The structural basis of the talin-KANK1 interaction that coordinates the actin and microtubule cytoskeletons at focal adhesions.

Adhesion between cells and the extracellular matrix is mediated by heterodimeric (αß) integrin receptors that are intracellularly linked to the contractile actomyosin machinery. One of the proteins that control this link is talin, which organizes cytosolic signalling proteins into discrete complexes on ß-integrin tails referred to as focal adhesions (FAs). The adapter protein KANK1 binds to talin in the region of FAs known as the adhesion belt. Here, we adapted a non-covalent crystallographic chaperone to resolve the talin-KANK1 complex. This structure revealed that the talin binding KN region of KANK1 contains a novel motif where a ß-hairpin stabilizes the α-helical region, explaining both its specific interaction with talin R7 and high affinity. Single point mutants in KANK1 identified from the structure abolished the interaction and enabled us to examine KANK1 enrichment in the adhesion belt. Strikingly, in cells expressing a constitutively active form of vinculin that keeps the FA structure intact even in the presence of myosin inhibitors, KANK1 localizes throughout the entire FA structure even when actomyosin tension is released. We propose a model whereby actomyosin forces on talin eliminate KANK1 from talin binding in the centre of FAs while retaining it at the adhesion periphery.

Evaluation of focal adhesion mediated subcellular curvature sensing in response to engineered extracellular matrix.

Fibril curvature is bioinstructive to attached cells. Similar to natural healthy tissues, an engineered extracellular matrix can be designed to stimulate cells to adopt desired phenotypes. To take full advantage of the curvature control in biomaterial fabrication methodologies, an understanding of the response to fibril subcellular curvature is required. In this work, we examined morphology, signaling, and function of human cells attached to electrospun nanofibers. We controlled curvature across an order of magnitude using nondegradable poly(methyl methacrylate) (PMMA) attached to a stiff substrate with flat PMMA as a control. Focal adhesion length and the distance of maximum intensity from the geographic center of the vinculin positive focal adhesion both peaked at a fiber curvature of 2.5 µm-1 (both ∼2× the flat surface control). Vinculin experienced slightly less tension when attached to nanofiber substrates. Vinculin expression was also more affected by a subcellular curvature than structural proteins α-tubulin or α-actinin. Among the phosphorylation sites we examined (FAK397, 576/577, 925, and Src416), FAK925 exhibited the most dependance on the nanofiber curvature. A RhoA/ROCK dependance of migration velocity across curvatures combined with an observation of cell membrane wrapping around nanofibers suggested a hybrid of migration modes for cells attached to fibers as has been observed in 3D matrices. Careful selection of nanofiber curvature for regenerative engineering scaffolds and substrates used to study cell biology is required to maximize the potential of these techniques for scientific exploration and ultimately improvement of human health.

A post-invasion role for Chlamydia type III effector TarP in modulating the dynamics and organization of host cell focal adhesions.

The human pathogen Chlamydia trachomatis targets epithelial cells lining the genital mucosa. We observed that infection of various cell types, including fibroblasts and epithelial cells resulted in the formation of unusually stable and mature focal adhesions that resisted disassembly induced by the myosin II inhibitor, blebbistatin. Superresolution microscopy revealed in infected cells the vertical displacement of paxillin and focal adhesion kinase from the signaling layer of focal adhesions, whereas vinculin remained in its normal position within the force transduction layer. The candidate type III effector TarP, which localized to focal adhesions during infection and when expressed ectopically, was sufficient to mimic both the reorganization and blebbistatin-resistant phenotypes. These effects of TarP, including its localization to focal adhesions, required a post-invasion interaction with the host protein vinculin through a specific domain at the C terminus of TarP. This interaction is repurposed from an actin-recruiting and -remodeling complex to one that mediates nanoarchitectural and dynamic changes of focal adhesions. The consequence of Chlamydia-stabilized focal adhesions was restricted cell motility and enhanced attachment to the extracellular matrix. Thus, via a novel mechanism, Chlamydia inserts TarP within focal adhesions to alter their organization and stability.

Intra- and intermuscular variations of postmortem protein degradation for PMI estimation.

