In a previous work, we showed that nearly 85% of the RAP-treated group showed pulp regeneration, the rest did not show pulp regeneration and none of the CH-treated group showed pulp regeneration as well [14]. Thus, a comparison of the nature of the remaining regenerated calcified tissues of the RAP-treated group that showed no pulp regeneration compared to the CH-treated group would be described below.
Calcium hydroxide-treated-canal cohort
The outcomes of calcium hydroxide treatment were more variable over the post-treatment time intervals. At 1-month post-treatment, only few samples showed histological evidence of complete apical bridging (Fig. 1a, Tables 1 and 2), whereas most of the treated canals had root apices that were either wholly open or showed signs of incomplete closure (Fig. 1b & c). Mild staining for Wnt10b was identified in the newly formed dentin at the opened root apex and in the granulation tissue that was at a distance from root apex (Fig. 1b). Mild to moderate staining for the intermediate filament stem/progenitor cell marker nestin was identified in the newly formed dentin at the root apex. Granulation tissue showed moderate immuno-reactivity and was found at a distance from the root apex (Fig. 1c).
At the 3-month interval, only few specimens of the canals showed complete root apex closure formed by cellular mineralized tissue (Fig. 2a, Tables 1 and 2). Most specimens showed incomplete root apex closure (Fig. 2c). No immune-reactivity was observed for Wnt10b either in the newly formed dentin or the granulation tissue (Fig. 2b). Mild staining for the intermediate filament stem/progenitor cell marker nestin was identified in the newly formed dentin and granulation tissue, while intense staining was observed in the odontoblast cell layer (Fig. 2c).
At 6-months post-treatment, two histological patterns were recognized. The first was comprised of a thin layer with either a histologically incomplete or complete apical bridge (Fig. 3a & b, Tables 1 and 2). Minor thickening of the lateral portions of the root was also observed. Newly formed cementum was observed with no attachments to the granulation tissue. Incomplete islands of regenerated bone were observed, which had no attachments to the apical portion of the tooth. Granulation tissue persisted until the 6-months post-operative period. In the second pattern, canals showed complete apical closure by a continuous layer of mineralized tissue with thickened lateral walls. Mineralized tissue was present inside the apical portion of the root canal. In addition, the newly formed cellular cementum was not attached to the granulation tissue, which was always found at a distance from the apical bridge (Fig. 3c).
Amelogenin-treated canal cohort
At one-month post-treatment, the canals treated with RAP showed the formation of dentin bridges, all of which were formed either by an incomplete or complete calcified tissue barrier (Fig. 1d & e, g, Tables 1 and 2). A consistent cellular response accompanying the apical closure was the appearance of a layer of cellular granulation tissue surrounding the root apex (Fig. 1d-f). The walls adjacent to the root apices appeared thickened due to the formation of newly formed dentin. Regenerated odontoblasts were found to be covering the newly formed dentin (Fig. 1d-e). Moderate to intense staining for Wnt10b was identified in the newly formed dentin at the root apex as well as among the odontoblast cell layer and in the granulation tissue (Fig. 1e). In addition, intense staining for nestin was recognized in the odontoblasts and in the condensed and regenerated cellular granulation tissue that completely surrounded the regenerating root apex (Fig. 1f).
At 3-months post-treatment, most of the amelogenin-treated teeth revealed complete root closure, as evidenced by a micro-anatomical analysis of the apical area, which showed cellular continuity and a dentin layer (Fig. 2d-f, Tables 1 and 2). Regeneration of the attachment apparatus was observed histologically in samples from this time interval (Fig. 2d). Wnt10b was recognized immunologically in the newly formed dentin and granulation tissue (Fig. 3e).
Mild to moderate to staining for nestin was recognized in the newly formed dentin bridge, and the granulation tissue (Fig. 2f).
At 6-months post-treatment with amelogenin, nearly all teeth showed histological evidence of the complete closure of the apical area by a thick layer of calcified tissue. A significantly higher percentage of amelogenin treated canals (70.9%) had a complete root closure in comparison to CH treated canals (10.8%) (p-value < 0.001) (Fig. 3d, Table 2). The presence of dentin-associated mineralized tissue was detected in a significantly higher percentage of roots treated with CH (43.3%) in comparison to none in roots treated with amelogenin (p-value< 0.001). The percentage of calcified islands were significantly higher in CH treated canals (23.3%) in comparison to amelogenin treated canals (5%) (P < 0.001). Dentin walls appeared to be thickened with newly formed dentine (Fig. 3d-i).
Moderate to intense staining for Wnt10b was recognized in the newly formed dentin and periodontal ligament (PDL) (Fig. 3e). Furthermore, intense staining to nestin was also recognized in these same structures (Fig. 3g).
In all of the teeth from the RAP group that did not show pulp regeneration, the newly formed dentin bridge was formed by tubular dentin (Figs. 2d-f, 3d-i).
