the strength of adhesive joints formed by bonding resins to etched enamel and/or conditioned dentin has resulted in greater application of these systems in everyday dental practice . dentin adhesives are classified as three step , two step and single step systems based on the manner in which the three fundamental steps of etching , priming and bonding to tooth substrate are accomplished . self - etch adhesive systems use non - rinse acidic monomers that eliminate the need for a separate rinsing step thus reducing the clinical application time . although contemporary dentin bonding systems show improvement in better handling and bonding characteristics , the achievement of a strong and stable bond to deep dentin still remains a challenge in restorative dentistry . dentin is a complex hydrated biological composite material with structural components and properties that vary with location . studies have shown that a reduction in bond strength occurs when resin composite is bonded to deep dentin . this can be attributed to the complexities in structure of deep dentin , such as an increase in the number of tubules and their diameters with much less intertubular dentin matrix as compared with superficial dentin . the durability of the bond between dentin and adhesive system depends largely on the structural integrity and mechanical properties of acid - demineralized collagen fibers . mechanical properties of collagen and its resistance to enzymatic degradation can be improved by an increase in the formation of intra - and inter - molecular and intermicrofibrillar cross - links . this can be achieved by the use of various collagen cross - linkers , both synthetic and natural , on the dentin substrate prior to the bonding procedure . naturally occurring collagen cross - linkers such as sodium ascorbate and proanthocyanidin have been reported to increase the collagen cross - linking in sound and caries - affected dentin.[68 ] but , the effect of these collagen cross - linkers on the bond strength of a self - etch adhesive to deep dentin has not been reported in the literature . hence , the aim of this in vitro study was to determine the shear bond strength of composite resin bonded to deep dentin using a self - etch adhesive , after treatment with two collagen cross - linking agents at varying time intervals . materials used in this study and their composition 
 10% sodium ascorbate solutionten grams of sodium ascorbate powder ( sdfinechem ltd . , mumbai , india ) were dissolved in 100 ml of distilled water to make 10% sodium ascorbate solution.6.5% proanthocyanidin solution6.5 g of grape seed extract in the form of powder ( puritans pride inc . , oakdale , ny , usa ) was collected from the capsules and dissolved in 100 ml of distilled water to make 6.5% proanthocyanidin solu tion . mumbai , india ) were dissolved in 100 ml of distilled water to make 10% sodium ascorbate solution . 6.5% proanthocyanidin solution 6.5 g of grape seed extract in the form of powder ( puritans pride inc . , oakdale , ny , usa ) was collected from the capsules and dissolved in 100 ml of distilled water to make 6.5% proanthocyanidin solu tion . thirty human maxillary central incisors extracted due to periodontal reasons were collected for the study . each tooth was sectioned longitudinally , parallel to the long axis of the tooth , into an equal mesial and a distal half by means of a low - speed diamond disc ( hi  di diamond precision tools ltd . , london , uk ) under copious water supply . the contents of the pulp chamber were then removed ultrasonically to determine its extent . using a diamond disc , the dentin in the proximal wall of each half of the crown incisal to the cemento - enamel junction was removed until the remaining dentin thickness was approximately 1 mm , as measured with a metal caliper ( iwanson spring metal caliper , i.d - tech , sialkot , pakistan ) , from the outer surface of the prepared proximal portion of the crown to the pulp chamber . these specimens ( n = 60 ) were randomly divided into three groups based on the surface treatment of dentin as follows : 
 group i ( n = 12 , control ) : no pre - treatment was done on the exposed proximal dentin surface . adhesive bonding and composite buildup was done according to the bonding protocol as described here . group i ( n = 12 , control ) : no pre - treatment was done on the exposed proximal dentin surface . adhesive bonding and composite buildup was done according to the bonding protocol as described here . the self - etch adhesive , xeno iii , was used according to the manufacturer 's instructions . an equal amount of liquid a and b ( 1:1 ) was mixed for 5 s and applied on the proximal dentin surface . the adhesive was left in place undisturbed for 20 s and then gently air dried and light cured ( astralis 3 light curing unit , ivoclarvivadent , schaan , liechtenstein ) for 40 s. composite buildup was done by placing two increments of 2-mm - thick composite resin ( ceram x nano ceramic restorative , dentsplydetrey gmbh , konstanz , germany ) using a 3 mm diameter plastic tube as a matrix , with each increment being light cured for 40 s. group ii ( n = 24 ) : 10% sodium ascorbate pre - treatment . this group was further subdivided into two subgroups based on the pre - treatment time as follows : 
