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Table of Contents
Abstract
Graphical Abstract
Introduction
Results
Discussion
Conclusion
Materials And Methods
Materials
Preparation And Characterization Of MoS 2 -TA Nanozyme
Construction And Characterization Of HAMA-M Hydrogel
Mechanical Property Of HAMA-M Hydrogel
Photothermal Property Of HAMA-M Hydrogel
BMSCs Isolation And Culture
Biocompatibility Of HAMA-M Hydrogel
Chondrogenic Differentiation Of BMSCs On HAMA-M Hydrogel
Evaluation Of Antioxidant Activity And Mitochondrial Function Of BMSCs On HAMA-M Hydrogel
Quantitative Real-Time PCR (QRT-PCR)
Western Blot Analysis
Animal Procedure
Histological Evaluation And Grading
Statistical Analysis
CRediT Authorship Contribution Statement
Ethics Approval And Consent To Participate
Consent For Publication
Declaration Of Competing Interest
Supplementary Data
Data Availability
Footnotes
Abstract
Tissue engineering has been used as a potential treatment strategy for articular cartilage regeneration, but current hydrogel scaffolds often fail to meet these criteria due to weak mechanical strength and unfavorable biocompatibility. Inspired by the metal ion-polyphenol redox system-initiated free radical polymerization (MPi-FRP), a novel molybdenum disulfide-tannic acid (MoS 2 -TA) dual-catalytic system was used as an initiator for the free radical polymerization of methacrylated hyaluronate (HAMA) to synthesize a HAMA-MoS 2 -TA (abbreviated as HAMA-M) nanocomposite hydrogel for cartilage repair under mild conditions. Compared to the pure HAMA hydrogel, HAMA-M hydrogels exhibited robust mechanical properties, and the adhesive strength was promoted for 81.32 %. Moreover, MoS 2 -TA endowed the hydrogel with excellent SOD and CAT-mimic activities and prominent photothermal conversion efficiency. Assisted by the mild photothermal therapy, the antioxidant HAMA-M hydrogel had excellent biocompatibility and effectively promoted chondrogenic differentiation of bone marrow mesenchymal stem cells by decreasing excessive ROS production, restoring mitochondrial function and promoting mitochondrial production. Further, the HAMA-M hydrogel alleviated the pro-inflammatory microenvironment and accelerated cartilage regeneration, with the ICCS score promoted to 77.75 % after 8-week therapy in vivo . This study provides a novel way for fabricating tissue engineering scaffolds and throws new light for cartilage repair.
Graphical abstract
In this paper, a methacrylated hyaluronate-molybdenum disulfide-tannic acid (HAMA-M) multifunctional hydrogel was prepared by the free radical polymerization of methacrylated hyaluronate (HAMA) using a novel molybdenum disulfide-tannic acid (MoS 2 -TA) dual-catalytic system as an initiator under mild conditions, and its role in promoting the chondrogenic differentiation of bone marrow mesenchymal stem cells and cartilage regeneration was verified by in vitro and in vivo experiments. Image 1
Introduction
Articular cartilage plays a crucial role in joint lubrication and load-bearing. Due to its lack of blood supply, nerves, and lymphatic vessels, articular cartilage has a limited capacity for spontaneous recovery following damage [ 1 ]. Although traditional treatments for cartilage defects—such as microfracture surgery, matrix-assisted autologous chondrocyte implantation, and osteochondral transplantation—have demonstrated significant improvements in alleviating cartilage degeneration, the regeneration of native tissue continues to pose a significant therapeutic challenge in orthopedics [ 2 ]. Tissue engineering utilizing scaffolds, cells, and signaling factors for the regeneration of injured tissues has emerged as a promising treatment strategy for articular cartilage repair [ [3] , [4] , [5] ]. An ideal tissue engineering scaffold should possess biocompatibility and biomimetic properties, facilitate cell attachment, promote tissue integration, and be practical for use [ 6 ]. Hyaluronic acid (HA) is a key component that contributes to the viscosity of synovial fluid and has been widely utilized in cartilage tissue engineering [ [7] , [8] , [9] ]. Previous studies have demonstrated that HA can improve the lubricity of cartilage boundaries, increase viscoelasticity, and promote cell adhesion and proliferation [ 10 ]. However, unmodified HA can hardly form crosslinked gels, being unsuitable for tissue engineering [ 11 ]. To create covalent and crosslinked three-dimensional scaffolds, the backbone of HA molecules is frequently modified and crosslinked using alkenyl, thiolated, phenolized, hydrazide, and other functional groups [ 12 ]. Among these HA derivatives, hyaluronic acid methacrylate (HAMA) is the most widely studied, as its double bonds can be readily crosslinked via photo- or thermal-initiated free radical polymerization (FRP) reactions [ 13 ]. Although the chemical modification of HA facilitates the formation of crosslinked matrices, most HA-based hydrogels exhibit slow gelation rates, weak mechanical properties, uncontrollable degradation rates, and limited efficiency in regulating the complex pro-inflammatory microenvironment, thereby restricting their applications in cartilage regeneration [ 14 ]. Moreover, the use of photo-initiators or ultraviolet irradiation can be detrimental to human health and may provoke host rejection. Therefore, there is an urgent need for the development of multifunctional and biocompatible HA-based hydrogels under mild conditions for cartilage repair. Recently, the metal ion-polyphenol redox system-initiated free radical polymerization (MPi-FRP) has been reported as a mild and rapid approach for the preparation of hydrogels intended for biomedical applications [ 15 ]. The documented reaction mechanism indicates that polyphenols reduce high-valent metal ions to low-valent metal ions, while being oxidized to semiquinone/quinone forms, thereby establishing a dual-catalytic system [ 16 ]. The dual-catalytic system can activate ammonium persulfate (APS) to produce sulfate radicals, which subsequently react with water to generate hydroxyl radicals and initiate the radical polymerization of vinyl monomers, leading to the rapid gelation of covalently crosslinked hydrogels. Moreover, the high efficiency of MPi-FRP often results in the production of high molecular weight polymers, which enhances the mechanical performance of the hydrogels. For instance, Wang utilized tannic acid (TA)-Fe 3+ self-catalytic systems to fabricate polyacrylic acid-based hydrogel strain sensors exhibiting outstanding mechanical strength and stable sensing properties [ 17 ]. Zong et al. employed a Cu-tannic autocatalytic strategy to fabricate conductive polyacrylamide hydrogel sensors and found that the Cu 2+ -TA redox catalytic system significantly shortens the gelation time [ 18 ]. Su et al. utilized a lignin-alkali metal ion system to produce an anti-freezing hydrogel electrolyte, demonstrating that the gelation time can be adjusted by varying the ratio of the components in the catalytic system [ 19 ]. As an alternative to metal ions in dual-catalytic systems, incorporating nanozymes into hydrogels can effectively enhance their multifunctionality. Nanozyme are nanomaterials that exhibit both enzyme-mimetic activities and unique physicochemical properties [ 20 ]. Among various nanozyme, molybdenum disulfide (MoS 2 ) nanozyme have garnered increasing attention in tissue engineering due to their excellent biocompatibility, antioxidant enzyme-mimetic performance, and strong absorption in the near-infrared (NIR) range. Xiao et al. reported that the Ag/MoS 2 nanozyme enhances the mechanical strength and adhesion of the hydrogel to skin [ 21 ]. Li et al. demonstrated that the MoS 2 TA/Fe nanozyme can impart excellent reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavenging capacities to the hydrogel, thereby preventing wound inflammation [ 22 ]. Lu et al. documented that the MoS 2 -dithiothreitol nanozyme not only improved the free radical scavenging ability but also enhanced the photothermal conversion efficiency of the hyaluronic acid-based hydrogel, thereby significantly accelerating wound healing [ 23 ]. However, these hydrogels remain unsuitable for cartilage reconstruction due to their slow gelation rate and low stiffness. Although the method of MPi-FRP has yet to be applied to HA hydrogels, it shows promise for the rapid synthesis of covalently crosslinked HA with enhanced mechanical strength, which is essential for cartilage tissue engineering. AMPK-SIRT1-PGC1α signaling pathway plays