The early distribution and possible role of nerves during odontogenesis.

Neural crest cells migrate along specific pathways to reach the mandibular and maxillary arches where they condense under specific areas of the ectoderm which will give rise to the primary and permanent dentition. In the mouse, the trigeminal ganglion becomes evident on E9 and the superior cervical sympathetic ganglion E13. Several studies have suggested that nerves in the vicinity of the developing teeth could influence the surrounding tissues and initiate tooth development, whereas other investigators have suggested that tooth development will proceed without an intact innervation. Innervation of the dental papilla has been reported as early as the cap stage in human teeth using an antibody to PGP 9.5. A large variety of putative neurotransmitters have been localized in the nerves of the dental pulp. Many of the putative neurotransmitters function in vasoregulation while others have unknown functions. A hypothesis is presented describing a possible signal transduction pathway between odontoblasts and nerve terminals.

H3-HRP analysis of the nerve supply to primate teeth.

Sensory, sympathetic and parasympathetic ganglia located in the head and neck of rhesus monkeys were histologically examined after injection of H3-HRP into the right mandibular premolars and molars. The results showed positive labeling of ganglionic cell bodies located in the ipsilateral trigeminal, superior cervical, and otic ganglia, plus the ipsilateral mesencephalic nucleus of the trigeminal nerve.

The effect of platelet-derived growth factor on the cellular response of the periodontium: an autoradiographic study on dogs.

Platelet-derived growth factor (PDGF) is a polypeptide growth factor considered to have a role in the proliferation and migration of fibroblasts at a wound healing site. The aim of this investigation was to determine if PDGF, when applied to root surfaces, would stimulate the proliferation of fibroblasts and further enhance regeneration. Six mongrel dogs with healthy periodontia were selected for this study. Using a closed wound surgical model, standardized 4 x 4 mm fenestration defects were created into dentin on the mid-facial of the mesial and distal roots of 4 mandibular posterior teeth in each quadrant. Each defect received either: 1) saline solution (C); 2) expanded polytetrafluoroethylene (ePTFE) membrane; 3) PDGF; or 4) ePTFE + PDGF. 3H-thymidine was administered 1 hour prior to animal sacrifice at 1, 3, and 7 days postsurgery. Each time period included 2 dogs with each dog undergoing the four different treatments. Slides were prepared for autoradiography. 3H-thymidine-labeled cells were counted and results were statistically analyzed using the Bonferroni (Dunn) t test on the SAS program. Results indicated PDGF enhanced fibroblast proliferation when compared to the groups without PDGF. Significant differences (P < 0.05) were noted at day 1 and 7 when PDGF and PDGF + GT were compared to C and GT groups. No significant differences were observed in labeled fibroblasts between the C and GT groups at any time period. These findings suggest that PDGF enhances fibroblast proliferation in early periodontal wound healing, whether used alone or in combination with the ePTFE membrane.

A clinical assessment of the effects of 10% carbamide peroxide gel on human pulp tissue.

Bleaching vital teeth with 10% carbamide peroxide gel is a routine procedure in which there has been no evidence of associated permanent pulpal damage. Synthesis of the enzyme heme oxygenase-1 (HO-1) is increased after exposure of eukaryotic cells to conditions of oxidative stress (including H2O2) as a defense against the damaging effects of free radicals. Dental pulps were evaluated for HO-1 (aka Heat Shock Protein 32) presence in teeth treated with 10% carbamide peroxide. Seventeen intact first premolars scheduled for orthodontic extraction were bleached for 4 h immediately preceding extraction. Fourteen additional premolars from the same individuals were not bleached. All 31 teeth were extracted, fixed, demineralized, frozen, sectioned, and immunostained with anti-HO-1 antibody using a standard ABC protocol. There was no significant difference in the presence of HO-1 between total bleached versus total unbleached teeth using the Fisher's Exact Test (p < or = 0.05). However, the histological findings could be interpreted to suggest that coronal odontoblasts and endothelial cells in the underlying pulp proper may have the potential to respond to oxidative stress by increasing the synthesis of HO-1 (HSP32). This could represent a component of an initial defensive response by specific cells in strategic locations in the pulp that precedes classical inflammatory pathways.

Autoradiographic analysis of odontoblast replacement following pulp exposure in primate teeth.

