Effect of different pH solvents on micro-hardness and surface topography of dental nano-composite: An in vitro analysis

Objective: Erosion of tooth surface is attributed to recent shift in diet pattern and frequent use of beverages. The aim of this research was to evaluate the effects of different beverages on surface topography and hardness of nano-filled composite material. Methods: Sixty flat disc shaped resin composite samples were fabricated and placed in distilled water for 24 hours. After 24 hours test samples were dried and divided into 4 groups. Group A (n=15) specimens were placed in tight amber bottle comprising 25 ml of artificial saliva. Similarly Group B, C and D were stored in equal amounts of orange juice, milk and coca cola drink respectively. Samples were checked for hardness and surface changes were evaluated with scanning electron microscopy. Results: There were strong significant difference observed in samples immersed in orange juice and artificial saliva. A strong significant difference was seen between Group D and Group A. Group A and Group C showed no significant difference. The micro-hardness test showed reduced values among all samples. Conclusion: Beverages consumed daily have a negative influence on hardness and surface degradation of nano-filled dental composite. Comparatively, nano-filled composites possess higher surface area to volume ratio of their fillers particle size may lead to higher surface roughness than other resin based dental biomaterials.

for treatment of patients, who are seeking for esthetic and therapeutic treatment. Presently, resin composite show properties that are near to natural teeth. 3 Since their improvement in the late 1950's resin composites are widely accepted for bearing acceptable aesthetics properties and bond directly to tooth structure by preserving healthy tissues. Due to its good bonding ability it is widely accepted in preventive and conservative dentistry. 4 Resin composite usually comprise of methacrylate polymers such as bisphenol Adiglycigyl methacrylate and tetraethylene glycol dimethacrylate. The filler particles and resin are bonded covalently by a coupling agent commonly known as 3-(trimethoxysilyl) propyl methacrylate (MPS), imparting better mechanical properties. 5 Degradation in the oral cavity is a complex phenomenon that is related to disintegration and dissolution of restorative materials in the oral cavity. The other types of chemical/physical degradation, such as wear and erosion occur due to food, chewing and microbes. Resin composites undergo a drastic change of physical and chemical conditions in the oral cavity, including temperature changes, different masticatory stresses and chemicals from food. These factors have immense effect in vivo degradation or failure of dental composites. Exposure to various kinds of acidic solutions results in restoration failure. Researchers have claimed that exposure of resin composite to low-pH liquids can have a deleterious effect on their mechanical properties. 6 To overcome these shortcomings of resin composite materials, a new class of dental composites have been introduced lately called nanocomposites. 7 Nano composites are considered to have advanced mechanical properties bearing good compressive strength, and better chemical and physical properties. 8 Hydrolysis is a chemical process that splits the covalent bonds between the polymers by adding water to ester bonds, leading in loss of resin mass and is quoted as one of the main reason of degradation of resin within the hybrid layer. 9 Resin composites possess polymer network that is mostly inclined to hydroscopic and hydrolytic effects to varying extents, that is dependent on their chemical structure. These effects will not only affect their service life but they may also result in short term release of unreacted components as well as long term leaching of degradation products in the oral cavity. 10 However, it was observed that leaching of different components combined with swelling and degradation of the cross linked network in resin composite and hydrolysis of filler-matrix interface finally results in decrease in mechanical properties. Degradation of resin composite may lead to formation of micro cracks via repeated sorption/ desorption cycles that in turn is related with hydrolytic degradation of polymeric network. 11 Average daily requirement to consume water is 2 to 3 liters. In developed nations more than half comes from soft drinks and fruit juices. Excessive contact of tooth with acidic drinks for a long period results in loss of hard tissues due to their low pH and highly acidic nature will eventually lead to change in surface texture. 12 The components of acids is also responsible for the surface nano-indentations of specimens. Orange juice comprise of citric acid whereas coca cola contains phosphoric acid. In a recent study by Fan et al., revealed that the surface features of specimens immersed in orange juice were more affected than those compared with specimen aged in distilled water or cola drink. 6 Erosion of tooth surface is attributed to recent shift in diet pattern and frequent use of beverages. 13 Researchers report that enamel micro-hardness lowered just after one hour immersion in coca cola. Surface hardness has been used as an indirect technique to measure polymerization adequacy. 14 In another study researchers 15 have quoted that the type of immersion solutions and the composition of soaked materials are important factors related to dissolution of dental composite materials. On the other hand, factors such as the solubility parameter, the cross-linking nature of resin matrix, and the solvent sorption uptake may directly influence polymer degradation rate. As milk, orange juice and cola drink are consumed by every other person of different age groups so the purpose of our study was to assess the erosive potential of these commonly consumed beverages on the surface of composite material using the micro-hardness testing and scanning electron microscopy. Although limited number of studies have been previously done yet the uniqueness of this study is the surface changes observation under scanning electron microscopy.

