flavonoid has the following structure: Formula I, Where n is an integer greater than or equal to 2. In the specific embodiment, n is 2, that is, in the form of a dimer.

[0023] In this invention, the concentration of the polymerizable flavonoid solution (including but not limited to a water / alcohol mixture) is not particularly limited, but is preferably 0.1-5 mg / mL, more preferably 0.1-2 mg / mL. The trivalent gallium ions are derived from gallium nitrate nonahydrate, and the molar ratio of the polymerizable flavonoid to the trivalent gallium ions is 1:(1-4). In one specific embodiment, the concentration of the polymerizable flavonoid solution is 0.5 mg / mL, the trivalent gallium ions are derived from gallium nitrate nonahydrate, and the molar ratio of the polymerizable flavonoid to the trivalent gallium ions is selected from 1:1, 1:2, or 1:4.

[0024] In this invention, the second functional layer is a hydrogel layer formed by oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CMCS) through dynamic Schiff base bonds, wherein the aldehyde group in the oxidized hyaluronic acid forms a dynamic Schiff base bond with the amino group in the carboxymethyl chitosan. The specific preparation of the hydrogel layer is not particularly limited; for example, the oxidized hyaluronic acid and the carboxymethyl chitosan can each be prepared into a 5% (w / w) solution with phosphate buffer, and then mixed to form the hydrogel layer.

[0025] In this invention, the roughness of the functionalized decellularized duck small intestinal mucosal scaffold is not less than 100 nm.

[0026] In this invention, the Janus extracellular matrix hydrogel scaffold has an antibacterial rate of nearly or greater than 90% against Staphylococcus aureus and Escherichia coli, and its hemolysis rate is less than 5%, preferably less than 2%.

[0027] Preparation method One aspect of the present invention provides a method for preparing a functionalized Janus extracellular matrix hydrogel scaffold, comprising the following steps: S1, Preparation of decellularized duck small intestinal mucosal scaffold dDIS; S2, dDIS is immersed in a composite solution containing polymerizable flavonoids and trivalent gallium ions and subjected to oscillatory loading. After washing away the unbound polymerizable flavonoids and free trivalent gallium ions, it is freeze-dried to obtain PCGa@dDIS. S3, oxidized hyaluronic acid (OHA) is prepared by oxidizing hyaluronic acid with sodium periodate; S4, mix the OHA solution with the carboxymethyl chitosan CMCS solution and uniformly coat it on one side (smooth surface) of PCGa@dDIS before gelation, so that an OHA / CMCS hydrogel layer is formed in situ, and the Janus extracellular matrix hydrogel scaffold is obtained. The side surface without hydrogel (i.e. rough surface) is used to contact the tendon to form a tendon contact surface, and the hydrogel layer forms an anti-adhesion barrier surface facing the surrounding tissue.

[0028] application In one aspect, the present invention provides the use of a functionalized Janus extracellular matrix hydrogel scaffold in the preparation of a medical implant material for tendon rupture repair, wherein the scaffold is capable of reducing peritendinous adhesions, improving tendon gliding, and increasing the mechanical strength of the repair site.

[0029] In specific flexor tendon repair applications, the PCGa@dDIS surface (i.e., the rough surface of the first functional layer) faces the tendon to be repaired, and the OHA / CMCS hydrogel surface (i.e., the smooth surface of the second functional layer away from the first functional layer) faces the tendon sheath or surrounding soft tissue, and is wrapped around the tendon repair site.

[0030] Example 1 This embodiment illustrates the preparation of a decellularized duck small intestinal mucosal scaffold.

[0031] Fresh duck small intestines were collected and transported to the laboratory under low temperature conditions. The intestinal contents and surface contaminants were removed, and the intestines were longitudinally dissected along the mesenteric margin and repeatedly rinsed with deionized water and phosphate buffer. Mucosal folds, mucus, and residual debris were gently removed, and excess adipose tissue, connective tissue, and visible muscle layer were excised. The resulting continuous duck small intestine mucosal sheet was designated as DIS.

