The response of subcutaneous connective tissue to newly developed calcium phosphate-based root canal sealers.

AIM: To evaluate the histopathologic biocompatibility of two new calcium phosphate-based sealers (CPS-1 & CPS-2) with a commercially available calcium hydroxide-based sealer (Acroseal). METHODOLOGY: Polyethylene tubes were filled with freshly mixed sealers and implanted subcutaneously in the dorsal connective tissue of rats. Empty tubes were used as controls. Histopathological examinations were conducted at 7, 15, 30, 60 and 90 days after the implantation procedure. The presence of inflammation and predominant cell types were analysed statistically with Mann-Whitney U and Kruskal-Wallis non-parametric tests. Fibrous connective tissue thickness adjacent to each sample was recorded. Differences were tested for significance using anova and 'Duncan's' multiple comparison test (P < 0.05). RESULTS: CPS-1 sealer was associated with severe inflammation and remained an irritation throughout the 90-day implantation period; the tissue reaction pattern was stromal fibrosis. The control, CPS-2 and Acroseal sealers had similar patterns of irritation, which were more severe initially and diminished with time creating a thin fibrous capsule around the implant with a complete absence of inflammatory cells. There was no difference in tissue reaction between the control, CPS-1, CPS-2 and Acroseal groups amongst the first two observation periods (P > 0.05). However, there was a highly significant difference between the same groups at the last two observation periods (P < 0.01). Also, there were highly significant differences between the observation periods within all four groups at 7, 15, 30, 60 and 90 days (P < 0.01). CONCLUSION: CPS-1 sealer was not biocompatible. CPS-2 sealer and Acroseal had a favourable biocompatibility level based on the histological findings.

Comparative study of biocompatibility of newly developed calcium phosphate-based root canal sealers on fibroblasts derived from primary human gingiva and a mouse L929 cell line.

AIM: To determine biocompatibility of three calcium phosphate cement (CPC) sealers, and to compare the cytotoxic response of human gingival fibroblasts (HGF) and one mouse fibroblast cell line (L929) to these materials. METHODOLOGY: Monocalcium phosphate, calcium oxide and synthetic hydroxyapatite were combined with one of three aqueous solutions: modified polyacrylic acid, glass-ionomer liquid or 35% w/w polymethyl vinyl ether maleic acid to obtain Types I, IIa and III CPCs, respectively. Commercial Ca(OH)(2) sealer was used as a control. The materials were packed into Teflon molds (5.5 x 3 mm), and cellular function was assessed using MTT assay. The specimens were placed immediately in contact with cells, then evaluated at (24 h, 1 week, 2 week, 3 week, 4 week, 5 week). RESULTS: All materials showed significant cytotoxicity for both L929 and HGF cells at 24 h except for Type III. Type I was severely toxic initially, but improved significantly (P < 0.05) over the 5 week evaluation. Types II and Ca(OH)(2) were both cytotoxic over the 5 weeks. Type III CPC was equivalent to Teflon the entire time. The results showed the same rank of cytotoxicity in both cultures. The cytotoxic response decreased in the order of Type II > Ca(OH)(2) > Type I > Type III overtime. L929 cells were generally more sensitive than HGF cells to the calcium hydroxide-based sealer (Acroseal). CONCLUSION: Types I and III have acceptable biologic properties for endodontic applications.

Induced premaxillary suture fusion: class III malocclusion model.

The etiology of class III malocclusion remains unknown. The present study investigates the relationship between craniofacial morphology and premaxillary suture fusion to test the hypothesis that class III malocclusion may be related to premaxillary suture fusion. Cyanoacrylate was applied to immobilize the left premaxillary suture in the experimental group. Sham surgeries in rats were used for controls. Dental impressions and radiographs were taken before and after surgery for comparison of craniofacial differences between groups. Overall cranial base lengths, craniofacial widths, and craniofacial angulations related to the anterior base showed significant differences between groups. At the end of the experiment, the growth of the snout in the experimental group was inhibited and deviated to the treated side, while no obvious change was seen in the control group. The results show that induced premaxillary suture fusion can affect craniofacial morphology and indicate that premature premaxillary suture fusion may result in class III malocclusion.