an important role in antioxidant stress, mitochondrial function and metabolism regulation [ 24 ]. PGC1α is a key transcriptional coactivator regulating mitochondrial biosynthesis and energy metabolism, and activated by AMPK phosphorylation and SIRT-1 acetylation [ 25 ]. PGC1α enters the nucleus and promotes the expression of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase, reducing intracellular ROS levels, thus regulating mitochondrial function. PGC1α is also recruited to chromatin as a pleiotropic regulator by interacting with nuclear receptors such as nuclear factor-related factor 2 (NRF2) or activating mitochondrial transcription factor A (TFAM), thereby promoting mitochondrial biogenesis [ 26 ]. Inspired by the switch between high- and low-valent metal ions during the catalytic process of nanozyme [ 27 ], we successfully replaced the metal ion-polyphenol redox system with a novel molybdenum disulfide-tannic acid (MoS 2 -TA) dual-catalytic system to initiate free radical polymerization (FRP) for HAMA, leading to the development of a multifunctional HAMA-MoS 2 -TA hydrogel (abbreviated as HAMA-M) for cartilage repair under mild reaction conditions. Specifically, the MoS 2 -TA nanozyme not only functions as an initiator but also imparts the hydrogel with excellent SOD and CAT mimetic activities, along with notable photothermal conversion efficiency ( Scheme 1 ). The in vitro and in vivo results demonstrated that, combined with mild photothermal therapy, the HAMA-M hydrogel exhibits significant effects on cartilage regeneration through the upregulation of chondrogenic factors and enhanced tissue integration. Additionally, this study presents a novel approach for fabricating tissue engineering scaffolds and offers new insights into combined therapies for cartilage repair. Scheme 1 Schematic illustration of the fabrication of HAMA-MoS 2 -TA hydrogel via the MoS 2 -TA dual-catalytic system and phototherapy for cartilage regeneration. Scheme 1
Results
Fabrication and characterization of the HAMA-M hydrogel
The MoS 2 -TA nanozyme were prepared by an ultrasonic exfoliation method and characterized by X-ray diffraction (XRD) and transmission electron microscope (TEM). The diffraction peaks around 14.5°, 32.8°, and 59.9° in the XRD pattern can be assigned to (002), (100), and (110) of 2H-MoS 2 (JCPDS no. 37–1492) ( Fig. 1 a) [ 28 ]. The TEM image confirmed the thin lamellar structure of MoS 2 -TA nanozyme ( Fig. 1 b), and lattice spacing showed in the high magnetified TEM images was calculated to be 0.27 nm ( Fig. 1 c), which was assigned to the (100) lattice plane of 2H-MoS 2 [ 29 ]. Furthermore, the absorption peaks at 610 nm and 670 nm ( Fig. S1 ) shown by the UV–vis absorption spectrum verified the successful exfoliation of the bulk MoS 2 [ 30 ]. The chemical structure of the HAMA was investigated by Fourier-transform infrared (FT-IR) spectroscopy and proton nuclear magnetic resonance ( 1 H NMR). The stretching band of the C=C bond at 1700 cm −1 in the FT-IR spectrum proved that the methacryl groups were decorated into the skeleton of HA ( Fig. S2 ). The 1 H NMR spectra revealed two new peaks at 5.7 and 6.1 ppm ( Fig. S3 ), confirming the successful methacrylation of the HA backbone. In addition, the degree of methacrylation was calculated to be 40 % according to the relative integration of the peak at 1.8 (methacrylate protons) and 1.9 (methyl protons on the HA backbone) [ 31 ]. Fig. 1 Characterization of MoS 2 -TA nanozyme and the HAMA-based hydrogels. (a) XRD spectra of bulk and exfoliated MoS 2 . (b) TEM and high magnified image(c) of MoS 2 -TA nanozyme. (d) EPR spectrum of MoS 2 nanozyme. XPS spectrum of C 1s (e,f) and Mo 3d (g,h) regions for MoS 2 -TA and MoS 2 -TA reacted with APS. (i) Gelation of the HAMA (i, ii) and HAMA-1.0M (iii, iv) hydrogels. (j) SEM image of lyophilized HAMA hydrogels (i); SEM image (ii) and Mo elemental mapping (iii, iv) of lyophilized HAMA-1.0M hydrogels. Stress-strain curves (k) and Young's modulus (l) of HAMA and HAMA-M hydrogels. (m) Rheological property of HAMA-M at varying concentrations. (n) Strength -extension curves and (o) adhesive strength of HAMA and HAMA-M hydrogels. (p) Degradation kinetics of the HAMA-M hydrogels. Fig. 1 The MoS 2 -TA dual-catalyzed polymerization process was investigated by electron paramagnetic resonance spectroscopy (EPR) and X-ray photoelectron spectroscopy (XPS). The EPR spectra of the reaction solution of MoS 2 -TA and APS are shown in Fig. 1 d. The quartet of signals with intensities of 1:2:2:1 was ascribed to the hydroxyl radical [ 27 ], and the signals of hydroxyl radical were found to be enhanced with the reaction time, confirming the nanozyme can react with APS to generate hydroxyl radical, which is similar to the metal ions-polyphenol redox catalytic systems [ 32 ]. As shown in C 1s XPS spectrum ( Fig. 1 e and f, Fig. S4a ), the peaks of 286.5 and 288.8 eV were assigned to C-O and C=O species, respectively [ 28 ]. It is obvious that the ratio of C=O to C-O groups in MoS 2 -TA was significantly increased with the reaction of APS, while it is almost the same as that of TA. The changes of the O 1s spectrum ( Fig. S4b–d ) showed a similar trend to that of the C 1s spectrum, indicating that APS was necessary for the conversion of catechol groups of TA into the semi-quinone/quinone group in the MoS 2 -TA dual catalytic system. The Mo 3d spectra MoS 2 -TA were shown in Fig. 1 g and h. The characteristic peaks at 229.7, 232.3, 232.9 and 235.5 eV can be attributed to Mo 4+ 3d 5/2 , Mo 4+ 3d 3/2 , Mo 6+ 3d 3/2 and Mo 6+ 3d 3/2 , respectively [ 27 ]. The Mo 3d XPS spectra also revealed that the Mo 6+ /Mo 4+ ratio increased when the MoS 2 -TA system reacted with APS, further confirming that APS facilitated the oxidation of Mo 4+ to Mo 6+ in MoS 2 . The HAMA-MoS 2 -TA hydrogels were assigned to be HAMA, HAMA-0.5M, HAMA-1.0M, and HAMA-2.0M, with the content of MoS 2 -TA nanozyme in hydrogels to be 0, 0.5 %, 1.0 %, and 2.0 %, respectively. As shown in Fig. S5 , the HAMA-M hydrogels could be cross-linked in about 1 min at room temperature, which is beneficial for in situ treatment of cartilage injury. And compared to the transparent HAMA hydrogel ( Fig. 1 i–i, ii), the HAMA-1.0M hydrogel exhibited a brown color ( Fig. 1 i–iii, iv). The microscopic morphology of the hydrogel was observed using scanning electron microscopy (SEM), revealing a partially collapsed porous structure of the HAMA hydrogel ( Fig. 1 j–i). In contrast, the porosity of the HAMA-1.0M hydrogel is more regular and shows a spatial network structure ( Fig. 1 j–ii). The EDS results reveal that the molybdenum and sulfur elements were evenly distributed in the hydrogel ( Fig. 1 j–iii, iv). The mechanical properties of HAMA-M hydrogel were further tested by a universal testing machine. As shown in Fig. 1 k and l, the pure HAMA hydrogel exhibited the lowest Young's modulus (26.14 ± 0.89 kPa) among four hydrogels. When MoS 2 -TA nanozyme was added, the Young's modulus of the HAMA-0.5M, HAMA-1.0M and HAMA-2.0M hydrogels were 51.10 ± 1.35 kPa, 86.73 ± 1.79 kPa, and 215.17 ± 8.39 kPa, respectively. And broken strain of HAMA-M hydrogel showed a similar trend with the Young's modulus, indicating that the increased concentration of MoS 2 -TA nanozyme evidently improved the mechanical strength of the hydrogels. Furthermore, the Rheological analysis showed that the storage modulus of the hydrogels was increased with the elevated concentration of MoS 2 -TA nanozyme ( Fig. 1 m), revealing that the incorporation of nanozyme could effectively increase the stiffness of the hydrogels. Next, the equilibrium swelling ratio of the hydrogels decreased with increasing proportions of MoS 2 -TA nanozyme ( Fig. S6 ), indicating nanozyme can increase the crosslinking density of the hydrogel. In addition, the adhesive properties of the hydrogels were determined through the lap-shear test. According to the results shown in Fig. 1 n and o, the adhesive strength of the HAMA-0.5M, HAMA-1.0M and HAMA-2.0M hydrogel was calculated to be 18.53 ± 0.69 kPa, 23.30 ± 2.36 kPa, and 26.63 ± 0.51 kPa, respectively, which is 44.20 %, 81.32 % and 107.24 % higher than that of the HAMA hydrogel (12.85 ± 0.97 kPa), indicating that cross-linking with MoS 2 -TA nanozyme significantly improved the adhesive strength of the hydrogels. Finally, the degradation experiments showed that most of the hydrogels would degrade in two months and the doping of MoS 2 -TA nanozyme can delay the degradation of the HAMA hydrogel ( Fig. 1 p).
