The Study On Biomodification Of Demineralized Dentin Matrix With Nordihydroguaiaretic Acid

Objective:The structural integrity of demineralized dentin matrix is essential to successful dentin remineralization as well as durable dentin-resin bonding.However,after demineralization,dentin collagen fibers are susceptible to degradation by exogenous collagenases and endogenous,activated,dentin-bound enzymes,such as matrix metalloproteinases(MMPs)and cysteine cathepsins(CCs).Crosslinking treatment has been documented as an effective strategy to preserve the structural integrity of demineralized dentin matrix by improving its mechanical properties and resistance to collagenase degradation.In this in vitro study,we firstly investigated the feasibility of using Nordihydroguaiaretic acid(NDGA),a natural di-catechol antioxidant from the creosote bush,as a dentin collagen crosslinker to biomodify the demineralized dentin matrix.Methods:Glutaraldehyde(GA),an efficient collagen crosslinker with known mechanism,was used for comparison.The resistance of crosslinked dentin matrix to exogenous Type I collagenase was characterized by dry mass loss,hydroxyproline release,the stability of apparent modulus of elasticity,scanning electron microscopy(SEM)and transmission electron microscopy(TEM),subsequently.In terms of biomechanical property,we applied swelling ratio and atomic force microscopy(AFM),respectively.In order to verify the mechanism underlying this,ninhydrin assay and fourier transform infrared spectroscopy(FTIR)were conducted,accordingly.Results:This investigation indicated that crosslinking with 20mg/ml NDGA for 5min significantly enhanced the tolerance of demineralized dentin matrix to collagenase degradation,reducing the loss of dry mass and hydroxyproline release as well as preserving the structural integrity of dentin collagen scaffold.With regard to ameliorating mechanical properties of demineralized dentin matrix,NDGA was capable of remarkably reducing the swelling ratio and augmenting the elastic modulus at both macroscale and nanoscale.Moreover,the application of NDGA biomodification did not destroy the triple helical structure of dentin collagen molecules.It was considered that the enhanced collagenase resistance of NDGA-crosslinked demineralized dentin matrix was attributed to decreased water content in the matrix,improved stiffness of collagen fibrils,physical masking of collagenase binding sites on collagen and limited mobility of collagenase molecules.Distinguished from GA crosslinking,the mechanism underlying dentin collagen crosslinking induced by NDGA was related with mussel inspired oxidation of ortho-catechol groups and oxidative coupling of ortho-quinone groups.Additionally,hydrogen bonds were probably involved between ortho-catechol groups and collagen molecules,which deserved more profound research.Conclusion:Accordingly,this investigation revealed the applicability and efficiency of NDGA as a dentin collagen crosslinker to improve biodegradation resistance and biomechanical properties.To our best knowledge,this is the first investigation concerning dentin collagen crosslinking with NDGA,which not only endows excellent evidence for mussel biomimetic chemistry in the protection of demineralized dentin matrix but also provides promising prospects for satisfactory dentin remineralization and durable dentin-resin bonding in the clinic.