Epidermal growth factor suppresses insulin-like growth factor binding protein 3 levels in human papillomavirus type 16-immortalized cervical epithelial cells and thereby potentiates the effects of insulin-like growth factor 1.

Human ectocervical epithelial cells are a primary target for infection by oncogenic papillomaviruses, which are strongly implicated as causative agents in the genesis of cervical cancer. Growth factors have been implicated as agents that stimulate proliferation and enhance the possibility of malignant transformation. In the present study we utilize several human papillomavirus (HPV) type 16-immortalized ectocervical epithelial cell lines to investigate the effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on cell proliferation and the production of IGF binding proteins (IGFBPs). ECE16-1 cells, an HPV16-immortalized/nontumorigenic cell line, maintained in defined medium, produce and release high levels of IGFBP-3 (38/42 kDa) as well as smaller amounts of a 24-kDa IGFBP. Supplementation of defined medium with EGF causes a dose-dependent increase in cell growth and a concomitant decrease in the levels of IGFBP-3 released into the culture medium. EGF suppression of IGFBP-3 is maintained even when EGF-stimulated cell growth is suppressed 67% due to the simultaneous presence of 3 ng/ml of TGF beta 1, indicating that EGF suppression of IGFBP-3 levels is independent of EGF effects on cell growth. EGF suppression of IGFBP-3 production is correlated with a reduction in IGFBP-3 mRNA level. In the presence of EGF, the growth response of the cells to ng amounts of IGF-I is significantly enhanced. Moreover, the simultaneous presence of both EGF and IGF-I reduces the level of IGFBP-3 more efficiently than EGF alone. We also observe that the IGFBP-3 level is decreased and the 24-kDa IGFBP level is increased in HPV16-positive tumorigenic versus nontumorigenic cell lines. This is the first report of EGF acting as a positive regulator of IGF-I action via the IGFBPs. On the basis of these findings, we propose that EGF stimulates ECE16-1 cell growth via a dual-action mechanism by (a) stimulating growth directly via the EGF mitogenic pathway and (b) stimulating growth indirectly by reducing the levels of inhibitory IGFBPs and thereby potentiating the effects of IGF-I. In addition, the observation that more highly transformed cell types produce lower levels of IGFBP-3 and higher levels of 24-kDa IGFBP suggests that tumor cells in more advanced cervical cancers may have an altered response to IGF-I.

Interferon and retinoic acid suppress the growth of human papillomavirus type 16 immortalized cervical epithelial cells, but only interferon suppresses the level of the human papillomavirus transforming oncogenes.

In the present study, we examine the effects of all-trans-retinoic acid (RA) and interferons-alpha and -gamma (IFN-alpha and IFN-gamma) on the growth of HPV16-immortalized cell lines, ECE16-1 and CaSki. Treating proliferating ECE16-1 cells with RA causes a concentration-dependent decrease in cell number. At 1 microM RA, cell growth is suppressed by 65% and the level of mRNA encoding cytokeratin K5, a biochemical marker of retinoid action, is also suppressed. In contrast, the level of transcript encoding the HPV16 oncogenes, E6 and E7, is reduced by only 5 to 10%. IFN-alpha at 1000 IU/ml or IFN-gamma at 200 IU/ml suppresses growth by 70%. This growth suppression by IFN-gamma is correlated with a > 90% reduction in E6/E7 mRNA levels. Additional growth suppression is observed upon simultaneous treatment with retinoid and interferon. Optimal suppression is observed in the presence of 200 IU/ml IFN-gamma and 1 microM RA. The rank order of effectiveness is IFN-gamma/RA > IFN-alpha/RA = IFN-gamma > RA > IFN-alpha. In contrast to the suppression of ECE16-1 cell growth, RA causes a concentration-dependent increase in CaSki cell number (50-60%) which is optimal at 1 microM RA. Cytokeratin K5 mRNA levels are markedly suppressed, and E6/E7 mRNA levels increased by 5% under these conditions. IFN-alpha at 1000 IU/ml or IFN-gamma at 200 IU/ml decreases CaSki cell growth by 20 and 45%, respectively, and 200 IU/ml of IFN-gamma reduce E6/E7 expression to undetectable levels. Addition of RA (1 microM) partially counters the IFN-dependent suppression of growth and E6/E7 mRNA levels. Our results suggest that retinoid-dependent changes in human papillomavirus-immortalized cervical cell proliferation are not always correlated with changes in E6/E7 transcript levels.

