ZnCe-LDO Nanozyme-Based Multifunctional Hydrogel Promotes Bone Regeneration by Inflammatory Macrophage Reprogramming and Piezo1 Activation

Abstract

Prolonged or excessive inflammation may lead to impaired vascularization and bone regeneration, hindering the normal repair process of bone tissue. Although the regulation of inflammation is crucial for promoting a conducive microenvironment for bone regeneration, individual anti-inflammatory interventions frequently are inadequate in facilitating effective bone repair. Here, a multifunctional hydrogel (GelMA-ZC-Yoda1) with multifaceted therapeutic strategy was designed by integrating Zinc/Cerium-layered double oxide nanozyme (ZnCe-LDO, with catalase-like activity) and Yoda1 (an activator of the mechanically sensitive Piezo1 ion channel) into photocurable GelMA hydrogel. The ZnCe-LDO nanozyme in the hydrogel promoted M2 macrophage polarization by reprogramming inflammatory macrophages, establishing a favorable microenvironment, while the sustained release of zinc and cerium ions facilitated osteo/angiogenesis. Additionally, the Yoda1 released from the hydrogel chemically simulated a mechanical signal to activate the Piezo1 channel, regulating osteo/angiogenesis via the Piezo1/YAP1 signaling pathway. In vivo findings indicated that the GelMA-ZC-Yoda1 hydrogel application improved the inflammatory microenvironment and effectively enhanced osteo/angiogenesis in a rat calvaria defect model. This study supports the advancement of injectable hydrogels with multifunction, including inflammatory macrophage reprogramming and promotion of osteo/angiogenesis.

Introduction

The regeneration and repair of bone tissue is a complex biological process regulated by numerous factors, with inflammation regulation being a pivotal aspect. Prolonged or excessive inflammation disrupts the microenvironment of tissue repair, thereby impeding both osteogenesis and angiogenesis. − Current studies predominantly focus on improving bone regeneration via immune modulation, yet achieving an effective balance between inflammation and tissue repair remains a significant challenge, limiting clinical efficacy. − Additionally, the regulation of inflammation is essential, while individual anti-inflammatory interventions are frequently inadequate in facilitating effective bone repair. Hence, it is imperative to explore intelligent and multifaceted collaborative therapeutic strategies to facilitate bone regeneration and repair.

Macrophages are crucial in orchestrating the immune response. They can differentiate into distinct polarization states, including proinflammatory (M1) and anti-inflammatory (M2) phenotypes. In the initial stages of inflammation and tissue repair, the priority is the activation of M1 macrophages. They recruit additional immune cells to the damaged region by secreting proinflammatory cytokines, which in turn trigger an inflammatory response. , As the inflammatory process advances, M1 macrophages undergo a gradual transition into M2 macrophages, which suppress inflammation through the secretion of anti-inflammatory and tissue repair cytokines. − Nevertheless, under pathological conditions influenced by chronic inflammation and aberrant immune responses, macrophages in the defect area encounter challenges in successfully transitioning from the M1 to the M2 phenotype. − This imbalance not only exacerbates the inflammatory response but also impedes the reparative processes of bone tissue. Hence, an appropriate M1/M2 ratio promotes the harmonious interaction between inflammation and repair processes and contributes to the creation of a conducive immune microenvironment for bone regeneration and repair.

Nanozymes, characterized by simulated enzymatic activity, participate in intricate biocatalytic processes. − The studies have shown that catalase-like nanozymes such as MnO₂ and prussian blue (PB) can promote the M2 macrophage polarization and orchestrate the inflammatory microenvironment by scavenging endogenous reactive oxygen species (ROS). Currently, layered double hydroxides (LDH), as nanocatalysts, have received considerable attention in the realm of biomedicine. − However, the therapeutic efficacy of LDH is compromised by its limited water dispersibility and physiological toxicity. , To address these issues, researchers have focused on LDH derivatives. Among them, layered double oxides (LDO) exhibit a larger surface area, an increased number of active sites, as well as excellent water dispersibility and biocompatibility, positioning it as a highly promising nanocatalyst. The enhanced performance of LDOs has rendered it a focal point of research and opened avenues for potential further applications. LDOs with multiple metal oxides exhibit multifunctional properties. To design therapeutic strategies that simultaneously regulate inflammatory responses and promote bone regeneration, this study selected zinc and cerium as the key metal elements. The studies have shown the characteristics of zinc ions in promoting osteo/angiogenesis. − An appropriate amount of zinc ions inhibited proinflammatory cytokines and induced anti-inflammatory cytokines, thus maintaining an anti-inflammatory microenvironment. , This property enables zinc to establish a more conducive microenvironment for bone regeneration. Cerium ions also play a critical role in promoting osteo/angiogenesis. − Cerium, typically present as trivalent or tetravalent ions, exhibits catalase-like activity and promotes the polarization of M2 macrophages by scavenging ROS, thereby helping to regulate the inflammatory microenvironment. − Therefore, this study combined zinc with cerium based on their respective biological functions and potential synergistic effects. Zinc regulates bone cell activity and inflammatory responses to positively influence osteo/angiogenesis, whereas cerium can scavenge ROS via its nanozyme activity, thereby stabilizing the immune microenvironment and facilitating osteo/angiogenesis. Considering the functions of both elements, this study speculated that zinc/cerium layered double oxide (ZnCe-LDO) nanozyme not only enhance osteo/angiogenesis through the release of zinc and cerium ions but also promote macrophage polarization toward the M2 phenotype through scavenging endogenous ROS, thereby improving the inflammatory microenvironment. This synergistic effect establishes more optimal regeneration microenvironment at the bone defect site, thereby accelerating tissue repair.

Mechanical stress signals are pivotal for maintaining bone homeostasis and remodeling. − The mechanically sensitive Piezo1 channel is regarded as the primary sensor of mechanical signals. , When cells are subjected to mechanical stress, the Piezo1 channel perceives diverse mechanical signals on the cell membrane, thereby contributing to bone development and homeostasis by activating intracellular signal transduction. , As a chemical activator of the mechanically sensitive Piezo1 channel, Yoda1 simulates bone and endothelial cells responses to mechanical stress, which are involved in the pivotal processes of osteo/angiogenesis. , However, Yoda1 administration on its own is unsustainable, and systemic Piezo1 activation may have unforeseeable adverse effects in other organs. − Thus, it is crucial to design a delivery system that slowly releases Yoda1 to precisely activate the Piezo1 channel in localized bone defect regions.

Therefore, in this study, the ZnCe-LDO nanozyme and Yoda1 were incorporated into a GelMA hydrogel to construct a multifunctional hydrogel platform (GelMA-ZC-Yoda1). Compared to traditional monotherapy, our study proposes a multifaceted therapeutic strategy that not only reprograms inflammatory macrophages to create a favorable microenvironment but also promotes osteo/angiogenesis by continuously releasing bioactive ions and simulating mechanical signals (Scheme ). This integrated approach, rarely reported in previous studies, utilizes the synergistic effect of ZnCe-LDO nanozyme and mechanical signal transduction, representing a therapeutic direction for inflammation-related bone defects. In summary, the multifunctional hydrogel incorporating ZnCe-LDO nanozyme may offer a promising therapeutic approach for bone defects associated with inflammation.

1. Schematic Illustration for Inflammatory Macrophage Reprogramming and the Coupling of Osteogenesis and Angiogenesis Regulated by the GelMA-ZC-Yoda1 Hydrogel.

Results and Discussion

Preparation and Characterization of ZnCe-LDO and Hydrogels

The ZnCe-LDO nanozyme was successfully synthesized using the hydrothermal synthesis method. Transmission electron microscopy (TEM) images showed that the ZnCe-LDO nanozyme was composed of layered nanoparticles with uniform size (Figure A). The particle size of the ZnCe-LDO nanozyme measured by dynamic light scattering (DLS) was 275 ± 11.6 nm (Figure B). Energy dispersive X-ray (EDX) elemental mapping confirmed the presence of Zn and Ce in the synthesized ZnCe-LDO nanozyme (Figure C, Figure S1A). The X-ray diffraction (XRD) results showed that the diffraction peaks corresponded to ZnO and CeO₂ (Figure D), confirming the successful preparation of the ZnCe-LDO nanozyme, comparable to what has been reported in previous studies. , Additionally, as shown in Figure E, a gradual increase in the dissolved oxygen content was observed with increasing concentrations of the ZnCe-LDO nanozyme. This suggests that ZnCe-LDO exhibits catalase-like (CAT) activity, promoting the decomposition of H₂O₂ into H₂O and O₂. The generated dissolved oxygen alleviates the hypoxic environment in the central region of the hydrogel materials and provides oxygen support for cell survival and differentiation preceding osteo/angiogenesis.

