In Vitro Study Of Human Enamel Biomimetic Remineralization Controlled By The Nacre Water Soluble Matrix From Perna Viridis

Dental enamel, which lies on the outermost layer of teeth, is the hardest mineralizedtissue in the human body. Enamel is composed of highly organized polymer/mineralcomposite, in which there is more than97%hydroxyapatite [HAP, Ca10(PO4)6(OH)2]crystals by weight. This structure provides enamel increased hardness, strength andanti-abrasive properties compared to monolith HAP. The prevailing theory is that theameloblasts secrete amelogenin, a major enamel protein constituting approximately90%ofall organic matrix material in developing enamel, and this protein plays a vital role inenabling crystallites to form a well-organized prism pattern. However, due to the acellularand protein-free composition of mature enamel, after the substantial mineral loss theregeneration can be hardly observed. Thus, many researchers study mineralized tissues inorder to gain inspiration for developing the approach to synthesize enamel prism-likestructure.Nacre, also known as mother-of-pearl, occurs in the inner layer of mollusc shells. Nacreis a natural composite material consisting of more than95wt%of calcium carbonate in thearagonite crystal form and1-5wt%of organic matter. The organic constituent can beseparated into two matrices: water-soluble (WSM) and insoluble. Many researchers havestudied the biomineralization of nacre with a particular focus on the WSM. It isdemonstrated that WSM can serve as a template for calcium carbonate crystallization andinfluence crystal size and crystal type. Nacre has outstanding mechanical properties, whichwere shown to be comparable to those of titanium. The fracture toughness of nacre is3000times greater than that of pure aragonite.Nacre and dental enamel have a number of similarities. Both nacre and enamel can beregarded as biogenic composite materials containing biological polymers and inorganiccrystals. Nacre has an organic component performing a function similar to that ofamelogenin present in enamel. Amelogenin serves as a template for HAP crystallization andcontrols the formation of hierarchical organized minerals, while the WSM of nacre serves asa template for calcium carbonate crystallization and influence crystal size and crystal type.Although the mineral phase of human enamel is calcium phosphate in the HAP form, whereas nacre is calcium carbonate in the aragonite form, this difference can be related tothe different environments surrounding sea shells and humans. Previous work has shownthat nacre can convert completely to HAP in phosphate buffer solution at room temperature,and can initiate bone formation both in vivo and in vitro. Hence, these similarities makenacre a promising template for enamel biomimetic remineralization though enamel andnacre differ in mineral composition and structure.Based on previous findings, we further implemented a modified biomimeticmineralization approach to rebuild the enamel structure on an acid etched enamel surface asan early caries model. Using a biomimetic mineralization method, we found that WSM had asignificant effect on the HAP crystal morphology in the remineralization of enamel crystals.The needle-shaped nanocrystals grew on acid etched enamel. In this report, we investigatethe effect of WSM on the HAP crystal growth on enamel, and demonstrate the successfulpreparation of bio-composite coating when oriented nano-sized apatite mineralization wasmediated by the presence of WSM. The present biomimetic synthesis is one of the primarysteps towards the development and design of novel biomaterial for future application inreparative and restorative dentistry.Objective: The aim of this study was to investigate the effects of nacre WSM on thecrystal morphology and organization of in vitro remineralized human enamel.Methods: Extracted human premolars were sliced thin and acid-etched to provide thedemineralized enamel surface. We extracted WSM from nacre of Perna viridis and utilized amodified biomimetic deposition method to remineralize the surface of demineralized enamel.The effect of nacre WSM on the crystal morphology and mineral phase of the remineralizedcoating were analyzed by scanning electron microscopy (SEM), atomic force microscope(AFM), attenuated total reflection-Fourier transformed infrared (ATR-FTIR), X-raydiffraction (XRD) and Vickers micro-hardness tester.Results: FE-SEM and AFM showed a layer covered with densely oriented prism-likecrystals. XRD indicated that the mineral phase of the crystal be an HAP phase. FTIR showedthat phosphate (PO4) bands appeared after remineralization. There were statisticallysignificant differences (p<0.05) among roughness and micro-hardness before and after enamel remineralization. However, remineralization with WSM increased micro-hardnessand induce the roughness (p<0.05), approximate to normal enamel.Conclusions: The results indicated that nacre WSM promoted the formation ofenamel-like HAP, which arrayed densely oriented. This study provides the basis for thedevelopment of novel dental materials for future application in reparative and restorativedentistry.