Dynamic Modulation of Immune-Neural Axis via Controlled Magnesium-Releasing Nanocapsules Accelerates Cranial Bone Regeneration

Dynamic Modulation of Immune-Neural Axis via Controlled Magnesium-Releasing Nanocapsules Accelerates Cranial Bone Regeneration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dynamic Modulation of Immune-Neural Axis via Controlled Magnesium-Releasing Nanocapsules Accelerates Cranial Bone Regeneration Yilin Mao, Qixuan He, Tianle Li, Jiusi Guo, Kelvin W.K Yeung, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8441498/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Critical-sized cranial defects present a significant challenge in the field of bone tissue regeneration. Despite the emergence of various approaches to promote new bone formation, the clinical outcomes remain suboptimal. In this study, we developed an inorganic-organic hybrid bioink suitable for 3D printing of photocurable scaffolds. This bioink incorporates our novel nanocapsules with a shell consisting of amorphous whitlockite and PEG coating, which endows the scaffolds with superior mechanical strength and osteogenic capacity. These nanocapsules enable a dual-phase Mg2⁺ release profile to facilitate the initial pro-inflammatory activation of macrophages followed by a seamless transition to a pro-regenerative phenotype. We further showed that this dynamic Mg²⁺ delivery strategy significantly outperformed traditional sustained-release approaches in supporting cranial bone regeneration. Moreover, the controlled immunomodulation through this tailored Mg²⁺ delivery more closely mimics the natural healing process, promoting the activation of sensory nerves, which is essential for effective bone regeneration. Overall, our study demonstrated the potential of our nanocapsules as a cost-effective approach for the dynamic modulation of the immune-neural axis, offering valuable insights for the future design of bioactive materials for cranial bone regeneration. Magnesium Immunomodulation Macrophage Bone regeneration Sensory nerve 3D printing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The repair of critical-sized cranial bone defects resulting from trauma, infections, and tumors remains a significant clinical challenge due to the lack of adequate structural support for cell migration and tissue regeneration [1, 2]. Although bone grafting materials and titanium mesh have been extensively used in clinical practice to address the issue, the regeneration process is often time-consuming, and the clinical outcomes can be suboptimal in some patients [3]. Cranial bone regeneration is a complex process involving coordinated interactions among multiple biological systems and cell populations, which are intricately orchestrated by sophisticated cellular crosstalk [4]. As the initial phase of bone healing, the acute inflammation stage plays a pivotal role in determining regenerative outcomes [5]. Both excessive inhibition and prolonged activation of the inflammatory response have been associated with impaired new bone formation. Therefore, there is increasing interest in targeting the innate immune response, particularly macrophages that dominate this process, to promote bone repair [6]. During the acute inflammatory stage, the infiltration of macrophages into the bone defect areas contributes to the secretion of various pro-inflammatory cytokines that are critical for recruiting bone-forming cells, such as mesenchymal stem cells (MSCs) and endothelial cells (ECs) [7-9]. Moreover, pro-inflammatory cytokines like prostaglandin E 2 (PGE 2 ) and interleukin-1 beta (IL-1β) have been shown to activate sensory nerves, which have recently been recognized as critically involved in bone regeneration [10, 11]. Following the initial pro-inflammatory polarization, macrophages within the bone defects need to shift toward anti-inflammatory phenotypes to resolve inflammation and establish a pro-regenerative microenvironment conducive to bone healing [12]. Therefore, instead of biasing macrophages towards a particular phenotype, next-generation orthopedic biomaterials should be designed to present bioactive cues that facilitate the pro-inflammatory activation and a seamless transition to anti-inflammatory states within the bone microenvironment [9]. Magnesium ions (Mg 2 ⁺) have been extensively studied as bioactive agents that promote bone tissue regeneration [13-15]. In our previous studies, we elucidated the biphasic role of Mg 2 ⁺ in modulating innate immune responses during bone regeneration [16]. Specifically, higher concentrations of Mg 2 ⁺ induce pro-inflammatory cytokine production, which recruits osteogenic progenitors and enhances their osteogenic activities. Conversely, lower Mg 2 ⁺ concentrations exert anti-inflammatory effects that facilitate osteogenic differentiation of progenitors and extracellular matrix mineralization [17]. Additionally, we previously demonstrated that Mg 2 ⁺ contributes to the activation of sensory nerves during bone healing [18]. On the one hand, calcitonin gene-related peptide (CGRP) released from activated sensory neurons can directly contribute to new bone formation [19]. On the other hand, sensitization of nociceptive neurons can trigger the interoceptive pathway to mediate the control of new bone formation in the central nervous system [18, 20]. Collectively, these findings underscore the importance of dynamically controlling Mg 2 ⁺ release throughout the bone healing process to maximize its regenerative potential. In this study, we developed novel polyethylene glycol (PEG)-coated nanocapsules with an amorphous whitlockite shell (PNC). These nanocapsules can be readily three dimensional (3D) printed into photocurable scaffolds with patient-specific shapes for cranial bone regeneration. The incorporation of PNC not only improved the mechanical properties of the scaffolds but also enabled sustained slow release of Mg 2+ , which significantly enhanced the osteogenic properties. More importantly, additional Mg 2+ can be encapsulated into these nanocapsules to achieve a two-stage Mg 2+ release profile. Our in vitro studies demonstrated that an initial burst release of Mg 2+ from Mg 2+ -loaded nanocapsules (Mg-PNC) promoted the pro-inflammatory activation of macrophages, while the subsequent mild and sustained Mg 2+ release facilitated the transition of macrophages toward anti-inflammatory phenotypes. In a rat cranial critical-sized bone defect model, this sequential immune modulation by Mg-PNC resulted in superior new bone formation compared to PNC, which predominantly exhibited anti-inflammatory effects throughout the healing process. Additionally, the dynamic immune microenvironment shaped by Mg-PNC enhanced reinnervation of trigeminal sensory neurons into the defect area and upregulated CGRP expression, contributing to inflammation resolution and osteogenesis. Our findings demonstrate that Mg-PNC is a cost-effective system for the controlled delivery of bioactive ions, with its superior osteogenic performance stemming from the dynamic modulation of the immune-neural axis. These results provide valuable insights for designing next-generation bioactive materials for cranial bone defect repair. Materials and Methods 2.1 Fabrication of PNC and Mg-PNC Calcium nitrate tetrahydrate (Ca(NO 3 ) 2 ·4H 2 O), and magnesium nitrate hexahydrate (Mg(NO 3 ) 2 ·6H 2 O) were sourced from Xihua (China). Magnesium chloride hexahydrate (MgCl 2 ·6H 2 O), trisodium phosphate dodecahydrate (Na 3 PO 4 ·12H 2 O), sodium dodecyl sulfate (SDS) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) were purchased from Sigma-Aldrich (USA). Polyethylene glycol (PEG, molecular weight 20000) and Poly (ethylene glycol) diacrylate (PEGDA, molecular weight 400) were obtained from Aladdin (China). Camphor quinone (CQ) was purchased from Esstech (USA). Nanocapsules (NCs) were prepared as follows: 8.86 g Ca(NO 3 ) 2 ·4H 2 O and 3.21 g Mg(NO 3 ) 2 ·6H 2 O were dissolved in 50 mL deionized (DI) water to form the salt solution. Simultaneously, dissolved 0.47 g SDS in 50 mL DI water for the SDS solution. Combined the SDS solution with the salt solution and stirred for 30 min. Then dissolved 11.4 g Na 3 PO 4 ·12H 2 O in 100 mL of DI water, added this solution to the mixture, adjusted the pH to above 10, then continued stirring at 60 °C for 24 h. Finally, centrifuged the mixture and washed until neutralization to harvest NCs. To synthesize PNCs, after preparing the salt solution and SDS solution, dissolved 5 g PEG in 50 mL of DI water, the PEG solution was added to the mixture, stirring for an additional 30 min. The subsequent steps were the same as those for NC nanoparticles. To encapsulate additional Mg²⁺ into the PNC nanoparticles, magnesium chloride (MgCl 2 ) solutions with mass fractions of 10%, 20%, 30%, and 40% were prepared using MgCl 2 ·6H 2 O, mixed with 2 g of PNCs and stirred for 4 h. Afterward, centrifuged the mixture and dried it overnight to obtain Mg-PNC nanoparticles containing varying MgCl 2 loadings. 2.2 Fabrication of PNC and Mg-PNC scaffolds For the synthesis of PNC scaffolds and Mg-PNC scaffolds, 0.025 g CQ and 0.025 mL DMAEMA were first mixed in 5 mL PEGDA and stirred for 1 h. Nanoparticles were then gradually added to the mixture under magnetic stirring at 350 rpm. To facilitate uniform dispersion, the suspension was bath-sonicated (Xinzhi, China) for 10 min, followed by continuous stirring for 10 h to obtain PNC and Mg-PNC bioinks. For 3D-printed scaffolds used in vivo experiments, PNC or Mg-PNC bioink was loaded into a biological 3D printer (REGENHU, Switzerland), equipped with a 30G nozzle. Printing parameters were set with a printing speed of 20 mm/s, gas pressure of 30 kPa, and the printing temperature of 25 °C. The scaffolds were printed as a single layer based on a pre-designed model with pore diameters of 2 mm. Following printing, the scaffolds were crosslinked using a 405 nm light-crosslinking lamp for 1 min. For PNC or Mg-PNC specimens used for in vitro study, the bioink was extruded into circular stainless-steel molds (6 mm × 6 mm × 1 mm) and subjected to photocrosslinking to ensure consistent shape. 2.3 Characterization of NC, PNC, Mg-PNC and 3D printed scaffolds The surface morphology and diameter of NC, PNC, Mg-PNC were observed using scanning electron microscope (SEM; SU-1500, HITACHI, Japan). Energy dispersive spectroscopy (EDS; SDD3310, iXRF, USA) was employed to analyze the elemental distribution and compositional purity of PNCs, Mg-PNCs and their 3D-printed scaffolds. Structural changes in NCs and PNCs with different MgCl 2 concentrations were characterized by X-ray diffraction (XRD; MiniFlex600, Rigaku, Japan; Cu Kα, 40 kV, 15 mA) and Fourier Transform Infrared Spectroscopy (FT-IR; Spectrum Two, PerkinElmer, USA). The structural morphology and lattice composition of NC, PNC and Mg-PNC were investigated using a high-resolution transmission electron microscope (TEM; FEI Tecnai G2 F20 S-Twin 200 kV, USA), with selected area electron diffraction (SAED) performed synchronously to further characterize their crystal structure features, including lattice parameters and crystalline orientation. Barrett-Joyner-Halenda (BJH) analysis was performed using a gas sorption analyzer (Autosorb 6100, Anton-Paar, Austria). Thermogravimetric analysis (TGA) was conducted by thermogravimetric analyzer (Q50 TGA, TA Instruments, USA). Mechanical testing of the scaffolds was conducted using a universal test instrument (E3000, Electropuls, USA). 2.4 Swelling, degradation, and ion release experiments of PNC and Mg-PNC For swelling, degradation and ion release studies, the prepared PNC and Mg-PNC discs were used as samples, and the initial weight of each disc was first measured and recorded as W 1 . The prepared PNC and Mg-PNC discs were immersed in 2 mL of phosphate-buffered saline (PBS; Thermo Fisher Scientific, USA) and incubated at 37 °C, with sampling conducted at predetermined time points: 1 h, 2 h, 6 h, 12 h, 24 h (1 d), 48 h, 72 h (3 d), 7 d, 14 d, and 28 d. For the swelling test, at each time point, the samples were taken out, blotted gently to remove surface water, and immediately reweighed to obtain the wet weight as W 2 . The swelling ratio (SR) was calculated using Formula (1): SR = (W 2 – W 1 )/W 1 × 100%. For the degradation test, the soaked discs were placed in a vacuum freeze dryer for 2 days, and the dry weight after degradation was recorded as W 3 . The degradation rate (DR) was calculated using Formula (2): DR = (W 3 – W 1 )/W 1 × 100%. Meanwhile, the supernatants were collected as the degradation solution according to the above timeline. The concentrations of calcium ions (Ca 2+ ), Mg²⁺, and phosphate ions (PO 4 3- ) in the collected degradation solutions were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES; Spectro Arcos, Germany). 2.5 Cell culture The human monocyte cell line THP-1 (TIB-202, ATCC, USA) was cultured in RPMI 1640 medium (Thermo Fisher Scientific, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, USA) and 1% (v/v) penicillin/streptomycin (P/S; Thermo Fisher Scientific, USA). To differentiate THP-1 cells into macrophages, 50 nM phorbol 12-myristate 13-acetate (PMA; MCE, USA) was used for 24 h induction, followed by switching to high-glucose Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher Scientific, USA) for further culture at 37 °C with 5% CO 2 to eliminate the continuous stimulation of PMA. hTERT-immortalized MSCs (hMSCs) were provided by Prof. Wei Qiao, and maintained in high-glucose DMEM with 10% FBS and 1% (v/v) P/S. Mouse dorsal root ganglion cell line (MED17.11 cell) was obtained from University of Sheffield, UK. For the proliferation phase, MED17.11 cells were cultured in DMEM/F12 medium supplemented with Glutamax (Thermo Fisher Scientific, USA), 10% FBS, 1% P/S, 5 ng/mL interferon gamma (IFN-γ; R&D, USA), and 0.5% chicken embryonic extract (Sera Lab, South Korea) in a 33 °C incubator with 5% CO 2 . For the differentiation of MED17.11 cells, the culture medium was switched to DMEM/F12 with Glutamax, supplemented with 10% FBS, 1% P/S, 10 ng/mL fibroblast growth factor 2 (FGF2, R&D, USA), 0.5 mM diButyrylcAMP (db-cAMP; Sigma-Aldrich, USA), 25 μM forskolin (Sigma-Aldrich, USA), 5 μg/mL Y-27632 (Cayman, USA), 100 ng/mL nerve growth factor (NGF; R&D, USA), and 10 ng/mL glial cell line-derived neurotrophic factor (GDNF; R&D, USA). These cells were cultured at 37 °C with 5% CO 2 . For the PGE2-treated MED17.11 group, PGE2 (MCE, USA) was added to the culture supernatant at a final concentration of 1 μM to stimulate cells, which were then maintained in the medium at 37 °C for 1 day. For co-culture studies between PNC/Mg-PNC and macrophages, THP1-derived macrophages were first directly co-cultured with specimens in complete medium under standard cell culture conditions (37℃, 5% CO₂) to simulate cell response to the material at early inflammation stage (day 0-3); then the specimens were incubated in fresh complete medium alone for an additional 3 days and used to culture new THP1-derived macrophages to simulate the cell response to material at late inflammation resolution stage (day 6-9). 