[0032] The DIS was initially sterilized and decontaminated by sonication in 75% ethanol for 20 min, and then thoroughly washed with PBS. It was then placed in a solution containing 0.5% SDS and 1% Triton X-100 and shaken for 24 h. After repeated rinsing with PBS, it was treated with DNase I at 37 °C to further remove residual nucleic acids, and then thoroughly washed with PBS and deionized water. Finally, it was frozen and lyophilized at -80 °C. The resulting scaffold was designated dDIS.

[0033] like Figure 1 As shown, after decellularization, the scaffold transformed from its original light yellow tissue into a thinner, translucent, and continuous membrane. Residual DNA decreased from 642.6 ± 10.4 ng / mg in DIS to 15.6 ± 3.0 ng / mg in dDIS. DAPI staining revealed almost no visible cell nuclei after decellularization, and agarose gel electrophoresis showed no obvious high-molecular-weight DNA bands. H&E, Masson, and Sirius Red staining showed a significant reduction in cellular components while maintaining the continuity of the collagen fiber framework; type I collagen, type III collagen, fibronectin, and laminin were still detectable, indicating that this treatment could remove major cellular and nucleic acid residues while preserving the main extracellular matrix structures.

[0034] Example 2 This embodiment illustrates the preparation and screening of polymeric flavonoids / gallium-functionalized dDIS.

[0035] A 0.5 mg / mL solution of a polymeric flavonoid was prepared, using gallium nitrate nonahydrate as Ga³⁺. + Sources, respectively based on polymeric flavonoids and Ga³ + PC / Ga composite solutions were prepared at molar ratios of 1:1, 1:2, and 1:4. Equal-sized dDIS samples were immersed in these composite solutions and incubated with shaking at room temperature in the dark to allow the polymerizable flavonoids and Ga³⁺ to react. + The polymeric flavonoids are loaded onto the scaffold surface and internal fibrous structure through coordination and non-covalent interactions such as hydrogen bonding between polymeric flavonoids and collagen. They are then gently rinsed with deionized water to remove unbound polymeric flavonoids and free Ga³⁺. + The samples were freeze-dried and labeled as PCGa1@dDIS, PCGa2@dDIS, and PCGa3@dDIS, respectively; unmodified dDIS served as a control.

[0036] like Figure 2 As shown in AI, scanning electron microscopy reveals that the original continuous layered and oriented fiber structure of dDIS remains intact after functionalization, with granular or film-like deposition appearing on the fiber surface and increasing with Ga³⁺. +The number of fibers increased with increasing feed ratio. The fiber sizes in each group exhibited a narrow, unimodal distribution, indicating that functionalization did not significantly disrupt the matrix structure. Atomic force microscopy revealed increased surface nano-ripples; the average roughness of dDIS was 50.56 ± 6.48 nm, PCGa2@dDIS was 89.60 ± 26.67 nm, and PCGa3@dDIS was 128.67 ± 11.00 nm.

[0037] XPS detected characteristic Ga signals in each PCGa@dDIS group. The FTIR spectrum of dDIS is at approximately 3307 cm⁻¹. - A broad peak for the stretching vibrations of OH and NH appears at ¹, and at approximately 1655, 1560, and 1240 cm⁻¹. - ¹ Collagenamide bands I, II, and III are retained; the peak shape and relative intensity change after functionalization, but the collagen characteristic bands are still clearly distinguishable, indicating the presence of polymerizable flavonoids and Ga³. + Successfully introduced without significantly damaging the collagen backbone.

[0038] In the DPPH experiment, dDIS only slightly lightened the purple solution, while all three PCGa@dDIS formulations significantly changed the solution from purple to light yellow and significantly reduced the absorbance near 517 nm, demonstrating that PC / Ga functionalization significantly enhances the free radical scavenging ability. Considering the moderate micro / nano roughness, functional component loading, and antioxidant properties, PCGa2@dDIS was used in subsequent examples and will be referred to as PCGa@dDIS for short.

[0039] Example 3 This embodiment illustrates the construction of the OHA / CMCS hydrogel and the Janus scaffold.

