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International Journal of Bioprinting 3D bioprinting of in vitro cartilage tissue model

chondrocyte-based cartilage models, such as the taken with an EVOS microscope to quantify the filament
chondrocyte 3D pellet model [32,33] , can be used when width using Fiji ImageJ software (1.53t version). Six
native tissue explants are not available. Here, we explored images were taken at different locations within the printed
peptide hydrogel bioinks, PeptiInks®, in terms of their structure. In each image, the filament width was quantified
application in 3D bioprinting using human primary three times using ImageJ and compared to the theoretical
chondrocytes for cartilage tissue modeling in vitro. We filament resolution.
compared its performance to a control 3D chondrocyte-
based pellet developed in vitro using previously optimized 2.3. Cell culture
manufacturing protocol . Human primary chondrocytes (HCHs; CellApplications,
[34]
San Diego, CA) were used in this study. HCHs between
In this study, we assessed the potential of PeptiInk passages 5 and 7 were consistently used. HCHs were
Alpha 1 (Manchester BIOGEL, Alderley Park, UK) as a cultured in chondrogenic growth media (Cell Applications,
material for 3D-bioprinting human cartilage tissue in vitro San Diego, CA), and they were incubated at 37°C in a
models. Primary human chondrocytes were encapsulated humidified atmosphere with 5% pCO . The media was
into PeptiInk Alpha 1, a neutrally charged peptide hydrogel, changed every 2 to 3 days. 2
and 3D-bioprinted structures were manufactured. Cell
viability, cell proliferation, and specific cartilage marker 2.4. Hydrogel cell encapsulation
production were assessed and compared to the current HCHs were manually mixed into Alpha 1 at a concentration
gold standard, chondrocyte cell pellets. of 1 × 10 cells/mL using a ratio of 1 mL of PeptiInk to
6
100 µL of cell culture media (1:10). Cell-laden PeptiInks
2. Materials and methods were loaded into 3-mL printing cartridges (Nordson, USA)
2.1. Hydrogel rheological characterization using a positive displacement pipette (Gilson Scientific,
Synthetic self-assembling peptide hydrogel Alpha 1 was Dunstable, UK). Loaded cartridges were centrifuged
obtained from Manchester BIOGEL (Alderley Park, UK). to remove air microbubbles for 2 min at 3500 rpm and
Alpha 1 was chosen due to its neutral charge, which has immediately printed into 12-well culture plates.
previously been shown to promote the chondrogenic 2.5. 3D bioprinting of HCH cell-laden Alpha 1
behavior of bovine chondrocytes in 3D in vitro cultures [28-30] The 3D-bioprinting process was performed using
and has a compressive modulus (E = 31 kPa ) in line the commercial printer BIOX6 (CELLINK, Sweden).
[29]
with previously used hydrogels for cartilage tissue Cylindrical structures with an outer diameter of 5 mm and
engineering [16,24] .
a thickness of 1 mm with 60% infill density were bioprinted
Oscillatory shear rheometry was performed using a using a 25G conical nozzle, an extrusion pressure between
Kinexus pro+ rheometer (Netzsh, Germany) with parallel 8 and 10 kPa, and a printing speed of 5 mm/s. The printed
sandblast plate geometry (40 mm, 0.5 mm gap size), structures were submerged in chondrogenic medium,
equilibrated to room temperature (25°C), and a solvent which was changed three times during the first hour to
trap to prevent the hydrogel from drying. Alpha 1 alone ensure pH equilibrium and twice each week over the
and Alpha 1 mixed with cell culture media in a 1:10 ratio culture period. Cell constructs were cultured for up to 14
were tested in triplicate to ensure reproducibility. For all days at 37°C in a humidified atmosphere with 5% pCO .
2
mixtures, rotational shear-viscosity measurements were
performed. Flow sweeps at 1% strain with a shear rate 2.6. Cell pellet formation and culture
ranging from 150 to 1 Hz were performed to assess the 3D control cell pellet cultures were formed using the
[34]
5
viscosity behavior under shear stress. methods of Yeung et al. Briefly, 4 × 10 cells were
centrifuged in 500 µL of culture media at 1750 rpm for 5 min
2.2. Printability tests in 15 mL tubes. After 72 h, the pellets were gently aspirated
Alpha 1 was loaded into a printer cartridge and printed, into ultra-low adhesion 24-well plates (ScienceCell, 0383),
using the BIOX6 bioprinter (CELLINK, Sweden), with with one pellet per well cultured in 1 mL of chondrogenic
multiple gauge (G) conical nozzles: 22G (400 μm— medium. Culture media were changed every 2 to 3 days.
extrusion diameter), 25G (250 μm—extrusion diameter), The cell pellets were cultured for up to 14 days at 37°C in a
and 27G (200 μm—extrusion diameter). 25G conical humidified atmosphere with 5% pCO .
nozzle was chosen as it gave a compromise between 2
structural accuracy and shear stress production. Different 2.7. Cell viability
pressures and printing speeds were combined to assess the Cell viability was assessed using a LIVE/DEAD assay.
filament continuity and width of Alpha 1 printed with a Bioprinted constructs were assessed 2 h post-printing (day 0),
25G conical nozzle. Images of the printed structures were on day 7 and day 14. Samples were cut in half to enable the

Volume 9 Issue 6 (2023) 452 https://doi.org/10.36922/ijb.0899