The anti-inflammatory effects of human recombinant copper-zinc superoxide dismutase on pulp inflammation.

Inflammation in the dental pulp is accompanied by release of a wide variety of highly oxidative molecules known as reactive oxygen species (ROS). ROS concentrations are controlled in vivo by an antioxidant enzyme scavenger system that may be overwhelmed by the increases in ROS production seen during inflammation. Supplementation of the antioxidant defense system, therefore, may limit the severity of the inflammatory response to injury due to this component. To test this hypothesis, this study examined the effects of superoxide radical scavenging on pulpal inflammation induced in rat molars by standardized cavity preparation. The extent of pulp inflammation was compared histomorphometrically between animals treated with exogenous administration of a human recombinant antioxidant enzyme, copper-zinc superoxide dismutase, conjugated to polyethylene glycol (hr-CuZn-SOD), versus saline-vehicle controls. There was a statistically significant reduction in area of inflammation involvement in those animals treated with hrCuZn-SOD, compared with controls. Although hrCuZn-SOD administration did not completely eliminate inflammation in all animals treated, there was a statistically significant lessening of the severity of the inflammatory response, as well as a greater degree of reparative dentin observed in the hrCuZn-SOD-treated animals.

Immunolocalization and adenoviral vector-mediated manganese superoxide dismutase gene transfer to experimental oral tumors.

The anti-oxidant enzyme system protects cellular macromolecules against damage from reactive oxygen species. One component of this system, manganese superoxide dismutase (MnSOD), has also been shown to display tumor suppressor gene-like activity. The purpose of this study was to examine changes in MnSOD expression during hamster cheek pouch carcinogenesis, and the effects of MnSOD overexpression using an adenoviral vector. Tumor induction was carried out using 7,12-dimethylbenz[alpha]anthracene. Animals were killed at periodic intervals, and cheek pouch tissues were excised and examined for MnSOD expression by immunohistochemistry and digital image analysis. We observed a reduction in MnSOD expression as early as 2 weeks after the start of carcinogen application. Low MnSOD expression persisted until the end of the 23-week experimental period. Solid hamster cheek pouch carcinoma xenografts were then established in nude mice. An adenoviral vector encoding the human MnSOD gene was delivered to the xenografts by direct injection. We observed high, immediate expression of MnSOD in the xenografts that persisted for 10 days following cessation of viral construct delivery. Delivery of the MnSOD construct resulted in a maximal 50% reduction in tumor growth compared with untreated controls. Our results suggest that MnSOD may be a tumor suppressor gene in the hamster cheek pouch model system.

Localization and changes in NADPH-diaphorase reactivity and nitric oxide synthase immunoreactivity in rat pulp following tooth preparation.

Inflammatory changes in the dental pulp are accompanied by release of a wide variety of chemical mediators. Nitric oxide, an oxidative free radical produced by the enzyme nitric oxide synthase (NOS), has been implicated in multiple inflammatory processes, which makes it a suitable marker for changes which likely occur following tooth pulp insult. Since limited information on nitric oxide in the pulp is available, it is necessary first to examine relative distributions of NOS in uninflamed and inflamed rat pulp. We accomplished this by characterizing regions of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity and the distribution of both macrophage NOS (macNOS) and neuronal NOS (nNOS) immunoreactivity in normal and inflamed rat molar pulp at multiple time points. The results showed that: (1) deep cavity preparation on the mesial surface of the molar produced a time-dependent inflammation, with acute inflammation early progressing to chronic, granulomatous inflammation with necrosis later that spread preferentially down the mesial root; (2) control (non-prepared) teeth showed a relatively faint and homogeneous distribution of NADPH-d and macNOS reactivity but no discernible nNOS reactivity; (3) inflamed teeth displayed localized increased intensity of NADPH-d and macNOS reactivity surrounding the inflamed area of pulp, but no increased nNOS activity; (4) pulp vessels supplying the inflamed area showed increased NADPH-d reactivity, but no increased macNOS or nNOS reactivity; and (5) neither NADPH-d, macNOS, nor nNOS reactivity was observed in pulpal nerves. Therefore, nitric oxide may mediate the pulpal inflammatory response through its effects on the paralesional pulp tissue and surrounding endothelial/vascular structures.

