Healthy Cementum Research Articles

Pre‐existing root cementum may promote cementoblast differentiation of human periodontal ligament cells

To observe whether preserved healthy cementum could promote differentiation of human periodontal ligament cells to cementoblasts. Symmetrical root slices from each healthy premolar were distributed into either the control group (cementum removed) or test group (cementum preserved). After isolation and characterization, human periodontal ligament cells were inoculated onto root slices for 7 days co-culture. Two slices per group were studied for cell morphology by scanning electronic microscopy. Twenty-three slices were detected for expression of cementum attachment protein and cementum protein 23, two putative cementoblast markers, by real-time polymerase chain reaction. Twenty slices were transplanted into nude mice and analysed using histology and immunohistochemistry for osteopontin and bone sialoprotein expression after 8 weeks. Cells of the test group had smoother fibroblast morphology and higher cementum protein 23 and cementum attachment protein expression than those of the control group (P < 0.01). In the test group, 14 root slices revealed cementum-like matrix formation resting on old cementum; no splits were observed between newly formed matrix and old cementum. In the control group, 17 specimens had fibrous tissue formation along the root surface and varying width of splits could be seen between new fibrous tissue and dentine surface. Only three specimens demonstrated presence of newly formed thin cementum-like matrix. Newly formed cementum-like matrix was positive for osteopontin and bone sialoprotein. The results demonstrate that healthy root cementum may promote differentiation of human periodontal ligament cells towards cementoblasts.

Comparative Effect of the Ionic Products from Bioactive Glass Dissolution on the Behavior of Cementoblasts, Osteoblasts, and Fibroblasts

The cementum, a mineralized tissue lining the tooth root surface, is required for development of a functional periodontal ligament. The presence of healthy cementum is considered to be an important criterion for predictable restoration of periodontal tissues lost as a consequence of disease. Despite the importance of cementum to general oral health, very little is known about the cells responsible for the formation of cementum, cementoblasts. The aim of this study was to examine the effect of the ionic products from the dissolution of bioactive glass with 60% of silica ( BG60S ) on the behavior of cementoblasts, osteoblasts and fibroblasts. The cell viability was tested by MTT assay based on mitochondria activity of the cell and Trypan Blue assay based on membrane cell viability. The membrane cell viability measured by Trypan Blue assay showed the beneficial effect on all the cell types tested. It was observed a higher proliferation in the presence of ionic products from dissolution of BG60S when compared to control. In the MTT assay we also observed increased cell viability on all the cell types, but proliferation of cementoblasts was higher (107%) than observed for the other cells (104%) compared to control. The results from this study suggest that Cementoblasts, osteoblasts and fibroblasts are important cells on events that control the development of mineralizing and not mineralizing tissues and the investigation of the comparative behavior of these cells can be a useful experimental model. The observed effect of the bioactive glass particles on cementoblasts shows that this material is an interesting alternative to be used in composite membranes for cementum tissue engineering.

The effect of chemical and/or mechanical conditioning on the Er:YAG laser‐treated root cementum: Analysis of surface morphology and periodontal ligament fibroblast attachment

This study compared the surface morphology as well as the biocompatibility of dental root cementum treated with Er:YAG laser irradiation alone and with the laser irradiation followed by chemical and/or mechanical conditioning. Healthy cementum plates were randomly assigned to the following control and treatment groups: (1) untreated control (C), (2) Er:YAG laser irradiation (L), (3) laser plus tetracycline HCl (TC) placement (L+TP), (4) laser plus TC burnishing (L+TB), (5) laser plus EDTA gel placement (L+EP), (6) laser plus EDTA gel burnishing (L+EB), (7) laser plus saline solution burnishing (L+SB), and (8) laser plus minocycline-HCl paste placement (L+MP). Specimens were subjected to scanning electron microscopy (SEM), histological observation and attachment assay using periodontal ligament (PDL) fibroblasts. The laser irradiation produced a thin affected layer (5.7 microm thickness) with a superficial microstructure on the cementum surface. The characteristic microstructures of the lased surface were fragile and could be removed by chemical and/or mechanical conditioning treatments. The L+TB group exhibited marked exposure of collagen fibers after removal of the microstructures on the lased surface. The L+EP group presented a peculiar, smooth surface without exposure of collagen fibers and a uniform arrangement of spherical microparticles on the ultra-high magnification of SEM. In cell attachment assay, the L+TB group exhibited the greatest number of attached cells among all the groups, followed by the L+EP, L+SB and control group. The laser alone group exhibited the lowest number of cells. The characteristic microstructure of the root cementum surface after Er:YAG laser irradiation has a tendency to hinder the early attachment of PDL cells. However, chemical and/or mechanical root conditioning treatment may improve and increase the biocompatibility of the Er:YAG laser-treated root cementum by removing the microstructures of the surface and/or further exposing the collagen fibers.