1.

Characterization of ZnCe-LDO and hydrogels. (A) TEM image of ZnCe-LDO (Scale bar: 50 nm). (B) Size distribution profile of ZnCe-LDO. (C) EDX elemental mapping of ZnCe-LDO (Scale bars: 5 nm). (D) XRD diffraction pattern of ZnCe-LDO. (E) Dissolved oxygen content of ZnCe-LDO nanozyme at varying concentrations over time. (F) Schematic illustration of the GelMA-ZC-Yoda1 hydrogel synthesis process. (G) Images before and after photo-cross-linking of the GelMA and GelMA-ZC-Yoda1 hydrogels. (H) Characteristics of the GelMA-ZC-Yoda1 hydrogel. (I) SEM images and (J) rheological test of the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels (Scale bars: 100 μm). (K) Variation of G′ and G″ with frequency in the GelMA-ZC-Yoda1 hydrogel, respectively. (L) Compressive modulus, (M) stress–strain curve, and (N) swelling index of the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels. (O) Cumulative release amounts of Zn and Ce ions, and (P) release curve of FITC in the hydrogel. (Q) Degradation behavior of the hydrogels under simulated physiological conditions. The data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

The ZnCe-LDO nanozyme and Yoda1 were incorporated into the GelMA hydrogel to form a multifunctional hydrogel (GelMA-ZC-Yoda1), as illustrated in Figure F. First, the different amounts of ZnCe-LDO were loaded into the hydrogel. To ensure the biocompatibility of ZnCe-LDO loaded hydrogel, we carried out cell vitality assay. The result showed that the ZnCe-LDO nanoparticles loaded in the hydrogel (0.05–0.4 mg/mL) have no obvious cytotoxicity, indicating that the concentration is appropriate in terms of biocompatibility (Figure S2A). Then, the ALP staining and ALP activity assays were carried out to determine the optimal concentration of ZnCe-LDO loaded in the hydrogel. As shown in Figure S2B and C, the hydrogel containing 0.2 mg/mL ZnCe-LDO showed the most obvious osteogenic differentiation, effectively promoting the osteogenic differentiation of BMSCs. Therefore, we chose 0.2 mg/mL ZnCe-LDO to be loaded into hydrogel in subsequent experiments.

The GelMA pregel solution is a translucent and slightly viscous liquid at room temperature (RT). After the incorporation of ZnCe-LDO and Yoda1, the resulting pregel mixture (GelMA-ZC-Yoda1) was a milky-white and slightly viscous liquid at RT. It formed into a hydrogel after exposure to ultraviolet light (UV), which was measured by tilting the bottle (Figure G). As shown in Figure I, the porous structures of the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels were observed using scanning electron microscopy (SEM). No significant differences were observed among the groups, suggesting that the incorporation of ZnCe-LDO and Yoda1 did not affect the structure of the hydrogels. Moreover, the GelMA-ZC-Yoda1 hydrogel exhibited excellent viscosity, firmness, and deformability, as shown in Figure H, which are beneficial for hydrogel implantation and tissue repair. Injection is an important and convenient method of local administration. The excellent injectability of GelMA-ZC-Yoda1 hydrogel was also demonstrated, allowing for the facile formation of letters using a syringe (Figure H).

The outstanding properties of hydrogel are profoundly related to cell adhesion, proliferation, and differentiation, which are necessary for tissue regeneration and repair. , Therefore, we evaluated the performance of the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels. To better understand the elastic properties of the hydrogel loaded with ZnCe-LDO and Yoda1, its rheological characteristics were first examined using a frequency sweep range of 0.1 to 100 Hz. The results, shown in Figure J, indicated that the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels have higher storage moduli than the GelMA hydrogel, indicating superior shape retention. Meanwhile, over the whole frequency range of 0.1–100 Hz, the GelMA-ZC-Yoda1 hydrogel’s storage modulus (G’) exceeds the loss modulus (G”), consistent with the solid-like behavior and affirming the GelMA-ZC-Yoda1 hydrogel’s strong stability (Figure K). Further compression tests were performed on the hydrogels. As shown in Figure L, the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels have higher compressive moduli, providing further evidence of their relatively stable structures and resistance to deformation. The stress–strain curve of the GelMA-ZC-Yoda1 hydrogel revealed a tensile strength of up to 133.9 kPa under 78.3% strain, which can provide sufficient structural strength for use as a scaffold in bone tissue engineering (Figure M).

The swelling index and equilibrium water content of the hydrogel, which directly affect nutrient and gas exchange, were further investigated. We found that the hydrogel loaded with ZnCe-LDO exhibited a lower swelling index and water content than the GelMA hydrogel, and a low concentration of Yoda1 had a negligible impact on the swelling index and water content (Figure N, Figure S1B). The incorporation of ZnCe-LDO and Yoda1 did not significantly affect the porosity of the hydrogel, which exchanged nutrients and gases. Moreover, ICP-MS was used to evaluate the Zn and Ce ion release capacity of the GelMA-ZC-Yoda1 hydrogel. Without a rapid release, the Zn and Ce ions were gradually released from the GelMA-ZC-Yoda1 hydrogel (Figure O). After 14 days, the cumulatively released amounts of Zn and Ce ions were 1.12 and 1.58 μg/mL, respectively, which are conducive to absorption by bone cells and contribute to effective bone regeneration. In addition, fluorescein isothiocyanate (FITC) was used to evaluate the release of Yoda1. After 14 days, the release rate of FITC reached 47.9% (Figure P). Given the crucial role of hydrogel degradation in bone regeneration, the in vitro degradation behaviors of the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels under simulated physiological conditions were examined. The results illustrated in Figure Q showed that the GelMA-ZC and GelMA-ZC-Yoda1 groups exhibited slower degradation in comparison to the GelMA group. Approximately 64% of GelMA-ZC and GelMA-ZC-Yoda1 hydrogels were retained, whereas only 47% of GelMA hydrogel was retained after 14 days of degradation. The gradual degradation rate observed in the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels facilitated sustained inward bone growth and provided enduring mechanical support within the bone defect region. Furthermore, comparable degradation behaviors were observed in the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels, suggesting that their degradation rates were slowed down by the incorporation of ZnCe-LDO, while the lower concentration of Yoda1 exhibited no significant effect on the degradation rate.

GelMA-ZC-Yoda1 Hydrogel Promoted M2 Macrophage Polarization by Suppressing the Activation of MAPK and TNF Signaling Pathways to Reprogram Inflammatory Macrophages

Regenerative processes in bone defect areas, including regeneration, repair, and healing, may be hindered by the inflammatory microenvironment. , According to the studies, nanozymes with CAT-like activity facilitated macrophage polarization toward the M2 phenotype and orchestrated the inflammatory microenvironment by scavenging endogenous ROS. − The M1 to M2 phenotypic transition in macrophages is pivotal in regulating oxidative stress and immune responses. To simulate oxidative stress conditions in realistic inflammatory environments, lipopolysaccharide (LPS) was employed to stimulate RAW 264.7 macrophages in this study. LPS, commonly used in immunological research, effectively induces a proinflammatory response in macrophages akin to bacterial infections, accompanied by significant ROS generation. ,

First, the hydrogels’ cytotoxicity toward RAW264.7 cells was evaluated by a CCK8 assay, and the results indicated the absence of evident toxicity across various hydrogels to RAW264.7 cells (Figure S4A). Subsequently, fluorescence staining and flow cytometry were conducted to assess the ability of the hydrogels loaded with ZnCe-LDO to scavenge intracellular ROS (Figure A). According to the results in Figure B, macrophages stimulated by LPS exhibited strong green fluorescence signals that were indicative of a considerable increase in ROS production. In contrast, a notable decrease in DCFH-DA fluorescence intensity was observed in macrophages from the GelMA-ZC and GelMA-ZC-Yoda1 groups, and flow cytometry results also confirmed a significant decrease in ROS levels in both groups (Figure C). Additionally, the presence of Yoda1 within the hydrogel did not yield discernible changes in ROS levels. In summary, the hydrogels loaded with ZnCe-LDO exhibited excellent ROS-scavenging capabilities, effectively shielding cells from environmental oxidative stress. The observed phenomenon in these experiments is associated with the polarization behavior of macrophages, providing insights into their functions across various stages of inflammation. The findings indicate that the GelMA hydrogels loaded with ZnCe-LDO effectively mitigate LPS-induced oxidative stress. This oxidative stress is typically accompanied by M1 macrophage polarization, which is indicative of the initial stages of inflammation. Nevertheless, as the inflammatory response diminishes, macrophages gradually transition toward M2 polarization, a process potentially related to the observed decline in ROS levels in our experiments.

