Materials and reagents

Ammonium hydroxide aqueous solution (NH3·H2O, 28%), ethanol, tetraethyl orthosilicate (TEOS), magnesium chloride hexahydrate (MgCl2·6H2O) and ammonia chloride (NH4Cl) were all purchased from Aladdin Biochemical Technology Co., Ltd. (China), and polyethyleneimine (PEI, mw = 25 000) was purchased from Sigma-Aldrich (USA).

Preparation and characterization of MSNs

Magnesium silicate nanospheres (MSNs) were synthesized via a two-step route in accordance with our previous studies.17 Monodispersed silica colloidal nanospheres (nano-SiO2) with an average diameter of 200 nmol/L were prepared with the modified Stöber method. MgCl2·6H2O (304.50 mg), NH4Cl (1.07 g) and NH3·H2O (2.00 mL) were dissolved in 20 mL of deionized water. Nano-SiO2 (200 mg) was dispersed in another 20 mL of deionized water with ultrasonic oscillation. Then, the two solutions were mixed and transferred into a reaction still and sealed to heat at 160 °C for 12 h. After naturally cooling to room temperature, the obtained MSNs were rinsed with deionized water and ethanol in turn and dried in vacuum at 60 °C overnight. The morphological and structural features of MSNs were examined by SEM (GeminiSEM 300, Zeiss, Germany) and TEM (JEM-1400flash, JEOL, Japan), and EDS was used to detect the main elements in MSNs.

Modification of MSNs and preparation of the MSN+miR-146a complex

MSNs were modified with PEI via electrostatic interactions.21 MSNs were resuspended in deionized water (1 mg/mL) and added to an equivalent PEI solution (1 mg/mL), and the mixed solution was stirred at room temperature at 400 r/min for 3 h. After centrifugation and thorough washing with deionized water, MSN-PEI was prepared and stored in deionized water at a 1 mg/mL concentration and examined by TEM. MiR-146a in this study is referred to as miR-146a-5p, and its sequence is shown in Table S1. MSN solutions (10 μL) were combined with miR-146a solutions (0.05 μg, 10 μL) at various weight ratios (MSN:miR-146a = 0:1, 25:1, 50:1, 75:1, 100:1, 125:1, 150:1, 200:1), mixed with a vortex mixer for 1 min and then incubated at 4 °C for 30 min. The optimal loading ratio was confirmed using a gel retardation assay with 1% agarose gel containing 1 × GelRed (Us Everbright, USA). The MSN+miR-146a complex was added to 10 × loading buffer, and electrophoresis was carried out at 100 V for 10 min in TAE running buffer (Mei5Bio, China). The miR-146a gel was visualized with a ChemiDoc MP Imaging System (Bio-Rad, USA). Then, the surface charge of MSNs adsorbing various amounts of miR-146a was estimated by a zeta potential analysis meter (Surpass, Anton Paar, Austria).

Cell culture

hDPSCs were isolated from third molars (clinical waste) or permanent teeth from adolescents following reported protocols,40 which were approved by the Ethical Committee of Stomatology Hospital of Zhejiang University School of Medicine (ethics approval number: 2023-027) in accordance with the Helsinki Declaration. Written informed consent was obtained from the subject or subject’s parents. Briefly, dental pulp was extracted with a dentinal excavator and then gently rinsed with phosphate-buffered solution (PBS) (Cienry, China). After being dissected into 1–2 mm3 pieces, the dental pulp tissue was planted into a 25 mm2 culture flask containing 1 mL of fetal bovine serum (FBS) (Gibco, USA) and cultured in a humidified incubator at 37 °C with 5% CO2 for 6 h. Then, 0.5 mL of minimum essential medium α (MEM α) containing 2 mmol/L L-glutamine (Gibco, USA) with 10% FBS and 100 U/mL streptomycin/penicillin (Cienry) (complete MEM α) was added into the flask for further culture. The medium was gently changed every 3 days, and the cells were passaged until 80% confluence using 0.05% trypsin containing ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher, USA). hDPSCs after P3 were collected for the study.

