Page 58 - IJB-6-1

Vascularization of printed bilayer skin grafts
skin [6-11] . A variety of commercial tissue-engineered demonstrate that the tissue-engineered skin graft
skins have been used clinically . However, the with vascular endothelial cells is significantly
[12]
majority of these products are designed for timely better in wound healing.
coverage and to promote wound healing, being
unable to satisfy every function of the skin. An 2 Materials and methods
ideal tissue-engineered skin would exhibit no 2.1 Construction of 3D printed bilayer skin graft
toxicity or immune rejection, have normal color,
include accessory structures, vessels, and nerves, 2.1.1 Cell culture and hydrogel preparation
while demonstrating flexibility and appropriate
mechanical strength, and able to exchange Normal human dermal fibroblasts (NHDFs), human
material and energy [13-15] . Vascularization of tissue- dermal microvascular endothelial cells (HMVECs),
engineered implants is a key issue that restricts the and normal human epidermal keratinocytes (NHEKs)
development of tissue-engineered skin products . were purchased from American type culture
[16]
At present, tissue-engineered skin has no vascular collection (ATCC), and maintained and subcultured
structure, its nutritional supply principally relies in accordance with the supplier’s protocol. NHDFs
on osmosis, and hence the thickness is limited. The were maintained in Dulbecco’s Modified Eagle’s
epidermis is unable to receive sufficient nutrients Medium (DMEM) supplemented with 10% fetal
quickly when the skin thickness is >1 mm, causing bovine serum (FBS) and 1% antibiotic/antimycotic
partial exfoliation and necrosis of the epidermis. solution, HMVEC in Roswell Park Memorial
In the present study, a 3D-printed artificial Institute (RPMI) 1640 medium supplemented with
skin was fabricated which included vascular 10% FBS, and 1% antibiotic/antimycotic solution
features and through a series of in vitro tests, and NHEKs in Iscove’s Modified Dulbecco’s
the cell-hydrogel hybrid material printed by an Medium (IMDM) supplemented with 10% FBS,
extrusion printing process was evaluated. The and 1% antibiotic/antimycotic solution. Cells were
rate of survival of keratinocytes, fibroblasts, incubated at 37°C in 5% CO .
2
and endothelial cells was found to be >90%. In Sodium alginate (Sigma-Aldrich) and gelatin
addition, the bilayer skin construct was evaluated (Sigma-Aldrich) were dissolved in deionized water,
in vivo by detecting the integration of bilayer skin heated in a water bath at 37°C, and then stirred with
transplantation with host tissue in a nude mouse a magnetic stirrer at 80 rpm for 24 h. The gelatin-
model. Nude mice are appropriate in the study of sodium alginate composite hydrogel solution with
wound healing because they do not suffer immune 4% (w/v) sodium alginate concentration and 10%
rejection. In this experiment, a full-thickness (w/v) gelatin concentration was prepared.
wound was created on the back of nude mice. 2.1.2 Cytotoxicity assay
The degree of wound healing contraction rate of
mice was close to 90% , significantly different The hydrogel constructs created in this study
[9]
from that of human wounds. However, the nude were composed of 10% gelatin and 4% sodium
mouse model exhibited the ability to support the alginate. The constructs were placed in DMEM
engineered skin transplantation, in addition to at a 1:10 volume ratio so as to prepare extracts
allowing measurement of the structural differences and cultured at 37°C for 24 h . An improved cell
[17]
between transplanted and normal skin following counting kit-8 (CCK-8) cytotoxicity assay (Dojin,
wound healing. Wound contraction is a part of the Japan) was used to determine cell activity, in
normal healing process, but when it is too large, accordance with the manufacturer’s instructions.
it may lead to dysfunction or esthetic problems in NHDFs were plated into the wells of a 96-well
the wounds of patients. The purpose of this study plate at a density of 5000 cells per well. Hydrogel
was to compare the in vivo response of a number of extracts were added and incubated with the cells
tissue-engineered skin grafts with different cellular in a humidified atmosphere containing 5% CO
2
components to non-transplanted skin grafts and to at 37°C for 24 h, 48 h, and 72 h. Cells without

54 International Journal of Bioprinting (2020)–Volume 6, Issue 1