2.

In vitro phenotypic transition of macrophages. (A) Schematic diagram depicting the hydrogel’s capacity to reduce ROS levels. (B) Representative fluorescence images (Scale bars: 100 μm) and (C) corresponding flow cytometry analysis of intracellular ROS levels. (D, E) The levels of TNF-α (D) and IL-10 (E) in the supernatant from macrophages using the ELISA assay. (F, G) The mRNA expression levels of M1 markers (TNF-α, IL-1β, and iNOS), M2 markers (TGF-β, IL-10, and Arg-1) and (H) Vegf and (I) Bmp2 in RAW264.7 cells. (J) Western blot images of iNOS and Arg-1 proteins and (K, L) quantitative analysis of the corresponding band intensity using ImageJ software. (M) Flow cytometry analysis of the M1 to M2 phenotypic transition. (N) Immunofluorescence images of iNOS and Arg-1 (Scale bars: 20 μm). The data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. (RAW264.7 cells cultured on the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels with LPS treatment were designated as the LPS, GelMA-ZC, and GelMA-ZC-Yoda1 groups, respectively, while the cells cultured on the GelMA hydrogel samples without LPS treatment served as controls.).

To better understand the correlation between the ROS level and macrophage phenotypic transition, this study further investigated the effects of the hydrogel loaded with ZnCe-LDO nanozyme on the regulation of the macrophage phenotypes in the LPS-simulated inflammatory microenvironment. Macrophage phenotypes were assessed using ELISA, qRT-PCR, and Western blot assays. RAW264.7 cells were inoculated on various hydrogels, with those on the GelMA hydrogel stimulated with LPS designated as the LPS group. RAW264.7 cells inoculated onto the GelMA hydrogel without LPS stimulation served as the control group. The TNF-α content in the LPS group was much higher than that in the control group, as shown in the ELLSA results, which successfully simulated the in vitro inflammatory microenvironment (Figure D). The GelMA-ZC and GelMA-ZC-Yoda1 groups exhibited a significant decrease in TNF-α content, accompanied by an increase in IL-10 content when compared to the LPS group (Figure D, E). This observation preliminarily suggested that the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels may facilitate the M1-M2 phenotypic transition of macrophages. Besides, the expression levels of inflammation-related genes in macrophages were analyzed using qRT-PCR assays. The data suggested that LPS stimulation markedly upregulated the expression levels of the M1-type related genes (IL-1β, TNF-α, and iNOS) in comparison to the control group, whereas this effect was substantially attenuated across the GelMA-ZC and GelMA-ZC-Yoda1 groups (Figure F). Meanwhile, the GelMA-ZC and GelMA-ZC-Yoda1 groups significantly upregulated the expression levels of M2-type related genes (TGF-β, IL-10, and Arg-1) in contrast to the LPS group (Figure G). These results corroborated the ELISA data, further confirming that the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels induced macrophage polarization toward the M2 phenotype in an inflammatory environment. Further validating the above conclusion, the Western blot showed that the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels both decreased the level of iNOS protein expression and increased the protein expression level of Arg-1 (Figure J-L). Notably, the mRNA expression levels of Bmp2 and Vegf in the GelMA-ZC and GelMA-ZC-Yoda1 groups, indicative of osteogenesis and angiogenesis, respectively, were higher than those in the LPS groups (Figure H, I), suggesting that these hydrogels may indirectly promote osteogenesis and angiogenesis through inflammatory macrophage reprogramming.

The alterations in macrophage phenotype were assessed via flow cytometry. The results suggested that the percentage of CD86-positive M1 macrophages in the GelMA-ZC and GelMA-ZC-Yoda1 groups was markedly reduced, whereas the percentage of CD206-positive M2 macrophages was significantly raised as compared to the LPS group (Figure M, Figure S5). Additionally, immunofluorescence staining was performed to evaluate the expression of iNOS and Arg-1. As illustrated in Figure N, weaker fluorescence of iNOS and notably stronger fluorescence of Arg-1 were observed in the GelMA-ZC and GelMA-ZC-Yoda1 groups than in the LPS group. All this collectively indicates that the ZnCe-LDO-loaded GelMA hydrogels exhibit a robust ability to induce M1 macrophages to polarize into M2 phenotype. Furthermore, no obvious variations occurred between the GelMA-ZC and GelMA-ZC-Yoda1 groups. This suggests that the immunomodulatory effect of the GelMA-ZC-Yoda1 hydrogel is predominantly facilitated by the ZnCe-LDO nanozyme, with the presence of trace amounts of Yoda1 exerting negligible influence on macrophage phenotypes.

To elucidate the potential mechanisms underlying ZnCe-LDO induced M2 macrophage polarization, RNA sequencing was performed on macrophages subjected to different treatments. A total of 1,412 differentially expressed genes (DEGs) were identified between the LPS and NC groups, comprising 802 upregulated and 610 downregulated genes (Figure A). Comparison of the GelMA-ZC and LPS groups revealed 11,528 DEGs, including 8,526 upregulated and 3,002 downregulated genes (Figure D). Notably, GO and KEGG enrichment analysis revealed that the MAPK and TNF signaling pathways were commonly enriched in both the LPS vs NC and GelMA-ZC vs LPS comparisons (Figure B–C, E-G), suggesting their potential involvement in ZnCe-LDO–mediated M2 macrophage polarization. Western blot analysis confirmed that LPS stimulation markedly increased the phosphorylation levels of p38 and JNK in macrophages, whereas ZnCe-LDO treatment (GelMA-ZC and GelMA-ZC-Yoda1 groups) significantly suppressed this phosphorylation, indicating inhibition of the MAPK signaling cascade (Figure H). Additionally, given that NF-κB is a well-established downstream effector of the TNF signaling pathway, we assessed the phosphorylation status of p65 and IκBα. Macrophages treated with LPS exhibited elevated levels of p-p65 and p-IκBα, accompanied by reduced total IκBα expression, consistent with activation of the NF-κB signaling pathway. Notably, ZnCe-LDO treatment (GelMA-ZC and GelMA-ZC-Yoda1 groups) reversed these effects, significantly reducing p-p65 and p-IκBα levels and restoring total IκBα expression (Figure I).

3.

RNA-sequencing analysis of macrophages with different treatment. (A) Volcano plot significantly up-regulated and down-regulated genes of macrophages cultured on the GelMA hydrogel with LPS treatment (LPS group). (B,C) GO and KEGG enrichment analysis of LPS vs NC group. (D) Volcano plot significantly up-regulated and down-regulated genes of macrophages cultured on the GelMA-ZC hydrogel. (E,F) GO and KEGG enrichment analysis of GelMA-ZC vs LPS group. (G) Venn diagram of KEGG analysis of LPS vs NC and GelMA-ZC vs LPS group. (H) Western blot images of p-P38, P38, p-JNK, and JNK in macrophages. (I) Macrophages lysates with different treatment were probed for p-P65, P65, P–IκBα, and IκBα. GAPDH was used as internal reference. (RAW264.7 cells cultured on the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels with LPS treatment were designated as the LPS, GelMA-ZC, and GelMA-ZC-Yoda1 groups, respectively, while the cells cultured on the GelMA hydrogel samples without LPS treatment served as controls.).

In summary, this study demonstrates that ZnCe-LDO-loaded GelMA hydrogels effectively mitigate LPS-induced oxidative stress and the inflammatory microenvironment by suppressing the activation of MAPK ang TNF signaling pathways, facilitating the transition of M1 macrophages into the M2 phenotype. This transition is closely associated with reduced ROS levels, indicating that ROS levels are vital for macrophage phenotypes. These findings provide new therapeutic strategies for inflammatory diseases and highlight the prospective value of nanozymes in regulating immune responses.

