Preparation of β-TCP
We used pure-phase β-TCP with a granulation size of 500–1000 µm, 65% ± 5% total porosity, 20% 5–50-µm, and 15% 50–200-µm pore diameter13 (CERASORB M, Curasan Co Ltd, Frankfurt, Germany); 200 mg control samples were prepared, with the same amount used for GDP treatment.
Gas discharge plasma treatment
We used the plasma jet device (AST Products Inc., North Billerica, MA, USA) for the argon GDP of the β-TCP test particles. The GDP treatment was set at 80 W using a radiofrequency of 13.56 MHz and 100 mTorr working pressure. The treatment time was 15 min with 10 mm argon plasma working distance from the β-TCP particles.
Surface morphological characterization
To analyze the surface morphologies of the scaffolds, β-TCP particles treated with argon gas discharge plasma were analyzed and compared with nontreated β-TCP. We used scanning electron microscopy (SEM; EX-250 SYSTEM, HORIBA, Kyoto, Japan) images to observe the microstructure and crystal size of the particles. These images were analyzed using ImageJ version 1.52 (NIH IMAGE, Bethesda, MD, USA). The arithmetical mean value roughness (Ra) was calculated for quantitative analyses.
For particle’s elements qualitative and quantitative analysis, energy-dispersive X-ray spectroscopy (EDS) was conducted. β-TCP test and control samples elemental composition analysis were performed using the same SEM. For the surface morphology evaluation, a machine that combines SEM and EDS was used (EX-250 SYSTEM, HORIBA, Kyoto, Japan).
X-ray photoelectron spectroscopy
Chemical analyses were achieved by a surface X-ray photoelectron spectroscopy (XPS) analysis technique with a depth profiling of approximately 50–70 Å from the surface using a monochromated 450 W Al Kα source (PERKIN-ELMER PHI ESCA 5500 SYSTEM). 220-W source power and 45° analyzer axis with angular acceptance of ± 7° were used for experiments recording. The charging shift was referred to the C1s line emitted from the saturated hydrocarbon at a binding energy of 285 eV. We recorded information on the chemical state of the core levels of the detected elements C1s, C1s, O1s, Ca2p, and P2p14.
X-ray diffraction analysis
β-TCP test and β-TCP control particles crystalline structures and chemical compositions were analyzed using powder X-ray diffraction (XRD). Pattern analyses were performed at a 60-kV and 45-mA current with Mo Kα λ = 0.71073 Å source. A diffractometer was used for all analyses in the range of 10° ≤ 2θ ≤ 70° (XPERT3 PRO, PANalytical Co. Ltd., Almelo, the Netherlands).
Fourier transform infrared spectroscopy
We measured 0.2-g powdered sample spectra from both the dry β-TCP test and β-TCP control on a Fourier transform infrared spectrometer (NICOLET iS50, Thermo Scientific, Madison, USA). Measurements were made in the wavelength range 4000–400 cm−1 with a resolution of 4 cm−1 at 25 °C and 65% ± 5% humidity. Three spectra were collected for each sample in the absorbance mode, including subtraction of a background scan, in order to reduce noise. Thus, the average of the three measurements were averaged to produce one spectrum15.
Human mesenchymal stem cells (hMSCs) were acquired from the Bioresource Collection and Research Center (Hsinchu, Taiwan). Moreover, they were maintained at 37 °C in humidified incubators with 5% CO2/95% air in specific culture media based on the protocol of our previous study, as described below16. Briefly, hMSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan, UT, USA) supplemented with l-glutamine (4 mmol/L), 10% fetal bovine serum, and 1% penicillin–streptomycin. The confluent cells were expanded until passage 3, using 0.05% trypsin–EDTA. The final concentration was adjusted to 1 × 104 cells/mL, and aliquoted into 24-well Petri dishes (NUNCLON; Roskilde, Denmark). On the same day, DMEM was mixed with β-TCP control or β-TCP plasma treated at a concentration of 1 g/10 mL. Twenty-four hours later, the medium was removed from each test well and substituted for the test media, consisting of the previously described DMEM + β-TCP control or β-TCP plasma treated. Same Dulbecco’s modified Eagle’s medium first described was used on control wells.
