The Effect Of Glutaraldehyde On Biomimetic Dentin Remineralization

BackgroundDentin caries is still one of the common diseases in clinical.The mineralized dentin collagen is the basic structural unit of dentin.The happening of dentin caries is a complex process that destructive demineralization along with reparative mineralization,accompanied by the structural collapse of collagen.Dentin caries is irreversible damage,if not detected or treated earlier,it will spread to the dental pulp,causing pulpitis or root periarthritis,even influencing patients' chewing function and facial aesthetics.Biomimetic mineralization is a concept strategy that mimic the biomineralization process by applying nanotechnology.The amorphous calcium phosphate(ACP)can penetrate into the collagen fibers to replace the moisture inside the demineralized collagen fiber,then translate into hydroxyapatite crystals,with the morphology and structure are similar to natural dentin.This dehydration mechanism simulated physiological process.In addition,the mineral of collagen fiber restored its mechanical performance,which can silence the endogenous matrix metalloproteinases(MMPs)and cysteine cathepsin to protect the demineralized collagen fibers.Therefore,biomimetic mineralization is a promising dentin repair methods.By the remineralization of demineralized dentin collagen,the remineralized dentin has similar morphology,structure and biological properties with natural dentin to keep the preservation tooth tissue maximum.In situ biomimetic mineralization has been studied,but it takes too long if applied in clinical.Therefore,to explore a time-saving remineralization technology to meet the clinical needs is highly recommended.Glutaraldehyde(GA)is a gold-standard cross-linking agent that has been widely accepted in the biomedical field and is considered to improve tissue function.It has been revealed that the collagen-based aldehyde-treated tissue has an affinity towards calcium ions,which can act as mineralization centers for calcium phosphate precipitation.For this investigation,inspired by the functional characteristics of the collagen matrix,GA was used as cross-linking and nucleation agent,which might result in the simultaneous molecular cross-linking of dentin collagen and nucleation of nanoapatite.The experiments were designed to combine the results from our previous studies with the GA-based modification of dentin collagen.ObjectiveThe poor mechanical properties and weak biological properties of unprotected dentin collagen present clinical challenges in current dentinal restoration practices.Here,we report a strategy of biomimetic dentin remineralization induced by GA to reconstruct the strength and biostability of demineralized collagen layer.By using GA,incorporation of nanominerals into the demineralized collagen in a time-saving manner was realized.Furthermore,by investigating the biomechanical behavior and enzymatic resistance of the remineralized collagen,mechanical strength and biostability were significantly enhanced due to mineral encapsulation.Our research highlights a combination of GA as a cross-linking agent with a biomimetic mineralization process and may provide a new protocol for dentists to improve the bonding durability and treat dental caries in clinical practice.Methods1.Sample preparation.Eighty recently extracted non-carious human third molars were obtained with the patients' consent under a protocol approved by the ethical committee of the First Affiliated Hospital of the Zhejiang University College of Medicine.The teeth were stored at 4 ? in a 0.1 wt%thymol solution.Dentin stubs were prepared by parallel cutting-2 mm below the cement-enamel junction and perpendicular to the longitudinal axis with a water-cooled slow-speed Isomet diamond saw.Enamel and pulp exposure was avoided for all specimens.The obtained dentin stubs were then cut into two different types of samples:dentin disks with dimensions of 10.0 mm × 8.0 mm×1.0 mm,and dentin beams with dimensions of 10 mm x 0.8 mm x 0.8 mm.A?3 ?m thick demineralized collagen matrix layer was produced on the surface of the dentin by etching with a 35%phosphoric acid at 25? for 10s.The specimens were divided into different experimental groups.2.Samples in the GA treated group were treated with a 5%GA solution for 3 min.Samples were cultivated in a remineralization solution for a designated period of time.Raman spectra of samples from both the control group and the GA-pretreated group were recorded to confirm the chemical modification of the dentin collagen by the GA.The remineralization was performed at a constant temperature of 37.5 ?.At designated time intervals,specimens were randomly retrieved from both the control group and the GA-pretreated group.The specimens were characterized by transmission electron microscopy(TEM),and scanning electron microscopy(SEM).3.Nanoindentation tests and enzymatic degradation experiments were used to evaluate the biomechanical properties and the biostability,respectively.Results1.The results of the Raman investigations indicate that a 3min pretreatment with 5%GA effectively produced cross-links in the dentin collagen while preserving the backbone conformation of collagen.2.The GA pretreatment couldsignificantlyshorten the period for the remineralization of a 3 ?m-thick dentin collagen matrix layer from 7 d to 2 d.The SEM micrographs illustrate the changes in micromorphology of the dentin collagen matrix after remineralization.The evolution of the remineralization process was further examined by TEM,which confirmed the promoting effect of GA-cross-linking.3.The mechanical properties of dentin collagen exposed to a GA pretreatment and a 2 day remineralization process were very similar to that of natural dentin.Obviously,both remineralization procedures resulted in an excellent recovery of the mechanical properties of the demineralized dentin.However,a much shorter time period was required for the GA cross-linking group.4.The enzymatic degradation experiments demonstrated the enhanced biological stability of dentin collagen after remineralization.It can be concluded that both GA cross-linking and the remineralization of demineralized collagen increases dentin resistance against enzymatic degradation,but the dentin collagen samples with intrafibrillar mineral support showed better results.ConclusionsThis investigation confirmed the dual function of GA,i.e.,its collagen cross-linking and calcium binding abilities.The demineralized dentin collagen matrices could be effectively cross-linked by treating demineralized dentin collagen with a 5%GA solution for 3 min.The modified collagen nets may provide a suitable scaffold and additional nucleation sites for the remineralization of the dentin to encapsulate the demineralized collagen,whereby the infiltrated amorphous precursors can evolve into the intrafibriller minerals.This GA-induced remineralization significantly promotes the dentin remineralization process,resulting in enhanced mechanical properties and an improved enzymatic resistance in a time-saving manner.We suggest that the combination of GA as a cross-linking agent with a biomimetic mineralization process may provide a new protocol for dentists to improve the bonding durability and treat dental caries in clinical practice.