Abstract Irregular alveolar bone defects pose persistent clinical challenges due to their complex morphology and the lack of biomaterials that simultaneously provide structural integrity, biocompatibility, and dynamic osteoinductive potential. Herein, we report a fiber-reinforced, dual-network hydrogel system (OHADN fiber@Yoda1 hydrogel) engineered to recapitulate mechanobiological cues for enhanced bone regeneration. This injectable hydrogel integrates oxidized hyaluronic acid (OHA) crosslinked with Yoda1-loaded PLGA-collagen fiber fragments and stabilized via catechol-Fe³⁺ coordination, forming a robust and self-healing structure. The fiber network enhances matrix stiffness and sustains Yoda1 release, promoting PIEZO1 activation in stem cells and enabling persistent mechanotransduction. In vitro, this system effectively regulates macrophage polarization, maintains cellular tension homeostasis, and significantly upregulates osteogenic markers via the PIEZO1–ITGα5 axis. Transcriptomic profiling and mechanistic validation revealed that focal adhesion and cytoskeletal signaling pathways are enriched upon hydrogel treatment. In a rat alveolar bone defect model, the OHADN fiber@Yoda1 hydrogel demonstrated superior bone volume restoration and trabecular architecture compared to conventional materials. This work presents a promising paradigm for spatiotemporal control of osteoimmune microenvironments through mechanoresponsive biomaterials. Graphical abstract [34]graphic file with name 12951_2025_3653_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12951-025-03653-y. Keywords: Fiber hydrogel, PIEZO1, Mechanotransduction, Tensional homeostasis, Bone repair Introduction Irregular alveolar bone defects from inflammation or trauma create significant restorative challenges [[35]1]. While autologous bone grafts remain the gold standard, donor site limitations and infection risks persist [[36]2]. Allogeneic alternatives face immune rejection concerns, driving demand for synthetic bone substitutes [[37]3]. Traditional bone substitute materials show good biocompatibility but suffer from slow degradation rates that mismatch bone regeneration timelines, ultimately compromising osteogenesis [[38]4, [39]5]. There is an urgent need to develop material systems that combine dynamic biological function with precise mechanical regulation to coordinate material degradation and osteogenic conversion to address the challenges posed by irregular bone defects. In recent years, the role of mechanical forces in bone development and remodeling has garnered increasing attention [[40]6, [41]7]. The physicochemical properties of biomaterials actively mediate mechanical stimulation, with matrix stiffness emerging as a critical factor influencing their osteogenic potential [[42]8, [43]9]. Biomaterial stiffness directly influences stem cell fate through cytoskeletal tension homeostasis mediated by mechanosensory protein [[44]10]. However, excessive tension may activate osteoclast signaling [[45]11], while insufficient matrix stiffness fails to sustain osteogenic spatial stability [[46]12]. This highlights the need to identify key balancing factors between functionalized osteogenesis and biomaterial matrix stiffness, with the mechanosensitive PIEZO1 channel emerging as a potential central hub for coordinating this mechanical equilibrium. The PIEZO1 channel plays a pivotal role in mechanical force transduction and skeletal development [[47]13, [48]14]. As a mechanically gated ion channel, PIEZO1 detects cell membrane tension changes, triggers Ca²⁺ influx, activates downstream signaling pathways (e.g., YAP/TAZ), and regulates stem cell fate [[49]15–[50]17]. Notably, its agonist Yoda1 enhances stem cell sensitivity to matrix stiffness [[51]18]. Our previous study demonstrated that Yoda1-induced PIEZO1 activation upregulated cytoskeleton-related gene expression and increased osteogenic protein production [[52]19], indicating PIEZO1’s essential role in maintaining cellular tension homeostasis and promoting bone regeneration. To actualize the mechanobiological strategy that modulates bone regeneration through PIEZO1-mediated regulation of cellular tensional homeostasis, we propose to engineer a biomaterial platform with spatiotemporal degradation profiles. Hyaluronic acid (HA), a fundamental component of the natural extracellular matrix, is widely used in injectable hydrogels for filling irregular bone defects [[53]20]. Its unique linear macromolecular structure ensures hydration and elasticity within the scaffold, providing excellent spatial adaptability. However, its low matrix stiffness often results in insufficient space maintenance, limiting its clinical translation [[54]21, [55]22]. Strategies such as enhanced chemical crosslinking and the incorporation of stiffer elements have been employed to address the insufficient stiffness of hydrogels [[56]2, [57]23]. Previous experimental results have shown that oriented fibers can achieve tensile strengths of up to 16 MPa, giving fiber-reinforced hydrogels a distinct advantage in modulating and enhancing matrix stiffness [[58]24, [59]25]. Additionally, Yoda1 lowers the threshold for cellular mechanical stimulation [[60]17], enabling effective osteogenesis even under lower stiffness conditions. So, in this study, fiber fragments with active functional groups (amine group) were used as crosslinking points. These fragments were crosslinked with the main chain—oxidized hyaluronic acid (OHA)—via an aldehyde-amine condensation reaction to form a typical hydrogel network structure. The Yoda1 was loaded in the fibers to exert a sustained release effect and avoid excessive drug release [[61]26]. The uniformly dispersed fiber fragments serve as a sustained source of high cellular traction forces, maintaining elevated osteogenic responses through tensional homeostasis feedback mechanisms, even after the faster-degrading hydrogel components have dissolved [[62]27]. Therefore, to address the clinical challenge of mandibular defects, this study aims to further enhance the stability of the hydrogel structure by introducing catechol groups that interact with iron ions to form a coordination network. This coordination network works in synergy with the covalent network formed by OHA and fiber fragments (loaded with Yoda1) to construct a dual-network hydrogel system (OHADN). This system creates a mechanotransductive microenvironment for stem cells, thereby achieving a balance between material degradation and osteogenic transformation. The fibrous reinforcement within this structure effectively maintains traction forces and controls the delivery of Yoda1, enhancing PIEZO1-mediated mechanosensing even at lower stiffness thresholds. Importantly, to elucidate the mechanobiological regulatory paradigm, we focused on the PIEZO1-integrin α5 (ITGα5) mechanosignaling hub, specifically analyzing how Yoda1-loaded fibrous reinforcement coordinates transcriptional activation and osteogenic differentiation through cytoskeletal tension homeostasis. These findings provide an important basis for designing bone substitutes through spatiotemporal mechanosignal regulation. Materials and methods Materials Poly(Lactic-co-Glycolic Acid) (PLGA, LA/GA = 50:50, Mw = 105 kDa) was procured from Jinan Daigang Biomaterial Co. Ltd. (Shandong, China). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was supplied by Aladdin Co. Ltd. (Shanghai, China). Collagen type I (C8060) was procured from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Yoda1 was purchased from Sigma–Aldrich (USA). HA (molecular weight 100 kDa − 150 kDa), sodium periodate (NaIO[4]), ethylene glycol, Ethyldimethylaminopropyl carbodiimide (EDC), N-hydroxysuccinimide (NHS), dopamine hydrochloride and iron(III) chloride were procured from Shanghai Macklin Biochemical Technology Co., Ltd. Fabrication of the OHADN fiber@Yoda1 hydrogel The fabrication of the Yoda1-loaded fiber fragments: Yoda1-loaded aligned fibers were fabricated by electrospinning technique as described in the previous study [[63]19]. 