Ha-cts-epa self-healing hydrogel and preparation method and application thereof

Abstract

The application discloses a kind of HA-CTS-EPA self-healing hydrogel and its preparation method and application, belong to medical technical field.The application grafts EPA to the amino group on the side chain of chitosan by amide bond, and obtains EPA grafting chitosan derivative (CTS-EPA);Then aldehyde group hyaluronic acid is crosslinked with CTS-EPA by dynamic schiff base reaction, and forms HA-CTS-EPA self-healing hydrogel, with injectability, self-healing, three-dimensional porous network structure and EPA sustained release capacity.Experiments in vitro and in vivo confirm that the hydrogel can effectively remove active oxygen, inhibit NF-κB signal pathway overactivation, protect mitochondrial function, reduce proinflammatory mediators and matrix degradation enzyme expression, provide immune shelter microenvironment for bone marrow mesenchymal stem cells (BMSCs), enhance the survival, retention and chondrogenic differentiation capacity of BMSCs in the pathological joint cavity of osteoarthritis, and can be used as BMSCs joint cavity delivery platform for the disease improvement treatment of osteoarthritis, with good clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, and specifically relates to the preparation of a self-healing hydrogel and its application in osteoarthritis. Background Technology

[0002] Osteoarthritis (OA) is a chronic degenerative disease characterized by the degeneration of articular cartilage, affecting the synovium, subchondral bone, and surrounding tissues. Its clinical burden and disability risk continue to rise with population aging. Traditional treatments, primarily focused on pain relief, anti-inflammation, and end-stage joint replacement, struggle to achieve structural reversal at the disease level. One fundamental reason is that OA is not simply mechanical wear and tear, but a systemic histopathological process driven by the breakdown of joint microenvironment homeostasis: the continuous release of inflammatory mediators, matrix metabolic imbalance, apoptosis, and abnormal subchondral bone remodeling mutually reinforce each other, resulting in a long-term suppression of local repair capabilities. Therefore, strategies centered on "remodeling the microenvironment—restoring tissue homeostasis" are considered a key direction for achieving disease-modifying treatment.

[0003] In the pathological network of osteoarthritis (OA), excessive accumulation of reactive oxygen species (ROS) and oxidative stress are considered crucial molecular events spanning the early, middle, and late stages. ROS not only directly induces membrane lipid peroxidation, DNA oxidative damage, and mitochondrial dysfunction, but also acts as a signaling hub, promoting the sustained activation of multiple pro-inflammatory pathways. In particular, there is a mutually amplifying positive feedback loop between ROS and NF-κB: ROS promotes IκBα phosphorylation and degradation and enhances p65 nuclear translocation, thereby driving the expression of inflammatory factors such as TNF-α and IL-1β, and matrix-degrading enzymes such as MMP-13; while the continuous release of inflammatory factors further exacerbates mitochondrial stress and ROS generation, ultimately forming a vicious cycle of "oxidative stress—inflammatory amplification—matrix degradation," driving irreversible cartilage degeneration. Disrupting this axis and restoring redox balance has a clear mechanistic rationale and interventional value.

[0004] Bone marrow mesenchymal stem cells (BMSCs) have been widely explored for regenerative osteoarthritis (OA) treatment due to their immunomodulatory paracrine effects, multi-lineage differentiation potential, and pro-repair capabilities. However, intra-articular injection of free BMSCs often faces the dilemma of "immediate decay upon entry": on the one hand, the high ROS and inflammatory factor environment within the OA joint cavity can rapidly damage mitochondria and membrane potential, inducing apoptosis and leading to a sharp drop in the effective cell count; on the other hand, the fluid shearing and dilution effects caused by joint activity make it difficult for cells to remain stably in the damaged area, further limiting the time window for their paracrine regulation and tissue repair effects. Therefore, improving the efficacy of BMSCs depends not only on the cells themselves but also on whether the local "soil" in which they are located is simultaneously improved.

[0005] Hydrogels, with their high water content, three-dimensional network, and biomimetic extracellular matrix characteristics, have become important candidate platforms for local delivery of bioactive molecules into the joint cavity. In particular, injectable self-healing hydrogels constructed based on dynamic covalent bonds can rapidly restore their network structure after shearing or damage, thus adapting to complex movements and repeated stresses within the joint cavity. Currently, self-healing hydrogels constructed based on aldehyde-modified hyaluronic acid and chitosan derivatives via a Schiff base reaction have been reported. For example, invention patent CN115192772A discloses an injectable conductive hydrogel constructed based on aldehyde-modified hyaluronic acid (OHA) and catechol-modified chitosan (DCS) via a Schiff base reaction for targeted repair of articular cartilage. This hydrogel imparts conductivity by introducing PDA-PAni-GO nano-conductive fillers and combines pulsed direct current stimulation to achieve targeted recruitment of cells and induction of chondrogenic differentiation. Furthermore, the invention patent with publication number CN118141976A discloses a hydrogel complex formed by carboxymethyl chitosan and aldehyde-modified hyaluronic acid through a Schiff base reaction, which can be used as a three-dimensional cell culture scaffold for wound repair. The aforementioned prior art demonstrates that aldehyde-modified hyaluronic acid and chitosan derivatives can be cross-linked via dynamic Schiff bases to construct injectable self-healing hydrogels.

[0006] However, the aforementioned hydrogel systems primarily focus on providing passive or physical functions such as physical support, cell encapsulation, and conductive stimulation, lacking the ability to actively regulate oxidative stress and inflammatory cascade responses in the pathological microenvironment of osteoarthritis (OA). Specifically, these hydrogels struggle to sustainably reverse the high ROS and pro-inflammatory factor microenvironment within the OA joint cavity, resulting in a more "coexistent" rather than "synergistic" relationship between the material and cells or drugs, thus limiting the depth of their intervention in OA disease progression.

[0007] Eicosapentaenoic acid (EPA), an ω-3 polyunsaturated fatty acid, has been proven to possess anti-inflammatory and antioxidant biological effects. Patent CN114886915A discloses a sodium hyaluronate injection solution incorporating EPA, which involves physically mixing an EPA nanoemulsion with a sodium hyaluronate solution for intra-articular injection to treat osteoarthritis (OA). This approach restores the normal viscoelasticity of synovial fluid through exogenous sodium hyaluronate, while simultaneously introducing EPA into the articular cartilage microenvironment to modulate the inflammatory response. Animal experiments showed that this composite injection solution increased knee cartilage thickness and reduced OARSI scores in mice, demonstrating a therapeutic effect on OA. However, in this approach, EPA is physically loaded into a sodium hyaluronate solution in the form of a nanoemulsion. Its release behavior is limited by the self-dispersion and metabolism of the nanoemulsion, making it difficult to achieve sustained release coupled with the degradation kinetics of the delivery carrier. Furthermore, the sodium hyaluronate solution system lacks physical protection and three-dimensional support for cells, making it unsuitable as a delivery platform for combined cell therapy.

[0008] Therefore, developing a delivery system that combines physical protection with active microenvironment regulation to break the vicious cycle of ROS / NF-κB and extend the therapeutic window of BMSCs in pathological joints is an important scientific issue for achieving improved treatment of OA. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention proposes a HA-CTS-EPA self-healing hydrogel, its preparation method, and its applications.