Photothermal effect of the HAMA-M hydrogel under NIR radiation
The photothermal effect of the HAMA-M hydrogels was further investigated by recording the temperature changes of the samples at varying concentrations of MoS 2 -TA nanozyme under 10 min of 808 nm NIR laser irradiation (0.78 W/cm 2 ). As shown in Fig. 2 a, the temperature of the HAMA-0.5M, HAMA-1.0M and HAMA-2.0M hydrogel increased to about 34.6 °C, 39.8 °C and 53.7 °C, respectively, while that of HAMA hydrogel only reached 30.6 °C under the same conditions. It was evident that HAMA-M hydrogels showed better photothermal performance than pure HAMA hydrogel. Besides, the equilibrium temperature of the gel shows a dependency on the nanozyme concentration, revealing that the MoS 2 -TA nanozyme can effectively improve the photothermal conversion efficiency of the HAMA hydrogel. Cyclical on/off irradiation was conducted to determine the photostability of the nanozyme-incorporated hydrogels ( Fig. 2 b and c). The results showed that the temperature change of the hydrogels was almost the same during five “heating/cooling” cycles, indicating the desirable photostability of the MoS 2 -TA nanozyme-incorporated hydrogels. Furthermore, HAMA-1.0M hydrogel was injected into cartilage defects in a rat osteochondral defect model and then treated with 808 nm NIR irradiation to evaluate photothermal effects in vivo . As shown in Fig. 2 d, the temperature rose to the required 40 °C in 3 min after NIR irradiation (0.78 W/cm 2 ). The in vivo irradiation results confirmed the suitability of the nanozyme-incorporated hydrogels for further applications in articular cartilage regeneration. Fig. 2 Photothermal properties of HAMA-M hydrogel under NIR radiation. (a) Temperature-increment curves of various concentrations of HAMA-M hydrogels under NIR irradiation of 0.78 W cm −2 over 15 min. (b) Thermal images of HAMA-1.0M under 808 nm NIR laser irradiation (0.78 W cm −2 ). (c) Thermal stability of HAMA-1.0M under repeated “heating/cooling” NIR laser irradiation (0.78 W cm −2 ). (d) Thermal images of the rat knee cartilage defect model after injecting HAMA-1.0M or HAMA with NIR laser irradiation over 15 min (0.78 W cm −2 ). (e,f) qRT-PCR assay of HSP47 and HSP70 gene expression in BMSCs. (g) Western blots and (h,i) quantitative analysis of the protein expression of HSP47 and HSP70. (n = 3, Mean values ± SD, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001). Fig. 2 It is reported that the mild heat generated in photothermal therapy can induce heat shock protein (HSP) expression, particularly HSP47 and HSP70, which play an important role in promoting osteogenesis through promoting synthesis of collagen and regulating inflammatory responses [ [33] , [34] , [35] ]. As a result, the expression of HSP47 and HSP70 of bone marrow mesenchymal stem cells (BMSCs) on HAMA-1.0M hydrogel under 808 nm NIR irradiation after 14 days of culture was investigated by qRT-PCR assay and western blot (WB) analysis. As shown in Fig. 2 e and f, the gene expression of HSP47 and HSP70 remained relatively unchanged in the pure HAMA group, while the gene levels of the two HSPs were observably significantly increased in the HAMA-1.0M + NIR group. The gene levels of HSP47 and HSP70 were up-regulated by 542.50 % and 439.49 %, respectively, as compared to the HAMA group. The protein expressions of HSP47 and HSP70 were consistent with the gene expression results. Compared to that in the HAMA group, the protein levels of HSP47 and HSP70 were increased by 76.73 % and 142.97 %, respectively ( Fig. 2 g–i). These results suggested that the nanozyme-incorporated hydrogels have excellent photothermal performance and show great potential in osteogenesis promotion.
Biocompatibility, viability, and proliferation of BMSCs on HAMA-M hydrogel
The cell biocompatibility of HAMA-M hydrogels was investigated by CCK8 assay and live/dead staining. The BMSCs were cultured in HAMA-M hydrogels for 3 days. The results of the CCK8 assay revealed that HAMA-1.0M group exhibited the highest cell viability among all HAMA-M hydrogels ( Fig. 3 a), thus this HAMA-1.0M hydrogel was chosen for subsequent experiments. To fix the optimal NIR irradiation time for maximum cell viability, the CCK8 assays and live/dead staining were performed in our preliminary experiments. The results showed that mild photothermal therapy within 15 min can prominently promote cell proliferation ( Fig. S7 and 8 ). Afterwards, the proliferation of BMSCs on HAMA-1.0M hydrogel under NIR radiation was assessed. As shown in ( Fig. 3 b), after culture for 7 days, the proliferation of BMSCs on the HAMA-1.0M hydrogel group and HAMA-1.0M hydrogel + NIR group was higher than that of the HAMA hydrogel group and HAMA + NIR group, which increased by 11.95 % and 43.64 %, respectively. Notably, the HAMA-1.0M + NIR group exhibited the highest proliferation rate, indicating that the mild heat stress induced by NIR irradiation on HAMA-1.0M hydrogel could further increase cell proliferation and viability. Fig. 3 Biocompatibility and cell proliferation of BMSCs on HAMA-M hydrogel. (a) Cell proliferation of BMSCs on HAMA-1.0M hydrogel was tested by CCK-8 assay. (b) Cell proliferation of BMSCs in the hydrogel of each group. (c) The cytoskeleton was investigated by using rhodamine-phalloidin staining. Cell spreading areas seeded on hydrogels were calculated by Image J (3 images, 10 cells in each group). (d) Cell morphology was observed with F-actin (red) and DAPI (blue) staining 1 day after seeding BMCs on the surface of hydrogels. (e) The BMSCs viability in each group of hydrogel was investigated by live/dead cell staining in 3D cultivation for 3, 7, and 14 days, AM for live cells (green) and PI for dead cells (red). (n = 3, Mean values ± SD, ∗p < 0.05, ∗∗p < 0.01,∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (f) H&E staining of hydrogel after 3, 7, and 14 days of 3D cultivation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 3 The cytoskeleton was investigated using rhodamine-phalloidin staining after culture on HAMA-1.0M hydrogels for 24 h ( Fig. 3 c and d). In the HAMA-1.0M group and HAMA-1.0M + NIR group, the cytoskeletal morphology of the BMSCs exhibited a wide branched network of elongated actin, flat and spindle-like characteristics, and larger spreading area, indicating that HAMA-1.0M hydrogel facilitates to achieve better cell growth and extension and cell affinity in the environment. The BMSCs viability on the HAMA-1.0M hydrogel was investigated by live/dead cell staining. As shown in ( Fig. 3 e), the BMSCs in the HAMA-1.0M hydrogel groups had more survival cells (green) than the HAMA hydrogel group for a three-dimensional culture of 14 days. Particularly, the HAMA-1.0M + NIR group supported a higher survivability of BMSCs. The proliferation of BMSCs cultured in the HAMA-1.0M hydrogel was detected by HE staining ( Fig. 3 f). The HAMA-1.0M hydrogel had a superior effect on supporting cell proliferation for 14 days, inducing that the HAMA-1.0M hydrogel has superior biocompatibility and low toxicity for cell culture, and provide an excellent 3D microenvironment for promoting cell survival and proliferation.
