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Biomedical subjects

D M Brunette

Publications and source records attributed to D M Brunette.

At least 19 recordsLinked to original sources
PubMed2006

Microfabricated discontinuous-edge surface topographies influence osteoblast adhesion, migration, cytoskeletal organization, and proliferation and enhance matrix and mineral deposition in vitro.

The fabrication of surfaces that stimulate increased adhesion, migration, and differentiated function of osteoblasts has been viewed as being desirable for many orthopedic applications. Previous studies have shown that microfabricated pits and grooves alter adhesion, spreading, matrix secretion, and production of mineral by rat calvarial osteoblasts (RCOs). The mechanisms underlying these effects are unknown, although microenvironment and cell alignment are considered to play a role. The aim of this work was to investigate the behavior of RCOs on microfabricated discontinuous-edge surfaces (DESs), which could provide an alternative means to control both the microenvironment and cellular alignment. Two types of discontinuous-type structures were employed, gap-cornered boxes and micron scale pillars. DES gap-cornered boxes and the pillars influenced the arrangement of F-actin, microtubules, and vinculin. Osteoblasts were guided in their direction of migration on both types of substrata. Both box DESs and pillars altered the staining intensity and localization pattern of phosphotyrosine and src-activated FAK localization. Cell multilayering, matrix deposition, and mineralization were enhanced on both discontinuous topographies when compared with smooth controls. This study shows that DESs alter adhesion, migration, and proliferative responses from osteoblasts at early time points (<1 week) and promote multilayering, matrix deposition, and mineral deposition at later times (2-6 weeks). Such topographical patterns could potentially be employed as effective surface features on bone-contacting implants or in membrane-based periodontal applications.

Actins↗
PubMed2005

"Gap guidance" of fibroblasts and epithelial cells by discontinuous edged surfaces.

Cell adhesion, shape, and directed migration are some of the fundamental processes underlying tissue development and organization. The setting of geometric limits on cellular behavior has led to the hypothesis that a continuous edge is required to elongate a cell and guide its direction of movement. The aim of this study was to examine the validity of this hypothesis by examining the response of human gingival fibroblasts and periodontal ligament epithelial cells, to microfabricated surfaces that incorporate discontinuous edges. Cell response was assessed through spreading, morphology, cytoskeletal organization, and time-lapse microscopy, on substrata with a pattern of repeated open boxes with gaps at the corners. Fibroblasts attached and spread within 6 h, adopting either a square, triangular, or diagonally elongated morphology. Epithelial cells took longer to adhere, but were observed to adopt morphologies similar to those of the fibroblasts. Addition of colcemid or cytochalasin-D attenuated the orientation and alignment of both fibroblasts and epithelial cells. Fibroblasts and epithelial cell migration was guided diagonally in their movement through gaps in the square pattern, demonstrating that a continuous edge is not a prerequisite for guided cell migration.

Animals↗
PubMed2005

Substratum roughness alters the growth, area, and focal adhesions of epithelial cells, and their proximity to titanium surfaces.

Epithelial (E) cells were cultured on smooth tissue culture plastic (TCP), TCP-Ti, polished Ti (P), and rough grit-blasted Ti (B), acid-etched Ti (AE), and grit-blasted and acid-etchedTi (SLA) surfaces and their growth, area, adhesion, and membrane-Ti proximity assessed. Rough surfaces decreased the growth of E cells compared to smooth surfaces in cultures up to 28 days. In general rough surfaces decreased the spreading of E cells as assessed by their area with the most pronounced affect for the SLA surface. On the other hand, the strength of E cells adhesion as inferred by immunofluorescence staining of vinculin in focal adhesions indicated that E cells formed more and larger focal adhesions on the smooth P surface compared to the rougher AE surface. As this finding indicates a stronger adhesion to smooth surfaces, it is likely that E cells on rough surfaces are more susceptible to mechanical removal. An immunogold labeling method was developed to visualize focal adhesions using back-scattered electron imaging with a scanning electron microscope (SEM). On rough surfaces focal adhesions were primarily localized on to the ridges rather than the valleys and the cells tended to bridge over the valleys. Transmission electron microscopy (TEM) measurements of membrane proximity to the Ti surface indicated that average distance of cell to the Ti increased as the Ti surface roughness increased. Therefore, the size and shape of surface features are important determinants of epithelial adhesive behavior and epithelial coverage of rough surfaces would be difficult to attain if such surfaces become exposed.

