Raw materials
MP was synthesized from a powder mixture of analytical grade MgHPO4·3H2O and Mg(OH)2 in the molar ratio of 2:1, which was sintered at 1150°C for 4 hours in a laboratory furnace30. The sintered material was ground by hand in an agate mortar to pass through a 63 µm sieve.
PEEK was obtained from Sigma Aldrich (USA) in the form of 6 mm granulate.
Cell line and cultivation
A cell line of mouse embryonic fibroblast (ATCC CRL-1658 NIH/3T3, USA) was used for cytotoxicity determination. Medium for NIH/3T3 was formulated by ATCC as follows: Dulbecco's Modified Eagle's Medium (PAA Laboratories GmbH, xAUS) containing 10% of calf serum (BioSera, FR) and 100 U·mL− 1 Penicillin/Streptomycin (GE Healthcare HyClone, UK), was used as the culture medium. Cells were incubated at 37°C in 5% CO2 in humidified air.
For osteoblast proliferation and differentiation, a mouse osteoblastic precursors cell line, MC3T3-E1, was obtained from the European Collection of Cell Culture (c.n. 99072810). Proliferation conditions were obtained by cultivation of the cells in MEM Alpha medium (Gibco, USA) enriched by 10% FBS (Sigma Aldrich, USA) and penicillin/streptomycin (100 U·mL− 1, 100 µg·mL− 1, BioSera, FR). The differentiation conditions were adjusted by cultivating the cells in a differentiation medium prepared as described above but with the addition of 10mM β-glycerolphosphate (βGP, Sigma Aldrich, USA) and 50 µg·mL− 1 ascorbic acid (AA, Sigma Aldrich, USA). Passages 3–7 were used for the experiments.
Preparation of the grafts
The composites were prepared by mixing MP powder with PEEK melt using a microextrusion machine (Xplore, USA) at 380°C. After 4 min of homogenization, the mixtures were pressed at 380°C for 3 min by the manual hot press into 1 mm thick sheets and cooled using a cooling device providing a constant temperature of 50°C to constant cooling for 10 minutes. Subsequently, the samples in the form of discs with a diameter of 2.5 cm were punched out. According to the percentage by weight of MP (0, 1, 5 and 10 wt. %), the samples were labelled as follows: PEEK_0, PEEK_1, PEEK_5 and PEEK_10.
Characterization of the raw materials
A Bruker D8 Advance diffractometer in Bragg–Brentano θ-θ geometry, operated at 40 kV and 40 mA was employed to collect X-ray powder diffraction (XRPD) data from the synthesized MP powder in the angular range 10–60° 2θ adopting Cu Kα radiation (λ = 1.5418 Å).
The particle mean diameter (D) of MP particles was obtained by laser granulometry adopting a CILAS 1090 (Orleans, France) instrument, by dispersing the powder in isopropyl alcohol.
Brunauer–Emmett–Teller (BET) specific surface area of the powder was measured using an instrument ASAP 2020 (Micromeritics, Norcross, GA, USA).
The microstructure of MP powder was observed with a scanning electron microscope (SEM) Quanta 450 FEG (FEI, CZ) in secondary electron mode at 20 kV accelerating voltage on a sample sputtered with a 7 nm thick gold film to reduce charging effects.
The spectra of the raw materials were measured on a Nicolet iZ10 secondary module equipped (Thermo Fisher Scientific Inc., USA) with diamond ATR crystal covering the range 4000–525 cm-1 at a resolution of 4 cm-1.
The thermal decomposition of PEEK granulate was determined by a combination of Differential thermal analysis (DTA)-Thermogravimetric analysis (TGA) using an STA 504 thermal analyzer (TA Instruments, DE). This method was used for real-time measurements of weight loss of the examined materials as a function of temperature. Measurements were performed under an N2 atmosphere at a heating rate of 20°C.min− 1 in the 30–1000°C temperature range. Samples were cut into small rectangular pieces weighing approximately 15 mg.
Physical-chemical and mechanical characterization of the grafts
Fourier transform infrared (FTIR) maps were acquired with a Nicolet iN10 FT-IR microscope (Thermo Fisher Scientific Inc., USA). The maps were obtained from the surfaces and cross-sections of all the samples over an area of 300×200 µm with a step size of 10 µm. Each spectrum was collected from a 10×10 µm area accumulating 64 scans with a spectral resolution of 8 cm-1 in the 4000–675 cm-1 spectral range.
A Keyence VHX-6000 confocal microscope (Keyence, Mechelen, BE) with a VHX-S600E free-angle observation system (Z-motorized) was used for the characterization of samples in terms of surface roughness (Sa: arithmetical mean roughness value and Sz: mean roughness depth) was determined using ISO 2517831. Samples were characterized before and after osteoblast proliferation and differentiation tests. Samples were thoroughly cleaned with isopropyl alcohol after osteoblast proliferation and differentiation tests.
The size of the contact angle was measured to determine the wettability of the samples before and after osteoblast proliferation and differentiation tests. Samples were thoroughly cleaned with isopropyl alcohol after osteoblast proliferation and differentiation tests. The measurements were performed using a See System instrument (Advex Instrument, CZ), where five measurements were performed for each sample, and the mean and standard deviation were calculated. Water was chosen as the liquid, the drop size was 10 µL, and the reading of the size of the contact angle was taken after 10 s. The measurement was performed at room temperature (RT, 23 ± 2°C).
