Dual-action bisphosphonate hydrogel preserves cortical bone integrity and restores healing cascade in osteoporotic fracture-related infection

Domain: chemistry
Published: 2026-01-10
Editorial outcome: major_revision

Abstract
In osteoporotic fracture-related infection (FRI), the coordinated bone healing is compromised by cortical bone lysis and persistent bacterial colonization. Current treatments primarily focus on infection control, but fail to preserve bone microarchitecture or restore disrupted healing cascade, particularly in fragile osteoporotic bone. Here, we develop a bioactive nanocomposite hydrogel based on bisphosphonate-modified hyaluronan (HABP) and either free bisphosphonate (BP) or pyrophosphate (TSPP), carrying antibacterial Ag⁺ ions and Vancomycin. Triggered by the pathological pH of biofilms, this hydrogel enables site-specific release of antimicrobials. In a rat osteoporotic FRI model, prominent cortical bone lysis and osteocyte apoptosis impair healing. HABP hydrogel effectively eradicates the Methicillin-resistant Staphylococcus aureus infection, preserves cortical bone integrity, and rescues osteocyte viability by inhibiting osteoclast activation and osteocyte apoptosis. The TSPP-based formulation, delivering a lower BP dose, enhances callus remodeling. Our study demonstrates the promising potential of the dual-action hydrogel for treating osteoporotic FRI via preventing bone lysis and rejuvenating coordinated healing cascades.

Introduction
Bone fractures caused by musculoskeletal trauma can be life-threatening events. While modern advancements in orthopedic fixation techniques and medical devices have significantly facilitated bone repair, one persisting complication, fracture-related infection (FRI), remains unresolved1. Normally, the healing of long bone fracture is a well-coordinated biological process, including the inflammatory phase, endochondral bone formation, and callus remodeling2. However, bacterial colonization at the fracture site generates a chronic inflammatory environment, triggering prominent bone lysis that destabilizes the fracture site and disrupts the natural healing process3. The clinical outcomes of FRI are further worsened in elderly patients with comorbidities such as osteoporosis, in which the cortical microarchitecture is more vulnerable, and the bone’s ability to regenerate is severely diminished3,4. This intersection of infection and osteoporosis not only worsens fracture instability and delays healing but also increases the risk of recurrent infection and mortality5. Despite these risks, the majority of current therapies for FRI are designed for otherwise healthy bone and fail to address the unique pathological synergy between infection and osteoporosis6,7.

A central pathological feature of FRI, particularly in osteoporotic bone, is cortical bone lysis, driven by excessive osteoclast activation and apoptosis of osteocytes8. It is known that the overactivated osteoclasts (OCs) are directly responsible for bone lysis and microarchitecture damage9, while osteocytes, the most abundant and major resident cells in cortical bone, serve as key regulators of bone remodeling10. Recent evidence suggests that osteocyte apoptosis, especially in inflammatory conditions like FRI, not only compromises bone integrity but also exacerbates inflammation and facilitates bacterial invasion through empty lacunae11. Yet, few therapeutic approaches have directly targeted both cell types in the context of osteoporotic FRI, underscoring a major gap in current treatment strategies8.

The current standard of care for FRI is to maintain stability and allow for bone healing through debridement, antibiotics, and implant retention (DAIR). However, systemic antibiotics often fail to eradicate bacteria completely, causing a high DAIR failure rate ranging from 21.4% to 43%12,13. Moreover, treatments aimed at enhancing bone formation (e.g., administration of BMP-2) lack specificity for infected and osteoporotic environments and do not prevent pathological bone lysis14,15. Therefore, an integrated therapy that addresses both microbial eradication and bone catabolism is urgently needed, particularly in osteoporotic FRI where bone fragility and immune dysregulation coexist.

Bisphosphonates (BP) are carbon-substituted analogs of pyrophosphates widely used to treat osteoporosis, bone metastasis, and Paget’s disease16,17. However, systemic application of BP is limited by poor bioavailability and gastrointestinal irritation18. BP inhibits OC maturation and induces OC apoptosis, thereby leading to a reduction in bone resorption. Notably, BP has been shown to reduce the porosity of cortical bone and promote bone mineralization in osteoporotic models19, and can also inhibit osteolysis and protect osteocytes from TNF-α-induced apoptosis20. These combined functions suggest the potential of BP in treating osteoporotic FRI. Besides, given the unique chemical structure of BP, comprising two phosphate groups bound to a central carbon atom, they can form dynamic coordination bonds with various bioactive cations, enabling the creation of dynamic hydrogels21. Our previous work has demonstrated a nanocomposite hydrogel that is capable of releasing BP, inhibiting osteoclast maturation, and facilitating bone regeneration22. In addition, BP-based hydrogel can also be engineered to load drugs and antimicrobial agents, offering the dual benefit of infection control and bone healing augmentation23. These features highlight the potential of BP-based hydrogels as a targeted therapy for osteoporotic FRI.

Here, we develop a BP-based bioactive nanocomposite hydrogel (HABP-BP) co-loaded with Vancomycin and Ag+ for the treatment of osteoporotic FRI. This hydrogel exploits the acidic microenvironment in infection sites to specifically trigger the release of antimicrobials, while the released BPs inhibit osteoclast activity and prevent osteocyte apoptosis, thereby preserving cortical bone integrity. Furthermore, using a rat model with osteoporotic FRI, we identify that the HBAP-BP-V-Ag+ hydrogel eradicates infection, prevents cortical bone lysis and osteocyte apoptosis, and consequently re-establishes the bone healing course through callus formation and mineralization. Interestingly, we demonstrate that the hydrogel with a high dosage of BP may delay fracture callus remodeling, which is ameliorated by using a modified formulation (HABP-TSPP-V-Ag+) with reduced BP concentration while maintaining therapeutic efficacy (Fig. 1). This refined system offers a tunable platform that balances infection control and bone regeneration, tailored for complex osteoporotic environments. Our findings suggest that BP-based bioactive nanocomposite hydrogels provide a disease-specific, dual-action therapy that directly addresses the overlooked intersection of osteoporosis and FRI. By targeting both pathogenic bacteria and bone-destructive mechanisms, it presents a comprehensive and translationally relevant therapy for infected fractures in osteoporotic patients.

Results
The BP-based bioactive hydrogels mediate pathological pH-specific release of antimicrobials
Leveraging the dynamic coordination between BP and bioactive cations, we synthesized HABP macromers (Supplementary Fig. 1) and fabricated the self-assembled nanocomposite hydrogel by simply mixing HABP, free BP, and Ca2+ (denoted as “HABP-BP”). To enhance its antibacterial activity, we further incorporated Ag+ into the hydrogel using the same coordination strategy, yielding the “HABP-BP-Ag+” formulation (Fig. 2A). To tailor the localized BP concentration, pyrophosphate (TSPP), a BP analog that shares a similar chemical structure but lacks anti-resorptive bioactivity, was used as a substitute for free BP, generating the “HABP-TSPP” and “HABP-TSPP-Ag+” hydrogels. Rheological time sweep tests showed that all hydrogels exhibited similar storage and loss moduli, ranging from 600 to 700 Pa, indicating that the inclusion of different components did not compromise their structural integrity (Fig. 2B and Supplementary Fig. 2). Alternating high (400%) and low (0.1%) shear strain rheological experiments demonstrated the self-healing properties of the HABP and HABP-BP-Ag+ hydrogels, which quickly reformed their structure after shear disruption. In contrast, the HABP-TSPP and HABP-TSPP-Ag+ groups showed slightly diminished self-healing capacity (Fig. 2C and Supplementary Fig. 3), reflecting the superior coordination dynamics of BP compared to TSPP, which enhances the remodeling and self-healing capacity of the hydrogels. Therefore, the HABP-BP-Ag+ hydrogel demonstrated robust injectability, successfully passing through a needle with an inner diameter of 0.45 μm and forming stable, moldable structures upon extrusion, making it suitable to fit the irregular bone defects (Supplementary Movie 1). The hydrogel fabrication process entails a straightforward mixing of precursor solutions and vortexing, resulting in a stable hydrogel formation within 10 seconds. Subsequent syringe-based injection enables direct delivery to the injury site, offering substantial advantages for clinical translation (Supplementary Fig. 4). SEM images revealed uniform porous structures and well-distributed BP/TSPP-Ag+/Ca2+ nanoparticles in all hydrogel formulations, confirming their stability and consistent morphology (Fig. 2D).

Next, to assess the release profiles of antimicrobial agents, Vancomycin was incorporated into the hydrogels (“HABP-BP-V-Ag+” and “HABP-TSPP-V-Ag+”). Both hydrogels exhibited an initial burst release of Vancomycin and Ag+ over the first 3 days, followed by a sustained release over 14 days. 1H NMR analysis showed chemical shift changes in vancomycin’s α-carbon hydrogens (amide, hydroxyl, carboxyl), suggesting hydrogen bonding with BP phosphates, which may account for the sustained release of Vancomycin (Supplementary Fig. 6). Notably, the release of both Vancomycin and Ag+ can be significantly accelerated under acidic environment (pH ~ 5.5), simulating the infected bone microenvironment (Fig. 2E–G and Supplementary Fig. 5). This pH-responsive release can be attributed to the protonation of BPs, which weakens the coordination bonds between BP and cations, leading to the disassembly of BP/TSPP-Ag+/Ca2+ nanoparticles and the subsequent release of antimicrobial agents. Besides, the HABP-BP-V-Ag+ hydrogel also showed a significantly higher cumulative release of BP compared to the HABP-TSPP-V-Ag+ group, as the latter formulation lacks free BP and can only provide BP through the degradation of the HABP macromers.