Monoclonal antibodies to human erythrocyte membrane Ca++-Mg++ adenosine triphosphatase pump recognize an epitope in the basolateral membrane of human kidney distal tubule cells.

Human calcium transporting tissues were examined to determine whether they contained a protein similar to the Ca++-Mg++ adenosine triphosphatase (Ca++-Mg++ATPase) pump of the human erythrocyte membrane. Tissues were processed for immunoperoxidase staining using monoclonal antibodies against purified Ca++-Mg++ATPase. In human kidneys, specific staining was found only along the basolateral membrane of the distal convoluted tubules. Glomeruli and other segments of the nephron did not stain. Staining of erythrocytes in human spleen was readily observed. Human small intestine, human parathyroid, and human liver showed no antigens that crossreacted with the antibodies to Ca++-Mg++ATPase. Specific staining of distal tubule basolateral membranes from the kidney of a chimpanzee was also noted. Our experiments show, for the first time, that basolateral membranes of the human distal convoluted tubule contain a protein that is immunologically similar to the human erythrocyte Ca++-Mg++ATPase. These observations suggest that the cells of the distal convoluted tubules of human kidney may have a calcium pump similar to that of human erythrocyte membranes.

Breaking biological barriers with a toothbrush.

Toothbrushing exposes epithelia and other tissues of the oral cavity to mechanical stress. Here, we investigated whether brushing induces cell wounding--plasma membrane disruption--in epithelial and other cell types in the oral cavity. Brushing of the gingivae and tongues of rats resulted in a striking increase in the number of cells positive for a marker of disruption injury. These cells included those in all strata of the gingival epithelium, and in the skeletal muscle of the tongue. Additionally, we found that brushing resulted in an increase in c-fos expression by junctional epithelial and skeletal muscle cells. Epithelial barrier function, however, was not overtly affected by brushing, despite the observed individual injuries to cells. We concluded that brushing disrupts cell plasma membrane barriers in the oral cavity and activates gene expression events that may lead to local adaptive changes in tissue architecture beneficial to gingival health.

Epitopes of the human erythrocyte Ca2+-Mg2+ ATPase pump in human osteoblast-like cell plasma membranes.

Human osteoblast-like cells were examined for the presence of the Ca2+-Mg2+ ATPase pump. The osteoblast-like cells had characteristic features of the osteoblast phenotype, including the presence of osteonectin, bone GLA protein, and type I collagen. The cells were able to mineralize matrix, their production of cAMP increased in response to PTH, and their alkaline phosphatase activity increased in response to 1,25-dihydroxyvitamin D3. Immunocytochemical staining of the osteoblast-like cells with a monoclonal antibody against human red cell Ca2+-Mg2+ ATPase demonstrated the presence of an epitope of the Ca2+-Mg2+ ATPase in these cells; staining of paraffin-embedded osteoblast-like cell sections demonstrated anti-Ca2+-Mg2+ ATPase staining only in cell plasma membranes. Western blot analysis of osteoblast-like cell homogenates showed that the monoclonal antibody to human erythrocyte Ca2+-Mg2+ ATPase bound to a major band at 140,000 mol wt, similar to the mol wt of known plasma membrane Ca2+-Mg2+ ATPases. The presence in the osteoblast-like cells of a Ca2+-Mg2+ ATPase similar to the human red cell calcium pump suggests that this enzyme may play a role in osteoblast intracellular calcium homeostasis.

Transforming growth factor beta 1 dysregulation in a human oral carcinoma tumour progression model.

A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.

Expression of plasma membrane Ca++ pump epitopes parallels the progression of enamel and dentin mineralization in rat incisor.