Tensile strength tests were conducted on the clear samples with dimensions 1×2.5×0.01 cm by means of an Instron 1122 (Instron, USA) instrument at a maximum load of 5 kN at a speed of 5 mm·min-1. Results have been reported as an average of five replicate measurements.
The Brinell hardness measurements were taken by a machine NEMESIS 9000 (INNOVATEST, NL) with a charge of 31.25 kgf for 10 s, and a minimum of 5 indentations were taken for each clear sample.
Cytocompatibility evaluation
Grafts cytotoxicity
The test was performed in accordance with the ISO standard 10993-532 Tested materials were extracted in a culture medium for 24 hours at 37°C with stirring. Extracts were prepared from sterilized samples (by ethanol for 1 hour), thus eliminating the need for filtration of extracts. The parent extracts (100%) were then diluted in a culture medium to obtain a series of dilutions. The extracts were used for up to 24 hours. Extracts were prepared according to ISO standard 10993 − 1233; 3 cm2·mL− 1 of media.
Mouse embryonic fibroblasts were seeded to pre-incubate in the 96-well plates (TPP, CH). The concentration of cells was 105 cells·mL− 1. The extracts were diluted with a medium to obtain the following concentration: 100, 75 and 50% of the parent extract. The medium was removed and replaced by individual extracts. All assays were performed in quadruplets. Cell viability was determined by MTT cell proliferation assay (Duchefa Biochemie, NL). The absorbance was measured at 570 nm with an Infinite M200 Pro NanoQuant instrument (Tecan, Switzerland) and the reference wavelength was adjusted to 690 nm.
Grafts bioactivity; Osteoblast proliferation and differentiation
Osteoblast was suspended using Trypsin (0.25%, Gibco, USA), followed by centrifugation to remove Trypsin and re-suspended in proliferation or differentiation media for further analyses. The PEEK composite discs were added into 6 well plates (TPP, CH) and washed with sterile 1x Phosphate Buffered Saline (PBS, BioSera, FR). 500 µL of cell suspension (106 cells·mL− 1) was added to the surface of the composite discs and allowed to adhere in the incubator for 2 hours. A control cell sample was seeded onto a Petri dish without the disc. They were then covered with fresh medium and cultured for several days. The medium was changed every 3 days.
RNA Isolation and qPCR
The cultured cells were harvested into 350 µL RLT lysis buffer (Qiagen, Valencia, CA) with β-mercaptoethanol (Sigma-Aldrich, USA) after 6 days of cultivation in proliferating media. Total RNA was extracted from the MC3T3-E1 cells using RNeasy Mini Kit (Qiagen, DE). RNA concentration and purity were assessed using a NanoDrop, and cDNA was synthesized using the reverse Master Mix (Generi Biotech, CZ). For cDNA synthesis, 500 ng of RNA was used, and biological replicates of each sample were diluted to a concentration of 100 ng/µl before performing a qPCR reaction. The qPCR was performed in a 10 µL final reaction volume containing the one-step Ideal PCR Master Mix (Generi Biotech, CZ) using Light-Cycler 96 (Roche, CHE) with preheating to 95 °C for 10 min. This was followed by 40 cycles of 95 °C / 15 sec and 62.5 °C / 1 min with mRNA probes for Col1a1, PCNA, Runx2, Spp1 (Mouse Col1a1, Mm00801666_g1; PCNA, Mm00448100_m1; Runx2, Mm00501584_m1; Spp1, Mm00436767_m1; TaqMan Gene expression Assay, Thermo Fisher Scientific, UK). Expression levels were calculated using the ΔΔCT method, with normalization against actin RNA levels (mouse Actb, Mm02619580_g1, TaqMan Gene Expression Assay, Thermo Fisher Scientific, UK). Three biological replicates were performed at each developmental stage.
Statistical Analysis: All results were expressed as mean ± standard deviations (SD) of three samples for each time point and compared using one-way ANOVA (One-way analysis of variance, Dunnett test: Compare all pairs of columns, Significant level = 0.05). Differences were considered as significant at p<0.05 indicated by the * symbol, respectively.
Cell Staining
To determine the impact of PEEK on the osteoblast behaviour, the three commonly used cell staining were used concretely
Mayer Haematoxylin was used for cell visualization. Cells cultivated in a proliferation medium for 10 days were fixed and stained with Haematoxylin (DiaPath, IT).
Levels of alkaline phosphatase were observed by staining ALP. Cells cultivated in a differentiation medium for 14 days were fixed and stained with 300 µL of Fast blue mixture containing 4 mg of naphthol AS-TR phosphate disodium salt (Sigma Aldrich, USA) in 150 µL of N,N-dimethylformamide (Fluka Chemicals, CH) and 12 mg of Fast blue BB Salt hemi(zinc chloride) salt (Sigma Aldrich, USA) in 15 mL of 0.1M Tris-HCl buffer (pH 9.6) for 4 hours in the dark.
Von Kossa staining was used to detect the presence of calcium deposits. Cells cultivated in differentiation media for 14 days were fixed and washed with distilled water. The water was removed, a 2% silver nitrate solution was added, and the plate was exposed to sunlight for 60 minutes, after which the plate was rinsed with distilled water (dH2O). Sodium thiosulfate (5%) was added for 10 minutes, the plates were then rinsed in dH2O, and nuclear red was added for 5 minutes. Finally, the plates were washed with dH2O.