Localization and changes in superoxide dismutase immunoreactivity in rat pulp after tooth preparation.

OBJECTIVE: To examine the distribution of superoxide in the uninflamed and inflamed dental pulp by characterizing the immunoreactivity of the detoxifying antioxidant enzymes, manganese and copper-zinc superoxide dismutases (MnSOD and CuZnSOD, respectively). STUDY DESIGN: In 12 rats, mesial cavity preparations were made on the maxillary right first molar; left molars were unoperated controls. After 5 days, the rats were killed, and histologic sections were processed by using MnSOD and CuZnSOD immunoreactivity, and the extent of inflammation was evaluated on alternate sections stained with hematoxylin and eosin. RESULTS: In the hematoxylin and eosin-stained sections, inflammation was consistent with round-cells: macrophages, lymphocytes, and plasma cells that coalesced into a distinct leukocytic "lesion", which obliterated portions of the underlying pulp. Both MnSOD and CuZnSOD immunoreactivity increased dramatically in inflammatory cells within the leukocytic lesion and in the tissue surrounding the lesion. CONCLUSIONS: The findings suggest that the protective role of SOD increases within pulp cells that are undergoing inflammatory stimulation. SOD immunoreactivity may be an early indicator of stress in pulp.

Splanchnic tissues undergo hypoxic stress during whole body hyperthermia.

Exposure of conscious animals to environmental heat stress increases portal venous radical content. The nature of the observed heat stress-inducible radical molecules suggests that hyperthermia produces cellular hypoxic stress in liver and intestine. To investigate this hypothesis, conscious rats bearing in-dwelling portal venous and femoral artery catheters were exposed to normothermic or hyperthermic conditions. Blood gas levels were monitored during heat stress and for 24 h following heat exposure. Hyperthermia significantly increased arterial O2 saturation, splanchnic arterial-venous O2 difference, and venous PCO2, while decreasing venous O2 saturation and venous pH. One hour after heat exposure, liver glycogen levels were decreased approximately 20%. Two hours after heat exposure, the splanchnic arterial-venous O2 difference remained elevated in heat-stressed animals despite normal Tc. A second group of rats was exposed to similar conditions while receiving intra-arterial injections of the hypoxic cell marker [3H]misonidazole. Liver and intestine were biopsied, and [3H]misonidazole content was quantified. Heat stress increased tissue [3H]misonidazole retention 80% in the liver and 29% in the small intestine. Cellular [3H]misonidazole levels were significantly elevated in intestinal epithelial cells and liver zone 2 and 3 hepatocytes and Kupffer cells. This effect was most prominent in the proximal small intestine and small liver lobi. These data provide evidence that hyperthermia produces cellular hypoxia and metabolic stress in splanchnic tissues and suggest that cellular metabolic stress may contribute to radical generation during heat stress.

A review of key research design and statistical analysis issues.

This article highlights some basic principles of the design and use of statistical tests, using a minimum of mathematics or statistical jargon. It is not the intent to summarize all the possible details involved with performing these tests, but instead to offer insight into evaluating the statistical methods found in published articles. Historically, the number of scientific articles published in which inappropriate statistical analyses were performed is alarming. Only when the reader understands the problem and demands change will this situation improve. The consequence of inaction is to be mired with an array of poorly designed articles that, at the very least, do not advance the field of study and, at worse, may influence practitioners not well versed in statistics to expose patients to useless, unnecessary, or even harmful procedures.

Induced hypoxia in rat pulp and periapex demonstrated by 3H-misonidazole retention.

