Evaluation of the biological properties of two experimental calcium silicate sealers: an in vivo study in rats.

AIM: To evaluate the biological properties of experimental sealers based on tricalcium silicate and dicalcium silicate, manipulated with polyethylene glycol (CE-1) and with the addition of calcium hypochlorite (CE-2) compared to AH Plus (AHP) and TotalFill BC Sealer (TBC). METHODOLOGY: The tissue reaction caused by the materials in the subcutaneous tissue of rats was evaluated after implantation of polyethylene tubes filled with the materials at 7, 15, 30 and 60 days. Sections were stained with haematoxylin and eosin (HE) for morphological analysis and to evaluated number of inflammatory cells/mm2 (ICs). Sections were used for immunohistochemical detection of interleukin-6 (IL-6) and osteocalcin (OC). The von Kossa method was used to identify calcium precipitation in the capsules. The data were submitted to anova and Tukey's tests, with 5% significance level. RESULTS: At 7 days, CE-1, CE-2 and AHP had higher numbers of ICs. AHP presented higher immunolabelling for IL-6. After 15 days, regarding IL-6, there was no difference between CE-2 and the control group. At 30 days, AHP exhibited the highest number of IC (P < 0.05) and CE-2 and the control group presented the lowest ICs and IL-6-positive cells. After 60 days, all materials exhibited decreases in ICs. CE-2, TBC and the control had the lowest values (P < 0.05). No significant difference was detected between CE-1 and TBC, and between CE-2 and control considering the immunoexpression of IL-6. In this period, AHP had the greatest number of IC and IL-6 (P < 0.05). In all periods, CE-1, CE-2 and TBC sealers had von Kossa-positive structures and OC-immunolabelled cells. CE-2 had higher number of OC-positive cells than the CE-1 and TBC sealers (P < 0.05), in all periods. OC immunolabelling was not observed in the capsules of AH Plus and the control groups. CONCLUSIONS: The experimental sealer and its association with calcium hypochlorite, in addition to TotalFill BC Sealer, were biocompatible and had bioactive potential.

Histomorphometric and immunohistochemical study shows that tricalcium silicate cement associated with zirconium oxide or niobium oxide is a promising material in the periodontal tissue repair of rat molars with perforated pulp chamber floors.

AIM: To evaluate the periodontium response to tricalcium silicate (TCS) with zirconium oxide (ZrO2 ) or niobium oxide (Nb2 O5 ) used in the sealing of perforated pulp chamber floors in rat maxillary molars. METHODOLOGY: In eighty rats, the perforations in right maxillary molars were filled with either TCS + ZrO2 , TCS + Nb2 O5 , White MTA (used as a gold standard material) or no repair material was placed (Sham Group, SG); the left molars of SG, were used as controls (CG). Sections of maxillary fragments following 7, 15, 30 and 60 days were used to evaluate the volume densities of inflammatory cells (VvIC) and fibroblasts (VvFb), width of the periodontal space, amount of collagen, number of osteoclasts and number of IL-6-immunostained cells. The data were subjected to two-way ANOVA followed by Tukey's test (P ≤ 0.05). RESULTS: At all periods, significant differences in VvIC were not detected among TCS + ZrO2, TCS + Nb2 O5 and MTA groups, which had values significantly lower (P < 0.05) than the SG. Significant differences in the number of IL-6-immunolabelled cells were not observed among TCS + ZrO2 , TCS + Nb2 O5 and MTA groups (P > 0.05) at 15, 30 and 60 days. At 7, 15 and 30 days, the number of osteoclast was significantly greater in TCS + ZrO2, TCS + Nb2 O5 and MTA (P < 0.05) than in the CG; no significant difference was detected after 60 days (P > 0.05). The width of the periodontal space and amount of collagen in TCS + ZrO2 and TCS + Nb2 O5 groups were similar to the CG at 30 and 60 days while SG specimens had a significant reduction (P < 0.05) in the amount of collagen and significant increase (P < 0.05) in the width of the periodontal space. CONCLUSIONS: TCS + ZrO2 and TCS + Nb2 O5 were associated with periodontium repair since these materials allowed the reestablishment of periodontal space width and collagen formation when used in the filling of uninfected perforations in the pulp chamber floor of maxillary rat molars. Furthermore, the significant reduction in the periodontal space of TCS + ZrO2 and TCS + Nb2 O5 specimens after 60 days confirmed that the experimental materials were associated with a more rapid recovery of the injured tissues than MTA.

