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.

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.

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.

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.

Tbx2 represses expression of Connexin43 in osteoblastic-like cells.

Tbx2 belongs to a family of developmental transcription regulatory factors. We evaluated whether the gap junction protein Connexin43 (Cx43), an important regulator of osteoblast function and bone development, may be a downstream target gene regulated by Tbx2. The Cx43 promoter contains direct repeats of the consensus T-box binding motif, TCACAC, and moreover, Tbx2 and Cx43 show overlapping expression domains in precursors to bone and in osteoblasts. In vitro analysis showed that the Cx43 promoter contains two Tbx2 binding sites, and this binding was dependent on the TCACAC consensus sequence. Transient transfection analysis with a Cx43 promoter-driven lacZ reporter construct revealed negative regulation mediated by these two Tbx2 binding sites in osteoblast-like cells. Thus, downregulation of Tbx2 led to de-repression of wild-type Cx43 promoter activity, whereas a promoter construct with mutated binding sites showed no de-repression. In stably transfected osteosarcoma cells in which expression of the endogenous Tbx2 gene was downregulated with a Tbx2 antisense construct, a marked de-repression of the endogenous Cx43 gene was observed. This was accompanied by a marked increase in the abundance of Cx43 gap junctions and increased functional gap junction-mediated cell-cell communication. Analysis of lacZ expression in transgenic mice containing the mutated Cx43 promoter-driven lacZ construct further suggested de-repression of the Cx43 promoter in limb buds, a region destined to give rise to long bones of the limbs. Taken together, these findings indicate that the promoter of Cx43 is repressible by Tbx2, both in cultured osteoblast-like cells in vitro and likely in the developing embryo.

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.

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.

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.

Extrinsic tension results in FGF-2 release, membrane permeability change, and intracellular Ca++ increase in immature cranial sutures.

There are numerous studies cataloging the temporal profiles of the various growth factors during the morphogenesis of cranial sutures. There are also many clearly documented mutations of the receptors of some of these growth factors such as fibroblast growth factor (FGF)R-2 and FGFR-3 in clinical craniosynostosis. It is obvious, and often concluded, that growth factors play a role or are involved in craniofacial development. However, precisely what that role is, what causes the changes in the growth factor levels, and why these changes occur in the particular temporal and spatial patterns observed remains elusive. Using simple physics, we applied a plasma membrane disruption model and the principles of complex adaptive systems to arrive at a conjecture of calvarial morphogenesis. The purpose of this article is to introduce the concept of complex adaptive systems, to propose our conjecture, and to provide experimental proof of some key steps in this conjecture: tension induces rapid and demonstrable physiological responses in some cells within the immature cranial sutures. These responses include increases of intracellular Ca++, plasma membrane permeability, and the release of growth factors, e.g., FGF-2. Paired coronal sutures from 1-week-old Sprague-Dawley rat pups were subjected to either 0.59 N of tensile force or no force for 5 minutes in a protein-free medium. FGF-2 levels in the media were measured by slot blot analysis. Western blot analysis was used to determine FGF-2 levels in the sutures. To determine cell membrane permeability changes, fluorescein-conjugated dextran, with a molecular weight of 10 kd, was added to the media during the 5 minutes with or without tensile force. Laser confocal microscopy was used to compare the amount of entry of this impermeant tracer and the pattern of permeability change at the tissue level. To determine the intracellular pCa++, the sutures were first loaded with a calcium indictor, FURA-2 AM, and then subjected isotonically to 0.059 N of tension. The intracellular pCa++ was expressed as ratio of Ca++-bound FURA-2 to Ca++-free FURA-2. The experimental findings were as follows: 1) Sutures, in response to tension, release FGF-2. 2) Sutures contain higher levels of FGF-2 when strained. 3) There is an increase in the sutural cell membrane permeability as a result of tensile strain. 4) The cells along the leading edges of the ossification fronts (at the insertion sites of Sharpey's fibers) demonstrated the maximum permeability increase. 5) There was an immediate (within seconds) increase in intracellular Ca++. and 6) This increase in intracellular Ca++ caused by tension was reversible and independent of the extracellular Ca++ ion availability. In summary, these data support, in part, the conjecture that growth of the brain places strain on the cells within the immature sutures, which causes the iteration of a set of cellular subroutines. These subroutines integrate to generate the emergent property of directed cranial expansion with dissipation of the initiating strains.

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.

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.

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.

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.

Chronic fluoride ingestion decreases 45Ca uptake by rat kidney membranes.