Effects of enamel matrix protein on the growth of human periodontal ligament cells on root cementum surfaces

To evaluate the effect of enamel matrix protein (EMP) on the attachment and proliferation of periodontal ligament cells (PDLC) on diseased cementum surfaces in vitro. Cementum chips were obtained from diseased roots exposed to periodontal pocket. Thirteen diseased root cementum chips were conditioned with EMP. Meanwhile, 13 diseased and 13 healthy cementum chips were treated with physiological saline as control. The growth and morphology of PDLC on the root surface were observed after 24 hours incubation by scanning electron microscope (SEM). PDLC attachment and proliferation were quantified using MTT assay at 16 or 72 hours. The cells on EMP treated roots under SEM were growing robust like the cells on healthy roots. By contrast, the diseased cementum surface without conditioned with EMP was only partly covered with spindle-shaped cells, with filopodia appearing short and thin. MTT assay indicated that the number of adhered and proliferated cells on diseased cementum chips treated with EMP was significantly greater than that on diseased chips treated with saline (adhesion: 0.45 +/- 0.03 vs. 0.37 +/- 0.05, P < 0.05; proliferation: 0.71 +/- 0.02 vs. 0.55 +/- 0.08, P < 0.01), but less than that on healthy chips (adhesion: 0.45 +/- 0.03 vs. 0.67 +/- 0.08, P < 0.05; proliferation: 0.71 +/- 0.02 vs. 1.05 +/- 0.09, P < 0.05). It was suggested that EMP could promote the growth of PDLC on the diseased root cementum surface.

Tetracycline Conditioning Augments the In Vivo Inflammatory Response Induced by Cementum Extracts

Studies have shown that extracts of cementum from periodontally involved teeth stimulated cytokine secretion from cultured human monocytes and that this stimulatory effect is inhibited by conditioning of the cementum with tetracycline. Using the subcutaneous chamber model in mice, the present study was designed to test the ability of cementum extracts from periodontally diseased teeth to induce an inflammatory response in vivo and to evaluate the effect of cementum conditioning with tetracycline. Subcutaneous chambers were implanted in 24 mice. Two weeks later, the animals received intrachamber injection of one of the following: diseased-cementum extract, healthy-cementum extract, diseased-cementum extract preconditioned with tetracycline, or medium alone. Chamber exudates were harvested and analyzed for leukocyte levels, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin-10 (IL-10). Injection of healthy- or diseased-cementum extracts increased the intrachamber levels of leukocytes. Extracts of diseased cementum were found to significantly increase the levels of TNF-alpha, IFN-gamma, and IL-10, compared with extracts of healthy cementum or media alone. Peak cytokine levels were observed 2 hours postinjection. Conditioning of diseased cementum with tetracycline before extraction resulted in augmented levels of TNF-alpha and IFN-gamma, and reduced levels of IL-10, compared with untreated diseased cementum. The present results demonstrate that conditioning of diseased cementum with tetracycline may induce an intense inflammatory response in a mouse model, and they suggest that local application of tetracycline for root conditioning should be carefully reinvestigated.

