Page 442 - IJB-10-2

International Journal of Bioprinting 3D-printed silicon nitride-PEEK implants

H × 01 180. ×  times in 70% ethanol for 30 min, and dried overnight in
OAD = intradiscal the biological safety cabinet. Samples were pre-incubated
H implant × π for 24 h in Minimum Essential Medium α (MEM α,
Gibco) supplemented with 10% fetal bovine serum (FBS;
The normality of the data distributions was assessed by Gibco) and 1% penicillin–streptomycin (Gibco) in ultra-
applying the Shapiro–Wilk test. Groups were compared low attachment plates (Corning) followed by seeding with
via analysis of variance (ANOVA), followed by a post-hoc 30,000 MC3T3-E1 mouse preosteoblasts (ATCC CRL-
Tukey Honest Significant Difference Test. Furthermore, 2593); tissue culture plastic (Corning) served as a positive
a 2 × 3 factorial ANOVA was conducted to evaluate the control. Mouse preosteoblasts were chosen because they
primary effects of material (PEEK vs. Si N -PEEK) and faithfully recapitulate the molecular events that occur
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design (Solid, Porous, and Porous Window). All statistical during osteoblast maturation and can be compared to
analyses were performed with a commercially available existing work due to their immortalized nature. 25,26 After 7
software program (SPSS version 28), using an alpha level days of incubation, cell culture media were supplemented
of 0.05. with 5 mM β-glycerophosphate (Sigma-Aldrich) and
50 µg/mL L-ascorbic acid (Sigma-Aldrich) to promote
2.3. Antibacterial testing
mineralization. To evaluate short-term cell attachment and
2.3.1. Antibacterial adhesion proliferation, BioVision’s MTT Cell Proliferation Assay Kit
PEEK, Si N -PEEK, and AFSN rods (1.75 × 12 mm) were was used after 24 and 72 h. To evaluate osteogenic activity, a
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prepared, with AFSN acting as a positive control due to combined approach described by Wilkesmann, Westhauser,
its demonstrated antimicrobial effect in vivo and it being and Fellenberg was used to simultaneously determine cell
the pure form of the antimicrobial component of the number with fluorescein-diacetate (FDA) and alkaline
Si N -PEEK composite material. Rods were placed in phosphatase activity with 4-methylumbelliferone-
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10% human serum, 1 × phosphate-buffered saline (PBS), phosphate (4-MUP) after 7, 14, and 21 days. Mineralization
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and 7 mg/mL dextrose (48-well plate, n = 6/condition) and was measured by staining with 2% alizarin red stain after 21
inoculated with 10 , 10 , or 10 colony-forming units per and 28 days and visualized, and then the stain was dissolved
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milliliter (CFU/mL) of Staphylococcus epidermidis (ATCC in 20% methanol and 10% acetic acid in deionized water,
14990). Samples were incubated at 37°C and 95 rpm for 24 followed by spectrophotometric quantitation (λ = 405 nm,
h, aseptically removed, gently rinsed 3 times in PBS, and TECAN Infinite M200).
placed in 10% trypsin to release adherent bacteria from Statistical significances for each set of results were
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the material surface. Serial dilutions were plated on 3M™ evaluated by ANOVA, followed by a post-hoc Tukey’s
Petrifilms™, and colonies were then counted (countable multiple comparisons test (n = 6, α = 0.05, Prism v9,
range 20–300 CFU). Each antimicrobial experiment was GraphPad, 2020).
independently performed 3 times and also repeated under
the same conditions for Escherichia coli (ATCC 25922). 3. Results
Statistical significance was tested using a Kruskal–Wallis
test (Prism v9, GraphPad, 2020). 3.1. Si N -PEEK cages and 3D printing
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A total of five cages per group of solid, porous, and
2.3.2. Scanning electron microscopy porous window designs for PEEK and Si N -PEEK were
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Scanning electron microscopy (SEM) samples were 3D-printed and examined for structural integrity (Figure
prepared by aseptic removal of samples, gentle rinsing in 2). Furthermore, weight measurements were conducted
PBS, fixing with 4% paraformaldehyde for 20 min, followed for each group of cages to identify outliers, addressing
by serial dehydrations (10 min in PBS, 20% ethanol, 40% the possibility of any infill/extrusion issues (Table S2 in
ethanol, 60% ethanol, 80% ethanol, and 100% ethanol). Supplementary File).
Samples were dried overnight, sputter-coated in 80/20 Pt/
Pd, and imaged by SEM (Zeiss Supra 50VP). 3.2. Mechanical testing
2.4. Cell culture 3.2.1. Compression and compression shear
PEEK, Si N -PEEK, AFSN, and Ti6Al4V samples (10 × 10 For compression, force–displacement curves of the cages
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× 1 mm ) were prepared, with Ti6Al4V acting as a positive that were tested above the 5th percentile ultimate force
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control due to its interaction with osteoblast-like cells being (6236 N) were plotted. For each group, the first linear
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well-studied, and it is known to support osseointegration in regions were defined separately, and the stiffness (Table
vivo. Samples were sterilized by sonicating in 70% ethanol 1) was computed by determining the slope of the linear
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for 30 min, transferred to a sterile environment, soaked 3 region for each sample.
Volume 10 Issue 2 (2024) 434 doi: 10.36922/ijb.2124