In recent years, protein decomposition has become of increasing interest for the use in forensic estimation of the postmortem interval (PMI). Especially skeletal muscle tissue has proven to be a prime target tissue, among other reasons, due to its large abundance in the human body. In this regard, it is important to know whether there are any intra- and intermuscular differences in the behavior of protein degradation. Thus, samples from different locations within several skeletal muscles as well as from cardiac and smooth muscle tissue samples were collected from three autopsy cases with varying degree of decomposition. Samples were analyzed by SDS-PAGE and Western blotting and compared for protein degradation patterns. Intramuscular variations turned out to be minimal and without major influence for the use of the method. Observed intermuscular differences provide possibilities for future improvement of the precision and temporal application range. The results of this study show the strengths and current limitations of protein degradation-based PMI estimation and provide a deeper understanding of intraindividual postmortem protein degradation processes.

Anti-correlation of HER2 and focal adhesion complexes in the plasma membrane.

Excess presence of the human epidermal growth factor receptor 2 (HER2) as well as of the focal adhesion protein complexes are associated with increased proliferation, migratory, and invasive behavior of cancer cells. A cross-regulation between HER2 and integrin signaling pathways has been found, but the exact mechanism remains elusive. Here, we investigated whether HER2 colocalizes with focal adhesion complexes on breast cancer cells overexpressing HER2. For this purpose, vinculin or talin green fluorescent protein (GFP) fusion proteins, both key constituents of focal adhesions, were expressed in breast cancer cells. HER2 was either extracellularly or intracellularly labeled with fluorescent quantum dots nanoparticles (QDs). The cell-substrate interface was analyzed at the location of the focal adhesions by means of total internal reflection fluorescent microscopy or correlative fluorescence- and scanning transmission electron microscopy. Expression of HER2 at the cell-substrate interface was only observed upon intracellular labeling, and was heterogeneous with both HER2-enriched and -low regions. In contrast to an expected enrichment of HER2 at focal adhesions, an anti-correlated expression pattern was observed for talin and HER2. Our findings suggest a spatial anti-correlation between HER2 and focal adhesion complexes for adherent cells.

Compliant substratum modulates vinculin expression in focal adhesion plaques in skeletal cells.

The biophysical properties of the extracellular matrix (ECM) dictate tissue-specific cell behaviour. In the skeleton system, bone shows the potential to adapt its architecture and contexture to environmental rigidity via the bone remodelling process, which involves chondrocytes, osteoblasts, osteoclasts, osteocytes and even peripheral bone marrow-derived stem/stromal cells (BMSCs). In the current study, we generated stiff (~1 014 ± 56) kPa, Young's modulus) and soft (~46 ± 11) kPa silicon-based elastomer polydimethylsiloxane (PDMS) substrates by mixing curing agent into oligomeric base at 1:5 and 1:45 ratios, respectively, and investigated the influence of substrate stiffness on the cell behaviours by characterizing cell spreading area, cell cytoskeleton and cell adhesion capacity. The results showed that the cell spreading areas of chondrocytes, osteoblasts, osteoclasts, osteocytes and BMSCs were all reduced in the soft substrate relative to those in the stiff substrate. F-actin staining confirmed that the cytoskeleton was also changed in the soft group compared to that in the stiff group. Vinculin in focal adhesion plaques was significantly decreased in response to soft substrate compared to stiff substrate. This study establishes the potential correlation between microenvironmental mechanics and the skeletal system, and the results regarding changes in cell spreading area, cytoskeleton and cell adhesion further indicate the important role of biomechanics in the cell-matrix interaction.

Effects of aligned and random fibers with different diameter on cell behaviors.

