In situ construction of flower-like nanostructured calcium silicate bioceramics for enhancing bone regeneration mediated via FAK/p38 signaling pathway | Journal of Nanobiotechnology

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Fabrication of nanostructure CS (nCS) bioceramics

CS powders were obtained from Kunshan Chinese Technology New Materials Co., Ltd. (China). The CS powders were mixed with 6 wt% polyvinyl alcohol (PVA), further compacted into discs under a pressure of 7 MPa in a stainless-steel die, and then heated at 1100 °C for 5 h to fabricate CS bioceramic discs with a diameter of 10 mm and a thickness of 2 mm. Subsequently, the CS discs were subjected to hydrothermal treatment (incubation in aqueous solutions with pH = 7 for 72 h at 180 °C) to generate flower-like nanostructure surfaces. The CS discs with nanostructure surfaces were labeled nCS and used in in vitro experiments.

To better observe bone regeneration in the animal experiments, porous CS bioceramic scaffolds were fabricated by the porogen method as described in a previous study [28]. Polyethylene glycol (PEG) with a diameter ranging from 300 to 600 μm was used as a porogen and mixed with CS powder in a suitable ratio with 6 wt% PVA. The mixture was pressed into a three-dimensional (3D) cylinder with a diameter of 5 mm and a thickness of 1 mm, and then, porous scaffolds were obtained after sintering at 1100 °C. Finally, CS bioceramic scaffolds with nanostructure surfaces were prepared after hydrothermal treatment and used in in vivo experiments.

CS bioceramic discs and scaffolds that were not subjected to hydrothermal treatment were considered control samples.

Characterization and protein adsorption performance of nCS bioceramics

The crystal phases of the CS and nCS discs were characterized by X-ray diffraction analysis (XRD, Rigaku, Japan), Fourier-transform infrared spectrometry (FTIR, Nicolet iS 10, Thermo, USA), and X-ray photoelectron spectroscopy (XPS, Thermo Kalpha, USA). The surface morphology of CS bioceramics was characterized by field emission scanning electron microscopy (FESEM, JEOL, Japan), transmission electron microscopy (TEM, FEI Tecnai F20, Japan), scanning transmission electron microscopy (STEM, FEI Tecnai F20, Japan) and energy dispersive spectrometer (EDS).

To evaluate the biodegradability of bioceramics, the CS and nCS discs were immersed in Tris–HCl buffer (pH = 7.4, 37 °C) at a ratio of 0.1 (mm3:ml), and the solution was refreshed every 2 days. At 1, 3, 7, 10, and 14 days, the pH value of Tris–HCl was measured with a pH meter (Mettler Toledo, Switzerland), and the concentrations of Ca and Si ions were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Varian, USA) [29]. In addition, the ions release of CS and nCS bioceramics in phosphate buffer solution (PBS, pH = 7.4, 37 °C, Gibco, USA) for 14 days also was measured as described above. After 14 days, the surface of CS bioceramics was observed by SEM–EDS mapping. The wetting of the CS and nCS samples was analyzed by a goniometer (SZ-CAM, SUNZERN, Shanghai, China). In brief, the CS and nCS samples were placed on the sample stage, and 10 μl Milli-Q water (Millipore, water purification system model Direct-Q 3 UV, Merck, USA) was added to each sample (n = 5) via syringe by a water dispensing system. The contact angle between the tangent to the liquid–vapor interface and the solid surface at the three-phase contact line was calculated by a computer system.

To assess the protein adsorption ability of CS and nCS, 15 mg/ml bovine serum albumin (BSA) solution was applied on both samples. After soaking both samples in 1 ml BSA solution (C0, 15 mg/ml) for 48 h at 37 ℃, the rest protein concentration (C1) was measured by Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Scientific TM, USA). The amount of BSA adsorption and the loading capacity of both samples were calculated according to the formulae below:

  • Protein adsorption amount (mg) = (C0−C1) × Vsolution.

  • Loading capacity (%) = (C0−C1)/C0 × 100%

  • C0: represented the original concentration of BSA solution.

