Immunoexpression of angiogenesis, nitric oxide synthase, and proliferation markers in gingival samples of patients with aggressive and chronic periodontitis.

BACKGROUND: In periodontal tissues, angiogenesis seems to be important for the maintenance of healthy tissues and in periodontal diseases. Angiogenesis is regulated through a complex interplay of molecular signals mediated by growth factors involving extracellular matrix remodeling, endothelial cell migration and proliferation, capillary differentiation and anastomosis. However, the influence of angiogenesis in the development, progression, and healing of periodontal lesions is currently under investigation. This immunohistochemical study evaluates the expression of vascular endothelial growth factor (VEGF), microvessel density (MVD), nitric oxide synthase (NOS) 1 and 3, and Ki-67 in gingival tissues of patients with aggressive and chronic periodontitis. METHODS: Twenty-seven human gingival biopsies were taken from patients with chronic periodontitis (n = 14 patients), generalized aggressive periodontitis (n = 6 patients), and healthy periodontia (n = 7 patients). The specimens were immunohistochemically stained for VEGF, MVD, NOS 1 and 3, and Ki-67. RESULTS: The levels of VEGF, MVD, NOS 1 and 3, and Ki-67 were found to be significantly different among groups (P >0.001). Patients with aggressive periodontitis had higher levels of these markers compared to those in patients with chronic periodontitis and healthy patients. CONCLUSIONS: The analysis demonstrates a higher expression of all immunologic markers particularly in subjects with aggressive periodontitis. In summary, the data from this pilot investigation suggests that VEGF is an important factor in the pathogenesis of the aggressive and chronic forms of periodontitis.

Cementogenesis examined from the viewpoint of microcirculation.

The purpose of this study was to perform a three-dimensional observation, via microvascular corrosion casts, of the microcirculation system during deposition of cementum after flap surgery and to investigate the permeable structure of the vascular endothelium. Two stages of wound healing after flap surgery were confirmed based on successive vascular changes. The transition between these stages occurred 3 weeks after surgery, at which time new blood vessels disappeared and an early stage of accumulation of new cementum was apparent. Hence, fibrous repair occurred during the first stage, and repair of hard tissue (ie, formation of cementum) occurred during the second stage. These findings suggest that metabolic activity in cementogenesis is low, based on the condition of the blood vessels, and therefore new cementum is not easily formed.

The vasculature in chronic adult periodontitis: a qualitative and quantitative study.

Blood vessel features in periodontal pocket soft tissues may be significant in the pathogenesis and progression of chronic periodontitis. The aim of this study was to make a quantitative histological assessment of the vasculature in soft tissue biopsies from patients with chronic adult periodontitis and patients with healthy periodontal tissues. We have also investigated changes in tissue morphology at both the histological and ultrastructural level. Twelve interdental biopsies were obtained, 6 from chronic adult periodontitis patients and 6 from healthy volunteers. The specimens were sliced, fixed in 3% glutaraldehyde, postfixed in 1% buffered osmium tetroxide, dehydrated, and embeded in araldite. One micron semithin sections were differentially stained with a dichromatic technique. The number of blood vessels (BV) for sub-epithelial, superficial and deep connective tissue layers were then assessed. Only in the sub-epithelial connective tissue layer was there a significant increase in the number of blood vessels (95% Confidence interval [CI]) in the chronic adult periodontitis specimens when compared to healthy specimens. The results of this study seem to indicate that a dichromatic staining technique facilitates the identification and quantification of blood vessels in epoxy resin embedded specimens at light microscope level, and that there is an increase in the number of blood vessels in the chronic adult periodontitis lesions.

An immunohistochemical and monastral blue-vascular labelling study on the involvement of capsaicin-sensitive sensory innervation of the junctional epithelium in neurogenic plasma extravasation in the rat gingiva.

Nerve fibres immunoreactive for substance P (SP) and calcitonin gene-related peptide (CGRP) were located preferentially at the base of the junctional epithelium. Occasional fibres were observed in close proximity to the subepithelial, small blood vessels. The vascular connective tissue papillae projecting into the epithelium were more densely surrounded by SP- and CGRP-immunoreactive fibres in the interdental col than in other regions of the gingiva. In some cases, hyperplasia of the junctional epithelium was noted in the interdental col where the connective tissue papillae were invaded by widened vessels, indicating severe irritation. SP- and CGRP-immunoreactive fibres around these papillae showed increases in their immunoreactivity and thickness, with some fibres terminating as large expansions. Double immunohistochemical staining revealed the co-existence of SP and CGRP in all nerve fibres within and under the junctional epithelium. Capsaicin pretreatment eliminated most of the immunoreactivity for both peptides. Intravenous infusion of capsaicin or SP caused increased permeability in vessels underlying the junctional epithelium, as indicated by Monastral blue labelling. Labelled vessels were arranged not only in a network extending under the epithelium but also in loops protruding into the connective tissue papillae. These labelled vessels were most abundant in the interdental col, where vascular loops with more complex configurations exhibited strong staining in their walls. In the case of hyperplasia of the junctional epithelium in the interdental col, widened vessels showing extensive labelling in their walls were observed. In capsaicin-pretreated animals, capsaicin-induced extravasation was abolished, while the effect of SP was still observed. These findings provide evidence that capsaicin-sensitive sensory nerves supplying the junctional epithelium are involved in neurogenic plasma extravasation in the rat gingiva. The enhancement of neurogenic plasma extravasation in the col may be vascular response associated with a higher susceptibility of this region to gingival inflammation.

