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Abstract: We have investigated the stability of the surface termination of ultrananocrystalline diamond/amorphous carbon (UNCD/a-C) composite films after their modification with UV/O3 treatments or NH3-containing plasmas. The films were prepared by microwave plasma chemical vapor deposition from CH4/N2 mixtures and possess the H-termination typical for all CVD grown diamond films. The treatment with UV/O3 or NH3/N2 plasma resulted in a change of the surface termination as revealed by contact angle measurements and X-ray photoelectron spectroscopy (XPS). Both processes rendered the as-grown hydrophobic UNCD surface hydrophilic; the influence of the treatment time as well as that of the carrier gas (N2 or Ar) for the plasma process were investigated. The main task of this investigation was to determine the stability of the surface termination with storage time and storage conditions and upon exposure to standard organic solvents, like acetone, ethanol and i-propanol. It was found that for the UV/O3 treated UNCD surfaces the storage in air, and after NH3/N2 plasma modification package under reduced pressure preserved to a great extent the achieved hydrophilic properties in a period up to three months. The changes that occur predominantly during the first days after the modification step were due mostly to interchange of surface groups with the ambient. Similar processes were observed after exposure of the UNCD surfaces to standard organic solvents. [Copyright &y& Elsevier]

Abstract: Ultrananocrystalline diamond/amorphous carbon nanocomposite films (UNCD/a-C) have been deposited by microwave plasma chemical vapour deposition at 600°C from 17% CH4/N2 mixtures. The as-grown films turned out to be hydrogen terminated and very stable. Photochemical amination of H-terminated diamond is a well-established route to attach functional groups to such surfaces for applications in biosensors. Here we report on experiments to aminate UNCD surfaces directly by exposure to ammonia plasmas. Thereafter the surfaces were reacted with the heterobifunctional crosslinker molecule SSMCC bearing a N-hydroxysuccinimide (NHS) ester group which should react with the surface NH2 groups. By means of X-ray photoelectron spectroscopy (XPS), contact angle measurements and fluorescence microscopy it is shown that both steps, plasma amination and SSMCC attachment lead to the desired aims. On the other hand, experiments to attach a thiol-bearing fluorescein molecule directly to H-terminated UNCD films turned out to be partially successful although according to literature such a reaction should be very unlikely. [Copyright &y& Elsevier]

Abstract: Amorphous thin films from the system As2Se3–Ag4SSe–SnTe were prepared by thermal vacuum evaporation from the corresponding bulk glassy samples. The film structure and surface morphology were investigated by scanning electron microscopy and atomic force microscopy; the results revealed uniform, smooth and homogeneous coatings. The amorphous chalcogenide films are transparent in a wide spectral range as shown by transmission and reflection measurements in the VIS and NIR regions. The optical band gap was determined and its compositional dependence is discussed in terms of structural considerations and the formation of charged defect centers. [Copyright &y& Elsevier]

Highlights: [•] UNCD and DLC films were modified by UV/O3 treatments, O2 or NH3-containing plasmas. [•] Surface composition, wettability and surface energy change upon modifications. [•] Higher efficiency of UNCD modifications was observed. [•] Cell attachment and growth were influenced by the surface termination and roughness. [ABSTRACT FROM AUTHOR]

Abstract: One-dimensional diamond nanostructures (diamond nanopillars) have been fabricated using nanocrystalline diamond films (NCD) as a starting material, and electron beam lithography (EBL) and reactive ion etching in an inductively coupled O2 plasma (ICP-RIE) as processing techniques. In a first step, the etch rates have been determined as a function of four major plasma parameters, namely the ICP power, the rf power applied to the substrate holder, the pressure, and the oxygen flow rate. These parameters have been varied in wide ranges. In order to get insight into the mechanisms of the etching process, etching experiments have been performed with unpatterned NCD films by varying the process times using rather short intervals. Finally, EBL has been applied prior to the etching to obtain one-dimensional pillars with diameters from 200nm to 1μm. Scanning electron microscopy has been employed to characterize the pillars. First results showed the process developed to be successful, and first examples will be presented. [Copyright &y& Elsevier]

