2.1. Materials
PEEK substrates were purchased from Xinye Plastic Materials Co., Ltd. (Dongguan, China). Low intensity pulse ultrasound (LIPUS) instrument was purchased from SXULTRASONIC Co., Ltd. (Shenzhen, China). Barium titanate, dopamine hydrochloride and sulfuric acid (H2SO4) were purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Alpha modified eagle medium (αMEM) and fetal bovine serum (FBS) were bought from Gibco (USA). β-glycerophosphate disodium salt, ascorbic acid, and dexamethasone were purchased from Sigma (USA). Cell viability staining kit, ghost pen peptide, alizarin red S (ARS) staining kit, nuclear protein separation kit, and laminB antibody were purchased from Solarbio Technology Co., Ltd. (Beijing, China). Cell counting kit-8 (CCK-8), calcein-acetoxymethyl ester (Calcein-AM) propyl iodide (PI) cell viability/cytotoxicity assay kit, and DAPI were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). LY294002 was purchased from Selleckchem (Houston, TX, USA). Akt, phosphorylated Akt (P-Akt), GSK3β, phosphorylated GSK3β (P-GSK3β), and β-catenin antibodies were purchased from Cell Signaling Technology Co., Ltd. (Danvers, USA). Primers were provided from Tsingke Biotech Co., Ltd. (Shanghai, China). Runt-related transcription factor 2 (Runx2) and alkaline phosphatase (ALP) antibodies were purchased from Proteintech Co., Ltd. (Wuhan, China).
2.2. Synthesis of PBSP
PEEK substrates were immersed in H2SO4 and stirred for 5 min, following a water bath (120℃, 12 h) to remove excess sulfuric acid to obtain sulfonated PEEK (SPEEK). 0.3 g of tris-base was weighed and dissolved in 50 mL of pure water firstly, then concentrated hydrochloric acid was added dropwise to adjust the pH of the solution to 8.5. Afterwards, 100 mg of dopamine hydrochloride (DA) was added and polymerized in a dark environment to obtain a PDA solution. For the immobilization of BTO, BTO powders (1.0 mg/mL) were added into PDA solution to obtain PDA@BTO suspension, then SPEEK substrates were soaked in PDA@BTO suspension and incubated with shaking at 37°C for 24 h at room temperature avoiding light. To remove the unattached PDA molecules, the reacted substrates were washed in an ultrasonic cleaner (SB25-12DT, SCIENTZ, China) with distilled water until the water became clear. Finally, the substrates were taken out and dry at room temperature to obtain PDA@BTO-SPEEK (PBSP).
2.3. Characterizations
The morphology and elemental composition of all synthesized samples were examined by scanning electron microscopy (SEM, Zeiss SIGMA, UK) and atomic force microscope (AFM, BRUKER Dimension Icon, Germany). The X-ray diffraction (XRD, BRUKER-D8 ADVANCED, Germany) and X-ray photoelectron spectroscopy (XPS, Thermo SCIENTIFIC ESCALAB250Xi, USA) were adopted to analyze the chemical structure of all samples. Water contact angles were measured by a contact angle measuring instrument (Dataphysics-OCA20, Germany). The piezoelectric properties of the samples were tested using a quasi-static d33 tester (ZJ-3AN, China). The output voltage of each group of samples was detected by a digital oscilloscope (DHO1000, China) under LIPUS triggering, and the following parameters were set: center frequency of 1 MHz, pulse repetition frequency of 1000 Hz, pressure intensity of 30 mW/cm2 and duty ratio of 50%. The concentration of Ba element in the extracts of PBSP after immersion in phosphate buffer saline (PBS) for 21 days was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 5110, USA). The hardness of samples was analyzed using a microhardness tester (HV-1000, China).
2.4. Cell Viability
Mouse embryo osteoblast precursor (MC-3T3-E1) cells were incubated in αMEM medium supplemented with 10% FBS and 1% streptomycin/penicillin at 37℃ in a CO2 incubator. CCK-8 assay was performed to evaluate the cytotoxicity of different samples when combined with LIPUS. The extracts were obtained according to ISO1099312:2007. When MC-3T3-E1 cells reach the logarithmic growth phase, they were seeded onto 96-well plates at a density of 2.0 × 103 cells per well, followed by 24 h of incubation in an incubator (37℃, 5% CO2). Then, the cells were irradiated with LIPUS stimulation for 10 min once a day. After 24, 48, and 72 h co-culture, the CCK-8 solution was added to each well (100 µL per well) and the cells were incubated for another 4 h. The absorbance value of each well at 450 nm was determined by a multifunctional microplate reader (VICTOR Nivo, Perkin Elmer, USA). The survival rate of cells was calculated by the following formula:
Where A was the absorbance of the experimental group, A0 was the absorbance of the PEEK group, and A1 was the absorbance of the negative group.
