The mean and standard deviation (SD) Vickers hardness values of sound dentin (negative control group), carious dentin (positive control group) and residual dentin following caries removal via papain-enzymatic based CMCR agent are shown in Table 2. The outcome of the Kolmogorov–Smirnov normality test showed that data followed the normal distribution and accordingly parametric statistical analysis was performed. The outcome of three-way ANOVA revealed that the variables; ‘teeth condition’, ‘material used’ and ‘site’ significantly affect the Vickers micro-hardness of the dentin (p < 0.05). Moreover, the interaction between the three variables exhibited significant effect on Vickers micro-hardness (p < 0.05).

Vickers hardness of Sound dentin -Bioactive-GIC group at junction site showed the highest value and was statistically significant different from all the test groups (p < 0.05). Vickers hardness of the Carious dentin-Glass-hybrid GIC group at 50 μm dentin showed the lowest value and had significant difference with all of the test groups except Carious dentin-Glass-hybrid GIC group at junction site and at dentin 100 μm, CMCR-Bioactive-GIC group at 100 and 150 μm dentin.

In Sound -Glass-hybrid GIC group Vickers hardness at junction was significantly higher than the three other groups (p < 0.05). While Vickers hardness values at dentin 50 μm, 100 μm and 150 μm showed insignificant difference between each other (p > 0.05). Similarly, Vickers hardness in Sound - Bioactive-GIC group at junction showed significant different with the three other groups, Vickers hardness at 50 μm, 100 μm and 150 μm dentin showed insignificant difference between each other (p > 0.05). Comparing the two sound groups showed insignificant differences between all groups (p > 0.05).

Vickers microhardness results of Caries- Glass-hybrid GIC group showed that, there is a significant difference between Caries- Glass-hybrid GIC group at 50 μm dentin and 150 μm dentin subgroup (p < 0.05). In addition, statistically insignificant difference was found between Caries -Glass-hybrid GIC group at junction and Caries -Glass-hybrid GIC group at 100 μm dentin (p > 0.05). No significant difference was found between Caries-Bioactive-GIC groups at junction, 50 μm dentin, 100 μm dentin and 150 μm dentin. Comparing the two caries groups, significant differences were found between Caries- Glass-hybrid GIC group at 50 μm and Caries -Bioactive-GIC groups at junction, 50 μm dentin, 100 μm dentin and 150 μm dentin (p < 0.05).

Groups identified by different superscripts were significantly different at p < 0.05, n = 30.

CMCR- Glass-hybrid GIC group at junction showed significant difference with all the CMCR sub-groups. While there were insignificant differences between the remaining groups. CMCR-Glass-hybrid GIC group at 100 μm dentin showed significant difference with CMCR-Bioactive-GIC group at 100 μm dentin (p < 0.05). CMCR-Glass-hybrid GIC group at 150 μm dentin showed significant difference with CMCR-Bioactive-GIC group at 150 μm dentin (p < 0.05).

The EDX elemental analysis of Calcium, Silica, Phosphorous and Aluminum is shown in Table 3; Fig. 4(a & b). The results of three-way ANOVA regarding Calcium levels revealed that substrate condition and material used had insignificant effect on Calcium levels (p > 0.05). While site factor had significant effect on Calcium level (p < 0.05). The interaction between “substrate-material” and “substrate- site” showed insignificant effect on Calcium levels (p > 0.05). While the interaction between “material-site” had significant effect on Calcium level (p < 0.05). The interaction between the three factors was insignificant (p > 0.05).

(a): Representing the elemental analysis results of the sound- Glass hybrid -junction group. (b): Representing the elemental analysis results of the CMCR- Bioactive GIC-junction group

Sound-Bioactive-GIC-Dentin, Caries-Bioactive-GIC-Dentin and CMCR-Bioactive-GIC-Dentin groups showed the highest Calcium ratio, these groups were significantly different with all remaining groups, except three groups that showed insignificant differences between them which were: Sound-Glass-hybrid GIC-Dentin, Caries-Glass-hybrid GIC-Dentin and CMCR-Glass-hybrid GIC-Dentin groups. While the lowest Calcium ratios were found in Sound-Bioactive-GIC-Junction, CMCR-Glass-hybrid GIC-Junction and CMCR-Bioactive-GIC-Junction groups these groups were significantly different with all test groups except three groups that were insignificantly different with them which are: Sound-Glass-hybrid GIC-Junction, Caries-Glass-hybrid GIC-Junction and Caries-Bioactive-GIC-Junction groups.

Comparing the sound groups: There were significant difference between Sound-Glass-hybrid GIC-Junction and Sound-Glass-hybrid GIC-Dentin groups, and between Sound-Bioactive-GIC-Junction and Sound-Bioactive-GIC-Dentin groups (p < 0.05). Insignificant differences were found between Sound-Glass hybrid GIC-Junction and Sound-Bioactive-GIC-Junction, and between Sound-Glass-hybrid GIC-Dentin and Sound-Bioactive-GIC-Dentin groups (p > 0.05). In caries groups: significant difference) was found between Caries-Bioactive-GIC-Junction and Caries-Bioactive-GIC-Dentin group (p < 0.05). CMCR groups showed: significant.

differences between CMCR-Glass-hybrid GIC-Junction and CMCR-Glass-hybrid GIC-Dentin, and between CMCR-Bioactive-GIC-Junction and CMCR- Bioactive-GIC- dentin group (p < 0.05). Insignificant differences were found between CMCR-Glass-hybrid GIC-Junction and CMCR-Bioactive-GIC-Junction, and between CMCR-Glass-hybrid GIC-Dentin and CMCR-Bioactive-GIC-Dentin groups (p > 0.05).

