Effect of Nano-Bioglass Addition to Calcium Hydroxide ... · 4. Calcium hydroxide-based cements,...

Global Scientific Research Journal of Dentistry

Effect of Nano-Bioglass Addition to Calcium Hydroxide Cements in Terms of Ions Release, Apatite Formation and Compressive Strength

Effect of Nano-Bioglass Addition to Calcium Hydroxide Cements in Terms of Ions Release, Apatite

Formation and Compressive StrengthShaymaa I Habib1*, Rania E Bayoumi2, Sabry A El-Korashy3 and Rasha M Abdelraouf1

1Assistant Professor of Dental Materials, Biomaterials Department, Faculty of Dentistry, Cairo University, Cairo, Egypt.2Lecturer of Dental Materials, Biomaterials Department, Faculty of Dentistry (Girls), Azhar University, Cairo, Egypt.

3Professor of Inorganic Chemistry, Faculty of Science, Ismailia, Suez Canal University, [email protected]

AbstractObjective: To evaluate the effect of the nano-bioglass addition to calcium-hydroxide cements regarding ions release, apatite formation and compressive strength.

Materials and Methods: Two calcium-hydroxide Ca(OH)2 liner materials were used; chemical and light-cured. Nano-bioglass (7wt%) was added to both cements. Specimens were prepared and classified as follows: Group I: Chemical-cured Ca(OH)2, and Group II: Light-cured Ca(OH)2. Each group was further subdivided according to the added bioactive glass into two subgroups a) control without nano-bioglass, and b) subgroup containing nano-bioglass. Forty disc-shaped-specimens were stored in distilled water at 37 oC (n = 10/subgroup). Half of the specimens in each subgroup were stored for 1 day, while the others for 7 days. The storage media was collected for pH measurement using pH-meter, and then calcium and phosphorus analysis using the atomic-absorption-spectrophotometry. For the apatite-forming ability, test was performed by soaking disc-specimens in phosphate-buffered-saline at 37 ºC (n = 6/subgroup). The endpoint times were 1 day and 7 days. Scanning-Electron-Microscope (SEM) and Energy-Dispersive X-ray-Analysis (EDXA) were used for microstructure and elemental analysis of the specimens’ surfaces. For the compressive-strength-test, twenty cylindrical specimens were prepared (n = 5/subgroup) and tested using universal-testing-machine. Statistical-analysis was performed and the significance-level was set at P≤0.05.

Results: After nano-bioglass addition, significant increases in pH values were recorded in both chemical-cured Ca(OH)2 and light-cured one in day 1 and day 7. Similarly, adding nano-bioglass caused significant increase in calcium-ions-release at day 1 and further increase at day 7 in both tested materials. Meanwhile, no significant difference in phosphorus-ions-release was recorded after nano-bioglass addition at days 1 and 7 in chemical cured Ca(OH)2 and at day 1 for light-cured one. SEM and EDXA showed different surface morphologies depending on the material type, addition of nano-bioglass and the immersion time. The compressive strength of the chemical-cured Ca(OH)2 was not significantly affected by nano-bioglass addition. Contrary, the light-cured one showed significant decrease in the compressive strength after nano-bioglass addition.

Conclusions: Although addition of nano-bioglass to Ca(OH)2 liner could decrease its compressive strength, yet, it improves the ions release and hence its bioactivity.

Keywords: Nano-bioglass, Calcium hydroxide, Ions release, Apatite formation, Bioactivity, Compressive strength

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Research Article Open Access

DentistryVolume 1 - Issue 1 - 14 pages

IntroductionPulp capping is a procedure which consists of the placement of biocompatible materials on pulp tissue

to preserve its vitality and stimulate the repair process via mineralized tissue formation1. It has long been rec-ognized that the traumatic exposure of the dental pulp



can be successfully treated with calcium hydroxide [Ca(OH)2] that produces calcified bridges across the exposed surfaces2. Conservative treatment of deep caries also has been performed for centuries using di-rect or indirect pulp capping3. The ultimate goal of pulp capping materials is to induce the formation of hard tissue via pulp cells4.

