The Impact of Ultrashort Pulse Laser Structuring of Metals on In-Vitro Cell Adhesion of Keratinocytes.

Besides the need for biomaterial surface modification to improve cellular attachment, laser-structuring is favorable for designing a new surface topography for external bone fixator pins or implants. The principle of this study was to observe how bioinspired (deer antler) laser-induced nano-microstructures influenced the adhesion and growth of skin cells. The goal was to create pins that allow the skin to attach to the biomaterial surface in a bacteria-proof manner. Therefore, typical fixator metals, steel, and titanium alloy were structured using ultrashort laser pulses, which resulted in periodical nano- and microstructures. Surface characteristics were investigated using a laser scanning microscope and static water contact angle measurements. In vitro studies with human HaCaT keratinocytes focused on cell adhesion, morphology, actin formation, and growth within 7 days. The study showed that surface functionalization influenced cell attachment, spreading, and proliferation. Micro-dimple clusters on polished bulk metals (DC20) will not hinder viability. Still, they will not promote the initial adhesion and spreading of HaCaTs. In contrast, additional nanostructuring with laser-induced periodic surface structures (LIPSS) promotes cell behavior. DC20 + LIPSS induced enhanced cell attachment with well-spread cell morphology. Thus, the bioinspired structures exhibited a benefit in initial cell adhesion. Laser surface functionalization opens up new possibilities for structuring, and is relevant to developing bioactive implants in regenerative medicine.

Synergistic effect of cold gas plasma and experimental drug exposure exhibits skin cancer toxicity in vitro and in vivo.

INTRODUCTION: Skin cancer is often fatal, which motivates new therapy avenues. Recent advances in cancer treatment are indicative of the importance of combination treatments in oncology. Previous studies have identified small molecule-based therapies and redox-based technologies, including photodynamic therapy or medical gas plasma, as promising candidates to target skin cancer. OBJECTIVE: We aimed to identify effective combinations of experimental small molecules with cold gas plasma for therapy in dermato-oncology. METHODS: Promising drug candidates were identified after screening an in-house 155-compound library using 3D skin cancer spheroids and high content imaging. Combination effects of selected drugs and cold gas plasma were investigated with respect to oxidative stress, invasion, and viability. Drugs that had combined well with cold gas plasma were further investigated in vascularized tumor organoids in ovo and a xenograft mouse melanoma model in vivo. RESULTS: The two chromone derivatives Sm837 and IS112 enhanced cold gas plasma-induced oxidative stress, including histone 2A.X phosphorylation, and further reduced proliferation and skin cancer cell viability. Combination treatments of tumor organoids grown in ovo confirmed the principal anti-cancer effect of the selected drugs. While one of the two compounds exerted severe toxicity in vivo, the other (Sm837) resulted in a significant synergistic anti-tumor toxicity at good tolerability. Principal component analysis of protein phosphorylation profiles confirmed profound combination treatment effects in contrast to the monotherapies. CONCLUSION: We identified a novel compound that, combined with topical cold gas plasma-induced oxidative stress, represents a novel and promising treatment approach to target skin cancer.

Discrimination between the effects of pulsed electrical stimulation and electrochemically conditioned medium on human osteoblasts.

BACKGROUND: Electrical stimulation is used for enhanced bone fracture healing. Electrochemical processes occur during the electrical stimulation at the electrodes and influence cellular reactions. Our approach aimed to distinguish between electrochemical and electric field effects on osteoblast-like MG-63 cells. We applied 20 Hz biphasic pulses via platinum electrodes for 2 h. The electrical stimulation of the cell culture medium and subsequent application to cells was compared to directly stimulated cells. The electric field distribution was predicted using a digital twin. RESULTS: Cyclic voltammetry and electrochemical impedance spectroscopy revealed partial electrolysis at the electrodes, which was confirmed by increased concentrations of hydrogen peroxide in the medium. While both direct stimulation and AC-conditioned medium decreased cell adhesion and spreading, only the direct stimulation enhanced the intracellular calcium ions and reactive oxygen species. CONCLUSION: The electrochemical by-product hydrogen peroxide is not the main contributor to the cellular effects of electrical stimulation. However, undesired effects like decreased adhesion are mediated through electrochemical products in stimulated medium. Detailed characterisation and monitoring of the stimulation set up and electrochemical reactions are necessary to find safe electrical stimulation protocols.

Selective adhesion inhibition and hyaluronan envelope reduction of dermal tumor cells by cold plasma-activated medium.

