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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;

^* corresponding author, trubiani{at}unich.it

	ABSTRACT

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Vasculogenesis describes the process by which endothelial precursor cells form new blood vessels. To characterize the topography and the cellular processes underlying vascularization of human dental pulp, we examiend the expression of the human hematopoietic progenitor cell antigen CD34. Dental pulps, obtained from deciduous and permanent teeth, were morphologically examined at light- and electron-microscope levels and by expression of CD34. The findings indicate that vasculogenesis of dental pulp is a complicated process starting from single CD34⁺ cells. These subsequently coalesce to form solid vascular cords inside the developing connective tissue, which later hollows. Pericytes were embedded within the fully formed microvessels’ basement membrane. The presence of CD34⁺ endothelial cells in permanent teeth reveals that the process of vasculogenesis persists into adult life, where it contributes to continuous adjustment of vessel and network structures in response to functional needs and dental tissue homeostasis.

KEY WORDS: CD34 • vasculogenesis • dental pulp • immunohistochemistry

	INTRODUCTION

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Vascular development entails vessel formation by vasculogenesis or by angiogenesis. Vasculogenesis involves the development of the vascular system in the embryo and is caused by in situ differentiation of endothelial progenitors or angioblasts (Reyes et al., 2002; Schmeisser and Strasser, 2002). During development, the expansion capacity of the vasculogenesis process implies that mesenchymal-derived angioblasts should be able to unite in the primitive mesenchyme, thus allowing the forming immature blood vessels to develop into a more mature stage. Angiogenesis is the process by which new blood vessels develop through the "sprouting" of pre-existing vessels. For many years, this was considered to be the sole mechanism of blood vessel formation in post-natal life (Moore, 2002). Both endothelial and hematopoietic cells are thought to originate from the same precursor cell, a blast-like bipotential cell, the hemo-angioblast. All of these cell types can be recognized from their cell structure, their function, and from cell-surface markers. In addition to some mature cells, virtually all stem and progenitor cells express an antigen—namely, the CD34 antigen—on their surfaces.

The CD34 antigen is a heavily glycosylated type I transmembrane protein (Sutherland and Keating, 1992; Lanza et al., 2001). The CD34 molecule’s full-length form is a monomeric structure exhibiting an apparent molecular mass of 110–120 kDa with an intracellular domain containing consensus sites for a variety of kinases (Krause et al., 1996). In normal conditions, CD34 is expressed on early lymphohematopoietic stem and progenitor cells, small-vessel endothelial cells, embryonic fibroblasts, and some fetal and adult nervous tissue cells. From observation of normal and pathological material, it has been suggested that CD34 is a signaling molecule involved in the maintenance of a phenotypically plastic state in undifferentiated cells. Recent studies suggest that endothelial stem cells might persist into adult life, contributing to the formation of new blood vessels (Moore, 2002; Reyes et al., 2002).

Dental pulp is a metabolically active tissue with a high capacity for regeneration and tissue turnover (Pinzon et al., 1966). Dental pulp develops from the primitive connective tissue forming the core of the developing tooth. The morphological characteristics of human dental pulp have been extensively analyzed (Matthews and Andrew, 1995; Yoshida and Ohshima, 1996), including the fact that dental pulp is almost completely surrounded by hard tissue, and there are few major vessels through the apical foramen supplying the human dental pulp. The microvascular bed arrangement of the dental pulp plays a major role in hard- and soft-tissue physiology, and thus its anatomy and histology have received considerable attention (Han and Avery, 1963; Rapp, 1992). However, the vascular distribution within primary tooth pulp has not been as well-studied as that within human permanent teeth. In particular, the developmental and chronological changes in the dental pulp’s vasculature and the relationship of these changes to tooth formation are insufficiently detailed. Since it has been reported that endothelial cell structure and functional integrity are important in the maintenance of vessel wall and circulatory function (Derringer et al., 1996; Sumpio et al., 2002), and perturbations of endothelial structure and function may also result in a pathological state, such information is useful to those who diagnose and treat dental conditions in normal and pathological states. Thus, an understanding of the human dental pulp’s cellular characteristics and of how vasculogenesis progresses is essential. This paper studies the presence and spatial arrangement of CD34 antigen during dental pulp microvasculature growth.

	MATERIALS & METHODS

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Intact dental pulp was obtained from 20 teeth extracted from 8- to 60-year-old patients, and from 10 deciduous teeth. All patients, including the parents of minor children, gave informed consent to the treatment protocol. After removal, the dental pulp was immediately processed for morphological and biochemical analyses. These 30 teeth had been extracted for orthodontic treatment and in accordance with the protocols approved by the Ethical Committee of the University of Chieti, Italy.

Morphological Analysis
    Light microscopy
Pulp samples were fixed in 10% formalin and embedded in paraffin. Morphological analyses were performed by standard procedures with hematoxylin-eosin staining. Masson’s dye staining was used for examination of the connective tissue and its collagen network.