Calcium hydroxide-based cements, first used by Her-mann in 1920, have been considered the “gold stand-ard”5 against which all new pulp protection materials are compared6. Calcium hydroxide Ca(OH)2 powder can be mixed with water or incorporated into a resin-ous matrix7. Calcium hydroxide cements are available as chemical and light cured pastes7. They are able to release hydroxyl (OH) and calcium (Ca) ions upon dis-solution8. This release of hydroxyl ions promotes heal-ing by increasing local pH and antimicrobial action9, while the calcium ions play an important role in tissue mineralization and hard tissue formation by activating Ca-dependent adenosine triphosphate (ATP) as well as stimulating fibronectin gene expression10. The re-lease of these Ca ions is necessary for cell migration, proliferation and differentiation11; however, these ma-terials have delayed bioactivity12 as it has been report-ed that calcium-phosphate deposits were identified on

the surface of calcium hydroxide after 28 days of stor-age in simulated body fluid12.

The invention of bioactive glass by Larry Hench in 1969 launched the field of bioactive ceramics13. Bio-glass is composed of minerals that occur naturally in the body (SiO2, Ca, Na, and P)14. The oldest bioglass was based on a silicate network (45 wt% SiO2) in ad-dition to 24.5 wt% Na2O, 24.5 wt% CaO and 6 wt% P2O5

15. Bioglass can bond chemically with tissues and form a hydroxyapatite layer16, and thus the use of bio-active glass may improve the bioactivity of different dental materials17.

Therefore, the objective of this study was to evaluate the effect of the addition of nano-bioglass to different calcium hydroxide cements, specifically regarding ion release, apatite formation and compressive strength at different immersion time.

Materials and MethodsTwo commercially available calcium-hydroxide liner materials were used in this study: chemical and light cured (Table 1). The chemical cured Ca(OH)2 was presented as a two pastes system and prepared by mixing equal volumes of base and catalyst pastes on

Table 1: Materials Used in this Study

Material Commercial Product and Man-ufacturer/Laboratory Prepared Chemical Composition Lot Number

Chemical cured Ca(OH)2

Dycal ivory, dentsply, Germany

Base paste: disalicylate ester of 1, 3, butylene glycol, calcium phosphate, calcium tungastate,

zinc oxide and iron oxide

Calalyst base: Calcium hydrox-ide, ethyl toluene sulfonamide, zinc stearate, titanium dioxide,

zinc oxide and iron oxide

023407

Light cured Ca(OH)2Calcimol LC, Voco, Cuxhaven,

Germany

UDMA, Ca(OH)2, Barium Sulfate, 2-dimethylaminoethyl methacry-

lates1605314

Phosphate Buffered Saline

Dulbecco’s Phosphate Buffered Saline, Lonza, BioWhittaker,

Belgium

K+ (4.18), Na+ (152.9), Cl- (139.5), PO4

3- (9.56, sum of H2PO4

- 1.5 mmol L-1 and HPO42-

8.06 mmol L-1)

1 MB160

Nano-bioglass 45S5Laboratory prepared at inorganic

Chemistry Department, Suez Canal University

46.1 mol% SiO2, 26.9 mol % CaO, 24.4 mol %Na2O, 2.6%

mol P2O5

------



a paper pad within 10 seconds until a uniform color emerged. In contrast, the light cured Ca(OH)2 was pre-sented as one paste and prepared in layers of 1mm thickness which was polymerized for 60 seconds as recommended by the manufacturer.

The nano-bioglass was prepared with the previously mentioned composition by the sol gel method, as re-ported by Wei Xia et.al.18.

A pilot study had been performed where different percentages of the nano-bioglass were added to the Ca(OH)2 pastes. The percentage of 7 wt% nano-bio-glass was selected.

