Effects of C-xylopyranoside derivative on epithelial regeneration in an in vitro 3D oral mucosa model

Figures & data

Fig. 1. Diagram showing the structure of β-D-xylopyranoside-n-propane-2-one (XPP)

Fig. 2. Viability measurement of oral keratinocytes and fibroblasts in a monolayer culture supplemented with 0 (control), 2, or 10 mM XPP.

Notes: Viability of oral keratinocytes cultured in EpiLife^® with (A) 0.06 mM Ca²⁺ and (B) 1.2 mM Ca²⁺, and (C) oral fibroblasts cultured in 10% FBS containing DMEM for 24 and 48 h determined by Cell Counting Kit-8 (n = 5).

Fig. 3. Measurement of sulfated-glycosaminoglycans (s-GAG) of oral keratinocytes and fibroblasts in a monolayer culture supplemented with 0 (control), 2, or 10 mM XPP.

Notes: Total amount of s-GAG in the culture medium secreted from oral keratinocytes cultured in EpiLife^® with (A) 0.06 mM Ca²⁺ and (B) 1.2 mM Ca²⁺, and (C) oral fibroblasts cultured in 10% FBS containing DMEM for 24 and 48 h, determined using a Blyscan assay kit (n = 5). Asterisks represent statistically significant differences compared with untreated cells (*; p < 0.05). The line in the middle of the box represents the median. The box shows all values between the 25% and 75% percentiles, referred to as the “interquartile range”. Whiskers indicate the minimum and maximum data values.

Fig. 4. Effect of XPP on epithelial regeneration in an in vitro 3D oral mucosa model (3DOMM).

Notes: (A) Histology of 3DOMMs treated with 0 (control), 2, or 10 mM XPP stained with hematoxylin and eosin. Bidirectional arrow indicates the epithelial thickness measured on this section. Original magnification 40×, scale bar = 50 μm. (B) Epithelial thickness of 3DOMMs shown by box plot. Values shown at the top of the boxes indicate mean ± SD (μm) of 11 independent experiments.

Fig. 5. Measurement of sulfated-glycosaminoglycans (s-GAG) in EpiLife^® with 1.2 mM Ca²⁺ of 3DOMMs treated with 0 (control), 2 and 10 mM XPP. (n = 11).

Notes: Asterisks indicate statistically significant differences compared with untreated cells (**; p < 0.01).

Fig. 6. Electrophoresis of GAGs on cellulose acetate membrane.

Notes: (A) Cell-associated GAGs from cells cultured with or without XPP. (B) cell-associated GAGs from cells cultured with 2 mM XPP. Untreated, Ch ABC lyase, Hep lyases, and HA lyase indicate untreated or treated with the indicated lyases. C, GAGs in the culture supernatant of the cells cultured with or without XPP. Lines indicate origins of electrophoresis. Indicated upper/lower positions of standards (Ch4S, Ch, Hep, DS, Ch6S, HA, and HS) below the electrophoresis images are the same as the positions of the bands.

Fig. 7. HPLC analysis of unsaturated disaccharides derived from cell-associated GAGs after lyase treatment.

Notes: A–D were without XPP and E–H were with 2 mM XPP. A–H were after treatment with Ch ABC lyase (A, E); Ch B lyase (B, F); Hep lyase II and Hep lyase III (C, G) and HA lyase (D, H). Arrows indicate the elution positions of standard unsaturated disaccharides with known structures: ΔDi-0S, ΔGlcUAβ1–3GalNAc; ΔDi-6S, ΔGlcUAβ1–3GalNAc(6S); ΔDi-4S, ΔGlcUAβ1–3GalNAc(4S); ΔDi-diS_D, ΔGlcUA(2S)β1–3GalNAc(6S); ΔDi-diS_E, ΔGlcUAβ1–3GalNAc(4S, 6S); ΔDi-diS_B, ΔGlcUA(2S)β1–3GalNAc(4S); ΔDiHS-0S, ΔGlcUAα1-4GlcNAc; ΔDiHS-NS, ΔGlcUAα1-4GlcNS; ΔDiHS-6S, ΔGlcUAα1-4GlcNAc(6S); and ΔDiHS-diS₂, ΔGlcUA(2S)α1-4GlcNS. 2S, 4S, 6S, and NS, represent 2-O-sulfate, 4-O-sulfate, 6-O-sulfate, and N-sulfate, respectively. The dashed lines indicate the NaH₂PO₄ gradient line.

Table 1 Unsaturated disaccharide compositions of cell-associated ChS chains.