  • C1: represented the rest concentration of BSA solution.

Cell culture

Sprague–Dawley (SD) rats (2 weeks old) were purchased from Shanghai Sippr-BK Laboratory Animal Co. Ltd. (China). Bone marrow stem cells (BMSCs) were extracted from the femur and tibia of the rats as previously reported [30]. Then, the BMSCs were cultured in α-minimum essential medium (α-MEM, Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% penicillin–streptomycin (Gibco, USA). The cultures were maintained in an incubator (37 °C, 5% CO2, and 95% relative humidity), and the medium was refreshed 3 times a week to remove the nonadherent cells. After reaching 80%–90% confluence, the BMSCs were passaged, and passages 2–4 were used in the in vitro experiments in the study. To decrease the alkalinity of CS bioceramics, we soaked all the samples in deionized water for two weeks and changed deionized water every day. After soaking in deionized water for two weeks, CS and nCS samples were directly used for subsequent in vitro and in vivo experiments.

Cell adhesion and morphology

BMSCs were seeded on CS and nCS discs (diameter of 10 mm) at a density of 2 × 104 cells per well in 24-well culture plates with 1.5 ml medium. Culture medium with CS and nCS bioceramics was also changed every day. After culturing for 6 h, the cells on the bioceramic discs were washed with PBS 3 times and fixed with 4% paraformaldehyde for 30 min according to the manufacturer’s instructions. Then, all the cells were incubated with phalloidin (Sigma, USA) for 30 min and 4′,6-diamidino-2-phenylindole (DAPI, Sigma, USA) for 5 min and then observed with a confocal laser scanning microscope (CLSM, Leica, Germany). At 24 h after incubation, the cells on CS and nCS bioceramics were fixed with 2.5% glutaraldehyde at 4 °C for 12 h. The samples were further fixed with 1% osmic acid solution for 2 h, dehydrated with gradient concentrations of ethanol solution (30%, 50%, 70%, 80%, 90% and 95%), coated with gold and finally observed by using scanning electron microscopy (SEM, JEOL, Japan).

Immunofluorescence staining was used to further evaluate the effect of nCS bioceramics with flower-like nanostructures on the expression of focal adhesion proteins in cells. BMSCs were seeded on CS and nCS bioceramics at a density of 2 × 104 cells per well in 24-well culture plates with 1.5 ml medium. After being cultured for 6 h, the BMSCs were fixed with 4% paraformaldehyde for 30 min, rinsed with PBS 3 times and permeabilized with 0.3% Triton-X 100. Then, the cells were further blocked in 1% bovine serum albumin (BSA) for 1 h. Both groups were incubated with primary rabbit anti-rat vinculin monoclonal antibodies (1:200, Abcam, USA) overnight at 4 °C on a table shaker to measure the expression level of focal adhesion proteins. Then, the samples were incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibodies (1:500, Thermo Fisher, USA) at 37 °C for 1 h. The cytoskeleton and cellular nuclei were stained with FITC-phalloidin and DAPI, respectively. Images were captured with an inverted fluorescence microscope (Olympus, Japan). All the experiments were repeated three times.

Cell proliferation and biocompatibility in vitro of CS bioceramic

The MTT assay was carried out to investigate the proliferation of cells cultured on CS and nCS bioceramics (diameter of 10 mm); the BMSCs were cultured at an initial density of 1 × 104 cells per well in 24-well culture plates in 1.5 ml culture medium, which was replaced every day. After 1, 3, and 7 days of culture, all the cells cultured on bioceramics were incubated with MTT solution (5 mg/ml) at 37 °C for 4 h. Subsequently, dimethyl sulfoxide (DMSO, Sigma, USA) solution was added, and the absorbance (OD value) was read at 490 nm with a microplate reader (Biotek, USA). All the experiments were repeated three times.

