Building biofilms in vital host tissues: a survival strategy of Actinomyces radicidentis.

OBJECTIVE: To investigate the ability of Actinomyces radicidentis to survive and establish in soft connective tissue that grew into subcutaneously implanted tissue cages in Sprague-Dawley rats. STUDY DESIGN: Known concentrations of A. radicidentis suspension, grown on blood agar and broth cultures, were inoculated into tissue cages in rats. The cage contents were retrieved after 7, 14, and 28 days for culturing and correlative light and transmission electron microscopy. RESULTS: Cell suspensions harvested from both types of cultures showed substantial decline in numbers in tissue cages during the observation period. However, correlative light and transmission electron microscopy revealed numerous aggregates of coccoid bacteria already by 7 days of observation compared with the formation of well established colonies with characteristic actinomycotic features by 14 days after inoculation. CONCLUSIONS: These results suggest that the pathogenicity of A. radicidentis is due to its ability to form large aggregates of cells held together by embedding themselves in an extracellular matrix in vital host tissues. Thus, A. radicidentis, like other pathogenic Actinomyces, existing in the protected biofilm-environment can collectively evade destruction and elimination by host defenses, including phagocytosis.

Experimental evidence supports the abscess theory of development of radicular cysts.

OBJECTIVE: The objective of this study was to experimentally induce inflammatory cysts in an animal model so as to test the hypothesis that radicular cysts develop via the "abscess pathway." METHODOLOGY: Twenty-eight perforated custom-made Teflon cages were surgically implanted into defined locations in the back of 7 Sprague Dawley rats. A week after the implantation of the cages, a known quantity of freshly grown, close allogeneic oral keratinocytes in phosphate buffer solution (PBS) was injected into each cage. One cage per animal was treated as the control that received only epithelial cells. The remaining 3 cages of each animal were trials. Seven days post epithelial cell inoculation; a suspension of 0.2 mL of Fusobacterium nucleatum (10(8) bacteria per mL) was injected into each of the 3 trial cages. Two, 12, and 24 weeks after the inoculation of the bacteria, the cages were taken out, and the tissue contents were fixed and processed by correlative light and transmission electron microscopy. Sixteen of the 21 trial cages could be processed and yielded results. RESULTS: Inoculations of epithelial cells followed 1 week later by F. nucleatum into tissue cages resulted in the development inflammatory cysts in 2 of the 16 cages. The 2 cages contained a total of 4 cystic sites. None of the control cages showed the presence of any cyst-like pathology. CONCLUSIONS: Inflammatory cysts were induced by initiating acute inflammatory foci (abscess/necrotic area) by bacterial injection that got enclosed by a proliferating epithelium. This finding provides strong experimental evidence in support of the "abscess theory" of development of radicular cysts.

Histological, ultrastructural and quantitative investigations on the response of healthy human pulps to experimental capping with mineral trioxide aggregate: a randomized controlled trial.

AIM: To investigate the pulpal response to direct pulp capping in healthy human teeth with mineral trioxide aggregate (MTA) as against calcium hydroxide cement (Dycal) as control. METHODOLOGY: Twenty healthy human third molars had iatrogenic pulpotomy and direct pulp capping with MTA. Another 13 teeth were capped with Dycal as controls. The teeth were restored, with IRM, clinically reviewed and extracted after a number of pre-determined intervals (1 week, 1 month and 3 months). The specimens were fixed, decalcified, subdivided axially into two halves in the oro-buccal (lingual-buccal) plane, embedded in plastic, serial sectioned and evaluated qualitatively and quantitatively by correlative light and transmission electron microscopy with appropriate statistical evaluation of the quantitative data. RESULTS: Iatrogenic pulpal wounds treated with MTA were mostly free from inflammation after 1 week and became covered with a compact, hard tissue barrier of steadily increasing length and thickness within 3 months following capping. Control teeth treated with Dycal revealed distinctly less consistent formation of a hard tissue barrier that had numerous tunnel defects. The presence of pulpal inflammation up to the longest observation period (3 months) after capping, was a common feature in Dycal specimens. CONCLUSIONS: The MTA was clinically easier to use as a direct pulp-capping agent and resulted in less pulpal inflammation and more predictable hard tissue barrier formation than Dycal. Therefore, MTA or equivalent products should be the material of choice for direct pulp capping procedures instead of hard setting calcium hydroxide cements.

On the causes of persistent apical periodontitis: a review.