[0010] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0011] This invention provides a method for preparing HA-CTS-EPA self-healing hydrogel, comprising the following steps:

[0012] (1) Hyaluronic acid was dissolved in deionized water and then sodium periodate aqueous solution was added in the dark. The reaction was carried out by stirring in the dark. Ethylene glycol was added to stop the reaction and stirring was continued. Then, solid OHA was obtained by dialysis and freeze drying.

[0013] (2) Dissolve CTS in an aqueous acetic acid solution to obtain a CTS mixture. Also, dissolve EPA, EDC·HCl and NHS in anhydrous ethanol to obtain an EPA mixture.

[0014] (3) The activated EPA mixture was added dropwise to the CTS mixture, and the reaction was carried out by stirring in the dark. The product was then dialyzed and freeze-dried to obtain CTS-EPA.

[0015] (4) Mix the OHA aqueous solution and the CTS-EPA acetic acid solution (pH=5.0-6.0) and carry out the Schiff base reaction to obtain HA-CTS-EPA self-healing hydrogel.

[0016] In step (1), 0.5 mmol of sodium periodate is added per gram of hyaluronic acid, and the initial concentration of the sodium periodate aqueous solution is 0.3-0.6 M; the molecular weight of hyaluronic acid is 0.8-2 × 10⁻⁶. 6 Da.

[0017] The stirring time is 12-36 h, and the amount of ethylene glycol added is based on the molar amount of sodium periodate. For every 5 mmol of sodium periodate, 1.5-3 mL of ethylene glycol is added to terminate the reaction.

[0018] The dialysis is performed in deionized water for 2-4 days, and the freeze-drying temperature is -50℃ to -20℃ for 48-96 hours.

[0019] In step (2), the mass ratio of CTS, EPA, EDC·HCl and NHS is 1:0.25-0.68:0.2-0.52:0.06-0.15; the volume percentage of the acetic acid aqueous solution is 0.5%-2%.

[0020] In step (3), the volume ratio of CTS mixture to EPA mixture is 3-8:1, and the stirring time in the dark is 12-36 h.

[0021] The dialysis process involves first dialyzing the EPA and byproducts in an aqueous solution containing different proportions of ethanol for 3 days using a dialysis bag, and finally dialyzing the EPA and byproducts in deionized water for 1 day.

[0022] In step (4), the mass concentration of the OHA aqueous solution is 2%-5%, and the mass concentration of the CTS-EPA acetic acid solution is 1%-3%; the volume ratio of the OHA aqueous solution to the CTS-EPA acetic acid solution is 1:1.

[0023] This invention provides a HA-CTS-EPA self-healing hydrogel prepared using the aforementioned preparation method.

[0024] The HA-CTS-EPA self-healing hydrogel is obtained by dynamically crosslinking aldehyde-modified hyaluronic acid with EPA-grafted chitosan to form Schiff base bonds.

[0025] The present invention also provides the application of the HA-CTS-EPA self-healing hydrogel in the preparation of drugs for treating osteoarthritis.

[0026] The beneficial effects of this invention are:

[0027] 1. The HA-CTS-EPA hydrogel prepared in this invention exhibits rapid gelation, injectability, shear thinning, and multiple self-healing dynamic network characteristics, forming a uniform porous structure and achieving continuous EPA release, while maintaining good adhesion and retention in in vitro flushing and motion simulation. Under oxidative and inflammatory stimulation, this material reduces intracellular and mitochondrial ROS load and alleviates mitochondrial dysfunction, accompanied by weakened NF-κB cascade activation and nuclear translocation, thereby suppressing pro-inflammatory mediators and matrix degradation phenotypes and promoting the return of cartilage homeostasis-related molecules. BMSCs exhibit better survival and anti-apoptotic ability in the three-dimensional microenvironment of the gel, and are better able to maintain chondrogenic expression and matrix synthesis under inflammatory background; ROS replenishment can weaken the above protective effects and partially restore the NF-κB activation and differentiation-impaired phenotype. In animal experiments, gel delivery significantly prolonged the retention of exogenous BMSCs in the joint cavity and reduced local apoptosis signals. Combined treatment showed more consistent improvement in both cartilage morphology and subchondral bone microstructure. At the same time, in situ oxidative stress and inflammatory markers decreased, accompanied by NF-κB pathway inhibition and enhanced antioxidant system.

[0028] 2. This invention proposes and validates a transformable combined treatment paradigm for osteoarthritis (OA): using an injectable self-healing HA-CTS-EPA hydrogel as a "structural delivery platform + active microenvironment modulator," it enhances the retention, survival, and chondrogenic function of bone mesenchymal stem cells (BMSCs) in the joint cavity by reducing ROS and inhibiting excessive NF-κB activation, ultimately improving cartilage and subchondral bone structure. Its scientific significance lies in amplifying the widely discussed but often fragmented oxidative stress and inflammation in OA, integrating them into an operable ROS / NF-κB axis, and strengthening the evidence chain that "ROS is a key upstream event" using causal complementation experiments, thereby establishing a closed-loop progressive mechanism of "materials-microenvironment-cell function-tissue repair." The HA-CTS-EPA hydrogel system prepared in this invention has the potential to be injection-friendly in the joint cavity, have strong retention capacity, and require no complex exogenous instruments, potentially improving the reproducibility of cell therapy and reducing the frequency of administration; simultaneously, EPA, as a bioactive fatty acid, has relatively favorable safety perceptions, and the hydrogel components and degradation pathways are more easily included in regulatory discussions. This strategy has the potential for clinical translation into injectable, locally administered, and cell-based combined therapies, and is expected to provide a more controllable and durable disease-improving intervention pathway for OA. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 Physicochemical structural characterization of CTS-EPA conjugate and HA-CTS-EPA self-healing hydrogel; where A represents the chemical groups detected by Fourier transform infrared spectroscopy (FT-IR); B represents the proton nuclear magnetic resonance spectroscopy (…). 1 C) Analyze the molecular structure of the material using H-NMR; D) Detect the residual primary amine retention rate using TNBS colorimetric method; E) Determine the gelation time using the inverted test tube method; F) Analyze the rheological storage modulus using rotational rheometer; G) Observe the microstructure and pore size using SEM and Cryo-SEM (scalebar = 50 μm); G) Determine the swelling rate of the hydrogel using the gravimetric method. All experiments were repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001.

[0031] Figure 2The dynamic rheological properties, injectability, and in vitro drug release behavior of the HA-CTS-EPA self-healing hydrogel were evaluated. Specifically, A represents the time-scan test of the hydrogel using a rotational rheometer; B represents the frequency-scan test of the hydrogel using a rotational rheometer; C represents the shear thinning and self-healing ability of the hydrogel using a step strain rheological test; D represents the injectability of the hydrogel using a macroscopic injection experiment; E represents the macroscopic self-healing of the hydrogel using a cut-and-adhesion experiment; F represents the in vitro degradation behavior of the hydrogel using an in vitro simulated degradation experiment; G represents the modulus retention rate of the hydrogel during degradation using a rheometer; H represents the in vitro EPA release curve of the hydrogel using reversed-phase HPLC; and I represents the retention capacity of the hydrogel in the isolated joint cavity using an isolated knee joint scour simulation. All experiments were repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001.