Cell Adhesion↗
PubMed2005

A PLGA membrane controlling cell behaviour for promoting tissue regeneration.

Barrier membranes are used in periodontal applications with the aim of supporting bone regeneration by physically blocking migrating epithelial cells. We report a membrane design that has a surface topography that can inhibit epithelial cell migration and proliferation on one side and a topography that guides osteoblast migration to a desired area. A PLGA copolymer (85:15) blended with MePEG, was cast to have surfaces with smooth, grooved or sandblasted-acid-etched topographies. Epithelial cells spread on smooth surfaces after 24 h, and cell numbers increased after 5 days. Cells on the smooth surface exhibited no preferred direction of migration. On the sandblasted-acid-etched topography epithelial cells spread but the cell number did not significantly increase after 5 days. Cell migration was inhibited on this surface. Osteoblasts spread on both grooved and smooth surfaces and cell number increased after 5 days on all surfaces. The cells that adhered in the grooves migrated preferentially in the direction of the grooves. Positive alkaline phosphatase staining was seen on all surfaces within 4 weeks and positive Von Kossa nodule staining within 6 weeks. These results suggest that surface topographies replicated on opposite sides of a biodegradable polymers membrane can inhibit proliferation and migration of the epithelial cells, and promote proliferation and directional migration of osteoblasts. Addition of appropriate surface topographies to membranes used in guided tissue regeneration has the possibility of improving clinical performance in periodontal tissue regeneration procedures.

Animals↗
PubMed2004

Effects of titanium-coated micromachined grooved substrata on orienting layers of osteoblast-like cells and collagen fibers in culture.

Osteogenic cells from newborn rat calvariae were cultured on titanium surfaces on which cell orientation could be manipulated. Substrata included smooth surfaces and substrata with smooth regions (gaps) flanked by grooves of 47-microm pitch and 3-, 10-, or 30-microm depth. Orientation angles of the cells were measured over time using propidium-iodide staining and confocal laser scanning microscopy. In addition, collagen fibers were identified using picro-sirius staining and reflected light polarization microscopy. Grooves proved effective in orienting cells, but their orienting ability decreased above the ridge level. Cells on the smooth surface showed no preferred orientation. Cells in the gaps became oriented as a result of cell-cell interactions with the cells on the flanking grooves. Cells in grooves produced oriented collagen fibers, but in the gaps, fibers could be parallel, perpendicular, or diagonal to the grooves. Collagen fibers on the smooth surfaces formed arrays of parallel fibers in a crisscross pattern. In long-term cultures, bone-like nodules were formed, but mostly above the ridge level. These data demonstrate that grooved surfaces can influence cell orientation both in cell populations above the cells in contact with the grooves and in cell populations adjacent to the grooves.

Animals↗
PubMed2003

Chemically patterned, metal oxide based surfaces produced by photolithographic techniques for studying protein- and cell-surface interactions I: Microfabrication and surface characterization.

Chemical patterns on smooth wafer substrates comprising areas with two different metals have been produced by vacuum metal deposition and photolithographic techniques. The combination of metals has been chosen from the series titanium (Ti), aluminium (Al), vanadium (V) and niobium (Nb), producing patterns (dots and stripes with dimensions of 50, 100 and 150 micrometer) with one of the metals as the background and with the second metal (foreground pattern) deposited on the background metal. The structure and chemical composition of the patterned surfaces were evaluated by scanning electron microscopy, X-ray photoelectron spectroscopy and imaging time-of-flight secondary-ion mass spectrometry. The surfaces proved to be geometrically well defined with the expected surface-chemical composition, i.e. a surface oxide (passive) film essentially composed of TiO(2),Al(2)O(3),V(2)O(5), or Nb(2)O(5). Ti/Ti patterned surfaces were produced as controls and found to show no chemical composition contrast. The surface roughness of the pattern was greater than that of the background by a factor of 2-3, but was still extremely smooth with Ra<2nm. The patterns serve as model surfaces for studying in vitro the behaviour of cells as well as the adsorption of serum proteins on different metal oxides, which will be reported in a companion paper. These surfaces can be used to compare and contrast the response of osteoblasts to Ti and other alloy components, such as Al, V, or Nb, which are used in load-bearing medical implants.