Mast cells and immunoexpression of FGF-1 and Ki-67 in rat subcutaneous tissue following the implantation of Biodentine and MTA Angelus.

AIM: To compare the formation of fibrous capsules around Biodentine and MTA Angelus implants as well as the participation of fibroblast growth factor-1 (FGF-1) and mast cells in the tissue response to these endodontic materials. METHODOLOGY: Sixty polyethylene tubes filled with Biodentine or MTA, and empty tubes (control group) were implanted into the dorsal subcutaneous tissues of male rats. After 7, 15, 30 and 60 days, the specimens were embedded in paraffin and the number of fibroblasts and mast cells was quantified in the sections stained with Masson's trichrome or Alcian Blue, respectively. FGF-1 and Ki-67 were detected by immunohistochemistry, and the number of immunolabelled cells was computed. The collagen content was estimated in the picrosirius red-stained sections. The data were subjected to two-way ANOVA followed by Tukey's test (P ≤ 0.05). RESULTS: The capsules were associated with a significant increase (P < 0.0001) in the number of fibroblasts and mast cells, and in the collagen content over time. A significant decrease (P < 0.0001) in the immunoexpression of FGF-1 and Ki-67 was observed in all groups from the 7th-60th day. At 60 days, the number of fibroblasts (P = 0.0226) and the collagen content (P < 0.0001) were significantly greater in MTA than Biodentine specimens, while the greatest number of mast cells and FGF-1-immunolabelled cells was observed in Biodentine specimens (P < 0.0001). A significant difference in Ki-67 immunoexpression was not detected between specimens of Biodentine and MTA. CONCLUSIONS: The collagen-rich capsule formed slowly around Biodentine in comparison with MTA. FGF-1 and mast cells participated in capsule remodelling, stimulating fibroblast proliferation and subsequent collagen production, in response to subcutaneous implants.

Biocompatibility of mineral trioxide aggregate flow and biodentine.

AIM: To evaluate the influence of powder-to-gel ratio (0.19 g powder to 50 µL of gel, thick MTA Flow, and 0.06 g powder to 50 µL of gel, fluid MTA Flow) on biocompatibility of MTA Flow (Ultradent Products Inc., South Jordan, UT, USA, lot: 2015122901) and compare it with Biodentine (Septodont Inc., Saint-Maur-des-Fossés, France, lot: B18542A). METHODOLOGY: The materials were manipulated and inserted into polyethylene tubes for implantation in twenty rats. After 7, 15, 30 and 60 days, the specimens were removed and embedded in paraffin. Haematoxylin and eosin sections were used to count the number of inflammatory cells (IC) and fibroblasts mm-2 (Fb). In the Masson's trichrome-stained sections, the fibrous capsule thickness was measured; picrosirius red-stained sections were used for birefringent collagen quantification. The data were submitted to two-way ANOVA and Tukey test (P ≤ 0.05). RESULTS: A significantly lower number of IC and consequently higher number of Fb were observed in the capsules adjacent to thick MTA Flow at all periods, in comparison with other materials (P ≤ 0.05). At 60 days, the quantity of birefringent collagen was significantly greater in the tissue in contact with thick MTA Flow, when compared with fluid MTA Flow and Biodentine. CONCLUSIONS: Although thick MTA Flow induced a less intense inflammatory response, all evaluated materials are biocompatible because they allowed regression of this process after 60 days.

Zirconium oxide and niobium oxide used as radiopacifiers in a calcium silicate-based material stimulate fibroblast proliferation and collagen formation.

