Type I collagen alone cannot initiate tissue mineralization. Sodium trimetaphosphate (STMP) is frequently employed as a chemical phosphorylating reagent in the food industry. This study examined the feasibility of using STMP as a functional analog of matrix phosphoproteins for biomimetic remineralization of resin-bonded dentin.
Equilibrium adsorption and desorption studies of STMP were performed using demineralized dentin powder (DDP). Interaction between STMP and DDP was examined using Fourier transform-infrared spectroscopy. Based on those results, a bio-inspired mineralization scheme was developed for chemical phosphorylation of acid-etched dentin with STMP, followed by infiltration of the STMP-treated collagen matrix with two etch-and-rinse adhesives. Resin–dentin interfaces were remineralized in a Portland cement-simulated body fluid system, with or without the use of polyacrylic acid (PAA) as a dual biomimetic analog. Remineralized resin–dentin interfaces were examined unstained using transmission electron microscopy.
Analysis of saturation binding curves revealed the presence of irreversible phosphate group binding sites on the surface of the DDP. FT-IR provided additional evidence of chemical interaction between STMP and DDP, with increased in the peak intensities of the P O and P–O–C stretching modes. Those peaks returned to their original intensities after alkaline phosphatase treatment. Evidence of intrafibrillar apatite formation could be seen in incompletely resin-infiltrated, STMP-phosphorylated collagen matrices only when PAA was present in the SBF.
These results reinforce the importance of PAA for sequestration of amorphous calcium phosphate nanoprecursors in the biomimetic remineralization scheme. They also highlight the role of STMP as a templating analog of dentin matrix phosphoproteins for inducing intrafibrillar remineralization of apatite nanocrystals within the collagen matrix of incompletely resin-infiltrated dentin.
Dental caries is a chronic infection in which hard tooth structures (enamel, dentin and cementum) are progressively broken down by bacteria, producing dental cavities. Tooth-colored resin-based composites have found increasing popularity for restoring dental cavities. The use of acid etching prior to the application of resin adhesives is a prerequisite for enhancing the mechanical retention of resin composites. However, exposed collagen fibrils remain within the bonded interface as a result of the discrepancy between the depths of acid demineralization and resin infiltration . These naked collagen fibrils are prone to hydrolysis after water sorption . Activation of endogenous matrix metalloproteinases (MMP) further results in breakdown of incompletely resin-infiltrated collagen matrices and degradation of resin–dentin bonds .
As one of the most intriguing processes in nature , biomineralization is essential for the formation of hard tissues like bone and teeth. Dentin is a mineralized collagenous tissue formed by matrix-mediated mechanisms . Collagen fibrils encapsulated by apatites in natural mineralized dentin do not degrade over time . As contemporary tissue engineering technologies are not yet capable of creating artificial dentin, remineralization of incompletely resin-infiltrated dentin collagen matrices appears to be the logical approach for extending the longevity of resin–dentin bonds. Type I collagen, accounting for 90% of the total protein content in the organic matrix of dentin , provides the framework and spatial constraint for ordered crystal deposition . However, in the absence of apatite seed crystallites, collagen by itself does not possess the thermodynamic and kinetic mechanisms for induction of apatite nucleation . Acidic non-collagenous proteins (NCPs) promote nucleation and post-nucleation growth of apatite crystallites within and around the gap zones of collagen fibrils and are essential for regulation of matrix mineralization .
Immobilization of protein molecules on intact collagen substrates promotes apatite nucleation because such molecules possess multiple polyanionic functional groups that act as favored sites for homogeneous nucleation . The inductive effect of phosphoproteins on the formation of hydroxyapatite is a well-known example . Phosphophoryn, the major NCP present in dentin, has been shown to bind calcium ions with high affinity after its highly phosphorylated post-translational modification . Grafting of molecules containing phosphate groups to polymeric biomaterials with limited or no mineralizing tendency has been reported as a successful procedure for converting those substrates into mineralized moieties . Binding of biomimetic molecules containing multiple phosphate groups to collagen fibrils produces phosphorylated and negatively charged surfaces that attract calcium ions by electrostatic interaction. The phosphorylated collagen serves as a scaffold for homogeneous nucleation of apatite crystallites . This biomimetic process may, in part, recapitulate the function of phosphate groups in naturally occurring phosphoproteins.
Trimetaphosphate (P 3 m) derived from volcanic eruptions has a cyclic structure that contains three phosphorus atoms in a six-membered ring. It plays an important functional link in prebiotic chemistry due to its purported roles as a phosphorylation reagent for bioorganic compounds and a coupling reagent for oligomerization of amino acids and nucleosides . Among its various salt forms, sodium trimetaphosphate (STMP, Na 3 P 3 O 9 ; Fig. 1 a ) is commonly used in the food industry as a chemical phosphorylation reagent . At alkaline pH, STMP can chemically react with the hydroxyl groups on proteins, thereby introducing phosphate functional groups to protein molecules . Although there are ample examples of the application of STMP as an effective phosphorylation agent for edible plant proteins in the food industry, few studies have been conducted on its use on Type I collagen, the major structural protein in bone and dentin. As a result, there is very limited understanding of the interactions between STMP and collagen and particularly its adsorption characteristics. A recent study that utilized STMP as a chemical phosphorylation agent for bovine collagen demonstrated extrafibrillar deposition of large spherical hydroxyapatite clusters around STMP-phosphorylated collagen matrices after the latter were incubated in a simulated body fluid (SBF) . In that study, there was no sequestering mechanism, such as the use of polyaspartic acid or polyacrylic acid, for rendering the formation of apatite in a nanoscale . The apatite clusters created in that study were large and could not fit inside collagen fibrils . More importantly, in the absence of an amorphous calcium phosphate sequestering mechanism, the exact role played by STMP in inducing homogeneous nucleation of apatite within collagen fibrils (i.e. intrafibrillar mineralization) is unknown. While the binding of large apatite spherical clusters via STMP to collagen may produce composite materials for filling of bone defects which can eventually be resorbed and replaced by new bone generated by osteocytes, those materials cannot be used as a restorative material to replace dentin, as the latter is a non-regenerable substrate produced by terminally differentiated odontoblasts. Thus, the use of STMP as a chemical phosphorylation agent for inducing in situ mineralization of collagen fibrils appears to be a more pragmatic approach. However, in the absence of adjunctive polycarboxylic acid analogs as sequestering agents, the use of STMP alone as a collagen phosphorylaton agent has limited potential for in situ remineralization of dentin in which the occurrence of intrafibrillar mineralization is perceived to be responsible for its biomechanical properties . As the concentration of STMP employed for phosphorylating collagen has not been optimized in the previous study , examining the interaction between collagen and STMP and determining its optimal binding concentration will bring to fruition the more efficient use of STMP as a chemical phosphorylation agent for remineralization of dentin collagen matrices in the presence of additional biomimetic analogs with sequestering function.