Page 238 - IJB-10-6

International Journal of Bioprinting DIW of concave hydroxyapatite scaffolds

To calculate the permeability of the porous scaffold, a were used for each sample type, and control samples,
simplified version of Darcy’s law was used : consisting of cells seeded directly in wells without scaffolds,
17
were included. A calibration curve with a known cell
number was used to calculate adhesion percentages. The
µ qL number of adhered cells was normalized by the available
K = (V)
AP ∆ area for each scaffold, as measured by micro-CT, and
expressed as a percentage relative to the control samples.
where K is the longitudinal permeability, μ the BMF 2.8.2. Cell proliferation, differentiation,
viscosity, q the flow rate, L the scaffold’s height in the and mineralization
longitudinal direction, A the scaffold’s cross-sectional area, To assess the effect of geometry on cell behavior, both cell
and ∆P the previously calculated applied pressure. proliferation and differentiation were studied on days 1, 7,
2.7. Mechanical properties in compression 14, and 21, while mineralization was quantified on days
The scaffolds were tested in uniaxial compression (10 14 and 21. Cells were seeded on the scaffolds at the same
samples per group) under wet conditions using a universal concentration used in the adhesion experiment for 1 h in
testing machine (BIONIX; MTS Systems Corporation, the incubator and subsequently covered with complete
USA). A 0.5 mm/min cross-head speed was used, following media. At each time point, the scaffolds were transferred
the ISO 13175-3 standard. The strength was determined to new well plates, rinsed with warm PBS, and lysed
as the maximum stress; the elastic modulus as the slope using 500 µL M-PER. Cell lysates were stored at −80°C
in the elastic region; and the strain energy density (SED), until measurement.
i.e., the work of fracture normalized by the sample volume, Cell proliferation was quantified using an LDH kit.
as the area under the stress versus strain curve. Moreover, Briefly, 100 µL of the cell lysates were added to the 96-
the Weibull modulus was calculated through the Weibull well plates; 100 µL of the reaction mixture was added
statistical analysis of the strength values: and incubated at room temperature for 15 min; 50 µL of
stop solution was added; the absorbance at 490 nm was
measured in a microplate reader (BioTek Instruments,
  1  USA). Cell proliferation was normalized by the area of

ln ln   = m( ln S () − ln σ 0 ) (VI) each scaffold and expressed as a percentage of proliferation
( )
   P s   relative to the control sample on day 1. Cell differentiation
was investigated by ALP quantification of the same cell
where P is the probability of survival, m is the Weibull
s
modulus, σ is the stress, and σ is a constant corresponding lysates. Briefly, 50 µL of cell lysates were pipetted into 96-
well plates; 100 µL of ALP buffer was added and incubated
0
to the stress for which 37% of the samples survive.
at room temperature for 15–20 min; 50 µL of stop solution
2.8. In vitro study was added; the absorbance was recorded at 405 nm. A
calibration curve with known ALP concentrations was
2.8.1. Cell adhesion used for calculations, with ALP concentrations expressed
The scaffolds were sterilized by immersion in 70% ethanol as ng/mL. These values were normalized by the cell number
for 2 h, followed by rinsing in PBS three times, and incubated (quantified by LDH) and expressed as ng/cell. Triplicate
overnight with complete McCoy’s 5A media (10% FBS, 1% samples for each type of geometry were used.
Pen/Strep, 2% HEPES, and 1% sodium pyruvate). The next Cell mineralization was investigated using AR-S
day, the scaffolds were dried using sterilized filter paper and quantification after 14 and 21 days. The cell-laden
before seeding to optimize cell droplet sedimentation.
scaffolds were fixed using 4% paraformaldehyde (Aname
Approximately 50,000 SaOs-2 (ATCC® HTB 85TM) Instrumentación Científica, Spain) for 20 min at room
pre-OB osteosarcoma cells were seeded per scaffold using temperature. Subsequently, the scaffolds were rinsed with
a 10 µL droplet, and incubated for 1 h at 37ºC and 5% PBS and stained with 40 mM Alizarin Red (AR) at pH
CO . Thereafter, 2 mL of complete media were added, and 4.2 for 10 min at room temperature. The scaffolds were
2
cells were further incubated for 6 h. The scaffolds were then rinsed under agitation in water and later with PBS to
then washed twice with warm PBS and transferred to remove any unbound AR. The scaffolds were decolorized
new well plates. Subsequently, 500 µL of 10% Presto Blue using 10% cetylpiridinium chloride in sodium hydrogen
diluted in complete media was added and incubated for 1 phosphate buffer at pH 7 for 20 min under agitation.
h. Cell adhesion was quantified by the fluorescence signal The supernatant containing the dye was collected and
(excitation: 560 nm; emission: 590 nm). Quadruplicates measured in a microplate reader at 564 nm. Control

Volume 10 Issue 6 (2024) 230 doi: 10.36922/ijb.3805