AIM: To evaluate the influence of the addition of microparticulate (micro) and nanoparticulate (nano) zirconium oxide (ZrO2 ) and niobium pentoxide (Nb2 O5 ) to a calcium silicate-based cement (CS) on the subcutaneous healing process in rats compared with MTA Angelus™. METHODOLOGY: In each rat, two polyethylene tubes filled with the following materials: (i) MTA; (ii) CS + ZrO2 micro; (iii) CS + ZrO2 nano; (iv) CS + Nb2 O5 micro or (v) CS + Nb2 O5 nano were implanted subcutaneously; empty polyethylene tubes were used in the Control group. After 7, 15, 30 and 60 days, the specimens (n = 5 per group in each period) were fixed and embedded in paraffin. Masson's trichrome sections were used to obtain the volume density of the inflammatory cells (VvIC) and fibroblasts (VvFb). The sections were also stained with Picrosirius-red to calculate the birefringent collagen content. Fibroblast growth factor-1 (FGF-1) was detected by immunohistochemistry, and the number of immunolabelled cells was obtained. The data were subjected to two-way anova followed by Tukey's test (P ≤ 0.05). RESULTS: At all periods, the VvIC was significantly lower (P < 0.001) in all the CS and Control groups than in the MTA group. At all periods, the VvFb was reduced significantly (P = 0.023) in the MTA group in comparison with the other groups. In addition, the number of immunolabelled cells in the capsules of the CS groups was significantly higher (P < 0.001) than in the MTA group at all time-points. CONCLUSIONS: The experimental materials (CS + ZrO2 and CS + Nb2 O5 ) induced fibroblast proliferation and accelerated the regression of the inflammatory reaction. However, the addition of nanoparticulate radiopacifiers did not improve the biological properties of a calcium silicate-based cement when compared to microparticulate agents.

In vivo evaluation of the inflammatory response and IL-6 immunoexpression promoted by Biodentine and MTA Angelus.

AIM: To evaluate the inflammatory process induced by Biodentine and mineral trioxide aggregate (MTA) in rat subcutaneous tissues. METHODOLOGY: A polyethylene tube filled with Biodentine (n = 20) or MTA (n = 20) was placed into the dorsal subcutaneous of forty male rats; in the control group (CG; n = 20), empty tubes were implanted. After 7, 15, 30 and 60 days, the polyethylene tubes surrounded by connective tissue were fixed and embedded in paraffin. The number of inflammatory cells was estimated in HE-stained sections; numerical density of interleukin-6 (IL-6)-immunolabelled cells was also performed. The differences amongst the groups were analysed statistically by Tukey's test (P ≤ 0.05). RESULTS: A high number of inflammatory cells and IL-6-positive cells were observed at 7 days, in all groups; however, in the Biodentine group, the number of inflammatory cells and IL-6-immunolabelled cells was significantly higher (P ≤ 0.05) in comparison with the other groups at 7 and 15 days. In the capsules of animals from all groups, a gradual and significant reduction (P ≤ 0.05) of these parameters was seen over time. At 60 days, the capsules exhibited numerous fibroblasts and bundles of collagen fibres; in addition, the number of IL-6-positive cells was not significantly different amongst Biodentine, MTA and control groups. CONCLUSIONS: There was a significant regression in the inflammatory reaction in the capsules indicating, therefore, that Biodentine is a biocompatible material.

Phytocystatin CsinCPI-2 Reduces Osteoclastogenesis and Alveolar Bone Loss.