Transforming growth factor beta 1 regulation of metalloproteinase production in cultured human cervical epithelial cells.

Collagenase levels are regulated in a cell type-specific manner by a variety of growth factors and cytokines, and increased type IV collagenase activity in tumor cells has been linked to metastatic growth. In this study we compare the effects of epidermal growth factor (EGF) and transforming growth factor beta 1 (TGF beta 1) on gelatinase production in cervical epithelial cell lines. EGF is a strong mitogen for cervical epithelial cells and TGF beta 1 suppresses growth. Metalloproteinase zymograms of conditioned medium from normal human ectocervical cells reveal two major bands of metalloproteinase activity at 72 and 92 Kd. In contrast, the level of the 92-Kd activity is greatly reduced in the human papillomavirus type 16-positive ECE16-1 and CaSki cells. EGF treatment produces minimal changes in metalloproteinase levels. Treatment of CaSki cells with 20 ng/ml of EGF reduces by 30 to 50% the level of both activities. In ECE16-1 cells, EGF decreases the 72-Kd activity by 50% and the 92-Kd activity slightly. TGF beta 1 treatment, in contrast, increases the 72-Kd activity 3- to 10-fold and the 92-Kd activity by > or = 25-fold in each cell type. In CaSki and ECE16-1 cells, the changes in metalloproteinase level are mediated by changes in level of the corresponding mRNAs. In each case, the metalloproteinases are secreted as inactive proenzymes which can be activated by in vitro treatment with organomercurials. Tests of a series of additional cervical cell lines reveal that metalloproteinase levels are generally higher in normal cervical cells and in cells immortalized by transfection with HPV16, whereas lower levels are observed in cells derived from human tumors. Moreover, a higher percentage of cell lines derived from human tumors do not respond to TGF beta 1 regulation of metalloproteinase levels. Parallel studies indicate that the TGF beta 1-stimulated increase in the 72- and 92-Kd activities is correlated with enhanced chemotactic and chemoinvasive behavior in both ECE16-1 and CaSki cells.

Retinoid X receptor-specific retinoids inhibit the ability of retinoic acid receptor-specific retinoids to increase the level of insulin-like growth factor binding protein-3 in human ectocervical epithelial cells.

The hormones derived from vitamin A and related synthetic ligands (retinoids) are important regulators of differentiation and development and have been shown to be therapeutically useful in the treatment of cervical cancer. All-trans-retinoic acid exerts its effects by activation of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers. These heterodimers bind to the retinoic acid response elements of target genes to regulate gene expression. RXR ligands act through RXR homodimers to regulate gene expression. In the present study, we describe the effects of RAR- and RXR-specific ligands on the regulation of insulin-like growth factor binding protein-3 (IGFBP-3) production and cell proliferation in human ectocervical epithelial (ECE) cell lines. Treatment of ECE16-1 cells with a RAR-specific ligand (TTNPB) or a ligand that interacts with both RAR and RXR receptors (9-cis-retinoic acid) increases IGFBP-3 levels and suppresses cell proliferation. In contrast, RXR-specific ligands (AGN191701, SR11217, and SR11237) do not regulate proliferation and slightly suppress the IGFBP-3 level. Cotreatment with increasing concentrations (0.01-1000nm) of RXR-specific ligand antagonizes the growth suppressive and IGFB-3-increasing effects of 1000 nM TTNPB. Similar results are observed in two other ECE cell lines, ECE16-D1 and ECE16-D2. These results indicate that RXR-specific ligands can antagonize RAR responses in these cell lines and suggest that a RAR-specific retinoid may be superior to one with mixed RAR/RXR binding activity for inhibiting cervical cancer cell proliferation. Moreover, the antagonism of RAR-dependent responses by RXR-specific ligands is consistent with a squelching model in which the RXR-specific ligand drives formation of RXR/RXR homodimers at the expense of the more active RAR/RXR heterodimers.

Insulin-like growth factor binding protein-3 does not mediate the interferon-dependent suppression of human ectocervical epithelial cell proliferation.