Besides, the live/dead staining was conducted to verify the viability of MC-3T3-E1 cells. Briefly, MC-3T3-E1 cells were seeded into 6-well plates at a density of 1.0 × 105 cells per well and then co-cultured for 24 h. The live cells were stained green by Calcein-AM, and the dead cells were stained red by PI. The stained cells were photographed in the dark with an inverted fluorescence microscope (IX73, Olympus, Japan).
2.5. Cell Adhesion
To detect the adhesion of cells after co-culture with the different substrates, MC-3T3-E1 cells (1.0 × 105 cells) were inoculated on the surface of each sample (with a diameter of 30 mm) combined with LIPUS. After 24 h, the culture medium was removed, and cells were fixed with 4% paraformaldehyde. Then the cells were permeated by 1% TritonX-100 solution. After that, TRITC and DAPI solution (100 nM) were added successively. The images of stained cells were captured by a confocal laser scanning microscope (CLSM, TCS SP8, Leica, Germany) in a dark environment.
2.6. Osteogenesis Induced Cell Culture
To detect the cell osteogenic differentiation phenotypes, MC-3T3-E1 cells were cultured with complete medium, in which included 10 mM β-glycerophosphate disodium salt, 50 µg/mL ascorbic acid, and 10 nM dexamethasone. MC-3T3-E1 cells (1.0 × 105 cells per well) were seeded in 6-well plates and incubated with the extract of different samples. The cell medium was changed and LIPUS stimulation was given to the corresponding group every three days.
2.7. ARS Staining
To assess extracellular matrix mineralization, the MC-3T3-E1 cells were immersed in ARS solution and photographed by an inverted fluorescence microscope (IX73, Olympus, Japan) on day 7 and day 14. Then, to conduct semiquantitative statistical analysis, the cells were co-incubated with 10% cetylpyridinium chloride (PHR1226, Sigma, Missouri, USA), for 30 min with shaking. After centrifugation for 15 min, the optical density (OD) values of collected supernatant were measured at a wavelength of 562 nm using a multifunctional microplate reader (VICTOR Nivo, Perkin Elmer, USA).
2.8. ALP Staining
The ALP activity of MC-3T3-E1 cells was evaluated after 7 days and 14 days by BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime, China), according to the manufacturer’s instructions. At the pre-set timepoints, the ALP staining solution was added to each well. The cells were photographed by an inverted fluorescence microscope (IX73, Olympus, Japan). The spread area and integrated optical density (IOD) of different groups of stained cells were analyzed by Image-pro Plus J software.
2.9. Expression of Osteogenesis-Related Genes
The osteogenic induction solution and cell culture procedure used were the same as above. After culture for 14 days, MC-3T3-E1 cells on various samples were harvested by TRIzol (Solarbio, China). The extracted RNA was then reverse transcribed into cDNA using a reverse transcription system (Toyobo, Japan). The expression of osteogenic genes including runt-related transcription factor 2 (Runx2), collagen type I alpha 1 chain (COL1A1), and osteopontin (OPN) were examined with quantitative reverse transcription polymerase chain reaction (qRT-PCR). GAPDH was selected as the internal control. The forward and reverse primers for the selected genes were listed in Table S1.
2.10. Expression of Osteogenesis-Related Proteins
The expression of osteogenesis-related protein was evaluated by applying the western blotting method at a preset time point. The total protein was extracted from Radio Immunoprecipitation Assay (RIPA) lysis buffer (Solarbio, China) with 1% protease inhibitor mixture (Beyotime, China) and 1% phosphatase inhibitor (Solarbio, China). The protein sample (30 µg) was then subjected to 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then blotted onto polyvinylidene fluoride (PVDF) membranes according to standard protocols. After blocking with 5% (w/v) BSA, the membranes were incubated with primary antibodies at 4℃. After washing, the membrane was incubated with the secondary antibodies coupled with horseradish peroxidase (HRP) at room temperature for 1 h, and the immune response signal was detected with ECL reagent (Yatase, China). β-actin was selected as the internal control to detect the protein expression levels of ALP and Runx2. The results were analyzed and quantified by Image-pro Plus J software.