2.6 Biocompatibility of PNC and Mg-PNC The biocompatibility of PNC and Mg-PNC was evaluated in accordance with ISO 10993-5:2009. THP-1-induced macrophages and hMSCs were cultured with sterile 0.1 g/mL material extracts for 3 or 7 days. Biocompatibility assessments were performed using 96-well plates at a cell density of 5 ×10 3 cells per well. Cell proliferation was assessed by the Cell Counting Kit-8 (CCK-8 assay; MCE, USA). The optical density (OD) values of CCK-8 treated cells were measured at a wavelength of 450 nm by a microplate spectrophotometer (SpectraMax 340, Molecular Devices, USA). 2.7 Cell adhesion assay The adhesion and morphology of THP-1 cells and hMSCs on PEGDA, PNC, and Mg-PNC were observed via SEM. The PEGDA discs were fabricated using the same mold as the PNC and Mg-PNC discs. Cells were seeded onto the surfaces of these discs in 24-well plates at a density of 1×10 5 cells per well and allowed to settle naturally. After 1 day of co-culture, each sample was gently rinsed with PBS and fixed overnight at 4°C using 2.5% glutaraldehyde. The samples were then dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%). Subsequently, all samples were sputter-coated with platinum (Pt) prior to SEM imaging. 2.8 Alkaline phosphatase (ALP) assay The p-nitrophenyl phosphate (p-NPP) method was conducted to assess the impacts of Mg 2+ or macrophage-derived conditional medium on the ALP activity of hMSCs. At day 3 or day 7, hMSCs were subjected to lysis with 0.2% Triton X-100 at 4 °C for 2 h. The cell lysate was centrifuged, and the resulting supernatant was collected for subsequent assays using an ALP detection kit (Sigma-Aldrich, USA). Total cellular protein content was determined via the BCA Protein Assay Kit (ThermoFisher Scientific, USA). Relative ALP activity was normalized against the total protein content of each sample, with final results expressed as units per minute per gram of total protein (U/min/g·protein). 2.9 Real Time-qPCR (RT-qPCR) RT-qPCR was used to evaluate the expression levels of h IL10, h PTGS2, h IL8, h TNF, h CCL5, h TGFB1, h IL1B, h MRC1, h RUNX2, h ALP, h COL1A1, m Calca, m Trpv1, m Ep4, m Tubb3, m Gap43. Primer sequences were provided in Table S1 of the supplementary information. Briefly, total RNA was extracted from harvested hMSCs, THP-1 induced macrophages or MED17.11 cells using RNeasy Mini Kit (Qiagen, Germany). Complementary DNA (cDNA) was obtained via reverse transcription kit (Takara, Japan) with thermal cycler (ProFlex PCR System, Thermo Fisher Scientific, USA). cDNA was then amplified by a RT-qPCR system (QuantStudio 6 Flex Real-Time PCR System, Thermo Fisher Scientific, USA), with each reaction performed in a final volume of 10 µL. Housekeeping genes h GAPDH (for human samples) or m Gapdh (for mouse samples) were used as internal references for data normalization. 2.10 Western blot To obtain total protein, cells were lysed in ice-cold RIPA lysis buffer (Thermo Fisher Scientific, USA) and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA), centrifuged for 10 min at 4°C to remove debris. Protein concentrations were determined by using BCA protein assay kit (Thermo Fisher Scientific, USA), and the protein extracts were heat-denatured in sodium dodecysulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime, China). Then the protein samples were separated by 10% SDS-PAGE (ACE, China) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with tris-buffered saline with Tween-20 ( TBST) containing bovine serum albumin ( BSA, Goldbio, USA) for 2 h, then incubated with primary antibodies: anti-ALP (Abcam, ab307726, 1:1000, UK), anti-OPN (Abcam, ab8448, 1:1000, UK), anti-Runx2 (Abcam, ab76956, 1:1000, UK), and anti-GAPDH (CST 2118S, 1:1000, USA). After overnight incubation with the primary antibodies on a shaker at 4°C, the membranes were washed 3 times with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, which included Anti-rabbit IgG, HRP-linked Antibody, (CST 7074P2, 1:5000, USA) and Anti-mouse IgG, HRP-linked Antibody (CST 7076P2, 1:5000, USA) for 1 h. Finally, the membranes were immersed in HRP chemiluminescent substrate (Thermo Fisher Scientific, USA) for 1 min, protein signals were visualized using a chemiluminescence imaging system (Amersham ImageQuant 800, China). 2.11 Neurite tracing and measurement MED17.11 neuron images were captured using an inverted microscope (Eclipse Ti-S, Nikon, Japan) with 4× and 10× objective lenses. ImageJ software equipped with the Simple Neurite Tracer plugin was employed to trace and measure neurites. 3 fields of view were randomly selected for counting, with at least 50 neurons randomly selected per view. To avoid the interference of neuronal interaction, neurons in contact with others were excluded from analysis. Neurites with a length of 100 μm or longer were defined as elongated neurites. The average axon length, the percentage of neurons with elongated neurites and the average length of the longest neurites per neuron were calculated to assess innervation capacity. 2.12 Animal surgery All animal protocols in this study were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong (No. 5959-21). Eight-week-old female Sprague-Dawley (SD) rats weighing 200–270 g were provided by HKU CULATR and kept in an environment with a 12h light/dark cycle, constant temperature of 22 °C and relative humidity of 50–60%. Throughout the experiment, the rats had unrestricted access to food and water. Critical-sized cranial bone defect models with a diameter of 6 mm were established in SD rats to study the osteogenesis effects of scaffolds. Rats were anesthetized with ketamine (67 mg/kg, Alfamine, Alfasan International B.V., Holland) and xylazine (6 mg/kg, Alfazyne, Alfasan International B.V.). After exposing the surgical field, a 6 mm diameter trephine (Meisinger, Germany) was used to create the defect. To evaluate the osteogenic capacity of PNC, rats with cranial bone defects were randomly assigned to the sham group or PNC group. To assess the osteogenic capacity of Mg-PNC, the rats were divided into the PNC scaffold group or Mg-PNC scaffold group. In scaffold groups, circular scaffolds with a diameter of 6 mm were implanted into the defect area. After wound closure, the rats were immediately administered subcutaneous injections of Oxytetracycline (30 mg/kg) and Burtemgenic (0.05 mg/kg). The rats were sacrificed at week 1 and week 8 post-surgery for further micro computed tomography (μCT) and histological analysis. 2.13 Fluorochrome labeling To assess bone regeneration and remodeling in the defect areas of PNC scaffolds, calcein green and xylenol orange were administered sequentially. Calcein green (5 mg/kg, Sigma-Aldrich, USA) was injected subcutaneously into the surgical region of rats at week 1 post-surgery, followed by xylenol orange (90 mg/kg, Sigma-Aldrich, USA) at week 3 post-surgery. The fluorescent signals from these labels were visualized using a fluorescence microscope (Nikon ECLIPSE 80i, Japan). 2.14 μCT analysis Following sacrifice, cranial specimens were collected and scanned using high-resolution μCT (Skyscan 1276, Bruker, USA). The scan parameters were set to 80 kV, 190 µA, with a resolution of 13 µm, averaging 2 images. The reconstruction data were obtained using NRecon software. Based on these data, a region of interest (ROI) corresponding to the 6 mm diameter defect area was selected for analyzing new bone formation. Bone volume/tissue volume (BV/TV) was analyzed using CT-Analyzer software. Additionally, 3D reconstructed micro-CT images and a 3D model of the newly formed bone tissue in the defect area were generated using CTvol software. 2.15 Histological analysis Following the μCT scan, cranial bone specimens were subjected to decalcification with ethylenediaminetetraacetic acid (EDTA), gradient dehydration, and paraffin embedding. The paraffin-embedded specimens were sectioned into 5-μm-thick slices using a rotary microtome (RM2155, Leica, Germany). Hematoxylin and eosin (H&E) staining and Masson's trichrome staining (Solarbio, China) were performed to evaluate bone regeneration. Stained sections were imaged using a stereomicroscope (SMZ18, Nikon, Japan). 2.16 Immunohistochemistry (IHC) analysis IHC staining was conducted to assess immune modulation, nerve innervation, and osteogenic effects. The relevant antibodies included anti-Sp7 (Abcam, ab22552, 1:200, UK), anti-CGRP (Abcam, ab81887, 1:200, UK), anti-CD68 (Abcam, ab283654, 1:200, UK), anti-OPN (Abcam, ab183910, 1:200, UK), and anti-OCN (Abcam, ab93876, 1:200, UK). For the cranial bone paraffin sections, after routine deparaffinization, antigen retrieval was performed using proteinase K (Sigma-Aldrich, USA) at 37 °C for 10 min, followed by blocking with donkey serum at room temperature for 1 h. After overnight incubation with the primary antibody at 4°C, secondary antibodies conjugated to Alexa Fluor 488 donkey anti-goat IgG (Thermo Fisher Scientific, 230114, 1:400, USA) or Alexa Fluor 647 donkey anti-rabbit IgG (Thermo Fisher Scientific, 150067, 1:400, USA) were applied, along with Hoechst 33342 (Thermo Fisher Scientific, 2747528, 1:500, USA) for nuclear staining. Trigeminal ganglia (TG) were harvested from the sacrificed rats, fixed in 10% formalin solution, dehydrated in gradient sucrose solutions, and embedded in optimal cutting temperature (OCT; Sakura Finetek, USA) compound for cryosectioning. The samples were then sectioned to a thickness of 10 μm using a cryostat (CM1860 UV, Leica, Germany) and were subjected to IHC staining with the anti-CGRP primary antibody (Abcam, ab81887, 1:200, UK). Confocal fluorescence images were acquired using the confocal laser scanning microscope (LSM 900, Zeiss, Germany). 2.17 Statistical analysis All quantitative data are presented as mean ± standard deviation (SD), and statistical analyses were performed using GraphPad Prism software. Comparisons between two groups were conducted using paired or unpaired t-tests, while comparisons among multiple groups were assessed using one-way or two-way ANOVA, followed by Tukey’s or Sidak’s post hoc test respectively. Statistical significance was indicated as follows: ns (not significant, p ≥ 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Results 3.1. Engineering hybrid nanocapsules for 3D-printable scaffold To synthesize capsule-shaped nanoparticles for controlled release, SDS was added as a foaming agent during the self-assembly of whitlockite precursors. Simultaneously, PEG was added to modify the surface of the amorphous whitlockite shell (Figure 1A). Unlike unmodified nanocapsules (NC), which tend to aggregate and precipitate, PEG-coated nanocapsules (PNCs) can be homogenously dispersed in the PEGDA (Figure 1B). TEM and SEM revealed that both NC and PNC exhibited characteristic capsule-like morphologies with diameters ranging from 30 to 60 nm (Figure 1C). The shell structure, approximately 10 nm thick, was confirmed to be weakly crystalline via SAED. The pore size measured from N 2 adsorption-desorption isotherms using the BJH method indicated that PNC exhibited a more prominent porous structure, with pore diameters between 20 and 50 nm (Figure 1D). XRD analysis identified the characteristic diffraction peaks of NC and PNC to both locate at 2θ = 32°, with the peak for PNC being broader and less sharp than that of NC, suggesting lower crystallinity influenced by PEG addition during hydrothermal synthesis (Figure 1E). TGA revealed a more significant mass loss of PNC between 100 °C to 150 °C, indicating the decomposition of PEG moieties (Figure 1F). The FT-IR spectra confirmed the major component of the nanocapsules as whitlockites, with characteristic P–O bending vibration peaks at 603.70 cm -1 and 563.20 cm -1 observed in NC (Figure 1G). In PNC, these peaks merged into a single peak at 572.84 cm -1 , suggesting the presence of PEG coating. Next, we tested the printability of PNCs by mixing them with PEGDA, which is generally considered not suitable for extrusion-based 3D printing due to its low viscosity and high fluidity [21]. We showed that PEGDA incorporated with PNC enabled 3D printing of complex structures (Figure 1H) and multilayer scaffolds (Figure 1I). The surface topography of the scaffold observed by SEM showed the homogeneous distribution of nanocapsules in the printed scaffold, resulting in increased surface roughness (Figure 1J). Mechanical testing using three-point bending, tensile, and compressive tests demonstrated that the incorporation of nanocapsules improved the mechanical properties of the printed structures (Figure 1K). 3.2. The incorporation of PNCs promotes osteogenic performance Despite being approved by the FDA for biomedical applications, the bioinert nature of PEGDA is known to hinder protein adsorption and cell attachment [22, 23]. We therefore evaluated the biological performance of the printed scaffolds modified with PNC. The cell viability of hMSCs was significantly higher in the PNC group compared to the control or pure PEGDA group (Figure 2A). SEM and confocal microscopy revealed that, unlike on pure PEGDA surfaces, cells attached to PNC-modified PEGDA exhibited a spindle shape with more prominent extensions of lamellipodia and filopodia (Figure 2B). Moreover, the addition of PNC also contributed to enhanced osteogenic differentiation of hMSCs, evidenced by significantly increased ALP activity (Figure 2C), upregulated expression of ALP, OPN, and RUNX2 at the protein level (Figure 2D), as well as increased expression of osteogenic marker genes, such as ALP , OPN , and COL1A1 (Figure 2E). Using ICP-OES, we confirmed that PNCs provided a slow and sustained Mg 2+ release (Figure S1), which likely contributes to its improved osteogenic effects. To further assess the in vivo performance of PNC, 3D-printed PNC scaffolds were implanted into rat critical-sized cranial defects (Figure S2). Calcein green/xylenol orange labeling showed that PNC scaffolds induced significant new bone formation at the early stage of bone healing, whereas minimal regeneration was observed in defects without scaffolds (Figure 2F). Additionally, immunofluorescent staining showed increased expression of osteopontin (OPN) and osteocalcin (OCN) in the PNC group than in the sham controls (Figure 2G). The enhanced new bone formation was further confirmed by H&E staining (Figure 2H) and μCT analysis (Figure 2I), which showed nearly double the bone volume in the defects grafted with PNC-incorporated scaffolds relative to sham controls (Figure 2J). 