Chondrogenic capacity of BMSCs in HAMA-M hydrogel
To evaluate chondrogenic capacity, the BMSCs were three-dimensional(3D) culture in HAMA-1.0M hydrogel under NIR radiation for 7 days. Safranine O ( Fig. 4 a) and toluidine blue staining ( Fig. 4 b) were used for cartilage extracellular matrix (ECM) evaluations, and their substantial staining indicates successful induction of chondrogenic differentiation of BMSCs. The HAMA-1.0M + NIR group facilitated the deposition of ECM and chondrogenesis with superior results. ACAN and COL2A1 play a key role in chondrogenesis. Here immunofluorescence staining was used to detect ACAN and COL2A1 levels in NIR stimulated BMSCs in the HAMA-1.0M hydrogel to evaluate ECM in the early stage of chondrogenesis. As shown in Fig. 4 c–f, the expression level of ACAN and COL2A1 was greatly augmented on HAMA-1.0M hydrogels by NIR stimulation. However, the augmentation increased in the order of HAMA, HAMA + NIR, HAMA-1.0M, and HAMA-1.0M + NIR, implying that HAMA-1.0M + NIR is the most capable of promoting chondrogenic differentiation of BMSCs and enhancing ECM secretion. Fig. 4 Chondrogenic capacity of BMSCs in HAMA-1.0M hydrogel after 3D cultivation in vitro . Histological profiling of chondrogenesis in HAMA-1.0M: (a) Safranine O staining and (b) toluidine blue staining of hydrogel after 14 days. Immunofluorescence and relative fluorescence intensity analysis of COL2A1 (c, d) and Aggrecan(e, f)after 14 days. (g–j) The chondrogenic-related gene expression of BMSCs (SOX9, ACAN, COL2A1and COL1A1) was tested by qRT-PCR at different time points. (k) Quantitative analysis of GAG/DNA (μg/μg) ratio after 21 days. (l) Western blots and (m,n) quantitative analysis of chondrogenesis-related protein levels after 21 days. (n = 3, Mean values ± SD, ∗P < 0.05, ∗∗P < 0.01,∗∗∗P < 0.001 ∗∗∗∗P < 0.001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 4 This finding was further corroborated by qRT-PCR analysis ( Fig. 4 g–j). The chondrogenic-related gene expression of BMSCs including SOX9, ACAN, and COL2A1 was significantly upregulated. The expression of chondrogenic-related genes (SOX9, ACAN, and COL2A1) increased by 304.39 %, 593.16 %, and 334.42 % in HAMA-1.0M + NIR group for 21 days, respectively, as compared with HAMA group. Moreover, the expression of the fibrocartilage factor COL1A1 was downregulated by 8.79 %, 38.06 %, and 64.90 % in HAMA + NIR, HAMA-1.0M, and HAMA-1.0M + NIR group, respectively. Glycosaminoglycans (GAG) is a major ECM component of cartilage, which is also a key marker for identifying the chondrogenesis process. GAG deposition of BMSCs was calculated to be increased by −11.58 %, 68.02 % and 135.55 % in HAMA + NIR, HAMA-1.0M, and HAMA-1.0M + NIR group for 21 days, respectively ( Fig. 4 k). Furthermore, the expression levels of chondrogenic markers ACAN and COL2A1 were performed by WB ( Fig. 4 l–n). The results showed that the levels of chondrogenic markers were significantly upregulated in the HAMA-1.0M group as compared with the HAMA group. Particularly, ACAN and COL2A1 in the HAMA-1.0M + NIR was increased by 959.74 % and 378.04 %, respectively. The results testified that HAMA-1.0M hydrogel can provide a favorable 3D microenvironment to effectively promote chondrogenic differentiation in BMSCs and have a positive effect on ECM secretion with the aid of NIR irradiation.
Antioxidant activity and mitochondrial function of BMSCs on HAMA-M hydrogel
Mitochondria production is essential for maintaining cellular homeostasis and function. MoS 2 nanozyme have attracted considerable attention in the biomedical field because of their antioxidant nanozyme-mimicking performance. The antioxidant activities of MoS 2 -TA nanozyme were assessed through the SOD and CAT activity assay kit. The results showed that the SOD- and CAT-mimicking antioxidant activity of the nanozyme was proportional to its concentration ( Fig. S9a and b ). Furthermore, their enzymatic activity could be significantly enhanced by NIR irradiation. The antioxidant activity of HAMA-1.0M in BMSCs with LPS exposure (24 h) was then tested by using DCFH-DA and DAF-FM DA probes ( Fig. 5 a and b). LPS prominently induced increase of intracellular ROS in BMSCs, which can react with DCFH-DA to produce 2′,7′-dichlorofluorescein with green fluorescence. The bright green fluorescence was observed in HAMA and HAMA + NIR group, suggesting that pure HAMA hydrogel can not effectively restrain ROS level in BMSCs under oxidative stress. Notably, the fluorescence intensity of HAMA-1.0M and HAMA-1.0M + NIR group was reduced by 35.48 % and 62.65 %, respectively, as compared to HAMA hydrogel, indicating that HAMA-1.0M hydrogel has excellent ability to scavenge ROS. The results were further confirmed by RNS detection. The DAF-FM DA probe is a NO-sensitive fluorescent dye. As shown in Fig. 5 c and d, HAMA-1.0M hydrogels were proved to reduce RNS generation in BMSCs, especially under NIR irradiation. Next, the cell apoptosis of LPS-induced BMSCs on HAMA-1.0M hydrogel was detected by flow cytometry. As shown in Fig. 5 e, the apoptosis rate of the HAMA-1.0M group (4.71 %) and the HAMA-1.0M + NIR group (4.30 %) was significantly lower than that of the HAMA group (20.80 %) and HAMA + NIR group (20.20 %). The results indicate that HAMA-1.0M hydrogel has an outstanding antioxidant effect to resist oxidative stress. Fig. 5 HAMA-1.0M regulates mitochondrial function. Intracellular ROS(a, b) and RNS (c, d) of BMSCs were tested by using DCFH-DA and DAF-FM DA probes. (e) Cell apoptosis rate was assessed by flow cytometry in 2D cultivation treated with LPS after 24 h. (f, i) The mitochondria-associated ROS levels, (g,j) mitochondrial membrane potential, and (h.k) mitochondrial mass of BMSCs in 2D cultivation treated with LPS after 24 h were examined by MitoSOX red, JC-1, and MitoTracker staining. (n = 3, Mean values ± SD, ∗P < 0.05, ∗∗P < 0.01,∗∗∗P < 0.001 ∗∗∗∗P < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 5 The overproduction of ROS causes BMSCs to undergo apoptosis and mitochondrial malfunction, ultimately inhibiting progression in chondrogenic differentiation of BMSCs [ 36 ]. To evaluate the effect of HAMA-1.0M on mitochondrial function, MitoSOX red, JC-1, and MitoTracker staining were applied to examine mitochondria-associated ROS levels, mitochondrial membrane potential, and mitochondrial mass, respectively. As shown in Fig. 5 f and i, mitochondria-associated ROS levels in the HAMA-1.0M groups were significantly lower than those in the HAMA groups, and the fluorescence intensity of the HAMA-1.0M + NIR group was reduced by 45.98 % compared to that of HAMA group. Noteworthy, mitochondrial membrane potential prominently was decreased in BMSCs after LPS stimulation. The fluorescence intensity ratio of JC-1 was increased by −3.65 %, 91.80 %, and 172.69 % in HAMA + NIR, HAMA-1.0M, and HAMA-1.0M + NIR ( Fig. 5 g and j), indicating that HAMA-1.0M hydrogel has an excellent ability to restore of mitochondrial membrane potential. These results were further confirmed by assessing mitochondrial mass using MitoTracker staining. More strong fluorescence was observed in the HAMA-1.0M and HAMA-1.0M + NIR groups than in the other two groups, and the fluorescence intensity in the HAMA-1.0M + NIR group was increased by 107.56 % compared with that in the HAMA group ( Fig. 5 h and k), suggesting that HAMA-1.0M hydrogel is capable of enhancing mitochondrial mass. Overall, these results indicate that HAMA-1.0M hydrogels combined with NIR treatment improved the function of mitochondria by increasing their energy metabolism and contributing to the reduction of apoptosis.