The results of three-way ANOVA regarding Silica levels revealed that a significant effect of substrate condition, material used and the site on Silica levels (p < 0.05). The interaction between “substrate-material”, “substrate-site” and “material-site” had also a significant effect on Silica levels (p < 0.05). The interaction between the three factors was also highly significant (p < 0.05).

Sound-Bioactive-GIC-Junction group had the highest ratio of Silica which showed statistically significant differences with all groups (p < 0.05). There were significant differences between Sound-Glass-hybrid GIC-Junction and Sound-Glass-hybrid GIC-Dentin groups and between Sound-Bioactive-GIC-Junction and Sound-Bioactive-GIC-Dentin groups (p < 0.05). In Caries group there was significant difference between Caries-Bioactive-GIC-Junction and Caries-Bioactive-GIC-Dentin groups while insignificant difference was found between Caries-Glass-hybrid GIC-Junction and Caries-Glass-hybrid GIC-Dentin groups. CMCR group showed: significant difference between CMCR-Glass-hybrid GIC-Junction and CMCR-Glass-hybrid GIC-Dentin, and between CMCR-Bioactive-GIC-Junction and CMCR-Bioactive-GIC-Dentin groups (p < 0.05). Significant differences were found between Sound-Glass-hybrid GIC-Junction group and Sound-Bioactive-GIC-Junction group, between Caries-Glass-hybrid GIC junction group and Caries-Bioactive-GIC-junction and between CMCR-Glass-hybrid GIC-junction group and CMCR-Bioactive-GIC-junction group (p < 0.05). Insignificant differences were found between CMCR-Glass-hybrid GIC-Junction and CMCR-Bioactive-GIC-Junction and between CMCR-Glass-hybrid GIC-Dentin and CMCR-Bioactive-GIC-Dentin (p > 0.05).

The results of three-way ANOVA regarding Phosphorous levels revealed an insignificant effect of substrate condition and material used on Phosphorous levels (p > 0.05), while the site “substrate-material”, “substrate-site” had an insignificant effect on Phosphorous levels (p > 0.05), while the interaction between “material-site” had a significant effect on Phosphorous levels (p < 0.05). The interaction between the three factors wasn’t significant (p > 0.05). There were no significant differences between all the groups (p > 0.05).

The results of three-way ANOVA regarding Aluminum levels revealed that substrate condition, material and site factor had a significant effect (p < 0.05) on Aluminum levels, The interaction between “substrate-material”, “substrate-site” and “material-site” had a significant effect on aluminum levels (p < 0.05). The interaction between the three factors was also high significant (p < 0.05). Sound-Glass-hybrid GIC-Junction and CMCR-Glass-hybrid GIC-Junction groups showed the lowest Aluminum levels and they showed significant differences with all test groups ((p < 0.05). Insignificant differences were found between Carious-Glass-hybrid GIC-Junction and Carious-Glass-hybrid GIC-Dentin groups and between CMCR-Bioactive-GIC-Junction and CMCR-Bioactive-GIC-Dentin groups (p > 0.05).

The major bands or parameters that can describe the mineral composition through micro-Raman spectroscopy corresponded to the phosphate (PO43−) and carbonate (CO32−) groups a shown in Fig. 5. Phosphate had two vibrations frequencies: major vibration frequency of v1 PO43− was near 960 cm− 1 and minor vibration frequencies of v2 and v4 PO43− vibrations were detected near 431 and 589 cm− 1, respectively. The vibration of v1 CO32− was detected near 1072 cm− 1. The high intensity of the v1 PO43− band is a positive sign and indicated high level of remineralization, while high intensity of the v1 CO32− is a negative sign and indicated high level of demineralization.

Regarding micro raman results: Bioactive-GIC specimens showed higher frequencies of v1 PO43− (higher than 960 cm− 1) and showed lower frequencies of v1 CO32−(lesser than 1072 cm− 1) compared to Glass-hybrid GIC specimens. CMCR-Bioactive-GIC specimens showed the highest phosphate vibration frequency (971 cm− 1) and the lowest carbonate vibration frequency (1046 cm− 1). While Caries-Glass-hybrid GIC specimens showed the lowest phosphate vibration frequency (913 cm− 1) and the highest carbonate vibration frequency (1088 cm− 1). Comparing Sound-Glass-hybrid GIC specimens with Sound-Bioactive-GIC specimens: Sound-Bioactive-GIC specimen showed higher phosphate vibration frequency and lower carbonate vibration frequency (960,1070 cm− 1) than Sound-Glass-hybrid GIC specimens (940,1073 cm− 1). In addition, comparing Caries-Glass-hybrid GIC specimens with Sound-Bioactive-GIC specimens: Sound-Bioactive-GIC specimens showed higher phosphate vibration frequency and lower carbonate vibration frequency (960,1058 cm− 1) than Caries-Glass-hybrid GIC specimens (913,1088 cm− 1).

Micro-Raman spectroscopy of specimens showing the intensity of phosphate (v1 PO43−) and carbonate (v1 CO32−) bands at three different conditions