Periodontal disease (PD) is a polymicrobial chronic inflammatory condition of the supporting tissues around the teeth, leading to the destruction of surrounding connective tissue. During the progression of PD, osteoclasts play a crucial role in the resorption of alveolar bone that eventually leads to the loss of teeth if the PD is left untreated. Therefore, the development of antiresorptive therapies targeting bone-resorbing cells will significantly benefit the treatment of PD. Here, we demonstrate the inhibitory effect of CsinCPI-2, a novel cysteine peptidase inhibitor from the orange tree, on periodontitis-induced inflammation, alveolar bone loss, and osteoclast differentiation. Using the ligature-induced periodontitis model in mice, we show that treatment with CsinCPI-2 (0.8 µg/g of body weight) significantly reduced inflammatory cell infiltrate in the connective tissue and prevented the loss of alveolar bone mass (BV/TV) caused by PD, effects associated with diminished numbers of TRAP-positive multinucleated cells. Furthermore, CsinCPI-2 significantly downregulated the numbers of inflammatory cells expressing CD3, CD45, MAC387, and IL-1ß. In vitro, CsinCPI-2 inhibited RANKL-induced TRAP+ multinucleated osteoclast formation in mouse bone marrow macrophage cultures in a concentration-dependent manner. This effect was not due to cytotoxicity, as demonstrated by the MTT assay. CsinCPI-2 inhibited RANKL-induced mRNA expression of Acp5, Calcr, and Ctsk, as well as the RANKL-induced upregulation of Nfatc1, a crucial transcription factor for osteoclast differentiation. Based on our findings, CsinCPI-2 prevents bone loss induced by PD by controlling the inflammatory process and acting directly on osteoclastogenesis, suggesting an interesting potential for CsinCPI-2 in the strategy for PD treatment.

Histological and histomorphometrical evaluation of furcation perforations filled with MTA, CPM and ZOE.

AIM: To evaluate the biological response of the periodontium adjacent to furcation perforations in rat molars filled with Endo-CPM-Sealer (CPM), MTA-Angelus (MTA) or zinc oxide-eugenol cement (ZOE). METHODOLOGY: The pulp chamber floors of maxillary right first molar teeth were perforated and sealed with CPM, mineral trioxide aggregate (MTA) or ZOE; the left first molars, without any treatment, were used as controls (CG). After 7, 15, 30 and 60 days, fragments of maxilla were fixed, decalcified and embedded in paraffin. Sections were stained with H&E, Masson's trichrome and submitted to tartrate-resistant acid phosphatase (TRAP) reaction, used as an osteoclast marker. The width of the periodontal space, the numerical density of inflammatory cells and the number of TRAP-positive osteoclasts in the bone surface were measured, and statistical analyses were performed using analysis of variance and Tukey test (P ≤ 0.05). RESULTS: In all experimental groups, the greatest number of inflammatory cells was observed at 7 days, especially in the ZOE group. In this group, the intense inflammatory process was related to a significant increase (P ≤ 0.05) in the number of osteoclasts and, thereby, in an increase in the width of the periodontal space. At 60 days, no significant differences in osteoclast numbers amongst CPM, MTA and CG groups occurred; the periodontal space was also significantly reduced in the experimental groups in comparison with the initial periods. However, in the ZOE group, the periodontal space was significantly larger (P ≤ 0.05) in comparison with MTA-based materials. CONCLUSIONS: The periodontium adjacent to perforations filled with MTA and CPM exhibited clear evidence of re-establishment and thus better biocompatibility than ZOE.

Cell death and quantitative reduction of rests of Malassez according to age.

BACKGROUND AND OBJECTIVE: Rests of Malassez are clusters of epithelial cells that remain in the periodontal ligament throughout life. However, it has been reported that the number of these structures decreases with age, and some epithelial cells undergo apoptosis in rests of Malassez of young and adult rats. Therefore, the purpose of the present study was to investigate the incidence of epithelial cell death and the quantitative changes in the rests of Malassez in rat molars of different ages. MATERIAL AND METHODS: Fragments containing the upper molars of rats aged 29, 45 and 120 d were fixed, decalcified and embedded for analysis by light microscopy. In the sections stained by hematoxylin and eosin, the number of rests of Malassez and the number of nuclei of these epithelial structures were obtained. Moreover, the nuclei exhibiting typical features of cell death were also counted in each rest of Malassez. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method for detection of cell death was also carried out. RESULTS: In all groups examined, some rests of Malassez exhibited epithelial cell nuclei with typical features of apoptosis and some of them were also TUNEL positive. From 29 to 120 d of age in rats, the quantitative analysis showed a significant decrease in the total number of rests of Malassez in the cervical, middle and furcation regions of the periodontal ligament. Moreover, a significant decrease of epithelial cell nuclei was concomitant to an increase in the frequency of cell death in the oldest rats. CONCLUSION: These results suggest that epithelial cell death by apoptosis may be, at least in part, responsible for the reduction in the number of rests of Malassez according to age.