We investigated the expression of Ca++ pump epitopes during enamel and dentin mineralization in the rat incisor. Secretory and maturation ameloblasts were studied as well as odontoblasts, using a monoclonal antibody (5F10) against human erythrocyte plasma membrane Ca++, Mg(++)-ATPase. A progressive increase in staining intensity in ameloblasts and the odontoblasts was observed beginning with the onset of mineralization. The mainly membrane-related labeling of ameloblasts showed variable intensity depending on the stage of enamel formation, whereas that of the odontoblasts showed even intensity during continued dentinogenesis. Staining of papillary cells was evident only during enamel maturation. Western blot analysis of freeze-dried ameloblasts was also used to determine the molecular weight of the Ca++ pump epitopes as well as the distribution and relative concentration of epitopes at each stage. An immunoreactive band of MW 140 KD and lower molecular weight bands that are more intense in late than in early maturation were demonstrated. Our studies suggest that the expression of plasma membrane Ca++ pump parallels the progression of mineralization in rat incisor enamel and dentin.

Microarray analysis of Tbx2-directed gene expression: a possible role in osteogenesis.

Tbx2 is a member of the developmentally important transcriptional regulatory T-box gene family, whose target genes have not been well characterized. In an attempt to identify genes that may be regulated by Tbx2, mouse cDNA microarrays were used to analyze differential gene expression profiles, comparing stably transfected NIH3T3 cells overexpressing Tbx2 and vector-transfected controls. Among 8734 genes, 107 genes were up-regulated by 2-fold or greater, and 66 genes were down-regulated by 2-fold or greater. Caveolin, pleiotrophin (osf-1), osteoblast-specific factor-2 (osf-2) and collagen type I alpha were among the genes upregulated in the Tbx2-overexpressing cells, whereas cadherin 3, tenascin C, and insulin-like growth factor binding protein 10/CYR61 (IBP10) were among the genes downregulated. Northern blot analysis confirmed the correlation of expression of several genes, including IBP10 and osf-2, in fibroblast NIH3T3 and rat osteosarcoma ROS17/2.8 cells differentially expressing Tbx2. In ROS17/2.8 cells transfected with antisense Tbx2, osf-2 was downregulated, whereas transfection of sense Tbx2 upregulated this gene. Interestingly, the expression of pleiotrophin (osf-1) and collagen I alpha with Tbx2 transfection showed an inverse regulatory correlation between NIH3T3 and ROS17/2.8 cells. Thus, Tbx2 can act as both a repressor and activator, and the cellular context can influence the effect on gene expression. Although the data do not address whether Tbx2 directly mediates the transcriptional effect, a number of candidate genes possess putative T-box gene regulatory elements. The results support the hypothesis that Tbx2 may be an important modulator of bone development. Further functional cluster analysis indicates that Tbx2 might also be involved in the regulation of cell cycle and cell adhesion.

Cerebrospinal fluid calcium homeostasis: evidence for a plasma membrane Ca2+-pump in mammalian choroid plexus.

A major unanswered question in central nervous system physiology concerns the mechanism by which cerebrospinal fluid (CSF) Ca2+ homeostasis is maintained in the face of hypo- or hypercalcemia. To address this question, we sought and found a protein of Mr approximately 140,000 in choroid plexus plasma membranes that forms a phosphorylated intermediate with characteristics of a plasma membrane Ca2+-pump. A choroid plexus plasma membrane protein of this molecular weight also bound to a monoclonal antibody prepared against the human erythrocyte plasma membrane Ca2+-Mg2+ ATPase Ca2+-pump. When this monoclonal antibody was used for immunohistochemical localization, the plasma membrane Ca2+-pump was found primarily in the CSF-facing membranes of choroid plexus cells from rats, cats, and man. The localization of a plasma membrane Ca2+-pump in the CSF-facing membranes of the choroid plexus suggests that the choroid plexus, by mechanisms including this pump, may regulate CSF Ca2+ concentrations.

Plasma membrane disruption in orthodontic tooth movement in rats.