Apical periodontitis is a chronic inflammatory disorder of periradicular tissues caused by aetiological agents of endodontic origin. Persistent apical periodontitis occurs when root canal treatment of apical periodontitis has not adequately eliminated intraradicular infection. Problems that lead to persistent apical periodontitis include: inadequate aseptic control, poor access cavity design, missed canals, inadequate instrumentation, debridement and leaking temporary or permanent restorations. Even when the most stringent procedures are followed, apical periodontitis may still persist as asymptomatic radiolucencies, because of the complexity of the root canal system formed by the main and accessory canals, their ramifications and anastomoses where residual infection can persist. Further, there are extraradicular factors -- located within the inflamed periapical tissue -- that can interfere with post-treatment healing of apical periodontitis. The causes of apical periodontitis persisting after root canal treatment have not been well characterized. During the 1990s, a series of investigations have shown that there are six biological factors that lead to asymptomatic radiolucencies persisting after root canal treatment. These are: (i) intraradicular infection persisting in the complex apical root canal system; (ii) extraradicular infection, generally in the form of periapical actinomycosis; (iii) extruded root canal filling or other exogenous materials that cause a foreign body reaction; (iv) accumulation of endogenous cholesterol crystals that irritate periapical tissues; (v) true cystic lesions, and (vi) scar tissue healing of the lesion. This article provides a comprehensive overview of the causative factors of non-resolving periapical lesions that are seen as asymptomatic radiolucencies post-treatment.

Biocompatibility of Beta-tricalcium phosphate root replicas in porcine tooth extraction sockets - a correlative histological, ultrastructural, and x-ray microanalytical pilot study.

This investigation studies porcine tissue response in tooth extraction sockets treated with root replicas made out of Beta-tricalcium phosphate (Beta-TCP; Beta-Ca(3)(PO(4))(2)) granules, molded and held together by thermal fusion of a thin film of polyglycolic-polylactic acid copolymer. Six left mandibular third incisors (n (1)/4 6) of experimental pigs are treated with the root replicas and four contralateral incisors are used as nontreated controls (n (1)/4 4). Two animals each were killed at 20, 40, and 60 weeks of observation periods. The mandibular jaw segments were prepared in toto for light microscopy by resin embedding and serial ground sectioning. Additionally, one Beta-TCP-treated socket at 60 weeks was thoroughly investigated by correlative light, electron microscopic and electron probe X-ray microanalysis to assess the bio-absorbability and host removal of the replica material from the implant site. The extraction wounds of the animals healed satisfactorily with very little histologically observable differences in the healing pattern of the test and control sites. The Beta-TCP was completely removed from extracellular sites, but at 60 weeks, remnants of it were found in the cytoplasm of multinucleated giant cells. The root replicas made out of Beta-TCP were biocompatible and bioabsorbable. Osseous healing occurred both in the test and control sockets, but the healing process was delayed due to the presence of Beta-TCP particles.

Quantitative histological and ultrastructural features of opercula of normally erupting human teeth.

Tooth eruption across the mucosa in humans has been studied rarely, although there are disturbances of eruption that are attributed specifically to failure of the supraosseous eruptive migration. The aim of this study was to analyze the soft tissues covering normally erupting teeth so as to get an insight into the supraosseous phase of tooth eruption and to provide the basis for comparison with cases of eruption disturbances. Six opercula covering normally erupting permanent molars (primary opercula) and six of succedaneous teeth (secondary opercula) were surgically removed from 10 patients aged 7.5-17.5 years. Specimens were examined light and electron microscopically and analyzed morphometrically. All opercula contained strands and islands of odontogenic epithelium, prominent numbers of high endothelial venules, nerves, and mast cells. Nerves comprised normally structured, 1.5-3.5 microm thick myelinated (Adelta) and thinner unmyelinated (C) fibers. In primary opercula, the proportions of blood vessels and nerves were three- and sevenfold higher than the respective values for the secondary opercula. Furthermore, primary opercula contained multinucleated, fibroblast-like giant cells that were not observed in secondary opercula. As all teeth under investigation were erupting normally, neither the presence of the giant cells nor the atypical proportions of blood vessels and nerves appeared to be decisive in the eruption process. These conspicuous tissue components of opercula seem merely to accompany the eruptive tooth movement.

Quantitative histological features and ultrastructure of opercula of human teeth showing normal and delayed eruption.