Staining methods applied to glycol methacrylate embedded tissue sections.

The use of glycol methacrylate (GMA) avoids some technical artifacts, which are usually observed in paraffin-embedded sections, providing good morphological resolution. On the other hand, weak staining have been mentioned during the use of different methods in plastic sections. In the present study, changes in the histological staining procedures have been assayed during the use of staining and histochemical methods in different GMA-embedded tissues. Samples of tongue, submandibular and sublingual glands, cartilage, portions of respiratory tract and nervous ganglion were fixed in 4% formaldehyde and embedded in glycol methacrylate. The sections of tongue and nervous ganglion were stained by H&E. Picrosirius, Toluidine Blue and Sudan Black B methods were applied, respectively, for identification of collagen fibers in submandibular gland, sulfated glycosaminoglycans in cartilage (metachromasia) and myelin lipids in nervous ganglion. Periodic Acid-Schiff (PAS) method was used for detection of glycoconjugates in submandibular gland and cartilage while AB/PAS combined methods were applied for detection of mucins in the respiratory tract. In addition, a combination of Alcian Blue (AB) and Picrosirius methods was also assayed in the sublingual gland sections. The GMA-embedded tissue sections showed an optimal morphological integrity and were favorable to the staining methods employed in the present study. In the sections of tongue and nervous ganglion, a good contrast of basophilic and acidophilic structures was obtained by H&E. An intense eosinophilia was observed either in the striated muscle fibers or in the myelin sheaths in which the lipids were preserved and revealed by Sudan Black B. In the cartilage matrix, a strong metachromasia was revealed by Toluidine Blue in the negatively-charged glycosaminoglycans. In the chondrocytes, glycogen granules were intensely positive to PAS method. Extracellular glycoproteins were also PAS positive in the basal membrane and in the region occupied by the lamina externa and reticular fibers surrounding each smooth muscle cells of the blood vessels. In the epithelial cells of the respiratory tract, acid and neutral mucins were histochemically detected by AB and PAS methods, respectively. Moreover, granules containing acid and neutral mucins were revealed in purple by AB and PAS concomitantly. In the sublingual gland sections, a distinct affinity of acid mucins by AB (in turquoise-blue) and collagen fibers by Picrosirius (in red) was obtained when these methods were combined. Although some routine dyes used in paraffin sections have showed a weak stain in historesin sections, our results showed that different dyes could be applied in GMA sections if modified staining procedures were assayed. Therefore, appropriate staining contrast and, thus, detection of one or different substances in a same section can be acquired in association to the good morphological resolution provided by GMA.

Apoptotic bone cells may be engulfed by osteoclasts during alveolar bone resorption in young rats.

