Immunohistological study of the effect of vascular Endothelial Growth Factor on the angiogenesis of mature root canals in rat molars

Abstract Tissue bioengineering has been applied to Endodontics to seek a more biological treatment. The presence of blood vessels is crucial for cell nutrition during tissue formation. Objective This study analysed the application of vascular endothelial growth factor (VEGF) in the angiogenesis of mature root canals. Material and methods Upper first molars of twelve 13-week old Wistar male rats were used. The root pulp of the mesiobuccal canal was removed and the root canal instrumented with K-files up to size #25. Periapical bleeding was induced into the root canal by introducing a #15 K-file beyond the apex. The teeth on the right side of the arch were filled up with blood clot (G1), whereas those on the left side were filled up with blood clot plus 50 ng/ml of VEGF (G2). Teeth were sealed with light-curing glass-ionomer cement and the animals were sacrificed after 60 days. The maxilla was dissected and fixed before obtaining serial sections for histological processing with haematoxylin-eosin (HE) and immunohistochemical factor-VIII. Immunohistochemical labelling was evaluated using scores for statistical analysis. Results Immunohistological analysis demonstrated the presence of angiogenesis in both groups, but with higher angiogenic maturation in G2 during the experimental period (p<0.05). HE staining showed connective tissue with absence of odontoblasts in all specimens. Conclusions It can be concluded that it is possible to obtain angiogenesis in mature root canals with or without the use of VEGF, although the latter tends to accelerate blood vessel formation.


Introduction
Regenerative dentistry is one of the investigative fields that promises to change current paradigms by relying on bioengineering principles. Since the discovery of stem cells in the dental pulp, Endodontics has been highlighted in this area, and the use of an intracanal blood clot for pulp revascularisation has been proposed 3,16 . However, most experiments have focused on the treatment of the permanent immature teeth, while the therapeutic approach on mature root canals has been little explored.
Although bioengineering comprises the triad of cells, scaffold and growth factors 23 , vascularisation is crucial for tissue development 9,26 . Thus, angiogenesis is a key element in the new tissue formation so that cells may receive the nutrients necessary to their proliferation and differentiation.
Among the several growth factors, the vascular endothelial growth factor (VEGF) has been indicated as one of the main ones involved in angiogenesis 14 .
In addition, VEGF promotes cell proliferation, differentiation and has chemotaxis towards endothelial cells and homing cells 13,18,28  The apex was opened using a #8 K-file and then widened it to a #20 size. Periapical bleeding was induced by inserting a #15 K-file beyond the apex.

Results
The morphological analysis has shown formation of loosen connective tissue without presence of odontoblasts in all specimens ( Figure 1B), differently from pulp in non-instrumented root canals ( Figure 1C).
In both groups, most specimens showed necrotic tissue surrounded by collagen fibres in all thirds, although necrotic spots were more evident in group 1 than in group 2 ( Figure 2).
Some specimens showed formation of highly cellular and well-developed tissue with presence of fibroblasts and functional vessels filled with blood cells. Such a situation was seen in the three thirds of the specimens from the group treated with VEGF, even though this finding was not observed in any section in the cervical third in the group treated with blood clot only.
Immunohistological analysis has demonstrated positivity in the newly formed tissue in most of specimens from both groups and thirds.
A prevalence of severe labelling in the coronal and middle thirds was observed in the group treated with blood clot only ( Figure 3A). Negativity was present in the middle and apical thirds of only one specimen in this group, which was not observed in the coronal third.
There was predominance of weak labelling in all thirds in the group treated with VEGF ( Figure 3B), although severe and negative labelling were also observed. Negativity was present in more than one   Table 1.
The statistical test did not demonstrate significant difference between the thirds in both groups (p>0.05), but the group treated with VEGF showed higher angiogenic maturation than the group treated with blood clot only (p<0.05).
Highly cellular and vascularised tissue in the specimens presenting negativity was confirmed by HE staining (Figure 4).

The immunohistological control showed negativity
and a well-organised pulp tissue with presence of functional mature vessels, fibroblasts spread in the pulp and layer of odontoblasts at the peripheral zone.

Discussion
The immunohistochemical results showed that angiogenesis takes place in mature root canal when periapical bleeding was provoked by instrumentation beyond the apex. The findings also showed that angiogenesis was faster when the VEGF was used to establish a blood clot in the root canal. Immunohistological study of the effect of vascular Endothelial Growth Factor on the angiogenesis of mature root canals in rat molars angiogenesis-related receptors 10 , and the VEGF-165 subtype because of its higher mitogenic potential on endothelial cells 10,14 . In addition, it followed previous assays reporting positive results regarding the formation of blood vessels by using this type and subtype of VEGF 4,9, The presence of necrotic spots in the connective tissue may be related to remnants of non-reabsorbed blood clots, as reported by other authors 11,22 . However, another logical explanation might be the presence of remnants of debris from the apical widening. Thus, other authors reported dentine chips or spicules within the newly formed tissue following revascularisation treatment 3,16 . In this study, evidence for this scenario was observed in the coronal third, which is a region in which repair is more complex and longer because of its apical distance, given the source of revascularisation 6,26 .
The immunohistological analysis showed positivity in the majority of the treated root canals, representing an active process of angiogenesis in both groups. This finding is in accordance with studies reporting positive angiogenesis with or without the use of VEGF applied to bioengineering 4,5,7,9,13,21 . Vascular formation without application of VEGF may be explained by proteins of extracellular matrix in the dentine, which induce cell proliferation and/or differentiation 15,24,29 . Studies have indicated that etching dentine with 17% EDTA, as it was performed herein, promotes the release of such proteins and produces better results regarding pulp regeneration 8,12 . In addition, growth factors are also present in blood cell concentrates, thus yielding tissue formation 16 .
Both severe and weak labelling patterns were identified and correlated to the stage of angiogenic On the other hand, weak labelling seems to be related to the final stage of angiogenesis, when blood vessels are almost mature. This scenario can be understood in the specimens with negativity, in which mature and functional vessels were present in the HE-stained sections. In fact, these specimens had action of both VEGF and signalling molecules, resulting in angiogenesis and further mature vessel.
The immunohistochemical control group confirms that formation of new tissues is due to the treatment applied, since these specimens have no odontoblasts as compared to non-instrumented root canals. Similarly, as for the angiogenic maturation stage, we observed that severe labelling was prevalent in the middle and coronal thirds in the group treated only with blood clot, whereas weak labelling was predominantly seen in these thirds in the group treated with VEGF.
In addition, a greater number of specimens with immunohistological negativity and well-vascularised tissue were observed in the VEGF group. No specimens from the group treated only with blood clot achieved this level of maturation at the coronal third.
The fact that this study used a vital pulp condition is noteworthy. Then, further studies using necrotic conditions should be considered. Therefore, the option of a biological endodontic treatment free of synthetic materials seems to be closer to reality. Consequently, it can state that changes in the endodontic paradigm are real.