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International Journal of Bioprinting Nanomaterial-bioinks for DLP bioprinting

In this present study, GO and CaP displayed slightly The nanomaterials CaP and GO have both been
different effects on the vitality and morphological associated with the ability to enhance mechanical
appearance of cells in the DLP-printed constructs. Besides properties, such as stiffness and compression strength
the influence of the nanomaterials, the GelMaBB bioink in hydrogels. 46,47,50,52–54,69,92,101–105 Despite the lower
alone had a significant impact on the cells compared to concentration of GO (0.5 mg/mL) compared to CaP
commercial GelMa bioink. The viability of hMSCs in (50 mg/mL), GelMaGO exhibited the highest value for
commercial GelMa bioink was surprisingly low, with Young’s modulus. These data from cell-free scaffolds
high rates of rounded and dead cells, while cell viability verified the stabilizing effect of GO on the material, i.e., the
and proliferation were the highest in GelMaBB amongst overall constructs, as previously reported in the literature.
all tested bioinks, as indicated by morphological data and It may be surmised that GO acts as a photo-absorber and
DNA quantification. Both added nanomaterials resulted in facilitates proper crosslinking of the bioink during printing
diverse effects on the cell morphology or viability staining. and subsequent storage.
For GelMaBB-CaP, we observed the presence of some The mechanical properties of bioinks are typically
dead cells morphologically, but none on the quantitative evaluated based on the materials or constructs themselves.
level; many cells still maintained a typical elongated hMSC Although this is necessary to assess the impact of material
morphology. Cells within GelMaGO were, especially composition without other influencing factors, integrated
in the later phase, characterized by a more broadened cells or physiological processes (e.g., degradation,
morphology with distinct stress fibers, probably associated differentiation, or matrix deposition) can actively change
with the increased stiffness of these samples. Stress fibers, as the biomechanical properties in bioprinted constructs.
a result of the higher stiffness of hydrogels containing GO, However, measuring Young’s modulus in biological-
have been reported by other groups, probably associated based soft samples is a complex endeavor to determine
with the need for a robust actin skeleton to facilitate the points of the first contact. Therefore, the algorithm for
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stretching or movement within a stiffer matrix. Besides fitting a linear curve can only be based on the linearity of
these morphological differences, DNA quantification compression curves, which may lead to a large scatter in
revealed no clear trends regarding the negative or positive the evaluation of Young’s modulus. In the investigated time
effects of CaP or GO on cell growth over time. In addition, frame, we did not observe significant differences in the
the inherent donor-to-donor variation of hMSCs, such as biomechanical properties between samples without cells
differences in cell growth or population heterogeneity, will (~20–60 kPa) or integrated with cells (~16–50 kPa). In
contribute to variations in DNA assessment and overall this context, it was demonstrated that mechanical stability
biological evaluations. 94,95 increases with calcium deposition in cell-laden hydrogels.
Hydrogels based on the fibrillating peptide FEFEFKFK or
Although nanoparticles might be taken up by hMSCs, alginate in combination with gelatin and GO display an
DNA quantification did not reveal significant cytotoxic increase in elasticity, compressive modulus, or stiffness
effects caused by the nanomaterials in the investigated over time, particularly when cells are encapsulated. These
time frame. Previous studies have demonstrated that studies suggest that the production of extracellular matrix
concentrations exceeding 50 µg/mL of GO can induce proteins and mineralization correlate with mechanical
cytotoxic effects in hMSCs. Our data, however, indicate properties. 80,83,106 Although in our study samples from
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that the much higher concentration of 500 µg/mL in our donors with a high calcification also displayed a temporal
bioink did not cause severe negative effects in comparison increase in the mechanical properties, the donor-to-donor
to the unmodified GelMaBB or the commercial GelMa variation was too high to validate this effect in our studies.
bioink. In our previous study, we have demonstrated The compressive strength of cancellous bone ranges
that the same CaP nanoparticles used in this study and between 0.1 and 16 MPa, and Young’s modulus ranges
integrated into polycaprolactone (PCL) can induce an between 0.05 and 0.5 GPa. 107,108 In contrast, hydrogel-based
increase in lactate dehydrogenase as an indicator for bioprinted constructs containing living cells, typically
cellular cytotoxicity in the early phases of culture after have significantly lower compression strength than that of
seeding of hMSCs. In the context of our present study, no bone, i.e., 1–30 kPa. 83,84,106 Thus, they are unable to act as
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quantitative cytotoxic effects were indicated. In addition load-bearing implants, if applied on their own. During the
to cytotoxicity, differentiation processes, often attenuating physiological processes of cellular differentiation and bone
cellular proliferation, can contribute to differences in formation in vivo, collagen deposition and calcification
cell numbers over time. 97–100 Thus, we can also assume processes will continuously change the biomechanical
that differentiation processes affect the results of DNA properties from soft to hard tissue in long-term processes
quantification in the present study. over several weeks or months. Thus far, a mechanically

Volume 10 Issue 6 (2024) 489 doi: 10.36922/ijb.4015