Page 309 - IJB-9-5

International Journal of Bioprinting Scaffold for engineering enthesis organ

differentiation was initiated by the replacement of 2.8. Statistical analyses
the media with tenogenic differentiation medium Statistical analyses were performed using Prism 8
(DMEM-F12 supplemented with 5% FBS, 1% software version 8.2.1 (GraphPad software, United
penicillin/streptomycin, 100 ng/mL CTGF, 50 µg/ States). Data were analyzed using one-way ANOVA
mL ascorbic acid, 100 ng/mL BMP-12, and 50 ng/mL followed by Sidak multiple comparisons post hoc test.
TGF-β3). The medium was replaced every 3 days for All data are expressed as means ± standard deviation
21 days. The aniline blue staining was used to quantify (SD), and a p value of 0.05 was considered significant
the collagen deposition. Aniline blue staining was for all experiments.
performed using the trichrome staining kit (HT15,
Sigma-Aldrich, St. Louis, Missouri, United States) 3. Results and discussions
following the manufacturer’s instructions. Briefly, cells 3.1. Polymers’ ability to support MSC adhesion and
were treated with a phosphomolybdic/phosphotungstic growth
acid solution at a ratio of 1:1, stained with the dye, Scaffolds fabricated by solvent casting (N = 30 per
and fixed with an acetic acid solution, and the dye polymer type, diameter Ø = 13 mm, and thickness
was extracted from the cells using DMSO. Absorbance h = 0.5 ± 0.1 mm) were biologically tested. Additional
was read at 670 nm (Ensight, PerkinElmer, Waltham, information can be found in Figure S1 (Supplementary
Massachusetts, United States). Pre-differentiated cells File). The results of the biocompatibility tests are shown
were also used with the enthesis scaffold. Specifically, in Figure 2. MSCs’ ability to adhere and grow on different
cells were differentiated into tenocytes for 7 days, as types of solvent-casting scaffolds was first evaluated
described above, then seeded on the scaffold or plastic using the MTS assay (Figure 2A and B). MSCs were
for further 21 days and maintained in a growth medium seeded on polymers or plastic culture plates (CTRL) and
for the experiment. maintained in a growth medium for 3 or 7 days. Results

2.7. Fabrication of clinically-relevant scale scaffold demonstrated the ability of all the tested solvent-casting
Three enthesis scaffolds were manually braided to fabricate scaffolds to sustain the MSC adhesion after 3 (Figure 2A)
a clinical-scale scaffold and demonstrate the versatility and 7 (Figure 2B) days. All the constructs showed an
and potentiality of scaling up this novel biofabrication average of five times lower number of adherent cells
approach. In this sense, scaffolds should replicate the T/Ls with respect to CTRL. However, they were all able to
in vivo morphology, structure, and size, but they should sustain the MSC growth rate, as evidenced by the lack
also present a region that optimizes insertion into the of difference in the percentage of cell proliferation after
bone and reduces the risk of failure after surgery. These 3 or 7 days of culture with respect to CTRL. The PLGA
clinically-relevant scale scaffolds were mechanically ones demonstrated the best ability to promote MSC
characterized by performing the same uniaxial tensile tests adhesion and growth among all the scaffolds. Based on
conducted for the enthesis scaffolds. The elastic modulus these results and literature data [35,36] , PLGA and PCL were
was calculated by introducing the packing factor (PF) . selected for other experiments. The ability of MSCs to
[34]
This parameter considers the air gaps that form during the adhere to PLGA and PCL solvent-casting scaffolds was
braiding process. The PF is defined as follows: further investigated by immunofluorescence analysis
A (Figure 1C). MSCs were seeded on scaffolds (or glass as
PF n s (I) control) and maintained in a growth medium for 3 days;
A
b at the end, cells were stained with anti-CD90 (red) as a
where n is the number of enthesis scaffolds used in the marker of MSCs [37] and anti-actin (green) as a marker
braiding process, and A and A are the cross-sections of the of cytoskeleton organization. Both PLGA and PCL were
s
b
single enthesis scaffold specimen and the braided scaffold able to support the MSC adhesion, in accordance with
approximated with a cylindrical shape, respectively. The PF the MTS results. Interestingly, the two solvent-casting
is used to calculate the effective elastic modulus E of the polymers differently modified the MSC cytoskeleton
b
3D braided scaffolds with the following relationship: organization. Cells grown on PLGA displayed overall
cell elongation and parallel cytoskeletal conformation,
E
E = (II) as evidenced during the MSC differentiation to
b PF tenocytes [38] . Conversely, several studies have reported
where E is the elastic modulus calculated from the stress– that MSCs exhibit a star shape during osteogenic
strain curve without considering air gaps. At this stage, lineage commitment [39,40] . In this respect, as evidenced
no biological tests were conducted. Scaffolds were only by immunofluorescence images, PCL mainly supported
mechanically characterized. star shape actin organization. These results prompted us

Volume 9 Issue 5 (2023) 301 https://doi.org/10.18063/ijb.763