Saira Khalid  ( Department of Science of Dental Materials, de'Montmorency College of Dentistry, Lahore, Pakistan )
Muhammad Aamir Rafique  ( Department of Prosthodontics, Faisalabad Medical University, Faisalabad, Pakistan )
Abdul Samad Khan  ( Department of Restorative Dental Sciences, College of Dentistry Imam Abdulrahman Bin Faisal University Dammam, Saudi Arabia. )

Objective: To investigate the effect of natural crosslinkers proanthocyanidin, genipin and glutaraldehyde on shear bond strength at the composite resin-dentin interface .

Method: The in-vitro study was conducted at the Postgraduate Medical Institute, Lahore, Pakistan, from June to September 2018. Exposed dentin surfaces of extracted teeth were conditioned and randomly divided into proanthocyanidin, genipin, glutaraldehyde and control groups according to the type of surface treatment. The dentin surfaces were treated with 6.5% of primers proanthocyanidin, genipin, glutaraldehyde in the relevant groups, while teeth in the control group did not receive any primer application. After thorough rinsing, surfaces of all teeth were restored with a bonding agent and a restorative composite. After 24h, shear bond strength was tested at the Pakistan Council of Scientific and Industrial Research laboratories in Lahore. Pattern of fractures and quality of interface were investigated microscopically at the Lahore campus of COMSATS University, Islamabad. Data was analysed using SPSS 22.

Results: Of the 80 teeth, there were 20(25%) in each of the 4 groups. Surface treatment in the three intervention groups significantly raised the shear bond strength at the composite resin-dentin interface compared to the control group (p<0.05).

Conclusion: Chemical modification with collagen crosslinkers improved bond strength at the composite resin-dentin interface.

Keywords: Collagen crosslinkers, Shear bond strength, Resin-dentin interface, Dentin primers. (JPMA 70: 1363; 2020).

https://doi.org/10.5455/JPMA.17870

 

Introduction

 

Restorative dentistry deals not only with the treatment of diseased tooth, but also looks to principally restore the function as well as aesthetics without compromising the biology.¹ Dental adhesive flows into the microporosities and forms a hybrid layer. The quality of the hybrid layer determines the durability of the bonded interface.² Different strategies have been undertaken to improve the durability of the hybrid layer, such as the introduction of bi-functional molecules, which can enhance resin penetration by preventing collagen collapse.³ Recently, the role of collagen-based cross-linkers has been found to strengthen dentin. They serve to produce new crosslinks between and within the delicate collagen mesh. Moreover, they have also been found to increase the stability of connective tissue, thus enhancing the strength of interface formed between dentin and composite resin.⁴ Glutaraldehyde (GDL) is a synthetic crosslinker⁵ which has a great chemical affinity with collagen and 2-hydroxyethyl methacrylate (HEMA). The mechanical properties of demineralised dentin have been improved with the application of GDL.⁶ Due to the cytotoxic concerns, the efficacy of naturally occurring crosslinkers has been studied on collagen-based tis su es, in cluding d entin.^7,8 Proanthocyanidin (PA) is a plant metabolite available in fruits, vegetables, nuts, seeds, flowers, leaves and bark. Grape seed is a rich source of PA where the seeds belonging to vitis vinafera grape have the highest yield. PA enhances resin-dentin bonds by improving the mechanical properties of dentin.^9,10 Grape seed PA has been found to be the most effective collagen biomodifier¹¹ that increases the resistance at the dentin-adhesive interface to enzymatic degradation by matrix metalloproteinases and cathepsins, enhances the bond strength of endodontic sealers¹² and reduces caries.¹³ PA renders demineralised dentin more receptive towards resin uptake and encapsulation around exposed collagen fibres after acid demineralisation.¹⁴ Genipin (GE) is another collagen crosslinker isolated from the fruit of jasmine. GE residues are not found to elicit cytotoxicity in tissues and are found to produce a low degree of inflammation compared to GDL, and also increase tissue regeneration.¹⁵ GE increases the mechanical properties of different collagen-based biomaterials and provides better strength to gelatin compared to GDL.¹⁶ Much work has been put forth in order to make use of different collagen crosslinkers in a clinically feasible manner, but it needs to be further worked out. The current study was planned to investigate the effect of PA, GE and GDL on shear bond strength (SBS) at the composite resindentin interface.

