Regulation of RAW264.7 macrophage polarization on smooth and rough surface topographies by galectin-3.

Recognition of topographical features induces phenotypic changes in macrophages although the receptors and signaling pathways are not completely characterized. As integrin molecules in focal adhesions/podosomes are in intimate contact with topography and topography modulates the NFkB pathway through cholesterol enriched raft-associated adhesive signaling structures we hypothesized that a cell-surface signaling complex comprised of galectin-3 together with its ligand CD98 and integrinß1 is important for topography-directed lineage determination. This study used polished, sand blasted and acid etched (SLA) surfaces and two novel grooved topographies (G1 and G2) produced by anisotropic etching of Si <1 1 0> to evaluate the role of galectin-3 in macrophage polarization in RAW 264.7 macrophages, as determined by gene expression and morphology. In the presence of the galectin-3 inhibitor, lactose, the M2 marker (mannose receptor) was down-regulated while the M1 marker (iNOS) was up-regulated on smooth and rough surfaces. This skewing of phenotype suggests a role for galectin-3 in macrophage polarization towards the M2 phenotype. Additionally, we evaluated the role of PI3K on polarization using PI3K inhibitor LY294002. We found that the M2 marker was down-regulated on both PO (surface polished) and G1 surfaces implicating PI3K in lineage determination. We also found that surface topography altered cell morphology; macrophages had a larger area on G2 surfaces. Lactose treatment significantly reduced the cell area on all topographies suggesting that the galectin-3 is also involved in signaling complexes triggering the rearrangement of the actin cytoskeleton. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2499-2509, 2017.

Microtopographical regulation of adult bone marrow progenitor cells chondrogenic and osteogenic gene and protein expressions.

Microtopographic features affect diverse cell behaviors. Adult bone marrow progenitor cells (AMPCs) constitute a multipotent heterogeneous population. We hypothesized that microtopographies could direct AMPCs lineage-specific differentiation. AMPCs isolated from Sprague-Dawley rats were CD45 depleted, expanded, and plated at 10(5) cells/cm2 on epoxy-microfabricated: (1) 60-microm-deep grooves with 95-microm pitch (D60P95), (2) 55-microm-wide and 10-microm-deep squares (W55D10), (3) 30-microm-deep grooves with 45-microm pitch (D30P45), (4) 17-microm-wide and 10-microm-deep pillars (W17D10), and (5) smooth control. AMPCs were cultured using expansion, chondrogenesis, or osteogenesis supporting media. Cell cultures were examined by scanning electron microscopy, qRT-PCR, and immunostaining at 2, 9, 16, and 23 days after plating. Expressions of osteogenesis-related genes, such as Runx-2, alkaline phosphatase, osteopontin, osteocalcin, and parathyroid hormone-related protein receptor (PTHr), and chondrogenesis-associated genes, such as Sox-9, type II collagen, and aggrecan, were determined. In expansion medium, W55D10 induced a transient increase of Sox9 expression. Compared with smooth surfaces, type II collagen mRNA and protein expressions in chondrogenic medium were significantly upregulated on W55D10 by day 23. In contrast, osteocalcin and PTHr expressions were significantly increased on D30P45 in osteogenic medium. We have demonstrated that W55D10 and D30P45 enhanced AMPCs chondrogenic and osteogenic terminal differentiation with appropriate culture conditions.

Hexagonal micron scale pillars influence epithelial cell adhesion, morphology, proliferation, migration, and cytoskeletal arrangement.