[0040] Hyaluronic acid was dissolved in deionized water, and sodium periodate solution was added. The reaction was carried out under light-protected conditions, which oxidized the vicinal diol structure on the hyaluronic acid backbone and introduced aldehyde groups. Ethylene glycol was added to terminate the oxidation after the reaction. Unreacted small molecules were removed by dialysis, and lyophilization was performed to obtain oxidized hyaluronic acid (OHA).

[0041] A 5% (w / w) solution of OHA and a 5% (w / w) solution of CMCS were prepared using PBS. When the two solutions were mixed, the aldehyde groups in OHA and the amino groups in CMCS underwent a dynamic Schiff base reaction to form an OHA / CMCS hydrogel. In the inverted bottle experiment, no visible flow occurred in the mixture, indicating the formation of a self-supporting gel. After the pre-prepared gel was cut and reassembled, it could restore its continuous structure and withstand tweezer pull, demonstrating that dynamic bonds can drive interfacial reconstruction.

[0042] After mixing the OHA / CMCS precursors but before complete gelation, they were uniformly coated onto one side of PCGa@dDIS and allowed to gel in situ to obtain the PCGa@dDIS / OHA-CMCS Janus scaffold. The uncoated side retained the PCGa@dDIS fiber surface as a tendon contact surface; the coated side formed a hydrogel surface, serving as an anti-adhesion barrier surface towards the surrounding tissues.

[0043] like Figure 3 As shown, OHA is at approximately 1730 cm. - ¹A new peak of aldehyde C=O stretching vibration was observed, and a new signal consistent with the structure of hydrated aldehyde or hemiacetal appeared in the proton NMR spectrum at 4.7–5.0 ppm. Cross-sectional scanning electron microscopy showed that the hydrogel layer was confined to one side of PCGa@dDIS, with continuous interlayer interfaces and no obvious pores, delamination, or peeling; the scaffold remained intact after significant bending and torsion.

[0044] Example 4 This embodiment demonstrates the degradation, swelling, antioxidant, and responsive release properties of the Janus scaffold.

[0045] The initial dry weights W0 of lyophilized dDIS, PCGa@dDIS, and Janus scaffold were weighed and placed in PBS at 37 ℃. Samples were removed at a predetermined time, surface liquid was aspirated, and the wet weight Wt was measured. The swelling rate was calculated as (Wt-W0) / W0×100%. All three materials underwent rapid liquid aspiration and entered a relatively stable swelling range within 24 h. The Janus scaffold did not exhibit sustained excessive swelling.

[0046] Samples were placed in an enzyme-containing degradation medium at 37 °C, removed, washed, freeze-dried, and weighed at predetermined times. Degradation was evaluated as the percentage of remaining dry weight to initial dry weight. The Janus scaffold experienced rapid mass loss in the early stages, followed by a slower degradation rate, maintaining a continuous structure throughout the experimental period.

[0047] like Figure 3 J and Figure 3 As shown in K, the DPPH radical scavenging ability imparted by PCGa@dDIS was retained after unilateral deposition of the hydrogel. PCGa@dDIS or Janus scaffolds were placed under neutral conditions, pH 4.5 acidic conditions, and conditions containing reactive oxygen species, respectively, with periodic sampling and replenishment of an equal volume of fresh medium; polymerizable flavonoids were quantified using UV-Vis spectrophotometry, and Ga³... + Quantitative analysis was performed using inductively coupled plasma atomic emission spectrometry. Figure 3 L and Figure 3 M shows that both active components exhibited rapid early release followed by a gradual plateau; the cumulative release under acidic and reactive oxygen species environments was higher than that under neutral environments, indicating that the stent has microenvironment-responsive release characteristics.

[0048] Example 5 This embodiment illustrates the evaluation of blood compatibility.

[0049] Fresh anticoagulated whole blood was collected, and red blood cells were separated by centrifugation and resuspended after repeated washing with PBS. Equal masses of dDIS, PCGa@dDIS, and Janus scaffolds were incubated with the red blood cell suspension at 37 °C; PBS was used as a negative control, and deionized water was used as a positive control. After incubation, the cells were centrifuged, the color of the supernatant was recorded, and the absorbance was measured at 541 nm. The hemolysis rate was calculated as (A sample - A negative) / (A positive - A negative) × 100%.

