Page 443 - IJB-10-6

International Journal of Bioprinting Bioprinted skin scaffolds with GNP exposure

transplant. The mice’s skin was removed 6 days later and In addition, live-dead staining was conducted to
used for further experiments. examine the cell viability under selected conditions with
skin cells (Figures S2 and S3, Supplementary File). The
2.8. Inductively coupled plasma-optical range of internal needle diameter had no significant
emission spectrometry influence on cell viability. As days progressed, the ratio
Three mice were euthanized at 6 days post-injection. of dead cells gradually increased, consistent with those
Transplanted skin and other organs were collected and cultivated directly in vitro.
weighed. A section of each part was minced into small
pieces, digested, and dissolved in nitric acid and a small 3.2. Bioprinting of cell-laden hydrogel
amount of hydrochloric acid. The amount of gold was Figure 2A illustrates the steps used for the bioprinting
determined using inductively coupled plasma-optical of the composite 3D skin tissue. To better imitate the
emission spectrometry (ICP-OES; Optima 7300 V ICP- spatial skin structure, we designed and investigated three
OES; PerkinElmer, USA). Natural skin was obtained from different cell-laden scaffold composites (Figure 2B). After
the nearby site of the transplanted collagen scaffold in the the bioprinting process, the skin tissue was immediately
same mouse. imaged by confocal microscopy and cultured in media
under submerged conditions. Figure 2C–E features the
2.9. Statistical analysis uniformly distributed GFP-HEKs in the epidermis and
Results are presented as mean ± standard deviation unless RFP-HDFs in the dermis. The cells were sparsely but
otherwise indicated. All of the data were analyzed using uniformly distributed in the collagen matrix in pattern 3,
SPSS Statistics 17.0 (IBM, USA) and processed using whereas in other patterns, the cells were too dense. The
GraphPad Prism 7 (GraphPad, USA). Differences between bioprinted cells maintained their shape and viability, and
two groups were analyzed using a two-tailed unpaired the boundary between layers was distinct, indicating no
t-test. Differences were considered significant at p < 0.05. cell invasion across the dermal and epidermal layers.
3.3. In vivo transplant
3. Results Over an 11-day period (Figure 3A), the borders of
3.1. Bioprinting parameters estimate the bioprinted skin construct tightly adhered to the
Prior to 3D cell-laden scaffold bioprinting, we examined surrounding mouse skin. As time progressed, the
the utility of the bioprinter by testing serial bioprinting previously shining surface of the constructs became matte,
conditions and cell viability. Firstly, different internal yet the wounds gradually reduced in size (Figures 3B–M
needle diameters and air pressures were used to assess and S4A, Supplementary File). The healing of the wound
the performance of the bioprinter. As anticipated, the injected with GNPs and normal saline was also analyzed on
number of pulses required to extrude 1 mL of ultrapure day 11 (Figure S4B, Supplementary File). No interruption
of the epidermis could be observed at the junction between
water decreased with the internal needle diameter and air the skin scaffolds and the mouse skin (Figures 4, 5, and
pressures. However, little correlation was observed when S5, Supplementary File). The structure of bioprinted
the air pressures exceeded 12 psi (Figure 1A–F). These scaffolds retained its shape and dimensions. The absence
results indicated that the bioprinter functioned effectively of color merging indicated no cell migration between
over a broad range of internal needle diameters and layers (Figure 4). The HEK layers in patterns 1 and 2
air pressures. were much thicker than those in pattern 3 (Figures 2C–E
In skin bioprinting, achieving a representative cell and 4), suggesting that the cell density in patterns 1 and
density within each layer (epidermis and dermis) is 2 was too high for the optimal growth, proliferation, and
crucial for obtaining morphologically and functionally differentiation of the HEKs.
representative tissue. Various bioprinting air pressures and 3.4. Vascularization
internal diameter of needles were examined with different Platelet endothelial cell adhesion molecule-1 (PECAM-1,
types of cells (Figure 1G–L). Both sets of results displayed CD31) is a member of the immunoglobulin gene
high cell viability across various pressures and diameters, superfamily of cell adhesion molecules. CD31 is a
indicating the stability of the bioprinter under different transmembrane glycoprotein that is highly expressed on
conditions. The viability of the bioprinted cells appeared the surface of the endothelium, making up a large portion
to be equivalent to or slightly higher than that of the cells of its intercellular junctions. Thus, CD31 can be used as a
cultivated in the Petri dish. The cell density was optimized marker of vascular endothelial and a hint for endothelial
by controlling the key bioprinting parameters, such as cell formation, consequently aiding in tissue development.
suspension densities. Here, the increased expressions of CD31 were found in

Volume 10 Issue 6 (2024) 435 doi: 10.36922/ijb.4692