| 1.BACKGROUNDIn the past two decades,the progress in adhesive dentist plays a vital role in practice of conservative and aesthetic dentistry.Although successful immediate bonding to enamel has been achieved,the bonding to dentin remains unsatisfactory due to its limited longevity.Current dentin bonding system always consists of small molecule etchant such as phosphoric acid and wet bonding technology.During dentin bonding procedure,small molecular acid etchants such as phosphoric acid can enter the intrafibrillar space of collagen fibers,resulting in complete collagen demineralization.Collagen fibers losing the support of extrafibrillar and intrafibrillar minerals will suffer a sharp decrease in mechanical property and are prone to collapse.The dissolution of intrafibrillar minerals will release endogenous matrix metalloproteinases,which can be activated under acidic conditions and can mediate the degradation of bonding interface.Water-wet bonding technique with high technical sensitivity was introduced to maintain the three-dimensional structure of collagen scaffold for resin infiltration.However,the introduced water by this technique can’t evaporate completely in a clinic setting,rising the concern of degradation of bonding interface in a humid microenvironment.In addition,the adhesive monomers cannot enter the intrafibrillar space of the demineralized collagen fiber even with the assistance of wet bonding technology.The curing of the adhesive in the water-bearing interface is also challenged.Therefore,the present dentin bonding system cannot form an ideal adhesive interface and is prone to degradation under the action of water,enzymes and other biodegradable factors.To solve the aforementioned problems,one has to create a paradigm shift by restricting demineralization to the extrafibrillar spaces of the mineralized collagen matrix using a new demineralize agent with molecular weight greater than 40 k Da.By this way,adhesive monomer only needs to infiltrate into the demineralized extrafibrillar spaces within bonding interface,reducing the difficulty of hybridization between resin and collagen.The extrafibrillar demineralization paradigm reduces the propensity of structural defects within the resin-dentin interface,preserves the mechanical properties of the dentin matrices and enhances its resistance to proteolytic degradation.The paradigm also reduces the exposure of host-derived proteases,such as MMPs and cysteine cathepsins,and contribute to interfacial stability.In addition,because intrafibrillar minerals are retained with the collagen fibrils,the latter should not collapse during air-drying.Hence,there is no need to use a water-wet bonding technique.This should help reduce the entrapment of extrinsic water within the hybrid layer.Our previous studies have synthesized methacrylate-based collagen-reactive monomer（CRM）.The reaction between isocyanate groups of the CRM and the-NH2 group of collagen introduces curable double bonds within collagen fibrils for preparation of high-quality hybrid layer via a copolymerization approach.Theoretically,CRM-based dentin reactive adhesive can improve dentin bonding performance via the dual"micromechanical interlocking&chemical adhesion"mechanism.2.OBJECTIVEIn this study,we propose the use of carboxymethyl chitosan（CMCS）as extrafibrillar demineralization agent instead of phosphoric acid-etching for creating microporosities in the dentin matrix for resin retention;a CRM-based adhesive（CBA）will be developed for additional chemical adhesion for dentin bonding.A new dentin bonding scheme will be constructed consequently.It will overcome the undesirable consequences associated with bonding systems that rely on phosphoric acid-etching（viz.over-demineralization of dentin bonding surface,incomplete adhesive resin infiltration,hydrolytic degradation by water sorption and enzymatic degradation by collagenolytic enzymes）.Successful completion of this project should improve the stability of the resin-dentin interface and foster the longevity of bonded clinical restorations.3.METHODS（1）New extrafibrillar demineralize agent consists of 1 wt%CMCS（Mw>40 k Da）was formulated and its calcium chelation efficiency was studied by Inductively Coupled Plasma Mass Spectrometry.The CMCS treated dentin was characterized by transmission electron microscopy,atomic force microscopy（AFM）,field emission scanning electron microscopy（FE-SEM）and confocal laser scanning microscopy.The inhibition effect of CMCS onⅣtype collagenase was also studied.Dentin bonding tests including micro tensile bond strength（μTBS）test,interface characterization and in situ zymography were performed to evaluate the dentin bonding performance of 1%CMCS-mediated extrafibrillar demineralization and to determine its technical parameters.（2）CRM with free-NCO group was synthesized via the reaction between hydroxyethyl methylacrylate and isophorone diisocyanate.CRM0 without free-NCO group was also synthesized and served as control monomer.The interaction between CRM and demineralized dentin collagen was studied by Micro Raman spectroscopy.CBA and a negative adhesive control（NA）were formulated with CRM and CRM0,separately.A commercial adhesive,One Step（OS;Bisco Inc.,Schaumburg,IL,USA）was used as commercial control.The degree of conversion（DC）,wetting ability,hydrophilicity and cytocompatibility of the adhesives were evaluated.Dentin bonding tests includingμTBS,FE-SEM,Micro Raman spectroscopy and AFM observation were performed to evaluate the immediate bonding performance and bonding durability of CBA.