Page 60 - IJB-3-1

Caroline Murphy, Krishna Kolan, Wenbin Li, et al.

netics, and cytokine expression profiles to BSCs. With Manassas, Virginia, USA), 1% 100× L-glutamine (GE
the addition of ASCs, the scaffold is expected to im- Life Sciences, Logan, Utah, USA), 2% 100× antibio-
prove its biomechanical and biological properties tic/antimycotic (GE Life Sciences, Logan, Utah, USA,
for better repair of the target tissue. In the current and minimum essential medium alpha modified
study, we investigate the feasibility of scaffold fabri- (α-MEM) (Sigma-Aldrich, St. Louis, Missouri, USA).
cation using a two syringe system with a PCL/borate After 24 hours, the media was removed and adherent,
glass composite dissolved in an organic solvent as a viable cells were washed twice with PBS, harvested
scaffold material, whilst simultaneously printing cells with 0.25% trypsin/1 mM EDTA (Gibco, Grand Island,
2
suspended in Matrigel, which is a gelatinous protein New York, USA), and replated at 100 cells/cm in
mixture representing basement membrane. Included in CCM. The media was changed every 3 to 4 days. For
this study are the effect of borate glass content on the all experiments, sub-confluent cells (≤70% conflu-
composite paste printability, the scaffold tempor- ent) between passages 2 and 6 were used. To prepare
al bioactivity, its degradation in culture media, and the bio-ink, ASCs were suspended at a concentration of
6
ASC viability in the scaffold. 10×10 cells per mL of Matrigel (Corning, Bedford,
Massachusetts, USA) diluted to 9 mg/mL in phos-
2. Materials and Methods phate buffered saline (PBS). The bio-ink was then
transferred to a tap ered nozzle tip (30G) which was
2.1 Preparation of PCL/13-93B3 Borate Glass stored on ice during the entire non-printing time.
Composite Material The bio-ink was gently pipetted to obtain a uniform
Polycaprolactone (Sigma-Aldrich, St. Louis, Missouri, distribution of cells just before printing. Matrigel was
USA) was dissolved in chloroform (CF) (Sigma-Ald- used in this work as it resembles the complex extra
rich, St. Louis, Misouri, USA) in a covered glass con- cellular environment found in many tissues.
tainer with the help of a stirrer at ~5 0 °C. The PCL 2.3 Scaffold Fabrication
weight to CF volume ratio (grams:mL) was varied from
1:1 to 5:4 to determine the ideal ratio for printing. An A square scaffold measuring 10 mm in length was
appropriate ratio was established by visually inspecting printed with 0°–90° orientation of the filaments in
the paste and through filament extrusion using a digital alternate layers. The schematic in Figure 1A shows
®
syringe dispenser (Loctite , Henkel North America, the printing set-up and Figure 1B depicts the printing
Rocky Hill, Connecticut, USA). Then, 13-93B3 glass process. A custom modified cartesian 3D printer
(Mo-Sci Corporation, Rolla, Missouri, USA) (nominal (Geeetech, Prusa I3 A Pro) with two additional sy-
composition – 53% B 2O 3, 20% CaO, 12% K 2O, 6% ringes controlled by digital dispensers was used for
Na 2O, 5% MgO, 4% P 2O 5 in weight percentage) with fabrication. The G-code for nozzle movement was
~20 µm particle size was added to the PCL:CF mix in written to print in a 0°–90° pattern to obtain rectangular
five different weight percentages in increments of 10, pores. The printing parameters such as air pressure,
ranging from 10% to 50%. A magnetic stirrer was used filament spacing, layer height, and printing speed were
to uniformly mix the composite paste, and no settling identified based on visual inspection and optical mi-
of the glass particle precipitate was observed before croscopic images after the first and second layer
transferring the paste to a syringe. Each ratio was tested printing for different paste compositions. To determine
using a digital syringe dispenser at air pressure ranging the printing parameters for Matrigel, an experiment
from 10 to 50 psi and with nozzle tip diameter ranging was conducted by varying the nozzle tip distance from
from 110 to 600 µm (32 G to 20 G). the glass slide, droplet dispensing time, and air pres-
sure. Fluorescent images of the droplets were taken
2.2 Preparation of Bio-ink and ImageJ software was utilized to quantify the
number of cells and cell distribution in each fluores-
6
Frozen vials of approximately 1 × 10 ASCs were ob- cent image. A tapered nozzle tip (30 G) with 160 µm
tained from three separate donors (LaCell, New Or- orifice provided the suitable droplet size (~400 µm) at
leans, Louisiana, USA). Vials were unthawed, plated 10 psi and 0.035 s dispensing time for deposition on the
2
on 150 cm culture dishes (Nunc, Rochester, New filament. While some droplets fell to the surrounding
York, USA) in 25 mL complete culture media (CCM), pores, most of the droplets stayed on the filament be-
and incubated at 37 °C with 5% humidified CO 2. The fore the Matrigel was allowed to cross-link at room
CCM contained 10% fetal bovine serum (Corning, temperature. The fabrication experiments were

56 International Journal of Bioprinting (2017)–Volume 3, Issue 1