The therapeutic agent such as propolis containing gel was prepared by dispersion of 0.2 gm in glycerol (5% w/w) (1 ml) using a mortar and a pestle following the earlier reported generic protocol. [6] Ten milliliters of glacial acetic acid (3% w/w) was then added with continuous mixing and finally nanodiamond:chitosan (10% nanodiamond:chitosan w/w) polymer was spread on the surface of the dispersion and mixed well to form the required gel. The strength of the prepared gel (10 gm) is 0.2 g of propolis in each gram of the base. The summary of the newly prepared materials was presented in Table 1.

Bioadhesion studies were done using Chatillon apparatus for force measurement. [7] This method determines the maximum force and work needed to separate two surfaces in intimate contact [8] . The hydrogels (0.1 g) were homogeneously spread on a 1 cm² glass disk and then the disks were fixed to the support of the tensile strength tester using double side adhesive. The gel was brought into contact with the contact with slice of dentin was established in order to imitate adhesion of the gel to the tooth structure, after a preset contact time (1 min) under contact strength (0.5 N) the 2 surfaces were separated at a constant rate of displacement (1 mm/s). The strength was recorded as a function of the displacement, which allowed to determine the maximal detachment force, Fmax, and the work of adhesion, W, which was calculated from the area under the strength-displacement curve.

The Folin Ciocalteu assay determination was carried out by enzymatic method spectrophometrically, according to the procedures previously described and results are expressed as catechin equivalents (CE) [9] .

One gram of the prepared gels was accurately weighed and dispersed in 10 ml of purified water. The pH of the dispersions was measured using pH meter (HANNA instruments, HI8417, Portugal).

The samples were prepared by freezing in liquid nitrogen for 10 min, and then were freezing-dried for 24 h. The prepared samples were fractured in liquid nitrogen using a razor blade. The fractured samples were attached to metal stubs, and sputter coated with gold under vacuum for SEM. The interior and the surface morphology were observed in scanning electron microscope (SEM, Hitachi S4800, Japan).

Stability of the gel formulations was also investigated. The organoleptic properties (color, odor), pH, drug content, and release profiles of the gels stored at 20˚C were examined on days (0, 15, and 30).

The known weight nanodiamond-containing dry gels and naproxen-containing dry gels were immersed in pH

Where: Hydrogels containing nanodiamond:chitosan (5%) are synthesized and characterized.

4.0, pH 9.0 buffer solutions, respectively, and kept at 25˚C for 48 h until equilibrium of swelling had been reached.

The swollen gels were taken out and immediately weighed with microbalance after the excess of water lying on the surfaces was absorbed with a filter paper. The equilibrium swelling ratio (SR) was calculated using the following equation:

where W_s and W_d are the weights of the gels at the equilibrium swelling state and at the dry state, respectively. [9] Experiments were repeated 6 times for each gel specimen and mean value was obtained.

Extracted non-carious, intact, human molars stored in water containing a few crystals of thymol at 4˚C were used within two months. Samples were checked before use for any damage caused by their removal. The roots of the teeth were removed with a separating disc and the occlusal enamel removed by grounding wet on 60-grit silicon carbide (SiC) paper. The teeth were embedded in PVC (Consjit Tubing, SA PVC, JHB, RSA) pipe containers with cold cure acrylic resin so that the grounded occlusal surfaces projected well above the resin. The 10 mm length pipes were put on a glass surface with one end blocked by the glass and the embedding done through the open end. Immediately after embedding the occlusal surfaces were ground wet with 180-grit followed by 600-grit SiC on a polishing machine to expose the superficial dentin. The samples were washed under a stream of tap water. A standardized zig (Ultradent ISO A2-70) with an internal diameter of 2.5 mm and height of 3 mm was used to shape the composite resin stud (SDR, Dentsply, CA, USA, Batch number 1105000609, Exp 2013-04). Two of these studs were then bonded to the polished dentin surface of each tooth via the bonding agent XP bond (Dentsply, New York, USA) using the following methodology: Groups C-E: Gels (1 - 4) (0.01 g, 10 seconds) + primer + Bonding immediately) or Groups K-N Gels (1 - 4) (0.01 g, 10 seconds) + Bonding immediately) The bonding agent contained: carboxylic acid modified dimethacrylate (TCB resin), phosphoric acid modified acrylate resin (PENTA), urethane dimetacrylate (UDMA), triethyleneglycol dimethacrylate (TEGDMA), 2-hydroxyethylmethacrylate (HEMA), butylated benzenediol (stabilizer), ethyl-4-dimethylamino-benzoate), camphorquinone, functionalized amorphous silica, t-butanol.