The sensitivity to cold plasma is specific to tumor cells while leaving normal tissue cells unaffected. This is the desired challenge in cancer therapy. Therefore, the focus of this work was a comparative study concerning the plasma sensitivity of dermal tumor cells (A-431) versus non-tumorigenic dermal cells (HaCaT) regarding their adhesion capacity. We found a selective inhibiting effect of plasma-activated medium on the adhesion of tumor cells while hardly affecting normal cells. We attributed this to a lower basal gene expression for the adhesion-relevant components CD44, hyaluronan synthase 2 (HAS2), HAS3, and the hyaluronidases in A431. Noteworthy, after plasma exposure, we revealed a significantly higher expression and synthesis of the hyaluronan envelope, the HAS3 gene, and the transmembrane adhesion receptors in non-tumorigenic HaCaTs.

Pulsed Electrical Stimulation Affects Osteoblast Adhesion and Calcium Ion Signaling.

An extensive research field in regenerative medicine is electrical stimulation (ES) and its impact on tissue and cells. The mechanism of action of ES, particularly the role of electrical parameters like intensity, frequency, and duration of the electric field, is not yet fully understood. Human MG-63 osteoblasts were electrically stimulated for 10 min with a commercially available multi-channel system (IonOptix). We generated alternating current (AC) electrical fields with a voltage of 1 or 5 V and frequencies of 7.9 or 20 Hz, respectively. To exclude liquid-mediated effects, we characterized the AC-stimulated culture medium. AC stimulation did not change the medium's pH, temperature, and oxygen content. The H2O2 level was comparable with the unstimulated samples except at 5 V_7.9 Hz, where a significant increase in H2O2 was found within the first 30 min. Pulsed electrical stimulation was beneficial for the process of attachment and initial adhesion of suspended osteoblasts. At the same time, the intracellular Ca2+ level was enhanced and highest for 20 Hz stimulated cells with 1 and 5 V, respectively. In addition, increased Ca2+ mobilization after an additional trigger (ATP) was detected at these parameters. New knowledge was provided on why electrical stimulation contributes to cell activation in bone tissue regeneration.

Synergistic effect of plasma-activated medium and novel indirubin derivatives on human skin cancer cells by activation of the AhR pathway.

Due to the increasing number of human skin cancers and the limited effectiveness of therapies, research into innovative therapeutic approaches is of enormous clinical interest. In recent years, the use of cold atmospheric pressure plasma has become increasingly important as anti-cancer therapy. The combination of plasma with small molecules offers the potential of an effective, tumour-specific, targeted therapy. The synthesised glycosylated and non glycosylated thia-analogous indirubin derivatives KD87 and KD88, respectively, were first to be investigated for their pharmaceutical efficacy in comparison with Indirubin-3'-monoxime (I3M) on human melanoma (A375) and squamous cell carcinoma (A431) cells. In combinatorial studies with plasma-activated medium (PAM) and KD87 we determined significantly decreased cell viability and cell adhesion. Cell cycle analyses revealed a marked G2/M arrest by PAM and a clear apoptotic effect by the glycosylated indirubin derivative KD87 in both cell lines and thus a synergistic anti-cancer effect. I3M had a pro-apoptotic effect only in A431 cells, so we hypothesize a different mode of action of the indirubin derivatives in the two skin cancer cells, possibly due to a different level of the aryl hydrocarbon receptor and an activation of this pathway by nuclear translocation of this receptor and subsequent activation of gene expression.

Laser Structured Dental Zirconium for Soft Tissue Cell Occupation-Importance of Wettability Modulation.

Various approaches are being pursued to physico-chemically modify the zirconia neck region of dental implants to improve the integration into the surrounding soft tissue. In this study, polished zirconia discs were laser microstructured with periodic cavities and convex waves. These zirconia samples were additionally activated by argon plasma using the kINPen®09. The surface topography was characterized by scanning electron microscopy and the surface wettability by water contact angle. The in vitro study with human gingival fibroblasts (HGF-1) was focused on cell spreading, morphology, and actin cytoskeleton organization within the first 24 h. The laser-induced microstructures were originally hydrophobic (e.g., 60 µm cavities 138.4°), but after argon plasma activation, the surfaces switched to the hydrophilic state (60 µm cavities 13.7°). HGF-1 cells adhered flatly on the polished zirconia. Spreading is hampered on cavity structures, and cells avoid the holes. However, cells on laser-induced waves spread well. Interestingly, argon plasma activation for only 1 min promoted adhesion and spreading of HGF-1 cells even after 2 h cultivation. The cells crawl and grow into the depth of the cavities. Thus, a combination of both laser microstructuring and argon plasma activation of zirconia seems to be optimal for a strong gingival cell attachment.