The BP-based bioactive hydrogels inhibit osteoclast differentiation and osteocyte apoptosis in vitro
To investigate the biological effects of the BP-based hydrogels on bone cellular functions, we first assessed their influence on the osteogenic differentiation of bone mesenchymal stem cells (BMSCs) in vitro. BMSCs were cultured with an osteogenic induction medium supplemented with 10% hydrogel extracts, in which the concentration of BP (9.8 μM) exhibited no significant cytotoxicity (Supplementary Fig. 7). The alkaline phosphatase (ALP) staining and ALP activity assays revealed comparable ALP activity in HABP-BP-V-Ag+ or HABP-TSPP-V-Ag+ hydrogel-treated groups. (Fig. 3A, B). Additionally, HABP-BP, HABP-TSPP, HABP-BP-V-Ag+, and HABP-TSPP-V-Ag+ hydrogels all exhibit higher mineralization capacity compared to the control group, which was accompanied by upregulated expression of Col1a1 and Bglap (Fig. S8). In contrast, TSPP showed no detectable effect on the mineralization of BMSCs, as confirmed by Alizarin Red S staining (Supplementary Fig. 9). Next, we evaluated the impact of hydrogel extracts on OC differentiation in primary bone marrow monocytes (BMM). The tartrate-resistant acid phosphatase (TRAP) staining showed that the cells exposed to the HABP-BP or HABP-BP-V-Ag+ hydrogel extracts had a significantly reduced number of multinuclear (>3) OCs compared to untreated controls. Consistently, phalloidin and DAPI staining revealed a significant reduction in actin ring formation in the HABP-BP and HABP-BP-V-Ag+ groups (Fig. 3A, C). These reductions was accompanied by the reduced expression of osteoclast-specific genes, including Ctsk, Nfatc1, and C-fos, particularly in the HABP-BP-V-Ag+ group, where the addition of Ag+ further inhibited OC activity (Fig. 3D)24. In contrast, the HABP-TSPP-based hydrogels, which lack free BP, demonstrated less pronounced inhibition of OC differentiation, suggesting that BP release plays a critical role in this effect. However, when Ag⁺ was incorporated into the HABP-TSPP-based hydrogels (HABP-TSPP-V-Ag⁺), the inhibitory effects on OC differentiation were enhanced, underscoring the combined anti-resorptive effects of BP and Ag⁺.

As the dominant cells in cortical bone, osteocytes play a key role in orchestrating bone homeostasis and maintaining the integrity of the cortical structure25. Osteoporotic FRI is often associated with elevated inflammatory cytokines such as TNF-α8, which can induce osteocyte apoptosis and compromise cortical bone integrity26. To simulate this condition in vitro, we treated MLO-Y4 osteocytes with TNF-α and evaluated apoptosis levels in the presence of hydrogel extracts. Flow cytometry analysis showed significantly elevated apoptosis induced by TNF-α, which was attenuated by treatment with HABP-based hydrogel extracts (Fig. 3E, F). The MLO-Y4 cells were further treated with BP or TSPP alone, confirming that this protective effect was mediated by BP rather than TSPP, as only BP treatment reversed TNF-α-induced changes in apoptotic gene expression, including upregulated Caspase-3 and downregulated Bcl-2 (Supplementary Fig. 7B, C). This protective mechanism is consistent with previous reports indicating that BP can inhibit osteoblast and osteocyte apoptosis through the opening of CX43 hemichannels and downstream Src/ERK signaling pathways27. Collectively, these findings demonstrate that both HABP-BP-V-Ag+ and HABP-TSPP-V-Ag+ can suppress OC maturation and resorptive activity while protecting osteocytes from TNF-α-induced apoptosis. Importantly, these hydrogels support osteogenic differentiation of BMSCs, highlighting their potential to mitigate cortical bone lysis and restore bone homeostasis in osteoporotic FRI.

The BP-based bioactive hydrogels exhibit bactericidal effects and inhibit biofilm formation
To examine the antimicrobial efficacy of the BP-based hydrogels in vitro, we performed a disc diffusion test against methicillin-resistant Staphylococcus aureus (MRSA). The HABP-BP-Ag⁺ hydrogel exhibited a significantly larger inhibition zone compared to the HABP-BP-V hydrogel, confirming the enhanced antimicrobial capacity with the addition of Ag+ (Fig. 4A, D). Given the propensity of bacteria to form biofilms on implants during FRI28, we next investigated the ability of the hydrogels to disrupt biofilms using live/dead staining. After 12-hour treatment, no visible reduction of SYTO-9 fluorescence was observed in the HABP-BP group, indicating limited intrinsic antimicrobial ability. In contrast, the HABP-BP-V-Ag+ and HABP-TSPP-V-Ag+ hydrogels effectively disrupted biofilms, as evidenced by the scattered propidium iodide (PI) signals interspersed with reduced SYTO-9 fluorescence (Fig. 4B). The confocal images further confirmed the enhanced biofilm reduction capacity of the HBAP-BP-V-Ag+ hydrogel compared to the HBAP-BP-V group (Fig. 4C). Furthermore, the bacterial colony-forming unit (CFU) assay was conducted to quantify the bactericidal capacity of the hydrogels. The HABP-BP-V hydrogel achieved an over 2-log reduction in bacterial load (99.9% reduction, from an average 8.7 to 5.9 log CFU/ml) after 24h of co-culture compared to Ctr group without intervention. This effect was further enhanced in the presence of Ag⁺, achieving a >5-log reduction (from an average 8.7 to 3.2 log CFU/ml) in bacterial load with the HABP-BP-V-Ag⁺ hydrogel compared to control group. (Fig. 4E). Notably, the bactericidal efficacy of the HABP-BP-V-Ag+ hydrogel was comparable to that of the HABP-TSPP-V-Ag+ one, confirming the robust antimicrobial effects of both systems. Further, we found that the co-culture with the HABP-BP-V-Ag+ and HABP-TSPP-V-Ag+ hydrogels significantly elevated the production of ROS in MRSA biofilms than the HABP-BP-V group, confirming that Ag+ release has the potential to trigger ROS generation that may synergize with vancomycin in biofilm eradication (Supplementary Fig. 10). Ag+ can disrupt bacterial cell walls and trigger bacterial apoptosis while simultaneously generating ROS. The ROS penetrates the biofilm matrix, compromising its structural integrity and facilitating Vancomycin access to embedded bacteria, thereby enhancing bacteria eradication (Fig. 4F)29. Collectively, these findings demonstrate that both HABP-BP-V-Ag+ and HABP-TSPP-V-Ag+ hydrogels achieve a clinically significant >5-log reduction in bacterial load, effectively disrupting biofilms and eliminating embedded bacteria. This highlights their potential as powerful therapeutic agents for managing FRI in clinical settings30.

BP-based bioactive hydrogels prevent cortical bone lysis and promote fracture healing in osteoporotic bone with FRI
The therapeutic potential of BP-based bioactive hydrogels was evaluated using an osteoporotic femur fracture rat model inoculated with MRSA (“OVX-M”) to simulate FRI (Fig. 5A)8. Ovariectomy surgery was performed on 6-month-old SD rats to induce osteoporosis over 3 months, followed by femur fracture surgery with bacteria inoculation. At week 4 post-surgery, the X-ray images showed extensive bone lysis and periosteal bone reaction in the OVX-M group, characterized by new bone formation with inflammatory irritation of the periosteal membrane. Quantitative analysis indicated significantly increased callus width and callus area in OVX-M group than those in the OVX control. Micro-CT analysis further confirmed substantial loss of high-density bone tissue at the fracture site, particularly in the axial view, indicating severe cortical bone resorption. This progressive bone loss at the fracture site likely compromised fracture stability, impairing callus bridging and creating dead space that exacerbated bacterial infection (Fig. 5B, C). Three-dimensional reconstructed images using different thresholds to distinguish newly formed low-density bone (blue) from the high-density bone tissue (gray), further revealed prominent cortical bone loss in the OVX-M group. BVh (threshold from 350 to 1000) represents mature cortical bone that is progressively eroded by osteoclast overactivation, while BVl (threshold from 165 to 350) is characterized by disorganized, poorly mineralized bone formation around lytic areas. By week 8, the cortical bone lysis becomes more prominent, with a dramatic reduction of high-density cortical bone in the OVX-M group compared to the OVX group (−88.3% in BV, p<0.0001). The coexistence of BVh loss and BVl reveals the uncoupling of normal remodeling where destructive processes outpace effective repair. Despite extensive formation of low-density bone tissue, no bridging of the fracture gap was observed in any of the rats in the OVX-M group (Fig. 5D, E), reflecting the impaired healing phenotype characteristic of osteoporotic FRI, which mimics the radiographic features observed in clinical scenarios1.

Notably, treatment with both HABP-BP-V-Ag⁺ and HABP-TSPP-V-Ag⁺ significantly increased bone volume (BVh) compared to untreated OVX-M controls by week 4, indicating ameliorated osteolysis. In contrast, no such protective effect was observed in the OVX-M-V group, underscoring the limitation of systemic antibiotic treatment. Besides, the unresolved bone lysis and non-union in the OVX-M-HABP-BP group further confirmed that the HABP-BP hydrogel alone was insufficient to support bone healing without effective infection control. By week 4, both OVX-M-HABP-BP-V-Ag⁺ and OVX-M-HABP-TSPP-V-Ag⁺ group showed reduced callus area compared to the OVX-M group (Fig. 5B, C). Notably, the HABP-BP-V-Ag⁺ group exhibited a substantial volume of high-density bone tissue in the fracture callus by the study endpoint (Fig. 5D, E). The increased callus area relative to OVX controls suggests delayed callus remodeling, consistent with reports that high systemic BP concentrations can delay the transition from woven bone to lamellar bone and impair callus remodeling31. To address this, the HABP-TSPP-V-Ag+ hydrogel was designed with a reduced BP concentration (20 mM), resulting in significantly lower callus area and improved callus remodeling compared to the HABP-BP-V-Ag+ group.

BP-based bioactive hydrogels restore disturbed formation of fracture callus in FRI and rejuvenate coordinated healing cascades
The integrity of cortical bone is the prerequisite for stabilizing the fracture site and facilitating the formation of a cartilage callus to bridge the fracture gap32. However, in osteoporotic FRI with severe bone lysis, the disturbance of callus formation has not been fully characterized. In the OVX-M group, H&E staining demonstrated extensive necrotic tissue and inflammatory cell infiltration at the fracture gap (Fig. 6A), while Safranin O staining showed no visible cartilage formation (Fig. 6B). Despite increased periosteal bone formation surrounding the cortical bone, the fracture gap remained unbridged. These pathological changes suggest that the loss of fracture site stability, compounded by ongoing infection, impaired the formation of a cartilage callus. Next, we performed IHC staining of osteocalcin (OCN) and collagen-1 (COL-1) to assess bone regeneration. In the non-infected control group, a brown-stained area adjacent to the fracture site indicated active new bone formation. Comparatively, the expression OCN and COL-1 was significantly decreased in the OVX-M group compared to that in the OVX group (Fig. 7A, C, D and Supplementary Fig. 11), highlighting impaired bone regeneration. In FRI, the overshoot of inflammatory cytokines, particularly TNF-α, is known to drive bone cell death and osteolysis9. Immunofluorescence staining demonstrated significantly increased TNF-α expression at the fracture site in the OVX-M group compared to the OVX control (Fig. 7B, E). At week 8, high-magnification H&E staining images demonstrated aggravated cortical bone erosion and empty osteocyte lacunae (Supplementary Fig. 12), accompanied by persistently elevated TNF-α levels (Fig. 7B, F). These findings elucidate that the normal healing process was diverted toward an infection-associated inflammatory phenotype in untreated osteoporotic FRI.