Mouse BMMs were harvested from the femur and tibia bones of 5-week-old male C57BL/6J mice (Zhang et al., 2008). The bone marrow was flushed out using a 25G needle and 1 mL syringe filled with cold MEM α until the bones turned pale. The turbid cell liquid was pipetted up and down and then filtered through a 70 μm filter. After centrifugation at 250 g for 5 min, the supernatant was discarded, and the cell pellet was resuspended and incubated in ammonium-chloride-potassium (ACK) lysing buffer (Amizona, USA) for 90 s to remove red blood cells. After PBS washes and centrifugation, the cells were resuspended in complete MEM α with 20 ng/mL recombinant mouse macrophage colony-stimulating factor (M-CSF) (Amizona) (MΦ-MEM α). The cells were counted and directly seeded at 1 × 105 cells/well into 24-well plates. The same BMMs were treated with 40 ng/mL M-CSF and an additional 40 ng/mL recombinant mouse receptor activator of nuclear factor kappa-B ligand (RANKL) (Amizona) to induce osteoclast differentiation. All animal procedures (including following in vivo experiments) were approved by the Institutional Animal Care and Use Committee of Zhejiang Center of Laboratory Animals (approval No. ZJCLA-IACUC-20010204).

Cytotoxic assay of MSNs

hDPSCs were seeded at 2 × 103 cells/well in 96-well plates. The next day, MSNs were added at various final concentrations (0–50 μg/mL). After coculture for 8 and 24 h, the CCK-8 assay was performed using a CCK-8 Cell Proliferation Kit (Beyotime, China) in accordance with the manufacturer’s instructions.

Cellular uptake assay

hDPSCs were seeded at 1.5 × 103 cells/well on glass coverslips in 24-well plates. The next day, 25 μg/mL MSN+miR-146a-FAM complex (MSN:miR-146a-FAM = 75:1) was added and cocultured with hDPSCs for 24 h. Then, the cells were fixed with 4% (v/v) paraformaldehyde (Haoke Biotech, China) and permeabilized with 0.1% (v/v) Triton X-100 (Servicebio, China). The cells were stained with rhodamine-labeled phalloidin (Invitrogen, USA) to label the cytoskeleton, a DiI probe (Beyotime) to label the cell membrane or LysoTracker Red (Beyotime) to label lysosomes before mounting with DAPI (Servicebio). Samples were imaged using a laser scanning confocal microscope (LSM980, Zeiss).

Transfection of miR-146a and in vitro cell experiments

All cells were divided into four groups: the miR-146a group and NC group (purely transfected with miRNA by lipidosome), MSN group (loading nonsense oligo) and MSN+miR-146a group. hDPSCs were seeded at 1.5 × 104 cells/well in 24-well plates. When cells became confluent, in the non-MSN groups, 20 nmol/L miR-146a or NC miRNA was transfected with Interferin, while the corresponding MSN+miR-146a or MSN-NC complex (MSN: miRNA = 75:1) was added to the MSN-treated groups. After 6 h of culture, all hDPSCs were thoroughly washed in PBS and incubated in fresh complete MEM α with 50 μg/mL ascorbic acid (Mecklin, China), 10 mmol/L β-glycerophosphate (Sangon Biotech, China) and 100 nmol/L dexamethasone (Mecklin) (osteogenic MEM α). The medium was changed every 3 days. The osteogenic differentiation of hDPSCs was evaluated by specific in vitro staining using an ALP staining kit (Beyotime, China), ALP activity assay kit (Beyotime) and SR staining kit (Phygene, China) on Day 7 followed by an ARS staining kit (Beyotime) on Day 14 according to the manufacturer’s instructions.

For BMMs, the medium was first changed 2 days after plating, and miR-146a and NC miRNA were transfected with Interferin or MSNs similarly for 6 h. Then, fresh MΦ-MEM α containing 1 μg/mL LPS (Mei5bio) was added to induce the polarization of BMMs. The supernatant was collected and directly added to another culture plate of hDPSCs (already received the same miRNA transfection as above) for 24 h to stimulate an inflammatory microenvironment for hDPSCs. For osteoclasts, the transfection of miRNA was performed on day 3 after M-CSF and RANKL treatment, and the medium was first changed on day 4. Osteoclastic differentiation was evaluated by a TRAP staining kit (Amizona).

