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Human Dental Pulp Vasculogenesis Evaluated by CD34 Antigen Expression and Morphological Arrangement

Dipartimento di Scienze Odontostomatologiche, Università di Chieti, Via dei Vestini 32, 66100 Chieti, Italy; and
¹ Istituto di Morfologia Umana Normale, Università di Ancona, Via Tronto 10/A, 60023 Ancona, Italy;

View larger version (111K): [in a new window]   Figure 1. Hematoxylin-eosin staining (A,B,C). (A) Deciduous tooth showing a range of cell types disseminated in the ground substance. Aggregates of sizeable cells form primitive vessels (arrows). (B) Pulp from 26-year-old subject. Spindle-shaped fibroblasts (light-red cells) are predominant in the condensed stroma. (C) Aged pulp composed of dense connective tissue with the presence of fibrocytes (dark red). Masson’s staining (D,E,F). Blue dye reveals the presence of collagen fibrils increasing with age. Cells stained red. (D) Deciduous tooth shows a few collagen fibrils around several forming vessels. (Large arrows, sizeable vessels; thin arrows, small vessels.) (E) Young pulp. Collagen fibrils encircle the definitive vessels (thin arrows), some of which contain peripheral blood. (F) Aged pulp. Densely packed collagen fibers are arranged in compact regular bundles (bright blue). Immunohistochemical determination of CD34 antigen (G,H,I). (G) Deciduous tooth with the presence of single CD34+ brown-stained cell (arrows), or clustered CD34+ brown-stained cells, to form a variety of microvessels. (H) Pulp from a 26-year-old subject showing the presence of differentiated CD34+ blood vessels (arrows). (I) Aged pulp containing blood vessels (arrow) not expressing the CD34 antigen. Bar: 100 µm.

View larger version (153K): [in a new window]   Figure 2. TEM view of deciduous teeth. (A) Presence of a cluster of mesenchymal-like cells in the ground substance. (Arrowsheads = nucleoli; large arrows = fibroblasts). The endothelial cells are identified by the presence of cytoplasmic Weibel-Palade bodies (thin arrows). (B) Microcapillary composed of a single endothelial cell layer enclosed in the connective tissue. The collagen fibrils are arranged in different directions, forming a loose meshwork. Bars: 2 µm.

View larger version (139K): [in a new window]   Figure 3. TEM view of an early-stage blood vessel formation. (A) One endothelial cell (E) just beginning a primitive lumen. A pericyte (P) wraps the vessel via the interposition of basement membrane. Collagen fibers exhibit longitudinal striations because of their fibrillar structure (arrows). (B) Longitudinal cut of an empty microcapillary. The endothelial cells are anchored by junction apparati (arrows) that, as reported at higher magnification in the insert, show the features of adherens junctions. Weibel-Palade "roundish" bodies are present at the cytoplasmic level. Bars: (A) 1 µm; (B) 2 µm.

View larger version (89K): [in a new window]   Figure 4. CD34 antigen investigation. (A) and (B) (higher magnification) report the immunolocalization of CD34 antigen. Gold particles present at the lumenal surface are disseminated or often aggregated (arrows) on the endothelial (E) cell surface. One pericyte (P) and the basement membrane (asterisk) are evident. The arrows in (A) indicate the presence of intercellular junctions among 3 different endothelial cells. (C) Western blot analysis of deciduous (lane A), a corresponding-aged permanent (lane B), 26-year-old (lane C), and 60-year-old (lane D) dental pulp. The higher levels of CD34 protein are detected in deciduous and permanent pulp in children, while a down-regulation is evident in adult patients; in old patients, CD34 expression is completely abolished. (D) The percentage (y-axis) of positive cells in deciduous (A), corresponding-aged permanent (B), 26-year-old (C), and healthy (D) dental pulp. The values were obtained by morphometric determination of light microscopy observations and are representative of 5 different experiments. (E) The levels of CD34 protein expressed in deciduous (A), corresponding-aged permanent (B), 26-year-old (C), and healthy (D) dental pulp, obtained by the Western-blotting technique and laser densitometry quantification and expressed as OD arbitrary units (y-axis). Results shown are representative of 3 different experiments. Bar: 1 µm. mw = molecular weight