In the OVX-M-V group, systemic vancomycin treatment did not mitigate bone lysis or improve healing outcomes, consistent with previous findings that systemic antibiotics have limited effects against FRI due to poor local drug penetration28. Similarly, the HABP-BP hydrogel, which lacks antimicrobial agents, demonstrated no therapeutic benefit either, as infection remained uncontrolled. While in the HABP-BP-V-Ag+ treated group, H&E staining demonstrated an intact cortical bone structure, in which a bony callus was formed surrounding the fractured bone. IHC staining showed increased expression of OCN and COL-1 compared to OVX-M. These observations suggest that HABP-BP-V-Ag+ hydrogels successfully mitigated inflammatory necrosis and preserved the cortical bone structure. However, Safranin O staining demonstrated a reduced area of cartilage callus compared to the OVX control (Fig. 6B, E). At week 8, the fracture gap in the HABP-BP-V-Ag+ group was connected by fibrotic tissue, around which a large volume of highly mineralized callus was formed (Fig. 6A). This suggests that while the hydrogel promoted fracture healing, high BP concentrations may have hindered callus remodeling, delaying the transition from woven bone to lamellar bone.

In contrast, the HABP-TSPP-V-Ag+ hydrogel demonstrated superior fracture healing outcomes. At week 4, Safranin O staining showed cartilage formation at the center of the fracture gap, which progressively mineralized into bone, leading to complete bridging of the fracture by week 8 (Fig. 6B, E, F). Further, we assess the histological bone healing with tissue quality score, which classifies callus from 0 (bony union, best) to 4 (mostly inflammatory cells, worst), with higher scores indicating poorer callus quality and impaired healing. The semi-quantitative score confirmed the restored healing in both OVX-M-HABP-BP-V-Ag+ and OVX-M-HABP-TSPP-V-Ag+ group at week 4, whilst by week 8, a more mature bony callus was observed in OVX-M-HABP-TSPP-V-Ag+ group compared to the fibrotic callus in OVX-M-HABP-BP-V-Ag+ group (Fig. 6C, D). Next, we performed 4-point bending test to assess the mechanical strength of the fracture callus. Compared to the OVX-M group, both hydrogel-treated groups showed enhanced ultimate load and stiffness. Notably, HABP-TSPP-V-Ag+ group exhibited even better ultimate load and stiffness compared to HABP-BP-V-Ag⁺ (Supplementary Fig. 12). These findings suggest that while a high dosage of topical BP in HABP-BP-V-Ag+ hydrogel does not impair bony callus formation, it may hinder callus remolding. Comparatively, a lower dosage of BP concentration in HABP-TSPP-V-Ag+ hydrogel yields a more coordinated healing process, promoting optimal callus remodeling and functional bone regeneration (Fig. 6G).

BP-based hydrogels eradicate the infection and prevent bacterial colonization of cortical bone
Eradicating bacteria at the fracture site is essential for improving bone healing outcomes in FRI. The biofilm-associated bacteria residing in the osteocyte-lacuno-canalicular network (OLCN) are highly recalcitrant and beyond surgical margins, causing persistent infection and treatment failure33. Here, we first evaluated bacterial contamination of bone tissue using Gram staining. Notably, the bacteria colonies were identified in the cortical bone tissue and OLCN in the OVX-M group at week 4, coinciding with the onset of cortical bone lysis. Reportedly, the dispersing of bacteria and colonization of OLCN is more prominent in osteoporotic bone with FRI due to the high cortical porosity8. In the OVX-M-V group, the presence of bacteria in bone tissue revealed limited therapeutic effect of systemic antibiotics against deep-seated infections (Fig. 8A). In contrast, negative CFU cultures and the absence of Gram-positive bacteria in bone tissue demonstrated that bacteria were eradicated in both the OVX-M-HABP-BP-V-Ag+ and OVX-M-HABP-TSPP-V-Ag+ groups, confirming the effect of pH-triggered release of Ag+ and antibiotics in vivo. In the OVX-M-V group, the bacterial load on bone tissue was significantly reduced, but such effects were not observed on the k-wire. (Fig. 8B, D, E).

Additionally, the bacterial biofilm formed on the implant is a major challenge in FRI management, serving as a persistent reservoir for reinfection34,35. SEM images of the K-wire surface in the OVX-M and OVX-M-V groups reveal extensive bacterial biofilm and necrotic tissue adherence (Fig. 8C). High-magnification images confirmed bacterial cells enclosed within the biofilm matrix. Notably, both HABP-BP-V-Ag+ and HABP-TSPP-V-Ag+ treatments prevented biofilm formation, and CFU plating confirmed the successful eradication of bacteria on the implant. These results collectively demonstrated the in vivo antimicrobial capacity of the HABP-BP-V-Ag+ and HABP-TSPP-V-Ag+ hydrogels.

BP-based hydrogels alleviate osteoclast activation and osteocyte apoptosis in vivo
As the key contributors to cortical bone damage, activated osteoclasts are major therapeutic targets in FRI9. By assessing osteoclast activity through immunohistochemistry (IHC) for cathepsin K (CTSK) (Fig. 9A), a large number of positively-stained osteoclasts was identified surrounding the cortical bone in the OVX-M group. Both HABP-BP-V-Ag+ and HABP-TSPP-V-Ag+ treatments significantly reduced CTSK expression, indicating inhibited osteoclast activation (Fig. 9C). The therapeutic effects were absent in the HABP-BP hydrogel-treated group. Notably, a similar reduction of osteoclast activation in HABP-TSPP-V-Ag+ groups indicates that a low dosage of BP can effectively prevent excessive bone loss in osteoporotic FRI. Additionally, elevated serum levels of CTSK in the OVX-M group also indicate bone catabolism, which was ameliorated by both HABP-BP-V-Ag+ and HBAP-TSPP-V-Ag+ treatments (Supplementary Fig. 13). Importantly, in the late stage of fracture repair, activated osteoclasts are indispensable for reducing the callus size and facilitating callus remodeling36. At week 8, we identified that the number of osteoclasts in the bone tissue around fracture site was significantly decreased in HABP-BP-V-Ag+ compared to HABP-TSPP-V-Ag+. (Fig. 9A, D). Consistently, in the OVX-M-HABP-BP-V-Ag+ group, the expression of bone matrix-deposited cytokines, including TGF-β1 and IGF1, was markedly reduced compared to the control group, whilst the expression of RANKL was remained comparable. In contrast, the OVX-M-HABP-TSPP-V-Ag⁺ group exhibited TGF-β1 and IGF1 levels similar to those of the OVX control, demonstrating preserved osteoclast-osteoblast coupling (Supplementary Fig. 14). These findings suggest that the enhanced callus remodeling observed in the HABP-TSPP-V-Ag⁺ group is primarily attributable to the reduced BP dosage, which prevents excessive osteoclast suppression and sustains the coupled remodeling process. In particular, osteoclasts play a critical role by first resorbing mineralized tissue and cartilage, then working in tandem with osteoblasts in coordinated cycles to rebuild strong, functional bone2. If this delicate balance is disrupted by excessive BP, it can hinder the timely restoration of proper bone structure and strength.

In cases of FRI, inflammatory cytokines such as TNF-α, and IL-1β are major initiators for inflammatory response9. Osteocyte apoptosis in inflammatory microenvironment is found to cause bone resorption and tissue damage26,37. Consequently, the increase of osteocyte-mediated cortical matrix degradation may exacerbate the spreading of the bacteria38, and the invasion of deeply embedded osteocytes and forming an escalated infection28. In our study, TUNEL staining showed significantly increased osteocyte apoptosis at the fracture site in the OVX-M group compared to the control group (Fig. 9B). The proportion of apoptotic osteocytes in cortical bone tissue was significantly reduced in the HABP-BP-V-Ag+ and HABP-TSPP-V-Ag+ groups (Fig. 9E), which can be attributed to the anti-apoptotic effects of BP on TNF-α-induced osteocyte death. Here, we found histological evidence of OLCN colonies in osteoporotic FRI, which was prevented by both HABP-BP-V-Ag+ and HABP-TSPP-V-Ag+ treatment. Taken together, we proposed that HABP hydrogel therapy can reduce osteoclast activation and osteocyte apoptosis, thereby strengthening cortical bone integrity, and preventing bacterial invasion of the cortical bone. With an appropriate dosage of BP, these effects will jointly provide a stable fracture site, promote the callus formation cascades, and facilitate healing in osteoporotic bone (Fig. 9F). Further we evaluated the presence of pro-inflammatory M1 macrophages in the bone tissue with IHC staining for iNOS. At week 8, neither the HABP-BP-V-Ag+ nor HABP-TSPP-V-Ag+ groups showed significant increases in CD68 or iNOS levels in the bone tissue compared to control group (Supplementary Fig. 15). ELISA test shows no significant elevation of TNF-α and IL-6 in both hydrogel treated groups, confirming that the HBAP hydrogel does not trigger prominent immune response (Supplementary Fig. 15). Moreover, H&E staining of the heart, kidney, and liver showed no signs of systemic toxicity following hydrogel treatment, confirming the biocompatibility and localized action of BP-based hydrogels (Supplementary Fig. 16). This underscores the safety and therapeutic potential of BP-based hydrogels as a targeted treatment for osteoporotic FRI.

Methods
Ethics statement
The animal study protocols were approved by the Institutional Animal Care and Use Committee of Nanjing Drum Tower Hospital (Permit number 2021AE01027).

Synthesis of the HA-BP
Referring to the previously reported synthesis method, slight modifications were made. One gram of hyaluronic acid (HA) was dissolved in 100ml of water, and stirred until dissolved, and then 2.15ml of methacrylic anhydride solution was added in an ice bath environment. The pH of the reaction system was maintained at 8.5–9 by dropwise addition of NaOH solution while stirring rapidly, and the reaction proceeded for three hours. The mixture was dialyzed against sodium chloride solution and deionized water for 3 days, then frozen at −80°C, and freeze-dried to obtain MeHA. 2.3g of 2.5mL of N-hydroxysuccinimide (NHS), 3.8g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 0.92g of mercaptoacetic acid were dissolved in 2ml of N,N-dimethylformamide (DMF), and after overnight reaction, 40ml of pyrophosphate buffer (1.12g) was added to maintain pH at 8.5, then reacted in the dark for 24h. After washing with anhydrous ethanol and drying, thiolated BP was obtained. Thiolated BP, MeHA, and tris(2-carboxyethyl)phosphine (TCEP) were dissolved in TEOA buffer with pH 8.5, reacted in the dark for 48h, dialyzed for three days, and then freeze-dried to obtain HABP. The grafting rate of BP onto HA was calculated as 21% by subtracting the double-bond grafting rate of HA-BP (6%) from that of MeHA (27%), representing the quantity of double bonds consumed by thiolated BP during the thiol-ene coupling reaction.