1.6 g PLGA was dissolved in 10 ml of HFIP. Then 15% (w/v) collagen and 4 µmol L^−1 of Yoda1 were added to the PLGA solution. Stir until fully dissolved. Aligned fibrous membrane was prepared by the electrospinning technique. A rotating cylinder was used to collect fibers to obtain aligned fibers. The flow rate of the solution was 100 µL min^−1. A voltage of 12 kV and 15 cm was applied between the needle (23 G) and collector. The rotation speed was 3000 rpm when a rotating cylinder was used. The membrane was vacuum-dried overnight to eliminate residual solvent. Aligned fibers were then cut into nanofiber fragments using a precision cryoslicer. Synthesis of OHA: The synthesis of OHA was based on previously reported methods [[64]28, [65]29] with modifications for aldehyde modification of HA. Briefly, HA (1.0 g) and sodium periodate (0.86 g) were separately dissolved in 100 mL and 5 mL of deionized water, respectively. Subsequently, the sodium periodate solution was added to the HA solution, and the mixture was stirred in the dark at room temperature for 6 h. Ethylene glycol (1 mL) was then added to the reaction solution to neutralize the excess sodium periodate. After an additional 1-hour reaction, the mixture was dialyzed against ultrapure water for 2 days and subsequently lyophilized to obtain OHA. Preparation of OHADN fiber@Yoda1 Hydrogel: To form the OHADN fiber@Yoda1 hydrogel, OHA was first dissolved in deionized water and activated with EDC and NHS. Dopamine hydrochloride was then added to introduce catechol groups onto the OHA backbone through an amidation reaction. The modified OHA was dissolved in deionized water to form solution (A) Meanwhile, ferric chloride and Yoda1-loaded fiber fragments were mixed in deionized water to form solution (B) Finally, solutions A and B were mixed and injected together, where imine bonds and iron ion coordination bonds were formed to create a dual-network hydrogel structure. Characterization The morphology of the aligned fibrous membrane and hydrogel was observed by scanning electron microscope (SEM, Zeiss Sigma 300, Germany). Energy dispersive spectroscopy (EDS) was performed using a Bruker Instruments EDS detector (Bruker, Quantax XFlash SDD 6, Germany). To determine the OHADN composition, Fourier-transform infrared spectroscopy (FTIR, Bruker, TENSOR27, Germany) was used to detect chemical functional groups. The scanning wavenumber range was 600–4000 cm^−1 at a resolution of 1 cm^−1. The hydrogels were injected to write “SYSU” to evaluate injectability. To evaluate the adhesiveness, the Yoda1-loaded fiber-reinforced hydrogel was injected into a piece of porcine skin, and then the other piece adhered to it. After ten minutes of stabilization at 37 ℃, the adhesiveness was assessed by observing the stability of the position against gravity. The self-healing ability was carried out by cutting one hydrogel into two pieces and reconnecting them. The tensile stress of the reconnected hydrogels was measured after 15 min. The rheological properties of hydrogels were evaluated on a rotary rheometer (Physica MCR301, Anton Paar). The hydrogels (Φ25 mm × 2 mm) were placed on a sample stage and tested with a 25 mm diameter flatbed device. The storage modulus (G’) and loss modulus (G”) were obtained from the frequency–modulus curves between 10 rad s^−1 and 100 rad s^−1 at a 5.0% strain amplitude. The viscosity and shear rate of the hydrogels were detected between 0.1 S^−1 and 100 S^−1 to assess the shear-thinning behavior of the hydrogels. For the degradability study, hydrogels were immersed in 5 mL of Hank’s balanced salt solution (HBSS, pH = 7.4) and put in a shaking incubator at 100 rpm and 37 ℃. The incubation medium was changed every week. At each predetermined time point, the samples were freeze-dried to a constant weight. The degradation rate was calculated using the following formula: graphic file with name d33e370.gif where M[0] is the mass of the hydrogels before incubation, and M is the mass of the hydrogels after incubation for different times. For release assay, hydrogels were immersed in 10 mL phosphate buffered saline (PBS) in 50 mL centrifugal tubes, which were placed in a cell incubator at 37 ℃. At specified time points (1, 2, 4, 6, 8, 10, 12, 16 and 18 days), releasing buffer was collected from the centrifugal tubes, stored at 4 ℃ before detection, and replaced with 10 mL fresh PBS. The concentration of Yoda1 in the release buffer was measured using high-performance liquid chromatography (HPLC; Agilent 1260, USA). Cell culture RAW264.7 (Mϕ, a murine-derived macrophage cell line, the Institute of Biochemistry and Cell Biology, Shanghai, China) and mouse bone marrow mesenchymal stem cells (mBMSCs, Cyagen Co., USA) were cultured in high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Gibco). The culture medium was replaced every alternate day for all cell culture cycles. In vitro evaluation of intracellular calcium ion and inflammatory factors The effect of hydrogel on the osteogenic microenvironment was assessed by intracellular calcium ion levels, inflammatory cytokine genes and secretion levels of RAW264.7. After incubating RAW 264.7 for 24 h, cells were washed once with PBS, adding 100 µl of fluo-4 staining solution prepared according to the instructions (Beyotime, Shanghai, China), and incubated with 37 ℃ for 30 min. The images were obtained using a confocal laser scanning microscope (CLSM; Olympus, Japan) and the relative fluorescence values was detected with a multi-functional microplate reader (BioTek Synergy H1, Agilent Technologies Inc., USA). The mRNA expression of M1 macrophage markers (interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-ɑ)) and M2 macrophage markers (arginase-1 (ARG1) and interleukin-10 (IL-10)) was measured by RT-qPCR. Briefly, total RNA was extracted using an RNA-Quick Purification Kit (ES science, Shanghai, China). Total RNA was reverse-transcribed into complementary DNA (cDNA) using a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). RT-PCR was performed using a QuantStudio 5 Instrument (ABI). The relative expression levels of the target genes were normalized based on the expression levels of the reference gene GAPDH. The primer sequences used for the RT-qPCR are listed in Table [66]S1 (Supplementary information). Immunofluorescence staining assays for inducible nitric oxide synthase (iNOS) and ARG1 were conducted to evaluate the immune reaction of hydrogel. Briefly, after incubating RAW 264.7 for 24 h, cells were fixed with 4% paraformaldehyde, treated with 0.1% Triton for 20 min, and sealed with 5% Bovine Serum Albumin (BSA) for 1 h. The primary antibodies anti-iNOS (1:100, Abcam) and anti-ARG1 (1:100, Affinity Biosciences), and the fluorescent secondary antibody (1:500, Absin, China) were incubated according to the manufacturer’s instructions, and cell nuclei were stained with DAPI. Fluorescence intensity was visualized by CLSM and analyzed using Image J software (V2.1.4.7, National Institutes of Health, USA). Inflammatory factors concentrations in culture medium supernatant were quantified by mouse inflammation antibody arrays C1 (C-series, Ray Biotech, USA) according to the manufacturer’s instructions. The inflammation array was processed and quantified by using the Ultra-high-sensitivity chemiluminescence imaging system (ChemiDoc, Bio-Rad Laboratories, USA) and membrane array extract