Tissue-engineered vascular grafts show promise as a treatment for patients with diseased or damaged arteries. The formation of a confluent endothelium lumen on scaffold is one of the key issues in vascular tissue engineering. The endothelial layer is composed of endothelial cells (ECs) aligned along the long axis of the vessel. The contact guidance effect is believed to fundamentally impact cell morphology, alignment, and behavior. By tuning the scaffold topography where the EC cells reside, the cell adhesion, shape, and other behaviors might be regulated. To this end, poly(ε-caprolactone) was electrospun to obtain different topographies by varying fiber diameter and orientation. The morphology and alignment of EC cells were investigated by analyzing fluorescence staining images. The results show that nano-sized fibers significantly enhance cell adhesion and proliferation due to the comparable geometric feature size of the fiber diameter. ECs align along the fiber oriented direction for both microfibers and nanofibers. It shows a better alignment of ECs on micro-aligned fibers than on nano-aligned fibers, which is due to the active size range of the contact guidance effect. These results indicate that scaffolds with ordered topographies might favor the formation of a confluent endothelium for vascular tissue engineering.

Analysis of a vinculin homolog in a sponge (phylum Porifera) reveals that vertebrate-like cell adhesions emerged early in animal evolution.

The evolution of cell-adhesion mechanisms in animals facilitated the assembly of organized multicellular tissues. Studies in traditional animal models have revealed two predominant adhesion structures, the adherens junction (AJ) and focal adhesions (FAs), which are involved in the attachment of neighboring cells to each other and to the secreted extracellular matrix (ECM), respectively. The AJ (containing cadherins and catenins) and FAs (comprising integrins, talin, and paxillin) differ in protein composition, but both junctions contain the actin-binding protein vinculin. The near ubiquity of these structures in animals suggests that AJ and FAs evolved early, possibly coincident with multicellularity. However, a challenge to this perspective is that previous studies of sponges-a divergent animal lineage-indicate that their tissues are organized primarily by an alternative, sponge-specific cell-adhesion mechanism called "aggregation factor." In this study, we examined the structure, biochemical properties, and tissue localization of a vinculin ortholog in the sponge Oscarella pearsei (Op). Our results indicate that Op vinculin localizes to both cell-cell and cell-ECM contacts and has biochemical and structural properties similar to those of vertebrate vinculin. We propose that Op vinculin played a role in cell adhesion and tissue organization in the last common ancestor of sponges and other animals. These findings provide compelling evidence that sponge tissues are indeed organized like epithelia in other animals and support the notion that AJ- and FA-like structures extend to the earliest periods of animal evolution.

Vinculin in cell-cell and cell-matrix adhesions.

Vinculin was identified as a component of focal adhesions and adherens junctions nearly 40 years ago. Since that time, remarkable progress has been made in understanding its activation, regulation and function. Here we discuss the current understanding of the roles of vinculin in cell-cell and cell-matrix adhesions. Emphasis is placed on the how vinculin is recruited, activated and regulated. We also highlight the recent understanding of how vinculin responds to and transmits force at integrin- and cadherin-containing adhesion complexes to the cytoskeleton. Furthermore, we discuss roles of vinculin in binding to and rearranging the actin cytoskeleton.

Talin and vinculin are downregulated in atherosclerotic plaque; Tampere Vascular Study.

BACKGROUND AND AIMS: Focal adhesions (FA) play an important role in the tissue remodeling and in the maintenance of tissue integrity and homeostasis. Talin and vinculin proteins are among the major constituents of FAs contributing to cellular well-being and intercellular communication. METHODS: Microarray analysis (MA) and qRT-PCR low-density array were implemented to analyze talin-1, talin-2, meta-vinculin and vinculin gene expression in circulating blood and arterial plaque. RESULTS: All analyzed genes were significantly and consistently downregulated in plaques (carotid, abdominal aortic and femoral regions) compared to left internal thoracic artery (LITA) control. The use of LITA samples as controls for arterial plaque samples was validated using immunohistochemistry by comparing LITA samples with healthy arterial samples from a cadaver. Even though the differences in expression levels between stable and unstable plaques were not statistically significant, we observed further negative tendency in the expression in unstable atherosclerotic plaques. The confocal tissue imaging revealed gradient of talin-1 expression in plaque with reduction close to the vessel lumen. Similar gradient was observed for talin-2 expression in LITA controls but was not detected in plaques. This suggests that impaired tissue mechanostability affects the tissue remodeling and healing capabilities leading to development of unstable plaques. CONCLUSIONS: The central role of talin and vinculin in cell adhesions suggests that the disintegration of the tissue in atherosclerosis could be partially driven by downregulation of these genes, leading to loosening of cell-ECM interactions and remodeling of the tissue.