Sublethal plasma membrane disruption (PMD) is an established mechanism for signaling in several cell types, including endothelial cells and skeletal muscle. We used a rat model of orthodontic tooth movement to test the hypothesis that periodontal ligament (PDL) cells communicate stretch to changes in bone cell activity in part via PMD. To produce PMD, we used a 50-g load from a spring activated in the buccal direction against the maxillary first molars for 5 min. Uptake of endogenous serum albumin was used as a PMD marker. Immunohistochemistry demonstrates albumin in PDL cells surrounding moved first molar tips. Image analysis shows significantly more albumin in cells of the buccal side (tension) of the moved teeth compared with those of the lingual, distal, and mesial sides, and those of the unmoved control. Albumin localization within cells of the PDL, after only 5 min of mechanical loading, suggests that PMD could promote uptake or release of signaling molecules.

Expression of connexin 43 in rat mandibular bone and periodontal ligament (PDL) cells during experimental tooth movement.

Bone remodeling in response to force requires the coordinated action of osteoblasts, osteoclasts, osteocytes, and periodontal ligament cells. Coordination among these cells may be mediated, in part, by cell-to-cell communication via gap junctions. This study tests the hypothesis that the regulation of expression of connexin 43, a gap junction protein, is part of the transduction mechanism between force as applied to bone during orthodontic tooth movement and bone remodeling. To test this hypothesis, we examined connexin 433 expression in a rat model system of experimental tooth movement. To establish the model, we extracted maxillary first molars to initiate supra-eruption of opposing mandibular molars. The rats were killed at 0, 6, 12, 24, and 48 hrs post-extraction. The mandibles were removed, demineralized, and embedded in paraffin. To localize connexin 43 protein and mRNA, we used a specific antibody for immunohistochemistry and a specific cDNA probe for in situ hybridization. Western and Northern blot analyses were used to assess the specificity of the connexin 43 antibody and cDNA probe, respectively. We found connexin 43 protein expressed by osteoclasts (++ ++) and periodontal ligament cells (++ +) in compression zones, and by osteoblasts (++ ++) and osteocytes (++ ++) in tension zones of the periodontal ligament. In addition, connexin 43 mRNA was found in some bone and periodontal ligament cells. Connexin 43 protein was found, by densitometric analysis, to be higher in the periodontal ligament after exposure to force compared with controls (P < 0.001). The number of osteocytes expressing connexin 43 48 hrs after molar extraction was also significantly greater in bone subjected to tension when compared with controls (P < 0.001). The results of this study support the hypothesis that connexin 43 plays a role in the coordination of events during experimentally induced alveolar bone remodeling.

Chemopreventive effects of green tea polyphenols correlate with reversible induction of p57 expression.

Green tea polyphenols are known to induce apoptosis in certain types of tumor cells. However, the mechanism(s) that enables normal cells to evade the apoptotic effect is still not understood. In this study, Western blot analysis combined with cycloheximide treatment was used to examine the effects of green tea polyphenols on the expression levels of p57, a cyclin-dependent kinase and apoptosis inhibitor, in normal human keratinocytes and in the oral carcinoma cell lines SCC25 and OSC2. The results showed that the most potent green tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), induced p57 in normal keratinocytes in a dosage- and time-dependent manner, while the levels of p57 protein in oral carcinoma cells were unaltered. The differential response in p57 induction was consistent with the apoptosis status detected by annexin V assay. The data suggest that the chemopreventive effects of green tea polyphenols may involve p57-mediated cell cycle regulation in normal epithelial cells.

Interleukin-1beta regulation of adhesion molecules on human gingival and periodontal ligament fibroblasts.