Aluminum↗
PubMed2003

Chemically patterned, metal-oxide-based surfaces produced by photolithographic techniques for studying protein- and cell-interactions. II: Protein adsorption and early cell interactions.

Protein adsorption and adhesion of primary human osteoblasts on chemically patterned, metal-oxide-based surfaces comprising combinations of titanium, aluminium, vanadium and niobium were investigated. Single metal samples with a homogeneous surface and bimetal samples with a surface pattern produced by photolithographic techniques were used. The physical and chemical properties of the samples have been extensively characterised and are presented in a companion paper. Here, we describe their properties in terms of cell responses during the initial 24h of cell culture. Regarding the cell number and activity there was no significant difference between any of the single metal surfaces. However the morphology of cells on vanadium surfaces became spindle-like. In contrast to the behaviour on single metal samples, cells exhibited a pronounced reaction on bimetallic surfaces that contained aluminium. Cells tended to stay away from aluminium, which was the least favoured metal in all two-metal combinations. An initial cell alignment relative to the pattern geometry was detectable after 2h and was fully developed after 18h of incubation. The organisation of f-actin and microtubules as well as the localisation of vinculin were all more pronounced on non-aluminium regions. We hypothesised that the differences in cell response could be associated with differences in the adsorption of serum proteins onto the various metal oxides. Protein adsorption experiments were performed using microscopy in conjunction with immunofluorescent stains. They indicated that both fibronectin and albumin adsorption were significantly greater on the non-aluminium regions, suggesting that differences in cellular response correlate with a modulation of the concentration of serum proteins on the surface.

Adsorption↗
PubMed2002

Effects of substratum surface topography on the organization of cells and collagen fibers in collagen gel cultures.

This study investigated the orientation of fibroblasts and collagen cultured on microfabricated grooved or smooth titanium surfaces, as well as on tissue culture polystyrene, in the presence or absence of collagen gels. The gels were first added either to the confluent fibroblast culture on the surface (cell-gel condition) or to the fibroblasts were suspended within the collagen gel and then placed onto the surface (gel condition). Cells and collagen were observed with differential interference, polarization, and confocal laser scanning microscopy. Although the smooth surfaces had no effect on cell orientation in the gel for the first 2 weeks of culture, cells did orient with grooves regardless of the culture conditions. There was evidence for orthogonal multilayering of cells under the cell-gel condition at 4 weeks, and collagen alignment reflected cell alignment. The interaction of the collagen gel with the surface depended on whether the cell-gel or the gel condition was employed. In the former condition, the gel contracted toward the substratum, whereas the gel condition resulted in the formation of a ring of collagen loosely attached to the substratum. These results suggest that the order in which fibroblasts encounter substratum and extracellular matrix can influence the eventual matrix-cell interactions, and that substratum topography can influence matrix and cell orientation in zones not immediately in contact with the surface.

Cell Adhesion↗
PubMed2002

Comparative investigation of the surface properties of commercial titanium dental implants. Part I: chemical composition.

The surfaces of five commercially available titanium implants (Brånemark Nobel Biocare, 3i ICE, 3i OSSEOTITE, ITI-TPS, and ITI-SLA) were compared by scanning electron microscopy, X-ray photoelectron spectroscopy, time-of-flight secondary ion mass spectroscopy. All five implant types were screw-shaped and fabricated from commercially pure (cp) titanium, but their surface properties differed both as regards surface morphology and surface chemical composition. The macro- and microstructure of the implant surfaces were investigated by scanning electron microscopy. The surfaces chemical composition was determined using the surface-sensitive analytical techniques of X-ray photoelectron spectroscopy and time-of-flight secondary ion spectrometry. Surface topographies were found to reflect the type of mechanical/chemical fabrication procedures applied by the manufacturers. The titanium oxide (passive) layer thickness was similar (5-6 nm) and typical for oxide films grown at or near room temperature. A variety of elements and chemical compounds not related to the metal composition were found on some implant types. They ranged from inorganic material such as sodium chloride to specific organic compounds believed to be due to contamination during fabrication or storage. The experimental findings are believed to make a contribution to a better understanding of the interplay between industrial fabrication procedure and physico-chemical implant surface properties.