Cell viability (WST-1)
Cell viability was measured at days 1, 3, and 7 after adding DMEM + β-TCP control or β-TCP plasma treated to the test wells. Cell viability was measured using a colorimetric assay for 96-well plates with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt reagent (WST-1 Kit, Roche Applied Science, Mannheim, Germany). Summarily, the cell medium was replaced with 500 µL fresh medium, and 100 µl were added into 96-well microtiter plate (5 × 104 cells/well) and incubated for 24 h. Later a 10 µl of cell proliferation reagent WST-1 was added to each well and incubated for 2 h. Cell viability was measured at 450 nm in an ELISA reader (Thermo Fisher Scientific Inc., USA) with a reference wavelength of 650 nm. The percentage viability was calculated from the following equation: % viability = (100 × (control − sample))/control17.
The hMSCs were prepared for immunofluorescence microscopy on days 1, 3, and 7, in 24-well Petri dishes (NUNCLON; Roskilde, Denmark) as previously described in cell culture. The hMSCs were washed using 2× phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 15 min. After fixation cells were washed three times with PBS for 10 min. Cells were permeabilized using 0.2% Triton X-100 for 20 min, washed three more times, blocked with 1% goat serum in PBS for 1 h, and incubated overnight with primary antibodies in 0.1% goat serum at 4 °C. The cells were washed three times and incubated with secondary antibodies in 0.1% goat serum for 2 h at ambient temperature. After 3× washes, samples were quenched with 0.5% (wt/vol) Sudan Black B (Sigma-Aldrich, St. Louis, MO, USA) for 10 min, nuclear counterstaining was performed using 300 μl of DAPI (0.1 μg/mL) for 10 min. When phalloidin staining was performed, 200 μl of phalloidin solution (methanol-based stock solution diluted in PBS once to obtain a 100-μl final concentration) was added for 15 min after staining with the secondary antibodies and two washing steps with PBS. Residual phalloidin removal was performed before mounting. The same methodology was utilized for three-dimensional (3D) immunofluorescence microscopy after cells were cultured in TCP control or β-TCP plasma-treated particles for 24 h. The immunofluorescent-labeled samples were placed on glass slides and viewed using the Olympus Fluoview FV-1000 confocal laser-scanning microscope (Olympus, Japan), equipped with a 40× oil objective. The fluorescence images from DAPI and Phalloidin were merged using Leica LAS X software.
Alkaline phosphatase assay
Alkaline phosphatase (ALP) activity was determined by modifying the previously reported methods16. After cell culture media was suctioned and DMEM + β-TCP control or β-TCP plasma treated media were added to the test wells. On days 1, 3, and 7, hMSCs were washed twice with PBS and resuspended in 300 μl of Triton-100 0.05%. The cells underwent three cycles of 5 min at 37 °C and 5 min at − 4 °C. Afterward, using the Thermo Scientific 1-Step p-nitrophenyl phosphate disodium salt (PNPP) protocol. 100 µL of the 1-Step PNPP was added to each 96-well plate and gently mixed. Following incubation at room temperature for 30 min. Next, the reaction was stopped by adding 0.4 M of NaOH, and the plate was read at a wavelength of 405 nm in the Multiskan GO microplate spectrophotometer assay reader (Thermo Fisher Scientific).
Real-time polymerase chain reaction
The cultures of hMSCs were completed on days 0 (only for control), 1, 3, and 7, as previously described, in cell cultures after the media were suctioned, and DMEM + β-TCP control or β-TCP plasma-treated media were added to the test wells. Total RNA was extracted using the Novel Total RNA Mini Kit (NOVELGENE, Molecular Biotech, Taiwan) under the conditions recommended by the manufacturer. The cells were trypsinized, harvested, and resuspended; subjected to cell lysis and RNA binding; and washed and eluted, as previously described18,19.