Fabrication of the HABP-BP-V-Ag+ hydrogel
HABP (4% w/v), free BP (200 mM), and Ca(NO3)2 (286 mM) were dissolved in PBS solution and vortexed to form the HABP-BP hydrogel. The HABP-BP-V-Ag+ hydrogel was prepared by adding AgNO3 (20 mM) and Vancomycin (5 mg/ml) to the mixture. The other two hydrogel groups, HABP-TSPP and HABP-TSPP-V-Ag+, were obtained by replacing free BP with TSPP (200 mM). The total volume of the HABP-BP-Ag+ hydrogel was 50 μL for in vivo application.

Rheological measurement
Using a hybrid rheometer, rheological tests were conducted on hydrogel samples with a volume of 100 μL and a thickness of 2 mm. For oscillatory time sweep experiments, the constant strain and frequency were fixed at 1% and 1 Hz, respectively. In the shear strain scan tests, the shear frequency was fixed at 1 Hz, and the shear strain ranged from 0.1 to 500%. For the high and low shear strain scan tests, three cycles of high shear and low shear were performed, with shear strains of 400% and 1%, respectively.

Scanning electron microscope
The hydrogel samples were rapidly frozen in liquid nitrogen and then placed in a freeze dryer for 6h. Afterward, they were rapidly frozen again in liquid nitrogen and sliced with a blade. The freeze-dried sample sections were mounted face-up on copper pillars and coated with a gold/palladium sputter for 60s. Subsequently, scanning electron microscopy (SEM) images were obtained using a field emission scanning electron microscope (Zeiss Merlin) equipped with an X-MaxN 20 EDS system.

Injectability test
Inject 200 μL of hydrogel into a luer-lock syringe with a needle inner diameter of 0.45 mm. Inject the hydrogel into a polytetrafluoroethylene mold in the shape of a leaf, then let it sit for 1 minute before demolding the hydrogel.

Quantification of the release of BP and Vancomycin from the hydrogel
At 37°C, 50 μL of hydrogel was immersed with 2ml PBS buffer. At designated time intervals, 1200 μL of the supernatant was replaced with a new release medium. The concentration of the BP in the release medium was determined through the formation with a Fe(III) ion and measured by UV/Visspectrophotometer at 293 nm wavelength51. The Vancomycin was detected by UV/Visspectrophotometer at the wavelength of 236 nm. The concentration of Ag+ was determined by inductively coupled plasma mass spectrometry (ICP-MS).

Preparation of hydrogel extracts
Briefly, the hydrogels were sterilized by submerging the samples in 70% v/v ethanol and then washed with PBS. Subsequently, 50 μL of the hydrogels was transferred to a 6-well culture plate and incubated with 2.5 mL of specific cell culture medium for BMSC, osteoblasts, and BMM-induced osteoclast for 24h (37°C, 5% CO2, and 95% air).

Primary bone marrow stem cell and osteogenic differentiation
The bone marrow stem cells from both tibiae of 1-month-old SD rats were flushed out and cultured in LDMEM containing 10% FBS and 1% PSN. The BMSC in the second passage was used in these experiments. Then the cytotoxicity was measured by CCK-852. Osteogenic differentiation was induced with dexamethasone (0.1 μM), ascorbate acid (50 μg/ml), and β-glycerophosphate (10 mM). ALP staining was performed on day 7, and ARS staining was performed on day 14. The ALP activity was assessed with the enzyme activity kit (Beyotime, P0321S).

Primary macrophage osteoclastogenesis, TRAP staining, and FITC-labeled phalloidin staining
The bone marrow cells from 6 weeks C57BL/6 mice were filtered through a cell strainer, and the cells were seeded in a T25-cm2 flask for proliferation. After 24h, the cells were collected and resuspended in 5 ml α-MEM with 30 ng/ml macrophage colony-stimulating factor (M-CSF; R&D System), and the adherent cells were classified as osteoclast. After 48h of culture, the cells were induced for osteoclast differentiation in α-MEM with 30 ng/ml and 50 ng/ml NF-κB receptor activators. After 5 days of culture, the cells were fixed with 4% paraformaldehyde and stained with TRAP working solution for 60 min. Then the TRAP-positive cells were observed and imaged by an optical microscope. The differentiated osteoclast was stained with actin labeled with DAPI, and fluorescein isothiocyanate to evaluate the formation of F-actin ring (G1248, Servicebio).

The in vitro antimicrobial effect of the HABP-BP-V-Ag+ hydrogel
The antibacterial was tested with MRSA (ST239). The bacteria strains were recovered and cultured in the TSB medium at 37°C for 24h. Then the bacteria suspension (OD600=0.25) was incubated with 50 μL hydrogel. Then the CFU in the medium was quantified by serial dilution and plate counting. Additionally, to observe the effect of hydrogel on the bacterial biofilm formation, the attached bacteria were cocultured with hydrogel in the confocal dishes for 24h and stained with LIVE/DEAD bacteria viability kit (L13152, Invitrogen). ROS accumulation was measured in 24-h mature MRSA biofilms treated with PBS or hydrogels. After washing and CFU quantification, biofilms were stained with 10 μM DCFHDA (1h, 37°C), and fluorescence intensity normalized to CFU was analyzed via spectrometry.

Quantitative polymerase chain reaction
The total RNA was extracted from the cultured cells with the Trizol reagent, and cDNA was synthesized according to the instructions from reverse transcription. RT-qPCR was performed with the ChamQ SYBR Color qPCR master Mix kit, and the premier sequence was supplemented in Table S1.

Flow cytometry
The MLO-Y4 cells were harvested according to the instructions with Annexin V FITC apoptosis Kit (Beyotime, C1062M). Briefly, the cells were digested and resuspended in a staining buffer, and 1×105 cells were incubated with Annexin V FITC in the dark for 15 min, propidium iodide was added and the apoptosis was detected by flow cytometry. (Accuri C6, BD Biosciences, Franklin Lakes, NJ, USA).

Animal study
Ovariectomy surgery was performed at month 6 and the rats were kept for another 3 months to induce osteoporosis. A 9-month-old Sprague-Dawley rat model with FRI was established with published protocol8. The animals were randomized into OVX, OVX-M (with MRSA), OVX-M-V (treated with systemic Vancomycin), or with hydrogel treatment, including OVX-M-HABP-BP, OVX-M-HABP-BP-V-Ag+, or OVX-M-HABP-TSPP-V-Ag+. The hydrogel treatment was applied 24h after surgery to allow for the infection development. Briefly, 50 μL of the hydrogel was injected with a pipette, and the treatment was applied at 24h post-surgery when the infection was established. Additionally, intraperitoneal injection of vancomycin was started until sacrifice (50 mg/kg body weight twice daily)53.

X-ray radiograph
The anterior-posterior X-rays were taken at week 4 and week 8 post-surgery. The callus morphology, including callus width and callus area was measured in the Image J software according to the established protocol8.

Micro-CT scanning
The femur was harvested and scanned with micro-CT (μCT-40, Scanco Medical, Switzerland). The region of interest was defined as 422 slices at the upper or lower than the fracture line. The image was visualized in the axial plane and the 3D reconstructed views. A separate reconstruction was used to differentiate the old cortical bone from the newly formed low-density bone, and the low-density bone volume (BVl) and high-density bone volume (BVh) were calculated accordingly8.

Histological and histomorphometric analysis
At weeks 4 and 8, femur samples were harvested, fixed in 10% neutral buffered formalin buffer, decalcified in 10% EDTA solution, and embedded in paraffin. A 5 μm slice was obtained for histological staining. A scoring system based on H&E staining was adopted to assess the histological component of the fracture callus54. Briefly, the bone union was defined as 5 grades. Bony union: 0; 1 the majority was new bone: 1; the majority was fibroblast with less new bone: 2; the majority was spindle with less inflammatory cells: 3; and the majority was inflammatory cells: 4. The modified gram staining was used to visualize the bacterial colonies in the bone tissue. The Safranin O-Fast green (SOFG) to quantify the proportion of cartilage area in the fracture callus.

Immunohistochemical staining and immunofluorescence staining
For the immunohistochemical staining, the prepared slides were rehydrated, heated in sodium citrate for antigen retrieval, and blocked with BSA. The primary antibodies of CTSK (1:200, DF6614, Affinity), Collagen I (1:200, AF7001, Affinity), Osteocalcin (1:200, DF12303, Affinity), and TNF-α (1:300, ab6671, Abcam), CD68 (1:200, DF7518, Affinity), iNOS (1:200, AF0199, Affinity), IGF1 (1:200, DF6096, Affinity), TGF-β1 (1:200, BF8012, Affinity), RANKL (1:200, AF0313, Affinity) were applied and incubated overnight, followed by the incubation of the Horseradish peroxidase (HRP)-conjuncted secondary antibody and visualized following reaction with 3,3’-diaminobenzidine (DAB). The positive expression of the protein at the target region of interest was quantified by ImageJ (NIH, USA). For immunofluorescent staining, the sections were incubated with corresponding secondary antibodies and stained with DAPI. The fluorescent images were captured and the fluorescence was quantified with Image-Pro Plus 6.0 software.

TUNEL staining
For TUENL staining, the slides were processed with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay kit (G1507; Servicebio), followed by diaminobenzidine staining, and were then counterstained with haematoxylin.

Bacteria observation and CFU quantification
Under the sterilized condition, the infected femur was harvested, weighed, and homogenized in sterilized PBS solution. The bacteria load in bone samples was calculated by plating in serial dilution and CFU counting. The bacteria load attached to the K-wire was sonicated, and the CFU load was determined in a similar method8. Additionally, the K-wire was carefully removed from the harvested femur and fixed with glutaraldehyde solution, air-dried, mounted, and subject to microstructure observation under SEM.

ELISA
Enzyme-linked immunosorbent assay test was performed to quantify bone turnover markers in the serum, including Cathepsin K, Trap5b, Balp, and Osteocalcin, TNF-α and IL-6.

Mechanical test
At eight weeks post-surgery, the femur was carefully dissected free of muscle. A four-point bending test was used to test the stiffness of the fracture callus. The ultimate load (N), stiffness (N/mm), were calculated based on the stress-displacement curves.