[0032] Figure 3 To demonstrate the excellent cell compatibility of HA-CTS-EPA hydrogel; A shows the CCK-8 assay for detecting the proliferation activity of SD rat BMSCs; B shows the CCK-8 assay for detecting the proliferation activity of SD rat primary chondrocytes; C shows Live / Dead staining to assess the survival status of SD rat BMSCs, live cells (green) / dead cells (red), scale bar = 100 μm; D shows Live / Dead staining to assess the survival status of SD rat primary chondrocytes, live cells (green) / dead cells (red), scale bar = 100 μm; E shows phalloidin / DAPI staining combined with CLSM to observe the cytoskeleton (red) and nucleus (blue) of 3D embedded BMSCs, scale bar = 50 μm; F shows the in vitro simulated shear-flushing experiment to detect the cell residue rate of BMSCs after flushing; G shows Live / Dead staining to detect the survival status of BMSCs after flushing, live cells (green) / dead cells (red), scale bar = 100 μm; all cell experiments were repeated at least three times, *P < 0.05. 0.05, **P < 0.01, ***P < 0.001.

[0033] Figure 4The results show the effects of HA-CTS-EPA hydrogel on the regulation of the osteoarthritis (OA)-like microenvironment. Specifically, A shows intracellular ROS levels detected by DCFH-DA fluorescence staining (green, scale bar = 50 μm); B shows mitochondrial ROS levels detected by MitoSOX fluorescence staining and flow cytometry (red, scale bar = 50 μm); C shows mitochondrial membrane potential detected by JC-1 staining (scale bar = 50 μm); D shows the expression of inflammation and matrix metabolism-related genes in a rat primary chondrocyte chronic inflammation model detected by RT-qPCR; EF shows the activity and apoptosis of BMSCs under IL-1β inflammatory conditions detected by CCK-8 assay and Annexin V-FITC / PI flow cytometry double staining; G shows the expression of chondrogenic genes analyzed by RT-qPCR; and H shows the level of GAG synthesis detected by Alcian Blue staining combined with DMMB assay (scale bar = 100 μm). All cell experiments were repeated at least three times. *P < 0.05, **P < 0.01, ***P < 0.001.

[0034] Figure 5 Results of the study on the molecular mechanism by which HA-CTS-EPA hydrogel improves the microenvironment; A shows the expression of NF-κB pathway proteins detected by Western blot in primary rat chondrocytes; B shows the detection of p65 nuclear translocation (p65 in red, DAPI in blue) by immunofluorescence staining in primary rat chondrocytes, scale bar=20 μm; CD shows the expression of oxidative damage markers 4-HNE and 8-OHdG by immunofluorescence staining in primary rat chondrocytes; E shows the activity of antioxidant enzymes SOD, CAT, and GSH-Px detected by biochemical reagent kits in primary rat chondrocytes; F shows the expression of apoptosis-related proteins detected by Western blot in the BMSCs loading system; G shows the expression of chondrogenic markers detected by Western blot in the BMSCs loading system; all cell experiments were repeated at least three times, *P < 0.05, **P < 0.01, ***P < 0.001.

[0035] Figure 6Results of ROS scavenging activity of HA-CTS-EPA self-healing hydrogel; A shows immunofluorescence staining to detect p65 nuclear translocation levels in chondrocytes, with p65 (red) and DAPI (blue) co-stained, scale bar = 20 μm; B shows Western blot detection of protein expression of p-p65, p65, TNF-α, MMP-13, and cle-Caspase-3 in chondrocytes; C shows Live / Dead staining to assess BMSCs activity and survival, with Live (green) and Dead (red) co-stained, scale bar = 50 μm; D shows Alcian Blue staining to detect GAG secretion in the chondrogenic matrix of BMSCs, scale bar = 100 μm; E shows immunofluorescence staining to assess Col II expression in the chondrogenic matrix of BMSCs, with Col II (green) and DAPI (blue) co-stained, scale bar = 50 μm. μm; F represents the relative mRNA expression levels of chondrogenic genes Sox9, Col2a1, and Acan in BMSCs detected by RT-qPCR; all cell experiments were repeated at least three times, *P < 0.05, **P < 0.01, ***P < 0.001.

[0036] Figure 7 The study aimed to prolong the in vivo retention and survival of BMSCs in the joint cavity of osteoarthritis (OA) using HA-CTS-EPA hydrogel. A represents the dynamic tracking of CM-Dil red fluorescence signals in the joint cavity of rats in each group using in vivo fluorescence imaging. B represents the quantitative analysis of total radiation flux at different time points after injection in each group of rats using in vivo fluorescence intensity analysis. C represents the survival and distribution of CM-Dil (red) and DAPI (blue) positive cells in rat joint tissue using frozen section fluorescence microscopy (scale bar = 100 μm). D represents the quantitative counting of CM-Dil positive cells under high-power fields. E represents the assessment of the expression of CM-Dil (red), cle-Caspase-3 (green), and DAPI (blue) in rat joint tissue using immunofluorescence co-staining (scale bar = 100 μm). F represents the quantitative analysis of the proportion of cle-Caspase-3 / CM-Dil double-positive cells using immunofluorescence positivity rate analysis. All animal experiments were n=10. *P < 0.05, **P < 0.01, ***P < 0.001.

[0037] Figure 8The results of synergistic therapy of HA-CTS-EPA hydrogel combined with BMSCs in vivo are presented. A shows the three-dimensional morphology of subchondral bone microstructure in an ACLT-induced OA rat model assessed by Micro-CT scanning; B shows the quantitative analysis of subchondral bone microstructure parameters assessed by Micro-CT scanning, including bar charts of BV / TV, Tb.Th, Tb.Sp, and Tb.N; C shows the routine morphology of articular cartilage assessed by H&E staining (scale bar = 100 μm); D shows the content of cartilage matrix proteoglycans assessed by Safranin O-Fixed Green staining (red represents proteoglycans), including bar charts with OARSI scores (scale bar = 100 μm); E shows the content of cartilage matrix polysaccharides assessed by toluidine blue staining (blue-purple represents polysaccharides), including bar charts with toluidine blue positive area percentages (scale bar = 100 μm). Animals n = 10, *P < 0.05, **P < 0.01, ***P < 0.001.