In this way were 80 teeth samples (each containing 2 studs) prepared and divided into 10 groups of 8 each, A-L (Table 2) and stored in a solution of artificial saliva. These groups were then treated as outlined in Table 2. After 24 hours one stud of each tooth was tested for shear bond strength and the other one after 3 months. An Instron Universal Testing Machine at a crosshead speed of 0.5 mm/minute was used to test the de-bonding strength. All data tests were analysed using the non-parametric ANOVA test.

Figure 3 and Figure 4 gives the shear bond strength values (MPa) after 24 hours (Figure 3) and after 3 month of storage of samples in artificial saliva (Figure 4) using conventional dental adhesive systems such as 3 bottle etch/prime/bond system (standard 1) and 2 bottle self etch/prime and bond system (standard 2).

Mean shear bond strength values and difference between the groups are summarized in Figure 3 for bonding to dentin after 24 hours and in Figure 4 for bonding after 3 month. In general there was an increase in bond strength of the dentin treated with the propolis containing nanodiamond:chitosan hydrogels compared to the bond strength of the conventionally bonded teeth. An increase in the shear bond strength was also previously reported [11] for chitosan containing hydrogels. Interestingly the increase in bond strength was also observed in the groups of hydrogen peroxide exposed samples suggesting that there additional benefits associated with nanodiamond:chitosan:propolis system are in need of further investigations [12] .

The results of this study suggests that the optimum results for the strengthening of dentin can be achieved throughout the immediate treatment with propolis:chitosan:nanodiamond with the increase of dentin bond strength. Also, impressively an almost immediately after the corresponding gel treatment and proceeding with bonding procedures is recommended with the significant increase in bond strength. The additional advantage of the system may suggest that, chitosan:propolis and nanodiamond interection with crystalline hydroxyapatite structure of the dentin layer increases the dentin bond strength observed especially in the case of the direct bonding between the hydrogel and the dentin interface. The increase of the bond strength is also attributed to the unique capacity of chitosan as well as biological properties of propolis to influence bioadhesion of the hydrogels and promote the formation of the reparative hydroxyapatite interface and therefore starting all important hydroxyapatite regeneration process making these type of designer bioactive hydrogels as important prototypes in development of bioactive restorative materials.

The additional benefit of using chitosan:antioxidant system as a bonding/pre-bonding to enamel and dentin system lies in its ability to show favorable immediate results in terms of bonding effectiveness as well as the durability of resin-dentin bonds for a prolonged time (up to 6 months) [13] . It is well documented that the hydrostatic pulpal pressure, the dentinal fluid flow and the increased dentinal wetness in vital dentin can affect the intimate interaction of certain enamel and dentin adhesives with dentinal tissue. There for the newly developed chitosan:antioxidant systems might at least be able to address the shortfalls in the current perspectives for improving bond durability through understanding factors affecting the long-term bonding performance of modern adhesives and addresses the current perspectives for improving bond durability.

On the basis of calibration curve for standard flavonoids, the concentration of these bioactive compounds in the hydroalcoholic extracts of the propolis sample used in the study was assessed. The content of flavonoids of the propolis was 1.73% ± 0.35% (Green Brazilian Propolis), 1.65% ± 0.25% (Uruguyan Propolis) and 1.28% ± 0.43% (Australian Propolis) which is consistent with the previously reported values for a hydroalcoholic extracts of the propolis in the literature [14] . The flavonoid content of the propolis extracts is the major contributing factor in biological activity of the propolis. The biological effects of flavonoids can be subdivided into 4 categories: 1) binding affinity to biological polymers, 2) binding of heavy metal ions, 3) catalyst of electron transport and 4) ability to scavenge free radicals [15] . The proposed mechanism of the propolis and its ability to mediate the inflammatory response in vivo have been previously explored in some details and suggested that the healing properties of propolis can be directly correlated with anti-oxidant potential as well as anti-inflammatory properties of flavonoids [16] . The work is currently on the way in our laboratories to evaluate the mechanism in vitro and in vivo in great details.