BACKGROUND: Failure of eruption of human permanent molars has been attributed to opercular lesions, although comparisons with specimens from normally erupting teeth are scarce. The aim of this study was to quantitatively analyse opercula associated with normal and delayed tooth eruption. METHOD: Twenty opercula covering permanent molars delayed in eruption were obtained from 13 patients aged 7.3-18.1 years. Six opercula from normally erupting molars of five 7.3-17.5-year-old subjects served as controls. Specimens were analysed light and electron microscopically and morphometrically. RESULTS: In addition to features recognized previously, prominent numbers of nerves, high endothelial-like venules and mast cells were observed. Ultrastructurally, large multinucleated cells did not reveal cell boundaries running between the nuclei, and mast cells seemed belonging to the MC(TC)-type. None of the features differed significantly between opercula from cases of delayed and normal tooth eruption. CONCLUSIONS: Disturbances of tooth eruption that are attributed to opercular lesions may represent retentions resulting from the failure of the eruption mechanism, rather than impactions because of a physical barrier.

Microbial status of apical root canal system of human mandibular first molars with primary apical periodontitis after "one-visit" endodontic treatment.

OBJECTIVE: To assess the in vivo intracanal microbial status of apical root canal system of mesial roots of human mandibular first molars with primary apical periodontitis immediately after one-visit endodontic treatment. The residual intracanal infection was confirmed by correlative light and transmission electron microscopy. STUDY DESIGN: Sixteen diseased mesial roots of mandibular first molars were treated endodontically, each in one visit. Mesio-buccal canals were instrumented using stainless steel hand files and mesio-lingual canals with a nickel-titanium rotary system. The canals were irrigated with 5.25% sodium hypochlorite (NaOCl) during the instrumentation procedures, rinsed with 10 mL of 17% ethylenediamine tetraacetic acid (EDTA), and obturated with gutta-percha and zinc oxide eugenol cement. Thereafter, the apical portion of the root of each tooth was removed by flap-surgery. The specimens were fixed, decalcified, subdivided in horizontal plane, embedded in plastic, processed, and evaluated by correlative light and transmission electron microscopy. RESULTS: Fourteen of the 16 endodontically treated teeth revealed residual intracanal infection after instrumentation, antimicrobial irrigation, and obturation. The microbes were located in inaccessible recesses and diverticula of instrumented main canals, the intercanal isthmus, and accessory canals, mostly as biofilms. CONCLUSIONS: The results show (1) the anatomical complexity of the root canal system of mandibular first molar roots and (2) the organization of the flora as biofilms in inaccessible areas of the canal system that cannot be removed by contemporary instruments and irrigation alone in one-visit treatment. These findings demonstrate the importance of stringent application of all nonantibiotic chemo-mechanical measures to treat teeth with infected and necrotic root canals so as to disrupt the biofilms and reduce the intraradicular microbial load to the lowest possible level so as to expect a highly favorable long-term prognosis of the root canal treatment.

Observations on pulpal response to carbon dioxide laser drilling of dentine in healthy human third molars.

Preservation of pulpal health is the primary prerequisite for successful application of laser systems in the hard tissue management of vital teeth. The purpose of this study was to investigate the short and long-term pulpal effects to cavity preparations in healthy human teeth using carbon dioxide (CO2) laser. A total of seven, healthy, third molars that were scheduled to be removed due to space problems were used. After the laser drilling, the occlusal cavities were closed temporarily, and the teeth were extracted 7 days (n=5) and 3 months (n=2) after the operation. The specimens were fixed, decalcified, subdivided and processed for light and transmission electron microscopy. Seven days postoperatively all the five teeth that had been irradiated with the CO2 laser did not reveal any pathological changes in the pulpo-dentine complex. Three months postoperatively the two teeth that were prepared with the laser showed subtle but distinct apposition of tertiary dentine that was lined with intact odontoblasts. One of the specimens at 3 months revealed the presence of a mild, but very circumscribed, pulpal infiltration of chronic inflammatory cells subjacent to the cavity preparation. The latter is unlikely to be due to a direct effect of the laser irradiation but a possible consequence of microleakage of oral antigens and/or other tissue-irritating molecules through the temporary restoration and the remaining dentine thickness (RDT). Although these preliminary histological results suggest that the CO2 laser under investigation induced only minimal response of the dentine-pulp complex when used as a hard-tissue drilling tool, with specific energy settings, pulse duration within thermal relaxation time and emitting radiations at 9.6 microm of wavelength, larger clinical trials involving various types of teeth are necessary to reach definite conclusions for large-scale clinical application of the laser device.

Pathogenesis of apical periodontitis and the causes of endodontic failures.