3.3. Dual-phase Mg 2+ release kinetics from Mg-PNC To further enhance the osteogenic performance of PNC, we managed to encapsulate additional Mg 2+ by soaking nanocapsules in MgCl 2 solution before printing (Mg-PNC, Figure 3A). This enabled a dual-phase Mg 2+ release kinetics: a rapid, burst Mg 2+ release from within the nanocapsule structure, followed by a sustained, mild Mg 2+ release resulting from the degradation of amorphous whitlockite shell. SEM-EDS mapping as well as the Mg/Ca ratio confirmed a uniform increase in magnesium content in Mg-PNC compared to that in PNC (Figure S3A-C). XRD, FT-IR, and TEM analyses indicated that Mg 2+ encapsulation did not alter the physicochemical properties or structures of PNC (Figure S3D-F). The surface topography of scaffolds printed using PNC and Mg-PNC was similar under SEM, displaying a hierarchical microstructure resembling natural bone units (Figure 3B) and uniform element distribution (Figure 3C and S4A & B). Quantitative analysis showed a significant increase in magnesium content in Mg-PNC scaffolds compared to PNC scaffolds (Figure 3C). We then employed ICP-OES to compare the ion release kinetics of the printed scaffolds. Unlike the constant Mg 2+ release profile of PNC, Mg-PNC exhibited a dynamic two-stage Mg 2+ release profile, with an initial burst release reaching approximately 5.4 mM within 6 h and a subsequent sustained release at a much lower level (Figure 3D). Interestingly, Mg-PNC also significantly elevated local Ca 2+ levels compared to those in PNC, likely due to hydration effects associated with Mg 2+ encapsulation [24]. Concomitantly, the concentration of PO 4 3- dropped significantly more slowly in the Mg-PNC group than in the PNC group. Moreover, the degradation and swelling rates of PNC and Mg-PNC only differed from each other within the first 2 h (Figure 3E and S4C), which might reflect the initial burst Mg 2+ release. Overall, the degradation was approximately 4% for PNC and 8% for Mg-PNC. Since the major difference between PNC and Mg-PNC lies in their Mg 2+ release kinetics in the first few days, which coincides with the macrophage-dominated inflammation phase [9], we then evaluated the cellular response of THP-1-derived macrophages to both PNC and Mg-PNC. Cell viability of the PNC group and the Mg-PNC group was both higher than 80%, with no significant difference between groups (Figure 3F). SEM images demonstrated that macrophages adhered better to nanocapsule-containing PEGDA than to PEGDA alone (Figure 3G). Indeed, the macrophages on the surface of Mg-PNC exhibited more pseudopodia extensions, suggesting their more activated status induced by high Mg 2+ level. 3.4. Tailored Mg 2+ release profile enhances bone regeneration through the modulation of macrophages To evaluate the bone regenerative efficacy, scaffolds incorporating PNC or Mg-PNC were implanted into rat cranial critical-sized defects. μCT data analysis at week 8 demonstrated significantly greater bone volume in the Mg-PNC group compared to that in the PNC group (Figure 4A & B). H&E staining and Masson's trichrome staining confirmed increased new bone formation with Mg-PNC scaffolds (Figure 4C). At week 1 post-operation, both PNC and Mg-PNC scaffolds led to significant immune cells accumulation at the bone defect area. Newly regenerated woven bone can be observed at the defect margin, especially in the Mg-PNC group. At week 8 post-operation, there was more mature bone in the Mg-PNC group compared with the PNC group. The enhanced osteogenic property of Mg-PNC compared to PNC was also testified by the significantly increased number of Osterix (Osx) positive osteogenic cells detected within the defect area at week 1 after the operation (Figure 5A & B). Additionally, IHC staining revealed an increased number of CD68-positive macrophages in the Mg-PNC group relative to the PNC group (Figure 5C & D), indicating a more active immune response triggered by dual-phase Mg²⁺ release. To investigate the dynamic modulation of Mg-PNC on macrophages, we cultured THP-1-derived macrophages with specimens immersed in culture medium for different durations. Specifically, specimens immersed for the first three days represent the early microenvironment at the active inflammatory phase, while specimens already immersed for six days were used to simulate the late microenvironment during the inflammation resolution stage [25, 26]. We found that the early-stage microenvironment created by Mg-PNC induced a more robust pro-inflammatory response than PNC, with a 7-fold increase in IL1B expression and a 3-fold increase in PTGS2 expression, alongside downregulation of anti-inflammatory genes, such as IL10 and MRC1 (Figure 5E). The expressions of CCL5 and IL8 , which are both known to be critical to cell recruitment and angiogenesis [17, 27], were also significantly elevated in the Mg-PNC group compared to the PNC group. Conversely, the late-stage microenvironment induced by Mg-PNC exhibited a more prominent anti-inflammatory effect than that induced by PNC, as it significantly upregulated the expression of IL10 , TGFB1 , and MRC1 without provoking excessive upregulation in the expression of IL1B and PTGS2. The distinct effects on macrophage polarization achieved by Mg-PNC demonstrated its capability to dynamically modulate the immune microenvironment throughout the bone healing process. Our data indicate that Mg-PNC facilitates the sequential pro-inflammatory activation of macrophages and their seamless transition to anti-inflammatory phenotypes for effective bone regeneration. 3.5. Mg-PNC triggers the activation of the immune-neuro axis In addition to the direct effects of macrophages on bone-forming cells, alterations in the local immune microenvironment may sensitize the sensory nerves present in the injured area, leading to the modulation of the immune-neuro axis [18]. We then asked whether dual-phase Mg 2+ release from Mg-PNC activates this axis. Immunofluorescent staining showed that CGRP-positive sensory nerves were highly co-localized with CD68 + macrophages and was significantly elevated in the Mg-PNC group relative to the PNC group (Figure 6A & B). Since the cranial bone defect area is primarily innervated by sensory neurons within the V1 (ophthalmic nerve) and V2 (maxillary nerve) branches of the TG [28] (Figure 6C), we then determined the expression of CGRP in TG. Immunofluorescent staining data revealed a significant increase in the number of CGRP + neurons within the V1 and V2 branches of TG in the Mg-PNC group compared to the PNC group (Figure 6D & E). To confirm whether Mg-PNC stimulates sensory neurons through the modulation of macrophages, we treated MED17.11-derived sensory neurons with supernatant harvested from the co-culture of THP-1-derived macrophages with or without scaffolds (Figure 6F). At 12 and 24 h, the average axon length of neurons (Figure 6G), the proportion of neurons with elongated axons (Figure 6H), and the length of the longest neurite (Figure 6I) were all significantly increased in the Mg-PNC group compared with the PNC and control groups. These findings confirmed that Mg-PNC promotes axon extension through the modulation of macrophages. Additionally, we examined the expression of genes associated with nociceptive sensitization (i.e., CALCA , TRPV1 , and EP4 ) and axon projection (i.e., GAP43 and TUBB3 ). These genes were significantly upregulated in the Mg-PNC group rather than in the PNC group when compared to the control group (Figure 6J). Given the recent discovery of the immunomodulatory role of neuropeptides, such as CGRP [18, 29], our data suggest that Mg-PNC might activate the sensory nerves through the early pro-inflammatory responses, with neuron-derived CGRP playing a key role in resolving inflammation (Figure 6K). Discussion 3D printed scaffolds have become a widely adopted strategy for bone tissue regeneration, particularly in cases involving critical-sized bone defects. Here in this study, we developed a novel hybrid bioink with excellent printability, mechanical strength, and osteogenic performance. More importantly, the capsule structure of the nanoparticles enables controlled release of bioactive agents, such as trace ions, small molecules, and nucleic acids. Through surface modification with PEG, we successfully integrated inorganic whitlockite nanoparticles with organic PEGDA polymer to form a cohesive bioink. Therefore, the implantation of scaffolds fabricated using this hybrid bioink did not induce any undesirable inflammatory response caused by nanoparticle agglomeration or phagocytosis by immune cells [30, 31]. Furthermore, compared to traditionally used hydroxyapatite nanoparticles, the nanocapsules developed in this study possess an amorphous whitlockite shell, which exhibits superior regenerative potential and biodegradability [32, 33]. As a result, the incorporation of PNC significantly enhanced the osteogenic properties of PEGDA, which is otherwise considered bioinert [34, 35]. In our study, we first tested the osteogenic property of PNC, which provides a single-phase sustained Mg 2+ release throughout the bone healing process. Despite the evident osteogenic properties observed in vitro and in vivo, the regeneration outcomes still seem suboptimal, with the critical-sized calvarial defects incompletely healed at week 8 after the injury. We hypothesized that the constant anti-inflammatory effects provided by this Mg 2+ delivery scheme might not be the most effective to accelerate bone regeneration. Indeed, depletion of macrophages during early bone healing stages blunted inflammatory responses and impaired new bone formation [36, 37]. Additionally, administration of non-steroidal anti-inflammatory drugs, which disrupt inflammatory responses, has also been associated with compromising fracture healing [38]. Therefore, we encapsulated fast-releasing Mg 2+ within nanocapsules to produce an additional wave of rapid Mg 2+ release, which was intended to promote the pro-inflammatory activation of macrophages. Our findings demonstrated that the dual-phase Mg 2+ release strategy, which enables dynamic immunomodulation, significantly accelerated the bone healing process to achieve complete closure of the critical-sized bone defects within 8 weeks. While our findings underscore the critical role of inflammatory response in bone repair, it is important to note that timely resolution of inflammation is equally crucial to this process, as prolonged presence of pro-inflammatory factors can inhibit osteogenesis [5]. In this study, we exploited the concentration-dependent effects of Mg 2+ on macrophage polarization to facilitate the transition from pro-inflammatory to anti-inflammatory phenotype. Our data demonstrated that the early microenvironment created by Mg-PNC upregulates various pro-inflammatory cytokines, including IL-1β, PGE2, IL-8, and CCL5. They positively contribute to the recruitment of host stem cells, an early event decisive to the outcomes of osteogenesis [9, 39]. In contrast, the late microenvironment created by Mg-PNC did not induce the same level of pro-inflammatory cytokine expression, which is beneficial considering their potential inhibitory effects on osteoblast differentiation [40, 41]. Moreover, the late-stage microenvironment shaped by Mg-PNC contributes to a dramatically different pro-regenerative niche for bone healing, manifested by the upregulation of IL-10, CD206, and TGF-β. Additionally, the controlled slow Mg 2+ release at the late bone healing phase is also preferred to prevent adverse effects, such as the inhibition of osteoblast activity and the mineralization of extracellular matrix [9, 42]. The cranial bone, especially the highly regenerative periosteum area, is densely innervated by sensory fibers [43]. Recent studies have highlighted their roles in cranial bone regeneration [44-46]. Our data demonstrated their close relationship with CD68 + macrophages in the cranial bone and their being sensitized by the pro-inflammatory microenvironment induced by Mg-PNC. This aligns with previous observations showing that macrophage and sensory nerves interact robustly to form an integrated neuro-immune axis involved in pain sensitization [47]. The pro-inflammatory cytokines upregulated by Mg-PNC, such as IL-1β and PGE2, have been reported to activate sensory neurons through actions on Transient Receptor Potential Vanilloid 1 (TRPV1) and Prostaglandin E receptor 4 (EP4) [48, 49]. As a result of the sensation of these inflammatory cytokines associated with pain, sensory neurons responsively secrete neuropeptides like CGRP [18, 50], which were more prominent in the Mg-PNC group compared to the PNC group. The activation of the sensory nerve during injury has been reported to be critical to tissue regeneration, as it contributes to inflammation resolution [51], revascularization [52], regulation of osteoblasts [53, 54], and the control of bone healing within the central nervous system [18, 55]. Therefore, our observation on the increased axon projection and upregulation of CGRP in the injured area and TG confirmed the involvement of the immune-neural axis in bone regeneration induced by Mg-PNC. Conclusion In summary, our study presents an inorganic-organic hybrid bioink featured by the incorporation of our innovative PEG-coated nanocapsules with an amorphous whitlockite shell. It can be used for 3D printing of scaffolds with superior mechanical and osteogenic properties for repairing critical-sized defects in the calvaria. Moreover, we showed that the nanocapsules enable a dual-stage Mg 2 ⁺ release to strategically promote the pro-inflammatory activation of macrophages and seamlessly facilitate their anti-inflammatory polarization. The osteogenic performance of this dynamic macrophage modulation induced by a tailored Mg 2 ⁺ release kinetics outperforms that achieved by constant anti-inflammatory modulation with a sustained, slow Mg 2 ⁺ release. Additionally, we demonstrated that the dual-phase Mg 2 ⁺-releasing Mg-PNC better mimics the natural inflammatory response in the bone healing process, contributing to the activation of sensory neurons to enhance new bone formation. The findings of our study underscore the potential of Mg-PNC as a cost-effective approach to promote cranial bone regeneration and provide valuable insights for the development of new bioactive materials that target the immune-neural axis. Declarations Ethical approval and consent to participate All animal protocols in this study were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong (No. 5959-21). Consent for publication Not relevant. Data availability statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no conflicts of interest. Funding This work was financially supported by funding from the Research Grants Council, the Government of the Hong Kong SAR (Collaborative Research Fund No.C7003–22Y and General Research Fund No.17118425 to W.Q.), the Food and Health Bureau, the Government of the Hong Kong SAR (No.09201466 to W.Q.), National Natural Science Foundation of China (No.82201124 to W.Q.), National Natural Science Foundation of China/Research Grants Council Joint Research Scheme (N_HKU721/23 to W.Q.); Hong Kong Innovation Technology Fund (ITS/256/22 to W.Q.), Shenzhen Science and Technology Innovation Committee Projects (Nos. SGDX20220530111405038 to W.Q.), Guangdong Basic and Applied Basic Research Foundation (2023A1515011963 to W.Q.). Authorship contribution statement Yilin Mao: Methodology, Investigation, Visualization, Data curation, Writing – original draft. Qixuan He: Methodology, Investigation, Visualization. Tianle Li: Methodology, Writing – review and editing. 