Interferon is a potential therapeutic agent for the treatment of cervical cancer. In the present study we examine the role of IFNgamma as a regulator of proliferation and production of IGFBP-3 expression in ectocervical epithelial cells. ECE16-1 cells are a model for studying early human papillomavirus-dependent cervical disease. IFNgamma produces a concentration-dependent inhibition of ECE16-1 cell proliferation that is associated with an increase in insulin-like growth factor binding protein-3 level. Growth suppression and IGFBP-3 increase is maximal at concentrations of IFNgamma >/=0.75 ng/ml. The increased IGFBP-3 expression is mediated via an increase in IGFBP-3 encoding mRNA. In contrast, IFNgamma inhibits proliferation of CaSki and SiHa cells, but IGFBP-3 is barely detectable and levels are not regulated by IFNgamma. These results suggest that the IFNgamma-dependent suppression of CaSki and SiHa cell proliferation is not mediated by secreted IGFBP-3. This result was confirmed when vector-mediated overexpression of immunoreactive IGFBP-3 in SiHa and CaSki cells did not consistently result in reduced cell proliferation rate.

Influence of amalgam, alloy, and mercury on the in vitro growth of Streptococcus mutans: I. Biological test system.

A procedure is presented for the in vitro growth of Streptococcus mutans in a dextrose-beef extract medium. Growth was estimated spectrophotometrically. The amount of amalgam, alloy, or mercury that was added to the sealed test tube influenced the rate and extent of growth.

Influence of amalgam, alloy, and Hg on the in vitro growth of Streptococcus mutans: III. Effect of specimen age and composition.

We will now summarize the conclusions from parts I, II and III of this study. A test procedure has been developed that provides a simple, quick, and nondestructive means of monitoring the in vitro growth of S mutans in the presence of amalgams and alloys. The spectrophotometric readings are related in a simple way to growth expressed as dry weight of bacteria and metabolic products. Results are expressed as growth relative to controls which represent bacteria growing under identical conditions but not in contact with metals. The %RA60 value that represents growth after 60 hours relative to controls is used as a measure of growth in the presence of alloys or amalgams. Spherical, fine cut, and dispersion alloys were studied as well as amalgams prepared from these alloys. The dispersion alloy inhibits growth less than the spherical alloy which in turn inhibits growth less than the fine cut alloy. The results for amalgams prepared from the alloys are reversed. At an aging time of two hours, dispersion alloy amalgams inhibit growth more than spherical alloy amalgams and fine cut alloy amalgams. Aging time of amalgams greatly influences the growth inhibition. Immediately after trituration growth is inhibited, but this inhibition is lost with aging. Different types of amalgams seem to lose growth inhibition at different rates. Hg composition in the range of 48 to 52% seems to have little effect on growth inhibition. It remains for clinical studies, which are concerned with the incidence of secondary caries associated with amalgams, to demonstrate that the age and nature of the amalgam are significant.

Influence of amalgam, alloy, and mercury on the in vitro growth of Streptococcus mutans: II. Comparison of amalgams and alloys.

The influence of three alloys and their amalgams on the in vitro growth of Streptococcus mutans has been estimated. Spherical, fine cut, and dispersion alloys were studied. Dispersion alloy amalgams seem to inhibit bacterial growth more than amalgams prepared from the other alloys in a similar fashion.

Human cervical cancer. Retinoids, interferon and human papillomavirus.

Our studies highlight the importance of dietary vitamin A (retinol) and other retinoids in maintaining normal cervical cell function and in inhibiting the growth of cervical tumors. Based on our results we conclude that 1) HPV 16-immortalization enhances cervical cell sensitivity to retinoids, 2) cytokeratin expression may be useful as a marker for evaluating the success of retinoid therapy in vivo, 3) retinoids do not necessarily act to inhibit proliferation of HPV-immortalized cervical cells via effects on HPV E6 and E7 RNA levels and 4) retinoids may act to inhibit cervical proliferation by "suppressing" the activity of the EGF and IGF signalling pathways. Based on these and other results, it is worth considering the possibility that vitamin A or related retinoids could be administered therapeutically, early in the neoplastic process (either systemically or locally), to inhibit the progress of the disease. These results also suggest that combined interferon/retinoid therapy may provide an enhanced beneficial effect to reduce cervical tumor size due to the fact that each agent is inhibiting cervical cell proliferation via distinct, but reinforcing, pathways (i.e., IFN gamma reduces E6/E7 expression, RA inhibits the function of the EGF and IGF1 signalling pathways).

