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Vascularization of printed bilayer skin grafts
A B
Figure 2. (A) Schematic diagram of transplantable printed skin. The top layer consisted of keratinocytes
and gel and the bottom layer fibroblasts, microvascular endothelial cells and gel. (B) A macroscopic
image of the printed skin graft.
medium used in vitro was the mixed solution of animal care used sterilized instruments and
DMEM: IMDM:RPMI = 1:1:1. the procedures were strictly sterile. Mice were
anesthetized with ketamine (40–90 mg/kg) and
2.2 Characterization of the in vitro 3D printed xylazine (5–10 mg/kg) by intraperitoneal injection
bilayer skin graft before surgery. The region for skin grafting was
NHEKs were labeled with cell Tracker™ Blue sterilized with iodophor and 75% ethanol. A full-
7-amino-4-chloromethylcoumarin dye, in thickness skin incision, 20 × 20 mm, approximately
accordance with the manufacturer’s protocol. 1 mm deep, was created on the backs of the nude
[20]
Briefly, the culture medium was removed and mice with ophthalmic scissors . The wounds
pre-warmed cell Tracker Blue (5 μM of blue represented approximately 30–40% of the back
dye in 2 ml of serum free media) added and of each animal. An aseptic gauze pad was used
incubated for 15 min at 37°C . The dye was to halt bleeding of the wound and prevent blood
[19]
then removed and the cells incubated with fresh loss exceeding 10–15% of total animal blood
culture medium at 37°C for 30 min. The medium volume during surgery. All animals were divided
was again removed and the cells washed with into four groups of eight nude mice each. The
phosphate-buffered solution (PBS) labeled cells printed skin graft was placed within wound so that
pass the dye to daughter cells but not adjacent it completely filled the defect in the experimental
cells. NHDFs were labeled with cell Tracker™ group of nude mice. The wound was not grafted
Green 5-chloromethylfluorescein diacetate dye in the control group. Tegaderm (3M, London, ON,
and HMVECs with cell Tracker™ Red CMTPX Canada, http://www.3m.com) was placed over the
dye. The labeling procedure was the same in each wound to protect the wound site and maintain a
[21]
case. moist environment (Figure 3). After surgery,
the animals were placed in an aseptic Individual
2.3 Characterization of the in vivo 3D printed ventilated cage (IVC) containing sterile surgical
bilayer skin graft gauze. The Tegaderm was in place for 8 – 10 days
to ensure that the printed skin had grafted to the
2.3.1 Animal surgery
tissues of the mice. During this period, infection
Thirty-two male nude mice were purchased from the and the dressings were monitored at least twice per
SPF Animal Room, Experimental Animal Center day. The mice were monitored until the experiment
of Xi’an Jiaotong University. All experiments had had completed, at which time the animals
been approved by the Institutional Animal Care were euthanized and tissue samples collected.
and Use Committee of the Ethics Committee of Tissues were fixed in 4% paraformaldehyde
Xi’an Jiaotong University Health Science Center, and then analyzed histologically and with
China. Surgical procedures and post-operative immunohistochemistry.
56 International Journal of Bioprinting (2020)–Volume 6, Issue 1