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Supplementary Files SupplementaryInformation.docx Graphicalabstract.docx Scheme1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 22 Mar, 2026 Reviews received at journal 22 Mar, 2026 Reviewers agreed at journal 16 Mar, 2026 Reviewers agreed at journal 13 Mar, 2026 Reviewers agreed at journal 12 Mar, 2026 Reviews received at journal 20 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers invited by journal 06 Jan, 2026 Editor assigned by journal 27 Dec, 2025 Submission checks completed at journal 27 Dec, 2025 First submitted to journal 24 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8441498","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":570950635,"identity":"2605b031-d7e7-4728-aa20-f3eeae03d551","order_by":0,"name":"Yilin Mao","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Yilin","middleName":"","lastName":"Mao","suffix":""},{"id":570950638,"identity":"041ef9c9-2b01-4b18-aa73-6f1b0370f180","order_by":1,"name":"Qixuan He","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Qixuan","middleName":"","lastName":"He","suffix":""},{"id":570950640,"identity":"1a6d38c0-7e7b-4502-a4a0-96b65bad893c","order_by":2,"name":"Tianle Li","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Tianle","middleName":"","lastName":"Li","suffix":""},{"id":570950643,"identity":"e4a13242-6420-401b-a568-d01f11cd8fb2","order_by":3,"name":"Jiusi Guo","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Jiusi","middleName":"","lastName":"Guo","suffix":""},{"id":570950645,"identity":"8d8999bf-753a-4142-9449-c9df3d22da63","order_by":4,"name":"Kelvin W.K Yeung","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Kelvin","middleName":"W.K","lastName":"Yeung","suffix":""},{"id":570950648,"identity":"886fe4c4-7e23-4d39-aba9-317f9074add9","order_by":5,"name":"Yuxiong Su","email":"","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Yuxiong","middleName":"","lastName":"Su","suffix":""},{"id":570950652,"identity":"9ba79425-4725-4927-862a-7255f4507dce","order_by":6,"name":"Jie Shen","email":"","orcid":"","institution":"Peking University Shenzhen Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Shen","suffix":""},{"id":570950657,"identity":"a00f0ca0-7e32-4dfe-9d45-292b0b6a6281","order_by":7,"name":"Xianglong Han","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xianglong","middleName":"","lastName":"Han","suffix":""},{"id":570950661,"identity":"ec18d619-0c47-4acb-a839-be840ad9c5a8","order_by":8,"name":"Jian Wang","email":"","orcid":"","institution":"Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Wang","suffix":""},{"id":570950664,"identity":"8899f4bc-c8ff-4379-b5bf-5163bcb6ebac","order_by":9,"name":"Wei Qiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAo0lEQVRIiWNgGAWjYDCCA2BkkyDBwMDG2ECCljQStQDBYRK08N0+vPHAj4rzeZIzEtgeziBGi+S5tIKDPWduF0tLJLAbbiBGi8EZHoMDvG23E+dJJLBJPiBWy8G/bedI1HKYt+1A4myQFqIcJnmGreCwzJnkxJk9D9skifI+3xnmzR/fVNglzjiefEyyhxgtILdBaSIjElnLKBgFo2AUjAIcAAAiWTjidVX10AAAAABJRU5ErkJggg==","orcid":"","institution":"The University of Hong Kong","correspondingAuthor":true,"prefix":"","firstName":"Wei","middleName":"","lastName":"Qiao","suffix":""}],"badges":[],"createdAt":"2025-12-24 09:53:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8441498/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8441498/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99781830,"identity":"936c6c1a-f005-4a64-b6aa-72d86c842748","added_by":"auto","created_at":"2026-01-08 10:47:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1124454,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioink modified by PEG-coated nanocapsule (PNC) exhibits superior printability and mechanical properties. \u003c/strong\u003eA) Schematic diagram for the synthesis of NC and PNC from whitlockite precursors. B) Photos showing the dispersion of NC and PNC in PEGDA; the black arrow indicates the aggregation and sedimentation of NC in PEGDA. C) Representative TEM (scale bar = 100 nm and 10 nm), SAED (scale bars = 10\u003csup\u003e-1\u003c/sup\u003e nm), and SEM (scale bar = 200 nm) images of as-prepared nanocapsules. D) N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption isotherms of NC and PNC; the inset shows the BJH analysis for pore size distribution. E) XRD patterns, F) TGA analysis, and G) FT-IR spectra of NC and PNC. H) Complex and tissue-shaped structures printed using PNC containing PEGDA. Scale bar = 5 mm. I) Single- and multi-layered scaffolds printed using PNC containing PEGDA. J) SEM images showing the surface morphology of photocured PEGDA with and without the addition of PNC. Scale bar = 2 µm. K) Three-point bending, tensile loading, and compressive strength tests comparing the mechanical properties of photocured PEGDA with and without the addition of PNC.\u003csup\u003e \u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e-values were calculated using a two-tailed unpaired t-test. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01,\u0026nbsp;\u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8441498/v1/87d084c851122686dff758b2.png"},{"id":99799320,"identity":"faf2504e-6255-45bb-a0bd-def69d124359","added_by":"auto","created_at":"2026-01-08 13:49:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2967414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePNC-containing bioink exhibits superior osteogenic efficacy\u003c/strong\u003e.\u003cem\u003e \u003c/em\u003eA) Cell viability of hMSCs cultured with medium extracts from PEGDA and PNC for 3 and 7 days. B) SEM and fluorescent staining images showing the attachment of hMSCs on the surface of PEGDA or PNC. Scale bars were 20 µm, 5 µm, and 50 µm, respectively. C) ALP activity of hMSCs cultured with medium extracts from PEGDA and PNC for 3 and 7 days. D) Western blots for the expression of osteogenesis-related proteins. E) RT-qPCR analysis for the expression of osteogenesis-related genes. F) Representative calcein green/xylenol orange labeling images showing the new bone formation. OB: old bone; NB: new bone; S: Scaffold. Scale bar = 2 mm. G) Immunofluorescent staining of OPN and OCN. B: bone; S: Scaffold. Scale bar = 100 µm. H) H\u0026amp;E staining images for the defect area in sham and PNC groups. B: bone; S: Scaffold. I) Representative μCT images and 3D reconstruction for the bone defect area. J) Quantitative measurement of bone volume at week 1, 2, 4, and 8 post-operation. For A \u0026amp; C, p-values were calculated by two-way ANOVA followed by Tukey’s \u003cem\u003epost\u0026nbsp;hoc\u003c/em\u003e\u0026nbsp;test. For E, p-values were calculated by one-way ANOVA followed by Tukey’s \u003cem\u003epost\u0026nbsp;hoc\u003c/em\u003e\u0026nbsp;test. For J, p-values were calculated by two-way ANOVA followed by Sidak’s \u003cem\u003epost\u0026nbsp;hoc\u003c/em\u003e\u0026nbsp;test.\u003csup\u003e *\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01,\u0026nbsp;\u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.001, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8441498/v1/844dc24c845dc8dbee4f2cae.png"},{"id":99798778,"identity":"d765b118-bfea-40fd-9d51-bd1b8a5d4253","added_by":"auto","created_at":"2026-01-08 13:48:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2084080,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEncapsulation of Mg\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e2+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e in PNC enables a dual ion release profile. \u003c/strong\u003eA) Schematic diagram for the encapsulation of additional Mg\u003csup\u003e2+\u003c/sup\u003e in PNC. B) SEM images of the microstructures of scaffolds printed from bioink incorporated with PNC or Mg-PNC. Insets depicted the elemental compositions of each scaffold. C) SEM-EDS mapping\u0026nbsp;showing the elemental distribution in PNC and Mg-PNC scaffolds. D) Cumulative Mg\u003csup\u003e2+\u003c/sup\u003e, Ca\u003csup\u003e2+\u003c/sup\u003e, and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3- \u003c/sup\u003erelease profiles of PNC and Mg-PNC determined \u003cem\u003ein vitro\u003c/em\u003e. E) Degradation rates of PNC and Mg-PNC as tested \u003cem\u003ein vitro\u003c/em\u003e by 28 days.\u003cstrong\u003e \u003c/strong\u003eF) Cell viability of THP-1-derived macrophages cultured with medium extracts from PNC and Mg-PNC for 72 h. G) SEM images of macrophages adhered to different surfaces for 24 h. Scale bar = 10 µm. For D-E, \u003cem\u003ep\u003c/em\u003e-values were calculated using one-way ANOVA followed by Tukey’s \u003cem\u003epost hoc\u003c/em\u003e test. For F, the \u003cem\u003ep\u003c/em\u003e-value was calculated using a two-tailed unpaired t-test. * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, “ns” indicates no significant difference.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8441498/v1/f0e2d9e6d56aac7fe26c51d7.png"},{"id":99781831,"identity":"38dbab26-2014-44e1-ac5f-ef342139c8e2","added_by":"auto","created_at":"2026-01-08 10:47:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3729054,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNanocapsules with dual-phase Mg²⁺ release promote new bone formation. \u003c/strong\u003eA) Representative μCT images and 3D reconstruction of the bone defect areas. B) Quantitative measurement of bone volume at week 1 and 8 post-operation. C) H\u0026amp;E staining and Masson's trichrome staining images for the defect area at week 1 and 8 post-operation. Red arrow: immune cells; S: scaffold; NB: new bone; OB: old bone. Scale bars = 500 µm for low magnification and 200 µm for high magnification. \u003cem\u003ep\u003c/em\u003e-values were calculated using a two-tailed paired t-test. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8441498/v1/62a4ad769ec5c5f50450560a.png"},{"id":99781833,"identity":"d52f604d-d442-4253-a776-4fe489353053","added_by":"auto","created_at":"2026-01-08 10:47:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1831800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMg-PNC facilitates the sequential polarization of macrophages\u003c/strong\u003e. A) Immunofluorescent staining showing the presence of Osx\u003csup\u003e+\u003c/sup\u003e cells in the cranial defects grafted with different scaffolds at week 1 post-operation. Scale bar = 100 µm. B) Quantitative analysis for the number of Osx\u003csup\u003e+ \u003c/sup\u003ecells. C) Immunofluorescent staining showing the presence of CD68\u003csup\u003e+ \u003c/sup\u003emacrophages in the cranial defects grafted with different scaffolds at week 1 post-operation. Scale bar = 100 µm. OB: old bone. D) Quantitative analysis for the number of CD68-positive cells. E) RT-qPCR data showing the expression of representative pro-inflammatory and anti-inflammatory genes in THP-1-derived macrophage treated with early-stage (0-3 day) and late-stage (6-9 day) hydrogel discs of PNC and Mg-PNC. \u003cem\u003ep\u003c/em\u003e-values were calculated using a two-tailed unpaired t-test. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01,\u0026nbsp;\u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.001, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8441498/v1/3268f86b1818167a9cd494b1.png"},{"id":99798941,"identity":"822474e5-019c-4787-820a-7f250e4ea9da","added_by":"auto","created_at":"2026-01-08 13:49:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3248761,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMg-PNC triggers the activation of the immune-neural axis. \u003c/strong\u003eA) Immunofluorescent staining images showing the co-localization of CD68 and CGRP in the cranial defect area at week 1 post-operation. Scale bar = 100 µm. S: scaffold; Green arrow: sensory nerve; OB: old bone. B) Quantitative analysis comparing the mean fluorescence intensity of CGRP in the Mg-PNC and PNC groups. C) Diagram illustrating the projections of TG and its branches in the craniofacial region of rats. D) Immunofluorescent staining images of CGRP in the V1 or V2 branch of TG at week 1 post-operation. Scale bar = 100 µm. E) Quantitative analysis for the number of CGRP\u003csup\u003e+\u003c/sup\u003e neurons within V1 and V2 branches of TG. F) Representative microscopic images showing MED17.11-derived sensory neurons treated with supernatant harvested from the co-culture of THP-1-derived macrophages with or without scaffolds. Scale bar = 50 µm. G) The quantitative analysis of average axon length, H) proportion of neurons with elongated axons, and I) length of the longest neurite. J) RT-qPCR data showing the expression of genes associated with nociceptive sensation and axon projection. K) At the early phase, the rapid Mg\u003csup\u003e2+ \u003c/sup\u003erelease from Mg-PNC induced the pro-inflammatory polarization of macrophages to activate sensory neurons, leading to the secretion of CGRP, which contributes to the resolution of inflammation to facilitate bone regeneration. For B and E, \u003cem\u003ep\u003c/em\u003e-values were calculated using a two-tailed unpaired t-test. For G-J, \u003cem\u003ep\u003c/em\u003e-values were calculated using one-way ANOVA followed by Tukey’s \u003cem\u003epost hoc\u003c/em\u003e test.