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Figures

Figure 1. Morphological analysis of single cellular spheroids derived from cell line

1-17 cells

(A) Phase-contrast microscopic images of line 1-17 cell-derived single cellular

spheroids. Line 1-17 cells (2.5 × 104 cells/well) were seeded on 96-U-well plates and

cultured in control medium for 1 day (I), 2 days (II), 3 days (III), and 4 days (IV).

Representative images are shown. Scale bars = 200 μm. (B) Diameter (I), roundness

(II), and surface roughness (III) of single cellular spheroids incubated for 1, 2, 3, and 4

days. Values are means ± SD from three independent experiments. **p < 0.01 vs. 1 day,

††

p < 0.01 (n = 96). n.s., not significant.

Figure 2. Three-dimensional (3D) tubular structure formation using singular

cellular spheroids (SCSs)

(A-F) Schematic images of 3D tubular structure formation. (A) Spheroids cultured in a

96-U-well plate are collected with a fine suction nozzle. (B) The spheroids are placed

onto an appropriate needle array according to a 3D design composed with the needle

array software. (C) The 3D-bioprinted spheroids are cultured on the needle array for

structural maturation. (D) After culture, the spheroids integrate with one another and

generate a cell-derived 3D tubular structure. Top (E) and side (F) images of the

software-composed 3D design are shown. (G, H) Representative images of the 3Dprinted SCSs on the needle array. Top (G) and side (H) images are shown. Scale bars =

1 mm.

Figure 3. Representative images of structures and evaluation of core materials

(A) Representative side (I) and top (II) images of spheroids incubated for 10 days on the

needle array. Top view of a 3D tubular structure (3DTB) just after being pulled out from

the needle array (III) and after a further 7 days of culture on a 10-cm dish without a core

material (WOC; IV). (B) Representative side (I) and top (II) images of a cylindrical

hydroxyapatite core and a top view of a 3DTB after 7 days of culture with a

hydroxyapatite core (HAC; III). (C) Representative side (I) and top (II) images of a

cylindrical titanium core and a top view of a 3DTB after 7 days of culture with a

titanium core (TIC; III). Scale bars = 1 nm. (D) X-ray diffractometer patterns of

hydroxyapatite powder (I) and a hydroxyapatite core (II). (E) Representative scanning

electron micrographs of a titanium core (I, II) and a hydroxyapatite core (III, IV) at

magnifications of ×200 and ×1,600. II and IV are highly magnified views of the boxed

areas in I and III, respectively.

Figure 4. Morphological and live cell distribution analyses in WOC, TIC, and

HAC

(A, B) Images of hematoxylin and eosin (A) and NucSpot (B) staining in WOC (I, II),

TIC (III, IV), and HAC (V, VI). II, IV, and VI are highly magnified views of the boxed

areas in I, III, and V, respectively. Experiments were performed in duplicate.

Representative images are shown. Green indicates NucSpot-positive living cells. Scale

bars = 500 μm. WOC: 3D tubular structure without a core material; TIC: 3D tubular

structure with a titanium core; HAC: 3D tubular structure with a hydroxyapatite core.

Figure 5. Picrosirius red and Masson’s trichrome staining in WOC, TIC, and HAC

(A, B) Images of picrosirius red (A) and Masson’s trichrome (B) staining in WOC (I,

II), TIC (III, IV), and HAC (V, VI). II, IV, and VI are highly magnified views of the

boxed areas in I, III, and V, respectively. Experiments were performed in duplicate.

Representative images are shown. After picrosirius red staining, a positive reaction was

confirmed in all areas of WOC, TIC, and HAC. After Masson’s trichrome staining,

blue-stained matrices were broadly distributed in WOC, TIC, and HAC. Red-stained

structures were confirmed in the outer areas. Scale bars = 500 μm. WOC: 3D tubular

structure without a core material; TIC: 3D tubular structure with a titanium core; HAC:

3D tubular structure with a hydroxyapatite core.

Figure 6. Gene expression analysis in line 1-17 cells, WOC, TIC, and HAC

PDL (COL3A1, PLAP1, and SDC1)-, angiogenesis (VEGFA and HGF)-, cementum

(CEMP1)-, and bone (OCN)-related gene expression in monolayer-cultured line 1-17

cells, WOC, TIC, and HAC. These gene expression levels were compared with realtime RT-PCR findings. Values are means ± SD from three independent experiments.

Line 1-17: monolayer-cultured line 1-17 cells; WOC: 3D tubular structure without a

core material; TIC: 3D tubular structure with a titanium core; HAC: 3D tubular

structure with a hydroxyapatite core. *p < 0.05, **p < 0.01 vs. line 1-17 cells (n = 3).

Figure 7. Immunohistochemical analysis for HGF, SDC1, and VEGF expression in

WOC, TIC, and HAC

(A-D) Images of immunohistochemical staining with anti-HGF (A), anti-SDC1 (B),

anti-VEGF (C), and rabbit control IgG (D) antibodies in WOC (I, II), TIC (III, IV), and

HAC (V, VI). II, IV, and VI are highly magnified views of the boxed areas in I, III, and

V, respectively. Brown indicates the presence of the target antigens. Nuclei were stained

with hematoxylin. No positive staining was observed in tissues incubated with rabbit

control IgG. Scale bars = 500 μm. WOC: 3D tubular structure without a core material;

TIC: 3D tubular structure with a titanium core; HAC: 3D tubular structure with a

hydroxyapatite core.