GelMA-ZC-Yoda1 Hydrogel Promoted the Osteogenic Differentiation of rBMSCs through the Piezo1/YAP1 Signaling Pathway

Here, we employed rat bone marrow-derived mesenchymal stem cells (rBMSCs) to examine the hydrogel’s osteogenic-promoting properties. First, primary rBMSCs were successfully isolated (Figure S3A). The rBMSCs at passage three, observed microscopically, exhibited classic fibroblastic and spindle-shaped morphologies (Figure S3B) and formed single-cell colonies (Figure S3C). The rBMSCs expressed surface markers of mesenchymal stem cells (CD29 and CD90), according to flow cytometric analysis in Figure S3D, and failed to express hematopoietic cell markers (CD11b and CD45). Moreover, the rBMSCs demonstrated pluripotency with the capacity to differentiate into osteocytes (Figure S3E), adipocytes (Figure S3F), and chondrocytes (Figure S3G), respectively. Thus, the isolated cells were identified as rBMSCs.

Second, cell compatibility serves as the cornerstone of applications in vivo. Hemolysis, proliferation, and cell viability are crucial indicators for assessing the biological characteristics of hydrogels. Initially, as illustrated in Figure S4B, the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels did not induce significant hemolysis, with hemolysis rates below 5% for each group, suggesting that the hydrogels exhibited outstanding biocompatibility. Subsequently, the cytotoxicity of rBMSCs cocultured with the hydrogels was determined by a CCK-8 assay. There are no obvious variations in the cell viability of rBMSCs among the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 groups (Figure S4C). The biocompatibility of the hydrogels was further investigated using calcein-AM/PI staining to examine the viability of the rBMSCs cocultured with the hydrogels for 1, 3, and 5 days. Almost all cells in each group are labeled with calcein-AM (green), suggesting high cell viability (Figure S4D). All these findings showed the excellent biocompatibility of the hydrogels.

The studies have shown that metal ions such as Zn and Ce significantly promote bone regeneration and repair. , The inherent ability of hydrogels to provoke bone regeneration in situ is of paramount importance. The osteogenic capacity of the hydrogel containing ZnCe-LDO and Yoda1 was assessed using alkaline phosphatase (ALP) and alizarin red staining (ARS) assays. ALP is an early osteogenesis-related indicator. After 7 days of culture in the osteogenic induction medium, the GelMA-ZC and GelMA-ZC Yoda1 groups exhibited stronger ALP staining and higher ALP activity than the GelMA group. Similarly, after 14 days of osteogenic culture, more calcium nodules were observed in the GelMA-ZC and GelMA-ZC-Yoda1 groups than in the GelMA group (Figure A, B). Notably, the GelMA-ZC-Yoda1 group exhibited the highest ALP activity and calcium level (Figure C, D).

4.

In vitro osteogenic properties of the hydrogels. (A) ALP and ARS staining and (B) representative microscopic images of rBMSCs cocultured with the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels (Scale bars: 200 μm). (C) Quantification of ALP activity for each experimental group. (D) Quantitative assessment of calcium nodules formation in ARS staining. (E) The gene expression levels of Alp, Runx2, Osx, and Ocn in rBMSCs evaluated by qRT-PCR analysis. (F) Western blot images of osteogenesis-related proteins in rBMSCs, and (G) quantitative analysis of the corresponding bands intensity using ImageJ software. (H) Western blot images of Piezo1 and YAP1 in rBMSCs and (I) quantitative analysis of the bands. (J) The gene expression levels of Piezo1 and Yap1 in rBMSCs cocultured with the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels. (K) Immunofluorescence images of RUNX2 and OCN (Scale bars: 50 μm). The data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Further, as analyzed in qRT-PCR and Western blot, higher mRNA and protein expression levels of osteogenic indicators, including ALP, Runx-related transcription factor 2 (RUNX2), Osterix (OSX), and osteocalcin (OCN), were found in the GelMA-ZC and GelMA-ZC-Yoda1 groups in comparison to the GelMA group, with the GelMA-ZC-Yoda1 group exhibiting the highest levels (Figure E-G). Immunofluorescence staining further confirmed these results (Figure K). The GelMA-ZC hydrogel showed excellent osteogenic potential in vitro compared to the GelMA hydrogel, primarily attributed to the ZnCe-LDO, whereas the GelMA-ZC-Yoda1 hydrogel exhibited greater osteogenic properties owing to the continuous release of Yoda1.

As a chemical activator of the mechanically sensitive Piezo1 ion channel, Yoda1 imitates the influence of mechanical stress on osteoblasts and participates in key processes of osteogenesis. The studies have shown that Piezo1 deficiency reduces the levels of Yes-related protein (YAP), thereby impairing the synthesis and secretion of type II and IX collagen. , Through its interactions with nuclear transcription factors involved in bone homeostasis, including RUNX2, STAT3, and β-catenin, YAP can further enhance the expression of osteogenic indicators. − Here, we investigated the significance of the Piezo1/YAP1 signaling pathway in the osteogenic differentiation of rBMSCs mediated by the GelMA-ZC-Yoda1 hydrogel. As analyzed by Western blot and qRT-PCR, the Piezo1 channel of cells in the GelMA-ZC-Yoda1 group was activated by Yoda1, resulting in a notable rise in the gene and protein expression levels of Piezo1 and YAP1 compared to the other groups (Figure H-J). , This indicates that the GelMA-ZC-Yoda1 hydrogel promotes the osteogenic differentiation of rBMSCs via the Piezo1/YAP1 signaling pathway. Overall, the data above demonstrates the outstanding osteogenic capability of the GelMA-ZC-Yoda1 hydrogel in vitro and establishes a robust foundation for further in vivo experiments.

GelMA-ZC-Yoda1 Hydrogel Promoted Angiogenesis through the Piezo1/YAP1 Signaling Pathway

Metal ions like Zn and Ce have been demonstrated to be involved in angiogenesis. , Human umbilical vein endothelial cells (HUVECs) were cocultured with various hydrogels to assess their angiogenic properties. We measured the cytotoxicity of the hydrogels on HUVECs. The CCK8 assay showed that a significant promotion of HUVEC proliferation was observed in the GelMA-ZC and GelMA-ZC-Yoda1 groups in comparison to the GelMA group, which suggested the hydrogels’ excellent biocompatibility (Figure S4E). Next, the cells were put through assays for scratch, transwell, and tube formation. The scratch assay suggested that uniform scratches with the same width were created at the base of each well at 0 h (Figure A). After incubation for 24 h, the GelMA-ZC group had a considerably narrower scratch than the GelMA group, with the GelMA-ZC-Yoda1 group exhibiting the smallest scratches. Subsequent quantitative analysis revealed a greater cell migration rate in the GelMA-ZC group than in the GelMA group, suggesting that the incorporated ZnCe-LDO promoted cell migration to some extent (Figure C). Moreover, the Yoda1-activated Piezo1 channel is also essential for angiogenesis. The findings revealed the highest HUVEC migration rate in the GelMA-ZC-Yoda1 group, suggesting a significant enhancement in cell migration following the incorporation of Yoda1. A transwell assay was conducted to further confirm the effect of the hydrogels on cell migration. The GelMA-ZC group had a notably greater number of migrated cells than the GelMA group, while the GelMA-ZC-Yoda1 group exhibited even more cell migration than the GelMA-ZC group (Figure B, D), further confirming that the ZnCe-LDO and Yoda1 coordinatively promoted cell migration.

5.

In vitro angiogenic properties of the hydrogels. (A) Scratch assay and (B) transwell assay assessing the migration activity of HUVECs. (C) Quantitative analysis of scratch assay and (D) transwell assay. (E) The optical, analytical, and calcein-stained microscope images of the Matrigel assay evaluating the tube formation ability of HUVECs. (F,G) Statistical analysis for the number of junctions (F) and total tube length (G) formed by HUVECs. (H–K) Relative expression levels of angiogenesis-related genes (VEGF, eNOS, HIF-1α, and MMP9) in HUVECs. (L, M) Relative mRNA expression levels of Piezo1 (L) and Yap1 (M) in HUVECs cocultured with the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels. (N) Western blot images of Piezo1 and YAP1 in HUVECs, and (O,P) quantitative analysis of the bands. (All scale bars: 100 μm). The data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

An assay known as tube formation was conducted to verify the angiogenic properties of different hydrogels, which directly reflected the extent of angiogenesis. A notable increase in tube structures was observed in the GelMA-ZC and GelMA-ZC-Yoda1 groups (Figure E). Subsequent quantitative analysis revealed that the GelMA-ZC-Yoda1 group outperformed the GelMA group in terms of junction number and total length, followed by the GelMA-ZC group (Figure F, G). Additionally, the mRNA expression levels of angiogenesis-related indictors (HIF-1α, VEGF, MMP9, and eNOS) were assessed in the HUVECs cocultured with various hydrogels. Notably greater expression levels of angiogenesis-related genes were observed in the GelMA-ZC and GelMA-ZC-Yoda1 groups, with the GelMA-ZC-Yoda1 group exhibiting the highest expression levels (Figure H–K), suggesting that the GelMA-ZC-Yoda1 hydrogel has superior angiogenic properties. In conclusion, the incorporation of ZnCe-LDO and Yoda1 in the GelMA-ZC-Yoda1 hydrogel expedited cell migration and induced angiogenic gene expression, thereby enhancing the angiogenic performance of the hydrogel.

