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International Journal of Bioprinting 3D cartilage induction and monitoring

before replacing with new media (DMEM Glutamax, 10% enhanced contrast images of a region of interest (ROI)
FBS, 1% P/S). Scaffolds with cells were cultivated for 1 week for both conditions could be observed. There was an
in a 6-well plate to ensure adequate cell-scaffold integration. observable increase in rugosity for quantity and size of
Thereafter, scaffolds were introduced inside BR for another grain in BR scaffolds compared to control samples. The
week, while control cell-loaded scaffolds were maintained presence of some fibrils (collagen) is noted in BR samples.
in the well plate for that week. Metabolic activity was The power spectral density (PSD) profile of the average
measured on days 1, 3, and 7 of BR experimental time. of three samples was computed to quantify the degree of
exchange in rugosity (Figure 4G). Curves have a higher
Results indicated a different response in cellular
metabolism as a function of flow rates (Figure 4A). signal power in BR samples than in control samples,
implying the presence of higher structures attached to
Following literature recommendations, a continuous flux the scaffold surface, as observed previously in Figure 4F.
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of 0.5 mL·min was probed with negative results. Next, a Finally, typical roughness parameters, the root mean
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discrete (1 h of work separated by each 6 h) form of the square gradient (Sqd) and interfacial ratio (Sdr), were
previous flow rate was applied to reduce the exposure stress also computed. Results indicated a statistical difference
over cells; negative results were obtained with a prominent between control and BR samples (p < 0.001), implying
reduction in cell metabolism at day 3. Several experiments that the rugosity of the scaffold increased with the exerted
from 0.5 to 0.8 mL·min were conducted (at a flow rate perfusion flow (Figure 4H).
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interval of 0.1 mL·min ) but reported no significant results
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(data not presented). Once the flow rate was increased to 0.8 Figure 5A and B corresponds to immunofluorescence
mL·min , the metabolism increased similarly to the control images without primary antibodies (negative) to validate
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scaffolds. At day 7 (with 0.8 mL·min ), the metabolism the lack of activity from the secondary antibodies. Alexa
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continued to increase compared to the other conditions, Deep Red was selected as the secondary antibody to
reaching almost double the rate at day 1. Consequently, avoid scaffold autofluorescence. Thus, each ECM protein
subsequent experiments were performed with a flow rate was represented separately (Figure 5C and D). Results
of 0.8 mL·min . Additionally, DNA quantification for BR indicated the abundant presence of all the common
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and control scaffolds was performed to detect changes AC ECM proteins and high cell viability in BR samples:
in metabolic activity associated with differences in cell collagen types I and II, and aggrecan. BR scaffolds
density. The results presented in Figure 4B displayed a presented much higher expressions of collagen type II
significant increase in BR samples compared to control and aggrecan, with a low expression of collagen type I. In
samples (p < 0.001) (Figure S5A, Supporting Information). contrast, control scaffolds presented much lower cellular
The results for ECM synthesis, i.e., collagen quantification, content, no aggrecan proteins, and a much higher content
are presented in Figure S5B, Supporting Information. of collagen type I. Conclusively, quantification assays,
Figure 4C displays similar results to DNA content and a SEM, and immunofluorescence demonstrated the active
much higher collagen presence in BR samples (p < 0.001). chondrogenic induction potential of BR for IPFP-MSCs.
In contrast, higher collagen type II concentration (Figure
S5C, Supporting Information) was noted in BR samples 3.4. Biomechanical correlation analysis
compared to control samples (p < 0.001) (Figure 4D). Ultrasound is a valuable non-invasive characterization
method. In this research, LIPUS (1 MHz) was employed to
Scanning electron microscopy (SEM) and study the mechanical evolution of the scaffold for 1 week
immunofluorescence techniques were applied to verify inside BR. Free-cell scaffolds (blank) were also evaluated
the increased amount of ECM in 3D scaffolds included in with ultrasound under the same conditions as ex vivo
BR. SEM results demonstrated that scaffold fibers in both experimentation to neglect any background information.
samples were covered by cells (Figure 4E). Nonetheless, Figure 6A and B displays arbitrary signals for cell-seeded
in the control scaffold, there were empty spaces between and blank scaffolds for each experimental day. In both
cells. Notably, in BR samples, the scaffold fibers were cases, a decrease in the V was observed after 24 h of
p
entirely covered by cells and ECM. Additionally, the shape treatment, along with a signal delay, implying a no-cell-
of IPFP-MSCs differed between control and BR samples; dependent behavior. In contrast, it was observed that V
p
BR IPFP-MSCs displayed a more circular cellular shape. increased from day 2 to day 7 in cell-loaded scaffolds (from
Nevertheless, cell viability and adequate cell-biomaterial 1.3 to 2.1 V), whereas, in blank scaffolds, no substantial
interaction were observed in both cases. variance was revealed (0.04–0.043 V).
In addition to SEM images, atomic force microscopy Figure 6C represents the evolution of signal amplitudes
(AFM) was applied to characterize the rugosity of the over time; in blank conditions, no significant difference
surface of the scaffold fibers between cells. In Figure 4F, was observed during 1 week of experimentation. Although

Volume 10 Issue 4 (2024) 377 doi: 10.36922/ijb.3389