Subsequently, gene expression levels were normalized to the expression of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. The analysis results were expressed as time-course gene changes relative to the cell’s genes cultured in DMEM only, and the calibrator sample representing the amount of transcript, was expressed on day 020. After the design of multiple primers, ALP, OC, CatK, Rank, RankL, OPG using the Primer-BLAST from the U.S. National Library of Medicine. Reactions were run using 2 μl of cDNA in a 20-μL reaction volume on LightCycler 96 system (Roche Molecular Systems, Inc., Pleasanton, CA, USA) with Fast SYBR Green Master Mix (Thermo Fisher Scientific, Vilnius, Lithuania) (Table 1). The reaction was repeated for 45 cycles; each cycle consisted of denaturing at 95 °C for 15 s and annealing, synthesis at 60 °C for 1 min and extension at 72 °C for 30 s, as per the manufacturer’s instructions. The relative amounts of the transcript of the tested genes were normalized using a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Posterior quantification was performed using the delta–delta calculation method16,21.
In vivo analysis
This in vivo animal experiment was conducted based on the ARRIVE guidelines22. The Experimental Animal Research of the Institutional Animal Care and Use Committee (IACUC) of Master Laboratory Co., Ltd. (IACUC no.: MI-201903–02, Hsinchu County, Taiwan) approved all experiments and animal care procedures. All surgical procedures were performed in accordance with the Animal Research: Reporting In Vivo Experiments guidelines. 15 adult male New Zealand white rabbits with a mean age of 3 months and a mean weight of 2.1 kg were ultimately enrolled in the present study. The animals were housed in separated cages in a climate-controlled Laboratory Animal Center, and the animals had ad libitum access to food and water.
Following an intramuscular anesthesia injection of Zoletil 50 (50 mg/mL) into the gluteal region at a dose of 15 mg/kg, the calvarial region was shaved and disinfected with iodine. Next, on the periphery of the calvaria for hemostatic and local anesthetic, 1.8 mL of 2% lidocaine with epinephrine 1/100,000 was injected.
A 2-cm long full depth incision was made in the linea media of the calvaria starting midway between the base of the ears. The pericranium was separated with a periosteal elevator from the outer table of the cranial vault23.
To prevent brain damage, the parietal bone was perforated three times using a 6.0-mm sterile trephine (3I Implant Innovation, Palm Beach Gardens, FL, USA)24. The first defect was filled with β-TCP control, and the second with β-TCP plasma-treated media. The third defect was left to heal as an unfilled control. Each animal was monitored closely until it fully recovery from anesthesia. The food intake, stool and urine output, and behavior of animals were monitored until they were sacrificed25,26,27.
Five randomly selected animals were sacrificed at 2, 6, and 8 weeks after surgery were euthanized with intramuscular injection of Zoletil 50 (50 mg/mL) at 15 mg/kg and later CO2 asphyxiation for 10 min. The monolithic blocks were extracted and immediately fixed in 10% formaldehyde for Micro CT, histological and histomorphometrical analysis.
Micro-computed tomography scanning of new bone formation
Sample blocks were prepared in formalin, and micro-computed tomography (micro-CT) scanning analyses were performed within 2 weeks using SkyScan 1076 (SKYSCAN, Antwerp, Belgium). After setting the micro-CT images, coronal images of the upper peripheral areas of the defect were saved in the database. To measure the tissue area/bone area, two-dimensional morphological analyses were performed. Thus, binary selections of samples from the morphometric analyses were made according to gray-scale density between units 20 and 80. The morphometric analyses were performed using SkyScan 1076 data-viewer software according to the manufacturer’s instructions.
Sections from all paraffin-embedded tissues were routinely stained with hematoxylin and eosin (H&E) and were simultaneously processed to reduce internal staining variations. The optical images of two mid-sections crossing the center of the calvaria defects were used to perform histomorphometrical analysis. For each histological section, the area occupied by the growing bone was identified and measured at a magnification of 200× using the ImageJ software, developed by the National Institutes of Health (NIH IMAGE, Bethesda, MD, USA). These values were used to calculate the percentage of bone area/tissue area.
All experiments shown in this study were conducted independently, and the results were presented as means ± standard deviation. Microsoft Excel Professional Plus 2016 (Microsoft Software, Redmond, WA, USA) was used for all quantitative statistical analyses. After the statistical analyses, the differences among the groups were compared and considered significant at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. The two-tailed Student t-test was used to compare between groups, respectively.