Abstract: Ultrananocrystalline diamond (UNCD) films have been deposited by microwave plasma chemical vapor deposition from 17% CH4/N2 mixtures. In order to change the original hydrogen termination of the UNCD surfaces, the films have subsequently been subjected either to the so-called UV/O3 treatment which leads to OH-terminated surfaces, or to NH3/N2 plasmas which introduces NH2 groups but also a certain amount of OH groups. These three types of surfaces have been characterized by X-ray photoelectron spectroscopy, contact angle and ζ-potential measurements. The contact angle measurements have shown that as-grown UNCD surfaces are highly hydrophobic but became highly hydrophilic after both treatments. The ζ-potential measurements revealed that the isoelectric point of the H-terminated as-grown surface is distinctively higher than that of either UV/O3 or NH3/N2 plasma treated surfaces. Finally, the interactions of these surfaces with neurons of the cockroach Leucophaea maderae have been investigated. These studies have shown that especially the two treated surfaces allow for a fast, strong attachment of these cells without compromising their viability and without changing their normal physiological responses. These results will be discussed in terms of those obtained with the different surface characterization techniques. [Copyright &y& Elsevier]

Abstract: Transformation from higher to ultra low friction coefficient was observed in ultrananocrystalline diamond film (UNCD) while changing the test atmospheric conditions. High friction coefficients were observed in dry argon and nitrogen atmosphere, however, low and ultra low friction coefficients were obtained in dry oxygen and in ambient atmospheric conditions, respectively. Wear rates follow the same trends as the friction coefficients. This fascinating behavior of friction and wear of UNCD film is explained by the chemical changes of sliding surfaces and extent of passivation of dangling covalent bonds. [Copyright &y& Elsevier]

Abstract: Thin (AsSe)100−x Ag x films have been grown onto quartz substrates by vacuum thermal evaporation or pulsed laser deposition from the corresponding bulk materials. The amorphous character of the coatings was confirmed by X-ray diffraction investigations. Their transmission was measured within the wavelength range 400–2500nm and the obtained spectra were analyzed by the Swanepoel method to derive the optical band gap E g and the refractive index n. We found that both parameters are strongly influenced by the addition of silver to the glassy matrix: E g decreases while n increases with Ag content. These variations are discussed in terms of the changes in the atomic and electronic structure of the materials as a result of silver incorporation. [Copyright &y& Elsevier]

Abstract: Amorphous thin films from the As2Se3–Ag4SSe–SnTe system were prepared by thermal vacuum evaporation from the corresponding bulk glasses. Their transmission, reflection, scattering and ellipsometric spectra were measured in the UV, VIS and NIR regions, and the results were mathematically simulated using the Tauc–Lorentz dispersion model in order to obtain the refractive index and extinction coefficient. The compositional dependence of the derived optical properties was found and discussed. [Copyright &y& Elsevier]

Thin amorphous Ge–S–AgI films were thermally evaporated on cantilever sensors and their sorption properties were investigated upon exposure to volatile analytes, such as water, ethanol, acetone, and ammonia vapours. The films were smooth and uniform in thickness as revealed by atomic force and scanning electron microscopies. The exposure to the analytes resulted in a change of the resonance frequency of the cantilever. Initially, the largest dynamic responses (frequency shifts) were observed towards acetone, i.e. the cantilever acted as a resonant microbalance. When the sensor was exposed to ammonia, its molecules were chemisorbed on the surface of the sensitive layer. This surface modification increased the sensor sensitivity towards water molecules by the creation of new interaction sites. [ABSTRACT FROM AUTHOR]

Nitrogen-vacancy (NV) color centers in diamond are excellent quantum sensors possessing high sensitivity and nano-scale spatial resolution. Their integration in photonic structures is often desired, since it leads to an increased photon emission and also allows the realization of solid-state quantum technology architectures. Here, we report the fabrication of diamond nano-pillars with diameters up to 1000 nm by electron beam lithography and inductively coupled plasma reactive ion etching in nitrogen-rich diamonds (type Ib) with [100] and [111] crystal orientations. The NV centers were created by keV-He ion bombardment and subsequent annealing, and we estimate an average number of NVs per pillar to be 4300 ± 300 and 520 ± 120 for the [100] and [111] samples, respectively. Lifetime measurements of the NVs' excited state showed two time constants with average values of τ 1 ≈ 2 ns and τ 2 ≈ 8 ns, which are shorter as compared to a single color center in a bulk crystal (τ ≈ 10 ns). This is probably due to a coupling between the NVs as well as due to interaction with bombardment-induced defects and substitutional nitrogen (P1 centers). Optically detected magnetic resonance measurements revealed a contrast of about 5% and average coherence and relaxation times of T 2 [100] = 420 ± 40 ns, T 2 [111] = 560 ± 50 ns, and T 1 [100] = 162 ± 11 μs, T 1 [111] = 174 ± 24 μs. These pillars could find an application for scanning probe magnetic field imaging.