Surface Engineering of Nanostructured Titanium Implants with Bioactive Ions.

Surface nanofeatures and bioactive ion chemical modification are centrally important in current titanium (Ti) oral implants for enhancing osseointegration. However, it is unclear whether the addition of bioactive ions definitively enhances the osteogenic capacity of a nanostructured Ti implant. We systematically investigated the osteogenesis process of human multipotent adipose stem cells triggered by bioactive ions in the nanostructured Ti implant surface. Here, we report that bioactive ion surface modification (calcium [Ca] or strontium [Sr]) and resultant ion release significantly increase osteogenic activity of the nanofeatured Ti surface. We for the first time demonstrate that ion modification actively induces focal adhesion development and expression of critical adhesion­related genes (vinculin, talin, and RHOA) of human multipotent adipose stem cells, resulting in enhanced osteogenic differentiation on the nanofeatured Ti surface. It is also suggested that fibronectin adsorption may have only a weak effect on early cellular events of mesenchymal stem cells (MSCs) at least in the case of the nanostructured Ti implant surface incorporating Sr. Moreover, results indicate that Sr overrides the effect of Ca and other important surface factors (i.e., surface area and wettability) in the osteogenesis function of various MSCs (derived from human adipose, bone marrow, and murine bone marrow). In addition, surface engineering of nanostructured Ti implants using Sr ions is expected to exert additional beneficial effects on implant bone healing through the proper balancing of the allocation of MSCs between adipogenesis and osteogenesis. This work provides insight into the future surface design of Ti dental implants using surface bioactive ion chemistry and nanotopography.

Identification and validation of potential new biomarkers for prostate cancer diagnosis and prognosis using 2D-DIGE and MS.

This study was designed to identify and validate potential new biomarkers for prostate cancer and to distinguish patients with and without biochemical relapse. Prostate tissue samples analyzed by 2D-DIGE (two-dimensional difference in gel electrophoresis) and mass spectrometry (MS) revealed downregulation of secernin-1 (P<0.044) in prostate cancer, while vinculin showed significant upregulation (P<0.001). Secernin-1 overexpression in prostate tissue was validated using Western blot and immunohistochemistry while vinculin expression was validated using immunohistochemistry. These findings indicate that secernin-1 and vinculin are potential new tissue biomarkers for prostate cancer diagnosis and prognosis, respectively. For validation, protein levels in urine were also examined by Western blot analysis. Urinary vinculin levels in prostate cancer patients were significantly higher than in urine from nontumor patients (P=0.006). Using multiple reaction monitoring-MS (MRM-MS) analysis, prostatic acid phosphatase (PAP) showed significant higher levels in the urine of prostate cancer patients compared to controls (P=0.012), while galectin-3 showed significant lower levels in the urine of prostate cancer patients with biochemical relapse, compared to those without relapse (P=0.017). Three proteins were successfully differentiated between patients with and without prostate cancer and patients with and without relapse by using MRM. Thus, this technique shows promise for implementation as a noninvasive clinical diagnostic technique.

Air atmospheric-pressure plasma-jet treatment enhances the attachment of human gingival fibroblasts for early peri-implant soft tissue seals on titanium dental implant abutments.