Journal Article↗
PubMed2001

Interaction between topography and coating in the formation of bone nodules in culture for hydroxyapatite- and titanium-coated micromachined surfaces.

Rat osteoblast cultures were maintained from 24 h to 6 weeks on hydroxyapatite (HA)- or titanium (Ti)-coated smooth and micromachined grooved substrata in medium supplemented with L-ascorbic acid-2-phosphate and beta-glycerophosphate to promote mineralization. The HA coatings, approximately 1 microm thick, were characterized using X-ray diffraction, surface roughness, and scanning electron microscopy (SEM). Osteoblasts elongated, aligned, and moved in the direction of the grooves on both Ti and HA grooved surfaces. HA surfaces produced significantly more bone-like nodules than Ti surfaces. All grooved substrata produced significantly more nodules than smooth surfaces. These results are consistent with the hypothesis that substrata can increase osteogenesis by formation of an appropriate microenvironment. There was also a statistically significant interaction between topography and chemistry in the formation of mineralized nodules. A strong correlation (r = 0.958) between alkaline phosphatase (Alk-P) at 2 weeks and nodule counts at 6 weeks was observed, suggesting that Alk-P may possibly be used as a leading indicator of osteogenesis on microfabricated surfaces. The results of this study indicate that surface topography and chemistry can affect osteogenesis, and that interactions between chemistry and topography can occur.

Animals↗
PubMed1999

The effects of the surface topography of micromachined titanium substrata on cell behavior in vitro and in vivo.

Surface properties, including topography and chemistry, are of prime importance in establishing the response of tissues to biomaterials. Microfabrication techniques have enabled the production of precisely controlled surface topographies that have been used as substrata for cells in culture and on devices implanted in vivo. This article reviews aspects of cell behavior involved in tissue response to implants with an emphasis on the effects of topography. Microfabricated grooved surfaces produce orientation and directed locomotion of epithelial cells in vitro and can inhibit epithelial downgrowth on implants. The effects depend on the groove dimensions and they are modified by epithelial cell-cell interactions. Fibroblasts similarly exhibit contact guidance on grooved surfaces, but fibroblast shape in vitro differs markedly from that found in vivo. Surface topography is important in establishing tissue organization adjacent to implants, with smooth surfaces generally being associated with fibrous tissue encapsulation. Grooved topographies appear to have promise in reducing encapsulation in the short term, but additional studies employing three-dimensional reconstruction and diverse topographies are needed to understand better the process of connective-tissue organization adjacent to implants. Microfabricated surfaces can increase the frequency of mineralized bone-like tissue nodules adjacent to subcutaneously implanted surfaces in rats. Orientation of these nodules with grooves occurs both in culture and on implants. Detailed comparisons of cell behavior on micromachined substrata in vitro and in vivo are difficult because of the number and complexity of factors, such as population density and micromotion, that can differ between these conditions.

Animals↗
PubMed1999

Observation of fibronectin distribution on the cell undersurface using immunogold scanning electron microscopy.

Immunogold staining followed by observation with scanning electron microscopy (SEM) has been quite effective in showing the distribution of proteins on dorsal cell surfaces. However, observation of proteins on the ventral cell surface using SEM has not been developed to the same extent. In this study, human gingival fibroblasts cultured on titanium-coated wafers were embedded in resin. After fracturing the wafers off the embedded cells, the undersurface of the cell was exposed by argon gas glow discharge etching. After 15 min of glow discharge etching, the resin covering the cell undersurface was completely removed. The distribution of fibronectin (FN) on the cell undersurface was demonstrated using an anti-FN antibody and colloidal gold (30 nm) conjugated with IgG. The undersurface was then coated with carbon or gold-palladium and observed by SEM. Using backscattered electron detection, gold beads could be identified in high contrast. On cells cultured for 5 hr, gold beads were distributed randomly on the entire cell undersurface. However, a line of gold beads was sometimes observed close to the edge of the cell. These results indicated that this immunogold/SEM etching method provides a powerful means for studying cell adhesion molecules on the cell undersurface. (J Histochem Cytochem 47:1487-1493, 1999)