Apical periodontitis is a sequel to endodontic infection and manifests itself as the host defense response to microbial challenge emanating from the root canal system. It is viewed as a dynamic encounter between microbial factors and host defenses at the interface between infected radicular pulp and periodontal ligament that results in local inflammation, resorption of hard tissues, destruction of other periapical tissues, and eventual formation of various histopathological categories of apical periodontitis, commonly referred to as periapical lesions. The treatment of apical periodontitis, as a disease of root canal infection, consists of eradicating microbes or substantially reducing the microbial load from the root canal and preventing re-infection by orthograde root filling. The treatment has a remarkably high degree of success. Nevertheless, endodontic treatment can fail. Most failures occur when treatment procedures, mostly of a technical nature, have not reached a satisfactory standard for the control and elimination of infection. Even when the highest standards and the most careful procedures are followed, failures still occur. This is because there are root canal regions that cannot be cleaned and obturated with existing equipments, materials, and techniques, and thus, infection can persist. In very rare cases, there are also factors located within the inflamed periapical tissue that can interfere with post-treatment healing of the lesion. The data on the biological causes of endodontic failures are recent and scattered in various journals. This communication is meant to provide a comprehensive overview of the etio-pathogenesis of apical periodontitis and the causes of failed endodontic treatments that can be visualized in radiographs as asymptomatic post-treatment periapical radiolucencies.

Pulpal response to Er:YAG laser drilling of dentine in healthy human third molars.

BACKGROUND AND OBJECTIVES: Maintenance of pulpal health is a critical prerequisite for successful application of light amplification by stimulated emission of radiations (lasers) in the hard tissue management of vital teeth. The purpose of this study was to investigate the short- and long-term pulpal effects to cavity-preparations in healthy human teeth using erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser. MATERIALS AND METHODS: A total of seven healthy third molars that were to be removed due to space-problem were used. Following the laser excavation, the cavities in dentine were closed temporarily and the teeth were extracted after 7 days (n = 5) and 3 months (n = 2) post-operation. The specimens were fixed, decalcified, subdivided, and processed for light and transmission electron microscopy. RESULTS: In the short-term group, four of the five laser-drilled teeth did not reveal any pathological changes in the pulp-dentine complex. One tooth showed mild disruption of odontoblasts (OB) and vascular dilatation subjacent to the deepest point of the cavity-preparation with a remaining dentine thickness (RDT) of less than 80 microm. The two teeth under long-term observation revealed distinct apposition of tertiary dentine (TD), lined predominantly with cuboidal cells on its pulpal aspect. CONCLUSIONS: These results would allow a conclusion to be drawn that the Er:YAG laser under investigation is a pulp preserving hard-tissue drilling tool when used with the specific energy settings and emitting radiation at a wavelength of 2.94 microm.

Ciliated epithelium-lined radicular cysts.

OBJECTIVE: This report describes 3 cases of ciliated epithelium-lined radicular cysts among 256 apical periodontitis lesions and also illustrates the occurrence of an Actinomyces-infected periapical cyst. STUDY DESIGN: Serial and step serial sections of 256 plastic-embedded root apices with attached apical periodontitis lesions that were prepared for a previous investigation were reviewed for the presence of ciliated epithelium-lined radicular cysts. The lesions that were found to have such epithelial lining were examined in a transmission electron microscope to elaborate the fine structure of the ciliated cells. RESULTS: A total of 3 ciliated columnar epithelium-lined cysts was found among the 256 apical periodontitis lesions examined. Two of the lesions also contained stratified squamous epithelium. All 3 lesions affected maxillary premolars. One of the lesions was a true cyst, and the other 2 were periapical pocket cysts. The lumen of 1 of the latter revealed the presence of typical "ray-fungus" actinomycotic colonies. CONCLUSION: Although the stratified squamous component of the epithelia that lined the radicular cysts reported here may be derived from the cell rests of Malassez, the ciliated epithelial cells may be of sinus origin. Microbial agents from diseased root canals can advance into radicular cysts, particularly in pocket cysts, with the possible threat of such infection in upper posterior teeth spreading into the maxillary sinus.

Glutathione protects chemokine-scavenging and antioxidative defense functions in human RBCs.