software (Ray Biotech, USA). The positive controls on each slide were used to normalize the intensity of the individual spots being compared on different slides. The complete list of inflammation in the arrays is summarized in supplemental Table S2 (Supplementary information). In vitro evaluation of cellular biocompatibility To estimate the in vitro cellular biocompatibility and corresponding biological functions of the hydrogels, the proliferation of mBMSCs on the hydrogels was assessed using a CCK-8 assay. Briefly, the optical density (OD) value of cells cultured for 1, 3, and 5 days was measured using a full-wavelength microplate reader (BioTek, USA) at 450 nm. For cell morphology observation, F-actin and nuclei were stained with actin-tracker Red and DAPI, respectively. Fluorescence images were obtained using a CLSM. In vitro evaluation of mBMSCs differentiation The activation of PIEZO1 channels was assessed by measuring the intracellular calcium level of BMSCs at the same step as above mentioned. The osteogenic ability of hydrogels was assessed by the expression of osteogenic genes and proteins. Briefly, cells were cultured in mineralizing medium (complete medium supplemented with 0.1 × 10^−6 M dexamethasone, 50 µg mL^−1 ascorbic acid, and 10 × 10^−3 M β-glycerophosphate) for 7 days. The expression of bone-associated genes, including osterix (OSX), runt-related transcription factor 2 (Runx2), osteopontin (OPN), and type I collagen (Col1), was evaluated. Primer sequences were determined using established GenBank sequences and are shown in Table [67]S1 (Supplementary information). Additionally, western blot analyses of alkaline phosphatase (ALP), RUNX2, OSX, COL1 and osteocalcin (OCN) were performed after osteogenic induction for 7 days. The proteins were probed with 1:1000 rabbit anti-ALP (Affinity Biosciences), 1:1000 rabbit anti-RUNX2 (Affinity Biosciences), 1:1000 rabbit anti-OSX antibodies (Affinity Biosciences),1:1000 rabbit anti-COL1 antibodies (Affinity Biosciences), 1:1000 rabbit anti-OCN antibodies (Affinity Biosciences), 1:1000 rabbit anti-GAPDH antibodies (Affinity Biosciences) and 1:2000 anti-rabbit/mouse HRP-conjugated IgG (CST). The antigen-antibody reaction was visualized using an enhanced chemiluminescence assay. Furthermore, IF staining for RUNX2 was performed on cells after 7 days of osteogenic induction, following the general IF protocol as previously described. Briefly, cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton, and blocked with 5% BSA. Cells were then incubated overnight at 4 °C with rabbit anti-RUNX2 primary antibody (Affinity Biosciences) diluted at 1:100 in blocking buffer. After washing, cells were incubated with an appropriate Alexa Fluor-conjugated secondary antibody (1:500, Absin, China) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI. Fluorescence images were captured by CLSM. Transcriptome sequencing There are two groups: Control and OHADN fiber@Yoda1 hydrogel. RNA sample quality was analysed, and the cDNA libraries were synthesized and sequenced using BGI technology. In brief, total RNA was extracted using RNeasy Micro Kit(QIAGEN, Germany)according to manual instruction and quantified using Agilent 2100 Bioanalyzer (Agilent, CA, USA) for concentration, purity, and integrity. Library preparation is performed using Optimal Dual-mode mRNA Library Prep Kit (BGI-Shenzhen, China). The sequencing data was filtered with SOAPnuke. The subsequent analysis and data mining were performed on Dr. Tom Multi-omics Data mining system ([68]https://biosys.bgi.com). Expression level of gene was calculated by RSEM (v1.3.1). The heatmap was drawn by pheatmap (v1.0.12) according to the gene expression difference in different samples. Essentially, differential expression analysis was performed using the DESeq2 (v1.34.0) with P < 0.05 and |log2 FC (fold change) ≥ 1. To take insight to the change of phenotype, GO and KEGG enrichment analysis of annotated different expression gene was performed by Phyper based on Hypergeometric test. The significant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value ≤ 0.05). In vitro evaluation of signaling pathways The signaling pathway differential genes identified in the previous step were verified by RT-qPCR and western blot. Briefly, cells were cultured in mineralizing medium for 7 days. The expression of differential genes was evaluated. Primer sequences were determined using established GenBank sequences and are shown in Table [69]S1 (Supplementary information). Additionally, western blot analyses were performed after osteogenic induction for 7 days. The antigen-antibody reaction was visualized using an enhanced chemiluminescence assay. In vivo bone regeneration assays in the rat alveolar bone defect model This study was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (approval no. SYSU-IACUC-2024-002595). Male Sprague - Dawley rats (SD, average weight of 300–350 g) were purchased from the Laboratory Animal Center of Sun Yat-sen University. SD rats were anesthetized with an intraperitoneal injection of 3% pentobarbital sodium. A gingival sulcus incision was made around the maxillary first molar using a micro-surgical blade. Bilateral maxillary molars were then removed using a sharpened dental explorer. A 2-mm-diameter implant drill was used to expand the extraction sockets to a standardized depth of 2 mm. The expansion of the defect was performed to remove periodontal support tissue that may facilitate bone regeneration without treatment. The rats were randomly divided into five groups (Control, OHADN hydrogel, OHADN fiber hydrogel, OHADN fiber@Yoda1 hydrogel, Hydroxyapatite). The materials (n = 4) were placed into each defect, and the blank defect control was untreated. After suturing of gingival tissue, penicillin was injected once a day postoperatively for 3 days. Rats were sacrificed at 1 month postoperatively, and the alveolar bone tissues with the hydrogels were harvested. The samples were stored in 4% paraformaldehyde for further analysis. Micro-CT was first used to evaluate osteogenesis on different materials. Samples were analyzed using a micro-CT scanner (Scanco Medical, µCT 50, Switzerland) at 70 kV, 114 µA, and 10 μm. The three-dimensional (3D) structures of the alveolar bone, bone volume over total volume (BV/TV%), bone trabecular thickness (Tb.Th) and bone trabecular separation (Tb.Sp) were evaluated using the µCT evaluation program V6.6. After alcohol-gradient dehydration and paraffin-embedded sections, the specimens were cut into 5 μm histological sections along the center of the alveolar bone defect and stained with hematoxylin-eosin (H&E) or Masson’s trichrome stain to evaluate the regeneration condition. To further analyze osteogenesis on different membranes, immunohistochemical staining was performed on the specimens harvested at 1 month with antibodies specific for RUNX2 (Affinity, 1:100 dilution, AF5186), OPN (Affinity, 1:100 dilution, AF0227), PIEZO1 (Affinity, 1:100 dilution, DF12083), ITGα5 (Proteintech, 1:50 dilution, 10569-1-AP), tensin 1 (TNS1, Proteintech, 1:100 dilution, 20054-1-AP). The sections were then scanned using a ScanScope XT tissue slide scanner (Aperio, Leica Biosystems, Buffalo Grove, IL, USA) for immunohistochemical examination. To investigate the spatial interaction between PIEZO1 and ITGα5, sequential immunohistochemical co-staining was performed using the Vari Fluor multiplex kit (MedChemExpress, USA) with rigorous microwave-mediated antibody stripping. The procedure was conducted as follows: After antigen retrieval in citrate buffer (pH 6.0) and endogenous peroxidase blocking with 3% H₂O₂, sections were first incubated with rabbit anti-ITGα5 (1:50) for 1 h at room temperature, followed