Isolation and culture of fetal porcine myogenic cells and the effect of insulin, IGF-I, and sera on protein turnover in porcine myotube cultures.

Porcine myogenic cells isolated from 50 to 55-d porcine fetuses were frozen and stored in liquid nitrogen until they were needed to establish cultures. Approximately 75.8 +/- .59% of the clonal cultures established from these frozen stocks produced myotubes and 60.8 +/- 2.3% of the nuclei in differentiated mass cultures were in myotubes. Differentiated cultures contained higher levels of creatine phosphokinase activity than undifferentiated cultures. Additionally, differentiated cultures incorporated [35S]methionine into putative myosin heavy chain, alpha-actinin, and actin more rapidly than did undifferentiated cultures. Insulin, insulin-like growth factor I, and sera stimulated total protein synthesis rate and decreased total protein degradation rate in myotube cultures. Based on our initial characterization, we believe that we have developed an effective and practical procedure for isolating and culturing fetal porcine myogenic cells.

Transforming growth factor beta-1 facilitates establishing clonal populations of ovine muscle satellite cells.

Myogenic cells isolated from lamb fetuses (approximately mid-gestation) exhibited a concentration-dependent decrease in myogenic cell proliferation in response to transforming growth factor (TGF) beta-1 (P < .001). Half-maximal inhibition of proliferation occurred at approximately .05 ng of TGF beta-1/mL and maximal inhibition of proliferation occurred at approximately .1 ng of TGF beta-1/mL. The specificity of this inhibition was confirmed by neutralization of the activity following exposure to a TGF beta antibody. The TGF beta-1 also suppressed proliferation of ovine satellite cells isolated from 5-d-old lambs (P < .0035), but to a lesser extent than observed for embryonic cells. In contrast, TGF beta-1 did not significantly suppress serum-stimulated proliferation of ovine satellite cells isolated from 30- or 150-d-old lambs. Similarly, TGF beta-1 did not suppress proliferation of skeletal muscle fibroblast-like cells isolated from either fetal lambs or 150-d-old lambs. In fact, proliferation of fibroblast-like cells derived from embryonic ovine muscle was enhanced by exposure to TGF beta-1 at all levels tested; however, a concentration-dependent response was not observed. Media transfer experiments showed that conditioning of culture media by postnatally derived cells did not render TGF beta-1 inactive. The studies described in this manuscript suggest that sensitivity of ovine myogenic cells to the antiproliferative effect of TGF-beta may vary with the stage of development.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured porcine myogenic cells produce insulin-like growth factor binding protein-3 (IGFBP-3) and transforming growth factor beta-1 stimulates IGFBP-3 production.

Insulin-like growth factor binding proteins (IGFBP) may act locally as autocrine or paracrine regulators of insulin-like growth factor activity in specific tissues such as muscle. Although secretion of IGFBP by cultured myogenic cell lines has been examined, little is known about secretion of IGFBP by primary myogenic cell cultures. This may be because primary myogenic cultures contain non-muscle cells (fibroblasts) that complicate interpretation of IGFBP determinations. We have circumvented this problem by subculturing nonfusing cells from extensively fused porcine myogenic cultures and comparing the IGFBP production of these nonfusing, porcine muscle-derived cells with that of primary porcine myogenic cell cultures. Immunoprecipitation with specific antibodies and 125I-IGF-I ligand blot analysis showed that myogenic cultures secreted IGFBP-3 (doublet band, 43 kDa and 39 kDa), IGFBP-2 (34 kDa), IGFBP-4 (30 and 24 kDa), and IGFBP-5 (30 and 28 kDa). Muscle-derived fibroblasts secreted no detectable IGFBP-3 but approximately 10 times more IGFBP-2 than did myogenic cell cultures. Treatment of myogenic cultures for 24 h with transforming growth factor (TGF) beta-1 caused a concentration-dependent increase in IGFBP-3 secretion with a maximum 1.5-fold increase occurring at .5 ng of TGF beta-1/mL. In contrast, TGF beta-1 treatment did not stimulate detectable IGFBP-3 secretion by muscle-derived fibroblast cultures. Northern analysis of total RNA using a porcine IGFBP-3 probe revealed that TGF beta-1 treatment resulted in a fourfold increase in the steady-state level of IGFBP-3 mRNA in myogenic cultures. Insulin-like growth factor binding protein-3 mRNA was not detectable in fibroblast cultures either before or after TGF beta-1 treatment. This is the first report of IGFBP-3 secretion by cultured myogenic cells.