High exposures to fluoride (F-) may occur in environments rich in F- from natural or industrial sources and from misuse of F--containing dental care products, particularly by children. Both acute and chronic exposures to elevated levels of F- have negative effects on several calcium-dependent processes, including kidney glomerular and tubular function. We examined the effect of chronic F- ingestion on ATP-dependent 45Ca uptake by rat kidney membrane vesicles to characterize the mechanism by which high F- alters Ca++ transport in the kidney. Twenty weanling female Sprague-Dawley rats were raised on low-F- (0.9 mg/L), semi-purified diet with a Ca++ concentration of 400 mg/100g diet. Rats were divided into four groups and were fed ad libitum deionized water containing F- at 0, 10, 50, or 150 mg/L added as NaF for 6 wk. This consumption produced plasma F- levels of <0.4, 2, 7, or 35 micromol/L, respectively. ATP-dependent 45Ca uptake was significantly lower in the 150 mg F-/L exposure group than in the 0 mg F-/L controls (P < 0.05). Studies with thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca++-pump, showed that the lower uptake was associated with significantly lower activities of both the plasma membrane Ca++-pump (P < 0.05, 150 mg F-/L group versus control) and endoplasmic reticulum Ca++-pump (P < 0.05 for both the 50 and 150 mg F-/L groups versus control). Slot blot analysis of kidney homogenates with specific Ca++-pump antibodies showed less (P < 0.05) endoplasmic reticulum Ca++-pump protein and plasma membrane Ca++-pump protein in all treatment groups than controls. Both Ca++-pumps are transport molecules of great importance in the regulation of Ca++ homeostasis. Our study suggests that chronic, high F- ingestion producing high plasma F- levels may occur in humans and may affect Ca++ homeostasis by increasing the turnover or breakdown or decreasing the expression of plasma membrane and endoplasmic reticulum Ca++-pump proteins.

Effects of chronic adult periodontitis and endotoxin (LPS) on gingival fibroblast plasma membrane Ca++-pump.

Gingival fibroblasts from patients with chronic adult periodontitis are known to produce cytokines in response to changing levels of bacterial lipopolysaccharide (LPS). Cytokine production is one of numerous cell processes that involve calcium dependent enzymes. It is possible that inflammation may induce changes in the amount of the Ca++-pump protein in gingival fibroblasts which could alter Ca++-dependent activities in these cells including the production and release of cytokines. The purpose of this study was to determine if differences exist in the amount of Ca++-pump protein in the gingival fibroblasts of periodontitis patients relative to control individuals without periodontal disease. Fibroblast explants from healthy tissue and from inflamed tissue from patients with chronic adult periodontitis, grown in culture, were analyzed for quantitative differences in the amount of Ca++-pump protein. Fibroblasts from chronic adult periodontitis patients exhibited significantly lower levels of Ca++-pump protein than fibroblasts from healthy subjects (p=0.0015). However, fibroblasts from chronic adult periodontitis patients, when activated with LPS, did not exhibit significant differences in the amounts of Ca++-pump protein as compared to untreated controls (p = 0.2177). Similarly, cells from healthy subjects did not show significant reduction in Ca++-pump protein following activation with LPS (p = 0.1732). Our results suggest that plasma membrane Ca++-pump is significantly reduced in fibroblasts derived from patients with chronic periodontitis. However, factors other than LPS may be involved in the down-regulation of Ca++-pump protein.

An isolation and in vitro culturing method for human intraoral bone cells derived from dental implant preparation sites.

In dental implantology, the biocompatibility of the osseous tissue to the implant surface and to local environmental factors plays an important role in the process of healing. Bone cells derived from intraoral osseous tissue proves to be an important source of the osteoprogenitor cells required for healing of the periodontium around implants. Historically, the rat calvaria model has been employed to study the effects of various dental treatments on bone in vitro. However, there are morphological and functional differences which exist between bone cells derived from rat calvaria and human intraoral osseous tissue that impose certain limitations on the usefulness of the rat calvaria model for dental implant applications. Therefore, an in vitro culturing method for the isolation, growth and maintenance of human intraoral bone cell cultures derived from osseous tissues is truly warranted. In addition, a method for the accurate characterization of these bone cells as osteoblasts is also vital. The specific objective of this study was to establish isolation and in vitro culturing methods utilizing human intraoral bone cells derived from dental implant preparation sites. This paper describes techniques for the harvesting of human bone cells from the intraoral derived osseous tissues and discuss the procedures for maintaining the primary intraoral bone cell culture. In addition, our studies utilize established protocols for the characterization of these cells as osteoblasts by means of alkaline phosphatase activity determination, identification of cellular osteonectin and osteocalcin antigens, establishing the presence of cells expressing type I collagen and determining the ability of cells to produce calcifications. The utilization of intraoral osseous tissue may prove useful for future dental implant research by providing an in vitro model system more closely related to conditions encountered clinically.

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.

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%.