Quantitative RT‒PCR

hDPSCs were collected after culture in osteogenic MEM α for 7 days or coculture with LPS-stimulated BMM-derived conditioned medium for 24 h, and BMMs were collected after 24 h of 1 μg/mL LPS stimulation. Total RNA was extracted with an RNA extraction kit (Vazyme, China) and quantified with a Nanodrop 3000 (Thermo Fisher). Reverse transcription (RT) of mRNA was performed with 400 ng RNA using a cDNA synthesis kit (Vazyme), and RT of miRNA was performed with 100 ng RNA using a miRNA synthesis kit (Accurate Biology, China). Then, qPCR was carried out on QuantStudio 7 Flex (Life Technology, USA) using HiScript II Q RT SuperMix for qPCR (Vazyme) in a 10 μL reaction volume. β-actin and U6 were used as the endogenous reference genes. The sequences of the primer pairs are presented in Table S1. The relative gene expression level of target genes was calculated using the ΔΔCt method.

Western blotting

HDPSCs after 7 days of osteogenic induction and BMMs after 24 h of 1 μg/mL LPS stimulation were lysed in RIPA lysis buffer (Beyotime) for 30 min on ice, ultrasonicated for 3 s, and then centrifuged at 15 000 × g at 4 °C for 5 min. The protein concentration of the supernatant was determined using a BCA protein kit (Beyotime). After the samples was mixed with 5 × SDS loading buffer (Mei5bio) to obtain 1 μg/μL protein solution, sodium dodecyl sulfate–polyacrylamide gel electrophoresis was conducted to separate proteins with 10 μg per lane. Then, the protein was transferred to a 0.2 μm polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich). After being blocked in 5% defatted milk for 50 min at room temperature, the membrane was incubated at 4 °C overnight with primary antibodies against the following proteins: β-actin (1:10 000, 66009-1-Ig) and GAPDH (1:10 000, 60004-1-Ig) (both from Proteintech, USA); Col1a1 (1:1 000, 720260S), TRAF6 (1:500, 67591), p65 (1:500, 8242) and p-p65 (1:500, 3033) (all from Cell Signaling, USA); OSX (1:1 000, ab209484) and VEGF-A (1:1 000, ab214424 (both from Abcam, USA); and RUNX2 (1:500, ET1612-47, Huabio, China). Following a 1 h incubation with secondary horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG antibodies (1:10 000, SA00001-1 and SA00001-2, Proteintech) at room temperature, the membrane was visualized with an enhanced luminol-based chemiluminescent (ECL) kit (Thermo Fisher) and exposed in a ChemiDoc MP Imaging System (Bio-Rad).

Flow cytometry

BMMs were collected after 24 h of 1 μg/mL LPS stimulation using 0.05% trypsin and resuspended in PBS after centrifugation. Flow cytometry staining was performed according to the manufacturer’s instructions. PE F4/80 antibody (123109) and PerCP/Cy5.5 CD11b antibody (101227) (both from BioLegend, USA) were used to mark mouse macrophages. BV421 CD40 antibody (562846, BD Pharmingen, USA), Alexa Fluor (AF) 488 Arg-1 antibody (53-3697-82) and SB436 CD163 antibody (62-1631-82) (both from Thermo Fisher) were used to stain BMMs as markers of M1 or M2 polarization for 30 min on ice. Data acquisition was performed using CytoFlex (Beckman Coulter, USA) and analyzed with CytoExpert software (Beckman Coulter).

Cell immunofluorescence assay

HDPSCs after 7 days of osteogenic induction and BMMs after 24 h of 1 μg/mL LPS stimulation were washed thoroughly with PBS, fixed in 4% (v/v) paraformaldehyde and permeabilized with 0.1% (v/v) Triton X-100. The hDPSCs were stained with RUNX2 antibody (1:5 000, 1256S, Cell Signaling) overnight at 4 °C and incubated with secondary AF488 anti-rabbit antibody (1:500, 21441, Thermo Fisher) for 2 h at room temperature. BMMs were stained with CD86 (1:500, 26903-1-AP, Proteintech) or AF488 Arg-1 antibody (1:500, 53-3697-82, Thermo Fisher) overnight at 4 °C followed by secondary CoraLite 594 anti-rabbit antibody (for CD86) (1:500, SA00013-4, Proteintech) for 2 h at room temperature. The cytoskeleton was stained with rhodamine-labeled phalloidin if needed before mounting with DAPI. Samples were imaged using an LSM980 laser scanning confocal microscope, and the mean IF intensity was quantitatively measured by ImageJ software.