The materials were divided into two main groups: group I: Chemical cured Ca(OH)2 paste, and Group II: Light cured Ca(OH)2 paste. Each group was sub-sequently subdivided according to the added nano-bioglass into two subgroups: a) control “without nano-bioglass”, and b) subgroup containing nano-bioglass, where the nano-bioglass powder was mixed manually with calcium hydroxide paste using a plastic spatula and glass slab.

Gp I: Chemical cured calcium-hydroxide (Dycal)

Gp Ia: Chemical cured Ca(OH)2 + 0% nano-bioglass (control group)

Gp Ib: Chemical cured Ca(OH)2 + 7% nano-bioglass

Gp II: Light cured calcium hydroxide (Calcimol LC)

Gp IIa: Light cured Ca(OH)2 + 0% nano-bioglass (con-trol group)

Gp IIb: Light cured Ca(OH)2 + 7% nano-bioglass

Hydroxyl ions (OH-), Calcium (Ca2+) and Phosphorus (PO4

3-) Ions Release Assessment

A total of 40 disc shaped specimens (n = 10/subgroup) (8 mm in diameter and 1.6 mm thick) of each mate-rial, chemical cured and light cured Ca(OH)2 ,were prepared using a Teflon mold according to the manu-facturers’ instructions.

The mold was filled with the different materials and covered with a polyester strip and a glass plate, in the case chemical cured Ca(OH)2, until the material com-pletely set. In the case of light cured Ca(OH)2, Light-Emitting Diode (LED) curing unit (Blue phase C5 Ivo-clar, Vivadent), was used.

A piece of dental floss was inserted into the liner pastes before setting so that an adequate length was projected from the mold in order to ensure the proper handling of specimens and complete immersion in the distilled water. After setting, the specimens were re-moved from the mold and any excess material was removed carefully.

Specimens were suspended individually in a ster-ile glass container with 10 ml triple distilled water at 37 ˚C in an incubator (CBM.Torre Picenardi (CR), Model 431/V, Italy), wherein half of the specimens of each subgroup (n = 5) were stored for 1 day, and the others for 7 days.

The storage water was collected for the analyses of hydroxyl ions (pH measurement), calcium and phos-phorus ion release at different time intervals (1, 7 days). The pH values of the holding water were deter-mined using a pH meter (HANNA instruments pH 212, USA), and the resulting solution from each period of analysis was used to quantify the release of Ca and PO4 ions (ppm) by the atomic absorption spectropho-tometry (Perkin Elmer A analyst 100, USA).

Apatite-Forming Ability (Bioactivity Test)

The apatite forming ability of the different investigated materials was evaluated via an in-vitro test by soaking the specimens in phosphate buffered saline. Six disc-shaped specimens were prepared from each sub-group material (8 mm in diameter and 1.6 mm thick).

Immediately after preparation, the specimens were soaked in Dulbecco’s Phosphate Buffered Saline (DPBS) in the incubator at 37 ºC. The DPBS is a physiological-like buffered (pH = 7.4) Ca and Mg-free solutions.

The endpoint times were 1 day and 7 days. At each endpoint, specimens were removed, washed with dis-tilled water, and then analyzed together with the con-trol specimens for each material for bioactivity by a Scanning Electron Microscope (SEM) (QuantaTM 250 FEG, FEI Company, Netherlands) with a magnification of 5000x and 20,000x. The latter was connected to a secondary electron detector for an Energy Dispersive X-ray analysis (EDXA) (Ametek, Materials Analysis Division, Netherlands) using an accelerating voltage of 20-25 kV.

The specimens were mounted on a copper holder and coated with a thin film of gold of about 200Å thickness



using a sputter coater (Baltec, SCD005, England) be-fore the scanning examination. The elemental analy-sis (weight % and atomic %) of the specimens was performed by applying the ZAF correction method.

Compressive Strength Test

Five specimens of each subgroup were prepared, as before, using a cylindrical Teflon mold with a diame-ter of 4 mm and a height 6mm according to the ISO 3107:2004 standard. The mold was then placed over a glass slab and filled with the mixed pastes. An addi-tional slab was subsequently placed on its surface up to the end of the curing. Before removal from the mold, the ends of the specimens were ground flat with car-bide paper in order to remove any excess materials.