[0038] Figure 9 To investigate the molecular mechanism of the in vivo repair effect of HA-CTS-EPA / BMSCs; where A represents the in situ ROS level in a rat OA model detected by DHE frozen section fluorescence staining (red scale bar = 50 μm); B represents the expression of the pro-inflammatory factor TNF-α in a rat OA model detected by immunohistochemistry (scale bar = 50 μm); C represents the expression of apoptotic clec-Caspase-3 in a rat OA model detected by immunofluorescence (scale bar = 50 μm); D represents the expression of the matrix-degrading enzyme MMP-13 in a rat OA model detected by immunohistochemistry (scale bar = 50 μm); E represents the expression of cartilage matrix protein ColII in a rat OA model detected by immunohistochemistry (scale bar = 50 μm); F represents the activation status of the NF-κB pathway in a rat OA model detected by Western blot; G represents the expression of antioxidant enzymes in a rat OA model detected by Western blot; n=10 animals, *P < 0.05, **P < 0.01, ***P < 0.001. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] A method for preparing HA-CTS-EPA self-healing hydrogel, the specific steps of which are as follows:

[0042] (1) Take 1g of hyaluronic acid (HA, Mw=1.0×10) 6 The hydroxyl group (OHA) was dissolved in 100 mL of deionized water. Then, 10 mL of a 0.5 M sodium periodate (NaIO4, Sigma-Aldrich, St. Louis, MO, USA, Cat#311448) aqueous solution was added in the dark. The mixture was magnetically stirred at room temperature in the dark for 24 h to allow the reaction to proceed. After the reaction was complete, 2 mL of ethylene glycol (Sigma-Aldrich, Cat#324558) was added to terminate the oxidation reaction, and stirring was continued for 1 h. The reaction solution was then placed in a dialysis bag (MWCO 3500 Da, Spectrum Labs, Rancho Dominguez, CA, USA) and dialyzed in deionized water for 3 days, changing the water 3 times a day. The dialyzed solution was pre-frozen at -80°C and then freeze-dried in a freeze dryer (Labconco, Kansas City, MO, USA) for 72 hours. The solid OHA was collected for later use.

[0043] (2) Dissolve 1 g of chitosan (CTS, degree of deacetylation ≥90%, Sigma-Aldrich, Cat#448869) in 50 mL of 1% (v / v) acetic acid aqueous solution to obtain CTS mixture. Separately, dissolve 0.46 g of eicosapentaenoic acid (EPA, Sigma-Aldrich, Cat#E2011), 0.36 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, Sigma-Aldrich, Cat#E1769) and 0.1 g of N-hydroxysuccinimide (NHS, Sigma-Aldrich, Cat#130672) in 10 mL of anhydrous ethanol to obtain EPA mixture.

[0044] (3) After activation for 1 hour, 10 mL of EPA mixture was slowly added dropwise to 50 mL of CTS mixture. The reaction mixture was stirred continuously at room temperature in the dark for 24 hours. The reaction product was gradually dialyzed for 3 days in aqueous solution containing different proportions of ethanol through a dialysis bag (MWCO 8000-14000 Da) to remove unreacted EPA and byproducts. Finally, it was dialyzed in deionized water for 1 day to obtain CTS-EPA conjugate. The grafting rate was defined according to the amino retention rate measured by TNBS method. After freeze drying, low grafting rate (CTS-L-EPA) and high grafting rate (CTS-H-EPA) products were obtained respectively.

[0045] (4) 5 mL of 3% (w / v) OHA aqueous solution and an equal volume of 2% (w / v) CTS-EPA acetic acid solution (pH about 5.5) were mixed at room temperature and spontaneously crosslinked using Schiff base reaction to form HA-CTS-L-EPA hydrogel and HA-CTS-H-EPA hydrogel, namely HA-CTS-EPA self-healing hydrogel.

[0046] The CTS-EPA conjugate, HA-CTS-L-EPA hydrogel, and HA-CTS-H-EPA hydrogel prepared in this embodiment were characterized, and the results are as follows: Figure 1 As shown. First, the chemical modifications of the material were characterized using FT-IR and 1H-NMR techniques. Figure 1 AB). The results showed that obvious amide bond characteristic signals appeared in the CTS-EPA spectrum. Further quantitative analysis of the effect of different EPA grafting degrees on the network-forming amino groups was performed using the TNBS method. Figure 1 C). Compared with the pure CTS group, the residual primary amine retention rates of both the CTS-L-EPA and CTS-H-EPA groups were significantly decreased; while compared with the CTS-H-EPA group, the retention rate of the CTS-L-EPA group was significantly higher, suggesting that the low-grafting strategy can retain sufficient gel-forming active sites while introducing functional groups. Subsequently, the physical gel-forming properties of hydrogels with different formulations were evaluated. Inverted tube experiment ( Figure 1 D) and rheological testing ( Figure 1 E) showed that compared with the HA-CTS group without EPA modification, there were no significant differences in the gel time and storage modulus of the HA-CTS-L-EPA constituent; however, compared with the HA-CTS-L-EPA group, the gel time of the HA-CTS-H-EPA constituent was significantly prolonged and the modulus was significantly reduced, indicating that excessive grafting degree would cause steric hindrance. SEM and Cryo-SEM observations ( Figure 1 F) and swelling rate analysis ( Figure 1 G) indicates that, compared to the HA-CTS group, the HA-CTS-L-EPA group exhibits a more regular three-dimensional porous network with a more uniform pore size distribution; the HA-CTS-L-EPA group maintained a higher swelling ratio, significantly higher than the HA-CTS-H-EPA group, suggesting that this structural parameter fully meets the material exchange requirements for subsequent three-dimensional culture of BMSCs. In subsequent experiments, HA-CTS-EPA refers to the selected optimal formulation of HA-CTS-L-EPA after screening.

[0047] To evaluate the mechanical and dynamic properties of HA-CTS-EPA hydrogel, a rotational rheometer was used to conduct systematic tests on the hydrogel. Time and frequency scan results showed that the storage modulus (G') of the HA-CTS-EPA hydrogel remained consistently higher than the loss modulus (G'') within the test range. Figure 2AB), indicating that it possesses a stable elastic network structure. In the step strain experiment, 1% and 300% strains were applied alternately ( Figure 2 C). When the strain increases to 300%, G' rapidly decreases below G'', and the gel network is disrupted; however, when the strain recovers to 1%, G' can rapidly recover to over 90% of its initial level in a very short time. This process can be repeated multiple times, fully supporting its excellent shear thinning and self-healing capabilities. In injectability and macroscopic self-healing tests ( Figure 2 (DE) This hydrogel can be smoothly extruded through a 26G needle and quickly returns to its original shape after extrusion; when two pieces of stained gel are cut and bonded together, they fuse together in a short time and do not break when stretched, directly demonstrating its seamless self-healing performance after breakage. These characteristics suggest that HA-CTS-EPA hydrogel can well adapt to the irregular space of the joint cavity and the complex mechanical movement environment.

[0048] Example 2

[0049] A method for preparing HA-CTS-EPA self-healing hydrogel, the specific steps of which are as follows:

[0050] (1) Take 1g of hyaluronic acid (HA, Mw=1.0×10) 6 The hydroxyl group (DA, Bloomage Biotechnology, Jinan, China) was dissolved in 100 mL of deionized water. Then, 10 mL of a 0.5 M sodium periodate (NaIO4, Sigma-Aldrich, St. Louis, MO, USA, Cat#311448) aqueous solution was added in the dark. The mixture was magnetically stirred at room temperature for 12 h in the dark to allow the reaction to proceed. After the reaction was complete, 1.5 mL of ethylene glycol (Sigma-Aldrich, Cat#324558) was added to terminate the oxidation reaction, and stirring was continued for 1 h. The reaction solution was then transferred to a dialysis bag (MWCO 3500 DA, Spectrum Labs, Rancho Dominguez, CA, USA) and dialyzed in deionized water for 2 days, changing the water 3 times a day. The dialyzed solution was pre-frozen at -80°C and then freeze-dried in a freeze dryer (Labconco, Kansas City, MO, USA) for 48 hours. The solid OHA was collected for later use.