To evaluate the blood compatibility of CS bioceramics in vitro, a hemolytic test was used to evaluate CS and nCS samples as previous study [31]. Fresh blood cells (BCs) were extracted from 2-week-old SD rats purchased from Shanghai Sippr-BK Laboratory Animal Co. Ltd., and the BCs were centrifuged at 2000 rpm for 5 min. The 4% BC solutions were obtained by mixing 0.2 ml BCs with 4.8 ml PBS solution, and both samples were incubated in PBS for 24 h to obtain the sample solution. Then, 0.5 ml BC solution was incubated in 0.5 ml sample solution, Triton X-100 and PBS at 37 °C for 1 h. Triton X-100 was used as a positive control, and PBS was used as a negative control. The supernatants were harvested by centrifuging the samples at 2000 rpm for 5 min, and the supernatants were photographed with a digital camera. Ultimately, the OD value of the supernatants were measured with a microplate reader at a wavelength of 540 nm.

Alkaline phosphate (ALP) activity and staining assay

BMSCs were seeded on CS and nCS discs at a density of 2 × 104 cells per well in 24-well culture plates in 1.5 ml culture medium, which was replaced every day. At 4, 7 and 10 days of culture, the cells cultured on bioceramics were rinsed with PBS, lysed with 1% Triton X-100, and centrifuged at 4 °C, and ALP activity was quantified with an ALP kit (Beyotime, China) according to the manufacturer’s protocol. The absorbance was read at 405 nm with a spectrophotometer, and the total cellular protein content was determined using a BCA protein kit (Beyotime, China). The ALP activity was normalized to the total protein concentration. To intuitively characterize the effect of nanostructures on ALP expression, an ALP staining assay was also carried out using an ALP staining kit (Beyotime, China) at Days 4, 7 and 10. All the experiments were repeated three times.

Quantitative real-time PCR (qRT–PCR) and immunofluorescence analyses of Runx2 expression

BMSCs were seeded on CS and nCS bioceramics (diameter of 25 mm) at a density of 1 × 105 cells per well in 6-well culture plates in 5 ml medium, which was replaced every day. After being cultured for 7 days, total RNA was isolated from each well using TRIzol Reagent and then reverse transcribed to complementary DNA (cDNA) by a PrimeScript™ RT Kit (TaKaRa, Japan). The expression levels of bone sialoprotein (Bsp), runt-related transcription factor-2 (Runx2), collagen type I alpha 1 (Col1a1), and osteopontin (Opn) were quantified, and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was used as a housekeeping gene for normalization. The primer sequences used in this study were presented in Additional file 1: Table S1.

BMSCs were seeded on both types of bioceramics (diameter of 10 mm) at a density of 1 × 104 cells per well in 24-well plates in 1.5 ml medium and were cultured for 7 days. Cell fixation with 4% PFA, cell permeability with 0.3% Triton X-100, and cell blocking with 1% BSA were performed. Both groups were incubated with primary rabbit anti-rat Runx2 monoclonal antibodies (1:5000, Abcam, USA) overnight at 4 °C on a table shaker to measure the expression level of Runx2. Furthermore, cell cytoskeleton and cellular nuclei staining were performed as described above. Images were captured with an inverted fluorescence microscope.

Western blotting experiments

Western blotting was used to assess the molecular mechanism underlying the effects of CS and nCS. The protein expression levels of focal adhesion kinase (FAK), phospho-focal adhesion kinase (p-FAK) and MAPK signaling pathway-related proteins (extracellular signal-related kinase, ERK; phospho-ERK, p-ERK; c-Jun-amino-terminal kinase, JNK; phospho-JNK, p-JNK; p38; phospho-p38, p-p38) were measured, and β-actin expression was used as a reference. BMSCs (3 × 105) were cultured on CS and nCS bioceramics (diameter of 25 mm) in 6-well plates in 5 ml medium per well for 48 h. Additionally, the FAK signaling pathway inhibitor PF573228 (1 μM, Sigma, USA) and p38 signaling pathway inhibitor SB203580 (10 μM, MedChemExpress, USA) were separately added to the culture medium for further studies. Total protein was extracted with RIPA buffer (Beyotime, China) supplemented with 1% phenylmethanesulfonylfluoride (PMSF, Beyotime, China) at 4 ℃ for 5 min, and the samples were centrifuged at 12,000 rpm for 15 min. Then, the cellular supernatants were collected, and the protein concentrations were quantified with a BCA protein assay kit (Thermo Fisher, USA). Protein samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) at 80 V for 20 min and 120 V for 50 min, and then, the separated proteins were transferred to polyvinylidene fluoride membranes (PVDF, Millipore, USA). After blocking with 5% skim milk, the PVDF membranes were incubated with each primary antibody (Abcam, UK) overnight at 4 °C. Then, the PVDF membranes were washed three times with Tris-buffer solution with Tween (TBST, Beyotime, China) and incubated with HRP-conjugated secondary antibodies (Abcam, UK) for 1 h at room temperature. The protein bands were visualized, and images were captured with an automated luminescent image analysis system (Tanon, China).