Evaluation of the BAX system for detection of Listeria monocytogenes in foods: collaborative study.

A multilaboratory study was conducted to compare the automated BAX system and the standard cultural methods for detection of Listeria monocytogenes in foods. Six food types (frankfurters, soft cheese, smoked salmon, raw, ground beef, fresh radishes, and frozen peas) were analyzed by each method. For each food type, 3 inoculation levels were tested: high (average of 2 CFU/g), low (average of 0.2 CFU/g) and uninoculated controls. A total of 25 laboratories representing government and industry participated. Of the 2335 samples analyzed, 1109 were positive by the BAX system and 1115 were positive by the standard method. A Chi square analysis of each of the 6 food types, at the 3 inoculation levels tested, was performed. For all foods, except radishes, the BAX system performed as well as or better than the standard reference methods based on the Chi square results.

Microleakage of several class V anterior restorative materials: a laboratory study.

The study was designed to evaluate the marginal leakage of abraded gingival areas in extracted teeth using five anterior composite resin acid-etch restorative materials and a glass ionomer cement, ASPA. In using three of the composite resin restorative materials, Simulate, Cervident, and Concise, there was a layer of unfilled resin between the etched tooth surface and the composite resin. Restodent and Enamelite were placed directly on the etched tooth surface. The results of the study indicate that there is a significantly greater degree of marginal leakage at the gingival margin than there is at the occlusal or incisal margin of composite restorations. In addition, greater marginal leakage was observed in those restorations where no layer of unfilled resin was placed between the etched tooth surface and the composite resin. The glass ionomer cement showed no marginal leakage at intervals of one day, three months, and six months; however, a small amount of leakage was observed at the incisal or occlusal and gingival margins at a year on half of the autoradiographs. A study has been initiated to determine leakage patterns around composite resin restorations placed in teeth with naturally occurring cervical erosion or abrasion.

The effect of incremental versus bulk fill techniques on the microleakage of composite resin using a glass-ionomer liner.

Incremental placement of composite resin has been suggested to reduce microleakage, particularly at the gingival margin of class 5 cervical restorations. It has become clinically advantageous to place a glass-ionomer liner over dentin to further minimize microleakage resulting from a bond between the dentin and glass ionomer, and glass ionomer and resin. The objective of this study was to compare the microleakage behavior of three hybrid composite/bonding agent systems using bulk and incremental filling techniques utilizing a glass-ionomer liner. This was accomplished in vitro using freshly extracted bovine incisors and a Ca45 radioisotope and autoradiography. Sixty bovine incisors were divided into six experimental groups of 10 specimens per group. Class 5 preparations were cut at the cementoenamel junction and restored with the appropriate combination of Herculite XR/Bondlite, P50/Scotchbond 2, or Pertac Hybrid/Pertac Bond. All teeth were lined with the glass ionomer Ketac Bond before the final restoration was placed. The samples were finished and stored for 24 hours in distilled water before thermocycling. The samples were tested for microleakage using a Ca45 radioisotope technique and autoradiography. Incisal (enamel) and gingival (dentin) margins were scored separately for microleakage but grouped for statistical analysis. Results were analyzed using the Kruskal-Wallis H test. Pertac Hybrid exhibited more leakage than Herculite XR or P50. The difference between microleakage of bulk and incremental filling techniques was only significant for P50.

Microleakage of new dentin bonding systems using human and bovine teeth.