The alveolar bone is a suitable in vivo physiological model for the study of apoptosis and interactions of bone cells because it undergoes continuous, rapid and intense resorption/remodelling, during a long period of time, to accommodate the growing tooth germs. The intensity of alveolar bone resorption greatly enhances the chances of observing images of the extremely rapid events of apoptosis of bone cells and also of images of interactions between osteoclasts and osteocytes/osteoblasts/bone lining cells. To find such images, we have therefore examined the alveolar bone of young rats using light microscopy, the TUNEL method for apoptosis, and electron microscopy. Fragments of alveolar bone from young rats were fixed in Bouin and formaldehyde for morphology and for the TUNEL method. Glutaraldehyde-formaldehyde fixed specimens were processed for transmission electron microscopy. Results showed TUNEL positive round/ovoid structures on the bone surface and inside osteocytic lacunae. These structures--also stained by hematoxylin--were therefore interpreted, respectively, as osteoblasts/lining cells and osteocytes undergoing apoptosis. Osteoclasts also exhibited TUNEL positive apoptotic bodies inside large vacuoles; the nuclei of osteoclasts, however, were always TUNEL negative. Ultrathin sections revealed typical apoptotic images--round/ ovoid bodies with dense crescent-like chromatin--on the bone surface, corresponding therefore to apoptotic osteoblasts/lining cells. Osteocytes also showed images compatible with apoptosis. Large osteoclast vacuoles often contained fragmented cellular material. Our results provide further support for the idea that osteoclasts internalize dying bone cells; we were however, unable to find images of osteoclasts in apoptosis.

Light and electron microscopic study of autologous implants of dental roots in the subcutaneous tissue of rats.

Maxillary first molars of rats were extracted. The roots were separated and subsequently implanted in the dorsal subcutaneous tissue of the same animal. In Experiment 1, the roots were implanted immediately after removal; in Experiment 2, the roots were subjected to freeze-thawing (devitalized) before implantation. After 4, 8, and 15 weeks of implantation, the roots were removed and processed for light and transmission electron microscopy. Roots of Experiment 1 showed an amorphous clear material filling depressions on the irregular surface of the cementum. This material consisted predominantly of collagen fibrils. Cells adjacent to the amorphous clear material exhibited numerous cytoplasmic processes surrounded by collagen fibrils. Globular structures with an electron-opacity similar to that of neighbouring cementum were observed within the bundles of collagen fibrils. Multinucleated cells with features of osteo/odontoclasts were often closely apposed to deep depressions of the cementum. In Experiment 2, only large multinucleated giant cells appeared around the surfaces of the roots. It seems therefore that, in Experiment 1, precementum formation and osteo/odontoclast mediated destruction occurred. It is likely that these activities may have originated from periodontal ligament cells carried to the implantation site. This conclusion is supported by the observation that such activities were absent in Experiment 2. Devitalized roots were unable to form precementum and were surrounded by foreign body giant cells.

Treatment with tacrolimus enhances alveolar bone formation and decreases osteoclast number in the maxillae: a histomorphometric and ultrastructural study in rats.

Recent studies have suggested that tacrolimus monotherapy is a beneficial therapeutic alternative for the normalization of cyclosporin-induced bone loss in animal models and humans. The mechanism accounting for this action is unclear at present. In the present study, we attempted to determine the effect of tacrolimus monotherapy on alveolar bone using histological, histomorphometric and transmission electron microscopy (TEM). Groups of rats (n=10 each) were treated with either tacrolimus (1mg/kg/day, s.c.) or drug vehicle for 60 days. Fragments containing maxillary molars were processed for light microscopy to investigate the alveolar bone volume, trabecular separation, number of osteoclasts and osteoblasts, and transmission electron microscopy to investigate their ultrastructural basic phenotype. Treatment with tacrolimus monotherapy during 60 days may induce increases in alveolar bone volume (BV/TV,%; P<0.05) and a non-significant decrease in trabecular separation (Tb.Sp,mm; P>0.05), represented by a decrease in osteoclast number (N.Oc/BS; P<0.05) and maintenance of osteoblast number (N.Ob/BS; P>0.05). Osteoblasts were often observed as a continuous layer of active cells on the bone surface. Osteoclasts appeared to be detached from the resorbed bone surface, which was often filled by active osteoblasts and collagen-rich matrix. Moreover, osteoclasts in the treated group were frequently observed as inactive cells (without ruffled border, clear zone and detached from the bone surface). Within the limits of the present study, we conclude that tacrolimus leads to an increase in alveolar bone formation, which probably exerts action on osteoclasts. Tacrolimus could, therefore, play a crucial role in the control of both early osteoclast differentiations from precursors, as well as in functional activation.