A desirable attribute of implants penetrating epithelium is the inhibition of downward epithelial migration. Simple grooved topographies can inhibit this migration either directly or indirectly by promoting connective tissue attachment, but few studies have focused on the direct effect of geometrically complex topographies on epithelial behavior. Therefore, we examined the influence of novel topographies comprising square floors surrounded by six-sided pillars on periodontal ligament epithelial cell adhesion, morphology, cytoskeletal organization, and migration. Relative to cells on smooth surface, epithelial cells on the pillar substrata adhered closely, exhibited reduced proliferation, had a reduced velocity, but higher persistence. Vinculin staining demonstrated that cells formed mature adhesions on the pillar tops, but smaller punctate adhesion in the gaps and on the pillar walls. Overall more mature adhesions were found on pillars compared to smooth surfaces, which may account for the reduced speed of migration limited on the pillars. F-actin stress fibers were predominantly found on pillar tops within 6 h, whereas microtubules (MTs) had a tendency to form in the gaps between the six-sided pillars. In conclusion, microfabricated pillars altered epithelial migration in ways that could prove useful in inhibition of epithelial downward migration on transmucosal implants.

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.

"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.

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.

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.

Synergistic interaction of topographic features in the production of bone-like nodules on Ti surfaces by rat osteoblasts.

The objective of this study was to study the responses of osteoblast-like cells to rough Titanium (Ti)-coated epoxy surfaces of differing topographic complexity. Four topographies were studied: polished (PO), coarse-blasted (CB), acid-etched (AE) and coarse-blasted+acid-etched (SLA). Rat osteoblasts were cultured on these surfaces and their morphology, thickness as well as the number and size of bone-like nodules measured. To determine cell shape and cell thickness, fluorescein-5-thiosemicarbazide was used to stain the cell components including the cell membrane, the stained cells were optically sectioned using epifluorescent microscopy and the optical sections were computationally reconstructed to obtain three-dimensional images in which cell volume and cell thickness could be determined. Similarly optical sections of bone-like nodules labeled with tetracycline were also reconstructed to determine their size. The different surface topographies were found to alter the thickness and morphology of osteoblasts cultured on these surfaces. Osteoblasts produced significantly more and larger nodules on SLA compared to other surfaces. Nevertheless and perhaps surprisingly, given the evidence in various cell populations that cell shape can affect cell differentiation, cell thickness was not directly correlated with an increase in bone-like nodule formation. Data were analyzed by factorial analysis of variance. In this way the primary effect of each surface treatment ( i.e. blasting and acid etching) could be assessed as well as their interaction. Both the acid etching and blasting processes significantly affected the number and size of bone-like nodules cultured on Ti surfaces. Moreover there were significant interaction effects indicating that surface topographic features can act synergistically to enhance bone formation. This result suggests that a useful approach to the optimization of surfaces for bone production could involve systematic investigation of combinations of processes each of which produces distinct surface topographical features.

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.

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.

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.

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.

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.

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.

Wavelength-dependent roughness: a quantitative approach to characterizing the topography of rough titanium surfaces.

Topographies of grit-blasted, etched, grit-blasted and etched, and microfabricated and etched surfaces of commercially pure titanium have been investigated. Such surface topographies vary across the scale range of interest for dental implants, extending from nanometers to millimeters. The complete characterization of topography requires the use of complementary methods. This study compared the topographic characterization methods of non-contact laser profilometry, interference microscopy, stereo-scanning electron microscopy (stereo-SEM), and atomic force microscopy. Non-contact laser profilometry was shown to be a useful method to characterize topographic features in the micron to millimeter range, whereas interference microscopy and stereo-SEM can be employed down to the submicron range. Stereo-SEM is particularly useful for quantifying topographies with complex, strongly corrugated ("sharp"), and high-aspect-ratio features and was shown to be complementary to non-contact laser profilometry and interference microscopy. Because of tip-related envelope problems, atomic force microscopy was not found to be suitable for the type of surfaces investigated in this study. Independent of the method used, the commonly used "integral" amplitude roughness parameters, such as Ra, Rq, or Rt, were often of limited value in the description of actual implant surfaces. The application of the wavelength-dependent roughness approach was shown to be an effective method for the description of surface topographies in the complete range of characteristic roughness and is also a useful means of examining the effects of surface treatment processes.

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)

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.

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.