Scaffolding Fabrication
BRT bioceramic scaffolds were fabricated as previously described. [13]. Briefly, Si-, Mg-, and Ca-containing BRT (Ca7MgSi4o16) Bioceramic powder was synthesized through a sol-gel process utilizing tetraethoxysilane (TEOS) and magnesium disitrate hexahydrate (Mg(NO)).three)2·6 hours2O) and calcium nitrate tetrahydrate (Ca(NOthree)2·4 hours2o). All chemicals were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). The synthesized powder was sieved through a 200 mesh screen and ground to particle size.
To prepare 3D printer bioink for BRT support, 0.15 g of sodium alginate powder and 5.0 g of BRT powder were added to 3.0 g of Pluronic F-127 (20.0 wt%). The ink was then extruded through a nozzle (inner diameter: 0.22 mm) and controlled using a Nano-Plotter.™ Software (GeSiM, Radeberg, Germany). Afterwards, the 3D printed primary BRT scaffold was calcined for 3 h (1,350°C, heating rate: 2°C min– One). According to the standard tessellation language file for 3D models, BRT scaffolds with aligned array structures (BRT-O) and BRT scaffolds with random shapes (BRT-R) were fabricated by placing filament patterns (0°/90°). . The fabricated scaffolds were then sterilized by UV irradiation for further in vitro and in vivo evaluation.
Scaffolding Characterization
The surface morphology of the BRT scaffolds was analyzed by scanning electron microscopy (SEM, Hitachi S-4800, Tokyo, Japan) at an acceleration voltage of 20 kV. Chemical composition and spatial matrix distribution were assessed by micro-Fourier transform infrared spectroscopy (Bruker, USA). The mechanical properties of the BRT scaffolds were analyzed using a universal mechanical testing machine (INSTRON 5566, Norwood, MA, USA) at a travel speed of 1.0 mm min.– One. Additionally, these scaffolds were placed in Tris-HCl solution (pH 7.40) for various periods of time to evaluate the ion release, pH value and weight change of scaffold decomposition in vitro. The samples were then incubated in a shaking incubator at 37°C. Afterwards, the solutions were collected and refreshed on days 3, 7, 14, 28, and 35. The concentrations of Ca, Mg, and Si ions were assessed using inductively coupled plasma atomic emission spectroscopy (ICP-AES, Varian Co., USA). , the pH value of the solution was monitored using a pH meter (Metrohm, Germany). To accurately measure weight, the support was removed, dried at 120°C for 24 hours, and then weighed using a digital scale. All experiments were performed in triplicate.
Evaluation of the effect of scaffolds on macrophage polarization
Murine macrophage RAW264.7 cells were obtained from the Cell Bank of the General Culture Preservation Committee of the Chinese Academy of Sciences (Shanghai, China). These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing 5% CO2, 10% FBS, and 1% penicillin/streptomycin.2 Incubator at 37 °C RAW264.7 cells were then cultured in six-well plates using various supports for the indicated periods.
Relative levels of iNOS and arginase 1 (Arg-1) gene mRNA transcripts to control GAPDH in different groups of RAW264.7 cells were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) using specific primers. (Table S1). The concentrations of cytokines (IL-10 and TNF-α) in the supernatants were determined using enzyme-linked immunosorbent assay (ELISA, Dakewe Bioengineering, China) according to the manufacturer's instructions. Additionally, the effect of scaffolds on the polarization of macrophages was analyzed by flow cytometry (BD Accuri C6, USA) using antibodies against CD86 (1:50 dilution, BioLegend, USA) and CD206 (1:400 dilution, BioLegend, USA). was analyzed. Additionally, immunofluorescence staining was performed 24 hours after culture. Briefly, primary antibodies against CD68, CD206 and iNOS (1:1,000 dilution, Abcam, USA) were dropped onto coverslips and incubated overnight at 4°C. Then, secondary antibodies Alexa Fluor 488 goat anti-mouse IgG (1:200, Abcam, USA) and Alexa Fluor 594 goat anti-rabbit IgG (1:200, Abcam, USA) were applied and mixed with the primary antibody for 1 h. I made it react. Nuclear staining was performed with 4',6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature. Afterwards, images were captured and visualized using a laser scanning confocal microscope (LSCM, Olympus, Japan). SEM was performed to observe the morphology of RAW 264.7 cells seeded on different supports for one day.
Assessing the impact of polarized macrophages on proliferation, migration and differentiation of bone marrow-derived mesenchymal stem cells (BMCSS) in vitro
To assess macrophages in response to the influence of scaffolds on BMSCS, conditioned medium (CM) was prepared by collecting supernatant samples from wells containing RAW264.7 cells seeded on scaffolds after 3 days of culture. . We then isolated, cultured, and identified BMSCS from the femur and tibia of male C57BL/6 N mice (6–8 weeks old) using well-established techniques, as previously described. [25]. Briefly, BMSCS were cultured in 96-well plates and treated with a mixture of DMEM and CM at a 1:1 ratio. BMSCS cultured with unconditioned medium were used as controls.