[0051] (2) Dissolve 1 g of chitosan (CTS, degree of deacetylation ≥90%, Sigma-Aldrich, Cat#448869) in 30 mL of 2% (v / v) acetic acid aqueous solution to obtain CTS mixture. Separately dissolve 0.25 g of eicosapentaenoic acid (EPA, Sigma-Aldrich, Cat#E2011), 0.2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, Sigma-Aldrich, Cat#E1769) and 0.06 g of N-hydroxysuccinimide (NHS, Sigma-Aldrich, Cat#130672) in 10 mL of anhydrous ethanol to obtain EPA mixture.

[0052] (3) After activation for 1 hour, 10 mL of EPA mixture was slowly added dropwise to 30 mL of CTS mixture. The reaction mixture was stirred continuously at room temperature in the dark for 36 hours. The reaction product was gradually dialyzed for 3 days in aqueous solution containing different proportions of ethanol through a dialysis bag (MWCO 8000-14000 Da) to remove unreacted EPA and byproducts. Finally, it was dialyzed in deionized water for 1 day to obtain CTS-EPA conjugate.

[0053] (4) 4 mL of 2% (w / v) OHA aqueous solution and an equal volume of 1% (w / v) CTS-EPA acetic acid solution (pH about 5.5) were mixed at room temperature and spontaneously crosslinked using Schiff base reaction to form HA-CTS-L-EPA hydrogel and HA-CTS-H-EPA hydrogel, namely HA-CTS-EPA self-healing hydrogel.

[0054] Example 3

[0055] A method for preparing HA-CTS-EPA self-healing hydrogel, the specific steps of which are as follows:

[0056] (1) Take 1g of hyaluronic acid (HA, Mw=1.0×10) 6The hydroxyl group (DA, Bloomage Biotechnology, Jinan, China) was dissolved in 100 mL of deionized water. Then, 10 mL of a 0.5 M sodium periodate (NaIO4, Sigma-Aldrich, St. Louis, MO, USA, Cat#311448) aqueous solution was added in the dark. The mixture was magnetically stirred at room temperature in the dark for 36 h to carry out the reaction. After the reaction was completed, 3 mL of ethylene glycol (Sigma-Aldrich, Cat#324558) was added to terminate the oxidation reaction, and stirring was continued for 1 h. The reaction solution was then placed in a dialysis bag (MWCO 3500 DA, Spectrum Labs, Rancho Dominguez, CA, USA) and dialyzed in deionized water for 4 days, changing the water 3 times a day. After pre-freezing at -80°C, the dialyzed solution was freeze-dried in a freeze dryer (Labconco, Kansas City, MO, USA) for 96 hours, and the solid OHA was collected for later use.

[0057] (2) Dissolve 1 g of chitosan (CTS, degree of deacetylation ≥90%, Sigma-Aldrich, Cat#448869) in 80 mL of 0.5% (v / v) acetic acid aqueous solution to obtain CTS mixture. Separately dissolve 0.68 g of eicosapentaenoic acid (EPA, Sigma-Aldrich, Cat#E2011), 0.52 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, Sigma-Aldrich, Cat#E1769) and 0.15 g of N-hydroxysuccinimide (NHS, Sigma-Aldrich, Cat#130672) in 10 mL of anhydrous ethanol to obtain EPA mixture.

[0058] (3) After activation for 1 hour, 10 mL of EPA mixture was slowly added dropwise to 80 mL of CTS mixture. The reaction mixture was stirred continuously at room temperature in the dark for 12 hours. The reaction product was gradually dialyzed for 3 days in aqueous solution containing different proportions of ethanol through a dialysis bag (MWCO 8000-14000 Da) to remove unreacted EPA and byproducts. Finally, it was dialyzed in deionized water for 1 day to obtain CTS-EPA conjugate.

[0059] (4) 5 mL of 5% (w / v) OHA aqueous solution and an equal volume of 3% (w / v) CTS-EPA acetic acid solution (pH about 5.5) were mixed at room temperature and spontaneously crosslinked using Schiff base reaction to form HA-CTS-L-EPA hydrogel and HA-CTS-H-EPA hydrogel, namely HA-CTS-EPA self-healing hydrogel.

[0060] Comparative Example 1

[0061] A method for preparing a hydrogel, the specific steps of which are as follows:

[0062] (1) Take 1g of hyaluronic acid (HA, Mw=1.0×10) 6 The hydroxychloroquine (OHA) was dissolved in 100 mL of deionized water. Then, 10 mL of a 5M sodium periodate (NaIO4, Sigma-Aldrich, St. Louis, MO, USA, Cat#311448) aqueous solution was added in the dark. The mixture was magnetically stirred at room temperature in the dark for 24 h to allow the reaction to proceed. After the reaction was complete, 2 mL of ethylene glycol (Sigma-Aldrich, Cat#324558) was added to terminate the oxidation reaction, and stirring was continued for 1 h. The reaction solution was then transferred to a dialysis bag (MWCO 3500 Da, Spectrum Labs, Rancho Dominguez, CA, USA) and dialyzed in deionized water for 3 days, changing the water 3 times a day. The dialyzed solution was pre-frozen at -80°C and then freeze-dried in a freeze dryer (Labconco, Kansas City, MO, USA) for 72 hours. The solid OHA was collected for later use.

[0063] (2) Dissolve 1 g of chitosan (CTS, degree of deacetylation ≥90%, Sigma-Aldrich, Cat#448869) in 50 mL of 1% (v / v) acetic acid aqueous solution to obtain CTS mixture. Separately, dissolve 0.36 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, Sigma-Aldrich, Cat#E1769) and 0.1 g of N-hydroxysuccinimide (NHS, Sigma-Aldrich, Cat#130672) in 10 mL of anhydrous ethanol to obtain EDC / NHS mixture.

[0064] (3) After activation for 1 hour, the EDC / NHS mixture was slowly added dropwise to the CTS mixture. The reaction mixture was stirred continuously at room temperature in the dark for 24 hours. The reaction product was gradually dialyzed for 3 days in aqueous solution containing different proportions of ethanol through a dialysis bag (MWCO 8000-14000 Da). Finally, it was dialyzed for 1 day in deionized water to obtain the CTS conjugate.

[0065] (4) Mix 5 mL of 3% (w / v) OHA aqueous solution and an equal volume of 2% (w / v) CTS acetic acid solution (pH about 5.5) at room temperature to crosslink and form HA-CTS hydrogel.