After 24 hours of incubation at 37 °C, each specimen was mounted in a loading fixture between the com-pression plates of a computer controlled Universal Testing Machine (LRX-plus; Lloyd Instruments Ltd., Fareham, UK) with a load cell of 5kN and a cross-head speed of 0.5 mm/min. The break load was recorded using computer software (Nexygen-MT; Lloyd Instru-ments) together with the specimens’ dimensions in order to calculate the compressive strength.

Statistical Analysis

Numerical data were analyzed for normality by checking the distribution of data and employing tests of normality

(Kolmogorov-Smirnov and Shapiro-Wilk tests). All data showed normal (parametric) distribution. Data were pre-sented as mean and Standard Deviation (SD) values.

Repeated Analysis of Variance (ANOVA) was used to study the effect of nano-bioglass, different materials and time intervals on mean pH, calcium and phos-phorus weight %. Bonferroni’s post-hoc test was also used for pair-wise comparisons when any ANOVA test was significant.

Further, a two-way Analysis of Variance (ANOVA) was used to study the effect of nano-bioglass and different materials on mean compressive strength. The signifi-cance level was set at P≤0.05, and the statistical anal-ysis was performed with IBM SPSS Statistics Version 20 for Windows.

ResultsHydroxyl Ions Release (pH)

The results of the study showed that chemical cured Ca(OH)2 recorded a significant higher mean pH than light cured Ca(OH)2 (P-value <0.001) before and after the addition of nano-bioglass at both tested time inter-vals. Moreover, the addition of nano-bioglass resulted in a significant increase in the pH values for both ma-terials (Table 2).

Calcium Ions Release

There was no significant difference in Ca ion release

Table 2: The Mean, Standard Deviation (SD) Values and Results of Repeated Measures ANOVA Test for Comparison Between pH of the Different Interactions

Chemical Curing Light Curing P-value (Between Dif-

ferent Materials)Mean SD Mean SD

Day 1

Control 10.5 0.6 9.0 0.4 0.004*

Bioglass 11.7 0.5 10.2 0.3 0.003*P-value (Between control

and Bioglass) 0.011* 0.011*

Day 7

Control 8.5 0.5 7.9 0.3 0.048*


and Bioglass) <0.001* <0.001*

P-value (Be-tween times)

Control 0.003* 0.056

Bioglass 0.690 0.022* Note: * Significant at P ≤ 0.05.



between chemical cured (Gp Ia) and light cured Ca(OH)2 (Gp IIa). However, with the addition of bio-active glass, results showed a significant increase in the mean calcium ion release at day 1 and a further increase at day 7 in both tested materials (Table 3).

Phosphorus Ions Release

There was no significant difference between chemical cured Ca(OH)2 (Gp Ia) and light cured Ca(OH)2 (Gp IIa)

at both tested time intervals. In turn, after addition of nano-bioglass, no significant difference in phosphorus ion release was recorded, except for the light cured Ca(OH)2 at day 7, which recorded the statistically low-est ion release (0.12 ± 0.03 ppm) (Table 4).

Apatite-Forming Ability (Bioactivity Test)

The SEM and EDX analysis showed different surface morphologies of the specimens depending on the type

Table 3: The Mean, Standard Deviation (SD) Values and Results of Repeated Measures ANOVA Test for Comparison Between Ca Ions Release (ppm) of the Different Interactions



Day 1

Control 32.6 2.1 26.5 1.1 0.118

Bioglass 44.9 7.8 37.2 2.6 0.059P-value (Between control

and Bioglass) 0.008* 0.016*

Day 7

Control 37.55 5.3 31.9 3.5 0.033*


and Bioglass) <0.001* <0.001*


Control 0.001* 0.008*

Bioglass <0.001* <0.001* Note: * Significant at P ≤ 0.05.

