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

<div><p>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.</p></div

Metabolism patterns.

<p>Metabolism patterns.</p

LPLI attenuates apoptosis.

<p>Bax and cleaved caspase-3 were assessed by immunohistochemistry. There is no expression of Bax or cleaved caspase-3 in control SMGs. Both markers were localized in SMGs from the diabetic rats, and were strongly stained in striated duct cell cytoplasm (arrows), but after LPLI there was no expression of Bax and only a faint expression of cleaved caspase-3. C, Control; D, Diabetes; DL, Diabetes + LPLI; ED, excretory duct; SD, striated duct. Scale bars, 50 μM.</p

LPLI attenuates inflammation via NF-κB.

<p>The expression of AGE, RAGE and phospho NF-κB p65 is observed only in diabetic rats (arrows), in striated and excretory duct cell cytoplasm, with little expression of AGE and RAGE in the DL group and no expression of phospho NF-κB after LPLI. C, Control; D, Diabetes; DL, Diabetes + LPLI; ED, excretory duct; SD, striated duct; ID, intercalated duct. Scale bars, 50 μM.</p

LPLI decreases the expression of cell death markers.

<p>Western blotting analysis showed that inflammatory markers (HMGB1, AGE, RAGE and phospho NF-κB p65) and apoptotic markers (Bax, p53, phospho-p53 (Ser15) and cleaved caspase-3) were increased in diabetic rats and were reduced by LPLI. Protein sizes were evaluated by standard protein markers, and their sizes were as follows: HMGB1 (25 kDa), AGE (non-specified), RAGE (46 kDa), phospho NF-κB p65 (65kDa), Bax (21 kDa), phospho-p53 (Ser15) (53 kDa), p53 (53 kDa), cleaved caspase-3 (17–19 kDa) and β-actin (42 kDa). TNF-α and Bad were assessed by immunohistochemistry. There was no expression of TNF-α or Bad in the control group. TNF-α and Bad were both strongly stained in striated duct cell cytoplasm (arrows) in SMGs from diabetic rats, but can barely be seen after LPLI (Fig 4). C, Control; D, Diabetes; DL, Diabetes + LPLI; ED, excretory duct; SD, striated duct. Scale bars, 50 μM.</p

Co-localization of Bax and HMGB1 assessed by double immunofluorescence staining.

<p>Double-positive cells were faintly localized in striated and excretory duct cells in the control SMGs, with intense staining in duct cells, as well as in endothelial cells of diabetic rat SMGs. These expression patterns are attenuated by LPLI. C, Control; D, Diabetes; DL, Diabetes + LPLI; ED, excretory duct; SD, striated duct; EN, endothelium. Original magnification 20X.</p

Experimental design.

<p>The rats were divided into 3 groups: control (C), diabetes (D) and diabetes + LPLI (DL). On day 1, diabetes was induced in the D and DL groups by injection of STZ. On day 29, the DL group received the LPLI. On day 30, all rats were sacrificed and their SMGs were collected. Each rat’s blood glycemia level was checked on days 3 and 30.</p

LPLI reduces HMGB1 accumulation in SMGs from diabetic rats.

<p>Morphology of SMGs from control, diabetic and diabetic rats treated with LPLI shows typical acinar and ducts cells in all 3 groups. Immunohistochemical analysis of HMGB1 in control, diabetic and diabetic rats treated with LPLI. HMGB1 expression was localized in the nuclei of striated duct cells (arrows), as well as, in the nuclei of endothelial cells in SMGs of diabetic rats. C, Control; D, Diabetes; DL, Diabetes + LPLI; ED, excretory duct; SD, striated duct. Scale bars, 50 μM. Diabetes increases RAGE and HMGB1 mRNA levels analyzed by qRT-PCR, and LPLI reduces those levels to patterns similar to the control group. Two independent measurements were averaged and relative gene expression levels were calculated as a ratio to β-actin expression of each sample.</p

List of primer sequences for the reverse (R) and (F) forward direction.

<p>List of primer sequences for the reverse (R) and (F) forward direction.</p

New bone formation area and periodontal ligament thickness for the studied materials.

<p>New bone formation area and periodontal ligament thickness for the studied materials.</p