Statistical analysis
The data was presented as mean ± standard deviation. Statistical analysis was performed with GraphPad Prism 8.0 software. An unpaired independent t-test was used for data comparison between the two groups. Data with more than two groups was analyzed by one-way ANOVA analysis with Tukey correction. A two-sided p-value less than 0.05 was considered as statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript presents a novel and clinically relevant strategy to address osteoporotic fracture-related infections (FRI)\nusing a disease-specific nanocomposite hydrogel. The work is thoughtfully designed, combining comprehensive in vitro and\nin vivo evaluations. The authors’ identification of prominent cortical bone lysis, osteocyte apoptosis, and cortical bone\nbacterial colonization in osteoporotic FRI meaningfully advances the field’s understanding of this high-risk condition.\nBuilding upon these findings, the design of a bisphosphonate-based hydrogel with low-dosage BP for targeted antimicrobial\ndelivery and bone preservation is highly translational. Notably, the inclusion of a low-dose BP group strengthens the clinical\nrelevance of the study. Overall, the manuscript is of high quality, and after addressing the points outlined below, it will be\nsuitable for publication in Nature Communications.\n1. Bisphosphonates are well-established in osteoporosis therapy and have been previously incorporated into hydrogels for\nbone regeneration and skin infection models. A comparison with prior BP-based hydrogel systems would help position the\nunique advances and disease-specific relevance of this work more clearly.\n2. The proposed mechanism involving reactive oxygen species (ROS) generation by Ag⁺ is intriguing, but direct\nexperimental evidence quantifying ROS levels in the medium would bolster this claim.\n3. In the bacterial colony counting assay, additional details on how the 3-log and 5-log reductions were calculated—along\nwith the specific control conditions used—would improve reproducibility and interpretation.\n4. One particularly interesting observation is the delayed callus remodeling in the HABP-BP-V-Ag⁺ group. A deeper\ninterpretation of the underlying reasons for this delay would provide valuable mechanistic insight.\n5. For the micro-CT analysis, clarifying how the thresholds for BV-high and BV-low were determined, and what these\ndistinctions mean biologically in the context of bone infection would help readers better understand the findings.\n6. Given the translational potential of this hydrogel system, a short discussion on practical challenges and opportunities for\nreal-world application would enhance the overall impact of the study.\nA few minor points also warrant attention:\n1. The semi-quantitative score mentioned in line 352 should be briefly explained for clarity.\n2. For figures 4 and other figure, labels the significance carefully, especially the comparison between HABP-BP-V-Ag+ and\nHABP-TSPP-V-Ag+ group.\n3. Details on the immunohistochemistry quantification method are needed.\n4. There appears to be a typo in line 605 (\"C57BL/c mice\"—should this be \"C57BL/6\"?).\n5. The spelling of \"MLOY-4\" should be checked and corrected throughout the manuscript.", "reviewer_2": "Thanks for reviewing this article. This study focuses on osteoporotic fracture-related infection (FRI) with significantly clinical\nimplications in orthopedics. To address the limitations of current treatments, the research innovatively developed a bioactive\nnanocomposite hydrogel based on bisphosphonate-modified hyaluronan (HABP). By loading bisphosphonates (BP),\nvancomycin, and silver ions (Ag⁺), the hydrogel achieves dual functions of infection control and bone protection. Overall, this\nstudy demonstrates a high degree of innovation, though some minor revisions are recommended before it can be accepted.\nThe comments and suggestions are detailed below:\n1. Do the degradation products of the hydrogel in vivo exert long-term effects on the organism or trigger immune responses?\n2. What is the grafting rate of BP in HABP?\n3. Since part of the BP in the hydrogel is grafted onto hyaluronic acid while the remainder exists in a free form, has the study\ninvestigated which fraction primarily contributes to its efficacy? Could a control group without BP addition be considered?\n4. What is the curing time of the hydrogel, and does it meet surgical requirements?\n5. Why were both vancomycin and Ag⁺ loaded to achieve antibacterial effects?\n6. Has the potential interaction between vancomycin and the hydrogel’s dynamic internal network (e.g., with BP-grafted\nhyaluronic acid) been considered?", "reviewer_3": "This comprehensive study on the use of dynamically PH-adapting Ag+-Vancomycin-releasing HA gel has numerous\nstrengths such as demonstration of design concepts; anti-bacterial effects; in vivo anti-infection effects; in vivo fracture\nhealing promotion in MRSA-infected fractures; fracture healing assessment by microCT; anti-osteoclastogenic effects.\nSample size, data presentations, Figure preparations are sutstanding.\nMinor concerns are osteoporotic rat fracture healing models because infected nonunions are most common in young active\nmales with tibia fractures (so-called open tibia fracture). Femur has more muscle and soft tissue coverage and infection is\nnot as serious as tibia. Regardless, the presented data sufficiently support the technical concepts and feasibility for future\nclinical use. Overall, The study is outstanding, rigorous, and clinically relevant."}, "author_response": "Response to the Comments from the Reviewers:\nWe sincerely appreciate the reviewers’ positive comments and the feedback provided to\nstrengthen the manuscript. Here we provide a point-by-point response to each of the\nreviewers. The corresponding changes to the revised manuscript have been highlighted\nin yellow font.\nReviewer #1:\nThe manuscript presents a novel and clinically relevant strategy to address osteoporotic\nfracture-related infections (FRI) using a disease-specific nanocomposite hydrogel. The\nwork is thoughtfully designed, combining comprehensive in vitro and in vivo\nevaluations. The authors’ identification of prominent cortical bone lysis, osteocyte\napoptosis, and cortical bone bacterial colonization in osteoporotic FRI meaningfully\nadvances the field’s understanding of this high-risk condition. Building upon these\nfindings, the design of a bisphosphonate-based hydrogel with low-dosage BP for\ntargeted antimicrobial delivery and bone preservation is highly translational. Notably,\nthe inclusion of a low-dose BP group strengthens the clinical relevance of the study.\nOverall, the manuscript is of high quality, and after addressing the points outlined below,\nit will be suitable for publication in Nature Communications.\n1. Bisphosphonates are well-established in osteoporosis therapy and have been\npreviously incorporated into hydrogels for bone regeneration and skin infection models.\nA comparison with prior BP-based hydrogel systems would help position the unique\nadvances and disease-specific relevance of this work more clearly.\nAuthor reply:\nWe appreciate the reviewer’s suggestions. We have now expanded our comparison with\nthe prior BP-based hydrogel systems and clarified the unique features and disease-\nspecific advances of our approach. While prior studies have incorporated BPs into HA-\nbased hydrogels for bone regeneration or infected skin wounds, most designs rely on\nex situ nanoparticles (e.g., calcium phosphate or magnesium silicate) for drug delivery\nor mechanical enhancement. In contrast, our hydrogel leverages in situ self-assembly\nof Ag⁺-Ca²⁺-BP nanoparticles directly within the hydrogel matrix, leading to improved\nmechanical integrity, homogeneity, and controlled antimicrobial release. Furthermore,\nunlike HA-BP/Ag⁺ hydrogels previously developed for wound healing (e.g., Shi et al.¹),\nwhich focus on surface infection control, our system is specifically engineered for\nosteoporotic fracture-related infection (FRI), which is a complex environment\ncharacterized by bone resorption, cortical lysis, and impaired healing. Moreover, we\nfurther enhance the hydrogel function by leveraging BP's inherent biological activity\nand incorporating TSPP for tunable dosage control, allowing for pH-responsive,\ntargeted antibiotic/Ag⁺ release.\nRevised manuscript now reads:\n“Unlike previous studies utilizing HABP with ex situ nanoparticles, including calcium\nphosphate or magnesium silicate 1,2, our work employs in situ self-assembled Ag⁺-Ca²⁺\n-BP nanoparticles within the hydrogel network, yielding superior mechanical\nproperties and stability. Compared with the previously reported HA-BP/Ag⁺ hydrogels\nsynthesized via BP-Ag⁺ coordination for infected wound healing3, our design further\nincorporates BP's intrinsic osteoprotective functions and uses TSPP to enable tunable\nBP dosage. This allows for pH-responsive, targeted antibiotic/Ag⁺ release, and more\nimportantly, positions BP as both a structural and bioactive agent tailored for\nosteoporotic FRI.”\n2. The proposed mechanism involving reactive oxygen species (ROS) generation by\nAg⁺ is intriguing, but direct experimental evidence quantifying ROS levels in the\nmedium would bolster this claim.\nAuthor reply:\nWe thank the reviewer for this valuable suggestion. We have performed quantitative\nanalysis of ROS production within MRSA biofilms treated with our hydrogels to provide\ndirect experimental support for the proposed mechanism. Specifically, we used\nDCFHDA staining to assess ROS accumulation in 24-hour mature MRSA biofilms\nfollowing co-culture with hydrogel formulations. Fluorescence intensity was\nnormalized to CFU counts to account for biomass variation. The results confirmed that\nboth HABP-BP-V-Ag⁺ and HABP-TSPP-V-Ag⁺ treatments significantly increased ROS\nlevels compared to the vancomycin-only formulation (HABP-BP-V), thus supporting\nthe role of Ag⁺-induced oxidative stress as a contributing factor in biofilm disruption\nand bacterial killing. We have now added this data to the revised manuscript.\nRevised manuscript now reads:\n“Further, we found that the co-culture with the HABP-BP-V-Ag+ and HABP-TSPP-V-\nAg+ hydrogel significantly elevated the production of ROS in MRSA biofilms than the\nHABP-BP-V group, confirming that Ag+ release has the potential to trigger ROS\ngeneration that may synergize with vancomycin in biofilm eradication (Figure S8).”\n“ROS accumulation was measured in 24-h mature MRSA biofilms treated with PBS or\nhydrogels. After washing and CFU quantification, biofilms were stained with 10 μM\nDCFHDA (1 h, 37°C), and fluorescence intensity normalized to CFU was analyzed via\nspectrometry. (methods)”\nFigure S8. (A) The representative images of ROS accumulation in MRSA biofilms\ntreated with hydrogels. (B) Quantitative analysis of the ROS fluorescence intensity.\n3. In the bacterial colony counting assay, additional details on how the 3-log and 5-log\nreductions were calculated—along with the specific control conditions used—would\nimprove reproducibility and interpretation.