Vascular adhesion molecules and initial development of inflammation in clinically healthy human keratinized mucosa around teeth and osseointegrated implants.

Expression of vascular cell adhesion molecules (HEV-CAMs) by capillary loops represents an early step necessary for leukocyte extravasation and subsequent migration to sites of inflammation. The purpose of this investigation was to compare, the presence and distribution of ICAM-1, ELAM-1, VCAM-1 and PECAM-1 expression in the microvasculature of healthy gingiva and peri-implant keratinized mucosa. The selected HEV-CAMs were detected by a three stage immunoperoxidase technique in serial sections from clinically characterized sites. Biopsies were qualitatively and quantitatively analyzed. All biopsies displayed a small inflammatory infiltrate subjacent to the most coronal extension of the junctional epithelium. HEV-CAMs were expressed both in the sulcular and oral aspects of biopsies. Intensity of ICAM-1, ELAM-1 and VCAM-1 staining, however, was consistently higher in the region of the infiltrated connective tissue subjacent to the most coronal extension of the junctional epithelium. Only a fraction of microvascular loops were positive for ICAM-1, ELAM-1 or VCAM-1 when compared to the constitutively expressed PECAM-1. No significant differences were observed between gingiva and keratinized peri-implant mucosa. It was concluded that in healthy gingiva or peri-implant mucosa 1. HEV-CAM expressing capillary loops were in close topographic association with the inflammatory infiltrate; 2. only a fraction of capillary loops are activated to express ICAM-1, ELAM-1 and VCAM-1 at any given time; and 3. HEV-CAM expression in the periodontium may be influenced by local factors.

Disturbances of gingival fibroblast population homeostasis due to experimentally induced inflammation in the cynomolgus monkey (Macaca fascicularis): potential mechanism of disease progression.

We have studied the relationship between perturbations of fibroblast turnover in inflamed gingiva of different severities. To perform detailed spatial analyses of gingival fibroblast progenitor cells, inflammatory cell infiltrates and blood vessels, 3 Cynomolgus monkeys with healthy periodontium and 2 with naturally occurring gingivitis and ligature-induced periodontitis were pulse-labeled with 3H-thymidine. Morphometric analyses of radioautographs from mid-sagittal supra-alveolar gingival connective tissues of incisors were performed in sites subjacent to junctional sulcular and oral epithelium, in the body of the lamina propria and just superior to the alveolar crest. The percentage of fibroblasts incorporating 3H-thymidine label, expressed as the labeling index (LI), was higher subjacent to the sulcular epithelium in periodontitis (1.73 +/- 0.37) than in healthy sites (1.06 +/- 0.22). This was not statistically significant (0.05 < p < 0.1) due to the small number of animals used. The sites subjacent to the sulcular epithelium also exhibited the largest increase in lymphocyte density from health to gingivitis (p < 0.01). In contrast, the LI of fibroblasts subjacent to the oral epithelium was 5-fold higher in healthy (0.82 +/- 0.17) compared to periodontitis sites (0.13 +/- 0.09; p < 0.05). Labeled fibroblasts were found close to blood vessels in all compartments and in all disease states; distance to blood vessels was reduced in inflamed sites (p < 0.10). There were increased numbers of blood vessels per unit area in the lamina propria of gingivitis compared to healthy sites. However, there were no regional differences with respect to blood vessel numbers or area in sites subjacent to junctional epithelium with different disease states. The results indicate that: 1) experimentally-induced inflammation in the gingiva of Cynomolgus monkeys is associated with site-specific perturbations of cell turnover; 2) fibroblast progenitors are preferentially situated adjacent to blood vessels as in the periodontal ligament; 3) the vascular response to inflammation is a generalized increase in blood vessel numbers, but not their size; 4) reactive proliferation of fibroblasts may compensate for cell death in the lamina propria but is not detectable at the site of connective tissue attachment loss subjacent to the junctional epithelium. Failure to maintain the fibroblast progenitor population may be an important component of attachment loss in progressive periodontitis lesions.

[Changes in microvascular patterns following the periodontal flap surgery].

Serial changes in the microvascular pattern beneath the inner epithelium and in the periodontal connective tissue attachment on the upper incisors in adult mongrel dogs, were studied by scanning electron microscopy to elucidate the process of reconstructing the vascular architecture following mucoperiosteal flap surgery. In the early stage of wound healing, capillaries beneath the epithelium in the marginal gingiva were transformed into glomeruli, in the course of the predominantly elongation type of angiogenesis. At the junctions, newly formed sinusoidal vessels were formed in the course of predominantly sprouting type of angiogenesis, and anastomosed with vasculatures between the flap and the periodontal membrane. In these newly formed vessels, vascular glomeruli in the marginal gingiva and newly formed sinusoidal vessels at the coronal side of the junctions were transformed into a capillary network beneath the inner epithelium accompanying the epithelization. At the apical side of the junctions, newly formed sinusoidal vessels showed a series of changes to cope with alterations of the tissue environment accompanying connective tissue attachment, but the vasculatures established after surgery showed a dense and irregular arrangement compared with the control side. These results show that the attachment mechanism obtained by wound healing differ from the original one.