\u003csup\u003e *\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.05,\u0026nbsp;\u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.01,\u0026nbsp;\u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.001, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026nbsp;\u0026lt;\u0026nbsp;0.0001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8441498/v1/03c853760e26454f68638973.png"},{"id":99805361,"identity":"7e3361b2-48a9-42a8-93f1-b85ee88f9e6e","added_by":"auto","created_at":"2026-01-08 14:16:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19412769,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8441498/v1/055b0847-6668-497c-abda-95ed28f54745.pdf"},{"id":99781838,"identity":"2ad26d83-f70c-434f-8333-84c6e9598089","added_by":"auto","created_at":"2026-01-08 10:47:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1912344,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8441498/v1/2c61b3d4b80fff17fa810f97.docx"},{"id":99799387,"identity":"fa8c1ff7-763a-4b45-99ef-c04296bbef4a","added_by":"auto","created_at":"2026-01-08 13:49:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":421084,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-8441498/v1/19a55156ff7afe96d43f5c0b.docx"},{"id":99781837,"identity":"69db3283-b86f-4048-85d8-5eb9429d5380","added_by":"auto","created_at":"2026-01-08 10:47:32","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":630114,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8441498/v1/0ad9197f5d3fc09435fa2a4c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDynamic Modulation of Immune-Neural Axis via Controlled Magnesium-Releasing Nanocapsules Accelerates Cranial Bone Regeneration\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe repair of critical-sized cranial bone defects resulting from trauma, infections, and tumors remains a significant clinical challenge due to the lack of adequate structural support for cell migration and tissue regeneration\u0026nbsp;[1, 2]. Although bone grafting materials and titanium mesh have been extensively used in clinical practice to address the issue, the regeneration process is often time-consuming, and the clinical outcomes can be suboptimal in some patients [3]. Cranial bone regeneration is a complex process involving coordinated interactions among multiple biological systems and cell populations, which are intricately orchestrated by sophisticated cellular crosstalk [4]. As the initial phase of bone healing, the acute inflammation stage plays a pivotal role in determining regenerative outcomes [5].\u0026nbsp;Both excessive inhibition and prolonged activation of the\u0026nbsp;inflammatory response have been associated with impaired new bone formation. Therefore, there is increasing interest in targeting the innate immune response, particularly macrophages that dominate this process, to promote bone repair [6]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDuring the acute inflammatory stage, the infiltration of macrophages into the bone defect areas contributes to the secretion of various pro-inflammatory cytokines that are critical for recruiting bone-forming cells, such as mesenchymal stem cells (MSCs) and endothelial cells (ECs) [7-9]. Moreover, pro-inflammatory cytokines like prostaglandin E\u003csub\u003e2\u003c/sub\u003e (PGE\u003csub\u003e2\u003c/sub\u003e) and interleukin-1 beta (IL-1\u0026beta;) have been shown to activate sensory nerves, which have recently been recognized as critically involved in bone regeneration [10, 11]. Following the initial pro-inflammatory polarization, macrophages within the bone defects need to shift toward anti-inflammatory phenotypes to resolve inflammation and establish a pro-regenerative microenvironment conducive to bone healing [12]. Therefore, instead of biasing macrophages towards a particular phenotype, next-generation orthopedic biomaterials should be designed to present bioactive cues that facilitate the pro-inflammatory activation and a seamless transition to anti-inflammatory states within the bone microenvironment [9].\u003c/p\u003e\n\u003cp\u003eMagnesium ions (Mg\u003csup\u003e2\u003c/sup\u003e⁺) have been extensively studied as bioactive agents that promote bone tissue regeneration\u0026nbsp;[13-15]. In our previous studies, we elucidated the biphasic role of Mg\u003csup\u003e2\u003c/sup\u003e⁺ in modulating innate immune responses during bone regeneration [16]. Specifically, higher concentrations of Mg\u003csup\u003e2\u003c/sup\u003e⁺ induce pro-inflammatory cytokine production, which recruits osteogenic progenitors and enhances their osteogenic activities. Conversely, lower Mg\u003csup\u003e2\u003c/sup\u003e⁺ concentrations exert anti-inflammatory effects that facilitate osteogenic differentiation of progenitors and extracellular matrix mineralization [17]. Additionally, we previously demonstrated that Mg\u003csup\u003e2\u003c/sup\u003e⁺ contributes to the activation of sensory nerves during bone healing [18]. On the one hand, calcitonin gene-related peptide (CGRP) released from activated sensory neurons can directly contribute to new bone formation [19]. On the other hand, sensitization of nociceptive neurons can trigger the interoceptive pathway to mediate the control of new bone formation in the central nervous system [18, 20]. \u0026nbsp;Collectively, these findings underscore the importance of dynamically controlling Mg\u003csup\u003e2\u003c/sup\u003e⁺ release throughout the bone healing process to maximize its regenerative potential.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this study, we developed novel polyethylene glycol (PEG)-coated nanocapsules with an amorphous whitlockite shell (PNC). These nanocapsules can be readily three dimensional (3D) printed into photocurable scaffolds with patient-specific shapes for cranial bone regeneration. The incorporation of PNC not only improved the mechanical properties of the scaffolds but also enabled sustained slow release of Mg\u003csup\u003e2+\u003c/sup\u003e, which significantly enhanced the osteogenic properties. More importantly, additional Mg\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003ecan be encapsulated into these nanocapsules to achieve a two-stage Mg\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003erelease profile. Our in vitro studies demonstrated that an initial burst release of Mg\u003csup\u003e2+\u003c/sup\u003e from Mg\u003csup\u003e2+\u003c/sup\u003e-loaded nanocapsules (Mg-PNC)\u0026nbsp;promoted the pro-inflammatory activation of macrophages, while the subsequent mild and sustained Mg\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003erelease facilitated the transition of macrophages toward anti-inflammatory phenotypes. In a rat cranial critical-sized bone defect model, this sequential immune modulation by Mg-PNC resulted in superior new bone formation compared to PNC, which predominantly exhibited anti-inflammatory effects throughout the healing process. Additionally, the dynamic immune microenvironment shaped by Mg-PNC enhanced reinnervation of trigeminal sensory neurons into the defect area and upregulated CGRP expression, contributing to inflammation resolution and osteogenesis. Our findings demonstrate that Mg-PNC is a cost-effective system for the controlled delivery of bioactive ions, with its superior osteogenic performance stemming from the dynamic modulation of the immune-neural axis. These results provide valuable insights for designing next-generation bioactive materials for cranial bone defect repair.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Fabrication of PNC and Mg-PNC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCalcium nitrate tetrahydrate (Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO), and magnesium nitrate hexahydrate (Mg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO) were sourced from Xihua (China). Magnesium chloride hexahydrate (MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO), trisodium phosphate dodecahydrate (Na\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026middot;12H\u003csub\u003e2\u003c/sub\u003eO), sodium dodecyl sulfate (SDS) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) were purchased from Sigma-Aldrich (USA).\u0026nbsp;Polyethylene glycol (PEG, molecular weight 20000) and Poly (ethylene glycol) diacrylate (PEGDA, molecular weight 400) were obtained from Aladdin (China). Camphor quinone (CQ) was purchased from Esstech (USA).\u003c/p\u003e\n\u003cp\u003eNanocapsules (NCs) were prepared as follows: 8.86 g Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;4H\u003csub\u003e2\u003c/sub\u003eO and 3.21 g Mg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO were dissolved in 50 mL deionized (DI) water to form the salt solution. Simultaneously, dissolved 0.47 g SDS in 50 mL DI water for the SDS solution. Combined the SDS solution with the salt solution and stirred for 30 min. Then dissolved 11.4 g Na\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026middot;12H\u003csub\u003e2\u003c/sub\u003eO in 100 mL of DI water, added this solution to the mixture, adjusted the pH to above 10, then continued stirring at 60 \u0026deg;C for 24 h. Finally, centrifuged the mixture and washed until neutralization to harvest NCs. To synthesize PNCs, after preparing the salt solution and SDS solution, dissolved 5 g PEG in 50 mL of DI water, the PEG solution was added to the mixture, stirring for an additional 30 min. The subsequent steps were the same as those for NC nanoparticles. To encapsulate additional Mg\u0026sup2;⁺ into the PNC nanoparticles, magnesium chloride (MgCl\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e\u0026nbsp;\u003c/sub\u003esolutions with mass fractions of 10%, 20%, 30%, and 40% were prepared using MgCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO, mixed with 2 g of PNCs and stirred for 4 h. Afterward, centrifuged the mixture and dried it overnight to obtain Mg-PNC nanoparticles containing varying MgCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eloadings.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Fabrication of PNC and Mg-PNC scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the synthesis of PNC scaffolds and Mg-PNC scaffolds, 0.025 g CQ and 0.025 mL DMAEMA were first mixed in 5 mL PEGDA and stirred for 1 h. Nanoparticles were then gradually added to the mixture under magnetic stirring at 350 rpm. To facilitate uniform dispersion, the suspension was bath-sonicated (Xinzhi, China) for 10 min, followed by continuous stirring for 10 h to obtain PNC and Mg-PNC bioinks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor 3D-printed scaffolds used \u003cem\u003ein vivo\u003c/em\u003e experiments, PNC or Mg-PNC bioink was loaded into a biological 3D printer (REGENHU, Switzerland), equipped with a 30G nozzle. Printing parameters were set with a printing speed of 20 mm/s, gas pressure of 30 kPa, and the printing temperature of 25 \u0026deg;C. The scaffolds were printed as a single layer based on a pre-designed model with pore diameters of 2 mm. Following printing, the scaffolds were crosslinked using a 405 nm light-crosslinking lamp for 1 min. For PNC or Mg-PNC specimens used for \u003cem\u003ein vitro\u003c/em\u003e study, the bioink was extruded into circular stainless-steel molds (6 mm \u0026times; 6 mm \u0026times; 1 mm) and subjected to photocrosslinking to ensure consistent shape.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Characterization of NC, PNC, Mg-PNC and 3D printed scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe surface morphology and diameter of NC, PNC, Mg-PNC were observed using scanning electron microscope (SEM; SU-1500, HITACHI, Japan). Energy dispersive spectroscopy (EDS; SDD3310, iXRF, USA) was employed to analyze the elemental distribution and compositional purity of PNCs, Mg-PNCs and their 3D-printed scaffolds. Structural changes in NCs and PNCs with different MgCl\u003csub\u003e2\u0026nbsp;\u003c/sub\u003econcentrations were characterized by X-ray diffraction (XRD; MiniFlex600, Rigaku, Japan; Cu K\u0026alpha;, 40 kV, 15 mA) and Fourier Transform Infrared Spectroscopy (FT-IR; Spectrum Two, PerkinElmer, USA). The structural morphology and lattice composition of NC, PNC and Mg-PNC were investigated using a high-resolution transmission electron microscope (TEM; FEI Tecnai G2 F20 S-Twin 200 kV, USA), with selected area electron diffraction (SAED) performed synchronously to further characterize their crystal structure features, including lattice parameters and crystalline orientation.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eBarrett-Joyner-Halenda\u0026nbsp;(BJH)\u0026nbsp;analysis was performed using a gas sorption analyzer (Autosorb 6100, Anton-Paar, Austria). Thermogravimetric analysis (TGA) was conducted by thermogravimetric analyzer (Q50 TGA, TA Instruments, USA). Mechanical testing of the scaffolds was conducted using a universal test instrument (E3000, Electropuls, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Swelling, degradation, and ion release experiments of PNC and Mg-PNC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor swelling, degradation and ion release studies, the prepared PNC and Mg-PNC discs were used as samples, and the initial weight of each disc was first measured and recorded as W\u003csub\u003e1\u003c/sub\u003e. The prepared PNC and Mg-PNC discs were immersed in 2 mL of phosphate-buffered saline (PBS; Thermo Fisher Scientific, USA) and incubated at 37 \u0026deg;C, with sampling conducted at predetermined time points: 1 h, 2 h, 6 h, 12 h, 24 h (1 d), 48 h, 72 h (3 d), 7 d, 14 d, and 28 d. For the swelling test,\u0026nbsp;at each time point, the samples were taken out, blotted gently to remove surface water, and immediately reweighed to obtain the wet weight as W\u003csub\u003e2\u003c/sub\u003e. The swelling ratio (SR) was calculated using Formula (1): SR = (W\u003csub\u003e2\u003c/sub\u003e \u0026ndash; W\u003csub\u003e1\u003c/sub\u003e)/W\u003csub\u003e1\u003c/sub\u003e \u0026times; 100%. For the degradation test, the soaked discs were placed in a vacuum freeze dryer for 2 days, and the dry weight after degradation was recorded as W\u003csub\u003e3\u003c/sub\u003e.