Successful osseointegration, i.e. the fully functional connection of patient's bone and artificial implant depends on the response of the cells to the direct contact with the surface of the implant. The surface properties of the implant which trigger cell responses leading to its integration into the surrounding bone can be tailored by surface modifications or coating with thin layers. One potential material for such applications is ultrananocrystalline diamond (UNCD). It combines the exceptional mechanical properties of diamond with good biocompatibility and possibility of coating as thin uniform films on different substrates of biological interest. In the current work we firstly deposited UNCD films on titanium-coated substrates and applied oxygen or ammonia plasma to modify their surface properties. The as-grown and modified UNCD exhibited relatively smooth surfaces with topography dominated by rounded features. The modifications induced oxygen- or amino-terminated surfaces with increased hydrophilicity. In addition, the UNCD coatings exhibited very low coefficient of friction when diamond was used as a counterpart. As-grown and modified UNCD samples were applied to study the responses of human osteoblast MG63 cells triggered by surfaces with various terminations assessed by proteomic analysis. The results revealed that the coating of Ti with UNCD as well as the plasma modifications resulting in O- or NH 2 -terminated UNCD induced upregulation of proteins specific for cytoskeleton, cell membrane, and extracellular matrix (ECM) involved in the cell-ECM-surface interactions. Proteins from each of these groups, namely, vimentin, cadherin and fibronectin were further studied immunocytochemically and the results confirmed their increased abundance leading to improved cell-to-surface adhesion and cell-to-cell interactions. These findings demonstrate the potential of implant coating with UNCD and its surface modifications for better osseointegration and bone formation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The development of quantum technologies is one of the big challenges in modern research. A crucial component for many applications is an efficient, coherent spin-photon interface, and coupling single-color centers in thin diamond membranes to a microcavity is a promising approach. To structure such micrometer thin single-crystal diamond (SCD) membranes with a good quality, it is important to minimize defects originating from polishing or etching procedures. Here, we report on the fabrication of SCD membranes, with various diameters, exhibiting a low surface roughness down to 0.4 nm on a small area scale, by etching through a diamond bulk mask with angled holes. A significant reduction in pits induced by micromasking and polishing damages was accomplished by the application of alternating Ar/Cl 2 + O 2 dry etching steps. By a variation of etching parameters regarding the Ar/Cl 2 step, an enhanced planarization of the surface was obtained, in particular, for surfaces with a higher initial surface roughness of several nanometers. Furthermore, we present the successful bonding of an SCD membrane via van der Waals forces on a cavity mirror and perform finesse measurements which yielded values between 500 and 5000, depending on the position and hence on the membrane thickness. Our results are promising for, e.g., an efficient spin-photon interface.

We introduce and demonstrate a new approach for nitrogen-vacancy (NV) patterning in diamond, achieving a deterministic, nanometer-thin, and dense delta-doped layer of negatively charged NV centers in diamond. We employed a pure nitridation stage using microwave plasma and a subsequent in situ diamond overgrowth. We present the highest reported nitrogen concentration in a delta-doped layer (1.8 × 10 20 cm -3 ) while maintaining the pristine diamond crystal quality. This result combined with the large optically detected magnetic resonance contrast can pave the way toward highly sensitive NV-based magnetometers. We further employed this delta-doping technique on high-quality fabricated diamond nanostructures for realizing a topographic NV patterning in order to enhance the sensing and hyperpolarization capabilities of NV-based devices.

Fly ash (FA) from lignite coal combusted in different Thermal Power Plants (TPPs) was used for the synthesis of zeolites (FAZs) of the Na-X type by alkaline activation via three laboratory procedures. FAZs were characterized with respect to their morphology, phase composition and surface properties, which predetermine their suitability for applications as catalysts and adsorbents. FAZs were subsequently modified with metal oxides (CuO) to improve their catalytic properties. The catalytic activity of non-modified and CuO-modified FAZs in the total oxidation of volatile organic compounds was investigated. FAZs were studied for their potential to retain CO 2 , as their favorable surface characteristics and the presence of iron oxides make them suitable for carbon capture technologies. Thin films of FAZs were deposited by in situ crystallization, and investigated for their morphology and optical sensitivity when exposed to pollutants in the gas phase, e.g., acetone. This study contributes to the development of novel technological solutions for the smart and valuable utilization of FA in the context of the circular economy and green energy production.