 

Materials and Methods

 

The experimental study was conducted at the Postgraduate Medical Institute (PMI), Lahore, Pakistan, from June to September 2018. After getting approval from the institutional ethics review committee, the sample size was calculated using World Health Organisation (WHO) formula.¹⁷ While keeping the expected SBS for control group at 10.8±4.9 and for PA group at 17.7±9.4, power of study at 80% and level of significance equal to 5%.

The sample was raised with extracted sound human premolar teeth collected from the Eexodontia Department, which were extracted due to orthodontic treatment. After cleaning by scrubbing, the extracted teeth were stored in normal saline and kept at 4OC to avoid unwanted changes in the teeth that can happen after extraction. All the teeth were brought back to room temperature at 24OC for sample preparation. The teeth were embedded in selfcure acrylic resin to make custom-made acrylic blocks. Tooth reduction was carried out using slow speed carbide saw (Jigsaw blade, type: U Length: 2-1/4, China) under constant water supply. After achieving a flat mid-coronal occlusal dentin surface, the occlusal and axial surfaces were reduced simultaneously until an occlusal diameter of 5mm and a height of 4mm for each tooth was achieved. A surface smear layer was created by grinding occlusal surfaces with 600 grit silicon-carbide abrasive papers. After the sample preparation, the dentin surfaces of sample teeth were conditioned with 10% phosphoric acid gel for 15s. Teeth were rinsed thoroughly with running tap water and dried using absorbent papers according to Kanca's wet bonding technique.18 Teeth were randomly divided into control, PA, GE and GDL groups. To prepare the experimental primers PA and GE, 6.5g of 95% grape seed extract powder (Italo Biological Technology Co., Limited. China) was dissolved in 100mL of the solvent (acetone-water 30:70) and 99% genipin powder in 100mL of the solvent respectively. Primer GDL was made by dissolving 6.5mL of 99% GDL in 100mL of the solvent. The potential of hydrogen (pH) of primers PA, GE and GDL were adjusted at 7 by dropwise addition of 1M sodium hydroxide (NaOH) (Sigma Aldrich, UK). The extracted samples were investigated with Fourier Transform Infrared Spectroscopy (FTIR) by using an attenuated total reflection (ATR) accessory (Thermo Nicolet 6700, USA). The resolution was 8cm-1 with 256 scan numbers and the range was 4000-525 cm-1 . Teeth in the control group did not receive any application of experimental primers, whereas the surface of teeth in groups PA, GE and GDL received 60s application of the respective primers by using micro-brush followed by thorough rinsing with running tap water after 60s and they were properly blot-dried using absorbent papers. Then all the teeth in the control and experimental groups entered the bonding phase which was carried out by applying two consecutive coats of bonding agent (Adper Single Bond 2, 3M-Exercise Science and Physical Education (ESPE) using a micro-brush with gentle application to improve penetration. The excess bonding agent was gently air-dried for 5s and photoactivated for 20s with a lightemitting diode (LED) curing unit (Woodpecker Medical Instruments, Co. Ltd..) as per the manufacturer's instructions. For the application of resin-based composite resin (Filtek Z250 XT 3M ESPE, USA; shade A1), a plastic ring with an internal diameter of 5mm and height of 2.5mm was used to build round cylinder of composite resin on the surfaces of prepared teeth as per BS EN 10477 specifications (Dentistry - Polymer-based crown and bridge materials).¹⁹ The composite resin was placed in increments and each increment was light-cured for 20s. The restored samples were then stored in deionised water at 37OC for 24h. SBS was determined with Universal Testing Machine (Shimadzu Corporation, Tokyo, Japan). The restored surface was positioned perpendicular to the 0.9mm thick loading shear blade having a chisel-like configuration. The shear blade was placed at the junction of the tooth and composite resin interface following International Organization for Standardization (ISO) standards for bond strength test protocols-TR 11405.²⁰ The crosshead speed was kept at 0.5mm.min-1 until the debonding occurred. Data was analysed using SPSS 22. Means and standard deviations were calculated for SBS, and expressed in MPa. One-way analysis of variance (ANOVA) post hoc Tukey's test was used to find significant difference in SBS in different groups. For pair-wise comparison, least significant difference (LSD) test was applied. Whereby, p<0.05 was taken as significant. After testing, specimens were examined under electronic zoom microscope (Olympus SZ x 7, Model SZ2-ILST Japan) and modes of failure were classified as adhesive (at the interface), cohesive (within the composite or tooth substrate) and mixed (combination of adhesive and cohesive failures). Representative samples of each group were prepared for optical microscopy analysis of the hybrid layer. Restored samples were kept under an electronic optical microscope (Optika B-600 MET, range 5X-1000X) at 100x magnification to observe the resin-restorative interface.