    Electron microscopy
Dental pulp fragments were fixed in 2.5% glutaraldeyde, post-fixed in 2.5% OsO₄, dehydrated in a series of ethanols, and embedded in Spurr’s resin. The thin sections were stained with uranyl acetate and lead citrate, and were then observed with a Philips CM10 TEM (Philips, Eindhoven, The Netherlands).

Immunohistochemistry
    Light microscopy
Paraffin-embedded preparations were exposed to mouse monoclonal primary antibody at the typical 1:25 working dilution, and further treated with a goat anti-mouse IgG peroxidase-conjugated secondary antibody at a 1:200 dilution. DAB (0.06%) in the presence of 0.03% H₂O₂ was used for visualization of peroxidase activity. The sections were counterstained with hematoxylin. We obtained negative controls by excluding the primary or secondary antibody, while the endogenous peroxidase activity and non-specific binding were inhibited by 1% H₂O₂ and 1% bovine albumin serum. The relative intensities of peroxidase-stained complexes were quantified with the use of LUCIA Image analysis software (Nikon Italia, Milan, Italy).

    Electron microscopy
Pulp tissues were fixed with 4% paraformaldehyde and 0.1 M cacodylate buffer (pH 7.4) for 30 min at 4°C, and then dehydrated and embedded in Unicryl at -20°C. Following this, thin sections were cut and incubated overnight with the appropriate anti-CD34 antibody at 4°C. After that, the sections were incubated for 2 hrs with a goat anti-mouse IgG, gold-labeled, stained with uranyl acetate, and observed under a Philips CM10 electron microscope operating at 60 kV.

Biochemical Analysis
Western-blotting analysis was performed as previously described (Fina et al., 1990; Lanza et al., 2001; Trubiani et al., 2002). The primary antibody was used overnight at a 1:50 dilution. This was followed by the secondary antibody of horseradish peroxidase-conjugated anti-mouse IgG at 1:500, which was left to react for a period of 2 hrs. We used DAB reaction to visualize the immune complex. Quantitative analysis was carried out by laser densitometry (Bio-Rad Laboratories, Milan, Italy).

Sources of Materials
Anti-CD34 monoclonal antibody clone QBEnd/10 was supplied by Immunotech a Coulter Company (Instrumentation Laboratory, Milan, Italy). This antibody detects the class II epitope of CD34. Sigma (Sigma-Aldrich, Milan, Italy) supplied immunochemical and reagent-grade materials, while the 20-nm gold-particle-conjugated goat anti-mouse IgG (H) (Human Abs) secondary antibody and Unicryl medium were supplied by BioCell (Inalco, Milan, Italy). Reagents for electron microscopy came from Polyscience (Warrington, PA, USA), and products for electrophoresis were supplied by Bio-Rad Laboratories (Milan, Italy).

	RESULTS

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Light Microscopy
Figs. 1A, 1B, and 1C show the hematoxylin-eosin staining results. Fig. 1A shows that deciduous pulp, obtained from a six-year-old child, is composed of ground substance and contains many cell types. Fibroblasts, blood cells, and aggregates of sizeable mononuclear cells are scattered within this mesenchymal-like stroma. Furthermore, aggregates of mononuclear cells tend to form cylinder-like structures, often delimiting a lumen (arrows). However, in a 26-year-old patient (Fig. 1B), sizeable cell aggregates have disappeared from the dental pulp, and the dental pulp shows an increase in the number of differentiated cells, such as spindle-shaped fibroblasts and fibrocytes. The fibrils and extracellular matrix are arranged to form a loose connective tissue. Fig. 1C shows a tooth slice from a 60-year-old patient in which the cell population, greatly decreased, appears to be exclusively composed of fibrocytes spread within the compact connective tissue.

View larger version (111K): [in this window] [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.

Immunohistochemistry
The expression of CD34 antigen is reported in Figs. 1G, 1H, and 1I. Fig. 1G proves the presence of several variously arranged CD34⁺ cells in the deciduous pulp. A consistent number of isolated positive cells is distributed within the stroma (arrows). In contrast, other isolated positive cells are arranged in a cord-like structure, some of which becomes tubular and forms the first capillaries. The 26-year-old pulp tissue in Fig. 1H shows a down-regulation of random CD34⁺ cells and the presence of well-defined microvessels recognizable from the intense expression of CD34 antigen on endothelial cell surfaces (arrows). Aged pulp (Fig. 1) does not show CD34 positivity; the vascular bed is unnoticeable and is essentially composed of full-sized blood vessels (arrow).

