Natural products can have an important role in caries control through their inherent biological abilities.
The aim of the study was to compare thel remineralizing potential of alcohol and freeze-dried aqueous miswak ( Salvadora persica ) extracts (M, MFD, respectively), propolis ethanolic extract (P) and chitosan-nanoparticles (Cs-NPs) based dental varnishes either without or with 5% NaF (MF, PF, CSF-NPs) to 5% NaF varnish in primary teeth enamel samples with artificial enamel lesions.
F − , Ca ++ , PO 4 −− ions release was assessed at 1,2,4 and 24 h. Surface microhardness, topography, and mineral content were assessed for primary teeth enamel before and after enamel lesion formation and after treatment and pH cycling using Knoop microhardness and SEM-EDX analysis.
F − was significantly released with NaF and MF, MFD varnishes; Ca ++ and PO 4 −− were significantly released by Chitosan followed by miswak varnishes, while propolis varnishes released the least amount of ions. After treatment of enamel lesions and pH cycling, F − was significantly recovered by NaF, MF, and CSF-NPs descendingly, while, Ca ++ , PO 4 −− and surface microhardness were significantly gained by chitosan-nanoparticles and miswak varnishes.
Chitosan-nanoparticles and miswak containing varnishes were most effective in remineralizing enamel lesions probably due to the release of F − , Ca ++ , PO 4 −− ions compared to NaF varnish that released F − only.
Fluoride varnishes have the advantage of delivering high fluoride concentration to tooth surfaces for long periods thus increasing fluoride uptake by enamel and amount of precipitated CaF 2 globules that act as mineral reservoirs releasing fluoride during cariogenic challenges. However, bioavailability of fluoride (F − ), inorganic phosphate (PO 4 −− ) and calcium (Ca ++ ) ions that constitute the hydroxyapatite crystals in the oral fluid pathing the teeth is essential to achieve complete remineralization. Hence, many of newly marketed fluoride varnishes contain calcium and phosphate ions . Natural products may have antimicrobial and/or remineralizing abilities. Moreover, their safeties, availability, lack of bacterial resistance and low cost, have attracted much attention to their potential uses . Those products may be useful for caries control in countries with limited financial resources where many of them are available at low costs.
Propolis, miswak and chitosan are natural products with many biological activities. Their antiplaque and antimicrobial effects against oral pathogens have been reported . Interestingly, those natural products may also have remineralizing potential. Miswak extract is rich in calcium, while its fluoride content is low . Propolis has different amounts of calcium and phosphorus according to its phytogeographic origin . While chitosan nanoparticles, due to their nontoxicity and biodegradability, are used as drug delivery vehicles through encapsulating nanoparticles of biologically active drugs. Chitosan degradation releases the loaded drug, this together with its film forming ability can lead to sustained release effect. Moreover, the nanosized structure may enhance the bioavailability and penetrability of the carried drug through biological membranes . Varnishes containing those natural products in combination with NaF inhibited bacterial demineralization when primary teeth enamel was pretreated and the varnishes were removed before inducing bacterial demineralization. Varnishes containing natural products without NaF also inhibited subsequent enamel demineralization to some extent, possibly due to release of minerals from those varnishes slowing down the demineralization process . Thus, in the present study, the ion release profile from those varnishes and their remineralizing ability were assessed.
Materials and methods
Fig. 1 represents a flow chart of the study methodology.
Preparation of experimental varnishes
Preparation of experimental varnishes as well as 5% NaF varnish was discussed in details in a previous study . The aqueous miswak extract was freezed dried and the powder was used in varnish preparation. While, the ethanolic misak extract required long periods of freeze drying and yielded small amount of powder so it was used in its aqueous form. Table 1 shows the ingredients of the eight experimental varnishes (V1 V8).
|Varnish||Solvent (mL)||Distilled deionized water (mL)||Colophony resin (g)||NaF (ALPHA CHEMIKA, Mumbai, India) (g)||Other ingredients (g)|
|V1 (M)||Miswak ethanolic extract varnish||75 mL of 10% miswak ethanolic extract||25||20||–||–|
|V2 (MF)||Miswak-fluoride varnish||75 mL of 10% miswak ethanolic extract||25||20||5||–|
|V3 (MFD)||Freeze dried aqueous miswak extract varnish||75 mL of 95% ethanol (Perfect Chemical, Cairo, Egypt)||25||20||–||10 g freeze dried aqueous miswak extract|
|V4 (P)||Propolis varnish||75 mL of 95% ethanol (Perfect Chemical, Cairo, Egypt)||25||20||–||10 g of 10% EEP|
|V5 (PF)||Propolis-fluoride varnish||75 mL of 95% ethanol. (Perfect Chemical, Cairo, Egypt)||25||20||5||10 g of 10% EEP|
|V6 (CS-NPs)||Chitosan-NPs varnish||25 mL of 2% acetic acid. (Perfect Chemical, Cairo, Egypt)
75 mL of 95% ethanol. (Perfect Chemical, Cairo, Egypt)
|–||20||–||10 g CS-NPs powder
(Nanotech for Photo Electronics, Giza, Egypt)
|V7 (CSF-NPs)||Sodium fluoride loaded chitosan-NP varnish||25 mL of 2% acetic acid. (Perfect Chemical, Cairo, Egypt)
75 mL of 95% ethanol.
|—||20||—||10 g CSF-NPs.