\u0026nbsp;The degradation rate (DR) was calculated using Formula (2): DR = (W\u003csub\u003e3\u003c/sub\u003e \u0026ndash; W\u003csub\u003e1\u003c/sub\u003e)/W\u003csub\u003e1\u003c/sub\u003e \u0026times; 100%. Meanwhile, the supernatants were collected as the degradation solution according to the above timeline. The concentrations of calcium ions (Ca\u003csup\u003e2+\u003c/sup\u003e), Mg\u0026sup2;⁺, and phosphate ions (PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u003c/sup\u003e)\u003csup\u003e\u0026nbsp;\u003c/sup\u003ein the collected degradation solutions were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES; Spectro Arcos, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human monocyte cell line THP-1 (TIB-202, ATCC, USA) was cultured in RPMI 1640 medium (Thermo Fisher Scientific, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, USA) and 1% (v/v) penicillin/streptomycin (P/S; Thermo Fisher Scientific, USA). To differentiate THP-1 cells into macrophages, 50 nM phorbol 12-myristate 13-acetate (PMA; MCE, USA) was used for 24 h induction, followed by switching to high-glucose Dulbecco\u0026rsquo;s modified Eagle medium (DMEM; Thermo Fisher Scientific, USA) for further culture at 37 \u0026deg;C\u0026nbsp;with 5% CO\u003csub\u003e2\u003c/sub\u003e to eliminate the continuous stimulation of PMA. hTERT-immortalized MSCs (hMSCs) were provided by Prof. Wei Qiao, and maintained in high-glucose DMEM with 10% FBS and 1% (v/v) P/S. Mouse dorsal root ganglion cell line (MED17.11 cell) was obtained from University of Sheffield, UK. For the proliferation phase, MED17.11 cells were cultured in DMEM/F12 medium supplemented with Glutamax (Thermo Fisher Scientific, USA), 10% FBS, 1% P/S, 5 ng/mL interferon gamma (IFN-\u0026gamma;; R\u0026amp;D, USA), and 0.5% chicken embryonic extract (Sera Lab, South Korea) in a 33 \u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. For the differentiation of MED17.11 cells, the culture medium was switched to DMEM/F12 with Glutamax, supplemented with 10% FBS, 1% P/S, 10 ng/mL fibroblast growth factor 2 (FGF2, R\u0026amp;D, USA), 0.5 mM diButyrylcAMP (db-cAMP; Sigma-Aldrich, USA), 25 \u0026mu;M forskolin (Sigma-Aldrich, USA), 5 \u0026mu;g/mL Y-27632 (Cayman, USA), 100 ng/mL nerve growth factor (NGF; R\u0026amp;D, USA), and 10 ng/mL glial cell line-derived neurotrophic factor (GDNF; R\u0026amp;D, USA). These cells were cultured at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. For the PGE2-treated MED17.11 group, PGE2 (MCE, USA) was added to the culture supernatant at a final concentration of 1 \u0026mu;M to stimulate cells, which were then maintained in the medium at 37 \u0026deg;C for 1 day.\u003c/p\u003e\n\u003cp\u003eFor co-culture studies between PNC/Mg-PNC and macrophages, THP1-derived macrophages were first directly co-cultured with specimens in complete medium under standard cell culture conditions (37℃, 5% CO₂) to simulate cell response to the material at early inflammation stage (day 0-3); then the specimens were incubated in fresh complete medium alone for an additional 3 days and used to culture new THP1-derived macrophages to simulate the cell response to material at late inflammation resolution stage (day 6-9).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Biocompatibility of PNC and Mg-PNC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe biocompatibility of PNC and Mg-PNC was evaluated in accordance with ISO 10993-5:2009.\u0026nbsp;THP-1-induced macrophages and hMSCs were cultured with sterile 0.1 g/mL material extracts for 3 or 7 days. Biocompatibility assessments were performed using 96-well plates at a cell density of 5\u0026nbsp;\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well. Cell proliferation was assessed by the Cell Counting Kit-8 (CCK-8 assay; MCE, USA). The optical density (OD) values of CCK-8 treated cells were measured at a wavelength of 450 nm by a microplate spectrophotometer (SpectraMax 340, Molecular Devices, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Cell adhesion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe adhesion and morphology of THP-1 cells and hMSCs on PEGDA, PNC, and Mg-PNC were observed via SEM. The PEGDA discs were fabricated using the same mold as the PNC and Mg-PNC discs. Cells were seeded onto the surfaces of these discs in 24-well plates at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well and allowed to settle naturally. After 1 day of co-culture, each sample was gently rinsed with PBS and fixed overnight at 4\u0026deg;C using 2.5% glutaraldehyde. The samples were then dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%). Subsequently, all samples were sputter-coated with platinum (Pt) prior to SEM imaging.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Alkaline phosphatase (ALP) assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe p-nitrophenyl phosphate (p-NPP) method was conducted to assess the impacts of Mg\u003csup\u003e2+\u003c/sup\u003e or macrophage-derived conditional medium on the ALP activity of hMSCs. At day 3 or day 7, hMSCs were subjected to lysis with 0.2% Triton X-100 at 4 \u0026deg;C for 2 h. The cell lysate was centrifuged, and the resulting supernatant was collected for subsequent assays using an ALP detection kit (Sigma-Aldrich, USA). Total cellular protein content was determined via the BCA Protein Assay Kit (ThermoFisher Scientific, USA). Relative ALP activity was normalized against the total protein content of each sample, with final results expressed as units per minute per gram of total protein (U/min/g\u0026middot;protein).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.9 Real Time-qPCR (RT-qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-qPCR\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ewas used to evaluate the expression levels of h\u003cem\u003eIL10,\u0026nbsp;\u003c/em\u003eh\u003cem\u003ePTGS2,\u0026nbsp;\u003c/em\u003eh\u003cem\u003eIL8,\u0026nbsp;\u003c/em\u003eh\u003cem\u003eTNF,\u0026nbsp;\u003c/em\u003eh\u003cem\u003eCCL5,\u0026nbsp;\u003c/em\u003eh\u003cem\u003eTGFB1,\u0026nbsp;\u003c/em\u003eh\u003cem\u003eIL1B,\u0026nbsp;\u003c/em\u003eh\u003cem\u003eMRC1,\u0026nbsp;\u003c/em\u003eh\u003cem\u003eRUNX2,\u0026nbsp;\u003c/em\u003eh\u003cem\u003eALP,\u0026nbsp;\u003c/em\u003eh\u003cem\u003eCOL1A1,\u0026nbsp;\u003c/em\u003em\u003cem\u003eCalca,\u0026nbsp;\u003c/em\u003em\u003cem\u003eTrpv1,\u0026nbsp;\u003c/em\u003em\u003cem\u003eEp4,\u0026nbsp;\u003c/em\u003em\u003cem\u003eTubb3,\u0026nbsp;\u003c/em\u003em\u003cem\u003eGap43.\u0026nbsp;\u003c/em\u003ePrimer sequences were provided in Table S1 of the supplementary information.\u003cem\u003e\u0026nbsp;\u003c/em\u003eBriefly, total RNA was extracted from harvested hMSCs, THP-1 induced macrophages or MED17.11 cells using RNeasy Mini Kit (Qiagen, Germany). Complementary DNA (cDNA)\u0026nbsp;was obtained via reverse transcription kit (Takara, Japan) with thermal cycler (ProFlex PCR System, Thermo Fisher Scientific, USA). cDNA was then amplified by a RT-qPCR system (QuantStudio 6 Flex Real-Time PCR System, Thermo Fisher Scientific, USA),\u0026nbsp;with each reaction performed in a final volume of 10 \u0026micro;L. Housekeeping genes h\u003cem\u003eGAPDH\u003c/em\u003e (for human samples) or m\u003cem\u003eGapdh\u003c/em\u003e (for mouse samples) were used as internal references for data normalization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10 Western blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain total protein, cells were lysed in ice-cold RIPA lysis buffer (Thermo Fisher Scientific, USA) and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA), centrifuged for 10 min at 4\u0026deg;C to remove debris. Protein concentrations were determined by using BCA protein assay kit (Thermo Fisher Scientific, USA), and the protein extracts were heat-denatured in sodium dodecysulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime, China). Then the protein samples were separated by 10% SDS-PAGE (ACE, China) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with tris-buffered saline with Tween-20\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eTBST) containing bovine serum albumin\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eBSA, Goldbio, USA) for 2 h, then incubated with primary antibodies: anti-ALP (Abcam, ab307726, 1:1000, UK), anti-OPN (Abcam, ab8448, 1:1000, UK), anti-Runx2 (Abcam, ab76956, 1:1000, UK), and anti-GAPDH (CST 2118S, 1:1000, USA). After overnight incubation with the primary antibodies on a shaker at 4\u0026deg;C, the membranes were washed 3 times with TBST and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, which included Anti-rabbit IgG, HRP-linked Antibody, (CST 7074P2, 1:5000, USA) and Anti-mouse IgG, HRP-linked Antibody (CST 7076P2, 1:5000, USA) for 1 h.\u0026nbsp;Finally, the membranes were immersed in HRP chemiluminescent substrate (Thermo Fisher Scientific, USA) for 1 min, protein signals were visualized using a chemiluminescence imaging system (Amersham ImageQuant 800, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.11 Neurite tracing and measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMED17.11 neuron images were captured using an inverted microscope (Eclipse Ti-S, Nikon, Japan) with 4\u0026times; and 10\u0026times; objective lenses. ImageJ software equipped with the Simple Neurite Tracer plugin was employed to trace and measure neurites. 3 fields of view were randomly selected for counting, with at least 50 neurons randomly selected per view. To avoid the interference of neuronal interaction, neurons in contact with others were excluded from analysis. Neurites with a length of 100 \u0026mu;m or longer were defined as elongated neurites. The average axon length, the percentage of neurons with elongated neurites and the average length of the longest neurites per neuron were calculated to assess innervation capacity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12 Animal surgery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal protocols in this study were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong (No. 5959-21). Eight-week-old female Sprague-Dawley (SD) rats weighing 200\u0026ndash;270 g were provided by HKU CULATR and kept in an environment with a 12h light/dark cycle, constant temperature of 22\u0026thinsp;\u0026deg;C and relative humidity of 50\u0026ndash;60%. Throughout the experiment, the rats had unrestricted access to food and water. Critical-sized cranial bone defect models with a diameter of 6 mm were established in SD rats to study the osteogenesis effects of scaffolds. Rats were anesthetized with ketamine (67 mg/kg, Alfamine, Alfasan International B.V., Holland) and xylazine (6 mg/kg, Alfazyne, Alfasan International B.V.). After exposing the surgical field, a 6 mm diameter trephine (Meisinger, Germany) was used to create the defect. To evaluate the osteogenic capacity of PNC, rats with cranial bone defects were randomly assigned to the sham group or PNC group. To assess the osteogenic capacity of Mg-PNC, the rats were divided into the PNC scaffold group or Mg-PNC scaffold group. In scaffold groups, circular scaffolds with a diameter of 6 mm were implanted into the defect area. After wound closure, the rats were immediately administered subcutaneous injections of Oxytetracycline (30 mg/kg) and Burtemgenic (0.05 mg/kg). The rats were sacrificed at week 1 and week 8 post-surgery\u0026nbsp;for further micro computed tomography (\u0026mu;CT)\u0026nbsp;and histological analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13 Fluorochrome labeling\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;To assess bone regeneration and remodeling in the defect areas of PNC scaffolds, calcein green and xylenol orange were administered sequentially. Calcein green (5 mg/kg, Sigma-Aldrich, USA) was injected subcutaneously into the surgical region of rats at week 1 post-surgery, followed by xylenol orange (90 mg/kg, Sigma-Aldrich, USA) at week 3 post-surgery. The fluorescent signals from these labels were visualized using a fluorescence microscope (Nikon ECLIPSE 80i, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.14\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u0026mu;CT analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing sacrifice, cranial specimens were collected and scanned using high-resolution \u0026mu;CT\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Skyscan 1276, Bruker, USA). The scan parameters were set to 80 kV, 190 \u0026micro;A, with a resolution of 13 \u0026micro;m, averaging 2 images. The reconstruction data were obtained using NRecon software. Based on these data, a region of interest (ROI) corresponding to the 6 mm diameter defect area was selected for analyzing new bone formation. Bone volume/tissue volume (BV/TV)\u0026nbsp;was analyzed using CT-Analyzer software. Additionally, 3D reconstructed micro-CT images and a 3D model of the newly formed bone tissue in the defect area were generated using CTvol software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.15\u003c/strong\u003e \u003cstrong\u003eHistological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the \u0026mu;CT\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003escan, cranial bone specimens were subjected to decalcification with ethylenediaminetetraacetic acid (EDTA), gradient dehydration, and paraffin embedding. The paraffin-embedded specimens were sectioned into 5-\u0026mu;m-thick slices using a rotary microtome (RM2155, Leica, Germany). Hematoxylin and eosin (H\u0026amp;E) staining and Masson\u0026apos;s trichrome staining (Solarbio, China) were performed to evaluate bone regeneration. Stained sections were imaged using a stereomicroscope (SMZ18, Nikon, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.16 Immunohistochemistry (IHC) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIHC staining was conducted to assess immune modulation, nerve innervation, and osteogenic effects. The relevant antibodies included anti-Sp7 (Abcam, ab22552, 1:200, UK), anti-CGRP (Abcam, ab81887, 1:200, UK), anti-CD68 (Abcam, ab283654, 1:200, UK), anti-OPN (Abcam, ab183910, 1:200, UK), and anti-OCN (Abcam, ab93876, 1:200, UK). For the cranial bone paraffin sections, after routine deparaffinization, antigen retrieval was performed using proteinase K (Sigma-Aldrich, USA) at 37 \u0026deg;C for 10 min, followed by blocking with donkey serum at room temperature for 1 h. After overnight incubation with the primary antibody at 4\u0026deg;C, secondary antibodies conjugated to Alexa Fluor 488 donkey anti-goat IgG (Thermo Fisher Scientific, 230114, 1:400, USA) or Alexa Fluor 647 donkey anti-rabbit IgG (Thermo Fisher Scientific, 150067, 1:400, USA) were applied, along with Hoechst 33342 (Thermo Fisher Scientific, 2747528, 1:500, USA) for nuclear staining.\u0026nbsp;Trigeminal ganglia (TG) were harvested from the sacrificed rats, fixed in 10%\u0026nbsp;formalin solution, dehydrated in gradient sucrose solutions, and embedded in optimal cutting temperature (OCT; Sakura Finetek, USA) compound for cryosectioning.