\nAuthor reply:\nThe suggested details of the bacterial CFU assay are added. The HABP-BP-V hydrogel\nachieved an over 2-log reduction in bacterial load (99.9% reduction, from average 8.7\nto 5.9 log CFU/ml) after 24 hours of co-culture compared to Ctr group without\n10\nintervention. This effect was further enhanced in the presence of Ag⁺, achieving a >5-\nlog reduction (from rom average 8.7 to 3.2 log CFU/ml) in bacterial load with the\nRevised manuscript now reads:\n“The HABP-BP-V hydrogel achieved an over 2-log reduction in bacterial load (99.9%\nreduction, from average 8.7 to 5.9 log CFU/ml) after 24 hours of co-culture compared\n10\nto Ctr group without intervention. This effect was further enhanced in the presence of\nAg⁺, achieving a >5-log reduction (from rom average 8.7 to 3.2 log CFU/ml) in\n10\nbacterial load with the HABP-BP-V-Ag⁺ hydrogel compared to control group.”\n4. One particularly interesting observation is the delayed callus remodeling in the\nHABP-BP-V-Ag⁺ group. A deeper interpretation of the underlying reasons for this delay\nwould provide valuable mechanistic insight.\nAuthor reply:\nWe thank the reviewer for highlighting this important observation. We have further\ninterpreted the potential mechanisms involved. Bisphosphonates (BPs) are effective in\nmanaging excessive bone resorption in severe conditions such as osteoporotic fracture-\nrelated infections (FRI). However, excessive dosing can overly inhibit osteoclast\nactivity, and disturb the coordinated osteoclast and osteoblast action cycles that\nremodel the fracture callus. In our model, the combination of grafted and free BP in the\nHABP-BP-V-Ag⁺ hydrogel likely resulted in prolonged osteoclast inhibition, which\ndisrupted this coordinated remodeling cycle, leading to an accumulation of mineralized\ncallus tissue without effective bridging or maturation.\nRevised manuscript now reads:\n“In particular, osteoclasts play a critical role by first resorbing mineralized tissue and\ncartilage, then working in tandem with osteoblasts in coordinated cycles to rebuild\nstrong, functional bone4. If this delicate balance is disrupted by excessive BP, it can\nhinder the timely restoration of proper bone structure and strength.”\n5. For the micro-CT analysis, clarifying how the thresholds for BV-high and BV-low\nwere determined, and what these distinctions mean biologically in the context of bone\ninfection would help readers better understand the findings.\nAuthor reply:\nWe appreciate the reviewer’s suggestions. In response, we have supplemented the\nthreshold of BVh and BVl, and further interpreted the meaning of BVh and BVl in FRI.\nIn bone infection, the distinction between BVh (highly mineralized bone) and BVl\n(newly formed, low-density bone) reflects the dynamic pathological process of\ninfection-driven bone destruction and repair. In addition, upon careful review, we\nidentified an inconsistency in the OVX-M-V group, where the 3D image did not match\nthe corresponding axial view. We have corrected this by replacing the 3D image with\nthe appropriate sample, ensuring consistency between the two visualization methods.\nThis revision provides a more accurate representation of BVh and BVl changes in the\ncontext of impaired fracture healing in FRI (Figure 4).\nRevised manuscript now reads:\n“BVh (threshold from 350 to 1000) represents mature cortical bone that is progressively\neroded by osteoclast overactivation, while BVl (threshold from 165 to 350) is\ncharacterized by disorganized, poorly mineralized bone formation around lytic areas.”\n“The coexistence of BVh loss and BVl formation reveals the uncoupling of normal\nremodeling where destructive processes outpace effective repair.”\n6. Given the translational potential of this hydrogel system, a short discussion on\npractical challenges and opportunities for real-world application would enhance the\noverall impact of the study.\nAuthor reply:\nWe thank the reviewer for the suggestion. We have further elaborated on the\nopportunities that our therapy can be applied to real practice, and the practical\nchallenges that may be countered.\nRevised manuscript now reads:\n“Our proposed adjunctive therapy can synergize with DAIR to improve infection\ncontrol and bone healing. This combined strategy may mitigate catastrophic bone lysis\nand non-union, ultimately enabling implant removal and functional bone regeneration.\nHowever, real-world application faces practical challenges including the optimal\ndosage and timing of intervention that integrates with DAIR, which demands precise\nsurgical and pharmacological coordination. Further preclinical and translational\nvalidation will be essential before advancing this strategy to clinical trials.”\nA few minor points also warrant attention:\n1. The semi-quantitative score mentioned in line 352 should be briefly explained for\nclarity.\nAuthor reply:\nWe have further elaborated on this scoring system.\nRevised manuscript now reads:\n“Further we assess the histological bone healing with tissue quality score, which\nclassifies callus from 0 (bony union, best) to 4 (mostly inflammatory cells, worst), with\nhigher scores indicating poorer callus quality and impaired healing.”\n2. For figures 4 and other figure, labels the significance carefully, especially the\ncomparison between HABP-BP-V-Ag+ and HABP-TSPP-V-Ag+ group.\nAuthor reply:\nWe have checked the labeling of significance, and figure details throughout the\nmanuscript, and labeled the p value between the HABP-BP-V-Ag+ and HABP-TSPP-V-\nAg+ groups accordingly.\n3. Details on the immunohistochemistry quantification method are needed.\nAuthor reply:\nWe have further supplemented the quantification methods in the methods section.\nRevised manuscript now reads:\n“The positive expression of the protein at the target region of interest was quantified by\nImage J (NIH, USA)”\n4. There appears to be a typo in line 605 (\"C57BL/c mice\"—should this be\n\"C57BL/6\"?).\nAuthor reply:\nThe typo has been corrected.\n5. The spelling of \"MLOY-4\" should be checked and corrected throughout the\nmanuscript.\nAuthor reply:\nThe typos have been corrected throughout the manuscript.\nReviewer #2:\nThis study focuses on osteoporotic fracture-related infection (FRI) with significantly\nclinical implications in orthopedics. To address the limitations of current treatments,\nthe research innovatively developed a bioactive nanocomposite hydrogel based on\nbisphosphonate-modified hyaluronan (HABP). By loading bisphosphonates (BP),\nvancomycin, and silver ions (Ag⁺), the hydrogel achieves dual functions of infection\ncontrol and bone protection. Overall, this study demonstrates a high degree of\ninnovation, though some minor revisions are recommended before it can be accepted.\nThe comments and suggestions are detailed below:\n1. Do the degradation products of the hydrogel in vivo exert long-term effects on the\norganism or trigger immune responses?\nAuthor reply:\nWe appreciate the reviewer’s insightful suggestions. To evaluate potential long-term\neffects of hydrogel degradation products in vivo, we performed both histological and\nimmunological assessments. Histologically, we observed that the fracture callus in\nhydrogel-treated groups exhibited normal bone architecture, with comparable\nmineralization and trabecular structure to the uninfected control group (no hydrogel\ntreatment). Importantly, no signs of chronic inflammation or excessive inflammatory\ncell infiltration were detected in the fracture gap at week 8 post-surgery.\nGiven that low-molecular-weight HA degradation products could potentially activate\npro-inflammatory macrophages, we further performed immunohistochemical (IHC)\nstaining for macrophages, including CD68 (pan-macrophage marker) and iNOS (M1\npro-inflammatory marker). Neither the OVX-M-HABP-BP-V-Ag⁺ nor the OVX-M-\nHABP-TSPP-V-Ag⁺ group showed elevated expression of CD68 or iNOS in the\nsurrounding bone tissue when compared to the control, indicating no local pro-\ninflammatory immune activation.\nAdditionally, we supplemented our analysis with ELISA to assess systemic\ninflammatory markers (TNF-α and IL-6), which are key indicators of foreign-body\nresponses. The results showed that levels of these cytokines remained within\nphysiological range and showed no significant difference between hydrogel-treated and\ncontrol animals. Together, these findings robustly demonstrate the safety of HABP-\nbased hydrogels for in vivo applications, with no evidence of notable immune activation.\nRevised manuscript now reads:\n“Further we evaluated the presence of pro-inflammatory M1 macrophages in the bone\ntissue with IHC staining for iNOS. At week 8, neither the HABP-BP-V-Ag+ nor HABP-\nTSPP-V-Ag+ groups showed significant increases in CD68 or iNOS levels in the bone\ntissue compared to control group. ELISA test shows no significant elevation of TNF-α\nand IL-6 in both hydrogel treated groups, confirming that the HBAP hydrogel does not\ntrigger prominent immune response (Figure S12).”\nFigure S12. (A) The IHC staining of CD68 and iNOS in the bone tissues at week 8. (B,\nC) Quantitative analysis of the expression levels. (D, E) The levels of TNF-α and IL-6\nin the rat serum at week 8. *p < 0.05, ****p < 0.001.\n2. What is the grafting rate of BP in HABP?\nAuthor reply:\nThank you for your question. The grafting rate was quantified by comparing the double-\nbond content of MeHA and HA-BP using ¹H NMR spectroscopy. Specifically, the\ndouble-bond grafting rate of MeHA was measured to be 27%, while that of HA-BP was\n6% after the thiol–ene reaction with thiolated BP. Therefore, the difference (21%)\ncorresponds to the fraction of double bonds consumed during BP grafting.\nRevised manuscript now reads:\n“The grafting rate of BP onto HA was calculated as 21% by subtracting the double-\nbond grafting rate of HA-BP (6%) from that of MeHA (27%), representing the quantity\nof double bonds consumed by thiolated BP during the thiol-ene coupling reaction.”\nFigure S1. The NMR spectroscopy of MeHA and HA-BP.\n3. Since part of the BP in the hydrogel is grafted onto hyaluronic acid while the\nremainder exists in a free form, has the study investigated which fraction primarily\ncontributes to its efficacy? Could a control group without BP addition be considered?\nAuthor reply:\nWe thank the reviewer for the valuable question. Our experimental design included two\nkey hydrogel formulations: (1) HABP-BP-V-Ag⁺: containing both grafted BP\n(covalently linked to HA) and free BP (coordinated to Ag⁺/Ca²⁺); (2) HABP-TSPP-V-\nAg⁺: containing grafted BP but replacing the free BP with sodium pyrophosphate\n(TSPP), a structural analogue of BP that lacks osteoclast-inhibiting and anti-apoptotic\nactivity. The in vivo radiographic, histological, and functional evaluation has\nconfirmed that the HABP-TSPP-V-Ag⁺ group yields better bone remodeling of the\nfracture callus compared to HABP-BP-V-Ag⁺. The delayed remodeling observed in the\nlatter group is likely due to over-suppression of osteoclast activity by the combined\naction of free and grafted BP, which may interfere with callus maturation in late-stage\nhealing. These suggest that grafted BP alone is sufficient to achieve the therapeutic\nbenefit, while excessive free BP may be counterproductive to long-term bone\nregeneration. Furthermore, our animal study included a group with the current\nstandard of care, systemic antibiotic treatment only without any BP. This group showed\npoor infection control and impaired fracture healing, underscoring the essential role of\nthe bioactive components in our hydrogel formulations.