BACKGROUND: Adhesion molecules have been implicated in the pathogenesis of rheumatoid arthritis and may also play a role in the pathogenesis of periodontal disease by promoting the recruitment and retention of leukocytes in gingival tissue. METHODS: The aim of the present study was to evaluate the capacity of interleukin-1beta (IL-1beta) to regulate adhesion molecule expression on clinically healthy human gingival (HGF) and periodontal ligament (PDL) fibroblasts. The HGF (n = 6) and PDL (n = 3) fibroblasts were treated with 1.0 ng/ml of IL-1beta for 24 hours and then incubated with primary intercellular adhesion molecule-1 (ICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1) antibodies followed by FITC-conjugated secondary antibodies. The expression of ICAM-1 and VCAM-1 was measured by immunofluorescence flow cytometry. RESULTS: The levels of ICAM-1 expression in IL-1beta treated HGF and PDL fibroblasts were statistically significant (P < or = 0.05) compared to normal untreated controls using log-transformed data and 3-way analysis of variance. Both cells expressed VCAM-1 after IL-1beta treatment, but the levels were not statistically different from controls. CONCLUSIONS: This study demonstrated that IL-1beta upregulated ICAM-1 expression in both HGF and PDL fibroblasts. Even though the level of VCAM-1 was not statistically different from both HGF and PDL fibroblasts treated with IL-1beta compared to controls, both cells do express the VCAM-1 molecules. These results suggest that ICAM-1 and VCAM-1 might be involved in the pathogenesis of periodontal disease.

The disintegration of superEBA cement in solutions with adjusted pH and osmolarity.

This study determined the disintegration of fast-set SuperEBA cement using ANSI/ADA Specification No. 30 (Spec #30) as well as modifications in pH, osmolarity, time before immersion, and duration of immersion that mimic the clinical, endodontic application of this material. After immersion intervals of 24 h, 1 wk, 1 m, 3 m, or 6 m, specimens were desiccated and weighed. The preimmersion and postdehydration weights were obtained to the nearest 1.0 mg and the percent weight loss from preimmersion levels was calculated. Adjusting the osmolarity to the physiologic level of 300 mOsm/kg resulted in significantly less weight loss (p < 0.05) than the control group in distilled water (no modification of Spec #30). The pH of the storage solution was found to be a significant factor in weight loss. As the acidity and the time of immersion increased, the weight loss also significantly increased with the greatest weight loss of 19.81% at pH 5.5 after 6 m storage. Immersion within 10 min of mixing was not significantly different (p > 0.05) in weight from the control of 1 h set-time. The 24 h weight loss measurements for the pHs of 5.5 and 6.4 were greater than the 1.5% allowed by Spec #30. All other 24 h measurements were less than the 1.5%.

Distribution of plasma-membrane Ca2+ pump in mandibular condyles from growing and adult rabbits.

Chondrocytes may control the mineralization of the extracellular matrix of condylar cartilage by several mechanisms including the release of microvesicles involved in the initial nucleation, the creation or modification of the local matrix to help propagate or restrict mineralization, and the regulation of the ionic environment at the calcifying foci within the matrix. The plasma membrane Ca2+-Mg2+ ATPase (Ca2+ pump) is known to play a part in the vectorial efflux of calcium in a variety of cells including chondrocytes. The purpose here was to study the distribution of Ca2+-pump protein in mandibular condyles from growing and adult rabbits, and compare the expression of that protein in progressively differentiating chondrocytes whose final stage is associated with a mineralized extracellular matrix. Ca2+-pump antigen was identified immunohistochemically in six growing and six adult rabbit mandibular condyles with a Ca2+ pump-specific monoclonal antibody. The presence of Ca2+-pump antigen was established in hypertrophic chondrocytes, and in osteoblasts and osteoclasts of subchondral bone. Slot-blot analysis of nitrocellulose-immobilized chondrocyte homogenates showed that the amount of Ca2+ pump in growing cartilage was more than twice that in adult cartilage (p < 0.05). The demonstration of Ca2+-pump antigen in the hypertrophic chondrocytes of growing rabbit condyles is consistent with a role for the plasma-membrane Ca2+ pump in the calcification of mandibular condylar cartilage.

Connexin-43 expression in oral-derived human osteoblasts after transforming growth factor-beta and prostaglandin E2 exposure.