OBJECTIVE: Although dental implants are commonly used for tooth restoration, there is a lack of studies of treatment regimens for preventing extra-oral infection and decreasing osseointegration failures by establishing early peri-implant soft tissue seals on titanium dental implant abutments. In this study, air atmospheric-pressure plasma-jet (AAPPJ) treatment was applied to titanium disks to assay the potential for early peri-implant soft tissue seals on titanium dental implant abutment. MATERIALS AND METHODS: After titanium disks were treated with AAPPJ for 10 s at 250, 500, 1000 and 1500 sccm, surface analysis was performed; the control group received air only or no treatment. Human gingival fibroblasts (HGF) were seeded onto the specimens for evaluating cell attachment and proliferation and adherent-cell morphology was visualized via confocal microscopy. RESULTS: In AAPPJ-treated specimens, the water contact angle decreased according to increased flow rate. Oxygen composition increased in XPS, but no topographical changes were detected. The effect of AAPPJ treatment at 1000 sccm was apparent 2 mm from the treated spot, with a 20% increase in early cell attachment and proliferation. Adherent HGF on AAPPJ-treated specimens displayed a stretched phenotype with more vinculin formation than the control group. CONCLUSIONS: Within the limitations of this study, the results indicate that AAPPJ treatment may enhance the early attachment and proliferation of HGF for establishing early peri-implant soft tissue seals on titanium dental implant abutments with possible favorable effects of osseointegration of dental implant.

A comparative study of BioAggregate and ProRoot MTA on adhesion, migration, and attachment of human dental pulp cells.

INTRODUCTION: The aim of the present study was to evaluate the effects of a novel bioceramic nanoparticular cement, BioAggregate (Innovative Bioceramix, Vancouver, BC, Canada), on the adhesion, migration, and attachment of human dental pulp cells (HDPCs) and to compare its performance with that of ProRoot mineral trioxide aggregate (MTA) (Dentsply, Tulsa, OK). METHODS: Primary cultured HDPCs were treated with various dilutions of BioAggregate and MTA extracts to assess the cell viability using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Cell adhesion assay was performed using type I collagen-coated plates. An in vitro scratch wound healing model was used to determine cell migration. Focal adhesion formation and cytoskeleton organization were further examined by double immunofluorescence labeling for vinculin and fibrous actin. To assess cell attachment, HDPCs were directly seeded onto the material surfaces and observed by scanning electron microscopy. RESULTS: HDPCs exposed to BioAggregate extracts showed the highest viabilities at all extract concentrations at 24 and 48 hours, whereas cells exposed to original MTA extracts displayed suppressed viabilities at 72 hours compared with the control. Treatment with BioAggregate extracts enhanced cellular adhesion and migration of HDPCs in a concentration-dependent manner, which was superior to the effects induced by MTA extracts. Immunofluorescence staining indicated that both BioAggregate and MTA optimized focal adhesion formation and stress fiber assembly. Furthermore, scanning electron microscopic analysis revealed that HDPCs attached onto BioAggregate were more flattened and exhibited better spreading than cells on MTA. CONCLUSIONS: BioAggregate is able to promote cellular adhesion, migration, and attachment of HDPCs, indicating its excellent cytocompatibility. Therefore, BioAggregate appears to be a possible alternative to MTA for pulp capping.

CAS directly interacts with vinculin to control mechanosensing and focal adhesion dynamics.

Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS-vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS-vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS-vinculin interaction in mechanosensing and focal adhesion dynamics.

Engineering three-dimensional constructs of the periodontal ligament in hyaluronan-gelatin hydrogel films and a mechanically active environment.