by HRP-conjugated goat anti-rabbit secondary antibody (10 min) and VF620 TSA working solution (1:200 in TSA buffer, 10 min in dark). After thorough washing, microwave stripping (citrate buffer, 95 °C for 10 min) was applied to completely remove bound antibodies. The sections were then re-blocked and sequentially incubated with rabbit anti-PIEZO1 (1:100), HRP-conjugated goat anti-rabbit secondary antibody, and VF488 TSA working solution (1:200, 10 min in dark). Nuclei were counterstained with DAPI before mounting. Fluorescence images were acquired using a CLSM at: VF570 (Ex/Em 550/570 nm) and VF620 (Ex/Em 590/620 nm) channels. Co-localization analysis was performed using Image J. Statistical analysis Statistical analysis was conducted using GraphPad Prism 8 (GraphPad Software). All experiments were conducted with a minimum of three replicates for each data point, and the data are expressed as the mean ± standard error of the mean. Differences between two groups were compared using the Student’s t-test. After the normal distribution test, multiple comparisons were performed using one-way analysis of variance (ANOVA), followed by a least significant difference (LSD) test. Statistical significance was set at P < 0.05. Results and discussion Synthesis and characterization of the OHADN fiber@Yoda1 hydrogel The OHADN fiber@Yoda1 hydrogel was fabricated using a dual-crosslinking mechanism involving catechol-Fe³⁺ coordination and imine covalent bonds, as shown in Fig. [70]1a. The oriented fibers loaded with Yoda1 were prepared using the well-established electrospinning technique [[71]19]. SEM analysis confirmed the uniform alignment of the fibers, as shown in Fig. [72]1b. This unidirectional orientation facilitates the precise cutting of the fibers into fragments. By controlling the thickness of the cryosectioning, we obtained fiber fragments of consistent size. The optimized segment length was approximately 20–50 μm, ensuring their uniform distribution within the hydrogel matrix without compromising injectability. As illustrated in the inset of Fig. [73]1c, a homogeneous translucent emulsion containing the fiber fragments was obtained. Considering that the tensile strength of the oriented fibers can reach up to 16 MPa, these fragments hold great potential for enhancing the mechanical properties of injectable hydrogels while maintaining their processability. Fig. 1. [74]Fig. 1 [75]Open in a new tab Synthesis and characterization of the OHADN fiber@Yoda1 hydrogel. (a) Schematic representation for fabricating the OHADN fiber@Yoda1 hydrogel. (b) SEM image of aligned fiber fabricating by electrospinning technique. (c) The image of fiber fragments, which the insets was PBS containing fiber fragments. (d) Vial inversion tests. (e) SEM image of OHADN fiber@Yoda1 hydrogel. The white arrows denote the fiber fragments (insets). (f and g) EDS elemental analysis of OHADN fiber@Yoda1 hydrogel. (h) FTIR spectra. (i) The storage modulus (G’) and loss modulus (G’’). (j) The viscosity-shear rate curves. (k) Degradation experiment. (l) The Yoda1 release curve In biomaterial science, the native extracellular matrix component HA has been strategically repurposed as a multifunctional hydrogel precursor, particularly advantageous for minimally invasive delivery through injectable formulations due to its inherent shear-thinning behavior [[76]30]. After oxidation, HA can be functionalized with aldehyde groups on its molecular chains, thereby offering enhanced possibilities for the construction of its hydrogels. OHA-based hydrogels have gained significant attention as filling materials for irregular bone defects due to their exceptional spatial adaptability [[77]21, [78]22]. The distinctive linear macromolecular architecture of OHA contributes to hydrogel moisture retention and elastic properties. In this study, hydrogel formation was confirmed through vial inversion tests, with representative results shown in Fig. [79]1d. The microstructure and elemental composition of the hydrogel were characterized using SEM and EDS. As shown in Fig. [80]1e, the OHADN fiber@Yoda1 hydrogel displayed a well-defined porous structure with a relatively uniform pore size distribution. This architectural feature is particularly beneficial for facilitating cell infiltration and nutrient transport, both of which are critical requirements for successful tissue engineering applications. Especially, interconnected fiber fragments were observed bridging the hydrogel walls, which is particularly significant for maintaining structural integrity during degradation. These residual fibers play a crucial role in preserving mechanotransduction signaling by sustaining cellular traction forces, thereby creating a favorable mechanical microenvironment that supports extended osteogenic differentiation processes. The EDS elemental mapping analysis (Fig. [81]1f and g) revealed that the OHADN fiber@Yoda1 hydrogel primarily consisted of carbon (C), nitrogen (N), oxygen (O), chlorine (Cl), and iron (Fe). The presence of these elements is consistent with the expected composition of the hydrogel system and suggests successful incorporation of the desired components. OHA was synthesized through NaIO[4]-mediated oxidation of the proximal hydroxyl (-OH) group in HA. FTIR analysis (Fig. [82]1h) confirmed the aldehyde group formation in OHA, evidenced by a characteristic peak at 1736 cm⁻¹ corresponding to the -CHO group. Comparative analysis revealed enhanced OHADN peaks at 1530 cm⁻¹ (C––N stretching), 1450 cm⁻¹ (aromatic C–C stretching), and 1280 cm⁻¹ (C–N stretching) in the arrow-marked regions. These observations indicate successful dopamine grafting onto HA via Schiff base formation between the aromatic amine (-NH[2]) and OHA’s aldehyde group, consistent with reported mechanisms [[83]29]. The matrix stiffness of hydrogels is a critical factor influencing cytoskeleton organization, cell migration, and differentiation [[84]31, [85]32]. To investigate whether the incorporation of cross-linked fiber fragments could enhance the mechanical properties of OHADN hydrogels, we conducted rheological experiments to evaluate the storage modulus (G’) and loss modulus (G”). It is well-established that a higher G’ value compared to G” indicates the successful formation of a stable hydrogel network [[86]29]. As shown in Fig. [87]1i, both the OHADN hydrogels and the OHADN fiber hydrogels exhibited G’ values that were consistently higher than their corresponding G” values across the frequency range of 10–100 rad s^−1, confirming the formation of stable hydrogel networks. Furthermore, the G’ values of the OHADN fiber hydrogels were significantly higher than those of the OHADN hydrogels, demonstrating that the incorporation of cross-linked fiber fragments effectively enhanced the mechanical strength of the hydrogel. The elevated G’ values in the OHADN fiber hydrogels indicate greater resistance to deformation under applied stress, a property that is essential for maintaining structural integrity in dynamic physiological environments. This improved mechanical robustness not only ensures the hydrogel’s stability during cellular activities but also better replicates the mechanical properties of native tissues, which is critical for supporting cell function and bone regeneration [[88]7, [89]33]. The adhesive properties of implanted materials are crucial for ensuring the retention of filling materials in irregular jaw defects. To evaluate the injectability and adhesion of OHADN fiber hydrogels, we analyzed their viscosity-shear rate curves using a rheometer. As shown in Fig. [90]1j, the viscosity of the OHADN fiber hydrogels decreased with increasing shear rate, demonstrating their shear-thinning