Transplantation studies have pinpointed the circadian clock of the Madeira cockroach to the accessory medulla (AME) of the brain's optic lobes. The AME is innervated by approximately 240 adjacent neuropeptidergic neurons, including 12 pigment-dispersing factor (PDF)-expressing neurons anterior to the AME (aPDFMEs). Four of the aPDFMEs project contralaterally, controlling locomotor activity rhythms of the night-active cockroach. The present in vitro Ca 2+ imaging analysis focuses on contralaterally projecting AME neurons and their responses to PDF, GABA, and acetylcholine (ACh). First, rhodamine-dextran backfills from the contralateral optic stalk identified contralaterally projecting AME neurons, which were then dispersed in primary cell cultures. After characterization of PDF, GABA, and ACh responses, PDF immunocytochemistry identified ipsilaterally and contralaterally projecting PDFMEs. All PDF-sensitive clock neurons, PDF-immunoreactive clock neurons, and the majority of ipsilaterally and contralaterally projecting cells were excited by ACh. GABA inhibited all PDF-expressing clock neurons, and about half of other ipsilaterally projecting and most contralaterally projecting clock neurons. For the first time, we identified PDF autoreceptors in PDF-secreting cockroach circadian pacemakers. The medium-sized aPDFMEs and all other contralaterally projecting PDF-sensitive clock cells were inhibited by PDF. The ipsilaterally remaining small PDF-sensitive clock cells were activated by PDF. Only the largest aPDFME did not express PDF autoreceptors. We hypothesize that opposing PDF signaling generates 2 different ensembles of clock cells with antiphasic activity, regulating and maintaining a constant phase relationship between rest and activity cycles of the night-active cockroach.

Diamond is a promising material for a number of bio-applications, including the fabrication of platforms for attachment and investigation of neurons and of neuroprostheses, such as retinal implants. In the current work ultrananocrystalline diamond (UNCD) films were deposited by microwave plasma chemical vapor deposition, modified by UV/O3 treatment or NH3 plasma, and comprehensively characterized with respect to their bulk and surface properties, such as crystallinity, topography, composition and chemical bonding nature. The interactions of insect circadian pacemaker neurons with UNCD surfaces with H-, O- and NH2-terminations were investigated with respect to cell density and viability. The fast and strong attachment achieved without application of adhesion proteins allowed for advantageous modification of dispersion protocols for the preparation of primary cell cultures. Centrifugation steps, which are employed for pelletizing dispersed cells to separate them from dispersing enzymes, easily damage neurons. Now centrifugation can be avoided since dispersed neurons quickly and strongly attach to the UNCD surfaces. Enzyme solutions can be easily washed off without losing many of the dispersed cells. No adverse effects on the cell viability and physiological responses were observed as revealed by calcium imaging. Furthermore, the enhanced attachment of the neurons, especially on the modified UNCD surfaces, was especially advantageous for the immunocytochemical procedures with the cell cultures. The cell losses during washing steps were significantly reduced by one order of magnitude in comparison to controls. In addition, the integration of a titanium grid structure under the UNCD films allowed for individual assignment of physiologically characterized neurons to immunocytochemically stained cells. Thus, employing UNCD surfaces free of foreign proteins improves cell culture protocols and immunocytochemistry with cultured cells. The fast and strong attachment of neurons was attributed to a favorable combination of topography, surface chemistry and wettability., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Osteomyelitis is a serious complication in oral and maxillofacial surgery affecting bone healing. Bone remodeling is not only controlled by cellular components but also by ionic and molecular composition of the extracellular fluids in which calcium phosphate salts are precipitated in a pH dependent manner., Objective: To determine the effect of pH on self-renewal, osteogenic differentiation and matrix mineralization of mesenchymal stem cells (MSCs)., Methods: We selected three different pH values; acidic (6.3, 6.7), physiological (7.0-8.0) and severe alkaline (8.5). MSCs were cultured at different pH ranges, cell viability measured by WST-1, apoptosis detected by JC-1, senescence was analyzed by β-galactosidase whereas mineralization was detected by Alizarin Red and osteogenic differentiation analyzed by Real-time PCR., Results: Self-renewal was affected by pH as well as matrix mineralization in which pH other than physiologic inhibited the deposition of extracellular matrix but did not affect MSCs differentiation as osteoblast markers were upregulated. The expression of osteocalcin and alkaline phosphatase activity was upregulated whereas osteopontin was downregulated under acidic pH., Conclusion: pH affected MSCs self-renewal and mineralization without influencing osteogenic differentiation. Thus, future therapies, based on shifting acid-base balance toward the alkaline direction might be beneficial for prevention or treatment of osteomyelitis., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Tendon tissues, due to their composition and function, are prone to suffer age-related degeneration and diseases as well as to respond poorly to current repair strategies. It has been suggested that local stem cells, named tendon stem/progenitor cells (TSPCs), play essential roles in tendon maintenance and healing. Recently, we have shown that TSPC exhibit a distinct age-related phenotype involving transcriptomal shift, poor self-renewal, and elevated senescence coupled with reduced cell migration and actin dynamics. Here, we report for the first time the significant downregulation of the ephrin receptors EphA4, EphB2 and B4 and ligands EFNB1 in aged-TSPC (A-TSPC). Rescue experiments, by delivery of target-specific clustered proteins, revealed that activation of EphA4- or EphB2-dependent reverse signaling could restore the migratory ability and normalize the actin turnover of A-TSPC. However, only EphA4-Fc stimulation improved A-TSPC cell proliferation to levels comparable to young-TSPC (Y-TSPC). Hence, our novel data suggests that decreased expression of ephrin receptors during tendon aging and degeneration limits the establishment of appropriate cell-cell interactions between TSPC and significantly diminished their proliferation, motility, and actin turnover. Taken together, we could propose that this mechanism might be contributing to the inferior and delayed tendon healing common for aged individuals.