 subgroup iia ( n = 12 ) : the exposed proximal dentin surface was treated with 10% sodium ascorbate solution for 5 min and rinsed with water , followed by the bonding / buildup procedure as described above.subgroup iib ( n = 12 ) : the exposed proximal dentin surface was treated with 10% sodium ascorbate solution for 10 min and rinsed with water . subgroup iia ( n = 12 ) : the exposed proximal dentin surface was treated with 10% sodium ascorbate solution for 5 min and rinsed with water , followed by the bonding / buildup procedure as described above . subgroup iib ( n = 12 ) : the exposed proximal dentin surface was treated with 10% sodium ascorbate solution for 10 min and rinsed with water . adhesive bonding and composite buildup followed as described above . this group was further subdivided into two subgroups based on the pre - treatment time as follows : 
 subgroup iiia ( n = 12 ) : the exposed proximal dentin surface was treated with 6.5% proanthocyanidin solution for 5 min and rinsed with water , followed by the bonding / buildup procedure as described above.subgroup iiib ( n = 12 ) : the exposed proximal dentin surface was treated with 6.5% proanthocyanidin solution for 10 min and rinsed with water . subgroup iiia ( n = 12 ) : the exposed proximal dentin surface was treated with 6.5% proanthocyanidin solution for 5 min and rinsed with water , followed by the bonding / buildup procedure as described above . subgroup iiib ( n = 12 ) : the exposed proximal dentin surface was treated with 6.5% proanthocyanidin solution for 10 min and rinsed with water . all the specimens were then stored in distilled water at 37c for 24 h. shear bond strength was determined using a universal testing machine ( lr 100 k , lloyd instruments , largo , florida , usa ) at a crosshead speed of 1 mm / min . the results were tabulated and statistically analyzed using statistical product and service solutions ( spss ) version 17.0 software ( spss inc . , chicago , il , usa ) . paired t - test was used to calculate the p value ( p < 0.001 ) . mumbai , india ) were dissolved in 100 ml of distilled water to make 10% sodium ascorbate solution.6.5% proanthocyanidin solution6.5 g of grape seed extract in the form of powder ( puritans pride inc . , oakdale , ny , usa ) was collected from the capsules and dissolved in 100 ml of distilled water to make 6.5% proanthocyanidin solu tion . mumbai , india ) were dissolved in 100 ml of distilled water to make 10% sodium ascorbate solution . 6.5% proanthocyanidin solution 6.5 g of grape seed extract in the form of powder ( puritans pride inc . , oakdale , ny , usa ) was collected from the capsules and dissolved in 100 ml of distilled water to make 6.5% proanthocyanidin solu tion . thirty human maxillary central incisors extracted due to periodontal reasons were collected for the study . each tooth was sectioned longitudinally , parallel to the long axis of the tooth , into an equal mesial and a distal half by means of a low - speed diamond disc ( hi  di diamond precision tools ltd . , london , uk ) under copious water supply . the contents of the pulp chamber were then removed ultrasonically to determine its extent . using a diamond disc , the dentin in the proximal wall of each half of the crown incisal to the cemento - enamel junction was removed until the remaining dentin thickness was approximately 1 mm , as measured with a metal caliper ( iwanson spring metal caliper , i.d - tech , sialkot , pakistan ) , from the outer surface of the prepared proximal portion of the crown to the pulp chamber . these specimens ( n = 60 ) were randomly divided into three groups based on the surface treatment of dentin as follows : 
 group i ( n = 12 , control ) : no pre - treatment was done on the exposed proximal dentin surface . adhesive bonding and composite buildup was done according to the bonding protocol as described here . group i ( n = 12 , control ) : no pre - treatment was done on the exposed proximal dentin surface . adhesive bonding and composite buildup was done according to the bonding protocol as described here . the self - etch adhesive , xeno iii , was used according to the manufacturer 's instructions . an equal amount of liquid a and b ( 1:1 ) the adhesive was left in place undisturbed for 20 s and then gently air dried and light cured ( astralis 3 light curing unit , ivoclarvivadent , schaan , liechtenstein ) for 40 s. composite buildup was done by placing two increments of 2-mm - thick composite resin ( ceram x nano ceramic restorative , dentsplydetrey gmbh , konstanz , germany ) using a 3 mm diameter plastic tube as a matrix , with each increment being light cured for 40 s. group ii ( n = 24 ) : 10% sodium ascorbate pre - treatment . this group was further subdivided into two subgroups based on the pre - treatment time as follows : 