 

Results

 

Total of the 80 teeth, there were 20 (25%) in each of the 4 groups. Surface treatment in the three intervention groups significantly raised the shear bond strength at the composite resin-dentin interface compared to the control group (p<0.05) (Figure 1).

Significantly more fractures occurred at the interface for the control group (p=0.002), while cohesive failure within the tooth was highest in PA group (p=0.001).

The number of mixed type of fractures were high in GE group (p=0.139) (Figure 2A-C).

Optical microscopy of the restored samples revealed that maximum resin penetration was observed for PA compared to the other groups, followed by GE and GDL groups (Figure 3).

FTIR spectra of the three experimental groups were also compared (Figure 4).

 

Discussion

 

In the present study, SBS was measured at the composite resin-dentin interface when PA, GE, and GDL experimental primers were applied on post-etch dentin surfaces. The alternate hypothesis was accepted that collagen crosslinkers (CCL) improved SBS at the composite resindentin interface when used as primer. To etch the surface, a concentration of 10% phosphoric acid as conditioner was used to avoid unnecessary shrinking of the delicate collagen mesh of dentin. The stability and expanded network of dentin is important for a good bonding with adhesive resin.²¹ The study followed wet bonding technique after post-etch rinsing. This was done to prevent exposed collagen network from collapsing that readily produces voids and reduce intertubular resin penetration.