Complementary to parameters established for cell-adhesion force curve analysis, we evaluated the slope before a force step together with the distance from the surface at which the step occurs and visualized the result in a two-dimensional density plot. This new tool allows detachment steps of long membrane tethers to be distinguished from shorter jumplike force steps, which are typical for cytoskeleton-anchored bonds. A prostate cancer cell line (PC3) immobilized on an atomic-force-microscopy sensor interacted with three different substrates: collagen-I (Col-I), bovine serum albumin, and a monolayer of bone marrow-derived stem cells (SCP1). To address PC3 cells' predominant Col-I binding molecules, an antibody-blocking β1-integrin was used. Untreated PC3 cells on Col-I or SCP1 cells, which express Col-I, predominantly showed jumps in their force curves, while PC3 cells on bovine-serum-albumin- and antibody-treated PC3 cells showed long membrane tethers. The probability density plots thus revealed that β1-integrin-specific interactions are predominately anchored to the cytoskeleton, while the nonspecific interactions are mainly membrane-anchored. Experiments with latrunculin-A-treated PC3 cells corroborated these observations. The plots thus reveal details of the anchoring of bonds to the cell and provide a better understanding of receptor-ligand interactions., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Tendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but also their healing. Despite of the acknowledged importance of the mechanical stress for tendon function and repair, the mechanotransduction mechanisms in tendon cells are still unclear and the elucidation of these mechanisms is a key goal in tendon research. Tendon stem/progenitor cells (TSPC) possess common adult stem cell characteristics, and are suggested to actively participate in tendon development, tissue homeostasis as well as repair. This makes them an important cell population for tendon repair, and also an interesting research target for various open questions in tendon cell biology. Therefore, in our study we focused on TSPC, subjected them to five different mechanical protocols, and investigated the gene expression changes by using semi-quantitative, quantitative PCR and western blotting technologies., Results: Among the 25 different genes analyzed, we can convincingly report that the tendon-related genes - fibromodulin, lumican and versican, the collagen I-binding integrins - α1, α2 and α11, the matrix metalloproteinases - MMP9, 13 and 14 were strongly upregulated in TSPC after 3 days of mechanical stimulation with 8% amplitude. Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC., Conclusions: Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC. Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.

Tenomodulin (Tnmd) is a well-known gene marker for the tendon and ligament lineage, but its exact functions in these tissues still remain elusive. In this study, we investigated Tnmd loss of function in mouse tendon stem/progenitor cells (mTSPC) by implicating a previously established Tnmd knockout (KO) mouse model. mTSPC were isolated from control and Tnmd KO tail tendons and their stemness features, such as gene marker profile, multipotential, and self-renewal, were compared. Immunofluorescence and reverse transcriptase-polymerase chain reaction analyses for stem cell-, tenogenic-, osteogenic-, and chondrogenic-related genes confirmed their stemness and lineage specificity and demonstrated no profound differences between the two genotypes. Multipotential was not significantly affected since both cell types differentiated successfully into adipogenic, osteogenic, and chondrogenic lineages. In contrast, self-renewal assays validated that Tnmd KO TSPC exhibit significantly reduced proliferative potential, which was also reflected in lower Cyclin D1 levels. When analyzing possible cellular mechanisms behind the observed decreased self-renewability of Tnmd KO TSPC, we found that cellular senescence plays a major role, starting earlier and cumulating more in Tnmd KO compared with control TSPC. This was accompanied with augmented expression of the cell cycle inhibitor p53. Finally, the proliferative effect of Tnmd in TSPC was confirmed with transient transfection of Tnmd cDNA into Tnmd KO TSPC, which rescued their proliferative deficit. Taken together, we can report that loss of Tnmd affects significantly the self-renewal and senescence properties, but not the multipotential of TSPC.