Meanwhile, we explored the significance of the Piezo1/YAP1 signaling pathway contributing to the tube formation of HUVECs mediated by GelMA-ZC-Yoda1 hydrogel. The results of Western blot and qRT-PCR revealed that the levels of Piezo1 and YAP1 in HUVECs regarding gene and protein were considerably upregulated in the GelMA-ZC-Yoda1 group compared to the other groups (Figure L-P). These findings suggest that the GelMA-ZC-Yoda1 hydrogel may promote HUVEC tube formation via the Piezo1/YAP1 signaling pathway.

Conditioned Medium Promoted Osteo/Angiogenesis through Inflammatory Macrophage Reprogramming

The above findings indicate that ZnCe-LDO-loaded hydrogels can directly promote osteogenesis and angiogenesis in BMSCs and HUVECs, independent of their immunomodulatory effects. To assess potential indirect effects mediated by immune regulation, we analyzed the influence of conditioned medium (CM) derived from macrophages cocultured with different hydrogels on osteogenic and angiogenic responses. The collected conditioned medium from macrophages cocultured with the GelMA, GelMA-ZC, or GelMA-ZC-Yoda1 hydrogels was employed in the culture of rBMSCs and HUVECs, as illustrated in Figure A.

6.

In vitro osteo/angiogenic properties of the conditioned medium through inflammatory macrophage reprogramming. (A) Schematic diagram illustrating the methodology for cell processing. (B) ALP staining images and (C) quantification of ALP activity in rBMSCs after 7 days of treatment with various conditioned medium (CM/NC, CM/LPS, CM/GelMA-ZC, and CM/GelMA-ZC-Yoda1). (D) ARS staining images and (E) quantitative assessment of calcium nodules formation in rBMSCs after 14 days of treatment various conditioned medium. (F) Scratch assay of HUVECs stimulated with various conditioned medium at 0 and 24 h. (G) Quantitative analysis of migration ratio (%) in scratch assay. (H) The optical and analytical microscope images depicting the tube formation ability of HUVECs in the Matrigel assay. (I) Quantitative analysis conducted to determine the number of junctions formed by HUVECs in each experimental group using ImageJ software. (All scale bars: 200 μm). The data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01.

ALP and ARS staining were performed to evaluate the osteogenic effects. As shown in Figure B-E, osteogenic differentiation of rBMSCs was suppressed in the LPS-induced inflammatory microenvironment (CM/LPS group), with significant downregulation in ALP and ARS staining as well as quantitative analysis. Compared to the CM/LPS group, ALP staining expression and ALP activity in the CM/GelMA-ZC and CM/GelMA-ZC-Yoda1 groups were noticeably increased (Figure B, C). Similarly, consistent results were observed in ARS staining, with more calcium nodules in the CM/GelMA-ZC and CM/GelMA-ZC-Yoda1 groups than the CM/LPS group (Figure D). The staining results were comparable with the quantitative assessment of calcium nodules (Figure E). These findings indicate that conditioned medium from macrophages cocultured with GelMA-ZC or GelMA-ZC-Yoda1 hydrogels promotes osteogenesis.

The angiogenic effect was assessed using a scratch assay and a Matrigel tube formation assay. The migratory ability and tube-forming capacity of HUVECs were both inhibited in the LPS-induced inflammatory microenvironment (CM/LPS group), as the results demonstrated in Figure F–I. In comparison to the CM/LPS group, the CM/GelMA-ZC and CM/GelMA-ZC-Yoda1 groups facilitated the migration of HUVECs (Figure F, G) and significantly promoted the formation of tubular structures by HUVECs (Figure H, I). These findings suggest the promotion of angiogenesis by conditioned medium from macrophages cocultured with the GelMA-ZC or GelMA-ZC-Yoda1 hydrogels.

Although GelMA-ZC-Yoda1 hydrogel exhibited excellent osteo/angiogenic properties compared to GelMA-ZC, no significant differences were observed in the osteogenic or angiogenic potential between CM/GelMA-ZC and CM/GelMA-ZC-Yoda1 groups. We speculate that this may be due to the low concentration of Yoda1 in the hydrogel, which does not significantly affect macrophage phenotype or cytokine secretion. In contrast, the immunomodulatory effect of GelMA-ZC-Yoda1 is primarily driven by ZnCe-LDO nanozymes, which enhance the secretion of cytokines such as BMP2 and VEGF, facilitating osteo/angiogenesis. In summary, the hydrogels loaded with ZnCe-LDO indirectly play an active role in promoting osteo/angiogenesis by improving the inflammatory microenvironment, which provides valuable insights for the development of immunomodulatory hydrogel materials.

GelMA-ZC-Yoda1 Hydrogel Promoted the Formation of Favorable Immune Microenvironment by Inflammatory Macrophage Reprogramming

To assess the immune response of the hydrogels in vivo within the bone defect, we used the widely adopted rat calvaria defect model in this study. Subsequently, precursor solutions of the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels were injected into the bone defect region, followed by gel induction for bone defect repair through UV irradiation. The mechanisms of early immunomodulation and the promotion of osteo/angiogenesis are illustrated in Figure A. The samples were collected for immunofluorescence and immunohistochemical staining 2 weeks after implantation of the hydrogels into the calvaria defect. As shown in Figure B, immunohistochemical staining was employed to assess TNF-α and IL-10 expression in the bone defect areas. The findings revealed a substantial presence of TNF-α-positive expression in the control group, concomitant with decreased IL-10-positive expression. In contrast, the GelMA-ZC and GelMA-ZC-Yoda1 groups exhibited notably raised IL-10 expression and reduced TNF-α expression (Figure D). These results suggested that the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels were able to effectively modulate the M2 macrophage polarization and suppress inflammatory reactions. Additionally, as analyzed by immunofluorescence results, more iNOS (M1 polarization marker, red) and fewer Arg-1 (M2 polarization marker, green) positive cells were found in the defect area of the control group (Figure C, F), suggesting an exacerbated inflammatory state with more M1 macrophage infiltration. Conversely, the GelMA-ZC and GelMA-ZC-Yoda1 groups showed the infiltration of more Arg-1-labeled M2 macrophages and fewer iNOS-labeled M1 macrophages in the bone defect regions. This further confirmed the robust immunomodulatory capacity of the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels to facilitate the transition of M1 to M2 macrophages, thereby alleviating the inflammatory response. Remarkably, the results of the in vitro studies were supported by the immunofluorescence and immunohistochemistry analyses, which showed no discernible variation in the expression levels of M1 or M2 macrophage markers between the GelMA-ZC and GelMA-ZC-Yoda1 groups. The above findings indicated that the incorporation of ZnCe-LDO nanozyme endowed the hydrogel with superior immunomodulation capability, whereas the addition of a small quantity of Yoda1 did not exert a noticeable effect on immunomodulation.

7.

In vivo immune responses. (A) The schematic diagram illustrates that the GelMA-ZC-Yoda1 hydrogel creates a favorable immune microenvironment within the defect area, facilitating the M1-M2 phenotypic transition, accompanied by pro-healing cytokines, significantly enhancing subsequent osteo/angiogenesis. (B) Representative immunohistochemical staining images of TNF-α, IL-10, BMP-2, and VEGF within the defect area 2 weeks post-treatment (Scale bars: 100 μm). (C) Representative immunofluorescence images (Scale bars: 100 μm) and localized magnified views (Scale bars: 20 μm) of iNOS and Arg-1 within the defect area 2 weeks post-treatment. (D, E) Semiquantitative analysis of immunohistochemical staining for TNF-α, IL-10, VEGF, and BMP2. (F) Semiquantitative analysis of immunofluorescence staining for iNOS and Arg-1. The data are shown as mean ± SD (n = 3). *p < 0.05, ***p < 0.001.