Mouse mandibular bone defect model

Eight-week-old male C57BL/6 mice were used to create mandibular bone defect models and divided into four groups: Blank, GelMA, MSN, MSN+miR-146a (n = 10 each). GelMA refers to a commercial gelatin methacryloyl hydrogel (EFL-GM 90, Engineering for Life, China). A 5% GelMA solution containing 1 mg/mL MSN with or without a corresponding amount of miR-146a was freshly prepared. Mice were anesthetized by intraperitoneal injection of 2,2,2-tribromoethanol (200 mg/kg; Sigma-Aldrich, USA), and the left mandibular region was shaved and disinfected. A 5-mm-long incision was made to expose the mandibular bone, and a 2 × 1.5 × 1 mm3 critical-size bone defect was created using a high-speed round bur. Approximately 5 μL of gel solution containing 50 μg/mL LPS and the appropriate biomaterial was injected per site and photocured with 405 nmol/L light, while the blank group was administered the same amount of PBS with LPS at the same time. The incision was carefully closed with a 5-0 silk suture. All mice were monitored every day, and no adverse effects were observed. After 2 and 4 weeks of healing, animals were euthanized using carbon dioxide, and mandible samples were collected and fixed in 4% (v/v) paraformaldehyde for 24 h followed by 70% ethanol at 4  °C.

Microcomputed tomography (CT) assay and biomechanical test

Mouse mandible samples were scanned using micro-CT (U-CT-XUHR, Milabs, The Netherlands) at a voltage of 55 kV and current of 0.17 mA with a 75 ms exposure time. After 3D volume rendering of scan data in Imalytics software (Gremse-IT, Germany), a virtual cylinder was created (1.2 mm diameter and 1 mm height) inside the mandibular bone defect area and designated as the region of interest (ROI). The bone grayscale threshold was set at 1 400 Hounsfield units (HU) for all samples. The BV/TV, BMD and Th. Sp were calculated to compare new bone formation among groups.

Another set of mice (n = 8) was euthanized after 4 weeks of healing. The paraformaldehyde-fixed mandibles were trimmed into 10 × 4 mm strip-like samples and subjected to a three-point bending test performed in an electronic universal material testing machine (5943, Instron, USA). The middle probe of the machine was directly pressed on the bone defect area, and the mean maximal force detected in the bending process was recorded for statistical analysis.

Histological and immunofluorescence analyses

The mandible samples were decalcified in a fast decalcified solution (Biotech, China) for 24 h at room temperature, dehydrated in graded ethanol and then embedded in wax. The tissue was cut into 5 μm sections using a microtome (Leica) and stained with HE, Masson’s trichrome and TRAP with commercial staining kits (Servicebio) to assess new bone formation and osteoclast function. After being blocked with bovine serum albumin (Servicebio), the sections were incubated with primary antibodies against Arg-1 (1:500, GB11285, Servicebio) and Runx2 (1:500, GB11264, Servicebio) as well as CD86 (1:500, 26903-1-AP, Proteintech) and Osx (1:500, ab209484, Abcam) at 4 °C overnight. Then, the sections were incubated with appropriate secondary antibodies (Servicebio) before mounting with DAPI. Samples were imaged using a DMI8 inverted fluorescence microscope (Leica Microsystems, Germany) and an LSM980 laser scanning confocal microscope with quantitative analysis by ImageJ.

Data analysis

Data were analyzed using Prism 8.0 software (GraphPad, USA) and are represented as the mean ± standard deviation (SD). Student’s t-test or one-way analysis of variance (ANOVA) was used to analyze the differences among groups followed by Tukey’s or Dunnett’s multiple comparison tests. The statistical significance level was set at P < 0.05.