（3）New dentin bonding scheme was constructed by the aforementioned CMCS-mediated extrafibrillar demineralization and dentin reactive adhesive CBA.Traditional bonding system containing phosphate acid and Solobond M（SM,VOCO,Germany）was served as control.Dentin bonding performance of these schemes combined either with wet bonding technique or with dry bonding technique was tested byμTBS.Thermocycling aging process was adopted to determine the bonding durability.The bonding interface was further characterized by AFM observation and in-situ zymography.4.RESULTS（1）1%CMCS extracted Ca2+from dentin in a time-dependent manner.CMCS conditioning for 30 s produced a 1μm partly extrafibrillar demineralized zone with remaining intrafibrillar minerals and the three-dimensional structure of the demineralized collagen network was maintained.The results of dentin bonding test showed that 1%CMCS demineralization treatment for more than 30 s combined both with wet bonding technique and dry bonding technique could form a continuous and complete bonding interface,and the bonding strength was better than that of traditional phosphoric acid-based bonding scheme.μTBS values remained stable before and after aging（p>0.05）.In addition,1%of CMCS processing can reduce the release of dentine endogenous enzymes and reduce the collagenase activity in the mixed layer.（2）After CRM treatment,the amide I peak of demineralized dentin collagen shifted and a new peak of urea-carbonyl group appeared.In addition,both the pyridine ring vibration and the ratio of pyridine/phenyl increased.CBA containing 35%CRM showed a DC of 60±1%and its contact angle on demineralized dentin was 20±1°.The water contact angle on cured VBA was significantly higher than that of OS（P<0.05）.There was no significant difference in the inhibitory effect of three adhesives on dental pulp stem cells（P>0.05）.CBA achieved comparable immediate bonding strength than OS（P>0.05）,while its bonding strength after aging was higher than OS and NA（P<0.05）.Continuous hybrid layer can be observed at the bonding interface of CBA group.The amide I shifted and new urea-carbonyl groups appeared within the bonding interface.Both the pyridine ring vibration intensity and the ratio of pyridine/phenyl increased within hybrid layer.Regardless of aging procedure,the dried bonding interface of CBA group was continuous with the lower nature tooth tissue without obvious depression,while OS group and NA group showed obvious contraction depression.The elastic modulus of the hybrid layer and the bottom of the hybrid layer in CBA group did not change significantly before and after aging（P>0.05）,which was significantly higher than that of other groups（P<0.05）.（3）Regardless of adhesive type（SM or CBA）,the"CMCS+dry bonding/wet bonding"achieved equivalent immediate bonding strength to that of"phosphoric acid+wet bonding"（P>0.05）.In the CMCS demineralization group,the two adhesives can form a continuous bonding interface and no obvious hybrid layer or resin tags can be observed.After drying,the bonding interface showed high resistance to shrinkage caused by water evaporation.There was no significant difference in the elastic modulus of"CMCS+CBA"in the two bonding modes（P>0.05）,but they were both higher than those of other groups（P<0.05）.The endogenous enzyme activity in the hybrid layer created by the traditional"phosphoric acid+dry bonding/wet bonding"was higher than those of CMCS groups（P<0.001）.In addition,the endogenous enzyme activity in the CMCS groups were independent of the type of adhesive and bonding mode（P>0.05）.After aging,theμTBS and the hybrid layer elastic modulus of"CMCS+dry bonding+CBA"group showed the lowest decrease and was higher than other groups（P<0.01）.In addition,This scheme showed the best interfacial sealing performance and the lowest endogenous enzyme activity（P>.05）.After aging,the adhesive interface in the CMCS groups contracted slightly after drying,while all the phosphoric acid groups（except for"wet bonding+CBA"）showed obvious shrinkage depression.5.CONCLUSION（1）1 wt%CMCS（Mw>40 k Da）treatment for 30 s can achieve chelation-based extrafibrillar demineralization,reducing the release and the activation of endogenous collagenase,creating stronger and durable dentin bonding effect than traditional phosphoric acid etching-based adhesion system.（2）The dentin collagen after CMCS-mediated extrafibrillar demineralization treatment showed high resistance against the dehydration contraction,which is more suitable for dry bonding technique with lower technical sensitivity,avoiding the risk of bonding failure caused by wet bonding technique.（3）The functional CRM in the dentin reactive adhesive CBA can covalently bond to the collagen within bonding interface and possesses collagen cross-linking effect.The quality and stability of the dentin bonding interface can be improved by CBA via dual"micro-mechanical chimerism&chemical bonding"mechanism,endowing excellent immediate bonding performance and bonding durability.（4）The novel“CMCS-mediated extrafibrillar demineralization+dry bonding+dentin-reactive adhesive CBA”bonding scheme can achieve excellent dentin bonding strength and durability.The dentin bonding interface of this novel bonding scheme showed high mechanical strength,sealing ability and low collagenase activity,reducing the risk of enzyme-mediated degradation.In conclusion,CMCS-based extrafibrillar demineralization technology can achieve stable and lasting dentin adhesion within a clinically acceptable time frame（30 s）.The dentin-reactive adhesive can improve the immediate dentin bonding performance and bonding stability by introducing additional covalent adhesion.The new bonding scheme consists of CMCS-based extrafibrillar demineralization,dentin-reactive adhesive and dry bonding technique will effectively eliminate structural defects within the resin-dentin interface,provide better protection for interfacial collagen fibrils against enzymatic degradation,via chemical bonding with partially-demineralized dentin,and enhance the mechanical properties of the bonded interface.It will play a key role in forming a more compact and stable bonded interface,significantly improving the durability of resin-dentin bonds,and prolonging the clinical longevity of bonded restorations.This scheme is different from the traditional demineralization theory based on acid-etching,and provides a foundation for further improvement of bonding durability and reduction of the technique sensitivity associated with bonding applications. |
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