Participation of Bacterial Biofilms in Refractory and Chronic Periapical Periodontitis

The aim of this study was to examine morphologically the participation of extraradicular biofilm in refractory periapical periodontitis. Six teeth and five extruded root filling gutta-percha points associated with refractory periapical periodontitis were investigated by scanning electron microscope. In nine of 11 samples examined, bacterial biofilms were seen at the extraradicular area. The gutta-percha surface was covered with glycocalyx-like structures, and filaments, long rods, and spirochete-shaped bacteria were predominant in the extraradicular sites. Planktonic cells, which were filaments and spirochete-shaped bacteria, emigrated from the glycocalyx structures in some spots. In the extracted teeth, biofilm consisting of both bacteria and glycocalyx-like structures were observed on the periapical root surfaces. Next to the residual periodontal ligament, a few filaments, rods, and fusiforms were attached on the healthy cementum surface. The present findings suggested that bacterial biofilms formed in the extraradicular areas were related to refractory periapical periodontitis.

Employing a transgenic animal model to obtain cementoblasts in vitro.

Proper formation of cementum, a mineralized tissue lining the tooth root surface, is required for development of a functional periodontal ligament. Further, the presence of healthy cementum is considered to be an important criterion for predictable restoration of periodontal tissues lost as a consequence of disease. Despite the significance of cementum to general oral health, the mechanisms controlling development and regeneration of this tissue are not well understood and research has been hampered by the lack of adequate in vitro experimental models. In an effort to establish cementoblast cell populations, without the trappings of a heterogeneous population containing periodontal ligament (PDL) cells, cells were obtained from the root surface of first mandibular molars of OC-TAg transgenic mice. These mice contain the SV40 large T-antigen (TAg) under control of the osteocalcin (OC) promoter. Therefore, only cells that express OC also express TAg and are immortalized in vitro. Based on results of prior in situ studies, OC is expressed by cementoblasts during root development, but not by cells within the PDL. Consequently, when populations are isolated from developing molars using collagenase/trypsin digestion, only cementoblasts, not PDL cells, are immortalized and thus, will survive in culture. The resulting immortalized cementoblast population (OC/CM) expressed bone sialoprotein (BSP), osteopontin (OPN), and OC, markers selective to cells lining the root surface. These cells also expressed type I and XII collagen and type I PTH/PTHrP receptor (PTH1R). In addition to expression of genes associated with cementoblasts, OC/CM cells promoted mineral nodule formation and exhibited a PTHrP mediated cAMP response. This approach for establishing cementoblasts in vitro provides a model to study cementogenesis as required to enhance our knowledge of the mechanisms controlling development, maintenance, and regeneration of periodontal tissues.

Root resorption and signs of repair in Papillon-Lefèvre syndrome. A case study

The aim of this investigation was to describe some tooth-related histological features of prepubertal periodontitis. Teeth extracted during treatment of two Papillon-Lefèvre syndrome patients were processed by means of the sawing and grinding technique. Light microscopy examination revealed little or no cementum in the coronal parts of the roots. Resorptions of various depths (0.02 to 1.5 mm) and to various extents (affecting up to 1/3 of the root surface) were observed in the 5 investigated teeth. Some resorptive defects on 1 of the examined incisors showed signs of spontaneous repair. Extrinsic fibers were inserted into the new cellular intrinsic fiber cementum which had formed directly on the bottom of the defect. Intact acellular extrinsic fiber cementum was found where fibers were still attached. Here, the characteristic of pristine cementum, a hyaline layer of peripheral dentin, could be identified. If resorption was not present, the cementum did not show any signs of hypoplasia. Thus, histological features of prepubertal periodontitis in the current material were (i) areas of extensive resorption, (ii) signs of spontaneous repair, and (iii) healthy cementum.

Human monocyte response to cementum extracts from periodontally diseased teeth: effect of conditioning with tetracycline.