Electron Microscopy
Figs. 2 and 3 report the transmission electron microscope study. Fig. 2A shows that the deciduous tooth contains a cluster of elongated mononuclear cells, as light microscopy also revealed. The oval nucleus contains nucleolar apparatus (arrowheads) and a large amount of less condensed chromatin. Close to these aggregates of mesenchymal-like cells, some differentiated fibroblasts and fibrocytes (large arrows) form the pulp’s stroma. Two endothelial cells, recognizable by the presence of specific cytoplasmic Weibel-Palade bodies (Weibel and Palade, 1964) (thin arrows), are so arranged that they form a primitive lumen, which is the site of initial vessel formation. Fig. 2B shows a subsequent stage in which one endothelial cell, immersed in the loose connective tissue, forms a well-defined microvessel. A few fibrils are dispersed in the ground substance. Fig. 3A reports the subsequent period of vessel formation, in which the collagen fibers, exhibiting the characteristic longitudinal striations as part of their component fibrils, envelop the microvessel. At this stage of vasculogenesis, another cell type becomes visible, one that encloses the forming vessel. These cells are consistent with features of pericytes. Pericytes, as peri-endothelial cells, progress alongside the forming vessels and may play a key role in vessel stabilization and vessel functions (Sims, 1991; Carlile et al., 2000). Fig. 3B shows a longitudinal section of a microvessel composed of a continuous single endothelial cell layer. Several intercellular junctions (arrows) are evident, and the dark streak between adjacent cells (insert), observable at higher magnifications, suggests the presence of transmembrane linker proteins, as described in endothelial adhesion belts (Tasman et al., 1999; Petzelbauer et al., 2000).

View larger version (153K): [in this window] [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 this window] [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 this window] [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

Quantitative Analysis
Fig. 4D illustrates the morphometric evaluation of an immunoperoxidase-stained slide, which shows the presence of more than 18% CD34⁺ cells in a deciduous tooth, 16% in an equivalent-aged permanent tooth, and falling to 3% in 26-year-old and disappearing entirely in 60-year-old dental pulp. Densitometric analysis of the level of CD34 protein (Fig. 4E) confirms the differing levels of CD34 in deciduous and same-aged teeth, compared with that in 26-year-old dental pulp, while it is unremarkable in the 60-year-old patient.

	DISCUSSION

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Vascular development involves vasculogenesis, in which endothelial cells form a primary tubular network. This has traditionally been thought of as playing a role only in embryonic development, but recent studies suggest that endothelial stem cells may persist into adult life, contributing to the formation of new blood vessels (Moore, 2002; Reyes et al., 2002). Moreover, the development and maintenance of the vascular system requires not only the formation of new vessels (vasculogenesis, angiogenesis) but also the continuous adjustment of vessel and network structures in response to functional needs. This angio-adaptation depends on the interplay of vascular responses to growth factors, the metabolic status of the tissue, and hemodynamic forces exerted by flowing blood. Therefore, the results presented in this paper suggest that dental pulp vasculogenesis originates from the synchronized aggregation of single CD34⁺ cells to form a tube-like structure. These structures represent the starting point of the future vascular bed. The extracellular matrix and the collagen fibril network play a key role in the spatial arrangement of the primary capillary plexus. Pericytes appear to wrap around the endothelium, partially covering the vessel circumference to various degrees. Moreover, the recognition of CD34⁺ cells proves that although vasculogenesis begins in the early period of tooth formation, it also continues for a very long time, persisting into adult life. The need to preserve vascular adaptability into adulthood may be explained by the fact that dental pulp is a metabolically active tissue with a high capacity for regeneration in response to different stimuli (Pinzon et al., 1966). In addition, it is important to emphasize that endothelial cell structure and functional integrity are essential to the maintenance of the vessel wall and circulatory function. Endothelial cells are dynamic and are capable of conducting a variety of metabolic and synthetic functions (Sumpio et al., 2002). These cells exert significant paracrine and endocrine actions, and the mediators are released in response to physical and chemical stimuli, as well as changes in hemodynamic forces such as alterations in blood pressure or blood flow. Endothelial cells play a key role in immune and inflammatory reactions by regulating lymphocyte and leukocyte movement into dental pulp (Sawa et al., 1998). Sepsi, or inflammatory stimuli, have profound effects on endothelial cells, provoking tissue factor synthesis, generation of adhesion molecules, inflammatory mediators, or anti-thrombotic and pro-coagulant factors. In conclusion, vasculogenesis may play a key role in causing the spatial arrangement of dental pulp formation, and the preservation of CD34⁺ cells into the early stages of adult life also makes the endothelium indispensable for tooth homeostasis.

	ACKNOWLEDGMENTS

 
We thank M. Piccirilli for expert technical assistance, and C. Prestt (Translator) for editorial assistance. This study was supported by grant no. FIRB-RBNE01N4Z9_003 from the Italian Ministry of Education, University and Research.

Received July 31, 2002; Last revision March 14, 2003; Accepted May 28, 2003

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