Cell migration and replication associated with odontoblast replacement occurring soon after pulp exposure in primate teeth were studied. Class 5 cavity preparations resulting in pulp exposures were restored with a calcium hydroxide-containing capping agent and amalgam. Eighty-four and 96 h after this the animals were injected with 0.5 microCi/g body wt tritiated thymidine (sp. act. 6.7 Ci/mM). Teeth were extracted 6, 8, 10 and 12 days after treatment. The number of labelled cells as well as the number of grains per labelled cell were counted for odontoblast-like, fibroblast-like and perivascular cells in three 60 x 260 microns zones. These zones represented the odontoblast and cell-free (zone 1), cell-rich (zone 2) and deep pulp (zone 3) areas of normal pulp tissue. Ten sections centred around the mid-point of the exposure were counted for each tooth. Matrix formation and labelled odontoblast-like cells were observed at the interface between the capping agent and the pulp as early as day 8. Other significant findings were: (1) an increase in labelled odontoblast-like cells in zone 1 over time, suggesting a continual influx of differentiating cells; (2) an increase in labelled cells in zone 1 over time with a concurrent decrease in zone 3, suggesting that the influx of cells in zone 1 was from the deeper pulp; and (3) differences in grain counts between zones, treatment times and cell types, indicating that at least two DNA replications had occurred between initial treatment and final odontoblast-like cell differentiation.

Tritiated thymidine autoradiographic study on the influence of sensory and sympathetic innervation on periodontal wound healing in the rat.

Understanding of wound healing mechanisms is important in designing preventive and therapeutic approaches to inflammatory periodontal diseases, which are a major cause of dental morbidity. In this study, cell proliferation was assessed after an experimental gingival wound; this was preceded by either resection of 3 mm of the inferior alveolar nerve, total extirpation of the superior cervical ganglion, trauma to those structures or sham operations. At different times, animals were pulsed with 0.5 microCi/g body weight of tritiated thymidine; histological sections were processed for quantitative autoradiography of different compartments of the periodontium. Wounding led to a significant increase in cell proliferation in the epithelial layer, the fibroblast compartment and the periodontal ligament, but not in the alveolar crest compartment. Sympathetic denervation significantly enhanced this response in the epithelial layer, the fibroblast compartment and the alveolar crest, whereas sensory denervation only modified the response in the fibroblast layer. Thus it appears that sympathetic innervation plays an important role in the regulation of cell proliferation in the periodontium and that pharmacological modulation of sympathetic activity should be further studied as a therapeutic approach in periodontal disease.

Autoradiographic study of the effects of pulsed electromagnetic fields on bone and cartilage growth in juvenile rats.

Application of pulsed electromagnetic fields (PEMF) has been used in growth and repair of non-union bone fractures. The similarities between the fibrocartilage callus in non-union bone fractures and the secondary cartilage in the mandibular condyle, both histologically and functionally, lead naturally to study the effects of PEMFs on growth in the condyle. The purposes of this study were: (1) to describe the effects of PEMFs on the growth of the condyle using autoradiography, [3H]-proline and [3H]-thymidine, and (2) to differentiate between the effects of the magnetic and electrical components of the field. Male pre-adolescent Sprague-Dawley rats (28 days old) were divided into three experimental groups of five animals each: (1) PEMF-magnetic (M), (2) PEMF-electrical (E) and (3) control, and were examined at three different times-3, 7 and 14 days of exposure. Each animal was exposed to the field for 8 h per day. Histological coronal sections were processed for quantitative autoradiography to determine the mitotic activity of the condylar cartilage and the amount of bone deposition. The PEMF (magnetic or electrical) had statistically significant effects only on the thickness of the articular zone, with the thickness in the PEMF-M group being the most reduced. Length of treatment was associated with predictable significant changes in the thickness of the condylar cartilage zones and the amount of bone deposition.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of root conditioning on periodontal wound healing with and without guided tissue regeneration: a pilot study. II. Autoradiographic evaluation.

This investigation deals with the proliferation and migration of the progenitor cells during the healing of closed periodontal wounds. Periodontal surgical defects affecting the bone and dentin were created in four mongrel dogs. The defects were treated with topical applications of citric acid, tetracycline, or sterile water with and without the placement of nonresorbable membranes. The dogs were killed at 1, 3, 7, and 21 days after surgery. One hour before they were killed, they were intravenously injected with tritiated thymidine. Tissues were processed and routinely prepared for autoradiographic studies. Labeled cells were counted at the apical, coronal, and central areas of the defects. Results suggested that the citric acid and tetracycline treatments inhibited cellular proliferation at the initial time periods of 1 and 3 days. At 7 and 21 days, differences between citric acid and tetracycline treatments were minimal, and neither showed any advantage over the application of sterile water. The placement of the nonresorbable membrane demonstrated a trend of increased labeling at 21 days for all three treatments.

Effect of root conditioning on periodontal wound healing with and without guided tissue regeneration: a pilot study. 1. Histologic evaluation.