Cellular hypoxia may be a useful indication of tissue distress in the dental pulp that could be used to investigate the early stages of pulpal responses. Tritiated misonidazole (3H-MISO) is a marker which preferentially labels cells with decreased oxygen tension (hypoxia). The experiments reported here were carried out to determine whether this agent could distinguish between hypoxic and normoxic pulp and periapical tissues. Rats were injected intra-peritoneally with either 3H-MISO, unlabeled MISO, or saline, then divided into normoxic, hypoxic, and control groups. Normoxic animals were maintained at ambient pressure. We induced hypoxia by maintaining animals in a hypobaric chamber at 0.5 atm for 24 hrs. 3H-MISO retention was assessed by quantitative analysis of tissue autoradiographs. 3H-MISO retention rates in normoxic animals showed little variation except for increased retention in mature ameloblasts and immature odontoblasts in the continually erupting incisor. In both incisor and molar pulps, hypobaric hypoxia significantly increased 3H-MISO retention when compared with normoxic controls. Hypobaric hypoxia also resulted in intense 3H-MISO retention in cellular cementum, periodontal ligament, osteocytes, and, occasionally, in molar pulp horn odontoblasts. This study demonstrated that, with standard autoradiographic techniques, 3H-MISO can label induced hypoxic disturbances in the pulp and surrounding periodontium.

Canal debridement: effectiveness of sodium hypochlorite and calcium hydroxide as medicaments.

The action of chemicals such as calcium hydroxide (Ca(OH)2) and sodium hypochlorite (NaOCl) that are used as tissue solvents may be enhanced by prolonged contact. The objective of this study was to determine if sealing Ca(OH)2 and NaOCl into the canal space would improve debridement of both the main canal and areas inaccessible to files. Mesial root canals of 75 freshly extracted mandibular molars were step-back hand-instrumented. Another six molars were controls. Either Ca(OH)2, NaOCl, or no medication was sealed in the canals for 1 or 7 days. Canals were finally irrigated with H2O and prepared for histological evaluation. The cleanliness of main canals and inaccessible areas (isthmi and fins) at the apical, middle, and coronal thirds was examined, scored, and compared by nonparametric statistical analysis. Results showed no significant differences among different groups in either the 1-day or 7-day time intervals in either the main canal or inaccessible areas. Instrumentation combined with NaOCl irrigation alone accounted for the removal of tissue in the main canal. In conclusion, in this system, prolonged contact with Ca(OH)2 and NaOCl was similarly ineffective; neither contributed significantly to canal debridement.

Anaerobic tissue-dissolving abilities of calcium hydroxide and sodium hypochlorite.

Closed root canals likely have an oxygen-free environment; most bacteria in canals are anaerobic. These bacteria and other debris are difficult to remove. Unknown is tissue dissolution with chemicals under these anaerobic conditions. This study evaluated and compared dissolving properties of calcium hydroxide (Ca(OH)2) and sodium hypochlorite (NaOCl) on bovine pulp tissue in aerobic and anaerobic environments. Sixty bovine pulp specimens were dried, then randomly divided into six groups. Groups A and B were immersed in Ca(OH)2 + water solution, whereas group C and D were in 2.5% NaOCl. Groups E and F (controls) specimens were placed in distilled water. Groups A, C, and E were incubated anaerobically, and groups B, D, and F were incubated under regular atmospheric conditions, all for 7 days. Percentages of weight loss were compared between groups. Results showed the following: (a) both chemicals partially dissolved pulp tissue, (b) anaerobic environment did not alter tissue-dissolving properties of Ca(OH)2 or NaOCl, and (c) Ca(OH)2 and NaOCl were equal and more effective than water.

Canal adaptation and coronal leakage: lateral condensation compared to Thermafil.

In a comparison of different obturating techniques and materials, lateral plus vertical compaction of gutta-percha had the least coronal leakage. Using increments of gutta-percha is indicated for tapered canals and those with irregular anatomy.

Characterization of cellular responses involved in reparative dentinogenesis in rat molars.

