Page 455 - IJB-10-3

International Journal of Bioprinting Biomimetic scaffolds for tendon healing

as the slope of the line tangent to the stress–strain 2.12. Determination of metabolic activity
curve at 50% of the ultimate stress. Work to break The metabolic activity of iMAT cells inside the tissue
39
was determined by calculating the area under the stress constructs was determined using the cell counting kit.
vs. strain curve until the breaking point was reached. Following the manufacturer’s instructions, a 1:10 mixed
Part (D) of the Supplementary File presents the results solution of CCK-8 reagent (500 µL) was added to each
of the force vs. displacement graph along with the tissue construct and was incubated for 4 h at 37°C.
corresponding parameters derived from it. The fluorescence of the samples was determined in an
Infinitive M200 microplate reader from TECAN Trading
2.9. Swelling study AG (Männedorf, Switzerland). The established parameters
In the swelling study, the acellular scaffolds were firstly were a detection wavelength of 450 nm and a reference
printed, then crosslinked, and finally dried using a wavelength of 650 nm. Acellular scaffolds were used as
lyophilization process. Once lyophilized, their initial blank. The results were represented as absorbance units vs.
weight was measured (W ). One milliliter of PBS was test condition (cell density and time). Five sampling times
0
added to each scaffold. They were incubated under shaking were established, and four replicates were performed in
conditions at 37°C for 24 h. At the established sampling each condition.
time points, the scaffolds were removed from the PBS,
slightly dried using a filter paper, and weighed (W ). Ten 2.13. Measurement of lactate
f
specimens were tested during the different time points. dehydrogenase activity
The swelling ratio was established using the following The effect of the bioprinting process on the tenocytes was
equation (Equation V): investigated using a lactate dehydrogenase activity assay
kit. Following the manufacturer’s instructions, 500 µL of
master reaction mix was added to each tissue construct
( W − W )
Swelling ratio = f 0 (V) and incubated for 4 h at 37°C. Finally, the fluorescence
W 0 of the samples was determined in an Infinitive M200
microplate reader. The established parameters were
2.10. Degradation study a detection wavelength of 490 nm and a reference
To establish the degradation rate, the acellular scaffolds wavelength of 680 nm. Acellular scaffolds were used as
were 3D-printed, crosslinked, dried with filter paper, and blank. The results were represented as absorbance signal
weighed (W ). One milliliter of DMEM medium was added vs. test condition (cell density and time) (part (E) of the
0
to each scaffold. Afterward, they were incubated under Supplementary File). Three replicates were performed in
shaking conditions at 37°C for 85 days. At the established each condition.
sampling time points, the scaffolds were removed from
the media, slightly dried, and weighed (W ). Twenty-five 2.14. Gene expression study
f
specimens were tested at each time point. The degradation The tissue constructs were cultured for 3 and 12 days under
percentage was established using the following equation the previously mentioned conditions. At the mentioned
(Equation VI): time points, four of these tissue constructs were collected,
enzymatically digested (using 1 mg mL of alginate lyase,
-1
at 37°C, and for 15–25 min), and centrifuged to separate
W
Degradation (%) = f ×100 (VI) the cells from the rest of the scaffold. The TRIzol method
W 0 was used to obtain the RNA of the samples. Finally, the
total mRNA quantity and purity were measured with a
2.11. Assessment of cell viability SimpliNano Nanodrop from GE Healthcare Life Sciences
The viability of iMAT cells inside the tissue constructs was (Madrid, Spain). The RNA was reverse-transcribed to
determined using the LIVE/DEAD™ Viability/Cytotoxicity cDNA using the Fast Gene Scriptase II, cDNA Synthesis
Kit assay. Following the manufacturer’s instructions, Kit. Real-time PCR (RT-qPCR) was performed using
live cells were stained with calcein-AM (excitation/ StepOnePlus Real-Time PCR Systems. To determine the
emission wavelength of 495/515 nm) and dead cells with expression of the target genes (glyceraldehyde-3-phosphate
ethidium homodimer-1 (excitation/emission wavelength dehydrogenase, Gapdh; Scleraxis BHLH transcription
of 495/635 nm). At the sampling time points, the tissue factor, Scx; Fos proto-oncogene, c-Fos; Tenascin C, Tnc;
constructs were imaged using a Nikon TMS microscope Collagen type I alpha 1 chain, Col1a1; Collagen type III
(Virginia, USA). alpha 1 chain, Col3a1; Fibronectin 1, Fn1), commercial

Volume 10 Issue 3 (2024) 447 doi: 10.36922/ijb.2632