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International Journal of Bioprinting 3D cell culture model for neural cell analysis

(p = 0.025), and ZTA (p = 0.012). No significant differences for evaluating the biological impact of wear debris. This
in ROS levels were observed between CoCrMo and novel approach offers a more physiologically relevant
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Ceridust (p = 0.85), CoCrMo and ZTA (p = 0.80), or alternative to traditional 2D culture systems, enabling
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between Ceridust 3615 and ZTA (p = 0.66). These findings improved simulation of the in vivo microenvironment
suggest a relatively higher oxidative stress response in cells and more accurate assessment of cellular responses to
exposed to materials other than PEEK-OPTIMA™. biomaterial-associated particles.
Quantitative analyses of C6 astrocyte-like cells showed We selected commercially available model particles
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that the positive control (cells with hydrogen peroxide) derived from PEEK-OPTIMA™, Ceridust 3615, ZTA
exhibited elevated ROS levels, as evidenced by the increased ceramic, and CoCrMo alloy to represent a broad spectrum
fluorescence intensity, compared to the cell-only negative of biomaterials commonly used in spinal implant
control (Figure A1 in Appendix). The average fluorescence applications. This selection ensured comprehensive
intensity of the positive control on Day 5 was significantly coverage of polymeric, ceramic, and metallic materials
higher (approximately 70 times; p < 0.05) than that relevant to current clinical practice. Prior to embedding
produced by all of the model particles (average fluorescence into neural cell-laden bioprinted GelMA hydrogels, the
intensity < 22 ± 3.9 a.u.). Notably, all model particles did particles were thoroughly characterized for their size
not induce significant ROS production compared to the and morphology.
cell-only negative control (average fluorescence intensity = The SEM analysis of PEEK-OPTIMA™ model particles
23.5 ± 5.4 a.u.). Similar results were observed for NG108- revealed irregular, granular morphologies. The particle
15 cells, where the positive control demonstrated higher size distribution showed a mode size between 0.1 and 0.8
ROS production (average fluorescence intensity = 37.1 μm, with an average diameter of 7.58 ± 3.97 μm. Notably,
± 2.6 a.u.) than the cell-only negative control (average 95% of the particles were smaller than 8 μm. These results
fluorescence intensity = 7.7 ± 5.8 a.u.) and any of the model are consistent with those of Du et al., who reported that
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particles (average fluorescence intensity < 2.1 ± 0.2 a.u.; p 99% of commercially available PEEK particles were under
< 0.05) (Figure S1, Supporting Information). However, 5 μm, although their mean particle size was smaller (1.05
no significant increase in ROS production was detected μm) compared to our findings. Furthermore, Hallab et
in NG108-15 cells exposed to any of the model particles al. reported that PEEK-OPTIMA™ particles exhibited
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compared to the cell-only negative control. granular to flake-like morphologies, with 95% of the
particles falling within the 1–10 μm size range, findings
The production of ROS by both C6 astrocyte-like that are consistent with our current observations. The
and NG108-15 cells, embedded in 3D-bioprinted model SEM analysis of Ceridust 3615 particles revealed a
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particles, was qualitatively assessed using the DCFDA predominantly granular morphology with noticeable
probe on Day 3. Production of ROS was proportional to agglomeration, aligning with observations reported by
the intensity of the green fluorescence (Figure 8a and b).
Liu et al. In our study, the average particle size was 8.75
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3.3.3. DNA integrity analysis ± 2.35 μm following filtration through an 8 μm filter,
The DNA integrity of C6 astrocyte-like cells exposed to and 5.27 ± 3.80 μm after passing through a 0.8 μm filter.
various model particles was assessed using a DNA damage These values are comparable to those reported by Green
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assay that quantifies γ-H2AX foci as a marker of DNA et al., who investigated murine peritoneal macrophage
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strand breaks. DNA damage was evaluated at the 1-h mark, responses to polyethylene particles using Ceridust 3615.
as genotoxic materials can induce immediate DNA strand Their work involved sequential filtration through 10, 1, 0.4,
breaks. Our findings revealed no detectable DNA damage and 0.1 μm filters, resulting in particle sizes of 7.2 ± 3.15,
in C6 astrocyte-like cells, as evidenced by the absence of 4.3 ± 1.89, 0.49 ± 0.11, and 0.21 ± 0.069 μm, respectively.
γ-H2AX foci (Figure 8b). ZTA particles in our study exhibited both granular and
polygonal morphologies, with a size range of 0.1–0.8 μm
4. Discussion and an average size of 3.69 ± 3.48 μm. These findings
are consistent with those of Asif, who also reported
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The primary objective of this study was to develop an agglomerated structures of ZTA particles. In comparison,
advanced 3D spinal cord cellular model using bioprinting Germain et al. investigated alumina powder and reported
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technology. While 3D bioprinting has been widely used a smaller mean particle size of 0.5 ± 0.2 μm. The observed
in different biomedical studies, 36–38 this is the first study size discrepancy may be attributed to differences in particle
to engineer a 3D-bioprinted model that integrates neural generation and filtration techniques, as Germain et al. used
cells with spinal implant wear particles and relevant a pin-on-plate wear simulator and employed finer filter
biomaterials, establishing an advanced in vitro platform membranes during particle separation. The CoCrMo
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Volume 11 Issue 4 (2025) 269 doi: 10.36922/IJB025180174