[0066] Example of implementation effect 1

[0067] The in vitro stability of the HA-CTS-EPA hydrogel prepared in Example 1 was tested as follows:

[0068] The in vitro stability of the gel was assessed using a simulated synovial fluid (containing hyaluronidase) degradation experiment. The degradation curves showed ( Figure 2 F), HA-CTS-EPA hydrogel exhibited controlled degradation, gradual reduction of residual mass, and swelling behavior, and G' remained within an effective range during the early stages of degradation (F). Figure 2 G), meeting the structural support requirements for use as a local delivery carrier. Reversed-phase HPLC was used to detect the release kinetics of EPA in the hydrogel ( Figure 2 (H) The results confirmed that HA-CTS-EPA has stable sustained-release characteristics in the simulated synovial fluid system, and the release curve conforms to the Ritger-Peppas model, indicating that the release of EPA is jointly controlled by gel network degradation and drug diffusion. Furthermore, in the isolated porcine knee joint cavity simulation experiment ( Figure 2 I) Fluorescently labeled HA-CTS-EPA hydrogel adhered to the cartilage surface, exhibiting strong local adhesion and significant retention even after repeated flushing and joint flexion and extension. (Summary) Figure 2 The results show that the HA-CTS-EPA hydrogel possesses the physical and functional properties to meet the local mechanical requirements of the joint cavity, ensure stable drug release, and allow for long-term retention, laying a material foundation for subsequent cell delivery.

[0069] To evaluate the potential of HA-CTS-EPA hydrogel as a cell delivery carrier, this invention employed both hydrogel extract and direct three-dimensional embedding methods to culture SD rat bone marrow mesenchymal stem cells (BMSCs) and primary chondrocytes, and systematically tested their cell compatibility. First, cell proliferation activity was detected using the CCK-8 assay. Figure 3 AB). Results showed that, during culture periods of 1, 3, 7, and 14 days, compared to the Control group, the proliferation activity of BMSCs and primary chondrocytes in the HA-CTS-EPA extract group was not significantly different, and both maintained a continuous proliferation trend, indicating that this material did not produce significant cytotoxicity. Live / Dead staining was further used to assess cell viability ( ). Figure 3(CD). Fluorescence images showed that, after 7 days of culture in HA-CTS-EPA extract, the vast majority of BMSCs and primary chondrocytes exhibited green fluorescence (live cells), while very few cells showed red fluorescence (dead cells). Quantitative analysis showed that the survival rate remained above 95%, with no significant decrease compared to the Control group, further supporting the excellent in vitro cell biocompatibility of HA-CTS-EPA hydrogel.

[0070] Subsequently, BMSCs were encapsulated in HA-CTS-EPA hydrogel for 3D culture to evaluate their support function for three-dimensional cell growth. Cytoskeleton morphology and distribution were observed using phalloidin / DAPI staining combined with confocal laser scanning microscopy (CLSM). Figure 3 E). The results showed that with the extension of culture time (days 3 and 7), BMSCs encapsulated in the gel network were evenly distributed, exhibiting a typical fibroblast-like extended scaffold morphology, and gradually establishing extensive intercellular connections, suggesting that the gel network can provide a suitable three-dimensional physical support microenvironment for BMSCs. Finally, to verify the cell protection function of the hydrogel under the mechanical stress environment of the joint cavity, this invention established an in vitro simulated shear scour system ( Figure 3 (FG). After mechanical flushing, compared with the free BMSCs group, the BMSCs loaded with HA-CTS-EPA gel showed a significantly higher cell residue rate, and the survival rate and morphological integrity of the remaining cells were significantly better than those in the free group. In conclusion, HA-CTS-EPA hydrogel not only possesses excellent cell compatibility but also provides a favorable 3D proliferation microenvironment for BMSCs and effectively resists cell loss caused by shear stress.

[0071] Example of implementation effect 2

[0072] The ability of the HA-CTS-EPA hydrogel prepared in Example 1 to regulate the osteoarthritis (OA)-like microenvironment was tested, as follows:

[0073] This invention utilizes H2O2 or IL-1β to stimulate primary rat chondrocytes to establish an in vitro injury model. DCFH-DA probe detection revealed that, compared to the Control group, the intracellular ROS level in the H2O2-stimulated group was significantly increased; while compared to the H2O2 group, the ROS green fluorescence intensity in the HA-CTS-EPA-treated group was significantly decreased. Figure 4 A). Further MitoSOX and JC-1 assays showed that, compared with the H2O2 group, the excessive production of mitochondrial ROS in the HA-CTS-EPA group was significantly inhibited ( Figure 4B), and the decrease in mitochondrial membrane potential was effectively reversed, with the red / green fluorescence ratio significantly increasing (B). Figure 4 C). Regarding inflammation and matrix metabolism, RT-qPCR results indicated that, compared with the IL-1β model group, HA-CTS-EPA significantly downregulated the expression of pro-inflammatory and matrix degradation genes such as TNF-α, IL-1β, and MMP-13, while significantly upregulating the expression of Sox9, Col2a1, and Aggrecan. Figure 4 D).

[0074] Subsequently, the protective effect of the hydrogel on BMSCs was evaluated in the IL-1β-induced inflammatory microenvironment. Cell viability and apoptosis assays showed that, compared with the IL-1β+ free BMSCs group alone, IL-1β+HA-CTS-EPA-loaded BMSCs exhibited significantly higher cell viability and lower apoptosis rate. Figure 4 EF). Furthermore, under chondrogenic induction conditions, compared to the free BMSCs group, the gel-loaded group maintained significantly higher levels of chondrogenic-related gene expression (Sox9, Col2a1) and glycosaminoglycan (GAG) synthesis. Figure 4 In summary, HA-CTS-EPA effectively protects BMSC function by alleviating oxidative stress and inflammation.

[0075] Example of implementation effect 3

[0076] The molecular mechanism by which the HA-CTS-EPA hydrogel prepared in Example 1 improves the osteoarthritis (OA)-like microenvironment was studied, as follows:

[0077] This invention focuses on the ROS / NF-κB signaling axis and has been systematically validated. In a rat primary chondrocyte model, control, IL-1β stimulation, IL-1β+HA-CTS, and IL-1β+HA-CTS-EPA groups were established. First, the activation status of the NF-κB pathway was detected by Western blot. Figure 5 A). The results showed that, compared with the Control group, the phosphorylation levels of p65 and IκBα (p-p65 / p65 and p-IκBα / IκBα ratios) were significantly increased in the IL-1β group; while compared with the IL-1β group and the IL-1β+HA-CTS group, the IL-1β+HA-CTS-EPA group significantly inhibited the abnormal phosphorylation of these two proteins, suggesting that the EPA-functionalized hydrogel effectively blocked the cascade amplification of NF-κB signaling. Subsequently, the nuclear translocation of p65 was observed using immunofluorescence staining ( Figure 5B). Compared with the Control group, p65 was significantly enriched in the nucleus in the IL-1β group, and the nuclear / cytoplasmic fluorescence ratio increased sharply; while in the IL-1β+HA-CTS-EPA group, the nuclear translocation of p65 was significantly blocked, and the nuclear / cytoplasmic ratio decreased significantly, further confirming its inhibitory effect on the activation of inflammatory signals.

[0078] In terms of mitigating oxidative damage, this invention assessed the expression of lipid peroxidation product 4-HNE and DNA oxidative damage marker 8-OHdG using immunofluorescence (IF) staining. Figure 5 CD). Compared with the control group, IL-1β stimulation led to a large accumulation of 4-HNE and 8-OHdG, with significantly enhanced fluorescence signals; compared with the IL-1β group and the IL-1β+HA-CTS group, the IL-1β+HA-CTS-EPA group significantly reduced the fluorescence intensity of these two biomarkers of oxidative damage. Simultaneously, antioxidant enzyme activity was detected ( Figure 5 E) indicates that the HA-CTS-EPA hydrogel significantly enhances the activity of SOD, CAT and GSH-Px, effectively restoring intracellular redox homeostasis.

