Cavity Preparation In Rat Molars Research Articles

Responses of pulp vasculature after cavity preparation in rat molars

ObjectivesOur recent study showed that cavity preparation increases the number of proliferative cells in the dental pulp during postoperative days 2–5. This study aimed to clarify pulp vascular changes following cavity preparation. MethodsGroove-shaped cavities were prepared on the medial surfaces of the upper first molars of 100-day-old Wistar rats. The animals were perfusion-fixed and tissues were collected during postoperative days 1–30, with subsequent India ink perfusion, immunohistochemistry for type IV collagen, CD31, and protein gene product (PGP) 9.5 and scanning electron microscopy with KOH digestion. The untreated upper first molars were used as controls. ResultsIn the controls, blood vessels with large diameters were located in the center of the pulp tissue and ramified to make capillary networks and PGP9.5-positive nerves were extensively arborized to form the subodontoblastic nerve plexus beneath the odontoblast layer. Cavity preparation induced disturbance in injured odontoblasts and subodontoblastic capillaries and nerves. Blood vessel density and thickness subsequently increased in the center and periphery of the pulp tissues with the exception of the subodontoblastic capillary network during postoperative days 3–5. PGP9.5-positive nerves overlapped with CD31-positive blood vessels in the mesial coronal pulp. Until day 30, when the tertiary dentin formation was completed, the pulp blood vasculature showed the same distribution and morphological features as that of the controls. ConclusionsThese results suggest that increased vascular flow under neuronal regulation plays an important role in cell proliferation in the dental pulp following cavity preparation.

Cell dynamics in the pulpal healing process following cavity preparation in rat molars

Odontoblast-lineage cells acquire heat-shock protein (HSP)-25-immunoreactivity (IR) after they complete their cell division, suggesting that this protein acts as a switch between cell proliferation and differentiation during tooth development. However, there are few available data concerning the relationship between cell proliferation and differentiation following cavity preparation. The present study aims to clarify the expression of HSP-25 in the odontoblast-lineage cells with their proliferative activity after cavity preparation by immunocytochemistry for HSP-25 and cell proliferation assay using 5-bromo-2'-deoxyuridine (BrdU) labeling. In untreated control teeth, intense HSP-25-IR was found in odontoblasts and some subodontoblastic mesenchymal cells. Cavity preparation caused the destruction of odontoblasts and the disappearance of HSP-25-IR was conspicuous at the affected site, although some cells retained HSP-25-IR and subsequently most of them disappeared from the pulp-dentin border by postoperative day 1. Contrary, some subodontoblastic mesenchymal cells with weak HSP-25-IR began to take the place of degenerated cells, although no proliferative activity was recognizable in the dental pulp. Interestingly, proliferative cells in the dental pulp significantly increased in number on day 2 when the newly differentiating cells already arranged along the pulp-dentin border, and continued their proliferative activity in the wide range of the pulp tissue until day 5. These findings indicate that progenitor cells equipped in the subodontoblastic layer firstly migrate and differentiate into new odontoblast-like cells to compensate for the loss of the odontoblast layer, and subsequently the reorganization of dental pulp was completed by active proliferation of the mesenchymal cells occurring in a wide range of pulp tissue.

Pulpal regeneration after cavity preparation, with special reference to close spatio-relationships between odontoblasts and immunocompetent cells.

The regeneration process of the odontoblast cell layer incident to tooth injury, especially its relationship with immunocompetent cells in pulp healing, has not been fully understood. The purpose of the present study was to clarify this relationship between odontoblasts and immunocompetent cells in the process of pulp regeneration following cavity preparation in rat molars by immunocytochemistry for heat shock protein (Hsp) 25 as well as class II major histocompatibility complex (MHC) molecules. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin, whereas class II MHC-positive cells were predominantly located beneath the odontoblast cell layer. Cavity preparation caused the destruction of the odontoblast layer to form an edematous lesion and the shift of class II MHC-positive cells with the injured odontoblasts toward the pulp core at the affected site. Some damaged odontoblasts without apparent cytoplasmic processes, round in profile, retained the immunoreactivity for Hsp25, suggesting the survival of a part of the odontoblasts against artificial external stimuli. Twelve hours after cavity preparation, numerous class II MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules. By postoperative 72 hours, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border, but the class II MHC-positive cells moved from the pulp-dentin border to the subodontoblastic layer. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts. The functional roles of Hsp 25-positive odontoblasts and immunocompetent cells such as class II MHC-positive cells in the process of pulp regeneration after cavity preparation are discussed in conjunction with our previous experimental data.