behavior and confirming their injectability. This property allows the hydrogel to be easily extruded through a syringe while maintaining its structural integrity, as evidenced by its ability to remain in a gel state post-injection (Fig. [91]S1a). Furthermore, Fig. [92]S1b illustrates that the OHADN fiber hydrogel exhibited strong tissue adhesion, a critical feature for effective defect filling and retention. It is worth mentioning that the adhesiveness of the OHADN fiber hydrogel can be attributed to its unique composition and crosslinking mechanism. The OHA molecular chains, rich in aldehyde and catechol groups, in combination with the coordination of Fe³⁺ ions and the covalent action of imine bonds, not only form a stable hydrogel network structure but also endow it with excellent interfacial adhesive properties. This makes the hydrogel system an ideal material for repairing irregular mandibular defects. When the hydrogel is injected to fill irregular bone defects, self-healing ability is essential to ensure the formation of a continuous barrier that protects against microbial invasion [[93]34, [94]35]. As demonstrated in Fig. [95]S1c, the OHADN fiber hydrogel exhibited remarkable macroscopic self-healing properties. When cut, the hydrogel rapidly healed without external intervention, and the healed hydrogel showed no visible cracks and could be lifted intact. This is the result of the combined action of dual dynamic chemical bonds. The self-healing capability not only ensures structural integrity but also enhances the hydrogel’s ability to maintain a protective barrier in dynamic physiological environments, making it highly suitable for applications in bone defect regeneration [[96]36]. To validate the crosslinking effect of fiber fragments in injectable hydrogels, we systematically evaluated the degradation kinetics of OHADN fiber hydrogel. As depicted in Fig. [97]1k, OHADN fiber hydrogel and OHADN hydrogel exhibited comparable degradation rates during the initial 3-day period. However, a distinct divergence emerged from day 5 onward, with the OHADN fiber hydrogel demonstrating a slower degradation profile. By day 14, quantitative analysis revealed a 16% reduction in degradation rate of the OHADN fiber hydrogel compared to OHADN hydrogel (p < 0.05). The result indicates that the covalent cross-linking network formed by the fiber fragments within the hydrogel system effectively retards the degradation kinetics of the hydrogel through a double-network reinforcement structure. Moreover, this observation aligns with established findings that cell-mediated hydrogel degradation facilitates cellular spreading and generates mechanical tension conducive to osteogenesis [[98]7]. Following partial hydrogel degradation, the persistence of slower-degrading fiber fragments establishes anchorage platforms supporting cell migration, microenvironments enhancing cellular proliferation and topographic guidance for collagen matrix organization [[99]26]. Enzymatic degradation experiment was conducted to analyze the release profile as shown in Fig. [100]1l. During hydrogel degradation (1–12 days), Yoda1 exhibited a release rate slope of 5.66%, which decreased to 0.53% post-hydrogel degradation, indicating that the drug-loaded fibrous fragments, when incorporated into the hydrogel matrix, exhibit an excellent sustained-release effect. Previous studies demonstrate electrospun fiber degradation durations up to 60 days [[101]19]. The hydrogel phase initially promotes stem cell migration and spreading through rapid stress relaxation, while the Yoda1-loaded fiber network subsequently sustains mechanical tension and stabilizes PIEZO1 channels via sustained Yoda1 release to enhance osteogenic differentiation. Research confirms Yoda1, a low-molecular-weight compound, selectively activates PIEZO1 by stabilizing its open conformation and reducing mechanical activation thresholds [[102]37]. Single-channel kinetic analysis reveals Yoda1-induced conformational changes: long-closed state occupancy decreased from 89.6 to 74.6%, long-open state increased from 7.3 to 22.5%, with slowed inactivation kinetics and accelerated recovery rates, ultimately achieving 2–3 times higher open probability [[103]18]. These physicochemical properties collectively emphasize the imperative for systematic cellular investigations to validate the therapeutic potential of this hydrogel scaffold system in guiding stem cell fate. Effect of OHADN fiber@Yoda1 hydrogel on immune microenvironment Macrophages, as versatile cellular components of the innate immune system, possess the remarkable capacity to dynamically respond to microenvironmental alterations and undergo polarization into diverse functional phenotypes, playing a pivotal role in tissue repair processes [[104]38]. Mechanical stimuli, particularly matrix stiffness, have been demonstrated to promote the pro-inflammatory activation of macrophages, thereby facilitating the secretion of wound-healing cytokines that contribute to tissue regeneration [[105]39]. However, excessive matrix stiffness may trigger severe foreign body responses and lead to fibrous capsule formation [[106]40]. Consequently, it is imperative to systematically evaluate the inflammatory responses elicited by biomaterials at the cellular level. Emerging evidence has demonstrated that PIEZO1 channel-mediated calcium influx plays a regulatory role in macrophage inflammatory responses and healing processes [[107]40]. To investigate this mechanotransduction pathway, we employed calcium-sensitive fluorescent probes to quantify intracellular calcium levels in macrophages cultured on OHADN fiber@Yoda1 hydrogel group. As illustrated in Fig. [108]2a and b, both immunofluorescence imaging and microplate reader analyses consistently revealed a moderate elevation in intracellular calcium concentration in macrophages cultured on OHADN fiber@Yoda1 hydrogel compared to control group. Fig. 2. [109]Fig. 2 [110]Open in a new tab Effect of OHADN fiber@Yoda1 hydrogel on immune microenviroment. (a) Immunofluorescence imaging of intracellular calcium ion staining. (b) Relative fluorescence value of intracellular calcium ion detecting by microplate reader. (c) Quantitative RT-PCR analysis of inflammatory cytokine. (d) Immunofluorescence and semi-quantitative analysis, red for iNOS, green for ARG1, and blue for nuclei. (e) Inflammatory factors concentrations in culture medium supernatant. (f) The schematic diagram of hybrid macrophage phenotype promoting by OHADN fiber@Yoda1 hydrogel. (*P<0.05, **P<0.01, ***P<0.001, ns: not significant, by Student’s t-test) To further characterize the macrophage polarization state, we performed comprehensive analysis of inflammatory cytokine profiles at both transcriptional and secretory levels. RT-qPCR analysis (Fig. [111]2c) demonstrated statistically significant upregulation of anti-inflammatory genes ARG1 (P < 0.05) and IL-10 (P < 0.01) in the OHADN fiber@Yoda1 hydrogel group, while maintaining comparable levels of pro-inflammatory genes IL-6 and TNF-α relative to control. Immunofluorescence results (Fig. [112]2d) revealed higher intensity of both ARG1 and iNOS in the OHADN fiber@Yoda1 hydrogel group compared to the control group; however, the iNOS/ARG1 ratio was significantly lower, indicating that the hydrogel induces a reparative microenvironment. Additionally, parallel assessment of secreted cytokines revealed a distinct pattern: substantial enhancement of Interleukin-4 (IL-4) and IL-10 production accompanied by significant suppression of multiple pro-inflammatory mediators, particularly Interleukin-1 (IL-1) and IL-6, though with a modest increase in Granulocyte-macrophage Colony-stimulating Factor (GM-CSF) and Interleukin-9 (IL-9) levels (Fig. [113]2e). These results suggest that OHADN fiber@Yoda1 hydrogel promotes a hybrid macrophage phenotype that combines regenerative anti-inflammatory properties with essential immune surveillance functions, potentially creating a balanced microenvironment conducive to tissue repair while maintaining necessary immune responsiveness, as shown in the schematic diagram in Fig. [114]2f. These experimental observations align with the mechanistic insights provided by Wang et al., whose work elucidated that PIEZO1-mediated mechanotransduction not only enhances macrophage phagocytic efficiency and apoptotic cell clearance capacity but also orchestrates a phenotypic shift from pro-inflammatory to anti-inflammatory states through precise regulation of inflammatory marker expression [[115]39]. Effect of OHADN fiber@Yoda1 hydrogel on cytocompatibility The cytocompatibility of biomaterials is crucial for their clinical application [[116]41]. To evaluate the biocompatibility of the hydrogels, BMSCs were seeded onto the hydrogel surfaces, and cell morphology was observed through cytoskeletal staining, while cell viability was assessed using the CCK-8 assay. As shown in Fig. [117]3a, cells in all groups exhibited healthy morphological characteristics. Notably, cells cultured on the OHADN fiber@Yoda1 hydrogel displayed a larger spreading area compared to those on the OHADN hydrogel and OHADN fiber hydrogel, with no statistically significant difference observed relative to the control group. The CCK-8 results revealed no significant differences in OD values among the groups on day 1 (Fig. [118]3b). By day 3 and 5, the OD value of the OHADN fiber@Yoda1 hydrogel group was higher than that of the OHADN fiber hydrogel groups, with no significant difference compared to the control group. These results collectively demonstrate that the OHADN fiber@Yoda1 hydrogel exhibits excellent cytocompatibility. Fig. 3. [119]Fig. 3 [120]Open in a new tab OHADN fiber@Yoda1 hydrogel possesses excellent cytocompatibility and osteogenic potential. (a) Cytoskeletal staining and cell-spread area analysis, blue for nuclei and red for F-actin. (b) The cytotoxicity of each group of materials was assessed by the CCK-8 assay. (c) Immunofluorescence imaging of intracellular calcium ion staining. (d) Relative fluorescence value of intracellular calcium ion detecting by microplate reader. (e) Quantitative RT-PCR analysis of osteogenic genes. (f) Western blot analysis of osteogenic proteins. (*P<0.05, **P<0.01, ***P<0.001, ns: not significant. The figure d was analyzed by Student’s t-test, while the remaining figures were analyzed using one-way ANOVA, followed by a LSD test.) The spreading area of cells on biomaterial surfaces is closely linked to the establishment and maintenance of cellular tensional homeostasis, a key factor regulating cell behavior and function [[121]10]. When cells adhere to a substrate, they generate cytoskeletal tension through the formation of focal adhesions and actin stress fibers, which are essential for maintaining mechanical equilibrium within the cell [[122]42]. The extent of cell spreading is directly influenced by the mechanical properties and surface characteristics of the biomaterial, such as stiffness, topography, and ligand density [[123]31, [124]43]. These factors collectively determine the ability of cells to sense and respond to their microenvironment, a process known as mechanotransduction [[125]44]. In the context of the OHADN fiber@Yoda1 hydrogel, the observed increase in cell spreading area suggests that this material provides an ideal mechanical microenvironment for cells to establish tensional homeostasis. The hydrogel’s composition and structural properties likely promote the formation of stable focal adhesions and actin stress fibers, enabling cells to generate and sustain the necessary cytoskeletal tension. This, in turn, supports cellular functions such as proliferation, differentiation, and migration, while enhancing the overall biocompatibility of the material. Furthermore, the role of mechanosensitive ion channels, particularly PIEZO1, cannot be overlooked in this process. PIEZO1 channels are directly gated by membrane tension and play a crucial role in cytoskeletal reorganization and tissue homeostasis, acting as key sensors of mechanical equilibrium [[126]15]. The low-molecular-weight compound Yoda1, which selectively targets PIEZO1 channels, enhances their sensitivity to mechanical stimuli [[127]37]. In the OHADN fiber@Yoda1 hydrogel, the incorporation of Yoda1 may further amplify the mechanotransduction signals, promoting a more robust cellular response to the mechanical microenvironment provided by the hydrogel. OHADN fiber@Yoda1 hydrogel possesses excellent osteogenic potential After confirming satisfactory cytocompatibility, BMSCs were seeded onto the surfaces of various materials and subjected to osteogenic induction for 7 days. Intracellular calcium levels were determined by staining with the fluo-4 probe as shown in Fig. [128]3c and d. The expression of osteogenic genes and proteins was evaluated using RT-qPCR, western blot and immunofluorescence staining, as shown in Fig. [129]3e and f and S2. Intracellular calcium ion staining revealed that the OHADN fiber@Yoda1 hydrogel group exhibited a significantly higher fluorescence intensity compared to the control group, suggesting enhanced calcium influx mediated by this hydrogel. This phenomenon is presumably attributed to the activation of the mechanosensitive PIEZO1 ion channel, which is specifically potentiated by the Yoda1 agonist incorporated in the hydrogel system. As a prototypical mechanosensitive ion channel, PIEZO1 coordinates mechanochemical transduction by detecting plasma membrane tension variations [[130]18]. This structural sensor initiates calcium ion influx through its characteristic trimeric propeller configuration, subsequently triggering mechanotransduction cascades involving Hippo pathway effectors YAP/TAZ^+ [[131]14]. The nuclear translocation of these transcriptional co-activators fundamentally governs the osteodifferentiation commitment of mesenchymal stem cells [[132]17, [133]45, [134]46]. RT-qPCR results revealed that the OHADN fiber@Yoda1 hydrogel group exhibited increased expression of osteogenic genes, including OSX, Runx2, OPN, and Col1, compared to the control group. However, the differences in osteogenic gene expression levels among the material groups were inconsistent. Specifically, Runx2 expression was higher in all material groups compared to the control, with no significant differences observed between the material groups. The expression of OSX was significantly elevated in the OHADN hydrogel and OHADN fiber@Yoda1 hydrogel groups compared to the control, while the OHADN fiber hydrogel group showed no significant difference relative to the control. Western blot results further supported these findings, showing that the OHADN fiber@Yoda1 hydrogel group exhibited higher protein levels of ALP, RUNX2, OSX, COL1, and OCN compared to the control group, consistent with the RT-qPCR data. Furthermore, immunofluorescence staining demonstrated that the expression of RUNX2 in the OHADN fiber@Yoda1 hydrogel group was significantly higher than in the other three groups (Fig. S2). These results collectively indicate that the OHADN fiber@Yoda1 hydrogel possesses excellent osteogenic potential, effectively promoting osteoblast differentiation and maturation. The observed upregulation of osteogenic genes and proteins in the OHADN fiber@Yoda1 hydrogel group highlights its superior ability to support osteogenic differentiation. RUNX2, a master regulator of early osteogenesis [[135]47], was consistently elevated