QCMC牙本质纤维外脱矿的疏水性粘接体系构建及机制研究

Hydrolytic degradation of adhesive resin and enzymatic degradation of exposed collagen fibrils within the bonded interface are known as the key factors contributing to bond failure. Based on the size-exclusion characteristic of collagen fibrils, we successfully achieved extrafibrillar demineralization of dentin using polyacrylic acid sodium salt (PAAN) and chitosan respectively. However, due to their low demineralization capacity, operating time required is still impractical for clinic. Compared with chitosan, quaternized carboxymethyl chitosan(QCMC), a derivative of chitosan, possesses better calcium binding ability, due to the presence of carboxyl in chitosan. Currently, the mechanisms of dentinal extrafibrillar demineralization induced by QCMC, as well as the effect of extrafibrillar demineralization on the durability of hybrid layer, have not been clarified. In this proposal, we aim to probe into the mechanism of dentinal extrafibrillar demineralization induced by QCMC, to formulate an original hydrophobic resin adhesive containing graphite oxide，accordingly to establish a dentinal hydrophobic bonding system based on extrafibrillar demineralization. By observing the effect of extrafibrillar demineralization on the activity of endogenous collagenase within the hybrid layer through in situ zymography, analyzing the permeability of resin monomer on hybrid layer with chromatograph and electron microscope, examining the resistance capacity of the system to secondary caries, we aim to evaluate the quality and durability of the hydrophobic hybrid layer of extrafibrillar demineralized dentin induced by QCMC from molecular biology, micromorphology and biomechanics. The success of the proposed hydrophobic bonding system based on extrafibrillar demineralization would benefit clinicians by providing a means for extending the durability of resin-dentin bonds via an original demineralization protocol.

牙本质混合层裸露胶原酶解和树脂水解是临床树脂充填失败的主要原因。课题组前期研究发现壳聚糖等高分子螯合剂可结合纤维间晶体Ca2+，实现牙本质纤维外脱矿。纤维内晶体的保留，能降低混合层酶活性，并可避免纤维网塌陷，利于干粘接条件下树脂单体渗入，减少混合层水残留。然而，壳聚糖受大分子影响，其与纤维间Ca2+配位的能力因物理空间阻碍作用而较弱。羧基的引入能提高壳聚糖的Ca2+结合能力及反应速度，我们推测对壳聚糖进行化学改性，可合成分子量合适且Ca2+配位能力强的QCMC，提高牙本质纤维外脱矿效率。因此，本课题拟分析QCMC与Ca2+的配位点及吸附动力学特征，获取Ca2+吸附性能最优的反应产物，并设计抗菌性疏水树脂粘接剂，建立QCMC纤维外脱矿的牙本质疏水性粘接体系，以原位酶谱法分析混合层内源性酶活性，从微观形态学和生物力学角度评价混合层的质量及稳定性，为该技术的转化应用、提高粘接耐久性提供理论支持。

本项目围绕“牙本质纤维外脱矿的疏水性粘接体系构建及机制研究”展开，主要研究内容为：1）明确壳聚糖致牙本质纤维外脱矿的能力，评估基于壳聚糖脱矿处理的牙本质粘接性能；2）制备羧甲基壳聚糖季铵盐（quaternized carboxymethyl chitosan，QCMC），评估QCMC的Ca2+螯合能力、抗菌性能及生物相容性；3）构建基于QCMC纤维外脱矿的树脂牙本质粘接模型，验证QCMC在保护胶原完整性、提高粘接耐久性中的作用。在项目支持下，我们获得以下结论：①1%壳聚糖（分子量50~190kDa）处理矿化牙本质60 s，可形成厚约150~200 nm的牙本质脱矿层，同时保留牙本质小管内玷污栓。②牙本质经1%壳聚糖处理60s后的干粘接和湿粘接强度与传统磷酸酸蚀湿粘接效果无明显差异，且均高于磷酸干粘接组。③经1%壳聚糖脱矿处理的干粘接和湿粘接样本，混合层水渗透性均低于磷酸酸蚀粘接组，提示纤维外脱矿技术可显著减少混合层残留水分，有利于抑制胶原酶解和树脂水解析出。④通过羧甲基壳聚糖CMC的季铵取代反应，将季铵基团引入CMC葡萄糖单元的C2氨基位点，成功合成了QCMC，季铵取代度约为64.7%~74.8%。⑤QCMC对Ca2+反应的结合常数为8.9×104 M-1，表现出中等强度的离子螯合能力；同时QCMC对变异链球菌及粪肠球菌的最小抑菌浓度均为0.312 mg/mL，具有一定的抑制口腔相关致病菌能力；且对人牙髓细胞表现出良好的生物相容性。⑥基于QCMC脱矿的牙本质干粘接和湿粘接系统可选择性溶解牙本质纤维外矿物，暴露纤维外间隙，获得与磷酸湿粘接相当的即刻微拉伸粘接强度，并在老化处理后始终保持良好的粘接效果。⑦经QCMC纤维外脱矿的牙本质干粘接和湿粘接样本，混合层内相对荧光强度在冷热循环处理前后均低于5.0%，提示QCMC纤维外脱矿处理可显著减少胶原降解，提高混合层胶原结构的稳定性，改善粘接耐久性。⑧采用原子力显微镜联合红外光谱技术，明确QCMC可通过选择性纤维外脱矿，保留牙本质纤维内矿物，改善其机械性能。本项目为改善粘接耐久性、提高树脂牙本质粘接修复远期成功率提供了新策略。