The immune microenvironment abundant in M2 macrophages regulates bone regeneration and repair processes by secreting various cytokines. Thus, an appropriate immune microenvironment is necessary for bone regeneration and repair. According to in vitro studies, the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels induced the M1 to M2 macrophage transition, subsequently resulting in the release of cytokines (BMP2 and VEGF) associated with osteo/angiogenesis. To substantiate the guiding effect of the hydrogel-mediated immunomodulation on osteo/angiogenesis in vivo, immunohistochemical staining for BMP2 and VEGF was carried out. Evidently, the GelMA-ZC and GelMA-ZC-Yoda1 groups had greater levels of BMP2 and VEGF expression in the bone defect areas than the control group (Figure B, E). These findings conclusively demonstrated that the GelMA-ZC and GelMA-ZC-Yoda1 hydrogels established an appropriate immune microenvironment by promoting the M1 to M2 macrophage transition in vivo. This contributes to the secretion of osteo/angiogenesis-related cytokines, which have a harmonious contribution to bone regeneration and repair.

GelMA-ZC-Yoda1 Hydrogel Promoted Bone Regeneration and Repair In Vivo

Eight weeks postimplantation, as depicted in Figure A, the biocompatibility of each hydrogel in vivo was assessed through routine blood examination, serum biochemical analysis, and histopathology of the major organs. The histopathological results of the major organs in all experimental groups, including the heart, liver, spleen, lungs, and kidneys, showed no notable damage or toxicity (Figure S6). Moreover, no significant alterations were observed in the routine blood examinations (Figure S7), and no damage to the liver or kidney function was found in the biochemical indicators (Figure S8). These findings indicated that the implanted hydrogels did not disrupt the biological functions of the major organs, establishing a crucial foundation for their potential biological applications. Simultaneously, three-dimensional images of the calvaria were reconstructed using micro-CT to further analyze the bone defect repair. The micro-CT data revealed the formation of new bone substantially increased in the GelMA-ZC-Yoda1 group, followed by the GelMA-ZC group, characterized by enhanced calcification and hard tissue formation in the calvaria defect region, which closely resembled the results of osteogenic differentiation in vitro. However, only a small amount of new bone was generated at the periphery of the bone defect area in the blank control group. The fact that a significant portion of the bone defect did not exhibit any indications of bone healing provides evidence that the critical-sized calvaria defect model was successfully established. The hydrogel-treated group guided the bone tissue to grow from the periphery of the defect area toward the center, as illustrated in Figure B. The GelMA-ZC-Yoda1 group showed more pronounced growth of new bone toward the center, and the new bone mostly covered the bone defect area at 8 weeks. Meanwhile, quantitative analysis of bone mineral density (BMD) and bone volume/total volume (BV/TV) in micro-CT images also confirmed these findings. Compared to the other groups, the BMD and BV/TV values increased in the GelMA-ZC and GelMA-ZC-Yoda1 groups, with the GelMA-ZC-Yoda1 group showing the highest values among all the groups (Figure C, D).

8.

In vivo bone regeneration in the rat calvaria defect model. (A) Schematic illustration depicting the implantation of hydrogels to evaluate the in vivo osteogenic potential. (B) Micro-CT images of calvaria defects from each group 8 weeks post-treatment. The red circles denote the defect region with a diameter of 5 mm. (C, D) Quantitative analysis of BV/TV (C) and BMD (D) within the defect regions. (E) H&E and Masson’s trichrome staining of the newly formed bone induced by the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels (Scale bar: 1 mm) with localized magnified views (Scale bar: 100 μm). FT: fibrous tissue. NB: newly formed bone tissue. Yellow, black, and red arrows indicate the newly formed central canal, bone lacunae, and blood vessels within the defect region, respectively. The data are shown as mean ± SD (n = 4). **p < 0.01, ***p < 0.001.

Histological analysis, including hematoxylin and eosin (H&E) and Masson’s trichrome staining, was performed on the bone defect areas. As illustrated in Figure E, more newly formed bone, collagen tissues, and neovascularization were shown in the defect regions of the GelMA-ZC and GelMA-ZC-Yoda1 groups than those in the other groups. These results indicated that the ZnCe-LDO-incorporated GelMA hydrogel induced in situ osteo/angiogenesis without the need for exogenous growth factors or stem cells. Importantly, the GelMA-ZC-Yoda1 group exhibited superior osteogenic effects across all groups, characterized by numerous bone lacunae, central canal, and neovascularization in the newly formed bone tissue.

Furthermore, 8 weeks postsurgery, immunofluorescence and immunohistochemical staining were conducted to evaluate osteo/angiogenesis in the bone defect areas, specifically targeting Runx2, OCN, and CD31. In Figure A-E, the substantial deposition of osteogenesis-related proteins (Runx2 and OCN) evident in the defect region of the GelMA-ZC-Yoda1 group, followed by the GelMA-ZC group, signifying the excellent osteogenic properties of the GelMA-ZC-Yoda1 hydrogel. Moreover, the GelMA-ZC-Yoda1 group exhibited significant angiogenesis at the defect site, as indicated by the increased expression of CD31, surpassing even the GelMA-ZC group. Nevertheless, the GelMA and control groups showed very little fluorescence expression of Runx2, OCN, and CD31. These results collectively confirmed the positive impact of the GelMA-ZC-Yoda1 hydrogel in promoting osteo/angiogenesis.

9.

Representative staining images of the rat calvaria after implantation. (A) Immunofluorescence images (Scale bar: 400 μm) and (B) immunohistochemical images of RUNX2, OCN, and CD31 (Scale bar: 50 μm). Red arrows indicate the newly formed blood vessels within the defect region. (C) Immunofluorescence images (Scale bar: 100 μm) and localized magnified views (Scale bar: 20 μm) of Piezo1 and YAP1. (D) Semiquantitative analysis of immunofluorescence staining and (E) immunohistochemical staining for RUNX2, OCN, and CD31. (F) Semiquantitative immunofluorescence analysis of Piezo1 and YAP1. The data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

In addition, we explored the significance of the Piezo1/YAP1 signaling pathway during osteo/angiogenesis in vivo facilitated by the GelMA-ZC-Yoda1 hydrogel. Piezo1 and YAP1 expression within newly formed bone in bone defects were considerably elevated by the GelMA-ZC-Yoda1 hydrogel, according to immunofluorescence staining, and a consistent trend in the expression and localization of Piezo1 and YAP1 was observed (Figure C, F). Therefore, in vivo results indicate that the GelMA-ZC-Yoda1 hydrogel activates Piezo1 to upregulate YAP1 expression, further inducing osteoblast differentiation and angiogenesis. The effect of GelMA-ZC-Yoda1 hydrogel on bone regeneration and repair in vivo is attributed not only to its capability to create an anti-inflammatory microenvironment but also to the synergistic effect of the released ions and Yoda1.

Currently, many studies on bone regeneration focus on improving bone regeneration through immune regulation. However, achieving an effective balance between inflammation and tissue repair remains a major challenge, thereby limiting clinical efficacy. Although the regulation of inflammation is essential, individual anti-inflammatory interventions are frequently insufficient to promote effective bone repair. Therefore, this study demonstrated an integrated approach by combining emerging fields such as immune regulation and mechanical signal activation, providing a multifaceted therapeutic strategy for bone regeneration and repair.

This study has demonstrated for the first time how ZnCe-LDO nanozyme (catalase-like activity) create an anti-inflammatory microenvironment conducive to bone regeneration by regulating immune responses, a critical factor for treating inflammatory bone defects. Furthermore, the activation of Piezo1/YAP1 signaling pathway promoted osteo/angiogenesis, which addresses a major challenge in bone regeneration, namely, how to effectively generate blood supply during the osteogenesis. In contrast to numerous studies concentrating on singular therapeutic method, this study adopted an integrated therapeutic strategy that mimics the natural bone regeneration process by combining multiple functions, a strategy scarcely found in existing literature. At present, it has not been reported in the literature that ZnCe-LDO nanozyme and Yoda1 are embedded together in the hydrogel matrix to form a comprehensive system integrating antioxidant properties, immune regulation and mechanical stimulation. This multifunctional hydrogel cooperatively enhanced osteogenesis and angiogenesis by the regulation of inflammatory responses, mechanical signal activation and release of bioactive ions, overcoming the limitations of monofunctional materials reported in previous studies.