Changes in the pattern of horseradish peroxidase diffusion into predentin and dentin after cavity preparation in rat molars.

The purpose of this study was to examine the process of reducing dentin permeability in adult rat molars after cavity preparation with horseradish peroxidase as a tracer. Class V cavities were prepared on the upper first molars of 18 rats. Horseradish peroxidase was injected into the vascular system at intervals of 3 hours and 3, 5, 7, 10, and 14 days after cavity preparation, and frozen sections were processed by the diaminobenzidine procedure. Horseradish peroxidase reaction products were noted in the dentinal tubules immediately beneath the cavity up until 14 days after cavity preparation. An abrupt stoppage of reactive products caused by impermeable junctional zone formation at the interface of the primary dentin and reparative dentin was observed at 14 days after cavity preparation. Reparative dentin formation and subsequent impermeable junctional zone formation participate in the reduction of dentin permeability after cavity preparation.

Responses of odontoblasts to cavity preparation in rat molars as demonstrated by immunocytochemistry for heat shock protein (Hsp) 25.

Responses of odontoblasts to cavity preparation in rat molars were investigated by immunocytochemistry for heat shock protein (Hsp) 25. In untreated control teeth, intense Hsp 25-immunoreactivity was found in the cell bodies of odontoblasts and their processes within the predentin. Confocal microscopy of Hsp 25-immunostained and rhodamine-labeled sections revealed that the immunoreactive odontoblasts were intensely labeled for phalloidin at the periphery of their cytoplasm and throughout their processes, but the reaction for phalloidin was limited within the inner half of the dentin. Cavity preparation caused an edematous reaction between the injured odontoblasts and predentin as well as a beaded swelling and successive destruction of the odontoblast processes. Immediately after cavity preparation, the odontoblasts beneath the edematous lesion showed an immunoreactivity for Hsp 25, which subsequently disappeared completely from the pulp-dentin border by 12 h after the operation. However, round cells without apparent cytoplasmic processes continued to be immunoreactive, suggesting the survival of a part of the odontoblasts against preparation stimuli. Numerous phalloidin-reactive but Hsp 25-immunonegative cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, probably categorized in a lineage of immunocompetent cells. By postoperative 72 h, newly differentiated odontoblasts with Hsp 25-immunoreactivity were arranged at the pulp-dentin border. These findings indicate that the time course of changes in the expression of Hsp 25-immunoreactivity reflects the regeneration process of odontoblasts, and suggest that this protein is a useful marker substance for differentiated odontoblasts.

The Re-Expression of Nerve Growth Factor Protein after Cavity Preparation in Rat Molars.

It is well known that nerve growth factor (NGF) is expressed in the odontoblasts of developing teeth, but not in completely grown teeth in rats. However, there have been no detailed studies of the expression of NGF in reactivated primary odontoblasts or in newly differentiated odontoblast-like cells during pulp repair. Therefore, to examine the involvement of NGF in odontoblasts during pulp repair, we immunohistochemically observed the localization of NGF in dental pulp after cavity preparation in rat molars. Based on the depth of the cavity, two types of pulp reaction were thus distinguished. 1) In shal low cavities, primary odontoblasts secreted a new dentin matrix, reactionary dentin, and also expressed NGF. 2) In deep cavities, odontoblast-like cells, differentiated from pulpal mesenchymal cells, formed a large amount of reparative dentin or osteodentin. Additionally, intense NGF-like immunoreactivity was observed in odontoblast-like cells. These results suggest that NGF not only acts as a neurotrophic factor of dental pulp, but is also involved in the differentiation and functional change of odontoblasts during pulp repair.

Fibrinogen/fibrin and fibronectin in the dentin-pulp complex after cavity preparation in rat molars

Objective. The purpose of this study was to examine changes in distribution of fibrinogen/fibrin and fibronectin in the dentinpulp complex after cavity preparation. Study design. Class V cavity preparations were prepared on maxillary first molars of 12 rats. The dentin and pulps were observed histologically and immunohistochemically for fibrinogen and fibronectin at 6 hours and 1, 2, and 3 days after the preparation. Results. At 6 hours and 1 day after cavity preparation, positive staining for fibrinogen was noted in the exudative lesion and in the dentinal tubules under the cavity preparation. Fibronectin staining in the exudate showed a pattern with close similarity to the fibrinogen staining. At 3 days after cavity preparation, the irregularly shaped predentin under the cavity preparation showed strong positive staining for fibronectin. Conclusions. Fibrinogen/fibrin and fibronectin are present during the healing process of the dentin-pulp complex after cavity preparation.