METHODS
The composite resin used for preparing the 60 samples of 7 mm diameter and 2 mm thickness was Te-Econom Plus (IvoclarVivadent, UK). Circular Teflon mold 7 mm in diameter and 2.0 mm in thickness was used. Mylar strip (Dentart, Polidental, Sao Paulo, Brazil) having a dimensions (10 x 120 x 0.05 mm) was placed over the top and bottom of the mold and pressed on top with a microscope slide 22 x 22 mm (BDH borosilicate glass) to form a flat surface of the sample. Light curing unit Quartz Tungsten Halogen (401™ Demetron Research Corporation, Danbury, CT, USA) with light intensity of 550 W/cm 2 was held rigidly and placed 1.0 mm over the glass slide for 40 seconds to cure down the dental composite samples. With the help of 3M Sof-Lex disks specimens were polished to get a clinical finish. These fabricated specimens were placed in distilled water for 24 hours for post irradiation hardness. After 24 hours specimens were dried and divided into 4 groups i.e. Group A (n=15) specimens were kept in tight amber coloured bottle containing 25 ml of artificial saliva; similarly Group B (n=15), C (n=15) and D (n=15) specimens were stored in equal amount of orange juice, milk and coca cola drink respectively. Before storing the specimens at 37±3°C for 14 days in an incubator (Sanfa DNP-9052, China), the pH of all the solvents were measured by using pH meter (Model no. 98129 Hanna Instruments, USA). The immersing solutions of all the groups were changed after every 24 hours. The quantity of 25ml solution was maintained in all the groups.
After 14 days specimens were removed from the solutions and dipped into cleansing solution containing 10 ml soap and 990 ml distilled water. Later flushed under running tap water and subsequently dipped into distilled water for one minute. Fluid on the surface of the specimens were removed by using absorbing paper. Specimens were ready for micro-hardness testing. Preparation of Artificial Saliva: The artificial saliva used in this study was of the following composition: Sodium chloride (NaCl) 0.400 g; Potassium chloride (KCl) 0.400 g; Calcium chloride monohydrate (CaCl 2 H 2 O) 0.795 g; Sodium dihydrogen phosphate (NaH 2 PO 4 ) 0.69 g; Disodium sulphide hydrate (Na 2 S x 9H 2 0) 0.005 g; Urea 1.0 g; Distilled water 1000 ml. The pH was then adjusted to 6.9 with Sodium hydroxide (NaOH) or Hydrochloric acid (HCl). Preparation of Experimental Solvents: Fresh orange juice, milk and coca cola soft drink was used.

Scanning Electron Microscopy (SEM) Analysis:
Surface changes were observed using the scanning electron microscopy (JEOL-JSM; 6460LV, Tokyo, Japan) at 20KV accelerating voltage. Vicker's Micro-hardness Test: Micro-hardness was measured for all the 40 samples of control and testing groups. Each sample of Group A i.e. samples immersed in artificial saliva; Group B immersed in orange juice; Group C immersed in milk; and samples of Group D immersed in coca cola were employed a 50 gm load for 30 seconds dwell time. Each sample was indented for 3 times to get the mean value of the sample. In this way mean value of each group obtained. Microhardness values of the specimens were recorded using Vicker's micro hardness tester (MMT -X7 Matsuzuwa, Japan). Statistical Analysis: Data was entered in Statistical Package for Social Sciences (SPSS) version 19. Descriptive analysis was executed in the form of mean ± standard deviation for surface microhardness. p-value of p < 0.05 were considered to be significant. Significant differences are represented by *P < 0.05. The level of significance (P) was calculated with the help of repeated measure ANOVA. For multiple comparisons, Tukey's multiple comparison test was used.