BACKGROUND AND OBJECTIVE: Periodontal ligament (PDL) cells in stationary two-dimensional culture systems are in a double default state. Our aim therefore was to engineer and characterize three-dimensional constructs, by seeding PDL cells into hyaluronan-gelatin hydrogel films (80-100 µm) in a format capable of being mechanically deformed. MATERIAL AND METHODS: Human PDL constructs were cultured with and without connective tissue growth factor (CTGF) and fibroblast growth factor (FGF)-2 in (i) stationary cultures, and (ii) mechanically active cultures subjected to cyclic strains of 12% at 0.2 Hz each min, 6 h/d, in a Flexercell FX-4000 Strain Unit. The following parameters were measured: cell number and viability by laser scanning confocal microscopy; cell proliferation with the MTS assay; the expression of a panel of 18 genes using real-time RT-PCR; matrix metalloproteinases (MMPs) 1-3, TIMP-1, CTGF and FGF-2 protein levels in supernatants from mechanically activated cultures with Enzyme-linked immunosorbent assays. Constructs from stationary cultures were also examined by scanning electron microscopy and immunostained for actin and vinculin. RESULTS: Although initially randomly distributed, the cells became organized into a bilayer by day 7; apoptotic cells remained constant at approximately 5% of the total. CTGF/FGF-2 stimulated cell proliferation in stationary cultures, but relative quantity values suggested modest effects on gene expression. Two transcription factors (RUNX2 and PPARG), two collagens (COL1A1, COL3A1), four MMPs (MMP-1-3, TIMP-1), TGFB1, RANKL, OPG and P4HB were detected by gel electrophoresis and Ct values < 35. In mechanically active cultures, with the exception of P4HB, TGFB1 and RANKL, each was upregulated at some point in the time scale, as was the synthesis of MMPs and TIMP-1. SOX9, MYOD, SP7, BMP2, BGLAP or COL2A1 were not detected in either stationary or mechanically active cultures. CONCLUSION: Three-dimensional tissue constructs provide additional complexity to monolayer culture systems, and suggest some of the assumptions regarding cell growth, differentiation and matrix turnover based on two-dimensional cultures may not apply to cells in three-dimensional matrices. Primarily developed as a transitional in vitro model for studying cell-cell and cell-matrix interactions in tooth support, the system is also suitable for investigating the pathogenesis of periodontal diseases, and importantly from the clinical point of view, in a mechanically active environment.

Development of a novel liquid crystal based cell traction force transducer system.

Keratinocyte traction forces play a crucial role in wound healing. The aim of this study was to develop a novel cell traction force (CTF) transducer system based on cholesteryl ester liquid crystals (LC). Keratinocytes cultured on LC induced linear and isolated deformation lines in the LC surface. As suggested by the fluorescence staining, the deformation lines appeared to correlate with the forces generated by the contraction of circumferential actin filaments which were transmitted to the LC surface via the focal adhesions. Due to the linear viscoelastic behavior of the LC, Hooke's equation was used to quantify the CTFs by associating Young's modulus of LC to the cell induced stresses and biaxial strain in forming the LC deformation. Young's modulus of the LC was profiled by using spherical indentation and determined at approximately 87.1±17.2kPa. A new technique involving cytochalasin-B treatment was used to disrupt the intracellular force generating actin fibers, and consequently the biaxial strain in the LC induced by the cells was determined. Due to the improved sensitivity and spatial resolution (∼1µm) of the LC based CTF transducer, a wide range of CTFs was determined (10-120nN). These were found to be linearly proportional to the length of the deformations. The linear relationship of CTF-deformations was then applied in a bespoke CTF mapping software to estimate CTFs and to map CTF fields. The generated CTF map highlighted distinct distributions and different magnitude of CTFs were revealed for polarized and non-polarized keratinocytes.

Fibroblast adhesion and activation onto micro-machined titanium surfaces.

OBJECTIVES: Surface modifications performed at the neck of dental implants, in the manner of micro-grooved surfaces, can reduce fibrous tissue encapsulation and prevent bacterial colonization, thereby improving fibrointegration and the formation of a biological seal. However, the applied procedures are technically complex and/or time consuming methods. The aim of this study was to analyse the fibroblast behaviour on modified titanium surfaces obtained, applying a simple and low-cost method. MATERIAL AND METHODS: An array of titanium surfaces was obtained using a commercial computerized numerical control lathe, modifying the feed rate and the cutting depth. To elucidate the potential ability of the generated surfaces to activate connective tissue cells, a thorough gene (by real time - qPCR) and protein (by western blot or zymography) expression and cellular response characterization (cell morphology, cell adhesion and cell activation by secreting extracellular matrix (ECM) components and their enzyme regulators) was performed. RESULTS: Micro-grooved surfaces have statistically significant differences in the groove's width (approximately 10, 50 and 100 µm) depending on the applied advancing fixed speed. Field emission scanning electron microscopy images showed that fibroblasts oriented along the generated grooves, but they were only entirely accommodated on the wider grooves (≥50 µm). Micro-grooved surfaces exhibited an earlier cell attachment and activation, as seen by collagen Iα1 and fibronectin deposition and activation of ECM remodelling enzymes, compared with the other surfaces. However, fibroblasts could remain in an activated state on narrower surfaces (<50 µm) at later stages. CONCLUSIONS: The use of micro-grooved surfaces could improve implant integration at the gingival site with respect to polished surfaces. Micro-grooved surfaces enhance early fibroblast adhesion and activation, which could be critical for the formation of a biological seal and finally promote tissue integration. Surfaces with wider grooves (≥50 µm) seem to be more appropriate than surfaces with narrow grooves (<50 µm), as fibroblasts could persist in an activated state on narrower grooved surfaces, increasing the probability of producing a fibrotic response.