HAMA-M hydrogel promotes mitochondrial biogenesis through the AMPK-SIRT1-PGC1α pathway
TFAM-mediated activation of the AMPK-SIRT1-PGC1α pathway is a key pathway regulating mitochondrial metabolism [ 37 ]. To assess the effects of hydrogels on mitochondrial biogenesis, the levels of the genes and proteins (SIRT1, PGC1α, TFAM, and NRF2) were determined by qRT-PCR and WB. As shown in Fig. 6 a–d, BMSCs in. Fig. 6 HAMA-1.0M hydrogel increased mitochondrial biosynthesis of BMSCs. (a–d) qRT-PCR detection of mitochondrial biogenesis genes in 3D cultivation hydrogels. (e)The ATP levels of BMSCs treated with LPS. (f) Western blots and (g–j) quantitative analysis of the mitochondrial biogenesis markers in 3D cultivation for 14 days. (n = 3, Mean values ± SD, ∗P < 0.05, ∗∗P < 0.01,∗∗∗P < 0.001 ∗∗∗∗P < 0.0001). Fig. 6 HAMA-1.0M and HAMA-1.0M + NIR groups exhibited a significant increase in the expression of mitochondrial metabolism factors including SIRT1, PGC1α, TFAM, and NRF2, as compared to HAMA and HAMA + NIR groups. ATP as the most important energy molecule, plays an important role in various physiological and pathological processes of cells. The ATP levels of BMSCs in HAMA-1.0M and HAMA-1.0M + NIR groups significantly recovered and improved after treatment with LPS ( Fig. 6 e). Meanwhile, the expression of mitochondrial biogenesis-related proteins was increased by 28.88 % and 62.06 % for SIRT1, 58.18 % and 109.38 % for PGC1α, 28.40 % and 49.37 % for TFAM, 75.95 % and 130.83 % for NRF2 in HAMA-1.0M and HAMA-1.0M + NIR groups, respectively, as compared to HAMA group ( Fig. 6 f–j).
HAMA-M hydrogel promotes cartilage regeneration and repair in vivo
The rat knee osteochondral defect model was utilized to evaluate the in vivo performance of HAMA-1.0M hydrogels for cartilage regeneration. Gross examination of knee joints with cartilage defects (diameter 2 mm × depth 1.5 mm) was carried out after implanting BMSCs-encapsulated hydrogels for 4 and 8 weeks. As shown in Fig. 7 a and b, the size of the defect area was still large in defect groups, and the defect site was filled with fiber tissue, indicating the cartilage can hardly regenerate once damaged. In contrast, all four hydrogel groups exhibited varied degrees of repair, as the defect area is gradually decreasing, demonstrating the regenerative capacity of the implanted HAMA-based scaffolds. The HAMA-1.0M and HAMA-1.0M + NIR groups exhibited better cartilage repair effects than the HAMA and HAMA + NIR groups. Specifically, the HAMA-1.0M + NIR group demonstrated superior cartilage regeneration, as evidenced by homogeneous formation, smooth morphology, and a well-integrated structure with the surrounding normal cartilage. The ICRS scores of HAMA, HAMA + NIR, HAMA-1.0M and HAMA-1.0M + NIR groups increased by 49.99 %, 57.14 %, 99.99 %, and 142.85 % respectively, as compared to the control group after 8 weeks treatment ( Fig. 7 c and e), indicating that the HAMA-1.0M hydrogels can significantly improve the cartilage regeneration under NIR irradiation. Fig. 7 HAMA-1.0M hydrogel promoted cartilage repair in vivo . (a, b) Representative pictures with macroscopic appearance, H&E, toluidine blue, and Safranin O& fast green staining at 4 and 8 weeks. (c–f) ICRS macroscopic scores and MODS histological scores at 4 and 8 weeks. (g,h) Immunohistochemical staining of COL2A1 at 4 and 8 weeks. (n = 3, Mean values ± SD,∗∗∗P < 0.001 ∗∗∗∗P < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 7 The microstructure and ECM production of the neonatal cartilage were analyzed by histological staining techniques, including H&E, toluidine blue, and Safranin O & fast green staining. In the HAMA-1.0M + NIR group, the cartilage structure was continuous without obvious gaps and cavities, chondrocytes were regularly and orderly arranged in the repair area, and glycosaminoglycans were uniformly deposited in the ECM. The results were also verified by the histological grade MODS scores ( Fig. 7 d and f). The histological score was 99.99 %, and 157.69 % in the HAMA-1.0M, and HAMA-1.0M + NIR group at 8 weeks post-surgery, respectively, which was higher than the other two groups. Immunohistochemistry staining was conducted to verify the levels of collagen type II within the repaired tissue. ( Fig. 7 g and h). A stronger positive staining in the HAMA-1.0M + NIR group indicated favorable tissue regeneration and had a primarily neonatal hyaline cartilage phenotype and extensive type II collagen deposition after 4 and 8 weeks post-transplantation as compared to other groups. Additionally, histological evaluation of the major organs after HAMA-1.0M hydrogels transplantation treated with 8 weeks indicated no pathological alterations in any group ( Fig. S10 ). These results indicate that HAMA-1.0M hydrogels serve as optimal scaffolds for the implantation and support of articular cartilage regeneration.