\nAs the reviewer proposed, we considered creating an additional hydrogel group without\nany BP (either grafted or free). However, technical limitations in grafting TSPP or\nsimilarly inert analogues onto HA due to the absence of appropriate reactive groups,\nthereby preventing us from generating a BP-free but chemically comparable control\nhydrogel. Thus, HABP-TSPP-V-Ag⁺ served as our closest functional control, allowing\nus to dissect the contribution of free versus grafted BP while maintaining consistent\nhydrogel properties.\nRevised manuscript now reads:\n“In this study, we observed significantly delayed callus remodeling in rats treated with\nHABP-BP-V-Ag+ hydrogel. In this group, both the free BP and grafted BP may exert\nbiological functions in vivo, and excessive BP may cause prolonged inhibition of\nosteoclasts, delaying callus maturation.”\n“Our study demonstrated that even a low dosage of BP in the TSPP-based formulation\nis sufficient in reducing excessive osteoclast activation and promoting bone healing,\nsuggesting that the grafted BP is sufficient to achieve desirable therapeutic effects in\nvivo.”\n4. What is the curing time of the hydrogel, and does it meet surgical requirements?\nAuthor reply:\nThe injectable hydrogel system we developed is designed for rapid intraoperative\napplication. It is formed by simply mixing two aqueous precursor solutions: one\ncontaining HA-BP, BP/TSPP, and vancomycin, and the other containing Ca²⁺ and Ag⁺\nions. Upon vortexing, the components self-assemble into a morphologically stable\nnanocomposite hydrogel within 10 seconds (Figure S4a). This rapid gelation process\nensures that the hydrogel can be readily prepared in the operating room without any\nexternal energy input or complex curing procedures. Moreover, for clinical simulation,\nwe demonstrated that this hydrogel can be loaded into syringes and injected directly\ninto irregular tissue defect sites, such as bone cavities. After injection into a mold, the\nhydrogel immediately adopted the shape of the defect, and upon demolding, it exhibited\nexcellent self-healing capacity, reforming into an integrated structure (Figure S4b-c).\nThis confirms that the hydrogel meets key criteria for injectability, conformability, and\nstructural stability, all of which are essential for surgical application.\nRevised manuscript now reads:\n“The hydrogel fabrication process entails a straightforward mixing of precursor\nsolutions and vortexing, resulting in a stable hydrogel formation within 10 seconds.\nSubsequent syringe-based injection enables direct delivery to the injury site, offering\nsubstantial advantages for clinical translation (Figure S4).”\nFigure S4. (A) A mixed solution of HA-BP, BP/TSPP, and Vancomycin quickly forms a\nwhite opaque hydrogel within 10 seconds after the addition of a mixed solution of\ncalcium and magnesium ions, and is quickly vortex-mixed. (B) The hydrogel loaded in\na syringe can be injected into a mold for rapid shaping. (C) The hydrogel removed from\nthe mold maintains its shape.\n5. Why were both vancomycin and Ag⁺ loaded to achieve antibacterial effects?\nAuthor reply:\nWe thank the reviewer for the critical comment. The rationale for co-loading\nvancomycin and Ag⁺ stems from the unique challenges of treating osteoporotic FRI.\nIn\nosteoporotic bone, reduced vascularity, impaired immune responses, and the presence\nof implant-associated biofilms significantly limit the efficacy of systemic or single-\nagent antibiotic therapy. Vancomycin, while effective against planktonic\nStaphylococcus aureus, exhibits limited penetration into mature biofilms, and its\nbactericidal efficiency is diminished in compromised tissue environments.\nAs we have illustrated in the in vitro antimicrobial test, we found that single antibiotics\nshowed a bacteria reduction activity of 2 log CFU, which may be insufficient to\n10\nsuccessfully eradicate the infection in vivo. In contrast, co-delivery of vancomycin and\nAg⁺ via the hydrogel achieved a 5 log CFU and significantly enhanced biofilm\n10\ndisruption. These effects are attributed to the ability of Ag⁺ to disrupt bacterial\nmembranes and enhance biofilm matrix permeability, thereby facilitating vancomycin\naccess to embedded bacteria. Therefore, we selected the combination of vancomycin\nand Ag+ to secure a thorough bacteria clearance in a highly resistant osteoporotic FRI\nmodel.\nRevised manuscript now reads:\n“The combination of vancomycin and Ag⁺ was used to enhance antibacterial efficacy\n(achieving a 5 log CFU reduction) and biofilm disruption, as vancomycin alone was\n10\ninsufficient for complete infection clearance in osteoporotic FRI due to compromised\nimmunity and biofilm resistance.”\n6. Has the potential interaction between vancomycin and the hydrogel’s dynamic\ninternal network (e.g., with BP-grafted hyaluronic acid) been considered?\nAuthor reply:\nWe thank the reviewer for this thoughtful and important question. In our initial design,\nwe anticipated that vancomycin would be encapsulated within the hydrogel through\nphysical entrapment, and its release would be primarily governed by the acid-triggered\ndissociation of the dynamic coordination network. However, to further explore whether\nspecific molecular interactions exist between vancomycin and bisphosphonate (BP)-\ngrafted hyaluronic acid, we conducted ¹H NMR spectroscopy in D O with both\n2\nvancomycin and BP present.\nThe analysis unexpectedly revealed chemical shift perturbations in the α-carbon\nhydrogen signals of vancomycin's functional groups, including amide, hydroxyl, and\ncarboxyl moieties, suggesting the presence of hydrogen bonding interactions with the\nphosphate groups of BP. These results indicate that, beyond physical encapsulation,\nnon-covalent interactions (likely hydrogen bonding) contribute to the loading and\nretention of vancomycin within the hydrogel matrix. These interactions may help\nexplain the sustained release profile observed in our release studies, as hydrogen\nbonding could reduce the diffusivity of vancomycin in the hydrogel. We have now\nincorporated these findings into the revised manuscript.\nRevised manuscript now reads:\n“1H NMR analysis showed chemical shift changes in vancomycin’s α-carbon hydrogens\n(amide, hydroxyl, carboxyl), suggesting hydrogen bonding with BP phosphates, which\nmay account for the sustained release of Vancomycin (Figure S6).”\nFigure S6. In the NMR spectrum (recorded in D O) of vancomycin mixed with BP, the\n2\nhydrogens on the α-carbons of functional groups prone to hydrogen bonding (amide,\nhydroxyl, and carboxyl groups) all exhibited chemical shift changes.\nReviewer #3:\nThis comprehensive study on the use of dynamically PH-adapting Ag+-Vancomycin-\nreleasing HA gel has numerous strengths such as demonstration of design concepts;\nanti-bacterial effects; in vivo anti-infection effects; in vivo fracture healing promotion\nin MRSA-infected fractures; fracture healing assessment by microCT; anti-\nosteoclastogenic effects. Sample size, data presentations, Figure preparations are\nsutstanding.\nMinor concerns are osteoporotic rat fracture healing models because infected\nnonunions are most common in young active males with tibia fractures (so-called open\ntibia fracture). Femur has more muscle and soft tissue coverage and infection is not as\nserious as tibia. Regardless, the presented data sufficiently support the technical\nconcepts and feasibility for future clinical use. Overall, The study is outstanding,\nrigorous, and clinically relevant.\nAuthor reply:\nWe sincerely thank the reviewer for their insightful comments. We fully agree that open\ntibial fractures in young, active males represent a clinically high-risk population for\ninfected non-union, largely due to limited soft tissue coverage and impaired vascularity.\nHowever, osteoporotic FRI, though less common, is also clinically significant,\nparticularly in elderly female patients, and has been associated with poorer outcomes\nand limited treatment options. Epidemiologically, large-scale clinical studies also show\nan increasing incidence of open fractures in elderly female patients5. Though less\ncommon, the osteoporotic FRI in aged females does occur clinically, and these cases\ntypically have worse clinical outcomes that warrant more effective interventions, which\nfurther underscores the importance of developing disease-specific therapies for this\nsubgroup.\nIn this study, we employed a well-established femur fracture model that closely mimics\nthe clinical phenotype of FRI, including key features such as bone lysis, periosteal\nreaction, and non-union. This model has been widely adopted in prior biomaterials and\northopedic infection studies6,7, ensuring its experimental consistency and translational\napplicability. Nonetheless, we acknowledge the reviewer’s important point and\nrecognize that gaps remain between preclinical FRI models and real-world clinical\nscenarios. We have revised the manuscript to reflect this limitation and to highlight the\nneed for future validation studies in open tibia fracture models.\nRevised manuscript now reads:\n“Additionally, while our femur fracture model effectively recapitulates key FRI features,\nit may not fully represent the higher-risk clinical scenario of open tibial fractures\ncommonly seen in young males, where the insufficient soft tissue coverage may further\nincrease the risk of infection and non-union. Future investigations are warranted to\nvalidate the therapeutic efficacy of our hydrogel in such challenging clinical settings.”\nReference:\nhyaluronan and calcium phosphate nanoparticles. Biomaterials 35, 6918-6929,\ndoi:10.1016/j.biomaterials.2014.05.003 (2014).\nNetwork. ACS Appl Mater Interfaces 11, 46233-46240, doi:10.1021/acsami.9b17627 (2019).\nCoordination Chemistry for Wound Healing. Adv Healthc Mater 7,\ndoi:10.1002/adhm.201700973 (2018).\nReviews Rheumatology 11, 45-54 (2015).\nThe epidemiology of open fractures in adults. A 15-year review. Injury 43, 891-897,\ndoi:10.1016/j.injury.2011.12.007 (2012).\nimprove bone-fracture healing in rats. Nat Med 22, 1160-1169, doi:10.1038/nm.4162 (2016).\nfurther delays healing. Bone & joint research 11, 49-60 (2022)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have fully addressed the concerns of reviewers and revised the manuscript accordingly. It is recommended for publication now.", "reviewer_2": "This study presents a novel bisphosphonate (BP)-based nanocomposite hydrogel (HABP-BP/TSPP-V-Ag⁺) designed to address the clinical challenge of fracture-related infection (FRI) in osteoporotic patients. The hydrogel serves dual purposes: 1) Anti-infective action: it is capable of releasing Ag⁺ ions and vancomycin under pathological pH conditions, thereby effectively eradicating MRSA biofilms; 2) Bone protection and healing promotion: the released BP suppresses osteoclast activation, prevents apoptosis of bone cells, preserves cortical bone integrity, and facilitates the restoration of the fracture healing process. The therapeutic efficacy of the hydrogel was validated in an osteoporotic FRI rat model. Notably, this approach innovatively incorporates pyrophosphate (TSPP) to partially substitute free BP, addressing the issue of delayed callus remodeling associated with high-dose BP administration and enabling controlled therapeutic delivery. The comments and suggestions are detailed below:\n1. Considering that the HABP-TSPP-V-Ag⁺ group demonstrates a more pronounced effect on callus remodeling, in addition to the reduction of BP dosage, it remains to be determined whether TSPP itself contributes to this process by promoting mineralization or through other mechanisms. Further investigation should be given to elucidate this precise role and underlying mechanism of TSPP in enhancing callus remodeling.