Responses of immunocompetent cells to cavity preparation in rat molars: an immunohistochemical study using OX6-monoclonal antibody.

Responses of immunocompetent cells, especially class II major histocompatibility complex (MHC) antigen-expressing cells, were investigated after cavity preparation in the erupted upper first molar teeth of rats, by immunohistochemistry using OX6-monoclonal antibody. In control teeth, OX6-immunopositive cells were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused an acute edematous reaction between the injured odontoblasts and predentin, and most of OX6-immunopositive cells in the affected site shifted away from the pulp-dentin border. After 12-24 hours, many OX6-immunopositive cells accumulated along the pulp-dentin border and extended their cytoplasmic processes into the exposed dentinal tubules. After 72 hours, newly differentiated odontoblasts replaced the degenerated odontoblasts, and few OX6-immunopositive cells remained along the pulp-dentin border. Our data suggest that some of the class II MHC antigen-expressing cells in the dental pulp participate in the initial defense reaction and presumably serve as a biological sensor for the external stimuli arriving through the exposed dentinal tubules.

Ultrastructural changes in odontoblasts and pulp capillaries following cavity preparation in rat molars.

Responses of odontoblasts and pulp capillaries to cavity preparation were investigated in the upper first molar teeth of rats, using light and transmission electron microscopy. At 100 days of age, the blood vessels of the pulp formed a subodontoblastic network consisting of continuous capillaries at a short distance from the odontoblast layer. Cavity preparation caused the displacement of some odontoblasts into the dentinal tubules, while others were separated from the predentin by rapid inflammatory exudation after drilling. The subodontoblastic capillary network under the injured dentin was shifted inwards together with the separated odontoblasts. The endothelium of the shifted capillaries showed a remarkable increase of pinocytotic vesicles, an event thought to be closely related to the formation of the exudative lesion. By one day after cavity preparation, most of the damaged odontoblasts had degenerated. Many cells with high nucleus/cytoplasm (N/C) ratios and prominent nucleoli accumulated around the subodontoblastic capillaries, some of which had many endothelial fenestrae facing these cells. These cells were suggestive of newly differentiating odontoblasts receiving nutritional supply from the capillaries. Three days after cavity preparation, newly differentiating odontoblasts took the place of the degenerated odontoblasts. They began to produce reparative dentin by five days after cavity preparation. Capillaries were located beneath the newly differentiating odontoblasts, but endothelial fenestrae gradually decreased in number. During the active reparative dentin formation, capillaries remained closely beneath the new odontoblast layer. Although the rate of reparative dentin deposition was not significantly lower than that in the primary dentin formation, one could not recognize an invasion of capillaries into the odontoblast layer nor a remarkable increase of endothelial fenestrae, both of which are common in active primary dentin formation. The results suggest that the function of capillaries differs between primary and reparative dentin formation.

Ultrastructural changes in odontoblasts and pulp capillaries following cavity preparation in rat molars.

Ultrastructural changes in odontoblasts and pulp capillaries following cavity preparation in rat molars.

Responses of pulpal nerves to cavity preparation in rat molars: An immunohistochemical study using neurofilament protein(NFP) antiserum.

The response of neural elements to a dentin injury was morphologically investigated in rat molars by use of immunostaining for neurofilament protein (NFP). An artificially formed cavity in dentin by drilling rapidly caused the displacement of some odontoblasts into the exposed dentinal tubules, while others were detached from the predentin. The subodontoblastic nerve plexus consisting of NFP-immunoreactive nerves shifted inward together with the separated odontoblasts, while a movement of the nerves into the exposed dentinal tubules was not recognized. The odontoblasts separated from the predentin degenerated and disappeared one day after the cavity preparation; at this time, the subodontoblastic nerve plexus underlying the drilled dentin was remarkably disrupted, presumably losing dentinal sensation of the drilled area. Three days after the cavity preparation, the destroyed odontoblastic layer began to be repaired by newly differentiating odontoblasts; the reparative dentin was produced from 5 to 7 days onward. Numerous NFP-positive nerves, beaded in type, gathered in the odontoblastic layer in accordance with the differentiation of the new odontoblasts. The increased beaded nerve fibers were suggested to represent peptide-containing nerves. In 10-15 days, the reparative dentin accumulated quite remarkably under the cavity area. The NFP-positive subodontoblastic nerve plexus was entirely reconstituted and also regained continuity to its surrounding plexus. The nerve fibers in the reconstituted plexus were mostly non-beaded in type as seen in the control teeth. Since dentinal tubules in the reparative dentin are not normally continuous to the primary dentinal tubules, dentinal sensation may not have been restored.