Theaflavins as a novel cross-linker quickly stabilize demineralized dentin collagen against degradation

Abstract

To investigate the ability of theaflavins (TF) from black tea to protect dentin collagen against enzymatic degradation via cross-linking effect under clinically relevant conditions. 10-µm-thick dentin films were microtomed from dentin slabs of human molars. Following demineralization, films or slabs were treated with TF at two concentrations (0.4% and 2%) for 30 s. A well-known collagen cross-linker grape seed proanthocyanidins (PA) was used as control. Collagen cross-linking interactions and stabilization against enzymatic degradation were investigated by Fourier transform infrared spectroscopy, weight loss, hydroxyproline release, and scanning/transmission electron microscopy. Data were analyzed by ANOVA, Tukey's and Student's T test (α = 0.05%). The results showed collagen cross-linking and stabilization efficacy was dependent on TF/PA concentrations. At 2.0%, TF and PA offered nearly full protection to collagen; at 0.4%, TF exhibited a significantly better collagen stabilization effect than PA (P < 0.05), while untreated collagen was completely digested. It's concluded that TF cross-links dentin collagen within a clinically relevant time (30 s) and offers excellent collagen protection against enzymatic degradation, with efficacy comparable to or better than PA. The study supports the potential use of TF as a novel, promising collagen cross-linker for degradation resistant, long-lasting dentin bonding in composite restorations.

Figure 1

Schematic diagrams of molecular structures of theaflavins (TF) from black tea (a) and proanthocyanidins (PA) from grape seed (b); representative FTIR spectra of TF and PA powders (c).

Figure 2

(a) Representative FTIR spectra of dentin collagen before (black line) and after the treatment of either 0.4% TF (red line) or 2.0% TF (blue line) for 30 s. Difference spectra of treated collagen films with the spectrum of untreated collagen subtracted (dashed lines) are shown in the middle and the spectrum of TF powder (green line) is shown in the bottom for comparison. Inset: the band ratios of A1145/A1235 for 0.4% TF and 2.0% TF treated collagen (*P < 0.001, n = 6). (b) Representative FTIR spectra of dentin collagen before (black line) and after the treatment of either 0.4% PA (red line) or 2.0% PA (blue line) for 30 s. Difference spectra of treated collagen films with the spectrum of untreated collagen subtracted (dashed lines) are shown in the middle and the spectrum of PA powder (green line) is shown in the bottom for comparison. Inset: the band ratios of A1445/A1235 for 0.4% PA and 2.0% PA treated collagen (*P < 0.001, n = 6).

Figure 3

Means and standard-deviations of dentin collagen resistance against collagenase digestion measured by (A) weight loss (%) and (B) hydroxyproline proline release (µg/mg film). Different lowercase letters demonstrate statistically significant differences among groups (n = 6, P < 0.05).

Figure 4

Secondary-electron (SE, left) and back-scattered electron (BSE, right) SEM images of acid-etched dentin without treatment (a) before and (b) after digestion (in 0.1% collagenase at 37 °C for 1 h); treated with 2.0% TF for 30 s followed by digestion (c); treated with 2.0% PA for 30 s followed by digestion (d); treated with 0.4% TF for 30 s followed by digestion (e); and treated with 0.4% PA for 30 s followed by digestion (f). The demineralized dentin layer of untreated control was evident as dark layer atop intact dentin under BSE mode before digestion (a), and was completely lost after digestion (b), while that of all treated groups (c–f) remained after digestion. DD demineralized dentin. ID intact dentin. T tubule.

Figure 5

TEM images of acid-etched dentin without treatment (a) before and (b) after digestion (in 0.1% collagenase at 37 °C for 1 h); treated with 2.0% TF for 30 s followed by digestion (c); treated with 2.0% PA for 30 s followed by digestion (d); treated with 0.4% TF for 30 s followed by digestion (e); and treated with 0.4% PA for 30 s followed digestion (f). Representative high magnification view of the demineralized dentin (DD) layer shown as inset (a–d); high magnification view of the bottom region (white arrow position) in DD layer (e1 and f1); high magnification view of the top region (black arrow position) in DD layer (e2 and f2). DD: demineralized dentin. ID intact dentin. T tubule.