RESULTS
The pH of different experimental solvents used in this study are given in Table-I. The details of artificial saliva and the ingredients present in the solvents used in this study are also present in Table-I. There was a strong significance difference between Group B and Group A; a strong significance difference was also observed between Group D and Group A, and there were no statistical significant difference seen between Group A and Group C.
The micro-hardness of the experimental material was reduced in all the groups. The micro hardness values from highest to lowest were found as follows: Group A followed by Group C then Group B and lowest in Group D. Table-II

DISCUSSION
Erosion of a resin based restorative material is a chemical process without the involvement of any micro-organisms. Erosion could be intrinsic, extrinsic or both. Consumption of easily and widely available beverages are the potential sources of resin based composite's erosion and degradation. 12 These beverages and drinks contain weak acids that are erosive to tooth structure. Our saliva normally has a pH of around 6.8. Whereas pure orange juice has a pH of 3.5 and that of coca cola 2.5. Milk's pH is somewhere close to pH of saliva. Studies have shown that any drink having a pH of 5.5 or below will wear away the enamel. 16 How long the drink stays in contact with the teeth, buffering power of the saliva, and the pH of the drink are the deciding factors in the erosion process of the tooth surfaces. Like natural tooth, effect of these acidic drinks on the restorative materials couldnot be undermined.
Sof-Lex finishing and polishing system was used because it provided the smoothest surface for composites. Before storing the specimens into experiemental solution they were stored in distilled water for 24 hours immediately after fabrication to achieve the maximum polymerization. 17 All specimens displayed colour changes after 14 days immersion. These colour changes were noticeable to the human eye without the aid of a UV-spectrophotometer. The specimens of Group B, C and D showed marked discolouration indicating surface roughness, degradation and penetration of water molecules; leading to sorption and hydrolysis of the material. The swelling of matrix eventually leads to hydrolysis of the material. 18 Immersion of samples in different kind of beverages containing weak acids had a profound effect on the surface deteriotion of a nano-filled composite particularly samples immersed into orange juice and cola cola drink. Matrix system present in experimental Effect of different pH solvents on dental nano-composites   material i.e. Te econom plus is of hydrophilicnature. The higher content of organic matrix in nano-filled composites could be the reason behind higher susceptibility to water absorption and material disintegration. 14 Since this nano-filled material had used the same polymeric matrix i.e. Bis-EMA, UDMA and TEGDMA, this result was expected and consistent with the other studies done. Irrespective of immersion medium, all specimens presented surface changes when observed under scanning electron microscopy at different magnifications. Several voids, cracks, roughness and protruding particles were observed in all specimens confirming the process of surface changes and erosion of the matrix. Although the pH of Group A specimens was 6.9 yet the artificial saliva is mainly composed of water and the effect of water uptake can degrade polymeric material. 18 The specimens of Group B & D showed strong significant difference comparing to Group A. This was expected as the specimens of Group B & D were stored in orange juice and coca cola drink for 14 days without the buffering factor of saliva. Saliva through buffering substances keep the pH near neutral and wash away acidic environment and limit the softening of the enamel surfaces. 19 Clinically, these beverages effects on polymeric composites may be different due to some factors such as acquired biofilm, dietary habits, and oral care products; which cannot be reproduced in vitro.These factors either acting alone or together may interfere with the physical and mechanical properties of the materials; influencing the durability of the polymeric resin composite. Although we daily consume acidic foods and drinks yet the composite materials show no immediate effect on their physical and mechanical properties. Excluding the samples of Group A, the samples of Group B, C and D all showed marked surface roughness and degradation (Fig. 2, 3 & 4). Samples of Group A shows surface roughness only (Fig.1). Morphological analysis of the composite material illustrates the values found in micro-hardness testing.
The findings of this study are in accordance with earlier studies by Maganur et al. 12 who found detrimental effects of low pH drinks on the longevity of a restorative material; significant reduction of microhardness of nano-filled composite materials when immersed in different beverages for 24 hours; 18 ZZ significant deduction in mean values of Vicker's hardness number due to immersion of composite specimens in cola, citric juice and wine. 20 In a recent Aftab Ahmed Khan et al.  Effect of different pH solvents on dental nano-composites study conducted by Cengiz et al., it was found out that acidic medium has the tendency to soften, thereby significantly roughening dental composite biomaterial; 21 and degradation of composite surface was also observed due to immersion in water, thermocycling and citric acid exposure. 22 In vitro environment cannot truly depict the natural conditions of the oral cavity. In our study the disregard for the effects of saliva buffering and thermocycling is a limitation. Future research needs to test more experimental designs that could depict the clinical behavior of the restorative material in vitro environment. This study at least confirms the erosive potential of certain weak acids present in different juices, drinks and milk; which are a potentially damaging factor that the public should be aware of.

CONCLUSION
Within the limitation of this study we can conclude that the weak acids present in different beverages consumed daily have a great effect on the hardness and surface roughness or degradation of the nanofilled composite material. Surface treatment, storage solution, material type and immersion time, light intensity, curing time and material thickness are important factors that influence the hardness value. Since the nano-filled composites contain greater surface area to volume ratio of their fillers particle system, this may cause them to suffer higher surface roughness or degradation as compared to other resin based materials.