Stathmin levels in growth plate chondrocytes are modulated by vitamin D3 metabolites and transforming growth factor-beta1 and are associated with proliferation.

Stathmin is a highly conserved, phosphorylated cytosolic protein that is found at decreased levels in all cells as they become more terminally differentiated, or when they decrease in their rate of proliferation. This study examined the hypothesis that stathmin levels in growth plate chondrocytes decreases as endochondral maturation increases. To test this hypothesis, we used a costochondral growth plate chondrocyte cell culture model. Cells derived from the resting zone (RC) express twice as much stathmin mRNA in culture and have twice as much stathmin protein as cells derived from the post proliferative growth zone ([GC]; prehypertrophic and upper hypertrophic cell zones). Stathmin levels in vivo were assessed by immunohistochemistry. To assess the effects of agents that modulate proliferation and differentiation, RC and GC chondrocytes were cultured in the presence of 10(-10) to 10(-8) M 1alpha,25-(OH)2D3, which regulates proliferation in both cell types but affects differentiation of only GC cells, or 10(-9) to 10(-7) M 24R,25-(OH)2D3, which regulates differentiation and maturation of RC cells but decreases proliferation of GC cells. In addition, RC cells were treated with 0.44 or 0.88 ng/mL of recombinant human transforming growth factor beta1 (rhTGF-beta1), which stimulates proliferation of RC cells and regulates proteoglycan production, but not alkaline phosphatase activity. Stathmin protein levels were determined using quantitative immunoblots, with recombinant human stathmin as a standard. The results show that stathmin levels are associated with proliferation. Proliferating chondrocytes in vivo exhibited higher levels of immunoreactive stathmin than either RC or GC cells in the growth plate. In culture, 1alpha,25-(OH)2D3 caused a dose-dependent decrease in stathmin in RC and GC cells within 24 h. 24R, 25-(OH)2D3 also reduced stathmin levels in GC cells within 24 h but only affected RC cells after prolonged exposures (96 h), at which time RC cells express a GC-like phenotype. rhTGF-beta1 caused an increase in stathmin levels in RC cells. Stathmin levels are sensitive to protein kinase C (PKC) in other cells. Inhibition of PKC with chelerythrine had no effect on the response of RC cells to 1alpha,25-(OH)2D3 but it blocked the effect of rhTGF-beta1, indicating that decreases in stathmin by vitamin D3 metabolites may not be modulated by PKC, whereas increases in stathmin via rhTGF-beta1 may be regulated via a PKC-dependent mechanism. These results support the hypothesis that constitutively expressed levels of stathmin are related to cell maturation state and that they are modulated by factors that regulate proliferation.

Expression and production of stathmin in growth plate chondrocytes is cell-maturation dependent.

Growth plate cartilage is comprised of linear columns of chondrocytes with the least differentiated cells at one end and the terminally differentiated cells at the other end. Rat costochondral chondrocytes can be divided into the resting cell zone (reserve cell zone), which contains relatively immature chondrocytes (RC cells), and the phenotypically more mature prehypertrophic and upper hypertrophic cell zones, which together may be termed the growth zone chondrocytes (GC cells). When grown separately in monolayer culture, they continue to express their zone-specific phenotype, providing a model for assessing cell-maturation-dependent expression of molecules associated with differentiation. Stathmin (also called prosolin, Op18, p19, 19K, and others) is a highly conserved, phosphorylated cytosolic protein with apparent ubiquitous expression. Although its exact function is unknown, stathmin is considered to be a messenger phosphorylated protein, it plays a role in tubulin stability, and it may participate in both general and specific regulatory pathways. One uniform observation is that the expression of stathmin protein decreases in all cells as they become more terminally differentiated in culture. There have been no published data regarding stathmin expression and production in chondrocytes. This study was based on the hypothesis that stathmin exists in chondrocytes and that the mRNA and protein levels decline in the GC cell with respect to the RC cell. Stathmin mRNA levels were determined and quantitated by reverse transcription-polymerase chain reaction (RT-PCR) and northern blots. Protein levels were determined using immunoblots. It was found that stathmin exists in chondrocytes and that RC cells express approximately twice the level of mRNA and protein to that found in GC cells. The results support the hypothesis and suggest that the level of stathmin expression and production in culture is related to the level of differentiation of RC and GC cells in vivo.