Fractures to the osteoporotic bone feature a delay in callus formation and reduced enchondral ossification. Human mesenchymal stem cells (hMSC), the cellular source of fracture healing, are recruited to the fracture site by cytokines, such as BMP-2 and BMP-7. Aim of the study was to scrutinize hMSC for osteoporosis associated alterations in BMP mediated migration and invasion as well as in extracellular matrix (ECM) binding integrin expression. HMSC were isolated from 18 healthy or osteoporotic donors. Migration was assessed using a collagen IV coated micro-slide linear gradient chamber and time-lapse microscopy. Invasion was analyzed utilizing an ECM coated transmembrane invasion assay. Quantitative real-time RT PCR was performed for the ECM binding integrins α1, α2, α3, α4, α5, α11, αv and β1. HMSC from osteoporotic patients showed a significant increase of migration upon BMP-2 or FCS stimulation, as well as a significant increase of invasion upon BMP-2, BMP-7 or FCS stimulation. Nevertheless, the migration and invasion capacity was significantly decreased compared to healthy controls. Out of all integrins analyzed, collagen binding integrin α2 was significantly downregulated in hMSC from osteoporotic patients. In conclusion, we here demonstrate for the first time osteoporosis associated alterations in BMP mediated hMSC recruitment. These findings may underlie the reduced healing of osteoporotic fractures. Nevertheless, the maintained migration and invasion response upon BMP stimulation illustrates the therapeutic potential of these clinically approved substances in the treatment of osteoporotic fractures. Another therapeutic target may be the downregulation of the collagen binding integrin α2 in hMSC from osteoporotic patients., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Integrins are cell surface receptors that connect extracellular matrix (ECM) components to the actin cytoskeleton and transmit chemical and mechanical signals into the cells through adhesion complexes. Integrin-activated downstream pathways have been implicated in the regulation of various cellular functions, including proliferation, survival, migration, and differentiation. Integrin-based attachment to the matrix plays a central role in development, tissue morphogenesis, adult tissue homeostasis, remodeling and repair, and disturbance of the ECM-integrin-cytoskeleton signaling axis often results in diseases and tissue dysfunction. Increasing amount of in vitro and in vivo evidences suggest that integrins are pivotal for proper development, function, and regeneration of skeletal tissues. In this paper, we will summarize and discuss the role of integrins in skeletogenesis and their influence on the physiology and pathophysiology of cartilage, bone, and tendon., (Copyright © 2014 Wiley Periodicals, Inc.)

