Nanostructured Ti surfaces and retinoic acid/dexamethasone present a spatial framework for the maturation and amelogenesis of LS-8 cells

Figures & data

Table 1 Primers used for real-time PCR

Gene	Accession number	Forward primer sequence (5′-3′)	Reverse primer sequence (5′-3′)	Product size (bp)
BMP2	NM_007553.3	TGACTGGATCGTGGCACCTC	CAGAGTCTGCACTATGGCATGGTTA	112
RUNX2	NM_001271627.1	TGCAAGCAGTATTTACAACAGAGG	GGCTCACGTCGCTCATCTT	188
SHH	NM_009170.3	GTTTATTCCCAACGTAGCCGAGA	CAGAGATGGCCAAGGCATTTA	200
AMELX	NM_009666.4	GATGGCTGCACCACCAAATC	CTGAAGGGTGTGACTCGGG	65
AMBN	NM_001303431.1	ACTGCTACCTGGAAACAA	TGCGGAAGGATAGTAAGT	161
ENAM	NM_017468.3	TGCAGAAATCCGACTTCTCCT	CATCTGGAATGGCATGGCA	114
KLK4	NM_019928.1	CCGGATCATACAAGGCCAGG	TGCGGATGCACCAAGACTC	105
MMP-20	NM_013903.2	ACACATTTCGACAATGCTGAGA	AAGTTGGGTACATCAGTGCTG	136
AMTN	NM_027793.1	GCACATACTCTCCCGTTCAC	AAGATTTGGGAGGCTAACGG	144
ODAM	NM_027128.2	TTGACAGCTTTGTAGGCACA	GACCTTCTGTTCTGGAGCAA	197
BCL-2	NM_009741.5	TGTCACAGAGGGGCTACGAGT	TCAGGCTGGAAGGAGAAGATG	94
BAX	NM_007527.3	CCAGGATGCGTCCACCAA	AAAGTAGAAGAGGGCAACCAC	196
CASP3	NM_001284409.1	CTAATCTGACGGTCCTCC	TCGCCAAATCTTGCTAAT	200
GAPDH	NM_001289726.1	CATGTTCCAGTATGACTCCACTC	GGCCTCACCCCATTTGATGT	136

Abbreviations: BMP-2, bone morphogenetic protein-2; RUNX2, runt-related transcription factor 2; SHH, sonic hedgehog; AMELX, amelogenin X-linked; AMBN, ameloblastin; ENAM, enamelin; KLK4, kallikrein related-peptidase 4; MMP-20, matrix metallopeptidase 20; AMTN, amelotin; ODAM, odontogenic, ameloblast-associated protein; BCL-2, B-cell lymphoma-2; BAX, BCL2-associated X protein; CASP3, caspase-3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Table 2 Surface roughness of nanostructured Ti surfaces

Group	Ra (nm)	Rq (nm)
P	1.85±0.47	2.62±0.89
NT5	4.85±0.93*	6.20±1.72*
NT20	11.40±3.05*,#	14.80±2.47*,#

* Note: p<0.01 vs P;

# p<0.01 vs NT5.

Abbreviations: Ra, average roughness; Rq, root mean square roughness.

Table 3 Elements composition of nanostructured Ti surfaces

Group	Relative atomic concentration (%)			Ti:O ratio
	Ti2p	O1s	C1s
P	25.41±1.47	63.07±2.63	9.27±0.28	2.47±0.27
NT5	24.85±2.93 (ns)	64.54±1.23 (ns)	8.68±0.52 (ns)	2.59±0.41 (ns)
NT20	23.40±1.05 (ns)	62.91±1.73 (ns)	11.26±1.41 (ns)	2.68±0.31 (ns)

Note: ns, no significance vs P or NT5.

Figure 1 Surface characterization of nanostructured Ti surfaces.