Existing bone regeneration therapies such as stem cell therapy, while capable of supplying osteogenic seed cells for bone defect repair, often suffer from high costs associated with in vitro expansion, storage, and transplantation, along with potential immune rejection and ethical concerns. These factors greatly hinder their clinical translation. Similarly, growth factor-based strategies (e.g., BMP-2 or VEGF delivery), though effective in activating osteogenic or angiogenic signaling pathways, face challenges such as rapid degradation, short half-lives, and the need for frequent high-dose administration. These drawbacks not only increase the risk of side effects but also complicate the synchronization of release kinetics with the natural course of tissue regeneration, ultimately escalating therapeutic costs. Therefore, the development of a multifunctional therapeutic platform that integrates immunomodulatory, antioxidative, and controlled-release capacity is of critical importance. In this context, GelMA-ZC-Yoda1 emerges as a promising candidate, offering multidimensional regulatory advantages. To further improve the effect of bone regeneration, our future studies will explore synergistic strategies that combine this hydrogel with stem cell therapy or growth factors-based application. The immunoregulatory microenvironment established by the GelMA-ZC-Yoda1 hydrogel may lower the required dosage of transplanted stem cells, while its three-dimensional porous architecture facilitates cell adhesion and committed differentiation, potentially reducing immune rejection risks and lowering treatment costs. Furthermore, when codelivering growth factors such as BMP-2 or VEGF, the hydrogel’s controlled-release properties can finely regulate the release kinetics, reduce dosing frequency, and minimize adverse effects associated with high local concentrations, thereby enabling a dynamic match with the tissue repair cascade. In addition to its application in bone regeneration, GelMA-ZC-Yoda1 hydrogel shows great potential in other clinical fields, particularly in the treatment of chronic diseases such as diabetes-related wound healing and periodontitis. Diabetes patients often struggle with wound healing, especially chronic diabetes ulcers. GelMA-ZC-Yoda1 hydrogel is an ideal candidate for promoting wound healing in diabetes due to its multifunctional properties in immunomodulation, angiogenesis and anti-inflammatory. Similarly, periodontitis, as a prevalent chronic oral inflammatory disease, presents significant treatment challenges. GelMA-ZC-Yoda1 hydrogel can significantly improve the local immune environment, reduce inflammation, and create a favorable microenvironment for alveolar bone regeneration by regulating macrophages reprogramming, thereby effectively promoting the repair and regeneration of periodontal tissue. Beyond diabetes-related wound healing and periodontitis, GelMA-ZC-Yoda1 hydrogel holds considerable promise in treating other chronic inflammatory conditions, such as traumatic injuries and arthritis. In conclusion, GelMA-ZC-Yoda1 hydrogel offers an integrated therapeutic strategy for tissue regeneration repair and the treatment of inflammatory diseases, positioning it as a promising material for future clinical applications.

Study Limitations

Although this study has demonstrated the therapeutic potential of GelMA-ZC-Yoda1 hydrogel in promoting bone regeneration and angiogenesis, several limitations should be acknowledged. The current evaluation of ZnCe-LDO nanozyme release is based primarily on diffusion kinetics, and potential interactions with the biological microenvironment, such as protein binding, enzyme degradation, or other biochemical processes, will be further extensively studied in the future. The bone defect model used in this study may not completely replicate the complexity of clinical conditions, such as different defect sizes or potential disease states like osteoporosis. In the future, the efficacy of the hydrogel needs to be tested in more complex disease models, and the long-term biocompatibility and potential systemic effects of GelMA-ZC-Yoda1 hydrogel need to be evaluated, which is crucial for future transformation applications. Although we suggest that GelMA-ZC-Yoda1 hydrogel promote osteo/angiogenesis via activating the Piezo1/YAP1 signaling pathway, the exact mechanism of mechanotransduction in vivo has not been fully explored. Future research will delve into the molecular interactions driving Piezo1 activation and its impact on bone regeneration during various stages of healing. This could reveal deeper insights into the therapeutic potential of modulating Piezo1 signaling. Additionally, this study did not include a systematic dose–response analysis of the Piezo1 agonist Yoda1. The selected dosage was based on prior literature and our preliminary experimental data. Although this concentration showed promising effects, its therapeutic window, long-term biosafety, and potential toxicity require further comprehensive evaluation. Accordingly, future studies will focus on optimizing Yoda1 dosage by systematically evaluating its effects on bone regeneration and immune modulation across a range of concentrations. In parallel, pharmacokinetic studies will investigate the distribution, metabolism, and excretion pathways of the in vivo degradation products of the GelMA-ZC-Yoda1 hydrogel, aiming to establish a solid theoretical foundation for its safe and effective clinical application.

Conclusions

In this study, we incorporated a CAT-like nanozyme, ZnCe-LDO, and an activator of the mechanically sensitive Piezo1 ion channel, Yoda1, into a GelMA hydrogel and successfully prepared a multifunctional photoresponsive GelMA-ZC-Yoda1 hydrogel with outstanding biocompatibility. The hydrogel exhibited excellent injectability, enhanced mechanical properties, and an appropriate swelling capacity. In vitro experiments demonstrated that the ZnCe-LDO nanozyme integrated into the GelMA-ZC-Yoda1 hydrogel effectively inhibited the secretion of proinflammatory cytokines and increased the secretion of anti-inflammatory cytokines by suppressing the activation of MAPK/TNF signaling pathways to reprogram inflammatory macrophage, thereby establishing a conducive immune microenvironment for bone regeneration and repair, and the continuous release of bioactive ions also promoted osteo/angiogenesis. Furthermore, Yoda1 was continuously released from the hydrogel to activate the Piezo1 channel through the chemical simulation of the mechanical signal, thereby enhancing the harmonized coupling of osteogenesis and angiogenesis via the Piezo1/YAP1 signaling pathway. The findings in vivo indicated that GelMA-ZC-Yoda1 hydrogel established a suitable immune microenvironment and exhibited notable characteristics of osteo/angiogenesis, suggesting its potential utility in the clinical treatment of bone defects.

Experimental Section

Synthesis of ZnCe-LDO Nanozyme and Hydrogels

The ZnCe-LDO nanozyme was synthesized following a procedure previously reported with some modifications. , ZnCl₂, and CeCl₃ in a 1:1 molar ratio was dissolved in deoxidized deionized (DI) water as solution A, while NaOH was dissolved in DI water as solution B. After that, solution A was gradually added to solution B while continuously stirring. The resulting precipitate was rinsed three times and further subjected to heat treatment for 16 h at 100 °C. The products were washed with DI water to obtain the final ZnCe-LDO nanozyme.

To prepare the hydrogels incorporated with ZnCe-LDO, 0.05 g of photoinitiated LAP powder was initially dissolved in 20 mL PBS solution at 50 °C. Afterward, 0.1 g of lyophilized GelMA was thoroughly dissolved in 1 mL of LAP solution at 50 °C. Simultaneously, 0.2 mg of ZnCe-LDO was incorporated, and the mixture was homogenized through vortex mixing to achieve a homogeneous blend, resulting in the formation of an LDO-incorporated hydrogel denoted as GelMA-ZC. For the preparation of the GelMA-ZC-Yoda1 hydrogel, 0.4 × 10^–6 M Yoda1 was incorporated into the GelMA-ZC pregel solution, thoroughly mixed, and then exposed to ultraviolet light.

Macrophage Polarization

To evaluate intracellular scavenging activity of reactive oxygen species (ROS), macrophages cocultured with various hydrogels were stained with the fluorescent probe DCFH-DA. After 24 h of coculture, the cells were exposed to 100 ng/mL LPS to simulate a ROS-rich microenvironment. The cells without LPS treatment were served as controls. After another 24 h of incubation, the samples were incubated in serum-free DMEM containing DCFH-DA at 37 °C for 20 min. Subsequently, the cells were counterstained with Hoechst 33342 and observed using fluorescence microscopy. Additionally, the cells were collected for flow cytometry analysis, and fluorescence intensity was analyzed using Flow Jo software.

To assess the phenotypic transition of RAW264.7 cells, the cells were cocultured with various hydrogel samples at 37 °C and 5% CO₂ for 24 h, followed by treatment with LPS to simulate an inflammatory microenvironment. Among them, the cells cultured on the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels with LPS treatment were designated as the LPS, GelMA-ZC, and GelMA-ZC-Yoda1 groups, respectively, while RAW264.7 cells cultured on the GelMA hydrogel samples without LPS treatment served as controls. After a 2-day culture period, the culture medium from each well was collected and subjected to low-speed centrifugation. The concentrations of TNF-α and IL-10 in the cell supernatant were determined using ELISA assay kits.