Blockout technique for impressions of teeth with increased open gingival embrasures.

A dental technique is described that blocks out enlarged gingival embrasures to eliminate distortion of an impression and the resultant cast for removable partial or fixed partial dentures. This procedure consists of injecting polyvinyl siloxane impression material in embrasures to form custom blockout wedges. This technique provides a simple method for clean, customized blockout of potentially damaging undercuts that can distort impressions and casts. The ultimate accuracy of the cast results in a precise definitive prosthesis.

Surface roughness modulates the local production of growth factors and cytokines by osteoblast-like MG-63 cells.

Titanium (Ti) surface roughness affects proliferation, differentiation, and matrix production of MG-63 osteoblast-like cells. Cytokines and growth factors produced in the milieu surrounding an implant may also be influenced by its surface, thereby modulating the healing process. This study examined the effect of surface roughness on the production of two factors known to have potent effects on bone, prostaglandin E2 (PGE2) and transforming growth factor beta 1 (TGF-beta 1). MG-63 cells were cultured on Ti disks of varying roughness. The surfaces were ranked from smoothest to roughest: electropolished (EP), pretreated with hydrofluoric acid-nitric acid (PT), fine sand-blasted, etched with HCl and H2SO4, and washed (EA), coarse sand-blasted, etched with HCl and H2SO4, and washed (CA), and Ti plasma-sprayed (TPS). Cells were cultured in 24-well polystyrene (plastic) dishes as controls and to determine when confluence was achieved. Media were collected and cell number determined 24 h postconfluence. PGE2 and TGF-beta 1 levels in the conditioned media were determined using commercial radioimmunoassay and enzyme-linked immunosorbent assay kits, respectively. There was an inverse relationship between cell number and Ti surface roughness. Total PGE2 content in the media of cultures grown on the three roughest surfaces (FA, CA, and TPS) was significantly increased 1.5-4.0 times over that found in media of cultures grown on plastic or smooth surfaces. When PGE2 production was expressed per cell number, CA and TPS cultures exhibited six- to eightfold increases compared to cultures on plastic and smooth surfaces. There was a direct relationship between TGF-beta 1 production and surface roughness, both in terms of total TGF-beta 1 per culture and when normalized for cell number. TGF-beta 1 production on rough surfaces (CA and TPS) was three to five times higher than on plastic. These studies indicate that substrate surface roughness affects cytokine and growth factor production by MG-63 cells, suggesting that surface roughness may modulate the activity of cells interacting with an implant, and thereby affect tissue healing and implant success.

Role of material surfaces in regulating bone and cartilage cell response.

Tissue engineering in vitro and in vivo involves the interaction of cells with a material surface. The nature of the surface can directly influence cellular response, ultimately affecting the rate and quality of new tissue formation. Initial events at the surface include the orientated adsorption of molecules from the surrounding fluid, creating a conditioned interface to which the cell responds. The gross morphology, as well as the microtopography and chemistry of the surface, determine which molecules can adsorb and how cells will attach and align themselves. The focal attachments made by the cells with their substrate determine cell shape which, when transduced via the cytoskeleton to the nucleus, result in expression of specific phenotypes. Osteoblasts and chondrocytes are sensitive to subtle differences in surface roughness and surface chemistry. Studies comparing chondrocyte response to TiO2 of differing crystallinities show that cells can discriminate between surfaces at this level as well. Cellular response also depends on the local environmental and state of maturation of the responding cells. Optimizing surface structure for site-specific tissue engineering is one option; modifying surfaces with biologicals is another.

Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63).