 subgroup iia ( n = 12 ) : the exposed proximal dentin surface was treated with 10% sodium ascorbate solution for 5 min and rinsed with water , followed by the bonding / buildup procedure as described above.subgroup iib ( n = 12 ) : the exposed proximal dentin surface was treated with 10% sodium ascorbate solution for 10 min and rinsed with water . subgroup iia ( n = 12 ) : the exposed proximal dentin surface was treated with 10% sodium ascorbate solution for 5 min and rinsed with water , followed by the bonding / buildup procedure as described above . subgroup iib ( n = 12 ) : the exposed proximal dentin surface was treated with 10% sodium ascorbate solution for 10 min and rinsed with water . adhesive bonding and composite buildup followed as described above . this group was further subdivided into two subgroups based on the pre - treatment time as follows : 
 subgroup iiia ( n = 12 ) : the exposed proximal dentin surface was treated with 6.5% proanthocyanidin solution for 5 min and rinsed with water , followed by the bonding / buildup procedure as described above.subgroup iiib ( n = 12 ) : the exposed proximal dentin surface was treated with 6.5% proanthocyanidin solution for 10 min and rinsed with water . subgroup iiia ( n = 12 ) : the exposed proximal dentin surface was treated with 6.5% proanthocyanidin solution for 5 min and rinsed with water , followed by the bonding / buildup procedure as described above . subgroup iiib ( n = 12 ) : the exposed proximal dentin surface was treated with 6.5% proanthocyanidin solution for 10 min and rinsed with water . adhesive bonding and composite buildup followed as described above . all the specimens were then stored in distilled water at 37c for 24 h. shear bond strength was determined using a universal testing machine ( lr 100 k , lloyd instruments , largo , florida , usa ) at a crosshead speed of 1 mm / min . the results were tabulated and statistically analyzed using statistical product and service solutions ( spss ) version 17.0 software ( spss inc . , chicago , il , usa ) . paired t - test was used to calculate the p value ( p < 0.001 ) . the results showed that the mean shear bond strength values of subgroup iia ( 21.43  0.58 ) , iib ( 21.97  0.48 ) , iiia ( 23.70  0.58 ) and iiib ( 23.82  0.39 ) were significantly higher than the mean shear bond strength value in the control group ( 13.31  0.87 ) ( p < 0.001 ) . both 5 min and 10 min pre - treatment times of 6.5% proanthocyanidin ( iiia [ 23.70  0.58 ] and iiib [ 23.82  0.39 ] ) yielded significantly higher shear bond strength values than the corresponding pre - treatment times of 10% sodium ascorbate ( iia [ 21.43  0.58 ] and iib [ 21.97  0.48 ] ) ( p < 0.001 ) . no significant difference in shear bond strength values was found between the two pre - treatment times in both the 10% sodium ascorbate and the 6.5% proanthocyanidin groups ( p > 0.05 ) . the two main strategies that are currently in use for adhesive bonding to enamel and dentin are the total - etch technique and the self - etch technique . although etch - and - rinse technique is still considered as the most effective approach to achieve efficient and stable bonding to enamel and dentin , the multiple application steps , the critical rinsing step and the frustratingly high incidence of post - operative sensitivity led to the evolution of more simplified , less technique - sensitive and user - friendly self - etch adhesives . the major shortcomings of dentin as a bonding substrate are its heterogeneous composition and hydrophilic nature . it presents an intricate composition comprised of 70% by weight of an inorganic phase of carbonate - rich hydroxyapatite crystals , 20% by weight of an organic phase of predominantly type i collagen and 10% water.[1012 ] covalent inter - and intra - molecular cross - links are the basis for stability , tensile strength and viscoelasticity of the collagen fibrils . various chemicals , both synthetic and natural , have the ability to increase these collagen cross - links in biological tissues . sodium ascorbate is an important component in the synthesis of hydroxyproline and hydroxylysine in collagen . hydroxyproline serves to stabilize the collagen triple helix and hydroxylysine is necessary for the formation of intermolecular cross - links in collagen . proanthocyanidins ( pa ) are naturally occurring bioflavonoids found in high concentrations in grape seed , pine bark , cranberries , lemon tree bark and hazel nut tree leaves . although pa from grape seed extract has been shown to effectively cross - link collagen in in vitro studies , their effect on the bond strength of resin composite bonded using a self - etch adhesive to deep dentin remains an unexplored area in restorative dentistry . in the present study , group i ( control ) recorded a mean shear bond strength value of 13.31 0.87 mega pascals ( mpa ) to deep dentin , which is lesser than the optimal bond strength of self - etch adhesive resins to superficial dentin ( 20 - 24 mpa).