(Nanotech for Photo Electronics, Giza, Egypt)
|V8 (NaF)||Sodium fluoride varnish||75 mL of 95% ethanol (Perfect Chemical, Cairo, Egypt)||25||20||5||—|
Preparation of enamel specimens
Seventy sound second primary molars (exfoliated or extracted for orthodontic reasons) were sectioned mesiodistally into 2 equal halves. The enamel and dentin surfaces of each halve were painted with an acid resistant varnish except for a 5 × 5 mm enamel window . Surface microhardness’ (SMH) specimens were embedded in polymethyl methacrylate and then enamel surface was ground flat and hand polished using aqueous slurries of progressively finer grades of silicon carbide papers up to 4000 grit prior to being coated with the acid resistant varnish.
Ion release analysis
Fifty enamel specimens were randomly assigned to 8 treatment groups (V1to V8) where specimens were coated with 10 μl of the corresponding varnish, and 2 control groups (n = 5). In (control 1), specimens were coated with varnish base only (ethanol, water and colophony); while in (control 2), uncoated enamel specimens served as negative control. After 1 min, each specimen was placed in a screw capped polyethylene tube containing 25 mL deionized water (pH 7.5) at room temperature . Specimens were transferred to new tubes containing 25 mL deionized water after 1, 2, 4, and 24 h of exposure. Ca ++ concentration was measured by atomic absorption spectroscopy (SavantAA, GBC Scientific Equipment, USA) , while PO 4 −− and F − were measured by ion chromatography (ICS-1100, Thermo Dionex, USA) . Ion concentration was determined by the average of two readings and expressed in mg/L. Both the rate and cumulative ion release were assessed.
Surface microhardness (SMH)
SMH specimens were randomly divided into 8 treatment groups and one control group which received no treatment (n = 5). Enamel lesions were made by immersing each specimen in10 mL of demineralizing solution (0.05 M acetic acid , 2.2 mM NaH 2 PO 4, 2.2 mM CaCl 2, pH was adjusted to 4.4 by 1 M KOH) for 96 h, followed by distilled water for 30 s . The specimens were then covered with 10 μl of the corresponding varnish, and then immersed in deionized water (10 mL/specimen) immediately. After 4 h (which is the maximum duration that is recommended by some manufacturers to wait before resuming eating to allow for F − transfer into enamel) , the varnishes were gently removed and specimens were subjected to pH cycling for 20 days . Each cycle involved 4 h of immersion in the demineralizing solution, 14 h of immersion in remineralizing solution (1.5 mM CaCl2, 0.15 M KCl, 0.9 mM NaH2PO4, at pH 7.0), followed by deionized water for 6 h. Fresh solutions were prepared daily and their pH was checked before each cycle. In each group, SMH was measured at three points; before formation of enamel lesion (SMH sound ), after enamel lesion formation (SMH lesion ), and after varnishes application and pH cycling (SMH Treatment ) . The specimens were indented with Vickers diamond indentor (NEXUS 400, INNOVATEST, Maastricht, The Netherland). Three indentations were performed per specimen at 200 g load with a dwell time of 15 s. The average of the three scores was obtained for each specimen. The percentage of SMH recovery (%SMHR) was calculated as follows : %SMHR = 100[(SMH Treatment – SMH lesion )/(SMH sound – SMH lesion )].
Scanning electron microscope (SEM) and energy dispersive X-ray (EDX)
Enamel specimens were randomly assigned into 8 treatment and one control (no treatment) groups; (n = 5). Enamel lesion formation, and remineralization with each of the test varnishes were the same as for SMH. Thereafter, a pH cycling protocol was carried out as described before. Specimens were examined with environmental SEM (Quanta 250 FEG, FEI company, Netherlands)) attached with energy dispersive X-ray analyzer (SEM-EDXA) with accelerating voltage of 30 KV, magnification 14× up to 10 6 and gun resolution of 1 nm. Surface Ca ++ , PO 4 −− and F − weight percent (wt%) were assessed for sound enamel (EDX sound ), after enamel lesion formation (EDX lesion ), and after treatment and pH cycling (EDX Treatment ). Three readings per specimen were recorded at each measurement and an average obtained .The percentage of mineral recovery for each ion as well as for the three ions together (total mineral recovery) was calculated as follows: % mineral recovery = 100[(EDX Treatment – EDX lesion )/(EDX sound – EDX lesion )].
Data were checked for normality using Kolmogorov – Smirnov and Shapiro-tests and were found to be normally distributed. Means were compared using One way Repeated measure ANOVA. While, post-Hoc Tukey test was performed for pairwise comparisons. The significance level was set at P ≤ 0.05.
