Page 307 - IJB-9-5

International Journal of Bioprinting Scaffold for engineering enthesis organ

2.2.2. Tendon/Ligament-like region After sterilization, scaffolds were incubated overnight
To mimic the physiological, morphological, and mechanical in DMEM-F12, supplemented with 10% FBS, 100 U/mL
characteristics of T/Ls, a scaffold must exhibit an anisotropic penicillin, and 100 mg/mL streptomycin. On the scaffolds
fiber orientation, as in native tissues. In T/Ls, collagen fabricated by solvent casting, the day after the soaking,
fibers are oriented along the applied stress direction . 20,000 cells were seeded in each well (24-multiwell
[29]
To replicate this characteristic, PLGA was processed by plate). For the tridimensional scaffolds, 250,000 cells
electrospinning technology. For this application, using and were seeded on 3D-printed PCL in a 24-multiwell plate;
manipulating a strong electric field allow the nanofibers 30,000 cells were seeded on an electrospun PLGA scaffold
to be extruded and collected in an adjustable manner. in a 24-multiwell plate. Similarly, for the multimaterial
To collect aligned fibers, a rotating drum collector was enthesis structure, 30,000 cells were seeded on the PLGA
used (drum diameter = 10 cm). PLGA nanofibers were side and 70,000 cells on the PCL side and used for further
obtained by electrospinning a solution of 10% (w/v) of experiments. As a control, 30,000 cells were seeded on
PLGA dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol plastic in a 24-multiwell plate.
(HFIP). This solvent possesses a dielectric constant almost
4 times higher than chloroform allowing thinner fibers 2.3.3. Viability tests
to be electrospun . The electrospinning process was The MTS assay (CellTiter 96 Aqueous One Solution Cell
[30]
performed using a Linari Engineering apparatus (Linari Proliferation Assay kit, Promega, Madison, Wisconsin,
Eng, Italy) featuring a commercial 10 mL syringe with a United States) was performed on cells seeded on the
21 G needle as its spinneret. Electrospinning parameters scaffolds fabricated by solvent casting in DMEM-F12,
were as follows: the applied voltage was 35 kV, the distance supplemented with 10% FBS, 100 U/mL penicillin, and
between the spinneret and the grounded collector was 10 100 mg/mL streptomycin, after 3 and 7 days of culture,
cm, the rotating speed of the collector was 800 RPM, and as described by the manufacturer. Briefly, 250 µL of fresh
the flow rate was 1 mL/h. The process was conducted for medium was added to each well with 37.5 µL of the MTS
2 h at room temperature, T = 21°C, and relative humidity, reagent and incubated for 2 h at 37°C in 5% CO . The
2
RH, = 45%. Samples of electrospun PLGA for cell culture supernatants were transferred in a 96-multiwell plate to
into a 24-multiwell plate were prepared by die cutting. avoid the interference of the materials, and the absorbance
was read at 490 nm (Ensight, PerkinElmer, Waltham,
2.2.3. Enthesis scaffold Massachusetts, United States).
The enthesis gradient in physical properties was replicated
by developing an ad-hoc biofabrication protocol in which 2.3.4. Immunofluorescence analysis
PCL was directly 3D-printed on electrospun PLGA mats. Cells were seeded on the scaffolds fabricated by solvent
Through the 3D printer, two layers of PCL (single layer casting (or on the glass as control) and maintained in
height 0.2 mm and oriented at 90 degrees to each other) DMEM-F12, supplemented with 10% FBS, 100 U/mL
were extruded, fabricating a grid structure on the PLGA penicillin, and 100 mg/mL streptomycin for 3 days. Then
strips. The extrusion parameters are the same as listed in cells were fixed in 4% paraformaldehyde (PFA) for 15 min,
Table 1. rinsed three times in PBS, permeabilized in PBS-BSA
2.5% with Triton X-100 0.1% for 7 min, and washed and
2.3. Biological validation of materials incubated in the blocking solution (PBS-BSA 2.5%) for
2.3.1. Cell culture 1 h at room temperature. Then, samples were incubated
MSCs (human mesenchymal stem cells (bone marrow), with primary antibody Anti-CD90 (HPA003733, Sigma-
SCC034, Millipore, Burlington, Massachusetts, United Aldrich, St. Louis, Missouri, United States) and Anti-
States) were purchased and were maintained in xeno- Actin (MAB1501, Millipore, Burlington, Massachusetts,
free medium (XF MSC expansion medium, cod: SCM045 United States) diluted in blocking solution overnight
Millipore, Millipore, Burlington, Massachusetts, United at 4°C. The day after, samples were rinsed in PBS-BSA
States). Cells were grown at 37°C in 5% CO and detached 2.5% and incubated with the secondary antibody (Anti-
2
using Trypsin with EDTA 1x (25-053-CI, Corning Inc., Rabbit Alexa Fluor 488, A-11001, and Anti-Mouse Alexa
New York, United States) at 80% confluence, used until Fluor 594, 1:500, A-11012, Thermo Fisher Scientific,
passage 6. Waltham, Massachusetts, United States) for 45 min at
room temperature. Finally, cells were rinsed in PBS and
2.3.2. Mesenchymal stem cells seeding protocol mounted with the Fluoroshield mounting medium with
All the tested scaffolds were sterilized by UV exposure DAPI staining (F6057, Sigma Aldrich, St. Louis, Missouri,
and then washed in phosphate-buffered saline (PBS); United States), and images were acquired using a Nikon
moreover, the three synthetic ones were rapidly washed E-Ri microscope with a magnification of 60x.
in ethanol (70% in H O) before the UV light exposure.
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Volume 9 Issue 5 (2023) 299 https://doi.org/10.18063/ijb.763