Discussion
Due to the complex pro-inflammatory microenvironment, the complete regeneration of native tissue remains a significant challenge in orthopedic therapy. Tissue engineering techniques show great potential for articular cartilage regeneration; however, current hydrogel scaffolds often suffer from limited mechanical strength and unfavorable biocompatibility. Recently, nanozyme has attracted increasing attention for the regulation of the inflammatory microenvironment [ 38 , 39 ]. Inspired by the metal ion-polyphenol redox system-initiated free radical polymerization, we developed the MoS 2 -TA nanozyme as a novel initiating system to prepare the multifunctional HAMA-M hydrogel aimed at regulating the microenvironment characterized by high oxidative stress and enhancing cartilage regeneration. The MoS 2 -TA nanozyme-catalyzed free radical polymerization can serve as a fast gelation strategy for preparing adhesive hydrogels, resulting in their formation under mild conditions [ 40 ]. The structure of the MoS 2 -TA nanozyme was characterized as few-layered nanosheets, with numerous exposed edge sites, which are favorable for catalysis. From the C 1s and O 1s XPS spectra ( Fig. 1 e and f, Fig. S4 ), we found that the ratio of C=O to C-O groups in MoS 2 -TA was nearly identical to that in TA and significantly increased upon the reaction with APS. This suggests that pure MoS 2 was of little help in converting the catechol groups of TA into the semi-quinone/quinone group, demonstrating that APS was necessary for activating the MoS 2 -TA dual catalytic system. The Mo 3d XPS spectra revealed that the Mo 6+ /Mo 4+ ratio was increased when the MoS 2 -TA system reacted with APS ( Fig. 1 g and h), further confirming that APS was beneficial for the oxidation of Mo 4+ to Mo 6+ in MoS 2 . According to the results of the XPS spectrum, the reaction mechanism of the MoS 2 -TA dual catalytic system can be proposed. Firstly, the Mo 4+ ion in MoS 2 was oxidized to Mo 6+ ion by APS. Then APS was rapidly decomposed to produce a large amount of sulfate radicals under the dual catalysis of the MoS 2 -TA redox catalytic system. Finally, the sulfate radicals reacted with water to produce hydroxyl radicals [ 27 ]. The EPR spectrum of the mixed solution of MoS 2 -TA and APS confirmed that hydroxyl radicals were generated during the activation ( Fig. 1 d). As a result, the hydroxyl radicals initiated the free radical polymerization of HAMA monomers and the multifunctional HAMA-M hydrogels were formed. The hydrogel was capable of being cross-linked within 1 min at room temperature, demonstrating the high efficiency of the MoS 2 -TA nanozyme catalytic system, which facilitates the applications of the products in tissue engineering. Compared to the pure HAMA hydrogel, the incorporation of MoS 2 -TA nanozyme obviously strengthened the mechanical properties of HAMA-M hydrogel. The SEM images showed that the nanozyme was distributed homogeneously in the gel ( Fig. 1 j), which contributes to the improvement of the mechanical properties of the hydrogel. The results of the mechanic test demonstrated that the incorporation of MoS 2 -TA nanozyme increases both the Young's modulus and the storage modulus of the HAMA hydrogel ( Fig. 1 k–m). Due to the multiple interactions of the catechol group of TA, including π-π interactions, cation-π interactions, hydrogen bonds, and others, these interactions enhance the functionality of the material [ 41 ], the adhesive strength of the HAMA-1.0M hydrogel was as strong as 23.30 kPa ( Fig. 1 n and o), which is critical for the scaffold-tissue integration in cartilage regeneration. With the increase in the concentration of MoS 2 -TA nanozyme, the swelling ratio and degradation rate of the hydrogels were reduced ( Fig. S6 and Fig. 1 p). Due to the abundant hydroxyl groups on the surface of the MoS 2 nanosheets, MoS 2 -TA nanozyme can effectively crosslink HAMA macromolecules through numerous hydrogen bonds, thereby increasing the crosslinking density and stiffness of the hydrogels. Moreover, the catechol groups of TA can bind with neighboring matrix materials through various interactions, such as hydrogen bonding, π-π interactions, hydrophobic interactions, and others, thereby further enhancing the adhesion capacity and tissue affinity of the HAMA hydrogel [ 15 ]. Besides, the integrated MoS 2 -TA nanozyme also enhances the photothermal efficiency of the HAMA-M hydrogels ( Fig. 2 a–d). It was evident that the hydrogel incorporated with MoS 2 -TA nanozyme exhibited a significant temperature increase under 808 nm NIR irradiation. MoS 2 has a large mass extinction coefficient in the NIR region, and the well-distributed nanozyme in the hydrogel is favorable for efficient photothermal conversion [ 42 ]. In addition, the results of the cyclic on/off irradiation tests shown that the nanozyme-incorporated hydrogels exhibit excellent photostability. Moreover, the incorporation of MoS 2 -TA significantly enhanced the antioxidant properties of the HAMA-M hydrogel. The MoS 2 -TA nanozyme has been demonstrated to exhibit excellent SOD- and CAT-mimicking activity that can be enhanced by NIR irradiation ( Fig. S9 ). The ability of the HAMA hydrogel incorporated with MoS 2 -TA nanozyme to scavenge intracellular ROS and RNS was assessed using DCFH-DA and DAF-FM DA dyes as fluorescent probes. The fluorescence in the MoS 2 -TA nanozyme-incorporated HAMA hydrogel group was significantly less bright than that in the HAMA hydrogel group and diminished further with NIR treatment, verifying the effective capacity of the MoS 2 -TA nanozyme-incorporated HAMA hydrogel to clear ROS and RNS. Given that TA is a plant-derived polyphenol with antioxidant and anti-inflammatory properties [ 43 ], we propose that TA and MoS 2 exhibit a synergistic ROS-scavenging effect, thereby significantly enhancing the antioxidant capacity of the hydrogel. The MoS 2 -TA nanozyme-incorporated HAMA hydrogel exhibited collaborative effects on cartilage regeneration through the regulation of the pro-inflammatory microenvironment, mild photothermal therapy, and enhancement of tissue integration. Additionally, the CCK-8 test demonstrated that the hydrogels exhibited no significant cytotoxicity ( Fig. 3 a). The results of live/dead staining confirmed the superior biocompatibility of the MoS 2 -TA nanozyme-incorporated hydrogels ( Fig. 3 e). Moreover, when assisted by NIR treatment, the MoS 2 -TA nanozyme-incorporated hydrogel further promoted chondrogenic differentiation and proliferation ( Fig. 3 b and c). Results from qRT-PCR and immunofluorescence assays indicated that the HAMA-1.0M + NIR group significantly up-regulated the expression of chondrogenesis-associated genes and proteins ( Fig. 4 ). Further studies suggested that HAMA-1.0M combined with NIR treatment enhanced mitochondrial function through increased energy metabolism ( Fig. 5 ), thereby contributing to the reduction of apoptosis [ 44 ]. In vivo , the results of H&E staining, toluidine blue staining, Safranin O & Fast green staining, and immunohistochemistry at 4 and 8 weeks further demonstrated that the MoS 2 -TA nanozyme-incorporated HAMA hydrogels were conducive to enhancing tissue integration ( Fig. 7 ). Combined with their anti-inflammatory capacity, photothermal conversion ability, and adhesive performance, the MoS 2 -TA nanozyme-incorporated HAMA hydrogels exhibited the most significant healing effect on damaged cartilage. The therapeutic mechanism of HAMA-M hydrogels on cartilage regeneration was further investigated. HSPs are a group of proteins that can improve cell tolerance under heat stress. There are many studies proved that HSPs play an important role in promoting osteogenesis under mild-heat stimulation. HSP47 and HSP70 are two kinds of key HSPs. HSP47 can help to promoting synthesis of collagen, and HSP70 was found to be benefited for reducing inflammatory responses. The gene and protein levels of HSP47 and HSP70 were up-regulated under NIR irradiation, indicating that the HSPs may play important roles in promoting osteogenesis under hyperthermia conditions in photothermal therapy. Besides, the AMPK-SIRT1-PGC1α pathway was reported to correlate with temperature-induced mitochondrial biogenesis [ 45 , 46 ]. The mild-heat stimulation can simultaneously upregulate both AMPK activity and SIRT1 expression, and further promoted the transcription of mitochondrial biogenesis mediators, notably PGC1α. On one hand, PGC1α activated antioxidant defense genes, attenuating intracellular ROS to maintain mitochondrial performance [ 47 ]. On the other hand, PGC-1α would promote mitochondrial biogenesis by NRF2 interaction and TFAM activation [ 37 ]. In this study, the expression of mitochondrial metabolism factors including SIRT1, PGC1α, TFAM, and NRF2 in HAMA-1.0M and HAMA-1.0M + NIR groups were all upregulated, indicating the AMPK-SIRT1-PGC1α pathway was activated to regulate mitochondrial function and accelerate mitochondrial production. It is worth mentioning that the outstanding SOD- and CAT-mimicking antioxidant activity of the HAMA-M hydrogels would certainly help to decrease excess ROS production and improve the function of mitochondria. As a result, we deduced that the HAMA-1.0M hydrogels may promote chondrogenesis through ROS scavenging and mild-heat stimulation.
Conclusion
In this paper, the MoS 2 -TA dual-catalytic system was used as a novel initiating system to prepare a biocompatible and multifunctional HAMA-M hydrogel for cartilage repair under mild reaction conditions. The MoS 2 -TA system not only serves as an initiator but also endows the hydrogel with robust mechanical properties, excellent photothermal conversion ability, and strong antioxidant capacity. The in vitro and in vivo results indicated that the HAMA-M hydrogel exhibited synergistic effects on cartilage regeneration by downregulating the pro-inflammatory microenvironment under mild photothermal therapy. This study provides a novel therapy for cartilage repair and offers new insights into cartilage tissue engineering.
Materials and methods
Materials
Sodium hyaluronate(HA, MW = 100 kDa, Yuanye, China), methacrylic hydride (MA, Macklin, China), molybdenum disulfide (MoS 2 , Energy Chemical, China), L-Ascorbic acid (AA, Beyotime, China), Tannic acid (TA; Macklin, China), ammonium persulfate (APS, Aladdin, China). All solvents and reagents were purchased from commercial sources and used according to instructions.