\n2. It is recommended to further supplement the analysis of variations in mineralization induction capacity and the expression profiles of key factors associated with bone remodeling across the hydrogel groups.\n3. The performance of the material in the open fracture model needs further evaluation. It is suggested to test the physical stability of the hydrogel in an environment with wound exudate, potential contamination, and mechanical stress, such as swelling, erosion resistance and degradation, etc.\n4. Please note the writing format: On line 645, CO2 should be modified to CO₂."}, "author_response": "Response to the Comments from the Reviewers:\nWe sincerely appreciate the reviewers’ positive comments and the feedback provided to\nstrengthen the manuscript. Here we provide a point-by-point response to each of the\nreviewers. The corresponding changes to the revised manuscript have been highlighted\nin yellow font.\nReviewer #1:\nThe authors have fully addressed the concerns of reviewers and revised the\nmanuscript accordingly. It is recommended for publication now.\nAuthor reply:\nWe sincerely thank the reviewer for the positive evaluation of our work and for\nrecommending it for publication. We greatly appreciate the constructive feedback\nprovided throughout the review process, which has significantly strengthened the\nquality and clarity of our manuscript.\nReviewer #2\nThis study presents a novel bisphosphonate (BP)-based nanocomposite hydrogel\n(HABP-BP/TSPP-V-Ag⁺) designed to address the clinical challenge of fracture-related\ninfection (FRI) in osteoporotic patients. The hydrogel serves dual purposes: 1) Anti-\ninfective action: it is capable of releasing Ag⁺ ions and vancomycin under pathological\npH conditions, thereby effectively eradicating MRSA biofilms; 2) Bone protection and\nhealing promotion: the released BP suppresses osteoclast activation, prevents apoptosis\nof bone cells, preserves cortical bone integrity, and facilitates the restoration of the\nfracture healing process. The therapeutic efficacy of the hydrogel was validated in an\nosteoporotic FRI rat model. Notably, this approach innovatively incorporates\npyrophosphate (TSPP) to partially substitute free BP, addressing the issue of delayed\ncallus remodeling associated with high-dose BP administration and enabling controlled\ntherapeutic delivery.\n1. Considering that the HABP-TSPP-V-Ag⁺ group demonstrates a more pronounced\neffect on callus remodeling, in addition to the reduction of BP dosage, it remains to be\ndetermined whether TSPP itself contributes to this process by promoting mineralization\nor through other mechanisms. Further investigation should be given to elucidate this\nprecise role and underlying mechanism of TSPP in enhancing callus remodeling.\nAuthor reply:\nWe thank the reviewer for this valuable suggestion. We totally agree with the reviewer\nand have conducted additional experiments to investigate the effects of TSPP on bone\nmineralization. Results from the Alizarin Red S Staining demonstrated that TSPP did\nnot promote mineralization in vitro, supporting its role as an inert control within our\nhydrogel formulation.\nIn the remolding process of fracture healing, active osteoclast initiate the process by\nresorbing early bone tissue1, thereby releasing matrix-stored cytokines such as IGF1\nand TGF-β1 that promote osteogenic differentiation. We supplement experiments to\nevaluate the expression of IGF1 and TGF-β1 in the fracture callus. In the OVX-M-\nHABP-BP-V-Ag+ group, we found significantly reduced expression of TGF-β1 and\nIGF1 compared to the OVX control group, consistent with excessive suppression of\nosteoclast activity at high BP dosage. In contrast, the OVX-M-HABP-TSPP-V-Ag+\ngroup displayed TGF-β1 and IGF1 expression comparable to that of the OVX control\ngroup. Concurrently, the expression of receptor activator of nuclear factor κB ligand\n(RANKL), a key cytokine that promotes osteoclast-driven bone resorption, exhibited no\nsignificant differences between the OVX-M-HABP-BP-V-Ag⁺ and OVX-M-HABP-\nTSPP-V-Ag⁺ groups. Taken together, these findings suggest that the enhanced callus\nremodeling observed in the OVX-M-HABP-TSPP-V-Ag⁺ group can be primarily\nattributed to the reduced dosage of BP, which preserves the activity of osteoclasts and\nallows for the physiological coupling between osteoclast-mediated resorption and\nosteoblast-driven bone formation, rather than any direct effect of TSPP.\nFigure S9. (A) Representative images and (B) quantification of ARS staining of bMSCs\ntreated with TSPP at day 14 post osteogenic differentiation.\nFigure S14. (A) IHC staining of RANKL, IGF1, and TGF-β1 in the fracture callus at\nweek 8. (B, C, D) Quantitative analysis of the expression levels. *p < 0.05, **p < 0.01,\n**p < 0.001, ****p < 0.0001.\nRevised manuscript now reads:\n“In contrast, TSPP showed no detectable effect on the mineralization of bMSCs, as\nconfirmed by Alizarin Red S staining (Fig. S9)”.\n“At week 8, we identified that the number of osteoclasts in the bone tissue around\nfracture site was significantly decreased in HABP-BP-V-Ag+ group compared to the\nHABP-TSPP-V-Ag+ (Fig. 8A, D). Consistently, in the OVX-M-HABP-BP-V-Ag+ group,\nthe expression of bone matrix-deposited cytokines, including TGF-β1 and IGF1, was\nmarkedly reduced compared to the control group, whilst the expression of RANKL was\nremained comparable. In contrast, the OVX-M-HABP-TSPP-V-Ag⁺ group exhibited\nTGF-β1 and IGF1 levels similar to those of the OVX control, demonstrating preserved\nosteoclast-osteoblast coupling (Fig S14). These findings suggest that the enhanced\ncallus remodeling observed in the HABP-TSPP-V-Ag⁺ group is primarily attributable\nto the reduced BP dosage, which prevents excessive osteoclast suppression and sustains\nthe coupled remodeling process.”\n2. It is recommended to further supplement the analysis of variations in mineralization\ninduction capacity and the expression profiles of key factors associated with bone\nremodeling across the hydrogel groups.\nAuthor reply:\nWe appreciate the reviewer's valuable suggestion. In response, we conducted additional\nexperiments to assess the mineralization-inducing capacity of the hydrogels. Our\nfindings reveal that HABP-BP, HABP-TSPP, HABP-BP-V-Ag⁺, and HABP-TSPP-V-\nAg⁺ hydrogels all significantly enhanced mineralization capacity compared to the\ncontrol group, with comparable mineralization induction capacity between HABP-BP-V-Ag⁺\nand HABP-TSPP-V-Ag⁺ groups.\nWe further analyzed the expression profiles of key osteogenic genes associated with\nbone remodeling using qPCR. The results indicate that the enhanced mineralization is\nassociated with the upregulated expression of Col1a1 and Bgalp (osteocalcin), which\ncan be attributed to the known osteogenic effect of BP reported previously2,3.\nAdditionally, we evaluated the expression of key coupling cytokines IGF1 and TGF-β1.\nReduced levels of IGF1 and TGF-β1 were detected in the HABP-BP-V-Ag⁺ group\nrelative to the control group, whereas expression was preserved in the HABP-TSPP-V-\nAg⁺ group. These findings demonstrate that the preserved activity of osteoclasts in the\nTSPP-based formulation enables osteoclast–osteoblast coupling, thereby facilitating\nmore effective callus remodeling.\nFigure S8. (A) The ARS staining test of bMSC treated with hydrogel extracts at day 14\npost osteogenic differentiation. (B) The quantification of the ARS staining. (C-G) The\nexpression of genes associated with bone remodeling at day 14.\nRevised manuscript now reads:\n“The alkaline phosphatase (ALP) staining and ALP activity assays revealed\ncomparable ALP activity in HABP-BP-V-Ag+ or HABP-TSPP-V-Ag+ hydrogel treated\ngroups. (Fig. 2A, B). Additionally, HABP-BP, HABP-TSPP, HABP-BP-V-Ag⁺, and\nHABP-TSPP-V-Ag⁺ hydrogels all exhibit higher mineralization capacity compared to\nthe control group, which was accompanied by upregulated expression of Col1a1 and\nBglap (Fig. S8). In contrast, TSPP showed no detectable effect on the mineralization of\nbMSCs, as confirmed by Alizarin Red S staining (Fig. S9)”.\n3. The performance of the material in the open fracture model needs further evaluation.\nIt is suggested to test the physical stability of the hydrogel in an environment with\nwound exudate, potential contamination, and mechanical stress, such as swelling,\nerosion resistance and degradation, etc.\nAuthor reply:\nWe sincerely appreciate the reviewer's insightful suggestion, which highlights an\nimportant consideration for clinical translation. We fully recognize that assessing the\nhydrogel's performance under real-world conditions is a critical aspect of its validation,\nwhile we were unable to provide additional data currently due to time and resource\nlimitations. In our subsequent research, we plan to systematically evaluate the\nhydrogels' properties, specifically examining their behaviors in environments that\nsimulate real wounds, including those with wound exudate, potential contamination,\nand mechanical stress. This will help us gain a more comprehensive understanding of\nits practical applicability.\nRevised manuscript now reads:\n“Further studies will examine the hydrogel's stability in clinically relevant\nenvironments, such as wound exudate, potential contamination, and mechanical stress,\nto further validate its applicability in open fracture scenarios.”\n4. Please note the writing format: On line 645, CO-2 should be modified to CO₂.\nAuthor reply:\nThe format error has been corrected.\nReferences\nrepair: The cellular picture. Semin Cell Dev Biol 19, 459-466,\ndoi:10.1016/j.semcdb.2008.07.004 (2008).\ndifferentiation of bone marrow stromal cells: a preliminary study. Clin Orthop Relat Res 467,\n3121-3128, doi:10.1007/s11999-008-0409-y (2009).\nof mesenchymal stem cells. J Bone Miner Res 22, 1603-1611, doi:10.1359/jbmr.070701 (2007)."}, {"round": 3, "reviewer_comments": {"reviewer_2": "In this work, authors developed a BP-based bioactive nanocomposite hydrogel for treating osteoporotic FRI. The authors systematically investigated the cascade cell effect of drug loaded hydrogel on infectious fracture healing, which is very interesting and significant. In addition, authors have made the point-by-point response and revised their manuscript accordingly. After moderate modification, I recommend its publication in Nature Communications.\nOn page 9, lines 194, bMSCs should be BMSCs"}, "author_response": "Author reply:\nWe thank the reviewer for the positive comments and valuable suggestion. The format\nerror has been corrected throughout the manuscript."}]

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