Cell Membrane↗
PubMed1998

Surface topography and serum concentration affect the appearance of tenascin in human gingival fibroblasts in vitro.

Tenascin is an extracellular matrix glycoprotein which affects cell behavior such as cell migration. This study was undertaken to investigate the time of appearance of tenascin (TN) in human gingival fibroblasts (HGF) and how it was affected by the surface topography of the titanium substratum or by serum concentration in the medium. HGF were cultured for 4 to 24 h and then processed for confocal immunofluorescence microscopy. Very few cells stained positive for TN 4 h after plating, but the number of TN-positive HGF gradually increased between 8 and 18 h after plating. The increase in the rate of the proportion of TN-positive cells on the grooved surface lagged behind that of HGF cultured on the smooth surface. The number of TN-positive cells in medium + 15% serum was significantly greater than that of cells in 5% serum or serum-free medium. The number of TN-positive cells was greater on the smooth titanium surface than on the grooved titanium surface in both 15% serum and 5% serum-containing medium. These findings suggest that TN production by fibroblasts in vitro can be modulated by factors in serum and by the surface topography of the substratum.

Blood Proteins↗
PubMed1998

Effects of titanium substratum and grooved surface topography on metalloproteinase-2 expression in human fibroblasts.

The chemical and topographic effects of commercially pure titanium on cell morphology and the regulation of matrix metalloproteinase-2 (MMP-2) gene expression, synthesis, and activity were investigated in early passage human gingival fibroblasts. Scanning electron microscopy showed that on smooth titanium (Ti), fibroblasts remained well spread and randomly oriented throughout the culture period. In contrast, cells on V-shaped grooved titanium (VTi) were oriented along the grooves by 16 h and proliferated in this organization throughout the culture period. The effects of substratum surface chemistry on MMP-2 expression were found to be distinct from those of topography. Northern hybridization analysis of fibroblasts cultured on Ti revealed an MMP-2 mRNA time-course expression pattern parallel to that observed on the tissue culture plastic (TCP) dishes, but at significantly lower levels at each time-point. The Ti mRNA levels were decreased by 34% at 16 h, 55% at 40 h, and 45% at 90 h relative to TCP. In contrast, MMP-2 mRNA expression on VTi showed both an altered time-course expression pattern and altered levels compared to Ti and TCP. Relative to TCP, VTi MMP-2 mRNA levels were approximately 80% less at 16 h and approximately 50% less at 40 h, but not significantly different at 90 h. Relative to Ti, VTi MMP-2 mRNA levels were approximately 75% less at 16 h, but approximately 40% greater at 40 h and approximately 70% greater at 90 h. These differences may be explained in part by the observed changes in MMP-2 mRNA half-life which decreased by approximately 40% on Ti but increased by over fourfold on VTi relative to TCP. The smooth Ti also showed an approximate twofold increase of MMP-2 secretion in the late cultures over TCP controls. These results indicate that substratum surface chemistry and topography-induced changes in cell shape can alter MMP-2 expression in normal fibroblasts. The molecular approach to investigating the major molecules involved in tissue degradation may provide sensitive indicators of tissue remodeling at the tissue-biomaterial interface.

Biocompatible Materials↗
PubMed1998

Alternative therapies: abuses of scientific method and challenges to dental research.