[0050] like Figure 6 As shown, the supernatants of the three stent groups were close to the PBS negative control, and no obvious red color was observed in the deionized water positive control. The hemolysis rates of dDIS, PCGa@dDIS, and Janus stents were all less than 2%, indicating that they have good blood compatibility.

[0051] Example 6 This embodiment illustrates the evaluation of antibacterial performance.

[0052] Gram-positive Staphylococcus aureus ATCC 25923 and Gram-negative Escherichia coli ATCC 25922 were selected. The bacteria were cultured to the logarithmic growth phase and adjusted to the same bacterial concentration, and then co-incubated with dDIS, PCGa@dDIS and Janus scaffolds at 37 °C for 24 h; the bacterial suspension without scaffolds served as a blank control.

[0053] After co-incubation, the bacteria were stained with DMAO / PI live and dead bacteria and observed under dark conditions. The bacteria were then fully eluted from the scaffold surface, serially diluted, and spread on agar plates. The plates were incubated overnight at 37 °C and colonies were counted. Simultaneously, the bacterial-scaffold samples were fixed, dehydrated with serial ethanol, dried, and sputter-coated with gold before being observed under a scanning electron microscope.

[0054] like Figure 4 As shown, the green DMAO signal was significantly reduced and the red PI signal was widely increased in the PCGa@dDIS and Janus groups; the number of bacterial colonies on plates was significantly reduced, and the average inhibition rate against both bacteria was close to or exceeded 90%. Under scanning electron microscopy, the bacteria in the PCGa@dDIS and Janus groups exhibited irregular outlines, surface depressions, wrinkling, and local collapse, while the bacteria in the blank group and dDIS group had relatively intact morphology. These results indicate that polymeric flavonoids and Ga³⁺... + The synergistic effect of these components can cause damage to the bacterial envelope and interfere with iron-dependent metabolism, giving the Janus scaffold broad-spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria.

[0055] Example 7 This example illustrates the repair of rat flexor tendon rupture and the evaluation of peritendinous adhesion resistance.

[0056] All animal experiments were approved by the Animal Ethics Committee of Nantong University, approval number S20250930-005. Experimental animals were randomly divided into a control group, a dDIS group, a PCGa@dDIS group, and a Janus group, with n=12 in each group. Under anesthesia and aseptic conditions, the skin and subcutaneous tissue of the rat paw were incised to expose the target flexor tendon. The tendon was then severed at a consistent anatomical location and repaired in situ using a modified Kessler suture technique.

[0057] In the material handling group, the repair site was wrapped with size-matched dDIS, PCGa@dDIS, or Janus scaffolds; the PCGa@dDIS side of the Janus scaffold faced the injured tendon, and the OHA / CMCS hydrogel side faced the surrounding soft tissue. No scaffold was implanted in the control group. The incision was closed layer by layer, and postoperative analgesia and feeding were performed according to the approved protocol. Materials were harvested at 2 and 3 weeks postoperatively.

[0058] While maintaining the integrity of the repair site and surrounding adhesions, the limb was immobilized. Loads of 0, 5, 10, 15, and 20 g were applied sequentially to the proximal flexor muscle belly. After the foot stabilized, photographs were taken and the plantar flexion angle was measured. Following the sliding test, the tendon was dissected while preserving the repaired area. Both ends were clamped in a universal testing machine and uniaxially stretched at a constant speed until fracture. The load-displacement curve and the maximum failure load were recorded. The repaired area, along with surrounding tissues, was fixed, embedded in paraffin, and stained with H&E and Masson staining.

[0059] like Figure 5 As shown, at 2 weeks post-operation, the blank group exhibited smaller plantar flexion amplitude under the same load, and more scar-like tissue was visible around the repair site. dDIS and PCGa@dDIS treatments improved tendon contour and gliding. The Janus group showed the greatest change in plantar flexion angle under each test load and the least peritendinous fiber bridging. The maximum failure load at 2 weeks post-operation in the Janus group was significantly higher than that in the blank group, indicating that anti-adhesion did not come at the expense of repair strength.