Although the link between altered stem cell properties and tissue aging has been recognized, the molecular and cellular processes of tendon aging have not been elucidated. As tendons contain stem/progenitor cells (TSPC), we investigated whether the molecular and cellular attributes of TSPC alter during tendon aging and degeneration. Comparing TSPC derived from young/healthy (Y-TSPC) and aged/degenerated human Achilles tendon biopsies (A-TSPC), we observed that A-TSPC exhibit a profound self-renewal and clonogenic deficits, while their multipotency was still retained. Senescence analysis showed a premature entry into senescence of the A-TSPC, a finding accompanied by an upregulation of p16(INK4A). To identify age-related molecular factors, we performed microarray and gene ontology analyses. These analyses revealed an intriguing transcriptomal shift in A-TSPC, where the most differentially expressed probesets encode for genes regulating cell adhesion, migration, and actin cytoskeleton. Time-lapse analysis showed that A-TSPC exhibit decelerated motion and delayed wound closure concomitant to a higher actin stress fiber content and a slower turnover of actin filaments. Lastly, based on the expression analyses of microarray candidates, we suggest that dysregulated cell-matrix interactions and the ROCK kinase pathway might be key players in TSPC aging. Taken together, we propose that during tendon aging and degeneration, the TSPC pool is becoming exhausted in terms of size and functional fitness. Thus, our study provides the first fundamental basis for further exploration into the molecular mechanisms behind tendon aging and degeneration as well as for the selection of novel tendon-specific therapeutical targets., (© 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Adhesion of metastasizing prostate carcinoma cells was quantified for two carcinoma model cell lines LNCaP (lymph node-specific) and PC3 (bone marrow-specific). By time-lapse microscopy and force spectroscopy we found PC3 cells to preferentially adhere to bone marrow-derived mesenchymal stem cells (SCP1 cell line). Using atomic force microscopy (AFM) based force spectroscopy, the mechanical pattern of the adhesion to SCP1 cells was characterized for both prostate cancer cell lines and compared to a substrate consisting of pure collagen type I. PC3 cells dissipated more energy (27.6 aJ) during the forced de-adhesion AFM experiments and showed significantly more adhesive and stronger bonds compared to LNCaP cells (20.1 aJ). The characteristic signatures of the detachment force traces revealed that, in contrast to the LNCaP cells, PC3 cells seem to utilize their filopodia in addition to establish adhesive bonds. Taken together, our study clearly demonstrates that PC3 cells have a superior adhesive affinity to bone marrow mesenchymal stem cells, compared to LNCaP. Semi-quantitative PCR on both prostate carcinoma cell lines revealed the expression of two Col-I binding integrin receptors, α1β1 and α2β1 in PC3 cells, suggesting their possible involvement in the specific interaction to the substrates. Further understanding of the exact mechanisms behind this phenomenon might lead to optimized therapeutic applications targeting the metastatic behavior of certain prostate cancer cells towards bone tissue.

Tendons and ligaments (T/L) are dense connective tissues of mesodermal origin. During embryonic development, the tendon-specific cells descend from a sub-set of mesenchymal progenitors condensed in the syndetome, a dorsolateral domain of the sclerotome. These cells are defined by the expression of the transcription factor scleraxis (Scx), which regulates tendon formation and several other characteristic genes, such as collagen type I, decorin, fibromodulin, and tenomodulin (Tnmd). In contrast to other mesenchymal progenitors, the genealogy and biology of the tenogenic lineage is not yet fully understood due to the lack of simple and efficient protocols enabling generation of progenitors in vitro. Here, we investigated whether the expression of Scx can lead to the direct commitment of mesenchymal stem cells (MSCs) into tendon progenitors. First, MSC derived from human bone marrow (hMSC) were lentivirally transduced with FLAG-Scx cDNA to establish 2 clonal cell lines, hMSC-Scx and hMSC-Mock. Subsequent to Scx transduction, hMSC underwent cell morphology change and had significantly reduced proliferation and clonogenicity. Gene expression analysis demonstrated that collagen type I and several T/L-related proteoglycans were upregulated in hMSC-Scx cells. When stimulated toward 3 different mesenchymal lineages, hMSC-Scx cells failed to differentiate into chondrocytes and osteoblasts, whereas adipogenic differentiation still occurred. Lastly, we detected a remarkable upregulation of the T/L differentiation gene Tnmd in hMSC-Scx. From these results, we conclude that Scx delivery results in the direct programming of hMSC into tendon progenitors and that the newly generated hMSC-Scx cell line can be a powerful and useful tool in T/L research., (© Mary Ann Liebert, Inc.)

Periodontal ligament (PDL) can be obtained from patients undergoing orthodontic treatment. PDL contains progenitor cells that can be expanded and differentiated towards several mesenchymal lineages in vitro. Furthermore, PDL-derived cells have been shown to generate bone- and PDL-like structures in vivo. Thus, PDL cells, combined with suitable biomaterials, represent a promising tool for periodontitis-related research and PDL engineering. Here, a new PDL cell line using lentiviral gene transfer of human telomerase reverse transcriptase (hTERT) was created. HTERT-expressing PDL cells showed similar morphology and population doubling time but an extended lifespan compared to the primary cells. In addition, PDL-hTERT cells expressed several characteristic genes and upon osteogenic stimulation produced a calcified matrix in vitro. When cultivated on two topographically different titanium scaffolds (MA and SLA), PDL-hTERT cells exhibited augmented spreading, survival and differentiation on smooth (MA) compared to rough (SLA) surfaces. These findings differ from previously reported osteoblast behaviour, but they are in agreement with the behaviour of chondrocytes and gingival fibroblasts, suggesting a very cell type-specific response to different surface textures. In summary, we report the testing of titanium biomaterials using a new PDL-hTERT cell line and propose this cell line as a useful model system for periodontitis research and development of novel strategies for PDL engineering.