[0079] Furthermore, in the BMSCs loading system, compared with the free cell group stimulated by IL-1β, the HA-CTS-EPA hydrogel encapsulation group significantly downregulated the expression of pro-apoptotic proteins Ccl-Caspase-3 and Bax, and upregulated the expression of anti-apoptotic protein Bcl-2 (…). Figure 5 F). Meanwhile, the protein expression levels of chondrogenic markers Sox9, Col2a1, and Acan were also significantly maintained (F). Figure 5 G). In summary, HA-CTS-EPA hydrogel effectively protects chondrocyte homeostasis and BMSC function by simultaneously alleviating oxidative damage and blocking NF-κB inflammatory signaling activation.

[0080] Example of implementation effect 4

[0081] The ROS scavenging of the HA-CTS-EPA hydrogel prepared in Example 1 was verified by the following experiment:

[0082] To further verify the key role of ROS in the biological effects of HA-CTS-EPA, this invention sets up a ROS replenishment experiment in the IL-1β-induced inflammatory microenvironment. Immunofluorescence analysis showed that, based on HA-CTS-EPA treatment, exogenous H2O2 intervention significantly enhanced the nuclear translocation level of p65 (…). Figure 6 A) suggests that increased ROS levels may weaken the inhibitory effect of HA-CTS-EPA on the NF-κB pathway.

[0083] Western blot results further showed that, compared with the IL-1β+HA-CTS-EPA group, H2O2 supplementation significantly upregulated the expression levels of p-p65, TNF-α, MMP-13, and cle-Caspase-3. Figure 6 B), indicating that the inflammatory response and apoptosis were reactivated. In the BMSCs loading system, CCK-8 and Live / Dead staining showed that H2O2 intervention significantly reduced cell viability (B). Figure 6 (C) indicates that increased ROS can weaken the microenvironmental protection effect of the HA-CTS-EPA construction.

[0084] Under chondrogenic induction conditions, Alcian Blue staining and GAG / DNA quantification showed that H2O2 replenishment significantly reduced matrix secretion capacity. Figure 6 D); Col II immunofluorescence signal was significantly weakened ( Figure 6 E); at the same time, the expression of Sox9, Col2a1 and Acan genes was significantly downregulated ( Figure 6 F).

[0085] In summary, elevated ROS levels can significantly weaken the anti-inflammatory and cartilage differentiation-promoting effects of HA-CTS-EPA, suggesting that its biological effects depend on the regulation of oxidative stress.

[0086] Example of implementation effect 5

[0087] The HA-CTS-EPA hydrogel prepared in Example 1 was subjected to in vivo cell protection and delivery efficiency experiments, as detailed below:

[0088] This invention uses an anterior cruciate ligament transection (ACLT) in rats to construct an osteoarthritis (OA) model, and performs intra-articular intervention 2 weeks post-surgery. Rat bone marrow mesenchymal stem cells (BMSCs) are labeled with CM-Dil fluorescent dye, and the in vivo cell retention, distribution, and relative survival status of the OA+BMSCs group, OA+HA-CTS+BMSCs group, and OA+HA-CTS-EPA+BMSCs group at different time points (1, 2, and 4 weeks) after injection are systematically compared and evaluated. First, the CM-Dil fluorescence signal in the joint cavity of each group is dynamically tracked using small animal in vivo fluorescence imaging technology. Figure 7A). The results showed that the fluorescence signal in the free BMSCs injection group decreased sharply after 1 week, and was almost undetectable by 4 weeks. Compared with the OA+BMSCs group, the fluorescence intensity of the OA+HA-CTS+BMSCs group was increased at all time points, and the cell retention time was partially prolonged, but the signal intensity still showed a significant decreasing trend over time. Compared with the OA+HA-CTS+BMSCs group, the CM-Dil red fluorescence signal of the OA+HA-CTS-EPA+BMSCs group remained at the highest level throughout the 4-week observation period. Figure 7 B), suggesting that HA-CTS-EPA hydrogel can significantly prolong the retention time of exogenous BMSCs in the joint cavity.

[0089] Subsequently, this invention further evaluated the survival and spatial distribution characteristics of BMSCs in the joint cavity through frozen sections of joint tissue and fluorescence microscopy. Figure 7 C). Histological section imaging results were highly consistent with in vivo imaging data: 4 weeks post-injection, almost no CM-Dil positive cells remained in the joint cavity of the OA+BMSCs group. Compared with the OA+BMSCs group, the OA+HA-CTS+BMSCs group showed a small amount of discrete red fluorescence signals around the cartilage and synovium, indicating that the basal hydrogel provided limited physical retention. However, compared with the OA+HA-CTS+BMSCs group, the OA+HA-CTS-EPA+BMSCs group not only showed a significantly increased cell viability density ( Figure 7 D), and a large number of CM-Dil positive cells showed a high-density, uniform distribution on the surface of the damaged cartilage and in the surrounding synovial region. To further assess the functional status of these surviving cells, the slides were subjected to immunofluorescence co-staining with the apoptosis marker cle-Caspase-3 (D). Figure 7 The results showed that the proportion of CM-Dil positive cells in the OA+BMSCs group was not included in the quantitative statistics due to the small number of cells. However, compared with the OA+HA-CTS+BMSCs group, the proportion of cle-Caspase-3 positive cells in the OA+HA-CTS-EPA+BMSCs group was significantly reduced, indicating that the hydrogel platform effectively inhibited apoptosis induced by the harsh microenvironment.

[0090] In summary, the HA-CTS-EPA self-healing hydrogel platform significantly prolongs the survival time of BMSCs in the joint cavity and optimizes their spatial distribution by actively improving the pathological microenvironment of OA, effectively broadening the in vivo action window of stem cell therapy.

[0091] Example of implementation effect 6

[0092] The synergistic therapeutic effect of the HA-CTS-EPA hydrogel prepared in Example 1 combined with BMSCs was investigated in vivo, as detailed below:

[0093] Micro-CT and multiple histological staining were used to systematically evaluate the remodeling of knee cartilage and subchondral bone in rats of each group. First, the microstructural changes of subchondral bone were analyzed by Micro-CT reconstruction. Figure 8 (AB). Compared with the Sham group, the OA group rats showed significantly reduced bone volume / total volume ratio (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), while significantly increased trabecular separation (Tb.Sp), suggesting that OA induced significant subchondral bone degeneration and bone loss. Compared with the OA group, treatment with BMSCs alone (OA+BMSCs group) or HA-CTS-EPA hydrogel (OA+HA-CTS-EPA group) improved the above bone parameters to some extent, with BV / TV, Tb.Th, and Tb.N showing some recovery, and Tb.Sp partially decreasing. Notably, compared with the single treatment groups, the OA+HA-CTS-EPA+BMSCs combined treatment group significantly reversed the abnormal bone remodeling process, with all microstructural parameters almost recovering to levels close to those of the Sham group, indicating that the hydrogel delivery system effectively amplified the protective effect of BMSCs on subchondral bone.