The amount of uncontrolled ROS is the main cause of the inability of the healing process to continue, and therefore, it would be ideal to utilize the antioxidant capacity of the “designer hydrogels” to detect and be able to “fight the free radical excess”. This has been assessed using a previously described model, where the HO radical can be generated from a reaction known as the biologic Fenton reaction, and this reaction requires the presence of H₂O₂ [17] . Bovine serum albumin (BSA), a completely water-soluble protein, was polymerized by hydroxyl radicals generated by the Fenton reaction system of Fe²⁺/EDTA/H₂O₂/ascorbate [18] . As a result, the protein loses its water-solubility and the polymerized product precipitates. The decrease in the concentration of the water-soluble protein can easily be detected.

We considered it worthwhile to study the chitosan: as a “built-in defence mechanism” for the in vitro-gener- ated free radical production and “site-specific” in vitro model counter reaction of the hydrogel. Therefore, we adopted a method for recording changes in the water solubility of the model protein, bovine serum albumin (BSA), exposed to free radicals generated by an inorganic chemical system. As clearly demonstrated by Figure 5 and Figure 6, upon exposure to standard H₂O₂ in the form of Fe²⁺/EDTA/H₂O₂/ascorbate solution as a baseline determinate for free radical generation under “prototypical in vitro free radical damage”, upon incorporation of the chitosan substituted hydrogels, the built-in antioxidant capacity and, therefore, free radical defence of the in vitro model have been activated and are of a significant value for which notice should be taken. This model represents the practical approach of in situ monitoring and testing of the amount of the free radical production and synergistic antioxidant defence of the system. Further investigations with fine-tuning of the system are currently on the way in our laboratory.

As clearly demonstrated by Figure 5 and Figure 6, upon exposure to standard H₂O₂ in the form of Fe²⁺/ EDTA/H₂O₂/ascorbate solution as a baseline determinate for free radical generation under “prototypical in vitro free radical damage”, upon incorporation of the chitosan substituted hydrogels, the built-in antioxidant capacity and, therefore, free radical defence of the in vitro model have been activated and are of a significant value for which notice should be taken. This model represents the practical approach of in situ monitoring and testing of the amount of the free radical production and synergistic antioxidant defence of the system. Further investigations with fine-tuning of the system are currently on the way in our laboratory.

The higher adhesiveness of the gels is desired to maintain intimate contact with the tooth structure, and the results are summarized in Table 3. Nanodiamond:chitosan hydrogels showed the highest adhesive force and work of adhesion, which can be expected, because of the well-known intrinsic bioadhesive properties of chitosan

[19] -[22] . The adequate water absorption capacity together with the cationic nature, which promotes binding to the negative surface of dentin structure, can also interpret these results.

Propolis containing hydrogels have shown the highest adhesive force and work of adhesion in comparison the nanodiamond:chitosan hydrogels which are not containing the propolis aditives. This can be expected, because of the well-known intrinsic bio-adhesive properties of chitosan and propolis. The adequate water absorption capacity together with the cationic nature, which promotes binding to the negative surface of skin and the dentin structure, can also be used to interpret these results. It is well documented that hydration of the polymer causes mobilization of the polymer chains and, hence, influences polymeric adhesion [23] . Appropriate swelling is important to guarantee adhesivity; however, over-hydration can form slippery non-adhesive hydrogels [23] . In addition, for the molecular arrangement of the polymeric chains, which are present in the new hydrogels, such as with propolis, the correlation between the force and work of adhesion is noticeable for all. Further experiments are to be conducted on skin samples to further evaluate the bio-adhesive capacity of the designer hydrogels.