Decrease in the number and apoptosis of alveolar bone osteoclasts in estrogen-treated rats.

BACKGROUND AND OBJECTIVE: Bone is a mineralized tissue that is under the influence of several systemic, local and environmental factors. Among systemic factors, estrogen is a hormone well known for its inhibitory function on bone resorption. As alveolar bone of young rats undergoes continuous and intense remodeling to accommodate the growing and erupting tooth, it is a suitable in vivo model for using to study the possible action of estrogen on bone. Thus, in an attempt to investigate the possibility that estrogen may induce the death of osteoclasts, we examined the alveolar bone of estrogen-treated rats. MATERIAL AND METHODS: Fifteen, 22-d-old female rats were divided into estrogen, sham and control groups. The estrogen group received estrogen and the sham group received corn oil used as the dilution vehicle. After 8 d, fragments containing alveolar bone were removed and processed for light microscopy and transmission electron microscopy. Sections were stained with hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP)-an osteoclast marker. Quantitative analysis of the number of TRAP-positive osteoclasts per mm of bone surface was carried out. For detecting apoptosis, sections were analyzed by the Terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL) method; TUNEL/TRAP combined methods were also used. RESULTS: The number of TRAP-positive osteoclasts per mm of bone surface was significantly reduced in the estrogen group compared with the sham and control groups. TRAP-positive osteoclasts exhibiting TUNEL-positive nuclei were observed only in the estrogen group. In addition, in the estrogen group the ultrastructural images revealed shrunken osteoclasts exhibiting nuclei with conspicuous and tortuous masses of condensed chromatin, typical of apoptosis. CONCLUSION: Our results reinforce the idea that estrogen inhibits bone resorption by promoting a reduction in the number of osteoclasts, thus indicating that this reduction may be, at least in part, a consequence of osteoclast apoptosis.

Apoptosis in the epithelial cells of the rests of Malassez of the periodontium of rat molars.

BACKGROUND AND OBJECTIVES: Epithelial rests of Malassez are clusters of cells derived from Hertwig's root sheath that remain in the periodontal ligament throughout life. Although it is known that the cells of Malassez proliferate, there are no studies showing that they undergo programmed cell death, i.e. apoptosis. In most tissues, proliferation is balanced by apoptosis. Thus we examined regions of the periodontium of young and adult rat molars in the hope of detecting apoptosis. METHODS: Wistar rats aged 29, 45 and 120 days were killed with chloral hydrate (600 mg/kg). Fragments containing maxillary molars were removed and fixed in formaldehyde, decalcified, and embedded in paraffin and glycol methacrylate. Sections were stained with hematoxylin/eosin and the Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) method for detection of apoptosis. Specimens were also fixed in glutaraldehyde-formaldehyde, decalcified and processed for transmission electron microscopy. RESULTS: Epithelial rests of Malassez containing round/ovoid basophilic dense bodies and TUNEL-positive structures were found in all specimens examined. Ultrastructural examination revealed that some cells of Malassez contained masses of condensed peripheral chromatin and a shrunken cytoplasm exhibiting intact organelles--images typical of apoptosis. Moreover, round/ovoid electron-opaque structures appeared to be in the process of being engulfed by neighboring epithelial cells of Malassez. CONCLUSIONS: Our results demonstrate that epithelial cells of Malassez's rests undergo apoptosis in the developing and adult periodontium. Apoptosis may, together with proliferation, be part of the mechanism of turnover/remodelling of the cells of Malassez.

Combined TUNEL and TRAP methods suggest that apoptotic bone cells are inside vacuoles of alveolar bone osteoclasts in young rats.

