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International Journal of Bioprinting Bioprinted plasma biocarriers for MSC delivery

module identifying pathway activation or inhibition based cluster primarily (but not exclusively) engages in PDGF,
on the Z-score algorithm. The comparison analysis module VEGF, and NGF signaling pathways. Additionally, these
was used to compare different experimental conditions factors participate in regulating collagen and hyaluronan
biosynthetic processes (Figure 1C).
2.8. Matrigel assays
Angiogenesis assays were performed using a 15-well 3.2. BMSC viability and adhesion within biocarriers
chambered coverslip with Matrigel (356231; Corning, We aimed to evaluate the effect of biocarriers on the
USA). Human umbilical vein endothelial cells (HUVECs) viability of BMSCs (Figure 2). We conducted live/dead
were added to the Matrigel bed, and conditioned media assays on bioprinted constructs after 96 h in culture
from cell-loaded biocarriers was added. Cells were (Figure 2A). Fluorescence microscopy of live cells
stained with CellMask™ Plasma Membrane Green Stain revealed that GelMa-based hydrogels are cytocompatible,
(C37608; Thermo Fisher, USA) and counterstained indicated by the high proportion of live cells after 96 h in
with Hoechst 33342 (PureBlu™ Nuclear Staining Dye; culture. Doping the biocarriers with sera (FFP and PRP)
BioRad, USA). A Zeiss microscope (Axio Observer.Z1 consistently improved viability and proliferation rates, as
Apotome 2.0, Axiocam 503M camera, Plan Neofluor 20×, evidenced by direct counts on the whole geometry of the
40×; Zeiss, Germany) was used for capturing live cell hydrogels after bioprinting (Figure 2B). Additionally, cell
fluorescence images. Angiogenesis was evaluated using morphology in the serum-doped hydrogels qualitatively
AngioTool software. displayed a more vital cell phenotype and increased cell-
to-cell contact (Figure 2A).
2.9. Statistical analysis
Differences in the distribution of Z-scores among The sera also promoted cell metabolism, as evidenced
biocarriers were compared using the Kruskal–Wallis test, by absorbance readings in XTT-type proliferation assays
followed by the Bonferroni adjustment. The data were (Figure 2C), exhibiting approximately twice the intensity
analyzed using the statistical program SPSS (version 18.0; compared to bare hydrogels. More importantly, comparing
IBM Corp., USA) the readings at 24 and 96 h reveals higher readings at 96 h
in the presence of sera. In their absence, cell metabolism
3. Results tends to decrease over time, suggesting hibernation of
cells in bare hydrogels. Additionally, IL-1β-mediated
3.1. Characteristics of the plasma products used for inflammation did not alter proliferation rates. Overall,
biocarrier functionalization these findings indicate that blood products, both FFP and
The soluble blood-derived product was obtained by PRP, enhance the proliferation and viability of BMSCs
coagulating PRP, which contained twice the platelet within hydrogels.
concentration of peripheral blood. Additionally, for
experimental validation, we prepared a soluble blood- Bioinformatic analysis of the secretome of cells cultured
derived product by coagulating plasma with a platelet in biocarriers doped with hemoderivatives (Table 1)
count three times lower than that of peripheral blood, provides further depth to the previous results. The
®
named FFP. PRP, the primary tested product, had a predictions made by IPA revealed that hemoderivatives
platelet concentration of 400,000 platelets/µL (twice that promote signaling pathways involved in cell survival and
of peripheral blood), while the platelet-poor plasma (FFP) viability and inhibit those related to necrotic and apoptotic
had a platelet concentration of 65,000 platelets/µL. In the events. Notably, these stimulatory effects are maintained
identification of key signaling proteins within the primary in the secretome regardless of whether the culture was
tested PRP, we classified 130 proteins that were at least performed under inflammatory conditions (IL-1β).
two times more concentrated in serum-converted PRP To further investigate cell adhesion in the different
compared to serum-converted FFP. Employing STRING hydrogel types, we seeded cells directly onto acellular
to unravel protein–protein interactions (with a minimum hydrogels. Figure 3 displays representative fields of BMSCs
required interaction score of the highest confidence level cultured on these hydrogels. After 96 h, the cells exhibit
at 0.9) and functions, Markov Cluster Algorithm (MCL) uniform growth on the hydrogels under both normal
clustering methodology revealed distinct clusters of (control) and inflammatory (IL-1β) conditions. However,
functional proteins. there are minor qualitative differences between the three
As displayed in Figure 1, we identified two main groups: types of hydrogels. Without serum in the formulation, cells
(i) GFs, including neurotrophins (Figure 1A); and (ii) a tend to grow without agglomeration or strong interactions.
molecular cluster of chemokines (Figure 1B), particularly In contrast, hydrogels doped with hemoderivatives display
involved in leukocyte chemotaxis (Figure 1D). The GF the formation of nodules connected by large bridging cells,

Volume 10 Issue 6 (2024) 304 doi: 10.36922/ijb.4426