Page 377 - IJB-10-4

International Journal of Bioprinting 3D cartilage induction and monitoring

Therefore, the flow rate could be modified as a function of transferred into new low-attachment 48-well culture plates
the ultrasound signal. with 1 mL of medium. All samples were incubated under
a 5% CO atmosphere at 37ºC for 14 days. The culture
2
2.2. Cells isolation and culture medium was replaced every 2 days in control scaffolds,
Infrapatellar fat pad mesenchymal stem cells (IPFP- whereas the medium was perfused for BR scaffolds.
MSCs) were directly extracted from patients with OA
after receiving approval from the Ethics Committee of 2.6. Metabolic activity
the Clinical University Hospital of Malaga, Spain (ethical The metabolic rate was assessed by colorimetric Alamar
approval number: 02/022010 Hospital Virgen de la Blue assay (Thermo Fisher Scientific, USA) following
Victoria, Málaga). Informed consent was obtained from the manufacturer’s instructions on days 1, 3, and 7 after
patients for all samples used in this study. Hoffa’s fat pads treatment (i.e., 8, 10, and 14 days after seeding). Cell-
were harvested from the inside of the capsule without free 3D scaffolds were used as controls, and data were
the vascular and synovial areas. The isolation and culture normalized to the appropriate control. The fluorescence
protocol of IPFP-MSCs were according to López-Ruiz intensity was measured using a plate reader (Synergy HT;
et al. IPFP-MSCs were characterized according to the BIO-TEK, USA).
33
established criteria of the International Society for Cell
Therapy (ISCT). 45 2.7. DNA and collagen quantification
Scaffolds (n = 3) were digested with papain (25 µL∙mL ) in
−1
2.3. Printing 3D scaffolds phosphated buffer EDTa (PBE) after 14 days in culture with
The required scaffold was designed using the Cura 3D Dulbecco’s Modified Eagle Medium (DMEM) Glutamax
program, and its printing was carried out in a class II (Thermo Fisher Scientific, USA), supplemented with 1%
laminar flow cabinet using the Monoprice Mini V2 printer P/S and 10% fetal bovine serum (FBS). Control scaffolds
(Monoprice, China). The printer was thoroughly cleaned were maintained inside a 6-well plate, where cell medium
with 70% ethanol and ultraviolet (UV) radiation overnight. was exchanged every 3 days. BR scaffolds were further
The holder was designed to fit inside a multi-well cultured inside the system for seven to ensure adequate cell
plate (i.e., a 6-well plate). Therefore, the holder adopted a adhesion. DNA content was estimated using a fluorometric
cylindrical geometry: 24 mm in diameter, 5 mm in height, marker (DAPI staining), and a DNA standard curve was
and 200 µm in layer height. The movement speed of the done using DNA from calf thymus (Sigma-Aldrich, USA).
extruder was set to 14 mm·s , and the working temperature For collagen quantification, scaffolds were digested
−1
was set to 230°C. Finally, the flow rate (the speed of the in pepsin (1 mg∙mL ) and buffered in acetic acid (0.5N)
−1
filament passing through the extruder) was determined for 48 h at 4ºC, followed by the addition of 1 mg∙mL
−1
to be 1 mm·s . The scaffold’s infill geometry and porosity pancreatic elastase solution at 4°C for 24 h. Finally,
−1
were extracted from previous work (PS: ≈375 µm). 46 samples were neutralized with 1 M Tris base, and the
To ensure complete sterility, the scaffolds were placed supernatant was collected for further assays. Collagen
in Petri dishes and washed with an increasing gradient of was quantified using Sirius Red assay (Sigma Aldrich,
20%, 50%, and 70% ethanol. After washing, scaffolds were USA). Samples were placed in microcentrifuge tubes and
irradiated by UV on both sides for 1 h and subsequently were embedded in Sirius Red buffer (0.1% in picric acid)
rewashed with 1% antibiotic (Penicillin-Streptomycin for 1 h RT. Then, the tubes were centrifuged for 15 min
[P/S]) in phosphate-buffered saline (PBS) to remove under 13,000 rpm, and the supernatant was discarded.
residual ethanol. Then, the pellet was resuspended in 250 µL of 0.1 M
NaOH. Finally, the absorbance of the supernatant was
2.4. Scaffolds functionalization measured in a microplate reader at 540 nm (Synergy HT;
Scaffolds were placed in a multi-well plate and immersed BIO-TEK, USA). For standard collagen, calfskin was used
in a 10% isopropanol solution of 1,6-hexane diamine for (Sigma-Aldrich, USA). On the other hand, to quantify
30 min at room temperature (RT). Thereafter, they were collagen type II, a commercially available collagen type
rinsed in PBA (Sigma-Aldrich, USA) at 5 mM dimethyl II ELISA kit (Chondrex, USA) was used according to
sulfoxide (DMSO) (Sigma-Aldrich, USA). Finally, the the manufacturer’s instructions, and its absorbance was
scaffolds were washed several times with PBS. measured at 490 nm on a microplate spectrophotometer
(Synergy HT; BIO-TEK, USA).
2.5. Seeding cells in scaffolds
The IPFP-MSCs suspension (1 × 10 cells·mL ) was 2.8. Immunofluorescence
−1
6
pipetted onto each scaffold and incubated for 4 h at 37°C Celltracker Green (1:1000; Thermo Fisher Scientific,
TM
to facilitate cell attachment. The cell-loaded scaffolds were USA) was added to the pellet before cell seeding for 30
Volume 10 Issue 4 (2024) 369 doi: 10.36922/ijb.3389