Monocyte inflammatory cytokines, such as TNF alpha and IL-1 beta, have been implicated in the pathogenesis of periodontal destruction. The present study was designed to test the ability of extracts of cementum from periodontally diseased teeth to induce the secretion of these mediators by monocytes, to evaluate the role of adsorbed endotoxin in this process, and to test the effect of cementum conditioning with tetracycline on the monocyte response. Human monocytes were incubated with varying concentrations of cementum extracts, and TNF alpha and IL-1 beta levels in the media were measured. The results showed that while extracts of healthy cementum had no effect on monocyte secretion, concentration as low as 0.5 mg/ml of cementum from diseased sites raised the levels of TNF alpha and IL-1 beta secretion 10-fold. This response was dose-dependent. Diseased cementum were found to contain 1.5 ng/mg endotoxin, while endotoxin was not detectable in the extracts of the healthy cementum. However, neutralization of the endotoxin by polymyxin B only partially reduced the monocyte secretory response by 50 to 70%, suggesting that other factors in the extracts are also involved in monocyte stimulation. To simulate the effect of root conditioning, cementum was first agitated in a tetracycline or control solution prior to its extraction in media. Pretreatment of diseased cementum with tetracycline (50 mg/ml) was found to block the secretion of TNF alpha from cementum-stimulated monocytes. Pretreatment of the diseased cementum with 10 mg/ml tetracycline was not more effective than saline and HCI controls, with all treatments reducing cytokine secretion by approximately 80%. The direct addition of tetracycline to cementum-stimulated monocyte culture was found to block TNF alpha secretion in a dose dependent manner. The results suggest that extracts from diseased cementum are potent stimulators of monocyte secretion, and that endotoxin as well as other factor(s) appear to be involved. These factors are partially extracted by washing and a 10 mg/ml tetracycline solution is not more effective than saline in achieving this goal. In addition, tetracycline was found to be a potent inhibitor of TNF alpha secretion by cementum-stimulated monocytes, suggesting a novel mechanism for this drug in periodontal therapy.

Nd: YAGレーザーの歯周治療への応用

A low power Nd: YAG laser. with a flexible fine optical fiber has been developed for dental use. To investigte the possibility of application in periodontal treatment, the following studies were made.The purpose of the first study was to determine which laser energy setting resulted in the best condition. Periodontally-involved extracted teeth with subgingival calculus were used. The tested energy settings were 1.5W, 2.0W, 2.5W and 3.0W. Results obtained were as follows: The efficiency at 1.5W irradiation was significantly lower than that at 2.0W, 2.5W and 3.0W. There was no significant difference in efficiency among 2.0W, 2.5W and 3.0W irradiation.The purpose of the second study was to reveal the effect of laser irradiation on inactivation of endotoxin artificially adsorbed by the healthy cementum and whether the inactivation of endotoxin spread or not by laser irradiation. After collagenase treatment, the healthy cementum was soaked in endotoxin solution. Energy settings were 30mJ for 2sec, 30mJ for 4sec, 50mJ for 2sec and 50mJ for 4sec. The results showed that endotoxin was inactivated at the irradiated spot at an energy setting of more than 30mJ for 2sec. Inactivation of endotoxin was also observed surrounding the spot.The purpose of the third study was to investigate the clinical effectiveness of laser irradiation in the periodontal pocket. 16 pairs of periodontal pockets with a probing depth were more than 5mm were selected for the study. One irradiated by laser and the other served as controll. Energy setting was from 1.75W to 2.25W at 20pps. The results showed that gingival crevicular fluid and probing depth were significantly redused in the experimental group. And pus discharge disappeared significantly in the experimental site.These results may confirm that laser treatment is effective in the treatment of periodontal disease.

Fluorine concentration changes in human periodontally diseased tooth roots following several treatment times with citric acid.

Citric acid conditioning of exposed cementum has become an important adjunct to the clinical management of periodontal disease, and aggressive root planing is recommended to remove hypermineralized and endotoxin-laden diseased cementum. A nuclear resonance reaction technique was used to examine fluorine concentration changes subsequent to application of citric acid (pH 1.0) to the periodontal disease-exposed cementum surfaces of human tooth roots. The technique does not require the test teeth to be sectioned, thereby permitting longitudinal assessments of changes in fluorine concentration and minimizing measurement errors due to the considerable biological variation found between individuals. Initial fluorine concentrations ranged from 0.9%-2.4%, and maxima occurred within 4-6 microns of the surface, suggesting the presence of a hypermineralized layer. Within 60 sec, the citric acid had effectively removed the hypermineralized layer and the previously observed fluctuations in fluorine concentration leveled out at 0.3%-0.5%. Although the results indicated rapid removal of the hypermineralized layer and establishment of fluorine levels normally found in healthy cementum, the experimental design did not permit appraisal of potential effects upon the organic components of periodontally-exposed cementum.