Data - Electrically Active KNN Bioceramics: Synthesis-Driven Modifications and their Influence on Stability and Osteoblast Response

The in vitro data from the study, "Electrically Active KNN Bioceramics: Synthesis-Driven Modifications and their Influence on Stability and Osteoblast Response," are published here [*]. The following dataset presents the results of the cell-biological in-vitro evaluation. Background: Piezoelectric ceramics, such as potassium sodium niobate (KNaNbO₃, KNN), are emerging as promising electrically active biomaterials for bone regeneration. KNN can generate and respond to electrical signals, which may help stimulate cells directly and support the healing of bone defects around implants. However, the properties and biocompatibility of KNN strongly depend on precursor chemistry and synthesis conditions. The objective was to evaluate synthesis-driven material optimization and its impact on osteoblast behavior. Materials: The following chemically undoped KNN ceramics were used: stoichiometric, S-KNN 0.2 mol% alkali-excess, A-KNN synthesis-driven modification - hybrid-atmosphere sintering, H-KNN KNN pellets (10 mm ⌀) were poled via corona discharge (30 kV, 30 min, 2.3 cm electrode spacing). All KNN compositions were characterized regarding surface roughness & microstructure, ion release, and piezoelectric stability in culture medium. Tissue culture polystyrene (TCPS) and glass substrates served as controls, while polished Ti6Al4V was used as a reference material representing commercial implant materials. Cell biological evaluation was performed using MG-63 human osteoblasts [1], cultured on both unpolarized and polarized specimens for 24 h. Cell spreading, morphology, cytoskeletal organization, metabolic activity, and reactive oxygen species (ROS) generation were analyzed. Cell Culture: MG-63 osteoblast-like cells (ATCC® CRL-1427™, Manassas, VA, USA) have been extensively studied for their morphological and physiological stability [1]. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, Renfrew, UK) with 10% fetal calf serum (FCS, Biochrom FCS Superior, Merck, Germany) and 1% gentamicin (Ratiopharm, Ulm, Germany) at 37 °C with 5% CO2. For cell biological investigations, MG-63 cells were seeded at 5 × 10⁴ cells per sample and grown for up to 24 hours, as specified for each experiment. Cell adhesion (60 min) & morphology (24 h) were analyzed using a field-emission scanning electron microscope (FE-SEM, Merlin VP compact, Carl Zeiss, Oberkochen, Germany; equipped with an InLens Duo detector, 5 kV). For the preparation, samples were washed after cultivation with HEPES (Sigma-Aldrich, Munich, Germany), fixed with 2.5% glutardialdehyde (GA, Merck, Darmstadt, Germany), dehydrated with an ascending ethanol concentration series (30%, 50%, 75%, 90%, 100% twice), dried in a critical point dryer (K850, Emitech, Taunusstein, Germany), and finally evaporated with carbon “C” under vacuum conditions (EM SCD 500, Co. Leica, Bensheim, Germany) [2]. To analyze the extent of cell spreading (90 min) by confocal laser scanning microscope (LSM 780, Carl Zeiss, Jena, Germany; C-Apochromat 40×/1.20 water objective, ZEN black software 2011 SP4), MG-63 cells were trypsinated, washed with PBS (Sigma-Aldrich, Darmstadt, Germany), and stained with the PKH-26 General Cell Linker Kit (Sigma-Aldrich, Darmstadt, Germany) for 5 min at 37 °C. After staining, cells were seeded onto the samples and cultivated for 90 min, then washed twice with PBS, fixed with 4% paraformaldehyde (PFA, Merck, Darmstadt, Germany), embedded with a coverslip with Fluoroshield™ containing DAPI (Merck, Darmstadt, Germany), and analyzed microscopically with the LSM 780. Cell areas in μm2 were measured for at least 40 cells per independent experiment and sample using ImageJ Version 1.46r [2]. The actin cytoskeleton organization of cells was determined using LSM780. Therefore, MG-63 osteoblasts were cultured on the samples for 24 h, washed three times afterward with PBS, fixed with 4% PFA (10 minutes), and permeabilization with 0.1% Triton X-100 (10 minutes). For actin staining, the cells were incubated with phalloidin-tetramethyl rhodamine (TRITC, Sigma-Aldrich; 1:15 in PBS) at RT in the dark for 1 