The effect of surface roughness on osteoblast proliferation, differentiation, and protein synthesis was examined. Human osteoblast-like cells (MG63) were cultured on titanium (Ti) disks that had been prepared by one of five different treatment regimens. All disks were pretreated with hydrofluroic acid-nitric acid and washed (PT). PT disks were also: washed, and then electropolished (EP); fine sandblasted, etched with HCl and H2SO4, and washed (FA); coarse sandblasted, etched with HCl and H2SO4, and washed (CA); or Ti plasma-sprayed (TPS). Standard tissue culture plastic was used as a control. Surface topography and profile were evaluated by brightfield and darkfield microscopy, cold field emission scanning electron microscopy, and laser confocal microscopy, while chemical composition was mapped using energy dispersion X-ray analysis and elemental distribution determined using Auger electron spectroscopy. The effect of surface roughness on the cells was evaluated by measuring cell number, [3H]thymidine incorporation into DNA, alkaline phosphatase specific activity, [3H]uridine incorporation into RNA, [3H]proline incorporation into collagenase digestible protein (CDP) and noncollagenase-digestible protein (NCP), and [35S]sulfate incorporation into proteoglycan. Based on surface analysis, the five different Ti surfaces were ranked in order of smoothest to roughest: EP, PT, FA, CA, and TPS. A TiO2 layer was found on all surfaces that ranged in thickness from 100 A in the smoothest group to 300 A in the roughest. When compared to confluent cultures of cells on plastic, the number of cells was reduced on the TPS surfaces and increased on the EP surfaces, while the number of cells on the other surfaces was equivalent to plastic. [3H]Thymidine incorporation was inversely related to surface roughness. Alkaline phosphatase specific activity in isolated cells was found to decrease with increasing surface roughness, except for those cells cultured on CA. In contrast, enzyme activity in the cell layer was only decreased in cultures grown on FA- and TPS-treated surfaces. A direct correlation between surface roughness and RNA and CDP production was found. Surface roughness had no apparent effect on NCP production. Proteoglycan synthesis by the cells was inhibited on all the surfaces studied, with the largest inhibition observed in the CA and EP groups. These results demonstrate that surface roughness alters osteoblast proliferation, differentiation, and matrix production in vitro. The results also suggest that implant surface roughness may play a role in determining phenotypic expression of cells in vivo.

Assessment of gingival margin thickness before margin placement.

Matrix material can be cut and placed beneath the gingival tissue to mimic a metal collar on a metal/ceramic crown restoration. If this metal matrix causes a discoloration of the gingival tissue, it would be wise to restore the tooth by using an all-porcelain margin.

Mercury in solution following exposure of various amalgams to carbamide peroxides.

Carbamide peroxide (CP) is an easily administered material for whitening teeth. Although toxicological research on CP alone has revealed no adverse health effects, possible oxidation and release of mercury from amalgams have not previously been investigated. This research evaluated the quantitative release of mercury from amalgams into solution by CP. CP preparations can generally be divided into two classes based on the presence or absence of carbopol, an oxygen-releasing inhibitor. Rembrandt (R), a 10% CP with carbopol and White and Brite (WB), a 10% CP without carbopol were used in this study. Four different types of amalgams [Dispersalloy (D), Sybraloy (S), Tytin (T) and Valiant Ph.D. (V)] were selected. Uniform samples of the four amalgams were prepared and stored at 37 degrees C for 1 week. Vials of saline (10 ml), R and WB were prepared. R and WB were mixed with saline to a 50:50 solution to reduce viscosity and facilitate stirring. Magnetic teflon coated stir bars were placed in all vials, and one amalgam specimen was placed in each non-control vial. After being stirred for 8 hours, solutions were analyzed for elemental mercury content using a Jerome Gold Film Mercury Analyzer. All background mercury levels were zero, but following the experiment there were significantly higher amounts of mercury in the CP solutions as compared to the 100% saline solutions. These results suggest there is an active oxidation of the amalgam releasing mercury ions into solution.