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International Journal of Bioprinting 3D-printed PCL-MNP multifunctional scaffolds

9T, Quantum Design, USA) outfitted with a vibrating- The Live/Dead staining kit (Thermofisher Scientific,
sample magnetometer (VSM). The machine operated at a USA) was used to qualitatively determine the number of
temperature of 310 K, with a magnetic field strength range live and dead cells at a given time point. Cell culture media
of −15,000 to 15,000 Oe. was drained out, and the scaffolds were submerged in 500
µL of staining media (calcein and ethidium homodimer)
2.4. Temperature profile test and incubated for 15 min. After incubation, the well plate
A Magnetherm (NanoTherics, United Kingdom [UK]) was containing the stained scaffolds was taken out, and the
used to measure the increase in temperature exhibited by scaffolds were imaged under a microscope. Image analysis
the scaffold reinforced with MNPs. Pure PCL and PCL- was carried out using ImageJ for cell viability using
MNP scaffolds (MNP concentration: 2.5–50%) were the equation:
placed in a 2 mL vial. These vials were gently lowered into
the Magnetherm, while a temperature probe measured Live cells
the increase in temperature. The apparatus was set at 30 V Cell viability = ×100 (II)
(the maximum allowed by the system), and the increase in Live cells + Dead cells
temperature over 15 min was measured.
2.5.3. Hyperthermia test
2.5. In vitro analysis The hMSCs (immortalized human bone marrow

2.5.1. Degradation test mesenchymal stromal cells, hTERT iMSC3, Catalogue No.
All fabricated scaffolds, from pure PCL to PCL with T0529; Applied Biological Materials Inc., Canada) and
50% MNP concentration, were submerged in 5 mL of HOS bone cancer cells (Catalogue No. 300449; Cytion,
phosphate-buffered saline (PBS) at 37°C in glass vials. The Germany) were seeded on all concentrations of PCL-
scaffolds were taken out of the glass vials at every time point MNP samples, followed by incubation at 37°C for 24 h.
Thereafter, AB reagent was supplied to these scaffolds,
and then dried to remove any excess weight due to PBS on and the supernatant was collected after 4 h for performing
their surface. Once dried, the weight of the scaffolds was the assay. The media in the vials with cell-laden scaffolds
measured. They were then placed back in the glass vial, and was replaced with fresh media. These glass vials were
a fresh supply of PBS was provided. The degradation was then placed in the Magnetherm (NanoTherics, UK)
measured using the following formula: and subjected to an external magnetic field for 2 h each.
The vials were taken out, supplied with AB reagent, and
collected after 4 h of incubation. Finally, cell viability was
(Finalweight − Initial weight ) measured for each scaffold using a Biotek Synergy H1
Wt% = ×100 (I)
Initialweight microplate reader (Agilent Technologies Inc., USA).
2.6. Statistical analysis
2.5.2. Cell viability assay All experiments were performed in triplicates. To
The Alamar Blue (AB) reagent (Invitrogen, USA) was determine whether cell growth was statistically significant
utilized for determining the cell growth of the human in the seeded scaffolds, a two-tailed t-test was utilized.
mesenchymal stem cells (hMSCs) on PCL-MNP samples. Results with p < 0.05 were determined to be statistically
By using the AB assay, we can quantitatively measure significant. Data are presented as mean ± standard
cell viability in vitro. The scaffolds were sterilized first by deviation. For cell viability percentage calculated from
submerging them in ethanol for 1 h and subsequently Live/Dead images, one-tailed student t-test was used and
washing them with PBS. The samples were then kept under p<0.1 was determined to be statistically significant.”
ultraviolet (UV) irradiation for 1 h. The samples were
placed in a 24-well plate, along with the respective 2D and 3. Results
3D controls and blanks consisting of just media. The AB 3.1. Characterization of magnetic nanoparticles
reagent was added at each time point and incubated for 4 The average particle size of the MNPs was determined
h. Thereafter, 200 µL of the solution was collected into a by TEM. Figure 1A and B presents the TEM images
96-well plate from each well of the 24-well plate. A Biotek of magnesioferrite nanoparticles at 100 and 50 nm
Synergy H1 microplate reader (Agilent, USA) was used magnifications, respectively. The average particle size
to determine the absorbance at wavelengths of 570 and was determined to be approximately 20 nm (Figure 1C).
600 nm. To minimize biological variability in the results, In addition to the particle size, the MNPs were also
experiments were performed in triplicates. characterized using XRD (Figure 1D) and FTIR

Volume 10 Issue 6 (2024) 394 doi: 10.36922/ijb.4538