Preparation and characterization of MoS 2 -TA nanozyme
The MoS 2 -TA nanozyme were prepared by a method of ultrasonic-assisted exfoliation. 100 mg TA was dissolved in 100 mL deionized water, and 200 mg bulk MoS 2 was added and stirred for 30 min. The MoS 2 dispersion was then ultrasonicated with 300 W power for 2 h and centrifuged at 5000 rpm for 15 min. The supernatant was collected and lyophilized. The morphology of MoS 2 -TA nanozyme was observed by transmission electron microscope (TED FEI, USA). The X-ray diffraction (XRD) pattern was obtained by X-ray diffractometer spectrum (Miniflex 600, Rigaku, Japan). The UV absorption curve was determined by a UV–Vis spectrophotometer (HORIBA, USA). The CAT activity of MoS 2 -TA nanozymes was measured using a total superoxide dismutase assay kit with WST-8 (Beyotime, China). Various concentrations of MoS 2 -TA nanozymes (5, 10 and 20 mg/mL) were tested according to the reagent supplier's instructions. Namely, WST-8 enzyme working solution (160 μL), working solution (20 μL), and sample or control blank (20 μL) were incubated at 37 °C for 30 min, Taken 20 μL of the mixture and added to a 96-well plate, and the absorbance was measured by a microplate reader at 450 nm, n = 3. Inhibition ratio = (A450 control 1 −A450 sample )/(A450 control 1 − A450 control 2 ) × 100 %. In addition, the CAT activity of MoS 2 -TA nanoenzymes was measured using a catalase assay kit (Beyotime, China). Briefly, the standard curve is calculated based on the instructions and the provided reagents. Then, the absorbance of samples at different concentrations is measured at 520 nm to calculate their corresponding catalase enzyme activity.
Construction and characterization of HAMA-M hydrogel
Hyaluronic acid methacrylate (HAMA) was synthesized by referring to previous methods [ 8 ]. Briefly, 1 % (w/v) HA solution was prepared by dissolving HA in deionized water. Then the methacrylic anhydride with the molar ratio of 10:1 to HA was added, and the pH of the reaction solution was maintained at 8 by 5 M NaOH. The solution was continuously stirred at 4 °C for 24 h and further purified by dialysis (molecular weight cut off, 8 kDa) against deionized water for 3 days. The purified product was flash-frozen and lyophilized. The degree of substitution (DS) of HAMA was determined by 1 H NMR (600 MHz Bruker, Switzerland). For fabricating the hydrogel, HAMA (5 % w/v) and MoS 2 -TA nanozyme were mixed and vortexed to get a homogeneous solution, then 10 mM ammonium persulfate (APS) was added to initiate the polymerization of HAMA. The formed nanozyme incorporated hydrogel with different ratios of MoS 2 -TA nanozyme (0.5 %, 1.0 %, and 2.0 % w/v) was abbreviated as HAMA-0.5M, HAMA-1.0M, and HAMA-2.0M, respectively. Microscopic morphology and elemental distribution of hydrogels were observed by scanning electron microscopy (SEM) and energy dispersion spectra (EDS) (FEI Quanta 600, USA). Fourier-transform infrared spectra (FT-IR) of hydrogels were recorded by an FT-IR spectrometer (IRAffinity1S, Shimadzu, Japan).
Mechanical property of HAMA-M hydrogel
Compression and adhesion strength were tested by a universal material testing machine (Instron, USA). For the compression test, the cylindrical hydrogels with a diameter of 5 mm and a height of 10 mm were compressed at a cross-head speed of 5 mm min −1 and Young's modulus was calculated at the strain ranging from 10 % to 15 %. The adhesive strength was evaluated by lap-shear tests [ 48 ]. Briefly, the pre-mixed solution was injected into amino-treated glass slices (bonded area = 25 mm × 25 mm). After being crosslinked in situ, the samples were measured at an across-head speed of 1 mm/min. The adhesive strength was calculated by dividing the maximum stress by the bonded area. Besides, the hydrogels' rheological properties were tested by a HAAKE MARS rheometer (Thermo Fisher Scientific, Waltham, MA, USA). In order to determine the swelling ratio, the hydrogels were immersed in phosphate-buffered saline (PBS) for a preset period, and the weight was recorded as W s . Subsequently, they were immersed in deionized water to remove excess salts, lyophilized and the weight was recorded as W d (n = 3). The hydrogels' swelling ratio (Rs) was calculated using the following equation. R s = W s − W d W d × 100 % For degradation ratio, all samples were kept in the PBS at 37 °C, 60 rpm shaken, and the remaining masses were regularly recorded. The initial weight of the scaffold was recorded as W 0 , and the lyophilized weight of the scaffold at different time points was recorded as Wt. The hydrogels' degradation ratio (R d ) was calculated using the following equation. R d = W 0 — W t W 0 × 100 %
Photothermal property of HAMA-M hydrogel
Near-infrared (NIR) laser (Leishi, China) (λ = 808 nm, output power = 50 mW, distance of the NIR source from hydrogels = 1 cm) was used to irradiate hydrogels with power density 0.78 W/cm 2 . Temperatures were recorded by thermal imaging cameras (FOTRIC, China).
BMSCs isolation and culture
The animal experimental protocols were approved by the Animal Ethics and Welfare Committee of Guangxi Medical University (Protocol Number: 202306011). Sprague Dawley (SD) rats (5-day-old) were supplied from the Experimental Animal Center of Guangxi Medical University. BMSCs were harvested from femoral and tibial bone marrow and cultured in α-MEM medium (Biosharp, China) supplemented with 10 % fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were incubated at 37 °C in an atmosphere containing 5 % CO 2 . The cells at passage 3 were used for all experiments.
Biocompatibility of HAMA-M hydrogel
The CCK-8 kit (Biosharp, China) was used to evaluate cell viability. Briefly, BMSCs were seeded on the hydrogel in a 24-well plate at a density of 1 × 10 5 cells per well, with or without periodic NIR irradiation(0.78 W/cm 2 ; 15 min per day). After culturing for 1, 3, and 7 days, the cells were washed with PBS. Subsequently, CCK-8 solution was introduced into the medium, followed by incubation at 37 °C for 2 h. The optical density (OD) was then assessed at a wavelength of 450 nm using a microplate spectrophotometer(Thermo Fisher, USA). The optimal concentration of hydrogels was selected for the following study based on cell proliferation. The cytoskeleton of BMSCs on HAMA-M hydrogels was tested by FITC-Phalloidin staining. After 24 h of incubation, the samples were washed and fixed with 4 % paraformaldehyde for 15 min. Subsequently, the cytoskeleton was stained with FITC-Phalloidin (Beyotime, China) for an hour, followed by staining the cellular nuclei with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 5 min. Images were taken with a confocal laser scanning fluorescence microscope (CLSM; Leica TCS SP8, Germany). Cell spreading area was calculated using Image J software. The cell viability of BMSCs on HAMA-M hydrogels was investigated by using a calcein/PI live/dead viability/cytotoxicity assay kit (Beyotime, China) according to the product instructions. The cell proliferation of BMSCs on HAMA-M hydrogels was performed by hematoxylin and eosin (HE) staining with HE staining kits (Solarbio, China).