Table 4: The Mean, Standard Deviation (SD) Values and Results of Repeated Measures ANOVA Test for Comparison Between P Ions Release (ppm) of the Different Interactions



Day 1

Control 0.35 0.07 0.23 0.06 0.341

Bioglass 0.37 0.04 0.28 0.04 0.338P-value (Between control

and Bioglass) 0.840 0.675

Day 7

Control 0.28 0.05 0.43 0.08 0.063


and Bioglass) 0.100 0.024*


Control 0.715 0.311

Bioglass 0.086 <0.001* Note: * Significant at P ≤ 0.05.



of materials used, the addition of bioactive glass, and the immersion time.

Chemical Cured Ca(OH)2

The surfaces of the specimens of the control group were similar at day 1 and day 7. Both showed an uneven appearance with irregular granules all over their surfaces (5000x) (Figure 1a). Some aggre-gates of micro-spherical crystals were detected with a high magnification (20,000x) (Figure 1b), while the EDX and elemental analysis also re-vealed the presence of C, N, O, Na, P, S, Ti, W, Zn and Ca (Figures 1c and 1d).

After the addition of bioglass and immersion 1 day in PBS, more spherical aggregates were clear, and clus-ters of different sizes formed (Figures 2a and 2b). The EDX and the elemental analysis displayed C, N, O, Na, Si, P, S, Ti, Zn and high Ca peaks with high Ca/P molar ratio = 3.6 (Figures 2c and 2d).

After 7 days storage in PBS, many noticeable spheru-lites were precipitated on the cement surfaces, and higher peaks of Ca, O and P denoted the presence of calcium phosphate crystals (Figure 3) with a Ca/P molar ratio of 2.4, close to carbonated apatite19.

Light Cured Ca(OH)2

The surfaces of the specimens of the control group showed plate-like crystals protruding on the surface with some spherical microcrystals in a homogenous glassy phase covering some of the specimens’ sur-faces (Figures 4a and 4b). The EDX and elemental analysis also showed the presence of high peaks of Si and minor peaks of C, N, O, Na, Al, Ca, P, S, and Ti in the material composition (Figures 4c and 4d). This was noticed in day 1 and day 7.

After the nano-bioglass addition and immersion for 1 day in PBS, some visible precipitates composed of spherical microcrystals were clearly covering the

Figure 1: Scanning Electron Micrograph of Chemical Cured Ca(OH)2 (Gp Ia; Control) (a) 5000x, (b) 20,000x, (c and d) EDX Pattern and Elemental Analysis Revealed C, N, O, Na, P, S, Ti, W, Zn and Ca Peaks in the Material Composition

(a) (b)

(d)(c)



surfaces (5000x) (Figure 5a). A higher magnification (20,000x) showed many spherulites that were packed to form clusters and networks of nanocrys- tals with needle-like projections (Figure 5b), while the EDX and elemental analysis showed high peaks of Ca and P with a Ca/P molar ratio of 1.52, denot-ing the formation of Ca-deficient hydroxyapatite19

(Figures 5c and 5d).

After 7 days of storage in PBS, densely packed clusters of mature spherical particles with noticeable spins were randomly distributed across the whole cement surfaces (Figures 6a and 6b). The EDX and elemental analysis showed higher Ca, O and P peaks with a Ca/P molar ratio of 1.57, denoting again theformation of Ca-deficient hydroxyapatite19. Moreover, the EDX showed an obvious reduction of Si peaks, denoting the increased thickness of calcium phosphate deposits which was able to mask the underly- ing components of the light cured Ca(OH)2 material(Figures 6c and 6d).

Compressive Strength

The results of the study showed that light cured Ca(OH)2 recorded the highest mean compressive strength before the addition of nano-bioglass (85.9 ± 3.4 Mpa) and a significant decrease in the compres-sive strength after the nano-bioglass addition (65.9 ± 9.9 Mpa), (p<0.001). However this remained higher when compared to the chemical cured one (14.6 ± 1.4 Mpa and 10.4 ± 2.3 Mpa before and after nano-bioglass addition respectively) (Table 5).