\u0026nbsp;The samples were then sectioned to a thickness of 10 \u0026mu;m using a cryostat (CM1860 UV, Leica, Germany) and were subjected to IHC staining with the anti-CGRP primary antibody (Abcam, ab81887, 1:200, UK). Confocal fluorescence images were acquired using the confocal laser scanning microscope (LSM 900, Zeiss, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.17 Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll quantitative data are presented as mean \u0026plusmn; standard deviation (SD), and statistical analyses were performed using GraphPad Prism software. Comparisons between two groups were conducted using paired or unpaired t-tests, while comparisons among multiple groups were assessed using one-way or two-way ANOVA, followed by Tukey\u0026rsquo;s or Sidak\u0026rsquo;s\u0026nbsp;\u003cem\u003epost hoc\u003c/em\u003e test respectively. Statistical significance was indicated as follows: ns (not significant, p \u0026ge; 0.05),\u0026nbsp;\u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1.\u003c/strong\u003e \u003cstrong\u003eEngineering hybrid nanocapsules for 3D-printable scaffold\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo synthesize capsule-shaped nanoparticles for controlled release, SDS was added as a foaming agent during the self-assembly of whitlockite precursors. Simultaneously, PEG was added to modify the surface of the amorphous whitlockite shell (Figure 1A). Unlike unmodified nanocapsules (NC), which tend to aggregate and precipitate, PEG-coated nanocapsules (PNCs) can be homogenously dispersed in the PEGDA (Figure 1B). TEM and SEM revealed that both NC and PNC exhibited characteristic capsule-like morphologies with diameters ranging from 30 to 60 nm (Figure 1C). The shell structure, approximately 10 nm thick, was confirmed to be weakly crystalline via SAED. The pore size measured from N\u003csub\u003e2\u003c/sub\u003e adsorption-desorption isotherms using the BJH method indicated that PNC exhibited a more prominent porous structure, with pore diameters between 20 and 50 nm (Figure 1D). XRD analysis identified the characteristic diffraction peaks of NC and PNC to both locate at 2\u0026theta; = 32\u0026deg;, with the peak for PNC being broader and less sharp than that of NC, suggesting lower crystallinity influenced by PEG addition during hydrothermal synthesis (Figure 1E). TGA revealed a more significant mass loss of PNC between 100 \u0026deg;C to 150 \u0026deg;C, indicating the decomposition of PEG moieties (Figure 1F). The FT-IR spectra confirmed the major component of the nanocapsules as whitlockites, with characteristic P\u0026ndash;O bending vibration peaks at 603.70 cm\u003csup\u003e-1\u003c/sup\u003e and 563.20 cm\u003csup\u003e-1\u003c/sup\u003e observed in NC (Figure 1G). In PNC, these peaks merged into a single peak at 572.84 cm\u003csup\u003e-1\u003c/sup\u003e, suggesting the presence of PEG coating.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we tested the printability of PNCs by mixing them with PEGDA, which is generally considered not suitable for extrusion-based 3D printing due to its low viscosity and high fluidity [21]. We showed that PEGDA incorporated with PNC enabled 3D printing of complex structures (Figure 1H) and multilayer scaffolds (Figure 1I). The surface topography of the scaffold observed by SEM showed the homogeneous distribution of nanocapsules in the printed scaffold, resulting in increased surface roughness (Figure 1J). Mechanical testing using three-point bending, tensile, and compressive tests demonstrated that the incorporation of nanocapsules improved the mechanical properties of the printed structures (Figure 1K).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. The incorporation of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePNCs\u0026nbsp;promotes osteogenic performance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDespite being approved by the FDA for biomedical applications, the bioinert nature of PEGDA is known to hinder protein adsorption and cell attachment [22, 23]. We therefore evaluated the biological performance of the printed scaffolds modified with PNC. The cell viability of hMSCs was significantly higher in the PNC group compared to the control or pure PEGDA group (Figure 2A). SEM and confocal microscopy revealed that, unlike on pure PEGDA surfaces, cells attached to PNC-modified PEGDA exhibited a spindle shape with more prominent extensions of lamellipodia and filopodia (Figure 2B). Moreover, the addition of PNC also contributed to enhanced osteogenic differentiation of hMSCs, evidenced by significantly increased ALP activity (Figure 2C), upregulated expression of ALP, OPN, and RUNX2 at the protein level (Figure 2D), as well as increased expression of osteogenic marker genes, such as \u003cem\u003eALP\u003c/em\u003e, \u003cem\u003eOPN\u003c/em\u003e, and \u003cem\u003eCOL1A1\u003c/em\u003e (Figure 2E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUsing ICP-OES, we confirmed that PNCs provided a slow and sustained Mg\u003csup\u003e2+\u003c/sup\u003e release (Figure S1), which likely contributes to its improved osteogenic effects. To further assess the \u003cem\u003ein vivo\u003c/em\u003e performance of PNC, 3D-printed PNC scaffolds were implanted into rat critical-sized cranial defects (Figure S2). Calcein green/xylenol orange labeling showed that PNC scaffolds induced significant new bone formation at the early stage of bone healing, whereas minimal regeneration was observed in defects without scaffolds (Figure 2F). Additionally, immunofluorescent staining showed increased expression of osteopontin (OPN) and osteocalcin (OCN) in the PNC group than in the sham controls (Figure 2G). The enhanced new bone formation was further confirmed by H\u0026amp;E staining (Figure 2H) and \u0026mu;CT analysis (Figure 2I), which showed nearly double the bone volume in the defects grafted with PNC-incorporated scaffolds relative to sham controls (Figure 2J).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Dual-phase Mg\u003csup\u003e2+\u003c/sup\u003e release kinetics from Mg-PNC \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further enhance the osteogenic performance of PNC, we managed to encapsulate additional Mg\u003csup\u003e2+\u003c/sup\u003e by soaking nanocapsules in MgCl\u003csub\u003e2\u003c/sub\u003e solution before printing (Mg-PNC, Figure 3A). This enabled a dual-phase Mg\u003csup\u003e2+\u003c/sup\u003e release kinetics: a rapid, burst Mg\u003csup\u003e2+\u003c/sup\u003e release from within the nanocapsule structure, followed by a sustained, mild Mg\u003csup\u003e2+\u003c/sup\u003e release resulting from the degradation of amorphous whitlockite shell. SEM-EDS mapping as well as the Mg/Ca ratio confirmed a uniform increase in magnesium content in Mg-PNC compared to that in PNC (Figure S3A-C). XRD, FT-IR, and TEM analyses indicated that Mg\u003csup\u003e2+\u003c/sup\u003e encapsulation did not alter the physicochemical properties or structures of PNC (Figure S3D-F). The surface topography of scaffolds printed using PNC and Mg-PNC was similar under SEM, displaying a hierarchical microstructure resembling natural bone units (Figure 3B) and uniform element distribution (Figure 3C and S4A \u0026amp; B). Quantitative analysis showed a significant increase in magnesium content in Mg-PNC scaffolds compared to PNC scaffolds (Figure 3C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe then employed ICP-OES to compare the ion release kinetics of the printed scaffolds. Unlike the constant Mg\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003erelease profile of PNC, Mg-PNC exhibited a dynamic two-stage Mg\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003erelease profile, with an initial burst release reaching approximately 5.4 mM within 6 h and a subsequent sustained release at a much lower level (Figure 3D). Interestingly, Mg-PNC also significantly elevated local Ca\u003csup\u003e2+\u003c/sup\u003e levels compared to those in PNC, likely due to hydration effects associated with Mg\u003csup\u003e2+\u003c/sup\u003e encapsulation [24]. Concomitantly, the concentration of PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3-\u0026nbsp;\u003c/sup\u003edropped significantly more slowly in the Mg-PNC group than in the PNC group. Moreover, the degradation and swelling rates of PNC and Mg-PNC only differed from each other within the first 2 h (Figure 3E and S4C), which might reflect the initial burst Mg\u003csup\u003e2+\u003c/sup\u003e release. Overall, the degradation was approximately 4% for PNC and 8% for Mg-PNC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince the major difference between PNC and Mg-PNC lies in their Mg\u003csup\u003e2+\u003c/sup\u003e release kinetics in the first few days, which coincides with the macrophage-dominated inflammation phase [9], we then evaluated the cellular response of THP-1-derived macrophages to both PNC and Mg-PNC. Cell viability of the PNC group and the Mg-PNC group was both higher than 80%, with no significant difference between groups (Figure 3F). SEM images demonstrated that macrophages adhered better to nanocapsule-containing PEGDA than to PEGDA alone (Figure 3G). Indeed, the macrophages on the surface of Mg-PNC exhibited more pseudopodia extensions, suggesting their more activated status induced by high Mg\u003csup\u003e2+\u003c/sup\u003e level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Tailored Mg\u003csup\u003e2+\u003c/sup\u003e release profile enhances bone regeneration through the modulation of macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the bone regenerative efficacy, scaffolds incorporating PNC or Mg-PNC were implanted into rat cranial critical-sized defects. \u0026mu;CT data analysis at week 8 demonstrated significantly greater bone volume in the Mg-PNC group compared to that in the PNC group (Figure 4A \u0026amp; B). H\u0026amp;E staining and Masson\u0026apos;s trichrome staining confirmed increased new bone formation with Mg-PNC scaffolds (Figure 4C). At week 1 post-operation, both PNC and Mg-PNC scaffolds led to significant immune cells accumulation at the bone defect area. Newly regenerated woven bone can be observed at the defect margin, especially in the Mg-PNC group. At week 8 post-operation, there was more mature bone in the Mg-PNC group compared with the PNC group. The enhanced osteogenic property of Mg-PNC compared to PNC was also testified by the significantly increased number of Osterix (Osx) positive osteogenic cells detected within the defect area at week 1 after the operation (Figure 5A \u0026amp; B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, IHC staining revealed an increased number of CD68-positive macrophages in the Mg-PNC group relative to the PNC group (Figure 5C \u0026amp; D), indicating a more active immune response triggered by dual-phase Mg\u0026sup2;⁺ release. To investigate the dynamic modulation of Mg-PNC on macrophages, we cultured THP-1-derived macrophages with specimens immersed in culture medium for different durations. Specifically, specimens immersed for the first three days represent the early microenvironment at the active inflammatory phase, while specimens already immersed for six days were used to simulate the late microenvironment during the inflammation resolution stage [25, 26]. We found that the early-stage microenvironment created by Mg-PNC induced a more robust pro-inflammatory response than PNC, with a 7-fold increase in \u003cem\u003eIL1B\u0026nbsp;\u003c/em\u003eexpression and a 3-fold increase in \u003cem\u003ePTGS2\u0026nbsp;\u003c/em\u003eexpression, alongside downregulation of anti-inflammatory genes, such as \u003cem\u003eIL10\u003c/em\u003e and \u003cem\u003eMRC1\u0026nbsp;\u003c/em\u003e(Figure 5E). The expressions of \u003cem\u003eCCL5\u003c/em\u003e and \u003cem\u003eIL8\u003c/em\u003e, which are both known to be critical to cell recruitment and angiogenesis [17, 27], were also significantly elevated in the Mg-PNC group compared to the PNC group. Conversely, the late-stage microenvironment induced by Mg-PNC exhibited a more prominent anti-inflammatory effect than that induced by PNC, as it significantly upregulated the expression of \u003cem\u003eIL10\u003c/em\u003e, \u003cem\u003eTGFB1\u003c/em\u003e, and \u003cem\u003eMRC1\u003c/em\u003e without provoking excessive upregulation in the expression of \u003cem\u003eIL1B\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePTGS2.\u0026nbsp;\u003c/em\u003eThe distinct effects on macrophage polarization achieved by Mg-PNC demonstrated its capability to dynamically modulate the immune microenvironment throughout the bone healing process. Our data indicate that Mg-PNC facilitates the sequential pro-inflammatory activation of macrophages and their seamless transition to anti-inflammatory phenotypes for effective bone regeneration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5. Mg-PNC triggers the activation of the immune-neuro axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to the direct effects of macrophages on bone-forming cells, alterations in the local immune microenvironment may sensitize the sensory nerves present in the injured area, leading to the modulation of the immune-neuro axis [18]. We then asked whether dual-phase Mg\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003erelease from Mg-PNC activates this axis. Immunofluorescent staining showed that CGRP-positive sensory nerves were highly co-localized with CD68\u003csup\u003e+\u003c/sup\u003e macrophages and was significantly elevated in the Mg-PNC group relative to the PNC group (Figure 6A \u0026amp; B). Since the cranial bone defect area is primarily innervated by sensory neurons within the V1 (ophthalmic nerve) and V2 (maxillary nerve) branches of the TG [28] (Figure 6C), we then determined the expression of CGRP in TG. Immunofluorescent staining data revealed a significant increase in the number of CGRP\u003csup\u003e+\u003c/sup\u003e neurons within the V1 and V2 branches of TG in the Mg-PNC group compared to the PNC group (Figure 6D \u0026amp; E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm whether Mg-PNC stimulates sensory neurons through the modulation of macrophages, we treated MED17.11-derived sensory neurons with supernatant harvested from the co-culture of THP-1-derived macrophages with or without scaffolds (Figure 6F). At 12 and 24 h, the average axon length of neurons (Figure 6G), the proportion of neurons with elongated axons (Figure 6H), and the length of the longest neurite (Figure 6I) were all significantly increased in the Mg-PNC group compared with the PNC and control groups. These findings confirmed that Mg-PNC promotes axon extension through the modulation of macrophages. Additionally, we examined the expression of genes associated with nociceptive sensitization (i.e., \u003cem\u003eCALCA\u003c/em\u003e, \u003cem\u003eTRPV1\u003c/em\u003e, and \u003cem\u003eEP4\u003c/em\u003e) and axon projection (i.e., \u003cem\u003eGAP43\u003c/em\u003e and \u003cem\u003eTUBB3\u003c/em\u003e). These genes were significantly upregulated in the Mg-PNC group rather than in the PNC group when compared to the control group (Figure 6J). Given the recent discovery of the immunomodulatory role of neuropeptides, such as CGRP [18, 29], our data suggest that Mg-PNC might activate the sensory nerves through the early pro-inflammatory responses, with neuron-derived CGRP playing a key role in resolving inflammation (Figure 6K).