[0094] Subsequently, H&E staining, Safranin O-Fast Green staining, and Toluidine Blue staining were used to further evaluate the morphological repair effect of the articular cartilage. Figure 8 (CE). Staining results showed that the articular cartilage surface in the Sham group was smooth, with uniform matrix staining and orderly cell arrangement; while the cartilage surface in the OA group was rough, showing severe wear, microcracks, and depletion of deep matrix. Compared with the OA group, all intervention groups showed varying degrees of histological improvement. However, the OA+HA-CTS-EPA+BMSCs group showed the most significant repair effect, with an intact and smooth cartilage surface, deep and uniform matrix staining, regular chondrocyte arrangement, and almost no obvious fibrosis. Combined with quantitative analysis of OARSI histological scores, the combined treatment group showed a significant reduction in scores compared with the OA group, and was significantly better than the single treatment group. In conclusion, the microenvironment regulation provided by HA-CTS-EPA hydrogel fully released the regenerative potential of BMSCs, and the two synergistically improved cartilage degeneration and subchondral bone pathological remodeling in the OA process.

[0095] Example of implementation effect 7

[0096] The molecular mechanism of the HA-CTS-EPA hydrogel prepared in Example 1 combined with BMSCs was verified in vivo, as follows:

[0097] To clarify the molecular mechanism of the in vivo repair effect, this invention performed pathological examinations on rat joint specimens. First, in situ oxidative stress in tissues was assessed using DHE fluorescence staining. Figure 9 A). Compared with the Sham group, the DHE red fluorescence of cartilage and synovium in the OA group was significantly enhanced; compared with the OA+BMSCs group, the fluorescence intensity of the OA+HA-CTS-EPA+BMSCs group was significantly reduced, indicating that the hydrogel can effectively clear local ROS. Subsequently, immunohistochemical detection of pro-inflammatory factor expression ( Figure 9 B). Compared with the Sham group, the OA group showed a significant increase in TNF-α positive staining; compared with the OA group, the combination therapy group showed a significant decrease in TNF-α expression. Furthermore, immunofluorescence analysis showed ( Figure 9 (C) Compared with the OA group, the expression of cle-Caspase-3 in the OA+BMSCs group was not significantly changed, while the positive rate of cle-Caspase-3 in the combined treatment group was significantly reduced, suggesting that this strategy effectively inhibited local cell apoptosis.

[0098] Regarding cartilage matrix remodeling, immunohistochemical results showed ( Figure 9 Compared with the Sham group, the OA group showed a significantly increased positive area for the matrix-degrading enzyme MMP-13 and a decreased Col II expression; compared with the OA+BMSCs group, the combination therapy group showed a significantly reduced MMP-13 expression and an increased Col II expression. Further Western blot analysis showed... Figure 9 Compared with the OA group, the expression of p-p65 and p-IκBα in the tissues of the combination therapy group was significantly downregulated, while the expression of antioxidant enzymes SOD1, SOD2 and CAT was significantly upregulated. These results indicate that HA-CTS-EPA / BMSCs effectively inhibit the in situ ROS / NF-κB axis and remodel stromal homeostasis.

[0099] In summary, this invention proposes and validates a transformable combined treatment paradigm for osteoarthritis (OA): using an injectable self-healing HA-CTS-EPA hydrogel as a "structural delivery platform + active microenvironment modulator," it enhances the retention, survival, and chondrogenic function of bone mesenchymal stem cells (BMSCs) in the joint cavity by reducing ROS and inhibiting excessive NF-κB activation, ultimately improving cartilage and subchondral bone structure. Its scientific significance lies in amplifying the widely discussed but often fragmented oxidative stress and inflammation in OA, integrating them into an operable ROS / NF-κB axis, and strengthening the evidence chain that "ROS is a key upstream event" using causal complementation experiments, thereby establishing a closed-loop progressive mechanism of "materials-microenvironment-cell function-tissue repair."

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a HA-CTS-EPA self-healing hydrogel, characterized in that, Includes the following steps: (1) Hyaluronic acid was dissolved in deionized water and then sodium periodate aqueous solution was added in the dark. The reaction was carried out by stirring in the dark. Ethylene glycol was added to stop the reaction and stirring was continued. Then, solid OHA was obtained by dialysis and freeze drying. (2) Dissolve CTS in an aqueous acetic acid solution to obtain a CTS mixture. Also, dissolve EPA, EDC·HCl and NHS in anhydrous ethanol to obtain an EPA mixture. (3) The activated EPA mixture was added dropwise to the CTS mixture, and the reaction was carried out by stirring in the dark. The product was then dialyzed and freeze-dried to obtain CTS-EPA. (4) Mix the aqueous solution of OHA and the acetic acid solution of CTS-EPA and carry out the Schiff base reaction to obtain HA-CTS-EPA self-healing hydrogel.

2. The method for preparing the HA-CTS-EPA self-healing hydrogel according to claim 1, characterized in that: In step (1), 0.5 mmol of sodium periodate is added per gram of hyaluronic acid, and the initial concentration of the sodium periodate aqueous solution is 0.3-0.6 M; the molecular weight of hyaluronic acid is 0.8-2 × 10⁻⁶. 6 Da.

3. The method for preparing the HA-CTS-EPA self-healing hydrogel according to claim 2, characterized in that: In step (1), the stirring time is 12-36 h, and the amount of ethylene glycol added is based on the molar amount of sodium periodate. For every 5 mmol of sodium periodate, 1.5-3 mL of ethylene glycol is added to terminate the reaction.

4. The preparation method of the HA-CTS-EPA self-healing hydrogel according to claim 3, characterized in that: In step (1), dialysis is performed in deionized water for 2-4 days, and the freeze-drying temperature is -50℃ to -20℃, and the time is 48-96h.

5. The method for preparing the HA-CTS-EPA self-healing hydrogel according to claim 4, characterized in that: In step (2), the mass ratio of CTS, EPA, EDC·HCl and NHS is 1:0.25-0.68:0.2-0.52:0.06-0.15; the volume percentage of the acetic acid aqueous solution is 0.5%-2%.

6. The method for preparing the HA-CTS-EPA self-healing hydrogel according to claim 5, characterized in that: In step (3), the volume ratio of CTS mixture to EPA mixture is 3-8:1, and the stirring time in the dark is 12-36 h.

7. The method for preparing the HA-CTS-EPA self-healing hydrogel according to claim 6, characterized in that: In step (4), the mass concentration of the OHA aqueous solution is 2%-5%, and the mass concentration of the CTS-EPA acetic acid solution is 1%-3%; the volume ratio of the OHA aqueous solution to the CTS-EPA acetic acid solution is 1:

1.

8. HA-CTS-EPA self-healing hydrogel prepared by the preparation method according to any one of claims 1-7.

9. The HA-CTS-EPA self-healing hydrogel according to claim 8, characterized in that: The HA-CTS-EPA self-healing hydrogel is obtained by dynamically crosslinking aldehyde-modified hyaluronic acid with EPA-grafted chitosan to form Schiff base bonds.

10. The use of the HA-CTS-EPA self-healing hydrogel according to claim 9 in the preparation of a medicament for treating osteoarthritis.

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