[0060] At 3 weeks post-surgery, gliding function improved in all groups compared to 2 weeks, but the Janus group still exhibited the greatest load-dependent plantar flexion change, the clearest tendon-surrounding tissue boundary, and the highest mean maximum failure load. H&E staining showed that the repaired tissue in the Janus group was more uniform, with fibers and tissue structures mainly arranged along the long axis of the tendon, and less disordered peritendinous tissue; Masson staining showed that its collagen continuity and longitudinal arrangement were superior to the other groups. These results demonstrate that the Janus scaffold can synergistically reduce extrinsic scar adhesions and improve the intrinsic structure and mechanical repair of the tendon.

[0061] Example 8 This embodiment illustrates a safety evaluation of major organs and tissues.

[0062] Animals were euthanized at the end of the experiment, and their hearts, livers, spleens, lungs, and kidneys were harvested. The tissues were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and stained with H&E. The changes in tissue structure, inflammatory infiltration, necrosis, and hemorrhage were observed under the same microscopic imaging conditions.

[0063] like Figure 7 As shown, no obvious material-related tissue damage, necrosis, hemorrhage or abnormal inflammatory infiltration was observed in the heart, liver, spleen, lungs and kidneys of the blank group, dDIS group, PCGa@dDIS group and Janus group, indicating that the scaffold did not cause obvious systemic tissue damage during the observation period of this experiment.

[0064] In summary, this invention utilizes decellularized duck small intestinal mucosa to construct a tendon contact layer that retains its natural fibrous structure. It endows the layer with antioxidant, antibacterial, and microenvironment-responsive release functions through polymeric flavonoid / gallium coordination functionalization, and constructs a dynamically Schiff base-crosslinked OHA / CMCS hydrogel barrier layer on one side of the layer. This spatially asymmetric structure can simultaneously reduce peritendinous adhesions, improve tendon sliding, and enhance repair strength in animal tendon rupture repair, while exhibiting good blood compatibility and short-term systemic tissue safety. It is suitable for preparing medical implant materials for tendon repair and postoperative anti-adhesion. Figure 8 ).

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A functionalized Janus extracellular matrix hydrogel scaffold, characterized in that, The device comprises a first functional layer and a second functional layer that are combined with each other. The first functional layer includes a decellularized duck small intestinal mucosal scaffold functionalized with polymeric flavonoids and trivalent gallium ions, which are based on benzopyran rings as the basic structural units. The second functional layer is an oxidized hyaluronic acid and carboxymethyl chitosan hydrogel layer disposed on one side surface of the first functional layer. The oxidized hyaluronic acid and the carboxymethyl chitosan are connected by dynamic Schiff base bonds. The side surface of the first functional layer without hydrogel is used for contact with tendons. The second functional layer constitutes an anti-adhesion barrier surface facing the surrounding tissues.

2. The functionalized Janus extracellular matrix hydrogel scaffold according to claim 1, characterized in that, The decellularized duck small intestine mucosal scaffold is prepared by the following steps: pre-cleaning of duck small intestine, ethanol treatment, decellularization with a combination of ionic and nonionic surfactants, nuclease treatment, washing, and freeze-drying.

3. The functionalized Janus extracellular matrix hydrogel scaffold according to claim 2, characterized in that, The ethanol treatment includes sonication with ethanol; the combined decellularization includes oscillation with a solution containing sodium dodecyl sulfate and Triton X-100. Preferably, the ethanol treatment includes sonication in 50%-90% ethanol for 5-40 min; the combined decellularization includes shaking treatment with a solution containing 0.1-5% sodium dodecyl sulfate and 0.1-5% Triton X-100 for 5-48 h. Preferably, the ethanol treatment includes sonication in 75% ethanol for 20 min; the combined decellularization includes shaking treatment with a solution containing 0.5% sodium dodecyl sulfate and 1% Triton X-100 for 24 h.