Dental implant placement stimulates a response in the supporting tissue; the response involves bone remodeling and release of wound-healing factors, including cytokines. Important factors such as transforming growth factor-beta (TGF-beta), which promotes matrix synthesis, and prostaglandin E2 (PGE2), a mediator of inflammation, have the potential to alter the communication between bone cells and interfere with implant site healing. Cells responsible for the formation of bone are interconnected to form a multicellular network. Cell-to-cell communication in this network occurs in part via gap junctions. In bone cells, the predominant gap junction protein is connexin-43. TGF-beta is a growth modulator produced by osteoblasts and released from the matrix in response to resorption and may influence the progression of periodontal disease. TGF-beta also promotes the synthesis of extracellular matrix proteins such as collagen, fibronectin, and adhesion molecules. PGE2 is a mediator of inflammation produced in response to periodontal pathogens. PGE2 levels in the gingival sulcular fluid have been correlated with attachment loss and bone resorption. The relationship between these factors and connexin-43 is unclear. Oral-derived (alveolar) bone was used because the phenotype of bone can differ between species and between different sites in the body. For our studies, explants of human osteoblasts were cultured on eight well plates and characterized by their expression of osteocalcin, osteonectin, alkaline phosphatase, type 1 collagen, and connexin-43. Cells were grown to near confluence on 12 well plates in 20% fetal bovine serum (FBS) Dulbecco modified Eagle medium (DMEM) and then cultured for 24 hours in 0.5% FBS DMEM before exposure to either 1, 5, or 10 ng/mL of TGF-beta in serum-free DMEM for 12 or 24 hours or to 20, 80, or 300 ng/mL of PGE2 in serum-free DMEM for 12 or 24 hours. After incubation, cells were removed from plates by scraping and assayed for connexin-43 protein, first by Western blot to confirm the specificity of the anti-connexin-43 antibody and then by slot blot analysis for quantitative comparison of connexin-43 expression. Our studies showed no significant changes in connexin-43 expression in response to either factor. These studies suggest that exogenous TGF-beta and PGE2 do not alter connexin-43 expression.

Characterization of rare mammary tumours appearing on the neck of RIII/Sa mice infected with mouse mammary tumour virus.

RIII/Sa and C3H mice harbour milk-borne mouse mammary tumour virus (MMTV) and develop mammary tumours at a high incidence. These mammary tumours usually arise ventrally and/or on the sides of the animals. In the present study, some mice of both strains were observed to have tumours in the dorsal neck area. Histological analysis of the tumours indicated their similarity to mammary tumours induced by MMTV oncogenesis. The neck tumours were found by thin-section electron microscopy to contain both type A and type B particles that are hallmarks of MMTV infection. In addition, the neck tumour DNA possessed insertion mutations of Wnt-1 and Fgf-3 proto-oncogenes, the activation of which play important roles in the development of mouse mammary tumours. These neck tumours appear to be mammary tumours that arise in the context of in-situ mammary tissue, similar to rare 'ectopic' human breast cancers that arise in the axillary region and other sites remote from the breast.

Temporary loss of plasma membrane integrity in orthodontic tooth movement.

In these studies, a rat model of orthodontic tooth movement was used to support the premise that periodontal ligament (PDL) cells experience plasma membrane disruption and resealing events upon application of mechanical stress. Immunoelectron microscopy, showed albumin in the cytoplasm of PDL and bone lining cells in the tension side of moved molars. The intracellular localization of this large molecule (60 KDa) suggests that these cells have undergone plasma membrane disruption and resealing. To further assess these and previous findings, fluorescent dyes (FITC-dextran and rhodamine-dextran) were delivered into the vascular system followed by application of 50 g of static load. These large dextran molecules (10 KDa) were preferentially taken up by PDL cells of the buccal (tension side) of moved molars. These cells were determined to be viable since dead cells do not retain these diffusible tracers. These studies provide evidence of a novel cellular mechanism for uptake and release of molecules and suggest a potential role for plasma membrane disruption in the mechanotransduction of orthodontic tooth movement.