across all material groups, suggesting that changes in extracellular matrix stiffness universally influence RUNX2 expression. However, the differential expression of OSX, a downstream transcription factor critical for osteoblast maturation [[136]47], indicates that not all materials can sustain the progression of osteogenic differentiation. The significant upregulation of OSX in the OHADN fiber@Yoda1 hydrogel group suggests that this material provides a conducive microenvironment for advanced osteogenic maturation. The role of mechanotransduction in osteogenesis is further underscored by the involvement of PIEZO1 channels, which are known to mediate mechanical signals during bone development [[137]13, [138]14]. Activation of PIEZO1 channels triggers cytoskeletal reorganization, which is essential for maintaining cellular tensional homeostasis and promoting bone regeneration. Our previous studies demonstrated that low-concentration Yoda1, a PIEZO1 activator, upregulates cytoskeleton-related genes and enhances osteogenic protein expression [[139]19]. This aligns with the current findings, where the OHADN fiber@Yoda1 hydrogel, likely through PIEZO1-mediated mechanotransduction, promotes a robust osteogenic response. The OHADN fiber@Yoda1 hydrogel not only enhances early osteogenic markers like RUNX2 but also supports the expression of late-stage markers such as OSX and OCN, indicating its potential to drive complete osteogenic differentiation. The integration of PIEZO1-mediated mechanotransduction further amplifies its osteoinductive properties, making it a promising candidate for bone tissue engineering applications. OHADN fiber@Yoda1 hydrogel promotes bone regeneration via PIEZO1 - ITGα5 axis To elucidate the molecular mechanisms underlying OHADN fiber@Yoda1 hydrogel-enhanced osteogenic differentiation of BMSCs, we conducted RNA sequencing analysis. High-throughput transcriptome sequencing was performed using the DNBSEQ platform, yielding an average of 6.44 GB clean data per sample across control and OHADN fiber@Yoda1 hydrogel groups. The alignment rates reached 98.96% for reference genomes and 80.38% for annotated gene sets, with 16,671 genes detected in total. To assess the inter-sample correlations in gene expression, Pearson correlation coefficients of all gene expression levels between every two samples were calculated. These coefficients were then visualized in a heatmap (Fig. [140]4a), with the majority of correlation coefficients falling within acceptable limits. Venn diagram analysis identified 761 and 698 unique differentially expressed genes (DEGs) in control and OHADN fiber@Yoda1 hydrogel groups, respectively (Fig. [141]4b). Volcano plot analysis revealed 2,226 upregulated and 2,343 downregulated genes with statistical significance (Fig. [142]4c). Hierarchical clustering demonstrated distinct separation of expression patterns between groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs highlighted significant enrichment in focal adhesion, regulation of actin cytoskeleton, and stem cell pluripotency-related signaling pathways in the hydrogel group (Fig. [143]4d). Gene Set Enrichment Analysis (GSEA) further confirmed substantial enrichment of the focal adhesion pathway (NES = 1.60, P < 0.05) in OHADN fiber@Yoda1 hydrogel-treated samples versus controls (Fig. [144]4e), suggesting its potential involvement in osteogenic differentiation. Fig. 4. [145]Fig. 4 [146]Open in a new tab Transcriptome analysis of the OHADN fiber@Yoda1 hydrogel. (a) Pearson correlation coefficients between every two samples. (b) Venn diagram analysis. (c) Volcano plot analysis. (d) KEGG pathway enrichment analysis. (e) GSEA analysis. (f) Heatmap of the represented up-regulated genes Notably, the OHADN fiber@Yoda1 hydrogel group exhibited significant upregulation of key osteogenic regulators within the focal adhesion pathway—Itgα5, Tns1, Spp1 (encoding OPN), and Id3 (a BMP signaling effector). This coordinated gene activation profile (Fig. [147]4f) reveals a mechanotransduction cascade where Yoda1-induced PIEZO1 activation primes mechanical signaling through the ITGα5-TNS1 axis, subsequently amplifying mineralization drivers (Spp1/OPN) and activating BMP-responsive transcription factors (Id3) to potentiate bone formation. RT-qPCR analysis demonstrated significantly upregulated mRNA expression levels of Itgα5, Tns1, Rhoa, Rock2, and Id3 in the OHADN fiber@Yoda1 hydrogel group compared to control groups (Fig. [148]5a). Western blot results in Fig. [149]5b and Fig. S3 further revealed that the OHADN fiber@Yoda1 hydrogel markedly enhanced protein expression of ITGα5, TNS1, RHOA, and ROCK2. These collective findings provide compelling evidence for the hydrogel’s dual functional mechanism, which concurrently activates mechanosensitive ion channels and orchestrates downstream osteogenic signaling pathways through spatiotemporal coordination. The schematic diagram in Fig. [150]5c delineates this complex working mechanism, highlighting the material’s capability to synchronize mechanical signal perception with biochemical pathway activation. Fig. 5. [151]Fig. 5 [152]Open in a new tab OHADN fiber@Yoda1 hydrogel promotes bone regeneration via PIEZO1 - ITGα5 axis. (a) Quantitative RT-PCR analysis of Itgα5, Tns1, Rhoa, Rock2, Id3. (b) Western blot assay of ITGα5, TNS1, RHOA, and ROCK2. (c) Schematic of OHADN fiber@Yoda1 hydrogel promoted bone regeneration through the PIEZO1 - ITGα5 axis. (*P<0.05, **P<0.01, ***P<0.001, analyzed by one-way ANOVA, followed by the LSD test.) Transmembrane communication plays a critical role in responding to extracellular stimuli and maintaining intracellular homeostasis [[153]48, [154]49]. The PIEZO1 channel serves as a key homeostatic sensor in shaping tissue homeostasis [[155]15, [156]16]. After disrupting actin filaments with cytochalasin D, the current amplitude of PIEZO1 channels decreases, indicating that the cytoskeleton is responsible for transmitting mechanical stimuli upon PIEZO1 channel activation [[157]15]. Atomic force microscopy colocalization experiments further reveal a strong interaction between PIEZO1 channels and the cytoskeleton [[158]50]. The function of PIEZO1 channels extends beyond transient transduction of mechanical signals; they achieve long-term homeostatic balance in the tissue microenvironment by dynamically regulating cellular tension, metabolic activity, and intercellular communication. On the other hand, ITGα5, a core receptor in cell-matrix interactions, transmits mechanical signals through focal adhesion complexes, while tensin regulates cytoskeletal reorganization. These two components synergistically maintain tension homeostasis [[159]51]. This suggests that PIEZO1 channels and the integrin system may exhibit cooperative regulatory mechanisms. As the primary mechanical sensor of the extracellular matrix, integrin signaling and the mechanical response of PIEZO1 channels are highly coupled in both spatial and temporal dimensions. Recent studies have revealed that PIEZO1 channels and integrin β1 on cardiac fibroblast surfaces mutually activate to form a positive feedback loop [[160]52, [161]53]. Stiffer extracellular matrices upregulate PIEZO1 channel activity via integrin β1 [[162]54], implying that integrins may indirectly modulate PIEZO1 channel activity by regulating cytoskeletal tension. v OHADN fiber@Yoda1 hydrogel promoted alveolar bone regeneration in vivo To investigate the in vivo osteogenic performance of OHADN fiber@Yoda1 hydrogel, we established a rat alveolar bone defect model [[163]55, [164]56] and implanted experimental materials into the bone defects (Fig. [165]6a). Bone regeneration efficacy was systematically evaluated using Micro-CT, H&E staining, Masson’s