Chondrogenic differentiation of BMSCs on HAMA-M hydrogel
For 3D cell cultures, BMSCs were collected, centrifuged, and resuspended with HAMA-M pre-polymerization solution at 1 × 10 7 cells/mL density, then injected into molds, and left to cross-link for 1 min. The BMSCs encapsulated hydrogels were cultured in a complete chondrogenic medium for 3, 7, and 14 days. The chondrogenic medium was composed of DMEM (Gibco, USA), 100 U/mL penicillin and 100 μg/mL streptomycin, 1 mmol/L sodium pyruvate, 40 μg/mL L-proline, 100 nmol/L dexamethasone, 50 μg/mL ascorbic acid 2-phosphate, 1 % (w/v) insulin-transferrin-selenium (ITS), and 10 ng/mL TGF-β1 [ 1 ]. For determination of the photothermal performance of the hydrogels on cell culture, petri dishes were placed in a biosafety cabinet and irradiated with an 808 nm NIR laser (Leshi, China) positioned 1 cm above the cell-encapsulated hydrogel. Irradiation was performed at an output power of 50 mW, with a power density of 0.78 W/cm 2 for 15 min per day. After 21 days of culture, the BMSCs were fixed using paraformaldehyde, embedded in paraffin wax, and then sectioned to a thickness of 5 μm. These sections were stained with Toluidine blue and Safranin O according to the standard protocol (Solarbio, China). The immunofluorescence staining was performed with aggrecan (ACAN), Col2A1 antibody (Affinity, China, 200:1), and secondary antibody goat anti-rabbit AF-488. Cellular nuclei were stained with DAPI. Images were captured with CLSM. To quantitative analysis of GAG, the samples were minced and digested with 1 mL of PBS with 20 mg/mL proteinase K (Beyotime, China). The quantitative analysis of GAG content was evaluated by DMMB (1,9-dimethyl methylene blue, USA) assay. The mixture was then incubated overnight at 60 °C, 800 rpm. The total DNA content was quantified through the use of the Hoechst33258 assay, employing a fluorescence microplate reader with excitation and emission wavelengths of 360 nm and 460 nm, respectively. Eventually, the GAG content of the sample was normalized to its DNA content.
Evaluation of antioxidant activity and mitochondrial function of BMSCs on HAMA-M hydrogel
BMSCs were seeded on the hydrogels in a 12-well plate at a density of 1 × 10 6 cells per well. After 24 h of culture, lipopolysaccharides (LPS) were added to the medium at a density of 1 μg/mL to induce an inflammatory response. 2′,7′ Dichlorofluorescein diacetate (DCFH-DA) and 3-Amino,4-aminomethyl-2′,7′-difluorescein, diacetate (DAF-FM DA) dyes were used to test the scavenging ability of hydrogels for ·OH and NO (Beyotime China). Images were taken with confocal laser scanning microscopy. The fluorescence intensity was calculated using Image J software. The cell apoptosis was detected using the Apoptosis Detection Kit (Yeasen, China). To evaluate the mitochondrial function, BMSCs were seeded at a density of 1 × 10 6 per well on the hydrogel in 6-well plates, treated with LPS (1 μg/mL), and cultured for 24 h. Mitochondria-associated ROS levels, mitochondrial membrane potential, and mitochondrial mass were tested by MitoSOX-Red, JC-1 kit assay, and MitoTracker Green; (Beyotime, China) according to the kit instructions.
Quantitative real-time PCR (qRT-PCR)
The total RNA was extracted by using the Total RNA Extraction Kit (Solarbio, China). The RNA was reverse-transcribed into cDNA with Prime Script RT kit (Takara, Japan), and the gene expression level was quantified with Universal SYBR Green Fast qRT-PCR Mix (ABclonal, China) by the Light Cycler 96 System (Roche, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to normalize other genes by the 2 −ΔΔCt method. All the primer sequences used in this study are listed in supplementary information ( Tables S1–3 ).
Western blot analysis
RIPA lysis buffer was used for protein extraction. Then, the extracted protein was separated with SDS-PAGE gels. After electrophoresis, polyvinylidene difluoride transfer membranes (Biosharp, China) were used for protein transfer. The non-specific antigens were blocked with a blocking buffer. After incubation with primary and secondary antibodies, the chemiluminescent signal was detected by an ultrasensitive multi-function imager (Amersham Imager 600, GE, USA). All primary antibodies were purchased from Affinity, China.
Animal procedure
The study was approved by the Animal Ethics and Welfare Committee of Guangxi Medical University (Protocol Number: SCXKGui-2023-0015). The adult SD rats (10 weeks of age, 250 g – 300 g) were purchased from the Animal Center of Guangxi Medical University. After intraperitoneal anesthesia using 2 % sodium pentobarbital at a dose of 30 mg/kg, a parapatellar longitudinal incision was used to expose the femoral trochlear, and a cylindrical osteochondral defect (diameter, 2 mm; depth, 1.5 mm) was produced with a dental drill as previously described [ 48 ]. The adult SD rats were randomly divided into 6 groups. Sham group: normal cartilage without any treatment. Defect group: the defects without any treatment. HAMA group: the defects treated with BMSCs encapsulated HAMA. HAMA + NIR group: the defects treated with BMSCs encapsulated HAMA hydrogel, and exposed to NIR irradiation regularly (0.78 W/cm 2 ; 15 min per day; 1 irradiation every 2 days up to day 14). HAMA-1.0M group: the defects treated with BMSCs encapsulated HAMA-1.0M hydrogel. HAMA-1.0M + NIR group: the defects treated with BMSCs encapsulated HAMA-1.0M hydrogel, and exposed to NIR irradiation regularly (0.78 W/cm 2 ; 15 min per day; 1 irradiation every 2 days up to day 14). The BMSCs were mixed with HAMA-1.0M (100 μL, 5 % w/v) solution or HAMA (100 μL, 5 %, w/v) solution with a cell concentration of 3 × 10 6 cells/mL. The BMSCs-encapsulated with hydrogel precursor solution were injected into the cartilage defects, followed by the repositioning of the patella and the suturing of the surgical incisions. Penicillin was injected intramuscularly postoperatively to prevent infection. The SD rats were euthanized by chloral hydrate anesthesia at weeks 4,8 respectively, and the distal femur was harvested, then decalcified, embedded, and sectioned according to the standard histological procedure.
Histological evaluation and grading
The SD rats were euthanatized after 4 or 8 weeks of treatment. The Samples were photographed with a digital camera for macroscopic evaluation. Histological evaluation was examined by H&E, toluidine blue, and Safranin O & Fast green staining under standard histological staining procedures. For immunohistochemical staining, sections were incubated with hydrogen peroxide for 30 min at 37 °C to block endogenous peroxidase and then heat-treated at 60 °C for 5 min to remove non-specific antigens. After blocking with goat serum, a monoclonal antibody against type II collagen (Affinity, China) was added and incubated overnight at 4 °C, followed by biotinylated goat anti-mouse IgG secondary antibody, and stained with 3,3′-diaminobenzidine (DAB, Boster, China). The cell nucleus was stained with hematoxylin. The pictures were captured with the orthographic microscope (Olympus, Japan). Evaluation of cartilage defect repair using the International Cartilage Repair Society (ICRS) scoring system and Modified O'Driscoll Scale (MODS) [ 49 , 50 ]. The scoring was done independently by three authors.
Statistical analysis
Determination of statistical significance between multiple groups was conducted via ANOVA with the Tukey method, while an unpaired two-tailed t -test was employed to analyze two independent groups. P values < 0.05 were regarded as significant. All analyses were conducted using GraphPad Prism.
CRediT authorship contribution statement
Qingbing Jiang: Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation. Yifeng Shang: Methodology, Investigation, Formal analysis. Hong Cheng: Writing – original draft, Methodology, Investigation, Data curation. Jinmin Zhao: Writing – review & editing, Validation, Supervision, Funding acquisition. Lerong Yang: Visualization, Validation, Investigation. Zhenzhen He: Formal analysis, Data curation. Jiyong Wei: Validation, Resources, Data curation. Ruiming Liang: Writing – review & editing, Supervision, Project administration, Funding acquisition. Wei Su: Writing – review & editing, Supervision, Resources, Project administration. Li Zheng: Writing – review & editing, Validation, Supervision, Funding acquisition, Conceptualization. Chuanan Liao: Writing – review & editing, Validation, Supervision, Funding acquisition, Conceptualization.
Ethics approval and consent to participate
All animal procedures were approved by the Animal Ethics and Welfare Committee of Guangxi Medical University.
Consent for publication
There is no conflict of interest in submitting this manuscript, and the manuscript is approved for publication by all authors.
Declaration of competing interest
No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. The authors declare no conflict of interest.
Supplementary data
The following is the to this article: Multimedia component 1 Multimedia component 1
Data availability
Data will be made available on request.
Footnotes
Supplementary data to this article can be found online at https://doi.org/10.1016/j.mtbio.2025.102056 .
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