Effect of Different Crosslinkers on Denatured Dentin Collagen’s Biostability, MMP Inhibition and Mechanical Properties

Abstract

Objective: Sound, natural dentin collagen can be stabilized against enzymatic degradation through exogenous crosslinking treatment for durable bonding; however, the effect on denatured dentin (DD) collagen is unknown. Hence, the ability of different crosslinkers to enhance/restore the properties of DD collagen was assessed. Methods: Demineralized natural and DD collagen films (7 mm × 7 mm × 7 µm) and beams (0.8 mm × 0.8 mm × 7 mm) were prepared. DD collagen was experimentally produced by heat or acid exposure, which was then assessed by various techniques. All specimens were then treated with 1 wt% of chemical crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/n-hydroxysuccinimide (EDC/NHS) and two structurally different flavonoids—theaflavins (TF) from black tea and type-A proanthocyanidins from cranberry juice (CR) for either 30 s or 1 h. The controls were untreated. Dentin films were assessed for chemical interaction and cross-linking effect by FTIR, biostability against exogenous collagenase by weight loss (WL) and hydroxyproline release (HYP), and endogenous matrix metalloproteinases (MMPs) activity by confocal laser microscopy. Dentin beams were evaluated for tensile properties. Data were analyzed using ANOVA and Tukey’s test (α = 0.05). Results: Compared with natural collagen, DD collagen showed pronounced structural changes, altered biostability and decreased mechanical properties, which were then improved to various degrees that were dependent on the crosslinkers used, with EDC/NHS being the least effective. Surprisingly, the well-known MMP inhibitor EDC/NHS showed negligible effect on or even increased MMP activity in DD collagen. As compared with control, cross-linking induced by TF and CR significantly increased collagen biostability (reduced WL and HYP release, p < 0.05), MMP inhibition (p < 0.001) and mechanical properties (p < 0.05), regardless of denaturation. Conclusions: DD collagen cannot or can only minimally be stabilized via EDC/NHS crosslinking; however, the challenging substrate of DD collagen can be enhanced or restored using the promising flavonoids TF and CR.

Theaflavin −3,3'-digallate/ethanol: a novel cross-linker for stabilizing dentin collagen

“…As depicted in Figure 7B , the characteristic peaks at 1,672, 1,530, and 1,234 cm −1 are the typical characteristic peaks of the three-dimensional spiral structure of collagen fibers ( Xu and Wang, 2012 ). These peaks were associated with the amide I band, amide II band, and amide III band, respectively, and exhibited sensitivity to the molecular conformation of the polypeptide chain, aligning with the results of infrared spectroscopy ( Antonakos et al, 2007 ; Nisar et al, 2023 ). The peak at 1,450 cm −1 and 2,930 cm −1 referred to the C-H bond, reflecting the organic components present in demineralized dentin ( Lazar et al, 2005 ).…”

“…Figure 7D is the N 1s fine spectrum. The peaks at 399.2 and 399.7 eV are associated with N-H and C-N bonds, respectively (Wang M. et al, 2023). The peak position of the ethanol group was close to that of the control group, indicating that the chemical structure of the demineralized dentin collagen was not influenced by the ethanol.…”

“…In the TF group, the peaks of N-H bond and C-N bond shifted significantly in the direction of high binding energy, indicating that TF3 could significantly change the chemical environment of demineralized dentin and affect its molecular structure. (Wang M. et al, 2023). The chemical state of O in different groups was also significantly different: the peak position of ethanol was close to that of control, indicating that ethanol did not change the chemical structure of demineralized dentin.…”

“…The absorption peak at about 1,450 cm −1 is caused by the shear vibration absorption of -CH2. At 1,235 cm −1 , the combination of C-N vibration and N-H bending vibration represents the amide III band ( Antonakos et al, 2007 ; Nisar et al, 2023 ). Compared with the untreated demineralized dentin collagen control, TF3 caused significant changes, including the absorption band of OH at 3,280 cm −1 at 30 s and 60 s of 100 mg/ml TF3 treatment became wider and the intensity increased.…”

“…Figure 7C is the C 1s fine spectrum. The peaks at 284.8 eV, 285.9 eV, and 287.1 eV represent C-C/C=C, C-N, and C=N/C=O, respectively (Wang M. et al, 2023). The binding state of C element in different groups was diverse: in ethanol and GA groups, the binding energy of C element was relatively low, indicating that the interaction between C atom and other elements was weak; in the TF3 group, the binding energy of C element was higher, indicating that the interaction between C atom and other elements was stronger.…”