Closed fenestration wounds in four mongrel dogs were used to study the source of fibroblast proliferation and extracellular matrix production during healing; the arrangement and attachment of newly formed collagenous fibers; and the cementogenesis and osteogenesis at healing sites. Fenestration wounds were made through the alveolar bone, periodontal ligament, cementum, and dentin, and citric acid, tetracycline, or sterile water was applied to the dentinal walls for 3 minutes. Nonresorbable membranes were randomly placed over half of the defects. Animals were killed at 1, 3, 7, or 21 days and routine histologic examinations with hematoxylin and eosin staining followed. Results of this pilot study suggest that the periodontal ligament and/or alveolar bone are the main source of fibroblast proliferation and migration as well as extracellular matrix formation at the initial stages of healing, and that at 21 days, citric acid stimulated more cementogenesis than tetracycline or sterile water. Also, while the tetracycline influenced the maximal deposition of alveolar bone, no differences in healing were found between the citric acid, tetracycline, and sterile water with and without the use of membrane barriers.

Preliminary report on the effects of propolis on wound healing in the dental pulp.

The purpose of this investigation was to determine the antimicrobial and healing potential of propolis on direct dental pulp exposures. This study used 25 adult male rats. Pulp exposures were performed and animals were allocated to propolis and calcium hydroxide (Ca(OH)2 groups. Animals were killed on days 5, 7, 10, and 14. The teeth were routinely processed for histological evaluation. Non-parametric tests were employed to analyze the data. No significant differences were found between study groups on the wound healing of the dental pulp. Both substances were comparable in exhibiting normal reorganization of the pulp and no increased vascularity, and were equally efficacious in maintaining a low inflammatory and microbial cell population as well as in stimulating the formation of reparative dentin.

An ultrastructural and autoradiographic analysis of primary and replacement odontoblasts following cavity preparation and wound healing in the rat molar.

Numerous studies using various animal and human models have reported changes in the morphology and metabolic activity of primary odontoblasts in the mature tooth pulp after perturbations of the tooth including cavity preparation and restoration, pulpal exposures and pulp capping with various capping agents. The first part of this study investigated changes in primary and replacement odontoblast activity after cavity preparation or pulpal exposure. Two groups of rats were used in this investigation. One group of rats had Class V cavities prepared to the DEJ of the first maxillary molars. These rats were immediately injected with 3H-proline and killed 15, 30 or 60 minutes later. Rats killed at day 1, 3, 5, 7, 10 or 14 were injected one hour prior to sacrifice. The second group of rats each had a pulp exposure that was capped with a calcium hydroxide containing material and restored with a composite resin. Rats were sacrificed as previously described. Tissue was processed routinely for ultrastructural analysis and E.M. autoradiography. The second part of this study consisted of an injection of 125I-fibrinogen one hour prior to a class V cavity preparation 1/2 the distance through dentin thickness. Rats were sacrificed at 5, 10, 15 and 30 minutes postsurgery. Differences in the location and distribution of the reduced silver halide grains were recorded as well as differences in the amount and distribution of the various organelles measured between primary and replacement odontoblasts. The results of this study suggests that primary and replacement odontoblasts were morphologically and physiologically dissimilar at the time periods tested in this study. 125I-fibrinogen was demonstrated within the dentinal tubules and in the floor of the cavity preparation as early as 5 minutes after completion of the cavity preparation. The preliminary results of the 125I-fibrinogen suggest that operative trauma can effect very rapid changes to the dental pulp leading to leakage of plasma proteins from the circulation, between odontoblasts, out of the tubules to the cut dentin surface.

Evaluation of the effects of sensory denervation on osteoblasts by 3H-proline autoradiography.

The inferior alveolar nerve was unilaterally resected in 30-day-old mice; other animals were unilaterally sham-operated. At 15, 30, 60, 90, or 150 days after surgery, the mice wee injected with 2 muCi of 3H-proline (sp. act. 1.0Ci/mM) per g of body weight and killed 15, 30, or 60 min later. Autoradiographs were prepared from 5 micron decalcified sagittal sections of mandibles and grain counts made over periosteal osteoblasts mesial to the first molar. In denervated mandibles, osteoblasts incorporated less isotope compared to controls with differences being maximal at the early intervals. These differences became attenuated with time, possibly due to an intrinsic compensatory mechanism, secondary to neurotrophic regulation.

Neuroregulation of protein synthesis in odontoblasts of the first molar of the rat after wounding.