	Unsaturated disaccharide (%)
Disaccharide structure	XPP (−)	2 mM XPP
ΔDi-0S^a	3.60	16.3
ΔDi-6S	21.0	25.1
ΔDi-4S	72.8	55.9
ΔDi-diSD	0.42	0.58
ΔDi-diSE	2.18	2.13
ΔDi-triS	N.D.^b	N.D.

Notes: Unsaturated disaccharide compositions of ChSs were determined by HPLC on a YMC-Pack Polyamine II column after treatment with Ch ABC lyase. The results are expressed as detected unsaturated disaccharide percentages of total unsaturated disaccharides.

^a ΔDi-0S, ΔGlcUA-β1–3GalNAc; ΔDi-6S, ΔGlcUAβ1–3GalNAc(6S); ΔDi-4S, ΔGlcUA β1–3GalNAc(4S); ΔDi-diS_D, ΔGlcUA(2S) β1–3GalNAc(6S); ΔDi-diS_E, ΔGlcUAβ1–3GalNAc(4S, 6S); ΔDi-triS, ΔGlcUA(2S)β1–3GalNAc(4S, 6S); and 2S, 4S, and 6S, represent 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively.

^b not detected.

Table 2. Unsaturated disaccharide compositions of cell-associated HS/Hep chains.

	Unsaturated disaccharide (%)
Disaccharide structure	XPP (−)	2 mM XPP
ΔDiHS-0S^a	76.9	74.1
ΔDiHS-NS	8.39	7.07
ΔDiHS-6S	13.9	18.1
ΔDiHS-diS1	N.D.^b	N.D.
ΔDiHS-diS2	0.82	0.78
ΔDiHS-triS	N.D.	N.D.

Notes: Unsaturated disaccharide compositions of HSs/Heps were determined by HPLC on a YMC-Pack Polyamine II column after treatment with Hep lyase II and Hep lyase III. The results are expressed as detected unsaturated disaccharide percentages of total unsaturated disaccharides.

^a ΔDiHS-0S, ΔGlcUAα1-4GlcNAc; ΔDiHS-NS, ΔGlcUAα1-4GlcNS; ΔDiHS-6S, ΔGlcUAα1-4GlcNAc(6S); ΔDiHS-diS₁, ΔGlcUAα1-4GlcNS(6S); ΔDiHS-diS₂, ΔGlcUA(2S)α1-4GlcNS; ΔDiHS-triS, ΔGlcUA(2S)α1-4GlcNS(6S); and 2S, 4S, 6S, and NS, represent 2-O-sulfate, 4-O-sulfate, 6-O-sulfate, and N-sulfate, respectively.

^b not detected.

Fig. 8. Expression of BMZ marker proteins. Immunoblotting detection of type IV collagen, laminin-5, nidogen-2, integrin α6, and integrin β1 in 3DOMMs treated with 0, 2, or 10 mM XPP.

Notes: Results shown are representative of four separate experiments. β-Actin is shown as a loading control. Values below the panels indicate the relative intensity of type IV collagen, laminin-5, nidogen-2, integrin α6, integrin β1 bands, which are normalized to the intensity of the bands of untreated 3DOMMs. Values with a >50% increase compared with a control are shown in italic bold.

Fig. 9. Expression of PGs. Immunoblotting detection of decorin, syndecan-1, and CD44 in 3DOMMs treated with 0, 2, or 10 mM XPP.

Notes: Results shown are representative of four separate experiments. β-Actin is shown as a loading control. Values below the panels indicate the relative intensity of decorin, syndecan-1, and CD44 bands, which are normalized to the intensity of the bands of untreated 3DOMMs. Values with a >50% increase compared with the control are shown in italic bold.

Fig. 10. Expression of Akt/mTOR signaling substrates. Immunoblotting detection of p-Akt, Akt, p-S6K, S6K, p-S6 and S6 in 3DOMMs treated with 0, 2, or 10 mM XPP.

Notes: Results shown are representative of four separate experiments. β-Actin is shown as a loading control. Values below the panels indicate the relative intensity of p-Akt, Akt, p-S6K, S6K, p-S6 and S6 bands, which are normalized to the intensity of the bands of untreated 3DOMMs. Values with a >50% increase compared with the control are shown in bold.

Fig. 11. Expression and immunolocalization of proteins used in immunoblot analysis in 3DOMMs treated with 0, 2, or 10 mM XPP.

Notes: (A) Immunostaining of type IV collagen. (B) Immunostaining of laminin. (C) Immunoreaction of integrin α6. (D) Immunoreaction of integrin β1. (E) Immunoreaction of syndecan-1. (F) Immunoreaction of CD44. (G) Immunostaining of p-S6. (H) Immunostaining of S6. Original magnification 40×, scale bar = 50 μm.