Oxidant stress, in vivo or in vitro, is known to induce oxidative changes in human red blood cells (RBCs). Our objective was to examine the effect of augmenting RBC glutathione (GSH) synthesis on 1) degenerative protein loss and 2) RBC chemokine- and free radical-scavenging functions in the oxidatively stressed human RBCs by using banked RBCs as a model. Packed RBCs were stored up to 84 days at 1-6 degrees C in Adsol or in the experimental additive solution (Adsol fortified with glutamine, glycine, and N-acetyl-L-cysteine). Supplementing the conventional additive with GSH precursor amino acids improved RBC GSH synthesis and maintenance. The rise in RBC gamma-glutamylcysteine ligase activity was directly proportional to the GSH content and inversely proportional to extracellular homocysteine concentration, methemoglobin formation, and losses of the RBC proteins band 3, band 4.1, band 4.2, glyceraldehyde-3-phosphate dehydrogenase, and Duffy antigen (P < 0.01). Reduced loss of Duffy antigen correlated well with a decrease in chemokine RANTES (regulated upon activation, normal T-cell expressed, and secreted) concentration. We conclude that the concomitant loss of GSH and proteins in oxidatively stressed RBCs can compromise RBC scavenging function. Upregulating GSH synthesis can protect RBC scavenging (free radical and chemokine) function. These results have implications not only in a transfusion setting but also in conditions like diabetes and sickle cell anemia, in which RBCs are subjected to chronic/acute oxidant stresses.

Aberrant expression and activation of insulin-like growth factor-1 receptor (IGF-1R) are mediated by an induction of IGF-1R promoter activity and stabilization of IGF-1R mRNA and contributes to growth factor independence and increased survival of the pancreatic cancer cell line MIA PaCa-2.

In the present study we investigated the mechanisms responsible for and the biological consequences of the constitutive activation of the insulin-like growth factor-1 receptor (IGF-1R) in the MIA PaCa-2 cells. An aberrant increase in the expression and activation of the IGF-1R was observed during the transition of growth states from exponential to quiescent. The increase in IGF-1R expression is preceded by an increase in IGF-1R mRNA transcript and is associated with an increase in the IGF-1R promoter activity. Inhibition of de novo transcription by actinomycin D increased the stability of IGF-1R mRNA in exponentially growing cells, thereby increasing the expression of IGF-1R to a level similar to that seen in quiescent cells. Increased IGF-1R signaling mediated the growth factor independence of quiescent MIA PaCa-2 cells through the constitutive activation of mitogen-activated protein kinase (MAPK). Exogenous IGF-1 increased cell proliferation and activated MAPK and AKT signaling pathways. The resistance of cells to apoptosis by IGF-1R signaling was mediated through MAPK and phosphatidylinositol 3-kinase (PI3K) pathways and a yet unidentified pathway(s). Thus, aberrant regulation of IGF-1R signaling is required for resistance to apoptosis and growth factor independence of MIA PaCa-2 cells. This likely protects cells from unfavorable conditions and allows cells to rapidly re-enter the cell cycle when conditions are favorable.

Apical inflammatory root resorption: a correlative radiographic and histological assessment.

AIM: To assess the reliability of routine single radiographs in the diagnosis of inflammatory apical root resorption by correlating the radiographic and histological findings. METHODOLOGY: The material comprised serial and step serial sections of plastic-embedded root-apices with attached apical periodontitis lesions that were prepared for a previous study and the diagnostic radiographs. The histological sections of 114 specimens were analysed by light microscopy and categorized into three groups: (i) those without any resorption (0); (ii) those with moderate resorption (+); and (iii) those with severe resorption (+ +). The radiographs were examined by a separate examiner and graded with a similar categorization of no resorption (0); moderate (+); and severe (+ +) apical resorption. RESULTS: Radiographically, 19% of the teeth were diagnosed as having apical inflammatory root resorption, whereas histologically, 81% of the teeth revealed apical inflammatory root resorption. A correlative radiographic and histological assessment (n = 104) revealed a coincidence of diagnosis in 7% of the specimens and noncoincidence of diagnosis in 76% of the specimens. CONCLUSIONS: The results indicate that routine single radiographs are not sufficiently accurate or sensitive to consistently diagnose apical root resorptive defects developing as a consequence of apical periodontitis.

Persistent periapical radiolucencies of root-filled human teeth, failed endodontic treatments, and periapical scars.

OBJECTIVE: This report describes 6 cases that demonstrate persistent periapical radiolucent lesions after conventional root canal treatment. STUDY DESIGN: Six teeth that had conventional root canal treatment or re-treatment with nonresolving periapical radiolucencies underwent periapical surgery. Biopsies were obtained and analyzed descriptively by correlative light and transmission electron microscopy for general features and microbial findings. RESULTS: Three findings were identified: periapical lesions with persisting infection in the apical root canal system (2 cases); a cyst (1 case); and periapical healing by scar tissue formation (2 cases). CONCLUSIONS: These results confirm previous observations that associated factors in the failure of endodontic treatment include persistent intraradicular infection and periapical cysts. In addition, unresolved periapical radiolucencies may occasionally be due to healing by scar tissue, which may be mistaken as a sign of failed endodontic treatment.