trichrome staining, and immunohistochemical analyses. Given the well-documented osteoinductive properties of hydroxyapatite, a material widely adopted in bone substitute applications, it was selected as the positive control [[166]57, [167]58]. A blank control group without material implantation was included for baseline comparison. Fig. 6. [168]Fig. 6 [169]Open in a new tab OHADN fiber@Yoda1 hydrogel promoted alveolar bone regeneration in vivo. (a) A schematic diagram of the animal experiment. (b) The 3D reconstruction of micro-CT and analysis of bone volume fraction (BV/TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). (c) Representative H&E and Masson’s trichrome staining images of various group at week 4. The white dashed box represents the defect location. Scale bars represent 1 mm. (**P<0.01, ***P<0.001, analyzed by one-way ANOVA, followed by the LSD test.) Initial biocompatibility assessment through H&E staining of visceral organs (heart, liver, spleen, lungs, kidneys) revealed no discernible pathological alterations in any experimental group after 4-week implantation, confirming the absence of systemic toxicity and further validating the biosafety profile of OHADN fiber@Yoda1 hydrogel (Fig. S3). Micro-CT analysis with three-dimensional reconstruction (Fig. [170]6b) demonstrated superior bone regeneration metrics in the OHADN fiber@Yoda1 hydrogel group. Specifically, this group exhibited a significantly higher bone volume fraction (BV/TV) compared to the blank control, OHADN hydrogel, and OHADN fiber hydrogel groups (P < 0.05), while showing comparable performance to the Hydroxyapatite positive control (P > 0.05). A similar trend was observed in Tb.Th measurements, suggesting enhanced osteogenic capacity. Notably, the OHADN fiber@Yoda1 hydrogel group displayed significantly lower Tb.Sp than the blank control and OHADN hydrogel groups (P < 0.05), with no statistical difference from either the OHADN fiber hydrogel or Hydroxyapatite group (P > 0.05). The reduced Tb.Sp observed in the experimental group aligns with previous reports that interconnected fiber networks in composite hydrogels can guide osteoblast migration and enhance matrix mineralization. These findings collectively indicate superior quality of neoformed bone in the OHADN fiber@Yoda1 hydrogel group. Consistent histological observations were obtained through H&E and Masson’s trichrome staining analyses (Fig. [171]6c). Analysis of newly formed bone tissue within the demarcated regions (white dashed lines) demonstrated superior bone formation in the OHADN fiber@Yoda1 hydrogel group compared to other experimental cohorts. These findings validate the outstanding osteogenic potential and structural guidance capabilities of the OHADN fiber@Yoda1 hydrogel. We further conducted immunohistochemical analysis to evaluate the expression levels of osteogenic proteins and signaling pathway components. As shown in Fig. [172]7a, the OHADN fiber@Yoda1 hydrogel group demonstrated significantly higher RUNX2 and OPN expression compared to control, OHADN hydrogel, and OHADN fiber hydrogel groups, while showing comparable levels to the Hydroxyapatite group. Figure [173]7b revealed that PIEZO1, ITGα5, and TNS1 expression in the OHADN fiber@Yoda1 hydrogel group markedly exceeded those in all other groups. Importantly, immunohistochemical co-staining analysis of OHADN fiber@Yoda1 hydrogel group demonstrated spatial co-localization of PIEZO1 and ITGα5 (Pearson correlation coefficient = 0.51, Fig. [174]7c), supporting their functional interaction in mechanotransduction. Fig. 7. [175]Fig. 7 [176]Open in a new tab OHADN fiber@Yoda1 hydrogel promoted alveolar bone regeneration in vivo. (a) Immunohistochemical staining and quantification of average optical density for RUNX2 and OPN of the alveolar bone defect. (b) Immunohistochemical staining and quantification of average optical density for PIEZO1, ITGα5 and TNS1 of the alveolar bone defect. (c) Immunohistochemical co-staining of PIEZO1 (green) and ITGα5 (red). Yellow indicates their spatial co-localization. Right panel: Quantitative curve of the fluorescence assay analyzed by Image J software. (**P<0.01, ***P<0.001, analyzed by one-way ANOVA, followed by the LSD test.) The observed upregulation of PIEZO1 expression may be attributed to Yoda1-induced activation of PIEZO1 channels through conformational changes, subsequently triggering increased ITGα5 and TNS1 expression which in turn regulates PIEZO1 channel expression, suggesting a potential positive feedback loop. This finding indicates a possible synergistic regulatory mechanism between PIEZO1 channels and the integrin system, which is structurally underpinned by their co-localization at focal adhesions. This mechanism is further supported by evidence of mechanical coupling: PIEZO1 colocalizes with integrins, and stiff matrices enhance PIEZO1 activity via integrin β1-mediated cytoskeletal tension [[177]50, [178]52–[179]54]. Recent finding also showed that PIEZO1 physically localizes to focal adhesions to regulate integrin-FAK signaling assembly [[180]59]. Specifically, PIEZO1 knockdown impairs focal adhesion formation and abrogates integrin-FAK pathway activation, confirming its essential role in mechanosensitive adhesion complexes. Leveraging these cooperative mechanisms, the OHADN fiber@Yoda1 hydrogel achieves spatiotemporal control of mechanosignaling through its biphasic architecture: the initial hydrogel phase facilitates stem cell migration via rapid stress relaxation, while the fiber network sustains mechanical tension and provides controlled Yoda1 release to stabilize the PIEZO1-ITGα5 signaling hub. Yoda1 sensitizes PIEZO1, enabling Ca²⁺ influx comparable to rigid substrates despite the hydrogel’s sub-rigid properties [[181]18]. This calcium influx activates RHOA/ROCK-mediated cytoskeletal contraction [[182]60], elevating ITGα5 expression and enhancing collagen binding for osteogenic differentiation [[183]61]. Collectively, our findings demonstrate that the OHADN fiber@Yoda1 hydrogel effectively promotes alveolar bone regeneration through the PIEZO1-ITGα5 mechanosignaling axis. While our data suggest possible crosstalk through membrane deformation when these mechanosensors are in proximity, we acknowledge that fully elucidating the direct molecular interplay within the PIEZO1-ITGα5 axis remains a limitation requiring further mechanistic validation in ongoing studies. Conclusion In this study, we developed a dual-network, fiber-reinforced hydrogel system that effectively integrates mechanical reinforcement with bioresponsive signaling to address the challenges of irregular alveolar bone regeneration. By incorporating Yoda1-loaded PLGA-collagen fiber fragments into an oxidized hyaluronic acid matrix, crosslinked via catechol–Fe³⁺ coordination and Schiff-base reactions, the hydrogel achieves enhanced structural integrity, injectability, and sustained mechanotransduction. This design maintains tensional homeostasis and activates the PIEZO1–ITGα5 mechanosignaling axis, thereby modulating immune responses and promoting osteogenic differentiation. Transcriptomic and protein-level analyses further confirm the enrichment of focal adhesion and cytoskeletal remodeling pathways. In vivo experiments demonstrate that this hydrogel significantly improves bone volume and quality in alveolar defects, rivaling traditional bone substitutes such as nano-hydroxyapatite. Collectively, this mechanoresponsive hydrogel offers a promising and clinically translatable strategy for treating complex bone defects through coordinated modulation of biomechanical and osteoimmune environments. Supplementary Information [184]Supplementary Material 1^ (1.2MB, pdf) Acknowledgements