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Tang, et al.
according to the same model and parameters as control GelMA solution without the process of 3D bioprinting. We
groups. All constructs were cultured for 4 days in vitro extracted the first and the second right maxillary molars,
before implantation experiments. and then prepared the alveolar socket into a 1.0 × 2.0 ×
1.5 mm bone defect with a dental implant machine.
3
2.6. Live/dead cell viability assay No implant was used to fill the bone defect in the blank
The viability of HERS or DPCs cells encapsulated in group while the four other groups implanted constructs
GelMA hydrogel was tested by Live/Dead Viability/ matching the alveolar socket defect before suture. The
Cytotoxicity Assay Kit for Animal Cells (KeyGEN, digital X-ray films were taken before and after surgery.
China) after 1, 4, and 7 days of incubation according After 8 weeks, samples were collected and treated with
to the manufacturer’s instructions (n = 4). Live and 4% paraformaldehyde for 24 h, then decalcified with 10%
dead cells were observed under laser scanning confocal ethylenediaminetetraacetic acid for 7 weeks. All specimens
microscope. The quantity of cells was counted through were embedded with paraffin for further analysis.
Image J software and the cell viability was quantified by 2.11. Histological and immunohistochemical
calculating the proportion of living cells among all cells. staining
2.7. Cell labeling 5 μm thick tissue sections were prepared for hematoxylin

Vybrant™ DiI and DiO cell-labeling solution (Thermo and eosin, Masson and immunohistochemical staining. All
Scientific, USA) are commonly used cell tracers in cell- samples were treated in accordance with manufacturer’s
cell fusion, cellular adhesion, and migration research due to recommended protocols. Primary antibodies used in this
their low cytotoxicity and high stability. To investigate the work consisted of COL-Ⅰ (1:200, ab270993, Abcam), OCN
interaction between HERS cells and DPCs loaded in GelMA, (1:500, ab93876, Abcam), RUNX-2 (1:500, ab76956,
the two kinds of cells were dyed by DiI and DiO solutions Abcam), and DSPP (1:500, 508413, Zen Bioscience).
separately according to the experimental protocols. First, Briefly, specimens were deparaffined and hydrated by
we added the cell-labeling solution into serum-free alpha- dimethylbenzene and ethanol, then permeabilized with
minimum essential medium with the concentration of 5 μL/ 0.1% Triton X-100, treated with citrate antigen retrieval
mL. Then cells were suspended by the working solution at solution and blocked with 10% goat serum before
a density of 1 × 10 /mL and incubated for 15–20 min at incubated with primary antibodies in a moist chamber
6
37°C. Finally, we resuspended cells in warm medium after overnight at 4°C. After incubating secondary antibodies
the treatment of centrifugation at 1200 rpm for 5 min and for 1–2 h at 37°C, DAB kit (Gene Tech, China) was used
repeated the procedure twice before viewing the cells under as a coloration solution and all slices were observed under
fluorescence microscope (Olympus, IX73, Japan). light microscope (BX43F Olympus, Japan).
2.8. Microscopic evaluation of constructs 2.12. Statistical analysis

GelMA scaffold was dehydrated by gradient alcohol All statistical analyses were performed using SPSS 24.0
to remove its aqueous phase. Then the scaffold was (SPSS Inc., Chicago, IL, USA). Student’s t-test was used
to compare the differences between two groups and one-
inspected under a scanning electron microscopy (SEM,
Inspect F, FEI, USA) for microscopic observation. way analysis of variance was performed to assess the
discrepancies between multiple groups. P < 0.05 was
2.9. Quantitative measurements of cell number considered statistically significant.
and cell confluence 3. Results
Cell convergence, the percentage of cell area in culture
dish, was one of the main parameters in cell culture in vitro 3.1. Immunofluorescence measurements
and usually used to assess cell proliferation [33,34] . In this The primary HERS cells grown in clusters with
study, we counted the cell number and cell confluence by a cobblestone appearance under light microscope
Image J software (n = 4). (Figure 2A, a). HERS cells expressed both epithelial cell
marker CK14 and mesenchymal cell marker Vimentin,
2.10. Alveolar socket transplantation in SD rats which were identified by immunofluorescence assay,
8-week-old female SD rats (210–240 g) were used for indicating that they had the properties of epithelial and
alveolar socket transplantation experiment (n = 4). The mesenchymal cells at the same time (Figure 2A, b and c).
experimental groups were designed as blank, DPCs 3.2. Viability of the cells encapsulated in GelMA
(3D bioprinting), HERS (3D bioprinting), HERS+DPCs
(3D bioprinting), and HERS+DPCs (mixed). The last The cell viability of HERS cells (Figure 2B) and DPCs
group mixed HERS cells and DPCs at a ratio of 1:2 in (Figure S2) was evaluated by Live/Dead assays after
International Journal of Bioprinting (2022)–Volume 8, Issue 3 143