Initial attachment, subsequent cell proliferation/viability and gene expression of epithelial cells related to attachment and wound healing in response to different titanium surfaces.

OBJECTIVES: A tight seal between the epithelium and the dental implant surface is required to prevent bacterial inflammation and soft tissue recession and therefore to demonstrate a long-term success. Surface hydrophilicity was recently shown to promote osseointegration. The aim of this study was to investigate the influence of surface hydrophilicity in combination with surface topography of Ti implant surfaces on the behavior and activation/differentiation of epithelial cells using a set of in vitro experiments mimicking the implant-soft tissue contact. METHODS: Hydrophobic acid-etched (A) and coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and modSLA surfaces were produced. The behavior of an oral squamous cell carcinoma cell line (HSC-2) grown on all surfaces was compared through determination of cell attachment and proliferation/viability (CCK-8 and MTT assay), time-lapse microscopy of fluorescence labeled cells and determination of gene expression by real time polymerase chain reaction. RESULTS: Within the surfaces with similar wettability cell spreading and cell movements observed by time-lapse microscopy after one day of incubation were most pronounced on smoother (A and modA) surfaces compared to rougher (SLA and modSLA) surfaces. Within the surfaces with similar roughness the hydrophilic surfaces (modA and modSLA) showed more cell spreading and cell activity compared to the hydrophobic surfaces (A and SLA). The relative gene expressions of cytokeratin14, integrin α6, integrin ß4, vinculin, transforming growth factor (TGF)-ß, TGF-ß1, and TGF-ß3 were decreased in HSC-2 on all four types of Ti surfaces compared to control surfaces (tissue culture polystyrene; p<0.01) and there was no significant difference of gene expression on the four different implant-surfaces. SIGNIFICANCE: We have demonstrated that for proliferation and spreading of HSC-2 cells the smoother and hydrophilic surface is optimal (modA). These results suggest that surface hydrophilicity might positively influence the epithelial seal around dental implants. All tested titanium surfaces downregulate cell attachment, cell proliferation, expression of adhesion promoters, and cytokines involved in wound healing in HSC-2 cells compared to control surfaces.

Translucent titanium coating facilitates observation of osteoblast migration behavior on a titanium surface.

PURPOSE: The appropriate surface composition and topography are crucial for osseointegration of titanium dental implants, and surface properties are known to enhance cell adhesion and promote expression of specific osteoblastic genes. In this study, a translucent titanium coating on glass coverslip (TiGlass) was introduced as a potential tool for direct observation of cell behavior on a titanium surface. MATERIALS AND METHODS: Scanning electron microscopy, energy-dispersive x-ray analysis, and atomic force microscopy were performed on TiGlass to provide information about its physical properties. Random migration, osteoblastic gene expression, and immunofluorescence cell staining on TiGlass were also examined and analyzed. RESULTS: The translucent titanium surface offered excellent optical characteristics that facilitated transmitted light observations under an optical microscope, transforming the opaque metal into an observable titanium matrix. Random migration analysis of the primary osteoblasts on TiGlass revealed that the titanium coating enhanced the migration speed of the osteoblasts and significantly shortened the time lag for the initial migration behavior. Further study of osteoblastic gene expression on this smooth titanium surface revealed no significant changes. Co-localization of actin filament and vinculin was found on TiGlass under epifluorescent microscopy. CONCLUSION: The application of a translucent titanium-coated coverslip in vitro altered the migration pattern of osteoblasts. Collectively, the results suggest that titanium promotes initial adhesion and accelerates osteoblast migration.