Notes: (A) Representative atomic force microscopy images showing the topography of the polished Ti (P) and nanostructured TiO₂ NT surfaces via anodization at 5 or 20 V (NT5 and NT20, respectively) TiO₂ surfaces. Scanning range=500×500×50 nm. (B) Representative field emission scanning electron microscopy images showing the morphology and topography of P, NT5, and NT20 surfaces from the vertical and lateral views (insets). Magnification=100,000×; scale bar=100 nm. (C) Representative images showing the water contact angle on modified Ti surfaces. Scale bar=1 mm; (D) contact angle analysis. *p<0.01 vs P; ^#p<0.01 vs NT5.

Abbreviation: NT, nanotubular.

Figure 2 Morphology of LS-8 cell attachment on nanostructured Ti surfaces in standard or retinoic acid (RA) medium.

Notes: (A and C) Representative images showing the morphology of LS-8 cells attached to the prepared Ti surfaces in standard or RA medium. Magnification=400×; scale bar=50 μm. (B and D) Image semi-quantitative analysis of attached LS-8 cells in standard or RA medium. *p<0.01.

Figure 3 Proliferation of LS-8 cells cultured on nanostructured Ti surfaces in standard medium (A) or RA medium (B). *p<0.01 vs P; ^#p<0.01 vs NT5.

Abbreviation: RA, retinoic acid.

Figure 4 Apoptosis/necrosis of LS-8 cell on nanostructured Ti surfaces in standard or RA media.

Notes: (A) Representative images of flow cytometry; (B) analysis of the proportion of viable cells (upper row), apoptotic cells (middle row), and necrotic cells (lower row). *p<0.01.

Abbreviation: RA, retinoic acid.

Figure 5 Expression of amelogenic genes in LS-8 cells cultured on nanostructured Ti surfaces in standard culture medium.

Notes: (A) BMP-2, (B) RUNX2, (C) SHH, (D) AMELX, (E) AMBN, (F) ENAM, (G) KLK4, (H) MMP-20, and (I) ODAM. *p<0.01 vs P; ^#p<0.01 vs NT5. Dotted line, gene expression of LS-8 cells on culture plate.

Figure 6 Expression of amelogenic genes in LS-8 cells cultured on nanostructured Ti surfaces with RA medium pretreatment.

Notes: (A) BMP-2, (B) RUNX2, (C) SHH, (D) AMELX, (E) AMBN, (F) ENAM, (G) KLK4, (H) MMP-20, and (I) ODAM. *p<0.01 vs P; ^#p<0.01 vs NT5. Dotted lines, gene expression of LS-8 cells on culture plate.

Abbreviation: RA, retinoic acid.

Figure 7 Extracellular matrix (ECM) mineralization of LS-8 cells on nanostructured Ti surfaces in standard or RA medium.

Notes: (A) Alkaline phosphatase (ALP) synthesis on modified surfaces in standard medium (upper row) and RA medium (lower row). (B) The formation of mineralized nodules on modified surface in standard medium (left column) and RA medium (right column), with the semi-quantitative analysis (C). Magnification=100×; scale bar=400 μm. *p<0.01.

Abbreviation: RA, retinoic acid.

Figure S1 Elements analysis on nanostructured Ti surfaces via X-ray photoelectron spectroscopy (XPS) scanning.

Abbreviation: CPS, counts per second; ev, electron volt.

Figure S2 Morphology of LS-8 cells cultured on nanostructured Ti surfaces in standard medium (A) and RA medium (B), the arrows indicate pseudopods of LS-8 cells. Magnification=400×; scale bar=25 μm.

Abbreviation: RA, retinoic acid; P, polished Ti surface.

Figure S3 The apoptosis-related genes expression of LS-8 cells on nanostructured Ti surfaces in standard medium and RA medium at 24 and 48 h. (A) BAX, (B) BCL-2, and (C) CASP3. *p<0.01.

Abbreviation: NS, no significance.

Figure S4 The alternation of amelogenic genes expression in LS-8 cells in response to RA medium 48-h pretreatment: (A) amelogenic genes expression on polished Ti surface in presence/absence of RA medium. (B) AMTN expression on nanostructured Ti surfaces in standard medium. (C) AMTN expression on nanostructured Ti surfaces with RA medium pretreatment. *p<0.01.

Abbreviations: EL, extremely low level; RA, retinoic acid.