CCL are tissue fixatives and PA, GE, and GDL post-etch application for 60s improved SBS. PA, GE, and GDL are capable of forming hydrogen bonding with amino acids of collagen within 60s of surface application. For an effective hybrid layer formation, the adhesion must be gap-free between the resin and dentin. Previously, researchers found crosslinkers effective in modifying properties of demineralised dentin by soaking samples in a solution of crosslinking agents.^22,23 Our study observed that the benefits of crosslinker could be best availed when used as a primer which was later rinsed thoroughly. It is expected that leaving an accessory layer over the conditioned dentin surface might hinder resin penetration. Another reason for rinsing was due to the antioxidant behaviour of PA and GE. Antioxidants are scavengers of free radicals which are the main source of the polymerisation reaction of the tooth-coloured adhesive restorative system. The quality of the hybrid layer and resin encapsulation depend upon the concentration of PA if it is added into the bottle of adhesive, as interference with the photoinitiator can result in a reduced degree of conversion and incomplete setting of the bonding agent.²⁴ Therefore, in the current study, the cross linkers were used as separate primers rather than adding in the bottle of bonding agent. The mean SBS of one minute post-etch application of GDL in the current study was 10.37MPa which was statistically higher than the control group (7.78MPa). The effective rise in bond strength was due to the ability of GDL to readily bond with alpha-amino groups of amino acids, Nterminal amino groups of peptides and sulfadryl groups of cysteine amino acid. A number of different amino acids, like lysine and hydroxylysine, drop after treatment with GDL which supports that GDL reacts with amino acids and engage free amino groups in new crosslinks.²⁵ The rise in bond stability is also explained by GDL inhibition to flow dentinal fluid and an increase in bond durability of post-etch dentin.²⁶ The concentration of GDL in present study was kept 6.5% and showed effective results. It is reported that up to 10% aqueous solutions of GDL are not harmful to dentin. However, GDL when used in combination with HEMA, as a single preparation (GLUMA bond), reduce cell viability and cell metabolism.²⁷ However, higher concentrations can damage human fibroblast cells.²⁸ The current study confirmed that the 6.5% PA was effective in raising bond strength from a value of 7.79MPa in the control group to 10.50MPa. PA strengthens collagen type I due to the presence of galloyl groups.²⁹ It has a strong potential to decrease activity of endogenous enzyme activity. It also reduce binding sites of degrading enzymes on collagen fibres irrespective of its application time.³⁰ Moreover, it removes unbound water from collagen molecules that hinder penetration of resin into intertubular space and result in microleakage around the restoration. It also tends to reduce hydrophilicity of dentin by crosslinking with collagen.³¹ Similarly, GE is an emerging crosslinking agent which is capable of forming bonds with amino groups of lysine, hydroxylysine and arginine. It is considered a safest alternative to synthetic crosslinking agents, unlike GDL, formaldehyde, and carbodiimide that have a tendency of leaving cytotoxic residues in the tissues.³² The comparison of mean shear strength done between the three available CCLs showed that GE had slightly low mean SBS value (10.37MPa) compared to 10.50MPa in PA group and 10.09MPa in GDL group. This might be due to the slow rate of crosslinking of GE, but the difference was nonsignificant. FTIR spectroscopy of grape seed PA in the present study revealed that crosslinking ability of PA was stronger compared to GE due to the presence of hydroxyl groups.³³ The hydroxyl groups readily form hydrogen bonds with carbonyl groups of amino acids of type 1 collagen. The acidity of hydroxyl groups results in the formation of negatively charged phenoxide ion which is capable of forming ionic and covalent bonds with amino acids.³⁴ Crosslinking of PA is superior in terms of multiple bond formation with collagen and reduction in hydrophilicity of dentin³⁵ whereas efficiency of GDL to form covalent bonds with amide groups in collagen is directly related to its concentration.³⁶ GE forms hydrogen bonds at the first instance. However, covalent bonds are also formed as a secondary mechanism that might explain the slow cross-linking behaviour of GE in which ester group of GE is substituted for amide group of collagen.³⁷ Study of fracture pattern revealed that PA group had maximum number of cohesive failure in the dentin. This might indicate that the resin-dentin interface was stronger than the dentin substrate itself. Similarly, an adhesive failure that occurred at the interface whether above or below the hybrid layer was maximum (75%) for samples in the control group. The resin-dentin interface of individual groups revealed that PA group showed maximum resin penetration among the four groups that were clearly evident by the presence of long uniform and continuous resin tags. The GE group showed very thin tags, while GDL showed very definite but shallow tags. On the whole, penetration of experimental groups was better than the control group. The above findings may be helpful in highlighting the role of different crosslinking agents for promoting efficient and stable bonding at the resin-dentin interface. There is much to explore and study regarding the role of GE in improving the quality of hybrid layer formed between demineralised dentin and dental composite system. Moreover, research must be carried out to develop an exact concentration of primers PA and GE that can be incorporated into resin restorative procedures to give maximum bond strength results with negligible issues of biocompatibility. The effect of available crosslinkers on a variety of dentin types, for example, carious, fluorosed and sclerotic dentin, should also be established.

 

Conclusion

 

Chemical modification with collagen crosslinkers improved bond strength at the composite resin-dentin interface. Benefits of long-term durability can be achieved after an application time of as short as 60s following acid conditioning of tooth surface during composite resin restorative procedures.

 

Disclaimer: The text is part of an MPhil thesis.

Conflict of Interest: None.

Source of Funding: None.

 

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