STATEMENT OF PROBLEM: Alternative health practitioners compete with established dentistry and medicine for the treatment of many conditions. Their popularity accrues because of deficiencies of current treatments, increased acceptance of self-treatment, and a highly individualized approach. Ineffective alternative therapies can appear effective because of the placebo effect, symptom variability, short-term observations, combination with established treatments, and reporting bias. Alternative therapies often lack a rational basis. A sound theory underlying a treatment enables the approach to be integrated with other areas of science and leads to progressive research. PURPOSE: This article argues that alternative therapies can best be considered bad science that commonly violates normal scientific norms or criteria for acceptance including comprehensiveness, falsifiability, openness, and objectivity. CONCLUSIONS: The claims of alternative therapists are useful in identifying perceived deficiencies in conventional treatments. Suggestions for countering alternative practitioners' claims include targeted research and improved access to and evaluation of the dental research literature.

Complementary Therapies↗
PubMed1998

The effects of dentifrice systems on oral malodor.

Chronic oral malodor is a serious concern for about one-fifth of the North American population, and a field of emerging research interest. The present three studies, one involving gas chromatography and two employing odor judge assessment, examined the efficacy of baking soda and other toothpastes in reducing breath odor. The most common cause of oral malodor is elevated levels of volatile sulfur compounds (VSC's), primarily hydrogen sulfide (H2S) and methyl mercaptan (CH3SH), in the breath. Gas chromatography, an accurate means of measuring breath VSC, was employed to evaluate the breath levels of VSC in 11 men after brushing with baking soda-containing dentifrices with or without the addition of Zn++. Dentifrices with either Zn++ or a concentration of baking soda 20% or greater significantly reduced VSC levels. The addition of Zn++ to baking soda dentifrices enhanced the anti-odor effects. In the first organoleptic study, dentifrices containing 20% baking soda and 30% baking soda demonstrated significantly greater ability to reduce breath odor than a standard sodium fluoride/silica dentifrice. The subjects' baseline mouth odor evaluations, initially rated as strong, declined after brushing with the baking soda toothpastes to a barely detectable level at one hour, then rising to a faint level at two hours and moderate levels at three hours. In the second organoleptic study, a dentifrice containing 65% baking soda demonstrated significantly greater ability to reduce breath odor than a standard sodium fluoride/silica tartar control dentifrice, but did not differ significantly from a standard dentifrice containing 0.76% sodium monofluorophosphate in a dicalcium phosphate dihydrate base. The results of these studies indicate that dentifrices containing 20% or more baking soda can confer a significant odor-reducing benefit for time periods up to three hours.

Adult↗
PubMed1997

Sensitivity of fibroblasts and their cytoskeletons to substratum topographies: topographic guidance and topographic compensation by micromachined grooves of different dimensions.

Fibroblasts alter their shape, orientation, and direction of movement to align with the direction of micromachined grooves, exhibiting a phenomenon termed topographic guidance. In this study we examined the ability of the microtubule and actin microfilament bundle systems, either in combination with or independently from each other, to affect alignment of human gingival fibroblasts on sets of micromachined grooves of different dimensions. To assess specifically the role of microtubules and actin microfilament bundles, we examined cell alignment, over time, in the presence or absence of specific inhibitors of microtubules (colcemid) and actin microfilament bundles (cytochalasin B). Using time-lapse videomicroscopy, computer-assisted morphometry and confocal microscopy of the cytoskeleton we found that the dimensions of the grooves influenced the kinetics of cell alignment irrespective of whether cytoskeletons were intact or disturbed. Either an intact microtubule or an intact actin microfilament-bundle system could produce cell alignment with an appropriate substratum. Cells with intact microtubules aligned to smaller topographic features than cells deficient in microtubules. Moreover, cells deficient in microtubules required significantly more time to become aligned. An unexpected finding was that very narrow 0.5-microm-wide and 0.5-microm-deep grooves aligned cells deficient in actin microfilament bundles (cytochalasin B-treated) better than untreated control cells but failed to align cells deficient in microtubules yet containing microfilament bundles (colcemid treated). Thus, the microtubule system appeared to be the principal but not sole cytoskeletal substratum-response mechanism affecting topographic guidance of human gingival fibroblasts. This study also demonstrated that micromachined substrata can be useful in dissecting the role of microtubules and actin microfilament bundles in cell behaviors such as contact guidance and cell migration without the use of drugs such as cytochalasin and colcemid.

Actin Cytoskeleton↗