Discussion Calcium hydroxide products have been accepted as the standard material for the conservative treatment of dental pulp exposures due to their therapeutic and biological potential, their ability to stimulate the forma-tion of sclerotic and reparative dentin, as well as the manner in which they protect the pulp against thermal stimuli20.

Figure 2: Scanning Electron Micrograph of Chemical Cured Ca(OH)2 with 7% Nano-Bioglass (Gp Ib) After 1 Day Immersion in PBS (a) 5000x, (b) 20,000x, (c and d) The EDX Pattern and Elemental Analysis Which Revealed C, N, O, Na, Si, P, S, Ti, Zn and Ca Peaks

(a) (b)

(d)(c)



Figure 3: Scanning Electron Micrograph of Chemical Cured Ca(OH)2 with 7% Nano-Bioglass (Gp Ib) After 7 Days of Immersion in PBS (a) 5000x, (b) 20,000x, (c and d) The EDX Pattern and Elemental Analysis Which Revealed C, N, O, Na, Si, P, S, Ti, Zn and Ca Peaks

(a) (b)

(d)(c)

Figure 4: Scanning Electron Micrograph of Light Cured Ca(OH)2 (Gp IIa; Control) (a) 5000x, (b) 20,000x, (c and d) The EDX Pattern and Elemental Analysis Which Revealed C, N, O, Na, Al, Si, P, S, Ti, and Ca Peaks in the Material Composition

(a) (b)

(d)(c)



Figure 5: Scanning Electron Micrograph of Light Cured Ca(OH)2 with 7% Nano-Bioglass (Gp IIb) After 1

Day Immersion in PBS (a) 5000x, (b) 20,000x, (c and d) The EDX Pattern and Elemental Analysis Which Re-vealed C, N, O, Na, Al, Si, P, S, Ti, and Ca Peaks

(a) (b)

(d)(c)

Figure 6: Scanning Electron Micrograph of Light Cured Ca(OH)2 with 7% Nano-Bioglass (Gp IIb) After 7 Days of Immersion in PBS (a) 5000x, (b) 20,000x, (c and d) The EDX Pattern and Elemental Analysis Which Revealed C, N, O, Na, Al, Si, P, S, Ti, and Ca Peaks

(a) (b)

(d)(c)



The success obtained with calcium hydroxide as a pulp capping agent is related to its high alkalinity, and it is suggested that the rise in pH is the most important factor in pulp healing21, 22.

In this research, chemical and light cured calcium hy-droxide cements were used. The light cured calcium hydroxide offered a command setting time and an easier application as an advantage over the chemical cured one7.

In this study, nano-bioglass was selected to be added to the different pulp capping materials investigated. Bioglass is a bioactive material composed of minerals that naturally occur in the body14. In the 1990’s, the sol-gel process emerged, providing a more versatile method to design bioactive glass nanoparticles23. This process is based on hydrolysis and the condensation of molecular precursors (alkoxydes or salts), which lead to the formation, at room temperature and ambi-ent pressure, of an inorganic network23

It was found that the bioactive glass nanoparticles pro-duced by sol-gel chemistry presented a high specific surface area and pore volume as well as being bio-compatible24. This makes them highly attractive in the fields of biomaterial due to the fact that bioactivity is strongly related to the specific surface area of the ma-terials as it has a direct impact on glass dissolution and apatite formation rates24.

This study was conducted to determine the effect of the addition of bioactive glass to calcium hydroxide (chemical and light cured) on pH, ion release, apatite-forming ability and compressive strength.

In the current research, a pilot study had been per-formed using 2, 5, 7 and 10 wt% nano-bioglass that was added to the calcium hydroxide pastes. These results showed no detectable apatite layer in the case of 2 and 5 wt%, while the addition of 10 wt% bioglass showed obvious apatite layer; however it caused a detrimental decrease in the compressive strength. Therefore, 7 wt% was selected to conduct this study.