Animal procedures and evaluation of bone regeneration

Six-week-old SD rats were purchased from Shanghai Sippr-BK Laboratory Animal Co., Ltd. to establish calvarial defects. Subsequent animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Ninth People’s Hospital Affiliated with Shanghai Jiaotong University, School of Medicine. All the rats were randomly divided into 3 groups. A 2-cm longitudinal incision was made after general anesthesia was achieved via the intraperitoneal injection of 3.5% pentobarbital sodium. The scalp was separated for exposition, and two symmetrical round defects 5 mm in diameter were made at the frontal bone of each rat using a drill. To avoid individual differences in the experiments, eighteen calvarial defects were constructed in nine SD rats. Three experimental modalities were randomly allocated to eighteen defects (n = 6), as follows: 1) blank control; 2) CS group; 3) nCS group. At 2, 4, and 6 weeks after the operation, all the rats were intraperitoneally injected with tetracycline hydrochloride (TE, 25 mg/kg), alizarin red (AL, 30 mg/kg), and calcein (CA, 20 mg/kg) to assess new bone formation and mineralization.

After being implanted for 8 weeks, the specimens (bone and scaffolds) were harvested, fixed in 4% paraformaldehyde for 3 days and then scanned with microcomputed tomography (PerkinElemer, QuantumGX, Japan) to analyze the bone volume fraction (BV/TV) in the defect area with auxiliary software (Analyze 12.0, Japan). On the one hand, half of the samples from all the groups were dehydrated with ascending concentrations of ethanol solution (75%–100%), embedded in polymethylmethacrylate (PMMA), sectioned using a microtome and further ground to a final thickness of 50 μm. Subsequently, fluorescent labeling of the samples was observed with CLSM (Leica, Germany), and the area of fluorochrome-stained bone and the distance between fluorescent stripes were measured with ImageJ (NIH; http://rsb.info.nih.gov/ij, USA). Next, the sections were stained with Van Gieson’s (VG) to observe the formation of new bone. The area of new bone formation in the VG-stained sections was measured with ImageJ. On the other hand, half of the samples from all the groups were decalcified with 10% EDTA for 1 month, dehydrated, embedded in paraffin, and stained using hematoxylin and eosin (H&E) and Masson’s trichrome staining [32,33,34].

To evaluate the biosafety in vivo of the CS bioceramics, CS and nCS bioceramics were implanted into the bilateral pockets of the perivertebral fascia lumbodraslis of SD rats under anesthesia (0.5 mg/kg of pentobarbital sodium). Briefly, the wounds were irrigated with normal saline and sutured in layers, and the animals were euthanized by carbon dioxide (CO2) asphyxia after 2 weeks. The implanted bioceramics and neighboring tissues were harvested for digital images. Subsequently, CS and nCS were removed and the subcutaneous tissue (Skin) was performed histological analysis, including H&E and immunohistochemical staining of tumor necrosis factor-a (TNF-α).

Statistical analysis

All the data are presented as the mean ± standard deviation (SD). Statistical analysis was performed by one-way analysis of variance using SPSS 22.0 software (IBM Inc., USA). A p value less than 0.05 was considered statistically significant.

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