Cumulative ion release
Except for the first hour, MF had the highest cumulative F − release, followed descendingly by NaF, MFD, CSF-NPs, PF, CS-NPs, M, P. For PO 4 −− , Chitosan containing varnishes had the highest release, followed by miswak varnishes, while propolis varnishes and NaF varnish released traces. Ca ++ was significantly released by Chitosan varnishes, followed by MFD. MF and M varnishes released comparable amounts of Ca ++ which were significantly lower than those of MFD at all time points. Propolis varnishes released the lowest Ca ++ followed by NaF, Table 2 . No ions were detected in both control groups.
|Ion (mg/L)||Varnish||Storage Time (h)||P-value|
|Mean (Sd)||Mean (Sd)||Mean (Sd)||Mean (Sd)|
|F −||Chitosan nanoparticles (CS-NPs)||0.17 (0.007) Ae||0.316 (0.015) bE||0.438 (0.022) Ce||0.438 (0.022) cE||0.04*|
|Chitosan Fluoride Nanoparticles (CSF-NPs)||0.7 (0.043) Ad||1.258 (0.083) bD||1.552 (0.142) Cd||1.722 (0.169) dD||0.001*|
|Miswak Alcohol Extract (M)||0.226 (0.011) Ae||0.265 (0.02) bE||0.289 (0.029) cE||0.289 (0.029) cE||0.006*|
|Miswak Fluoride (MF)||5.878 (0.229) aB||10.96 (0.385) bA||15.23 (0.498) Ca||18.142 (0.8) dA||0.001*|
|Miswak Freezed dried (MFD)||2.678 (0.284) aC||4.472 (0.463) bC||5.038 (0.745) cC||5.146 (0.777) dC||0.001*|
|NaF||6.5 (0.43) aA||8.82 (0.858) bB||10.14 (1.139) cB||10.98 (1.346) dB||0.033*|
|Propolis (P)||0.02 (0.01) aE||0.03 (0.016) bE||0.036 (0.021) bE||0.04 (0.025) bE||0.048*|
|Propolis Fluoride (PF)||0.152 (0.015) aE||0.494 (0.03) bE||0.718 (0.036) cDE||0.98 (0.355) cDE||0.002*|
|PO 4 −−||Chitosan nanoparticles (CS-NPs)||2.002 (0.057) aA||2.984 (0.069) bA||3.482 (0.103) cA||3.482 (0.103) cA||0.0001*|
|Chitosan Fluoride Nanoparticles (CSF-NPs)||1.76 (0.312) aB||2.59 (0.311) bB||2.702 (0.318) cB||2.71 (0.326) cB||0.011*|
|Miswak Alcohol Extract (M)||0.152 (0.019) aCD||0.282 (0.035) bCD||0.474 (0.061) cC||0.584 (0.078) dC||0.005*|
|Miswak Fluoride (MF)||0.184 (0.013) aCD||0.326 (0.037) bC||0.443 (0.062) cC||0.509 (0.085) dC||0.027*|
|Miswak Freezed dried (MFD)||0.257 (0.13) aC||0.467 (0.142) bC||0.565 (0.156) cC||0.615 (0.162) dC||0.005*|
|NaF||0.008 (0.008) aD||0.012 (0.013) aE||0.012 (0.013) aD||0.012 (0.013) aD||0.178|
|Propolis (P)||0.036 (0.004) aCD||0.064 (0.006) bDE||0.1 (0.024) cD||0.126 (0.028) dD||0.001*|
|Propolis Fluoride (PF)||0.056 (0.024) aCD||0.078 (0.037) bDE||0.08 (0.041) bD||0.08 (0.041) bD||0.038*|
|Ca ++||Chitosan nanoparticles (CS-NPs)||0.478 (0.019) aA||0.86 (0.055) bA||1.146 (0.072) cA||1.202 (0.112) cA||0.0001*|
|Chitosan Fluoride Nanoparticles (CSF-NPs)||0.498 (0.015) aA||0.896 (0.029) bA||1.21 (0.043) cA||1.312 (0.109) dA||0.0001*|
|Miswak Alcohol Extract (M)||0.27 (0.066) aC||0.474 (0.116) bC||0.614 (0.132) cC||0.714 (0.185) dBC||0.003*|
|Miswak Fluoride (MF)||0.27 (0.052) aC||0.486 (0.098) bC||0.664 (0.127) cC||0.788 (0.167) dB||0.019*|
|Miswak Freezed dried (MFD)||0.414 (0.021) aB||0.66 (0.047) bB||0.942 (0.09) cB||1.126 (0.113) dA||0.014*|
|NaF||0.008 (0.008) aE||0.01 (0.012) aE||0.01 (0.012) aE||0.01 (0.012) aE||0.374|
|Propolis (P)||0.136 (0.009) aD||0.248 (0.016) bD||0.376 (0.029) cD||0.54 (0.043) dCD||0.003*|
|Propolis Fluoride (PF)||0.108 (0.016) aD||0.198 (0.028) bD||0.354 (0.035) cD||0.454 (0.047) dD||0.007*|