Page 547 - IJB-10-3

International Journal of Bioprinting Engineered 3D-printed PVA vascular grafts

(iii) LDH release assay: Following the incubation period, Plasma-free hemoglobin and total blood hemoglobin
50 µL of the supernatant in each well was collected (TBH) were determined according to ASTM F756-13,
and added to a clean well in a new 96-well plate using Drabkin’s reagent. To adjust the TBH concentration
to measure LDH released by cells. To measure the to 10 ± 1 mg/mL, whole pooled blood was diluted with
released LDH following the cells’ exposure to the Ca/Mg-free PBS. Following this step, the diluted TBH
vascular graft, the CyQUANT LDH Cytotoxicity concentration was verified.
Assay (Invitrogen, USA) was used. Samples were The samples were prepared according to the guidelines,
prepared according to the producer’s specifications, and the corresponding quantities were suspended in
and the absorbance was measured at 490 nm. For 7 mL PBS. Negative control was prepared using 40%
non-adherent U937 monocyte cells, the sample poly(ethylene glycol) (PEG) 8000 and positive control
was removed from the well, and the cells and cell using 10% Tween-100 in PBS. Approximately 1 mL of
culture were collected in Eppendorf tubes to be diluted blood was added to each sample and then incubated
centrifuged at 400 rpm for 5 min. The supernatant at 37°C for 3 h with mixing every 30 min.
was collected, and LDH release was measured.
For all quantitative measurements, blank samples Following the incubation period, tubes containing
were prepared (extract/medium without cells and the samples were centrifuged at 700 rpm for 15 min, and
dilutions). The blank absorbance was subtracted the supernatant was carefully collected. The supernatant
from the cell samples’ absorbance to eliminate any of each sample was diluted to 1:1 with Drabkin’s reagent
possible interferences. and incubated for 15 min. Thereafter, the absorbance of
the samples was measured at 540 nm. The percentage of
All experiments were performed in triplicate. Cell hemolysis for each sample was determined as follows:
viability and LDH release were calculated relative to
negative control samples (untreated cells). Data are
−
reported as mean ± standard deviation (SD). Statistical Hemolysis(%) = Sample hemoglobin absorbance blankabsorbance ×100%
analysis was performed using Student’s t-test, with − TBH −− blankabsorbance (II)
Sample hemoglobin absorbance blankabsorbance
Hemolysis(%) =
statistical significance at *p < 0.05, **p < 0.01, and ***p < ×100%
TBH −− blankabsorbance
0.001.
by dividing the blank-corrected samples’ supernatant
2.9. Hemocompatibility evaluation hemoglobin absorbance by the blank-corrected TBH
Hemocompatibility evaluation of the optimized vascular concentration of the diluted blood and subsequently
grafts was performed on horse blood acquired from multiplying with 100. The samples’ acceptance criteria
the “Cantacuzino” National Medico-Military Institute described in the ASTM F756-13 were followed.
Research and Development (Romania), which is accredited
to produce and commercialize laboratory animals and 3. Results and discussion
serums. Whole blood from three healthy animal donors 3.1. Development of lysine-functionalized and
was collected on heparin. The testing was performed unmodified poly(vinyl alcohol) grafts
according to ASTM F756-13 (Standard practice for After crosslinking and drying, PVA channels are generally
assessment of hemolytic properties of materials), i.e., the regarded as grafts. The starting material, PVA filament,
direct method for hemoglobin determination. was subjected to thermal treatment to investigate its
Drabkin’s Reagent (Sigma-Aldrich, USA) was used to potential impact on the PVC channels post-printing. The
quantitatively determine hemoglobin release. The reagent findings indicated a notable increase in the crystallinity
was prepared according to the producer’s specifications of the PVA filament. However, there was no significant
and reconstituted in deionized water with 30% Brij L23 influence observed on the 3D-printed channels compared
solution (Sigma-Aldrich, USA). to the non-thermally treated filament. Henceforth,
the study proceeded with thermal-treated 3D-printed
A calibration curve was obtained using a channels, which demonstrate a significant impact on both
cyanmethemoglobin standard solution of 180 mg/mL crystallinity and crosslinking density. After crosslinking,
hemoglobin (hemoglobin equine; Sigma-Aldrich, USA) only PVA grafts, 3D-1H and 3D-3H, exhibited sufficient
in Drabkin’s regent. Serial dilutions of the standard were stability for biofunctionalization with lysine and
performed, and the absorbance was measured at 540 nm. subsequent mechanical flow testing (Table 1). Specifically,
The slope of the standard curve was determined using PVA graft 3D-3H (biofunctionalized and unmodified) was
Microsoft Excel as 0.000134 mg/mL (absorbance). successfully tested for mechanical flow and subsequently

Volume 10 Issue 3 (2024) 539 doi: 10.36922/ijb.2193