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e3D printed scaffolds have become a widely adopted strategy for bone tissue regeneration, particularly in cases involving critical-sized bone defects. Here in this study, we developed a novel hybrid bioink with excellent printability, mechanical strength, and osteogenic performance. More importantly, the capsule structure of the nanoparticles enables controlled release of bioactive agents, such as trace ions, small molecules, and nucleic acids. Through surface modification with PEG, we successfully integrated inorganic whitlockite nanoparticles with organic PEGDA polymer to form a cohesive bioink. Therefore, the implantation of scaffolds fabricated using this hybrid bioink did not induce any undesirable inflammatory response caused by nanoparticle agglomeration or phagocytosis by immune cells [30, 31]. Furthermore, compared to traditionally used hydroxyapatite nanoparticles, the nanocapsules developed in this study possess an amorphous whitlockite shell, which exhibits superior regenerative potential and biodegradability [32, 33]. As a result, the incorporation of PNC significantly enhanced the osteogenic properties of PEGDA, which is otherwise considered bioinert [34, 35].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn our study, we first tested the osteogenic property of PNC, which provides a single-phase sustained Mg\u003csup\u003e2+\u003c/sup\u003e release throughout the bone healing process. Despite the evident osteogenic properties observed in vitro and in vivo, the regeneration outcomes still seem suboptimal, with the critical-sized calvarial defects incompletely healed at week 8 after the injury. We hypothesized that the constant anti-inflammatory effects provided by this Mg\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003edelivery scheme might not be the most effective to accelerate bone regeneration. Indeed, depletion of macrophages during early bone healing stages blunted inflammatory responses and impaired new bone formation [36, 37]. Additionally, administration of non-steroidal anti-inflammatory drugs, which disrupt inflammatory responses, has also been associated with compromising fracture healing [38]. Therefore, we encapsulated fast-releasing Mg\u003csup\u003e2+\u003c/sup\u003e within nanocapsules to produce an additional wave of rapid Mg\u003csup\u003e2+\u003c/sup\u003e release, which was intended to promote the pro-inflammatory activation of macrophages. Our findings demonstrated that the dual-phase Mg\u003csup\u003e2+\u003c/sup\u003e release strategy, which enables dynamic immunomodulation, significantly accelerated the bone healing process to achieve complete closure of the critical-sized bone defects within 8 weeks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile our findings underscore the critical role of inflammatory response in bone repair, it is important to note that timely resolution of inflammation is equally crucial to this process, as prolonged presence of pro-inflammatory factors can inhibit osteogenesis [5]. In this study, we exploited the concentration-dependent effects of Mg\u003csup\u003e2+\u003c/sup\u003e on macrophage polarization to facilitate the transition from pro-inflammatory to anti-inflammatory phenotype. Our data demonstrated that the early microenvironment created by Mg-PNC upregulates various pro-inflammatory cytokines, including IL-1\u0026beta;, PGE2, IL-8, and CCL5. They positively contribute to the recruitment of host stem cells, an early event decisive to the outcomes of osteogenesis [9, 39]. In contrast, the late microenvironment created by Mg-PNC did not induce the same level of pro-inflammatory cytokine expression, which is beneficial considering their potential inhibitory effects on osteoblast differentiation [40, 41]. Moreover, the late-stage microenvironment shaped by Mg-PNC contributes to a dramatically different pro-regenerative niche for bone healing, manifested by the upregulation of IL-10, CD206, and TGF-\u0026beta;. Additionally, the controlled slow Mg\u003csup\u003e2+\u003c/sup\u003e release at the late bone healing phase is also preferred to prevent adverse effects, such as the inhibition of osteoblast activity and the mineralization of extracellular matrix [9, 42].\u003c/p\u003e\n\u003cp\u003eThe cranial bone, especially the highly regenerative periosteum area, is densely innervated by sensory fibers [43]. Recent studies have highlighted their roles in cranial bone regeneration [44-46]. Our data demonstrated their close relationship with CD68\u003csup\u003e+\u003c/sup\u003e macrophages in the cranial bone and their being sensitized by the pro-inflammatory microenvironment induced by\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eMg-PNC. This aligns with previous observations showing that macrophage and sensory nerves interact robustly to form an integrated neuro-immune axis involved in pain sensitization [47]. The pro-inflammatory cytokines upregulated by Mg-PNC, such as IL-1\u0026beta; and PGE2, have been reported to activate sensory neurons through actions on Transient Receptor Potential Vanilloid 1 (TRPV1) and Prostaglandin E receptor 4 (EP4) [48, 49]. As a result of the sensation of these inflammatory cytokines associated with pain, sensory neurons responsively secrete neuropeptides like CGRP [18, 50], which were more prominent in the Mg-PNC group compared to the PNC group. The activation of the sensory nerve during injury has been reported to be critical to tissue regeneration, as it contributes to inflammation resolution [51], revascularization [52], regulation of osteoblasts [53, 54], and the control of bone healing within the central nervous system [18, 55]. Therefore, our observation on the increased axon projection and upregulation of CGRP in the injured area and TG confirmed the involvement of the immune-neural axis in bone regeneration induced by Mg-PNC.\u003c/p\u003e\n"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, our study presents an inorganic-organic hybrid bioink featured by the incorporation of our innovative PEG-coated nanocapsules with an amorphous whitlockite shell. It can be used for 3D printing of scaffolds with superior mechanical and osteogenic properties for repairing critical-sized defects in the calvaria. Moreover, we showed that the nanocapsules enable a dual-stage Mg\u003csup\u003e2\u003c/sup\u003e⁺ release to strategically promote the pro-inflammatory activation of macrophages and seamlessly facilitate their anti-inflammatory polarization. The osteogenic performance of this dynamic macrophage modulation induced by a tailored Mg\u003csup\u003e2\u003c/sup\u003e⁺ release kinetics outperforms that achieved by constant anti-inflammatory modulation with a sustained, slow Mg\u003csup\u003e2\u003c/sup\u003e⁺ release. Additionally, we demonstrated that the dual-phase Mg\u003csup\u003e2\u003c/sup\u003e⁺-releasing Mg-PNC better mimics the natural inflammatory response in the bone healing process, contributing to the activation of sensory neurons to enhance new bone formation. The findings of our study underscore the potential of Mg-PNC as a cost-effective approach to promote cranial bone regeneration and provide valuable insights for the development of new bioactive materials that target the immune-neural axis.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal protocols in this study were approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong (No. 5959-21).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot relevant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by funding from the Research Grants Council, the Government of the Hong Kong SAR (Collaborative Research Fund No.C7003\u0026ndash;22Y and General Research Fund No.17118425 to W.Q.), the Food and Health Bureau, the Government of the Hong Kong SAR (No.09201466 to W.Q.), National Natural Science Foundation of China (No.82201124 to W.Q.), National Natural Science Foundation of China/Research Grants Council Joint Research Scheme (N_HKU721/23 to W.Q.); Hong Kong Innovation Technology Fund (ITS/256/22 to W.Q.), Shenzhen Science and Technology Innovation Committee Projects (Nos. SGDX20220530111405038 to W.Q.), Guangdong Basic and Applied Basic Research Foundation (2023A1515011963 to W.Q.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYilin Mao: Methodology, Investigation, Visualization, Data curation, Writing \u0026ndash; original draft. Qixuan He: Methodology, Investigation, Visualization. Tianle Li: Methodology, Writing \u0026ndash; review and editing. Jiusi Guo: Methodology, Writing \u0026ndash; review and editing.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eKelvin W.K.Yeung: Resources, Writing \u0026ndash; review and editing. Yuxiong Su: Writing \u0026ndash; review and editing. Xianglong Han: Writing \u0026ndash; review and editing. Jian Wang: Writing \u0026ndash; review and editing. Wei Qiao: Conceptualization, Investigation, Methodology, Visualization, Validation, Writing \u0026ndash; review and editing, Funding acquisition, Supervision, Project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank Tony Liu from the Department of Orthopaedics and Traumatology, School of Clinical Medicine, LKS Faculty of Medicine, The University of Hong Kong, for his invaluable support and insightful guidance on \u0026mu;CT scanning.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eE.A. Masters, B.F. Ricciardi, K.L.M. Bentley, T.F. Moriarty, E.M. Schwarz, G. Muthukrishnan, Skeletal infections: microbial pathogenesis, immunity and clinical management, Nat Rev Microbiol, 20 (2022) 385-400. \u003c/li\u003e\n\u003cli\u003eJ. He, J. Lu, F. Zhang, J. Chen, Y. Wang, Q. 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Yuan, J. Lv, J. Huang, J. Huang, C. Zhang, Z. Hong, H. Li, Y. Huang, J. Lin, L. Yuan, L. Lin, S. Yu, C. Zhang, J. Lin, W. Li, C. Chang, B. Yang, W. Zhang, X. Fang, Optogenetic activation of mechanical nociceptions to enhance implant osseointegration, Nat Commun, 16 (2025) 3093. \u003c/li\u003e\n\u003cli\u003eH. Chen, B. Hu, X. Lv, S. Zhu, G. Zhen, M. Wan, A. Jain, B. Gao, Y. Chai, M. Yang, X. Wang, R. Deng, L. Wang, Y. Cao, S. Ni, S. Liu, W. Yuan, H. Chen, X. Dong, Y. Guan, H. Yang, X. Cao, Prostaglandin E2 mediates sensory nerve regulation of bone homeostasis, Nat Commun, 10 (2019) 181. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Magnesium, Immunomodulation, Macrophage, Bone regeneration, Sensory nerve, 3D printing","lastPublishedDoi":"10.21203/rs.3.rs-8441498/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8441498/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Critical-sized cranial defects present a significant challenge in the field of bone tissue regeneration. Despite the emergence of various approaches to promote new bone formation, the clinical outcomes remain suboptimal. In this study, we developed an inorganic-organic hybrid bioink suitable for 3D printing of photocurable scaffolds. This bioink incorporates our novel nanocapsules with a shell consisting of amorphous whitlockite and PEG coating, which endows the scaffolds with superior mechanical strength and osteogenic capacity. These nanocapsules enable a dual-phase Mg2⁺ release profile to facilitate the initial pro-inflammatory activation of macrophages followed by a seamless transition to a pro-regenerative phenotype. We further showed that this dynamic Mg²⁺ delivery strategy significantly outperformed traditional sustained-release approaches in supporting cranial bone regeneration. Moreover, the controlled immunomodulation through this tailored Mg²⁺ delivery more closely mimics the natural healing process, promoting the activation of sensory nerves, which is essential for effective bone regeneration. Overall, our study demonstrated the potential of our nanocapsules as a cost-effective approach for the dynamic modulation of the immune-neural axis, offering valuable insights for the future design of bioactive materials for cranial bone regeneration.","manuscriptTitle":"Dynamic Modulation of Immune-Neural Axis via Controlled Magnesium-Releasing Nanocapsules Accelerates Cranial Bone Regeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-08 10:47:27","doi":"10.21203/rs.3.rs-8441498/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-23T00:58:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T00:07:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102242268585160071953795262511308116959","date":"2026-03-16T21:44:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"92902259523579206773235626138629991832","date":"2026-03-13T15:13:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279187611245738253444027600782750929457","date":"2026-03-12T11:11:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-21T01:13:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104436318752835809340286232712115293566","date":"2026-01-09T01:45:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285785862208450187490259230948718332028","date":"2026-01-07T15:10:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-06T23:58:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-27T12:16:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-27T12:15:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanobiotechnology","date":"2025-12-24T09:39:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanobiotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jnan","sideBox":"Learn more about [Journal of Nanobiotechnology](http://jnanobiotechnology.biomedcentral.com)","snPcode":"12951","submissionUrl":"https://submission.nature.com/new-submission/12951/3","title":"Journal of Nanobiotechnology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"87f573fe-d085-4bf9-a07e-4fc3882c0f91","owner":[],"postedDate":"January 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-10T22:54:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-08 10:47:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8441498","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8441498","identity":"rs-8441498","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}