2.11. Activation of Mechanically Sensitive Ion Channels
To investigate the mRNA expression levels of the mechanically sensitive ion channels markers, including piezo type mechanosensitive ion channel component 1 (Piezo1), transient receptor potential vanilloid (TRPV) 4 and TRPV6, qRT-PCR and western blotting were carried out. In short, MC-3T3-E1 cells (1.0 × 105 cells per well) were seeded in 6-well plates and incubated with the extract of different samples for 24 h. After that, total RNA and proteins were extracted respectively according to the methods described above. Then, the expression of target genes was tested using the same procedures mentioned before. The forward and reverse primers for the selected genes are listed in Table S1.
2.12. Intracellular Ca2+ Detection
After coculture, the culture medium was removed and the cells were washed with PBS. Fluo-4 AM working solution (1.0 µM) was added to cover the cells sufficiently. Then the cells underwent another incubation at 37℃ for 30 min for fluorescence probe loading. After washing with PBS for 3 times, the fluorescence of Fluo-4 AM was observed by an inverted fluorescence microscope (IX73, Olympus, Japan) to determine the change in intracellular Ca2+ concentration.
2.13. Transcriptome Analysis
To unveil the potential mechanism of PBSP + LIPUS mediated Ca2+ influx during osteogenesis, transcriptome sequencing (RNA-seq) analysis was performed. Specifically, MC-3T3-E1 cells (1.0 × 106 cells/mL) were co-cultured with PBSP and PBSP + LIPUS, respectively, in 6-well plates for 14 days, and the gene expression profile of each group was investigated. Total RNA of each group was collected using TRIzol reagent following the manufacturer’s instructions and stored at − 80°C before sequencing. RNA-seq was performed by the Novogene Bioinformatics Institute (Beijing, China). The differentially expressed genes (DEGs) were examined using volcano plots and heatmap. Meanwhile, gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted to determine potential signaling pathways.
2.14. Akt/GSK-3β/β-catenin Signaling Pathway Validation
To confirm the activation of Akt/GSK-3β/β-catenin signal by PBSP + LIPUS, western blotting was conducted to assess the protein expression of Akt, phosphorylated Akt (p-Akt), GSK3β, phosphorylated GSK3β (p-GSK3β), and β-catenin in MC-3T3-E1 cells. LY294002 was introduced to selectively inhibit the PI3K activation. The expression of target genes was tested using western blotting mentioned before. The results were analyzed and quantified via Image-pro Plus J software.
2.15. Establishment of Skull Defect Model in Rats
The animal experiments were approved by the Animal Care and Use Committee of Zhongnan Hospital of Wuhan University (No. ZN2022282). A total of twenty-four SD rats were utilized in the calvarial defect model as previously reported. Firstly, the rats were anesthetized by intraperitoneal injection of pentobarbital sodium solution (40 mg/kg). Then, two critical sized full-thickness bone defects (with a diameter of 4 mm) were created at the center of each parietal bone in the rats using a saline cooled ring drill, and different samples were implanted according to grouping. The surgical area was washed and sutured in layers and positions. LIPUS stimulation was performed on the second day after surgery.
2.16. Microcomputed tomography (Micro-CT) Test
Whole calvarias were dissected from the sacrificed rats for bone formation evaluation at week 4 and 8 post-implantation. Before the Micro-CT analysis, all the samples were fixed with a 4% paraformaldehyde solution for 48 h. Micro-CT (Skyscan 1276, Bruker, Germany) scanning was used to evaluate the healing conditions of each specimen. Then, the bone parameters including bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) were measured by three-dimensional reconstruction.
2.17. Histological Staining
Rat calvarias fixed with 4% paraformaldehyde were rinsed with water, dehydrated with ethanol, cleaned with xylene, and then embedded in methyl methacrylate. Each specimen was cut into slices and subsequently processed with immunofluorescent staining for evaluating the expression of Runx2. The Runx2-positive stained sites were observed by a fluorescence scanner (PANNORAMIC MIDI, 3DHISTECH, Hungary). Masson staining was also carried out for assessing collagen fibrogenesis. All the staining images were captured using a high-resolution microscope (ECLIPSE Ci, Nikon, Japan). The quantification was conducted via Image-pro Plus J software.
2.18. In Vivo Biosafety
For the evaluation of the in vivo biosafety of PBSP + LIPUS, hematoxylin and eosin (H&E) staining of major organs dissected from the above rats was performed at week 8 post-implantation. Meanwhile, the whole blood was collected for the blood routine examination.
2.19. Statistical Analyses
All data in graphs were presented as the means ± standard deviations (s.d.). Statistical analysis among groups was performed using one-way analysis of variance (ANOVA) followed by t test. All measurements were conducted at least in three replicates. The GraphPad Prism was employed to carry out the data processing and statistical analysis, in which asterisks indicated the significant differences (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).