The objective of this study was twofold: to evaluate the microleakage behavior of three dentin bonding systems and to determine if bovine teeth are comparable substrates to human teeth when studying the microleakage of various materials. The materials evaluated were Scotchbond Multi-Purpose Adhesive, Prisma Universal Bond 3, and All-Bond 2. All three bonding systems were used in combination with Prisma APH hybrid composite for comparison of microleakage behavior. Sixty class 5 preparations were cut at the cementoenamel junction for groups containing 30 human and 30 bovine teeth. A 1 mm 45 degree bevel was placed at the enamel margin. Teeth were grouped according to the dentin bonding system used and then restored according to the manufacturer's directions. After restoration, the teeth from each group were stored in distilled water at 37 degrees C for 3 days. The teeth were then thermocycled between 4 degrees C and 58 degrees C for 100 cycles and returned to distilled water at 37 degrees C for an additional 4 days. The teeth were then sealed with nail polish up to 1 mm from the margins of the restoration and placed in 45Ca isotope for 2 hours. The teeth were then sectioned and placed on x-ray film to produce autoradiographs. Microleakage was evaluated for the enamel and dentin margins separately using the following scale: 0 = no leakage, 1 = penetration of isotope to less than 1/2 the distance to the axial wall, 2 = penetration of isotope greater than 1/2 of the distance to the axial wall but short of the axial wall, and 3 = penetration of isotope to the axial wall or beyond. The materials were compared to each other using the Mann-Whitney U and Kruskal-Wallis tests. The gingival margins were compared to the incisal margins for all materials. No statistically significant differences in microleakage were revealed between the incisal and gingival location for human substrates, but there was statistically significant greater gingival microleakage for bovine substrates. All-Bond 2 leaked significantly more than Scotchbond Multi-Purpose for human substrates at the incisal margin. All-Bond 2 had significantly more microleakage than Prisma Universal Bond 3 at both dentin and enamel margins for the bovine substrate. There were no statistically significant differences in microleakage among the bonding systems for the human substrate. No statistically significant differences between the microleakage behavior of human and bovine substrates were found. These results support the use of bovine teeth for in vitro microleakage studies.

Microleakage of a new cavity varnish with a high-copper spherical amalgam alloy.

In a study in vitro Copalite and Barrier cavity varnishes were applied to the walls of class 1 prepared cavities before placing Tytin, a high-copper amalgam. Tests with 45Ca and autoradiographs showed that at six months only Copalite was effective in preventing microleakage. At one year neither Barrier nor Copalite was preventing leakage.

Microleakage of Gluma Bond, Scotchbond 2 and a glass ionomer/composite sandwich technique.

Bonding to dentin with long-term success has been a quest of dentistry for some time. Microleakage is a major problem that can lead to staining and discolorization of the restoration and postoperative sensitivity. Some of the newer dentin bonding systems include Gluma Bond and Scotchbond 2. A glass ionomer/composite "sandwich" technique has also been advocated for dentin bonding. Here a glass ionomer is first bonded to the dentin, bonding agent is applied to the glass ionomer and a composite restoration is then placed into the cavity preparation. The purpose of this study was to compare the microleakage of Gluma Bond, Scotchbond 2, the glass ionomer/composite sandwich technique along with a positive and negative control. This was accomplished, in vitro, using extracted human teeth and a Ca45 radioisotope technique. The teeth were examined for microleakage at periods of 1 week, 6 months and 12 months. The results of this study indicate that, in vitro, the glass ionomer sandwich technique does not stop microleakage. The results also indicate that, in vitro, Gluma Bond and especially Scotchbond 2 restorations can resist microleakage for short periods of time. However, at the end of the 12 months, all Gluma Bond and Scotchbond 2 specimens exhibited gross microleakage.

Comparisons of fit of CAD-CAM restorations using three imaging surfaces.

One of the factors that influences the overall fit of a Cerec restoration is the thickness of the optical powder. The objective of this study was to compare the measurement of the fit of glass inlays produced with the Cerec instrument after each preparation had been coated, in separate trials, with imaging powder applied with an aerosol; imaging powder applied with air from a dental unit; and a water-soluble paint applied with a brush. Ten inlay preparations were made in extracted teeth. Each preparation was coated with one of the three imaging media and an inlay was made. This was repeated until each preparation received each imaging surface. Each inlay was measured at eight different points by using an image analysis system interfaced to a stereoscopic measuring microscope. The inlays fabricated on the two powder surfaces were not significantly different, but the painted surfaces were found to result in a significantly better-fitting inlay.

Microleakage and thermal properties of hybrid ionomer restoratives.

The objective of this study was to compare the microleakage and thermal properties of two recently introduced hybrid materials to those of a conventional glass-ionomer cement. Class V preparations were prepared at the cementoenamel junctions in freshly extracted bovine incisor and evaluated for microleakage with a 45Ca radioisotope method. Thermal properties were evaluated with thermal mechanical analysis and differential scanning calorimetry. The degree of microleakage and the coefficient of thermal expansion of the conventional glass-ionomer cement were found to be significantly less than those of either hybrid.