Studies Of Guided Tissue Remineralization Of The Hybrid Layer Created In Caries-affected Dentin: Mechanism And Application

Objectives:Over the past few decades, scientific developments in dental materials and bond technology have changed dentistry's approach to manage carious teeth. The hallmark of minimally invasive dentistry (MID) is conservative treatment of caries to preserve their potential for remineralization. Therefore, the clinical bonding substrate is likely to be a combination of normal dentin and caries-affected dentin after caries excavation. It has been shown that bond strengths to caries-affected dentin are significantly lower than those to normal dentin and the hybrid layer created in caries-affected dentin is thicker and more porous compared with that in normal dentin. This may be the result of the changed structures of caries-affected dentin, such as occluded dentin tubules and decreased hardness. However, durability studies on bond strengths to caries-affected dentin are still limited. Our previous studies have shown that the poor-infiltrated hybrid layer (5-8μm) has been successfully remineralized via guided tissue remineralization (GTR), which restores the enzyme exclusion and fossilization properties of naturally mineralized dentin. This proof-of-concept strategy is capable of preserving the longevity of resin-dentin bonds. It has been proved that the collagen matrix of caries-affected dentin demonstrates intermolecular crosslinks and normal cross-banding. And therefore, caries-affected dentin is physiologically remineralizable. As caries-affected dentin can extend several hundred microns below the excavated surface, our objectives in this study is to test the effect of GTR on remineralizing caries-affected dentin and the hybrid layer created in caries-affected dentin. Materials and Methods:(1) A single-layer of typeⅠcollagen fibrils was reconstituted over formvar-andcarbon-coated 400-mesh Ni TEM grids and cross-linked with 0.3 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/0.06M N-hydroxysuccinimide (NHS). Thereafter, half of TEM grids containing cross-linked collagen fibrils were phosphorylated with 0.313-2.5 wt% hydrolyzed sodium trimetaphosphate (STMP) or sodium tripolyphosphate (TPP) for 5 min. Remineralization was performed initially for 4-24 h to identify the optimal STMP/TPP concentration for further remineralization for 48-72 h. They were floated upside-down over 30μL GTR remineralizing medium containing simulated body fluid (SBF)/Portland Cement with polyacrylic acid (PAA) as a sequestration analog. The templating analog control consisted of STMP/TPP-treated collagen and SBF (no sequestration analog). The sequestration analog control consisted of reconstituted collagen and PAA-containing SBF (no templating analog). The collagen negative control (no sequestration and templating analogs) consisted of reconstituted collagen and SBF. Grids were examined unstained using transmission electron microscopy (TEM) and selected area electron diffraction (SAED) at 110 kV.(2) Thirty extracted non-carious human third molars were obtained with patient-informed consent under a protocol approved by the Human Assurance Committee of the Medical College of Georgia. A 1 mm thick dentin disk was prepared by making two parallel cuts perpendicular to the longitudinal axis of each tooth using a low-speed Isomet saw under water cooling. A 250-300μm thick layer of partially demineralized dentin was created by pH-cycling procedure to mimic caries- affected dentin below the excavated surface. The samples were then randomly divided into two groups:half of them were remineralized using a classical top-down approach and the rest using non-classical bottom-up approach. Micro-computed tomography (μCT) and TEM were employed to examine mineral uptake and apatite arrangement within the mineralized collagen matrix.(3) Artificial carious lesions with lesion depths of 300±30μm were created by pH-cycling.2.5% hydrolyzed STMP was applied to the artificial carious lesions to phosphorylate the partially-demineralized collagen matrix. Half of the STMP-treated specimens were bonded with One-Step. The adhesive and non-adhesive infiltrated specimens were remineralized in a Portland cement-SBF system containing PAA to stabilize amorphous calcium phosphate (ACP) as nanoprecursors.μCT and TEM were used to evaluate the results of remineralization after a 4-month period.Results:(1) In the collagen negative control, there was no remineralization. And ACP phases were bigger than those in the sequestration analog control. In the sequestration analog control,1000μg/mL PAA was included in the remineralization medium but did not treat the reconstituted collagen with STMP or TPP.(2) For the templating analog control, no sequestration analog was included in the remineralization medium and needle-shaped apatite was randomly precipitated over the surface of collagen fibrils after 72 h. Cross-banding patterns produced by discrete apatite crystallites could be identified only from partially-mineralized collagen fibrils in presence of both templating and sequestration analog.(3) In the view of mineral uptake, the top-down approach which relied on the mineral density could remineralize only the base of the partially demineralized dentin. Conversely, the entire partially demineralized dentin, including apatite-depleted collagen fibrils, can be completely remineralized by the bottom-up approach.(4) In absence of PAA and STMP as biomimetic analogs (control groups), there was no remineralization irrespective of whether the lesions were infiltrated with adhesive. From ultrastructure, extrafibrillar remineralization was predominantly observed in the top-down approach, while the bottom-up approach showed evidence of both intrafibrillar and extrafibrillar remineralization.(5) For the STMP-treated experimental groups immersed in PAA-containing SBF, specimens without adhesive infiltration were more heavily remineralized than those infiltrated with adhesive. Statistical analysis of the 4-monthμCT data revealed significant differences in the lesion depth, relative mineral content along the lesion surface and changes in the intergrated mineral loss (AZ) between the non-adhesive and adhesive experimental groups for all the three parameters.(6) TEM examination indicated that collagen degradation occurred in both the non-adhesive and adhesive control and experimental groups after 4 months of remineralization. And even the denatured collagen fibrils could be remineralized via GTR.Conclusions:There is no difference between STMP and TPP in templating hierarchical intrafibrillar apatite assembly in reconstituted collagen via GTR. The ability of using simple non-protein molecules to reproduce different levels of structural hierarchy in mineralized collagen fibrils indicates the ultimate simplicity in Nature's biomineralization design principles. And this challenges the need for using more complex recombinant matrix proteins in bioengineering applications. The mechanisms involved in the classical top-down and non-classical bottom-up remineralization approaches appear to be different. The former probably relies on epitaxial growth of seed crystallites within the collagen fibrils. The latter involves transformation of ACP nanoparticles into apatite crystallites in the presence of biomimetic analogs. GTR using STMP as a biomimetic ananlog is a promising method to remineralize artificial carious lesions particularly in areas devoid of seed crystallites. Future studies should consider remineralizing the real caries-affected and caries-infected dentin via GTR. During remineralization process, MMP-inhibitors should be incorporated within the partially-demineralized collagen matrix to prevent collagen degradation.