4. The functionalized Janus extracellular matrix hydrogel scaffold according to claim 1, characterized in that, The polymeric flavonoid is a polyhydroxy polymer composed of flavan-3-ol units linked by carbon-carbon bonds, with a concentration of 0.1-5 mg / mL; the trivalent gallium ions are derived from gallium nitrate nonahydrate; the molar ratio of the polymeric flavonoid to the trivalent gallium ions is 1:(1-4); Preferably, the concentration of the polymeric flavonoid is 0.5 mg / mL, the source of the trivalent gallium ions is gallium nitrate nonahydrate, and the molar ratio of the polymeric flavonoid to the trivalent gallium ions is selected from 1:1, 1:2 or 1:

4.

5. The functionalized Janus extracellular matrix hydrogel scaffold according to claim 4, characterized in that, The molar ratio of the polymeric flavonoid to the trivalent gallium ions is 1:

2.

6. The functionalized Janus extracellular matrix hydrogel scaffold according to claim 1, characterized in that, The oxidized hyaluronic acid and the carboxymethyl chitosan were each prepared into a 5% (w / w) solution using phosphate buffer, and then mixed to form the hydrogel layer. Preferably, the hydrogel layer is capable of reforming a continuous interface through dynamic Schiff base bonds after cutting and maintaining its bond with the first functional layer when bent or twisted.

7. The functionalized Janus extracellular matrix hydrogel scaffold according to any one of claims 1-6, characterized in that, The hydrogel scaffold exhibits an inhibition rate of nearly or greater than 90% against Staphylococcus aureus and Escherichia coli, and its hemolysis rate is less than 5%.

8. A method for preparing a functionalized Janus extracellular matrix hydrogel scaffold according to any one of claims 1-7, characterized in that, Includes the following steps: S1, Preparation of decellularized duck small intestinal mucosal scaffold dDIS; S2, dDIS is immersed in a composite solution containing the polymeric flavonoid and the trivalent gallium ions for oscillatory loading, unbound polymeric flavonoid and free trivalent gallium ions are washed away, and then freeze-dried to obtain PCGa@dDIS; S3, oxidized hyaluronic acid (OHA) is prepared by oxidizing hyaluronic acid with sodium periodate; S4, mix the OHA solution with the carboxymethyl chitosan CMCS solution and uniformly coat it on one side of the PCGa@dDIS before gelation to form an OHA / CMCS hydrogel layer in situ, thus obtaining the Janus extracellular matrix hydrogel scaffold.

9. The preparation method according to claim 8, characterized in that, Step S1 includes: longitudinally dissecting the duck small intestine along the mesenteric margin, removing intestinal contents, mucus, adipose tissue, connective tissue, and visible muscle layer; repeatedly washing with deionized water and phosphate buffer; sonicating in 75% ethanol for 20 min; shaking in a solution containing 0.5% sodium dodecyl sulfate and 1% Triton X-100 for 24 h; thoroughly washing after treatment with DNase I at 37 °C, freezing at -80 °C, and freeze-drying. Preferably, step S2 includes: preparing a polymerizable flavonoid solution with a concentration of 0.5 mg / mL, providing trivalent gallium ions with gallium nitrate nonahydrate, and preparing a composite solution according to a molar ratio of polymerizable flavonoid to trivalent gallium ions of 1:1, 1:2, or 1:4; and incubating dDIS with shaking at room temperature and in the dark. Preferably, step S3 includes: dissolving hyaluronic acid in deionized water, adding sodium periodate and carrying out an oxidation reaction under light-protected conditions, adding ethylene glycol to terminate the reaction, removing small molecules by dialysis, and then freeze-drying to obtain OHA; Preferably, step S4 includes: preparing an OHA solution and a CMCS solution, each with a mass fraction of 5%, mixing the two and immediately coating them onto one side of PCGa@dDIS, leaving the other side exposed with the fibrous extracellular matrix surface.

10. The use of the functionalized Janus extracellular matrix hydrogel scaffold according to any one of claims 1-7 in the preparation of medical implant materials for tendon rupture repair; Preferably, the scaffold can reduce peritendinous adhesions, improve tendon sliding, and increase the mechanical strength of the repair site; Preferably, the medical implant material is used for flexor tendon repair, and is configured such that, in use, the PCGa@dDIS side faces the tendon to be repaired, the OHA / CMCS hydrogel side faces the tendon sheath or surrounding soft tissue, and is wrapped around the tendon repair site.

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