Page 209 - IJB-9-3

International Journal of Bioprinting Dual ions mixed GelMA for hair follicle regeneration

follicle regeneration whose mechanisms are considered the the other contains Zn/Si dual ions (Si 120 μg/mL and Zn
same as the development process during the embryonic 20 μg/mL). GelMA and photoinitiators were obtained
period . Determining and utilizing the mechanisms of in from EFL (Jiangsu, China). Hydrocolloid wound dressing
[5]
situ regeneration may contribute to novel approaches to was purchased from Roosin (Jiangsu, China).
scar-free wound healing and alopecia.
2.2. Studying the effects of ions on cells migration
Recently, bioactive ions have been extensively To determine the optimal concentration of ions, we
investigated in the areas of regenerative medicine and tissue prepared a series of gradient concentration of solutions
engineering. For therapeutic applications, bioactive ions (1/64, 1/32, 1/16, 1/8) diluted with corresponding media
have some advantages, such as being inexpensive, easily (DMEM, SH30243.01, Hyclone, USA; HUVEC specific
accessible, chemically stable, and potentially safer than medium, iCell-h110-001b, iCell, China). Then, scratch
products of genetic engineering [6,7] . Accumulating evidence wound healing assay was employed to assess the migration
has shown that bioactive ions have beneficial effects on of human skin fibroblasts (HSFs), human umbilical
regulating specific cell behaviors. Zinc (Zn) is an essential vein endothelial cells (HUVECs), human immortalized
trace element of hair follicles and can induce differentiation keratinocytes (HaCaTs), and hair follicle dermal papilla
of adipocytes, suggesting that Zn may function as a stimulus (DP) cells in media with different concentration of ions .
[14]
in promoting hair follicles growth in the future . Silicon Briefly, cells were seeded in six-well plates and cultured
[8]
(Si) ion has stimulatory effects on angiogenesis, and some up to about 80% confluent. Then, a plastic tip (200 μL)
bioactive materials based on silicate can regulate cell–cell was used to scrap the cell monolayer in each well, and
interactions and hair follicle stem cells (HFSCs) activities the original media was replaced with serum-free media
to accelerate wound healing [9,10] . The biological effects of the containing ions. At 0, 12, and 24 h, an optical microscope
two ions imply that they may be valuable for wound repair. (Leica DMI 4000 B, Germany) was used to capture optical
Based on the extensive effects of Zn and Si ions images. ImageJ was employed to analyze the migration rate,
in regenerative medicine, we hypothesized that the which was calculated as the percentage of the remaining
interactions between HFSCs and endothelial cells may area (A) to the initial wound area (A ).
0
be regulated by the two ions, which promote in situ hair A A
follicle regeneration and angiogenesis in the excisional Cell migration rate 0 A 100% (I)
wound. To confirm our hypotheses, we first investigated 0
the effects of Si and Zn ions on migration and proliferation where A is the wound area value of all groups at each time-
of different cells as well as blood vessel formation to point, and A is the wound area value of the corresponding
0
determine a suitable concentration of ion solution. group at 0 h.
However, bioactive ions such as Si or Zn ions have 2.3. Studying the beneficial effects of ions on cell
several limitations, such as the difficulty of being fabricated viability
into three-dimensional (3D) scaffolds and a rapidly Then, the viability of different cells was evaluated by a
scattered site which scarcely matches the demand of in situ Cell Counting Kit (CCK-8) assay (CK04, Dojindo, Japan)
regeneration. Integrating multiple biocompatible factors after treatment with different concentration of ions.
into 3D-printed hydrogel-based scaffolds is a practical and HSFs, HUVECs, HaCaTs, and DP cells were respectively
effective approach to improving skin repair efficacy [11,12] . seeded into 96-well plates at 3 × 10 cells per well and
3
Gelatin methacryloyl (GelMA) is one of the widely used cultured in the corresponding complete media for 24 h.
natural bioinks for 3D-printed hydrogel-based scaffolds Then, the original media was replaced by 100 μL ions
due to their excellent features of biocompatibility and solutions diluted by complete media. Then, the cells were
degradability, as well as desirable release profiles . In continuously cultured for 4 days, and a CCK-8 assay was
[13]
this study, we integrated the solution containing identified applied to cells at 1st, 2nd, 3rd and 4th day. A multimode
bioactive concentration of ions with 3D-printed GelMA microplate reader (Tecan Spark 10M, Switzerland) was
hydrogel and then treated excisional wound in mice to employed to measure the absorbance of the reaction
observe the synergetic effects on wound healing, perfusion product at a wavelength of 450 nm. The results were
recovery and in situ regeneration of hair follicle. calculated according to Equation II:

2. Materials and methods Cell viability rate OD OD b (II)
e
2.1. Materials OD b
We obtained two ion solutions from Chinese Academy The optical density (OD) in experimental groups (Si, Zn/Si)
of Sciences: one only contains Si ions (120 μg/mL), and was designated as OD , and that in control group was OD .
e b

Volume 9 Issue 3 (2023) 201 https://doi.org/10.18063/ijb.703