The expression levels of inflammation-related genes in RAW264.7 cells cocultured with various hydrogels for 2 days were assessed through qRT-PCR, with the primer sequences detailed in Table S1. The expression levels of iNOS (M1-type) and Arg-1 (M2-type) were examined by Western blot and immunofluorescence staining. Meanwhile, the cells in all experimental groups were collected for immunophenotype analysis using flow cytometry to assess the surface expression of CD86 (M1 phenotype) and CD206 (M2 phenotype), as previously described.

RNA Sequencing

After coculturing with various hydrogels with/without LPS treatment, total RNA was extracted from the cells using Trizol reagent. RNA-sequencing analysis was performed at Beijing Novogene Co., Ltd.,China.

In Vitro Osteogenic Differentiation of rBMSCs

The osteogenic differentiation of rBMSCs was induced using α-MEM complete medium supplemented with 10 mM β-GP, 50 μg/mL ascorbic acid, and 10 nM dexamethasone. The GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 pregel solutions were polymerized under UV light. Following a 24 h coculture period of rBMSCs with various hydrogel samples, the medium was replaced with osteogenic induction medium and refreshed every 3 days. ALP and ARS staining assays were performed at specified time points.

For ALP staining, rBMSCs were fixed in 4% PFA and then stained using BCIP/NBT solution following the manufacturer’s instructions. ALP activity was quantitatively analyzed using the ALP assay kit. Additionally, ECM mineralization was determined by ARS assay. The cells were fixed, stained with 1% alizarin red solution, and washed with DI H₂O to remove excess dye. Calcium nodules were quantified by adding 10% cetylpyridinium chloride (CPC) for dissolution, followed by measuring absorbance at 542 nm.

After 7 days of incubation, the expression levels of osteogenesis-related genes, including Piezo1 and YAP1, were analyzed using qRT-PCR analysis as previous description, with the primer sequences detailed in Table S2. The protein expression levels of osteogenesis-related indicators, as well as Piezo1 and YAP1, in rBMSCs were assessed by Western blot analysis. Additionally, the expression levels of RUNX2 and OCN were evaluated using immunofluorescence staining.

In Vitro Angiogenic Ability

HUVECs were cultured on a 6-well plate until reaching approximately 90% confluence for scratch assays. Scratches were created at the bottom of the well using sterile pipet tips. The cells were incubated in serum-free medium containing extracts from the GelMA, GelMA-ZC, and GelMA-ZC-Yoda1 hydrogels. Images of HUVEC migration were randomly captured at 0 and 24 h. The formula was used to calculate migration rate of HUVECs: migration rate (%) = (A₀-A₂₄)/A₀×100%, where A₀ and A₂₄ represent the initial and remaining scratch areas after 24 h, respectively.

The hydrogels were solidified in the lower chamber of a 24-well transwell plate (8-μm pore filters, Corning, NY, USA), pretreated with complete medium. HUVECs were inoculated in the upper chamber and incubated for 12 h in serum-free medium. After removal of the cells adhering to the filter membrane, HUVECs on the lower surface were stained with 0.1% crystal violet to evaluate their migratory activity.

50 μL of Matrigel was evenly spread onto precooled 24-well plate and then allowed to solidify at 37 °C for 60 min. HUVECs were seeded onto the Matrigel and exposed to different extracts. Tube formation was observed and photographed using a microscope after 8 h of incubation. Following PBS washing, the cells were stained with calcein-AM for 15 min, and fluorescent images were captured under a fluorescent microscope.

The expression levels of angiogenesis-related genes (VEGF, eNOS, HIF-1α, and MMP9) were analyzed by qRT-PCR, with the primer sequences detailed in Table S3. Moreover, qRT-PCR and Western blot were used to analyze the mRNA and protein levels of Piezo1 and YAP1 in HUVECs cocultured with hydrogels.

Preparation of Conditioned Medium (CM)

The conditioned medium collected from macrophages cocultured with various hydrogels was employed to promote osteo/angiogenesis. The macrophages were cultured on various hydrogels. After 24 h of incubation, the cells were exposed to 100 ng/mL LPS for another 24 h. The supernatants from each group were collected and filtered under sterile conditions to remove gel and cell debris. The resulting supernatants were subsequently mixed with complete culture medium at a 1:2 ratio to prepare conditioned medium for subsequent cultivation of rBMSCs and HUVECs.

Construction of Bone Defect Model and Micro-CT Evaluation

All in vivo procedures were approved by the Animal Ethics Committee of Nanjing Medical University. A critical-sized calvaria bone defect model in Sprague–Dawley (SD) rats was used to evaluate the immunomodulatory and bone healing effects of the GelMA-ZC-Yoda1 hydrogel. Four-week-old male SD rats, purchased from the Experimental Animal Center of Nanjing Medical University, were randomly assigned to four groups (n = 6): (1) untreated bone defects (control group), (2) GelMA group, (3) GelMA-ZC group, and (4) GelMA-ZC-Yoda1 group. After a one-week acclimation period in a specific pathogen-free (SPF) environment, the rats were anesthetized, and all surgical procedures were performed under sterile conditions. Subsequently, a circular bone defect model with 5 mm diameter was carefully constructed bilaterally along the calvaria suture using a dental trephine drill, with particular attention paid to avoiding any damage to brain tissue. Next, pregel solutions of the respective hydrogels were injected into the defect areas and cross-linked under visible UV light. Wounds were then sutured with 4–0 nylon sutures. All rats received intramuscular antibiotic injections daily for three consecutive days. At the eighth week postimplantation, rats from each group were euthanized with pentobarbital sodium, and their calvaria bone samples were analyzed through micro-CT scanning with parameters set at 55 kV, 70 μA, and a resolution of 15.6 μm, followed by 3D reconstruction by CTVol (SkyScan). The bone formation parameters were analyzed with DataViewer and CTAn program (SkyScan).

Biocompatibility Assessment and Histological Analysis

Following micro-CT evaluation, the calvaria bone specimens underwent further decalcification in 10% EDTA solution and were subsequently encased in paraffin. The samples were then stained with H&E as well as Masson’s trichrome for histopathological examination. To delve into the intricacies of bone regeneration, immunohistochemical and immunofluorescence staining were employed to detect the expressions of Runx2, OCN, and CD31 at the eighth week. Furthermore, the expression levels of Piezo1 and YAP1 in the processes of osteogenesis and angiogenesis were assessed by immunofluorescence staining. To gauge the in vivo biocompatibility of the hydrogels, vital organs were harvested at 8 weeks post-treatment and submitted to H&E staining for detailed analysis, while blood samples were collected for routine examination and serum biochemical analysis to ensure the overall well-being and response of the organism to the treatment.

In Vivo Immune Responses

To explore the potential immunomodulatory effects of the GelMA-ZC-Yoda1 hydrogel in vivo, rats subjected to various hydrogel treatments were euthanized 2 weeks postimplantation, and their calvarias were collected and then fixed in 4% PFA for subsequent assessments. After decalcification in a 10% EDTA solution, the samples were subjected to immunofluorescence (iNOS and Arg-1) and immunohistochemical (BMP2 and VEGF) staining to elucidate the macrophages phenotypic polarization. Simultaneously, immunohistochemical staining for TNF-α was carried out, along with IL-10, to assess the early inflammatory status within the implanted defect region.

Statistical Analysis

All data were plotted and analyzed using GraphPad Prism 8.0 software, presented as the means ± standard deviations (SD). Statistical differences were analyzed by the student’s t test and one-way ANOVA. Results demonstrating a significance level of P < 0.05 were deemed statistically significant.

All data is available from the corresponding author upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.5c10123.

• Characterization of hydrogels; in vitro cell viability, ALP staining and ALP activity of hydrogels loaded with different amounts of ZnCe-LDO; cell isolation, culture and identification; in vitro biocompatibility of hydrogels; macrophage phenotypic transition of hydrogels; H&E staining of major organs; analysis of biochemical parameters and routine blood parameters; and primer sequences for qRT-PCR analysis of gene expression (PDF)

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Y.X. and M.X. contributed equally to this work.

Ya Xiao and Mengran Xu performed the experiments and wrote the original draft. Yijia Shi, Jing Wang and Zehan Li revised the manuscript. Tong Xiao, Haowen Yu and Na Lv analyzed the data. Wei Jiang, Yexiang Sun, and Delin Hu checked the data. Yi Hu and Jinhua Yu designed the experiments and revised the manuscript. All authors reviewed and approved the manuscript.

All experimental animal procedures were approved by the Animal Ethics Committee of Nanjing Medical University (Ethical number: IACUC-2108030).

The authors declare no competing financial interest.