[1921 ] this is in accordance with previous studies done by tagami et al . , pashley et al . , kaaden et al . and yazici et al . , who showed that bond strength of self - etch adhesive resins to deep dentin can be as low as 10.3 - 16.7 mpa results of this in vitro study showed that when deep dentin was treated with sodium ascorbate and proanthocyanidin , the bond strength reached values ( 21.43 - 23.82 mpa ) comparable with the optimal bond strength values of superficial dentin . this can be attributed to improved dentin collagen stability obtained from an increase in the number of collagen cross - links , achieved by the use of these collagen cross - linkers . group iii ( 6.5% proanthocyanidin ) showed a significantly higher bond strength to deep dentin compared with group ii ( 10% sodium ascorbate ) in both the subgroups ( p < 0.001 ) . this is in accordance with the findings of macedo et al . , who showed that the application of 6.5% grape seed extract and 5% gluteraldehyde to caries - affected and sound dentin significantly improved the microtensile bond strength of composite to dentin . similarly , walter et al . showed that 0.5% proanthocyanidin efficiently stabilized collagen and increased its resistance to caries compared with 0.625% genipin and 5% gluteraldehyde . bedran - russo , et al . found that naturally occurring cross - linking agents such as 0.5% proanthocyanidin and 0.625% genipin are capable of stabilizing demineralized dentin collagen more effectively compared with 5% gluteraldehyde . this could be attributed to the specificity of proanthocyanidin to facilitate the enzyme proline hydroxylase , which catalyzes the hydroxylation of proline , an essential step in collagen biosynthesis . pa and proteins have been shown to interact in four different ways : covalent interactions , ionic interactions , hydrogen bonding interactions or hydrophobic interactions . castellan et al . showed that when demineralized , dentin is treated with pa , it results in improved mechanical properties and reduced water absorption as a result of the dense collagen network formed by the use of exogenous cross - linkers . unlike previous studies that employed longer application times , application times of 5 and 10 min were chosen in the present study , and the results showed no statistically significant difference in the shear bond strength values between the two pre - treatment times in both 10% sodium ascorbate and 6.5% pa groups . hence , dentin pre - treatment with collagen cross - linkers can be safely recommended as an effective chair side procedure to overcome the disadvantage of reduced bond strength of self - etch adhesive systems to deep dentin . under the limitations of this in vitro study , the following conclusions can be drawn : 
 a significant improvement in the bond strength of resin composite to deep dentin bonded using a self - etch adhesive was obtained when the dentin surface was pretreated with both 10% sodium ascorbate and 6.5% proanthocyanidin compared with the control groupthe use of 6.5% proanthocyanidin as a collagen cross - linker on deep dentin significantly improved the shear bond strength values compared with the use of 10% sodium ascorbatethere was no significant difference in the shear bond strength values between the 5 min and 10 min pre - treatment times in both 10% sodium ascorbate and 6.5% proanthocyanidin groups . a significant improvement in the bond strength of resin composite to deep dentin bonded using a self - etch adhesive was obtained when the dentin surface was pretreated with both 10% sodium ascorbate and 6.5% proanthocyanidin compared with the control group the use of 6.5% proanthocyanidin as a collagen cross - linker on deep dentin significantly improved the shear bond strength values compared with the use of 10% sodium ascorbate there was no significant difference in the shear bond strength values between the 5 min and 10 min pre - treatment times in both 10% sodium ascorbate and 6.5% proanthocyanidin groups .
objectives : to evaluate the shear bond strength of composite resin to deep dentin , bonded using a self - etch adhesive , after treatment with two collagen cross - linkers at varying time intervals.materials and methods : thirty extracted human incisors were sectioned longitudinally into equal mesial and distal halves ( n = 60 ) . the proximal deep dentin was exposed and the specimens were divided based on the surface treatment of dentin prior to bonding as follows : group i ( n = 12 , control ) : no prior dentin surface treatment ; group ii ( n = 24 ) : dentin surface pretreated with 10% sodium ascorbate ; and group iii ( n = 24 ) : dentin surface pretreated with 6.5% proanthocyanidin . 
 groups ii and iii were further divided into two subgroups based on the pre - treatment time of five and 10 min . 
 shear bond strength of the specimens was tested using universal testing machine and the data were statistically analyzed.results:significantly higher shear bond strength to deep dentin was observed in teeth treated with 10% sodium ascorbate and 6.5% proanthocyanidin compared to control group . 
 no significant difference was observed between 5 min and 10 min pre - treatment times.conclusion:dentin surface pre - treatment with both 10% sodium ascorbate and 6.5% proanthocyanidin resulted in significant improvement in bond strength of self - etch adhesive to deep dentin .