Influence of laminarin on adhesive bond stability, MMP activity, and collagen cross-linking in caries-affected dentin

The long-term stability of resin restorations is limited by the vulnerability of the hybrid layer and activity of matrix metalloproteinases (MMPs) within dentin. Natural collagen crosslinkers enhance the durability of an adhesive interface by inhibiting MMPs and stabilizing collagen. This study aimed to evaluate the dual functional effects of laminarin, which is a polysaccharide derived from brown algae, on the resin-dentin interface by assessing (1) microtensile bond strength (MTBS), (2) MMP-2 activity via molecular docking and in situ zymography, and (3) collagen cross-linking and stability via FTIR spectroscopy in sound (SD) and caries-affected dentin (CAD).

Human molars (n = 64) were used to obtain SD and CAD substrates. Following 35 % phosphoric acid etching, laminarin (3, 4, and 5 wt%) was applied for 30 s or 2 min (n = 16 per group). Distilled water served as the control. To evaluate long-term stability, specimens underwent collagenase aging using Clostridium histolyticum collagenase (Type Ⅰ, 1 mg/mL) for 6 days at 37 °C. Bond strength and leakage before and after collagenase aging were evaluated using microtensile bond strength (MTBS) and nanoleakage tests following resin restoration. Interactions between laminarin, MMP-2, and type I collagen were analyzed via molecular docking. The MMP activity was assessed using in situ zymography, and collagen cross-linking was evaluated using Fourier transform infrared. Cell compatibility was confirmed using the MTT assay using human dental pulp stem cells. Data were analyzed using Two-way ANOVA and Tukey's post-hoc test to evaluate the effects of laminarin concentration and aging, with the significance level set at α= 0.05.

In SD (2 min group), significant main effects were observed for concentration (F = 49.510, p < 0.001) and aging (F = 9.003, p = 0.003). The control group showed a significant MTBS decrease after aging (47.45 ± 3.71 MPa to 38.00 ± 9.62 MPa; p = 0.001), whereas laminarin groups remained stable (p = 0.076-0.659). In CAD, laminarin-treated groups maintained significantly higher MTBS than the control post-aging (p < 0.001). The molecular docking and zymography analyses indicated that laminarin exhibits a dual-functional effect by exerting potential inhibitory interactions with MMP-2 and enhancing collagen cross-linking. The cytotoxicity was low, with an average cell viability rate above 80 %.

Laminarin shows significant potential as a dual-functional natural crosslinker, especially at 4-5 wt% after over 2 min of application time.