Although it is generally accepted that osteoclasts breakdown and resorb bone matrix, the possibility that they may also be able to engulf apoptotic osteoblasts/lining cells and/or osteocytes remains controversial. Apoptosis of osteoblasts/ lining cells and/or osteocytes and interactions between these cells and osteoclasts are extremely rapid events that are difficult to observe in vivo. A suitable in vivo model for studying these events is the alveolar bone of young rats because it is continuously undergoing intense resorption/remodeling. Thus, sections of aldehyde fixed alveolar bone of young rats were stained by the combined terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and the tartrate-resistant acid phosphatase (TRAP) method for the simultaneous visualization of apoptotic cells and osteoclasts in the same section. The combined TUNEL and TRAP reactions, in the same section, greatly facilitated visualization of relationship between osteoclasts and apoptotic bone cells during alveolar bone remodeling. Our results showed that several TRAP-positive osteoclasts exhibited large vacuoles containing TUNEL positive apoptotic structures, probably derived from osteoblasts/lining cells and/or osteocytes. These results support the idea that alveolar bone osteoclasts are able to internalize dying apoptotic bone cells.

Apoptosis in the early developing periodontium of rat molars.

Development of the periodontium involves a series of complex steps that result in the formation of root dentine, cementum, bone and fibres of the ligament. These precisely controlled and timed events require the participation of the enamel organ derived epithelial cells of Hertwig's (HRS) and ectomesenchymal cells of the dental follicle. These events involve rapid turnover of the tissues and cells, including disappearance of epithelial cells of HRS. Thus, it seemed likely to us that programmed cell death (apoptosis) may play a role in the development of the periodontium. Fragments of first molars, obtained from 14- and 29-day-old rats, were fixed in glutaraldehyde-formaldehyde and processed for light and electron microscopy. For the TUNEL method for detection of apoptosis, specimens were fixed in 4% formaldehyde and embedded in paraffin. Results confirmed that epithelial cells of HRS maintain a close relationship with the forming dentine root, and that they may become trapped in the dentino-cemental junction. Some of the epithelial cells exhibited ultrastructural features which are consistent with the interpretation that they were undergoing programmed cell death, i.e. apoptosis. Periodontal fibroblast-like cells showed typical images of apoptosis and engulfed apoptotic bodies. TUNEL positive structures were present in all corresponding regions. It seems therefore that apoptosis of epithelial cells of HRS and fibroblast-like cells of the periodontal ligament constitutes an integral part of the developmental process of the tissues of the periodontium.

Effect of ibotenate lesions of the ventromedial hypothalamus on the water and salt intake induced by activation of the median preoptic nucleus in sodium-depleted rats.

In this study we investigated the influence of a ventromedial hypothalamus (VMH) lesion with ibotenic acid on water and sodium intake and pressor responses induced by combined treatment of the median preoptic nucleus (MnPO) with angiotensin II (ANG II) and adrenergic agonists (phenylephrine, norepinephrine, isoproterenol and clonidine). Male Holtzman rats with a stainless steel cannula implanted into the MnPO and bilateral sham (vehicle) or VMH lesions with ibotenic acid were used. The ingestion of water and sodium and mean arterial pressure (MAP) were determined in separate groups submitted to sodium depletion with the diuretic furosemide (20 mg/rat). ANG II (10 pmol) injection into the MnPO of sham-lesioned rats induced water and sodium intake and pressor responses. VMH-lesion reduced ANG II-induced water intake and increased saline intake. In sham rats phenylephrine (80 nmol) into MnPO increased, whereas norepinephrine (80 nmol) and clonidine (40 nmol) reduced ANG II-induced water intake while sodium intake was reduced only by clonidine into MnPO. In VMH-lesioned rats, phenylephrine reduced, noradrenaline increased and clonidine produced no effect on ANG II-induced water intake. In lesioned rats ANG II-induced sodium intake was reduced by phenylephrine and noradrenaline, whereas clonidine produced no change. ANG II-induced pressor response was reduced in VMH-lesioned rats, but the pressor response combining ANG II and phenylephrine or noradrenaline in VMH-lesioned rats was bigger than sham rats. These results show that the VMH is important for the changes in water and sodium intake and cardiovascular responses induced by angiotensinergic and adrenergic activation of the MnPO.