Both bioglass and calcium hydroxide have an alkaline nature14, 22. The use of materials with high alkalinity favors a high rate of tissue mineralization as well as offers good antimicrobial activity25. The alkalinizing power of a pulp capping material represents a key property for different alkaline-related biological prop-erties26. The release of hydroxyl ions during the hy-

dration reaction creates an adverse environment for bacterial survival and proliferation. These antibacterial properties are required primarily at the dentin/resto-ration interface where residual bacteria could further increase the risk of re-infection and secondary caries. Moreover, alkaline pH causes an inflammatory reac-tion with the formation of reparative dentin26.

The results of the study showed that chemical cured Ca(OH)2 recorded a significant higher mean pH than light cured Ca(OH)2 before and after the addition of nano-bioglass at both tested time intervals (Table 2). This may be attributed to the presence of methacrylate in the light cured calcium hydroxide’s composition at the expense of alkaline calcium hydroxide7.

Moreover, the results of the present study showed that the addition of nano-bioglass resulted in a signifi-cant increase in the pH values for both materials. This could be explained by the gradual dissolution of the nano-bioglass14.

The alkalinization and the calcium releasing ability of calcium hydroxide materials to the surrounding media play a great role in its physical and biological proper-ties27, 28. In addition, the released ions from bioglass promote the growth of a carbonated hydroxyapatite lay-er at its surface29, 30. Therefore, by analyzing the alka-linization and calcium releasing abilities, the mineraliza-tion potential of the material could be expected27, 28. To measure the ionic release, different methods are well established such as atomic absorption spectrometry, Ca ion selective electrode, fluorometry, etc.31.

The results of this study showed no significant differ-ence in Ca ion release between chemical cured and light cured Ca(OH)2. This suggested that the resin portion of light cured calcium hydroxide was able to sustain Ca and OH ion release within the wet environ-ment8. With the addition of bioactive glass, the results showed a significant increase in the mean calcium ion release at day 1, and a further increase at day 7 in both tested materials. This may be related to the chemical composition of the bioglass that was rich with calcium14. The concentration of the calcium ions increased over time due to the continuous dissolution of the bioglass14.

On the other hand, the recorded insignificant differ-ences in phosphorous ion release after the bioglass addition in both cements may be attributed to the low phosphorous concentration in the experimental



bioglass used (2.6% mol P2O5), compared to calcium (26.9 mol % CaO).

The bioavailability of calcium ions plays a key role in the various biological events of the cells involved in the new formation of mineralized hard tissues32. The Ca ions stimulate the expression of bone-associated proteins mediated by calcium channels, and large quantities of Ca ions can activate adenosine triphos-phate (ATP), which plays a significant role in the min-eralization process32. The Ca ions are also necessary for the differentiation and mineralization of pulp cells, and a Ca-rich medium induces both proliferation and differentiation into odontoblast-like cells33. In addi-tion, the release of Ca ions enhances the activity of pyrophosphatase, which helps to maintain dentin mineralization and facilitates the formation of a den-tin bridge33.

Since the introduction of bioactive glass by Hench in the 1970’s, it has attracted great attention due to its ability to chemically bond with host tissues, which was directly related to its atomic structure34, 35. Bioactive glasses are able to form carbonated hydroxyapatite layer on their surfaces once exposed to biological flu-ids34, 36. The ability of this bioglass to dissolve has been explained by the low connectivity of the SiO2 network due to the presence of sodium and calcium that act as network modifiers, which results in the formation of non-bridging silicon-oxygen bonds34. Surface Na, Ca and phosphate ions can be exchanged with the H+

from the biological fluid, creating Si-OH bonds34. Then, more Si-OH bonds are formed due to the hydrolysis of Si-O-Si bonds resulting from a pH increase34. When calcium and phosphate ions migrate to the surface, this leads to the formation of the precursor amorphous calcium phosphate that later crystallizes to form cal-cium deficient hydroxyapatite36.