After 1, 3, and 5 days of culture, proliferation of BMSCS was evaluated using the CCK-8 assay. Wound scratch assay and transwell assay were performed to evaluate the migration of BMSCS. For wound scratch analysis, BMSCS were seeded in 6-well plates with basal medium. When cell confluency reached 90–100%, a scratch was made in the cell layer using the head of a 200 μL pipette tip (Axygen, USA). Serum-free CM was then added, and cells were further cultured for 12 and 24 hours. Cells without added CM were used as a control. Afterwards, cell migration was observed using a light microscope (Olympus, Japan), and the healing area was calculated using ImageJ software (NIH, USA). Additionally, a Boyden chamber was applied for transwell analysis. BMSCS were placed in the upper chamber with basal medium, and RAW264.7 cells were seeded on different scaffolds in the lower chamber. After culturing for 24 hours, the infiltrated cells were fixed with 4% paraformaldehyde, stained with crystal violet solution, and analyzed under a light microscope (Olympus, Japan).
After incubation with the supernatants for 7 days, osteogenic markers bone morphogenetic protein 2 (BMP2) and RUNX2 in BMSCS were assessed by qRT-PCR and Western blot. For qRT-PCR, the primers used are listed in Table S1. For Western blot, anti-RUNX2 (1:1,000; ab23981, Abcam) was used as the primary antibody, and Conjugated Horseradish Peroxidase was used as the secondary antibody. Calcium nodule formation was assessed at day 21 using Alizarin red S (ARS). Washed BMSCS were fixed in 4% paraformaldehyde and stained using 2% ARS solution for 20 min at room temperature. Stained calcium precipitates were dissolved in 10% cetylpyridinium chloride (Sigma-Aldrich, USA) for 15 min, and dye release was quantified spectrophotometrically at 562 nm (Thermo Fisher Scientific, USA).
In vivo onlay graft regeneration
All animal procedures were approved by the Animal Experiment Ethics Committee of the Ninth People's Hospital Affiliated with Shanghai Jiao Tong University School of Medicine. The study design followed the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
Animal model for onlay implantation
A total of 27 New Zealand white male rabbits (weight 2.8 ± 0.2 kg) were purchased from the Laboratory Animal Center of the Ninth People's Hospital of China. All animals were maintained under standard conditions (temperature 22 ± 2 °C, humidity 55 ± 5%, 12/12 h light/dark cycle) with water and food. optional. These animals were given general anesthesia by intravenous injection of pentobarbital sodium (30 mg/kg, Sigma), and their skull fur was shaved and disinfected. A longitudinal incision was then made along the midline of the skull and the soft tissue flap was lifted to expose the skull region. After preparing the recipient area with embellishment, the scaffold was implanted into the corresponding bone bed on both sides of the midline and fixed using titanium screws. For the autologous bone graft group, a circular bone block (Φ: 8 mm) harvested from the skull was fixed to the defect site using a dental trephine bur. The incision was then closed using layer-by-layer sutures. Therefore, the rabbits were divided into three groups: (1) autograft group; (2) BRT-R group; (3) BRT-O group. These rabbits were routinely housed and fed under standardized conditions and euthanized at 2, 6, or 16 weeks post-operatively with an overdose of intravenous pentobarbital sodium (150 mg/kg). A skull specimen was taken for further evaluation.
Micro CT analysis
Samples were fixed with 4% paraformaldehyde and evaluated using a micro-computed tomography system (micro-CT, Scanco Medical, Bassersdorf, Switzerland) with the following parameters: 70 kV voltage, 114 mA current, and 700 ms integration time. The acquired images were analyzed using the μ-CT 80 system software for 3D construction. A cylinder with a diameter of 8 mm and a height of 1 mm was selected as the volume of interest (VOI). Bone mineral density (BMD) and bone volume/tissue volume (BV/TV) were then calculated for quantitative analysis of bone regeneration within each VOI.
Histological and immunohistochemical evaluation
For histological evaluation, a portion of the samples was dehydrated and embedded in polymethyl methacrylate (PMMA). Without decalcification, tissue blocks were cut into sections (thickness: 200 μm) using a hard tissue microtome (Leica, Germany) and then sequentially ground to a final thickness of 25 μm. These sections were then stained with toluidine blue staining solution for new bone regeneration analysis. Another part of the sample was decalcified in 10% ethylenediaminetetraacetic acid and prepared into 10-μm-thick sections. Histological evaluation of newly formed bone and residual scaffold was performed using hematoxylin and eosin (H&E) staining. For each slice, three fields of view were randomly selected. Images were observed under a microscope (Olympus dp51) and captured using a digital camera (DXM1200). The osteogenic properties of different scaffold materials were compared using Image-Pro Plus software (Media Cybernetics, Inc.).
For immunohistochemical staining, specimens 2 weeks after surgery were dewaxed and analyzed for RUNX2 (Abcam, USA), an osteogenic marker, CD68 (Abcam, USA), a pan-macrophage marker, and CD206 (Abcam, USA), an osteogenic marker. Incubation was performed with primary antibody. For M2 and M1 markers, iNOS (Abcam, USA) was incubated with secondary antibodies at a 1:100 dilution overnight at 4°C. Afterwards, the stained area was observed under a light microscope (Leica DMI 6000B Microsystems, Germany), and the percentage of positive cells was calculated at 40× magnification.
statistical analysis
All statistical analyzes were performed using analysis of variance with Tukey's post hoc test using GraphPad Prism 8.0 (GraphPad Software Inc., USA). Data are presented as mean ± standard deviation. all blood-Values <0.05 were considered statistically significant.