Odontoblasts respond to occlusal trauma by increased elaboration of a matrix which is subsequently calcified to form reparative dentin. The purpose of the present study was to analyze quantitatively and compare the ability of odontoblasts to synthesize collagen after wounding in rats with an intact innervation (baseline) and in rats with sensory (inferior alveolar nerve, IAN) and/or sympathetic (superior cervical ganglion, SCG) surgical denervation. Surgery was performed 7 days prior to wounding. All rats had 1 mm of enamel and dentin removed from the occlusal surface of the first mandibular molar (resected side) with the contralateral tooth serving as a control. Rats were killed 1 h after injection with 3H-proline on days 0, 5, 10 or 15 after wounding, and mandibles were removed and processed for autoradiography. Grain counts were performed over odontoblasts throughout the pulp horns for each time period and for control and experimental molars in intact (baseline) and denervated groups. When compared to the control baseline, the experimental baseline data showed increased 3H-proline uptake throughout the study with a peak at 5 days. When compared to the baseline data, IAN and SCG results demonstrated a delay or attenuation of the protein synthetic response. The results indicate that the sensory and sympathetic neural components may regulate odontoblastic response to wounding.

Presence and location of adrenergic nerve endings in the dental pulps of mouse molars.

In all, 30 adult (45-day-old) Swiss Webster mice were used for light and electron microscopic examination of the presence, number, and location of adrenergic endings in the first molar teeth. Prior to sacrifice, 10 animals received i.p. injections at 8, 6, 4, and 2 hours of 0.5 cc of 20 mg/kg solution of 5-hydroxydopamine (5-OH-DA) as a label for adrenergic endings. The animals were then anesthetized, perfused with Karnovsky's fixative, and the teeth were postfixed in Osmic acid, decalcified, embedded in methacrylate, and serial-sectioned. The sections were surveyed by light microscopy, and the number and location of nerve endings containing the reduced 5-OH-DA were recorded. Ten control mice were injected with the vehicle solution and prepared in the same manner. A third series of mice were given a single injection of 5-OH-DA, sacrificed, and prepared for ultrastructural study. The molar pulps were divided into four areas to facilitate examination: pulp horns, coronal pulp, bifurcation area, and root pulp. These four areas were further divided into three zones: odontogenic, vascular-related, and nonvascular-associated. The location and number of endings were evaluated, and an average of approximately 70 endings containing the 5-OH-DA were found in each tooth using light microscopy. These represented 35.5 +/- 5.2 in the pulp horns; 26.1 +/- 2.4 in the central coronal; 5.4 +/- 0.7 in the bifurcation, and 5.6 +/- 0.9 in the root pulp per tooth. Vascular related endings were found in greatest number, the odontogenic zone next, and free endings lease. Verification of location of 5-OH-DA by ultrastructural analysis revealed the false transmitter in vesiculated endings in the four areas and zones of the pulp.

Healing of primate dental pulps capped with Teflon.

The pulps of Rhesus monkey teeth were exposed and capped with three materials: Teflon, a commercial hard-set calcium hydroxide (Ca(OH)2) material, and Ca(OH)2 plus saline. Experimental test periods were 3, 10, and 21 days, and 5 and 8 weeks. After treatment, the teeth were removed and processed for routine histologic evaluation. Teeth treated with the two Ca(OH)2 materials showed resolution of the inflammatory response and hard tissue formation at the exposure site as early as 10 days postoperatively, with consistent healing at 21 days and longer. Teflon had a similar soft tissue healing pattern but at a slower rate. Hard tissue formation at the exposure site in the teeth treated with Teflon was infrequent at the early time periods and present in only 20% of the teeth treated for 5 and 8 weeks. By evaluating the soft and hard tissue responses of the Ca(OH)2-capped and Teflon-capped teeth it may be possible, in future studies, to identify events unique to odontoblast differentiation during pulpal healing.

Denervation-induced changes in cell proliferation in the rat molar after wounding.

The dental pulp has the capacity to initiate and maintain repair after trauma. The purpose of the present study was to quantitatively analyze the role of the peripheral nervous system in regulation of pulpal cell proliferation in response to wounding. Six groups of ten rats were used in these studies. There was one baseline group (wounded, but innervation intact) and five resection groups. The resection groups included rats with unilateral superior cervical ganglionectomy (SCG), unilateral inferior alveolar nerve resection (IAN), unilateral chorda tympani (CT) resection, IAN + SCG, or a complete unilateral nerve resection (IAN + SCG + CT). One millimeter of enamel and dentin was removed from the first mandibular molar on the experimental (resected) side. Therefore, each rat had an experimental and control molar. Rats were killed at various intervals from day 0 to day 15 after wounding and received 0.5 muCi/g b.wt. 3H-thymidine 1 hour before death. For the baseline (innervation intact) data a peak in 3H-thymidine incorporation occurred at 5 days after wounding. In the resected groups, there was a general increase in the number of labeled cells at the zero time point, and a suppression of the 5-day peak with a delay in the proliferative response to wounding. The SCG + IAN-resected group maintained the lowest number of labeled cells throughout the entire experimental period compared to the experimental baseline data and the two controls. At the initial and termination points the SCG + IAN-resected groups demonstrated the highest number of labeled cells.(ABSTRACT TRUNCATED AT 250 WORDS)