h, then embedded with Fluoroshield™ DAPI [3]. To analyze the early cell cycle activity, MG-63 cells (after 24 h) were fixed, permeabilized, and stained with recombinant Anti-Ki67 antibody (SP6; Abcam Inc., Cambridge, UK; dilution 1:25 in PBS) overnight. The secondary antibody anti-rabbit-IgG-AF488 (Invitrogen AG, Carlsbad, CA, USA; diluted 1:100 in PBS) was added at RT in the dark for 60 min, and the samples were embedded in Fluoroshield™ mounting medium containing DAPI. The cell imaging was done by the LSM780 using ZEISS 63× oil-immersion objectives Plan Neofluar (1.25 oil/0.17) or C-Apochromat 40×/1.2 water immersion objective with laser diode (excitation: 405; for nucleus), an argon laser (excitation: 488 nm; for actin), and a helium–neon ion laser (excitation: 543 nm; for Ki67, PKH-26). The 3-D overlay and bar setting were evaluated using Zen 2.3 software (version 2.12, Blue Edition) [3]. Metabolic activity and, thus, cell viability were determined by the MTS assay (CellTiter96® AQueous One Solution Cell Proliferation Assay, Promega Corporation, Madison, WI, USA). The MTS assay was performed in MG-63 cells, which were seeded in DMEM with eluted ions from KNN or on the corresponding KNN surface. After 22 h of incubation, the DMEM was discarded, and MTS solution (1:6 in medium) was added and incubated for another 2 h at 37 °C. For the analysis, 100 µL of the supernatant was transferred to a 96-well plate (in triplicate), and absorbance was measured at λ = 492 nm with a reference at 650 nm (Anthos 2010 Reader, Biochrom, Cambridge, UK) [3]. Cellular reactive oxygen species (ROS) were determined in MG-63 cells, which were seeded in DMEM with eluted ions from KNN or on the corresponding KNN surface using the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay (Abcam Inc.). For the measurements, MG-63 osteoblasts were detached with 0.05% trypsin-EDTA, washed with PBS, centrifuged, and the cell pellet of 1 × 10⁶ cells was resuspended in 20 µM DCFH-DA staining solution and incubated in the dark at 37 °C for 30 minutes. Afterward, stained cells were washed, centrifuged, resuspended in phenol red-free DMEM (without pyruvate; Thermo Fisher Scientific, Life Technologies) supplemented with 10% FCS and 1% gentamicin, and seeded onto surfaces or in the corresponding media (5% hydrogen peroxide as a positive control; only media as a blank). After a 30-minute incubation, DCF fluorescence was measured using a microplate reader (Infinite M200, Tecan, Grödig, Austria) at 485 nm excitation and 535 nm emission. Subsequently, all samples were further cultivated in the incubator at 37 °C and analyzed for ROS levels at additional time points: 1, 2, 4, and 24 hours. Background fluorescence from the blank was subtracted from all sample values to ensure accuracy [2]. Statistical analyses were performed by GraphPad Prism Version 7.02 for Windows (GraphPad Software Inc., La Jolla, CA, USA). Data were first assessed for normality by the Shapiro–Wilk test and for homogeneity of variances using Bartlett’s test. For normally distributed and homoscedastic data, a one-way ANOVA with post hoc Tukey’s multiple comparisons test was conducted. Non-parametric assumptions, the Kruskal–Wallis test followed by Dunn's multiple comparisons test was done. For the ROS analysis, a multiple t-test post hoc Holm-Sidak was used. Results: All KNN compositions retained measurable piezoelectric response during 14 days under physiological conditions. KNN ceramics maintain the viability of MG-63s, highlighting their favorable biocompatibility profile. Customized microstructures improved cell behavior and ensured functional stability. Polarization resulted in modest but reproducible improvements in osteoblast adhesion, spreading, cytoskeletal organization, and metabolic activity, accompanied by increased ROS signaling. Conclusion: Optimized KNN ceramics exhibit robust in vitro cellular behavior, supporting osteoblast viability and adhesion comparable to those of clinically established titanium alloys. Polarization-induced surface charges further enhance cell spreading and motility. These findings provide a platform for the development of KNN-based piezoelectric biomaterials for bone regeneration in tissue engineering.