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

Zhiyong Chen^{1, 2} Yingxian Wei^{3, 4} Likun Liang^{3, 4} Xu Wang^{3, 4} Fangfei Peng^{3, 4} Yiying Liang^{3, 4} Xin Huang^{3, 4} Kaiqi Yan^{3, 4} Yunxia Gao^{3, 4} Kangjing Li^{3, 5} Xiaoman Huang^{3, 4} Xinglu Jiang^{3, 6*} Wenxia Chen^{3, 5*}

• ¹Guangxi Key Laboratory of Oral and Maxillofacial Rehabilitation and Reconstruction, Guangxi Clinical Research Center for Craniofacial Deformity, China
• ²Department of Prosthodontics, College & Hospital of Stomatology, Guangxi Medical University, China
• ³Guangxi Key Laboratory of Oral and Maxillofacial Rehabilitation and Reconstruction, Guangxi Clinical Research Center for Craniofacial Deformity, China
• ⁴College & Hospital of Stomatology, Guangxi Medical University, China
• ⁵Department of Endodontics, College & Hospital of Stomatology, Guangxi Medical University, China
• ⁶Clinical Laboratory Medicine Department, College & Hospital of Stomatology, Guangxi Medical University, China

Objectives: To study the ability of theaflavin-3,3'-digallate (TF3)/ethanol solution to crosslink demineralized dentin collagen, resist collagenase digestion, and explore the potential mechanism.Methods: Fully demineralized dentin blocks were prepared using human third molars that were caries-free. Then, these blocks were randomly allocated into 14 separate groups (n=6), namely control, ethanol, 5 % glutaraldehyde (GA), 12.5, 25, 50, and 100 mg/ml TF3/ethanol solution groups. Each group was further divided into two subgroups based on crosslinking time: 30 and 60 seconds. The efficacy and mechanism of TF3's interaction with dentin type I collagen were predicted through molecular docking. The cross-linking, anti-enzymatic degradation, and biomechanical properties were studied by weight loss, hydroxyproline release, scanning/transmission electron microscopy (SEM/TEM), in situ zymography, surface hardness, thermogravimetric analysis, and swelling ratio. Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy were utilized to explore its mechanisms.Statistical analysis was performed using one and two-way analysis of variance and Tukey's test.Results: TF3/ethanol solution could effectively crosslink demineralized dentin collagen and improve its resistance to collagenase digestion and biomechanical properties (p < 0.05), showing concentration and time dependence. The effect of 25 and 50 mg/ml TF3/ethanol solution was similar to that of 5 % GA, whereas the 100mg/ml TF3/ethanol solution exhibited better performance (p < 0.05). TF3 and dentin type I collagen are mainly cross-linked by hydrogen bonds, and there may be covalent and hydrophobic interactions.TF3 has the capability to efficiently cross-link demineralized dentin collagen, enhancing its resistance to collagenase enzymatic hydrolysis and biomechanical properties within clinically acceptable timeframes (30 s/60 s).Additionally, it exhibits promise in enhancing the longevity of dentin adhesion.