Figure 8. Immunofluorescence histochemical analysis for CEMP1 and OCN

expression in WOC, TIC, and HAC

(A-C) Images of immunofluorescence histochemical staining with anti-CEMP1 (A),

anti-OCN (B), and rabbit control IgG (C) antibodies in WOC (I–III), TIC (IV–VI), and

HAC (VII–IX). Green indicates the presence of the target antigens. Nuclei were stained

with DAPI. No positive staining was observed in tissues incubated with rabbit control

IgG. The strong green color in the middle of HAC indicates autofluorescence of the

residual hydroxyapatite core. Scale bars = 500 μm. WOC: 3D tubular structure without

a core material; TIC: 3D tubular structure with a titanium core; HAC: 3D tubular

structure with a hydroxyapatite core.

Figure S1. Live cell distribution analysis in monolayer-cultured line 1-17 cells

(A, B) Images of NucSpot staining in monolayer-cultured line 1-17 cells. The cells were

treated with phosphate-buffered saline (PBS) (A) or 3% H2O2 in PBS (B) for 10 min.

Experiments were performed in duplicate. Representative images are shown. Green

indicates NucSpot-positive living cells. Scale bars = 300 μm.

Figure S2. PDL-related collagen gene expression analysis in line 1-17 cells, WOC,

TIC, and HAC

PDL-related collagen gene (COL1A1 and COL12A1) expression in monolayer-cultured

line 1-17 cells, WOC, TIC, and HAC is shown. Gene expression levels were compared

with real-time RT-PCR findings. Values are means ± SD from three independent

experiments. PDL: periodontal ligament; WOC: 3D tubular structure without a core

material; TIC: 3D tubular structure with a titanium core; HAC: 3D tubular structure

with a hydroxyapatite core.

Figure S3. Highly magnified images of immunofluorescence histochemical analysis

for CEMP1 expression in WOC, TIC, and HAC

(A-C) Highly magnified images of Figure 8A. They are images of immunofluorescence

histochemical staining with anti-CEMP1 in WOC (I–III), TIC (IV–VI), and HAC (VII–

IX). The highly magnified images (B, C) correspond to the outermost area (B) and

middle lamella (C) of the three-dimensional structure (A), respectively. Green indicates

the presence of the target antigens. Nuclei were stained with DAPI. The strong green

color in the middle of HAC indicates autofluorescence of the residual hydroxyapatite

core. Scale bars = 250 μm. WOC: 3D tubular structure without a core material; TIC: 3D

tubular structure with a titanium core; HAC: 3D tubular structure with a hydroxyapatite

core.

Figure S4. Highly magnified images of immunofluorescence histochemical analysis

for OCN expression in WOC, TIC, and HAC

(A-C) Highly magnified images of Figure 8B. They are images of immunofluorescence

histochemical staining with anti-OCN in WOC (I–III), TIC (IV–VI), and HAC (VII–

IX). The highly magnified images (B, C) correspond to the outermost area (B) and

middle lamella (C) of the three-dimensional structure (A), respectively. Green indicates

the presence of the target antigens. Nuclei were stained with DAPI. The strong green

color in the middle of HAC indicates autofluorescence of the residual hydroxyapatite

core. Scale bars = 250 μm. WOC: 3D tubular structure without a core material; TIC: 3D

tubular structure with a titanium core; HAC: 3D tubular structure with a hydroxyapatite

core.

Figure S5. Immuno-fluorescent histochemical analysis for OSX expression in

WOC, TIC and HAC

(A-C) Images of immunofluorescence histochemical staining with anti-OSX antibodies

in WOC (I–III), TIC (IV–VI), and HAC (VII–IX). B and C are highly magnified views

of the outer and inner boxed areas in A, respectively. Highly magnified images with

Roman numerals correspond to each original image. Green indicates the presence of the

target antigens. Nuclei were stained with DAPI. The strong green color in the middle of

HAC indicates autofluorescence of the residual hydroxyapatite core. Scale bars = 250

μm. WOC: 3D tubular structure without a core material; TIC: 3D tubular structure with

a titanium core; HAC: 3D tubular structure with a hydroxyapatite core.

Figure S6. Immuno-fluorescent histochemical analysis for BMP2 expression in

WOC, TIC and HAC

(A-C) Images of immunofluorescence histochemical staining with anti-BMP2

antibodies in WOC (I–III), TIC (IV–VI), and HAC (VII–IX). B and C are highly

magnified views of the outer and inner boxed areas in A, respectively. Highly magnified

images with Roman numerals correspond to each original image. Green indicates the

presence of the target antigens. Nuclei were stained with DAPI. The strong green color

in the middle of HAC indicates autofluorescence of the residual hydroxyapatite core.

Scale bars = 250 μm. WOC: 3D tubular structure without a core material; TIC: 3D

tubular structure with a titanium core; HAC: 3D tubular structure with a hydroxyapatite

core.

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