Mesenchymal stem cells (MSCs) can contribute to tissue repair by actively migrating to sites of tissue injury. However, the cellular and molecular mechanisms of MSC recruitment are largely unknown. The nuclear factor (NF)-kappaB pathway plays a pivotal role in regulating genes that influence cell migration, cell differentiation, inflammation, and proliferation. One of the major cytokines released at sites of injury is tumor necrosis factor-alpha (TNF-alpha), which is known to be a key regulator of the NF-kappaB pathway. Therefore, we hypothesized that TNF-alpha may lead to MSC invasion and proliferation by activation of the NF-kappaB pathway. TNF-receptor 1 and 2, NF-kappaB (p65), and IkappaB kinase 2 (IKK-2) are expressed in human MSCs (hMSCs). Stimulation of hMSCs with TNF-alpha caused a p65 translocation from the cytoplasm to nucleoplasm but did not change the expression profile of MSC markers. TNF-alpha strongly augmented the migration of hMSCs through the human extracellular matrix. Using lentiviral gene transfer, overexpressing a dominant-negative mutant of IKK-2 (dn-IKK-2) significantly blocked this effect. NF-kappaB target genes associated with migration (vascular cell adhesion molecule-1, CD44, and matrix metalloproteinase 9) were upregulated by TNF-alpha stimulation and blocked by dn-IKK-2. Moreover, using the bromodeoxyuridine assay, we showed that the inhibition of the NF-kappaB pathway caused a significant reduction in the basal proliferation rate. TNF-alpha stimulated the proliferation of hMSCs, whereas overexpression of dn-IKK-2 significantly blocked this effect. TNF-alpha led to the upregulated expression of the proliferation-associated gene cyclin D1. In conclusion, we demonstrated that the NF-kappaB pathway components, p65 and IKK-2, are expressed in hMSCs. Our data provide evidence that this signal transduction pathway is implicated in TNF-alpha-mediated invasion and proliferation of hMSCs. Therefore, hMSC recruitment to sites of tissue injury may, at least in part, be regulated by the NF-kappaB signal transduction pathway.

Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chondrogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

Within the bone lie several different cell types, including osteoblasts (OBs) and mesenchymal stem cells (MSCs). The MSCs are ideal targets for regenerative medicine of bone due to their differentiation potential towards OBs. Human MSCs exhibit two distinct morphologies: rapidly self-renewing cells (RS) and flat cells (FC) with very low proliferation rates. Another cell type found in pathological bone conditions is osteosarcoma. In this study, we compared the topographic and morphometric features of RS and FC cells, human OBs and MG63 osteosarcoma cells by atomic force microscopy (AFM). The results demonstrated clear differences: FC and hOB cells showed similar ruffled topography, whereas RS and MG63 cells exhibited smoother surfaces. Furthermore, we investigated how selected substrates influence cell morphometry. We found that RS and MG63 cells were flatter on fibrous substrates such as polystyrene and collagen I, but much more rounded on glass, the smoothest surface. In contrast, cells with large area, namely FC and hOB cells, did not exhibit pronounced changes in flatness with regards to the different substrates. They were, however, remarkably flatter in comparison to RS and MG63 cells. We could explain the differences in flatness by the extent of adhesion. Indeed, FC and hOB cells showed much higher content of focal adhesions. Finally, we used the AFM to determine the cellular Young's modulus. RS, FC and hOB cells showed comparable stiffness on the three different substrates, while MG63 cells demonstrated the unique feature of increased elasticity on collagen I. In summary, our results show, for the first time, a direct comparison between the morphometric and biophysical features of different human cell types derived from normal and pathological bone. Our study manifests the opinion that along with RNA, proteomic and functional research, morphological and biomechanical characterization of cells also reveals novel cell features and interrelationships.

The identification of mesenchymal stem cells (MSCs) in adult human tissues and the disclosure of their self-renew-al and multi-lineage differentiation capabilities have provided exciting prospects for cell-based regeneration and tis-sue engineering. Although a considerable amount of data is available describing MSCs, there is still lack of information regarding the molecular mechanisms that govern their adhesion and migration. In this work, we will review the current state of knowledge on integrins and other adhesion molecules found to be expressed on MSCs. The discussed topics include the characteristics of MSCs and their clinical applications, integrins and their central role in cell-matrix attachment and migration, and comments on mobilization, differentiation and contribution to tumour development. Finally, by understanding the complex and fundamental pathways by which MSCs attach and migrate, it might be possible to fine-tune the strategies for effective and safe use of MSCs in regenerative therapies.