During primary dentin formation, differentiating primary odontoblasts secrete an organic matrix, consisting principally of type I collagen and non-collagenous proteins, that is capable of mineralizing at its distal front. In contrast to ameloblasts that form enamel and undergo programmed cell death, primary odontoblasts remain metabolically active in a functional tooth. When dentin is exposed to caries or by operative procedures, and when exposed dentinal tubules are treated with therapeutic dental materials, the original population of odontoblasts is often injured and destroyed. The characteristics of the replacement pool of cells that form reparative dentin and the biologic mechanisms that modulate the formation of this matrix are poorly understood. Based on the hypothesis that events governing primary dentinogenesis are reiterated during dentin repair, the present study was designed to test whether cells that form reparative dentin are odontoblast-like. Cervical cavities were prepared in rat first molars to generate reparative dentin, and animals were killed at various time intervals. In situ hybridization with gene-specific riboprobes for collagen types I and III was used to study de novo synthesis by cells at the injured dentin-pulp interface. Polyclonal antibodies raised against dentin sialoprotein (DSP), a dentin-specific protein that marks the odontoblast phenotype, were used in immunohistochemical experiments. Data from our temporal and spatial analyses indicated that cells forming reparative dentin synthesize type I but not type III collagen and are immunopositive for DSP. Our results suggest that cells that form reparative dentin are odontoblast-like.

Characterization of induced pulpal hypoxia using 3H-misonidazole.

Tritiated misonidazole (3H-MISO) is a bioreductively activated marker which preferentially binds to cells with decreased oxygen tension (hypoxia). Detection of hypoxia may be an important indicator of metabolic imbalance. For 3H-MISO to be functional as a marker, normal oxygen (normoxic) levels must be distinguishable from increased hypoxic retention. This investigation characterized retention of 3H-MISO in: (a) normoxic pulp, (b) induced hypoxic pulp, and (c) reoxygenated hypoxic pulp. Rats were injected intraperitoneally with either 3H-MISO, unlabeled MISO, or saline, then divided into normoxic and hypoxic groups with appropriate controls. Normoxic animals were maintained at ambient pressure. Hypoxia was induced by placing animals in a hypobaric chamber at 0.5 atm. Pulps were then removed and prepared for liquid scintillation counting. Hypoxic pulps retained significantly more 3H-MISO than normoxic pulps (analysis of variance, p = 0.001). Hypoxic 3H-MISO retention was unaffected by subsequent, transient tissue reoxygenation. 3H-MISO detects changes in pulpal oxygen status that deviate from normal.

Measurement of blood flow to osseous tissue in dogs using the radiolabelled microsphere method.

1. The purpose of this study was to examine bone blood flow in various intra- and extra-oral sites. 2. The radiolabelled microsphere method was utilized to assess osseous blood flow in the following regions of 10 dogs: rib, long bone, and anterior and posterior regions of the maxilla and mandible. 3. Samples of cancellous and cortical bone were also obtained from each of these regions with the exception of the maxilla and the anterior mandible. 4. Mean blood flow ranged from 3.71 +/- 0.81 (SE) ml min.-1 100 g-1 in the mandibular posterior cortical bone to 22.7 +/- 4.66 ml min-1 100 g-1 in the cancellous rib samples. 5. Blood flow to the cancellous tissue of the rib was significantly greater (P < 0.05) than the other tissues with the exception of maxillary posterior bone and cortical rib. 6. Results from this study indicate that blood flow to the maxillary posterior bone is relatively high, but blood flow in other intraoral osseous sites is significantly less than that of cancellous rib bone.

Ultrasonic condensation of gutta-percha: an in vitro dye penetration and scanning electron microscopic study.

Lateral condensation of gutta-percha with and without ultrasonic activation of the spreader was compared by use of dye penetration analysis and scanning electron photomicrographs of the gutta-percha fills in extracted human incisors and canines. The root canal fillings in three groups of 10 teeth each were laterally condensed using an ultrasonically activated spreader, a fine finger spreader, or a non-activated ultrasonic spreader. Significantly less apical dye penetration occurred when teeth were obturated using an ultrasonically activated spreader as compared with manual condensation with fine finger spreaders. The ultrasonically condensed gutta-percha mass was more homogeneous with fewer voids compared with gutta-percha masses from the two groups that were condensed without ultrasonic activation. A correlation between apical microleakage and the appearance of the gutta-percha mass was seen. All teeth with well-condensed, more homogeneous fillings had low dye penetration. All teeth with a high extent of dye penetration had poorly condensed, less homogeneous fillings. However, not all teeth that had poorly condensed fillings had high dye penetration.