In our study, this bioactivity was studied via the immer-sion of the calcium hydroxide pulp capping materials into phosphate buffered saline (DPBS), where the re-lease of the calcium ions from the materials was the limiting parameter in the precipitation reaction, and the large amount of phosphate in this media represents the continuous replenishment of phosphate ions from tissue fluids37. A commercial medium was selected be-cause of its standardization and availability37.

The SEM and EDXA results of our study clearly dem-onstrate that the light cured Ca(OH)2 material pos-sessed higher bioactivity than the chemical cured Ca(OH)2 one and showed that the surface morphol-ogy and chemical composition were rapidly modified after the addition of nano-bioglass and immersion in DPBS. The significant finding was the fast formation of nano-spherulites of Ca-deficient hydroxyapatite with a Ca/P molar ratio of 1.52 that began after 1 day of im-mersion in the phosphate containing solution (Figure 5). Moreover, many spherulites were packed to form clusters of spheroidal bodies with needle-like projec-tions after 7 days of storage in DPBS (Figure 6).

It has been reported that the hydroxyl, ester and ether chelating groups(OH, C = O, C-O-C and C-O groups) of the resin component exposed on the surface of the light cured Ca(OH)2 material were the coordination sites for chelating calcium ions, forming resin-calcium chelate complexes that provided initiation sites for apatite nucleation38.

The evaluation of the mechanical properties of pulp capping materials is important in order to predict their clinical behavior during amalgam condensation or ap-plication of other materials39. The ISO standard (ISO 3107:2004) was selected in this study as the guideline for the evaluation of compressive strength40.

Table 5: The Mean, Standard Deviation (SD) Values and Results of the Two-Way ANOVA Test for the Com-parison of Compressive Strength of the Different Interactions

Chemical Curing Light Curing

P-value (Between Different Materials)Mean SD Mean SD

Control 14.6 1.4 85.9 3.4 <0.001*

Nano-bioglass 10.4 2.3 65.9 9.9 <0.001*P-value (Between control

and Nano-bioglass) 0.265 <0.001*

Note: * Significant at P ≤ 0.05.



Further, the results of this research were consistent with those obtained by previous studies that reported the compressive strength of Dycal in a range of 14.5 to 36 MPa39, 40. However, the light cured calcium hy-droxide showed a significantly higher compressive strength (85.9 ± 3.4 MPa) when compared to the chemical cured one (14.6 ± 1.4 MPa) (Table 5). This may be attributed to the methacrylate resinous part in the light cured type7,41.

Results also showed that the addition of nano-bioglass caused a significant decrease in the mean compres-sive strength of the light cured type (65.99 ± 9.9 MPa). This may be due to the loose attachment between the calcium hydroxide matrix and nano-bioglass particles that acted as fillers which had not been firmly adhered to the resin matrix, thus decreasing the compressive strength42. This was in agreement with a previous study which showed that bioglass added to glass iono-mer cement decreased its compressive strength42.

However, it should be noted that the compressive strength of chemical cured Ca(OH)2 without the ad-dition of nano-bioglass, and the light cured one with and without the addition of nano-bioglass, was much higher than the minimal strength required by the ISO 3107:2004. Ultimately, they were considered strong enough to resist an average stress of 10.5 MPa, which is applied through an amalgam condensation cycle43.

Conclusion1. Although the addition of nano-bioglass to Ca (OH)2

liner negatively affected its compressive strength, it is still within or significantly higher than the ac-ceptable clinical limit.

2. The addition of nano-bioglass to Ca(OH)2 liners improve their ion release (OH-, Ca2+) and, hence, their bioactivity.

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Citation: Shaymaa I Habib, Rania E Bayoumi, Sabry A El-Korashy and Rasha M Abdelraouf, ”Effect of Nano-Bioglass Addition to Calcium Hydroxide Cements in Terms of Ions Release, Apatite Formation and Compressive Strength”, Global Scientific Research Journal Dentistry, 1(1), 2018, pp. 1-14.

Copyright © 2018 Shaymaa I Habib et al., This is an open access article distributed under the Creative Commons Attribution License, which permits wwunrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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