Local Inflammatory Response after Intramuscularly Implantation of Anti-Adhesive Plasma-Fluorocarbon-Polymer Coated Ti6AI4V Discs in Rats.

Orthopaedic implants and temporary osteosynthesis devices are commonly based on Titanium (Ti). For short-term devices, cell-material contact should be restricted for easy removal after bone healing. This could be achieved with anti-adhesive plasma-fluorocarbon-polymer (PFP) films created by low-temperature plasma processes. Two different PFP thin film deposition techniques, microwave (MW) and radiofrequency (RF) discharge plasma, were applied to receive smooth, hydrophobic surfaces with octafluoropropane (C3F8) or hexafluorohexane (C6F6) as precursors. This study aimed at examining the immunological local tissue reactions after simultaneous intramuscular implantation of four different Ti samples, designated as MW-C3F8, MW-C6F6, RF-C3F8 and Ti-controls, in rats. A differentiated morphometric evaluation of the inflammatory reaction was conducted by immunohistochemical staining of CD68+ macrophages, CD163+ macrophages, MHC class II-positive cells, T lymphocytes, CD25+ regulatory T lymphocytes, NK cells and nestin-positive cells in cryosections of surrounding peri-implant tissue. Tissue samples were obtained on days 7, 14 and 56 for investigating the acute and chronical inflammation (n = 8 rats/group). Implants with a radiofrequency discharge plasma (RF-C3F8) coating exhibited a favorable short- and long-term immune/inflammatory response comparable to Ti-controls. This was also demonstrated by the significant decrease in pro-inflammatory CD68+ macrophages, possibly downregulated by significantly increasing regulatory T lymphocytes.

The nanomorphology of cell surfaces of adhered osteoblasts.

The functionality of living cells is inherently linked to subunits with dimensions ranging from several micrometers down to the nanometer scale. The cell surface plays a particularly important role. Electric signaling, including information processing, takes place at the membrane, as well as adhesion and contact. For osteoblasts, adhesion and spreading are crucial processes with regard to bone implants. Here we present a comprehensive characterization of the 3D nanomorphology of living, as well as fixed, osteoblastic cells using scanning ion conductance microscopy (SICM), which is a nanoprobing method that largely avoids mechanical perturbations. Dynamic ruffles are observed, manifesting themselves in characteristic membrane protrusions. They contribute to the overall surface corrugation, which we systematically study by introducing the relative 3D excess area as a function of the projected adhesion area. A clear anticorrelation between the two parameters is found upon analysis of ca. 40 different cells on glass and on amine-covered surfaces. At the rim of lamellipodia, characteristic edge heights between 100 and 300 nm are observed. Power spectral densities of membrane fluctuations show frequency-dependent decay exponents with absolute values greater than 2 on living osteoblasts. We discuss the capability of apical membrane features and fluctuation dynamics in aiding the assessment of adhesion and migration properties on a single-cell basis.

Phenotypic stability of the human MG-63 osteoblastic cell line at different passages.

One of the most popular cell lines in osteogenesis studies is the human osteoblastic line MG-63. For cell biological investigation, it is important that the cells remain stable in their phenotype over several passages in cell culture. MG-63 cells can be used to provide fundamental insights into cell--material interaction. The aim of this study is to present a systematic characterization of the physiological behavior of MG-63 cells in the range of passages 5-30. Significant cell physiology processes during the first 24 h, including cell morphology, availability of adhesion receptors, cell cycle phases, as well as the expression of the signaling proteins Akt, GSK3a/b, IkB-α, ERK1/2, p38-MAPK, and intracellular calcium ion mobilization, remained stable over the entire range of passages P5-P30. Due to these stable characteristics in a wide range of cell culture passages, MG-63 cells can be considered as a suitable in vitro model to analyze the biocompatibility and biofunctionality of implant materials.

First evidence of SGPL1 expression in the cell membrane silencing the extracellular S1P siren in mammary epithelial cells.

The bioactive lipid sphingosine-1-phosphate (S1P) is a main regulator of cell survival, proliferation, motility, and platelet aggregation, and it is essential for angiogenesis and lymphocyte trafficking. In that S1P acts as a second messenger intra- and extracellularly, it might promote cancer progression. The main cause is found in the high S1P concentration in the blood, which encourage cancer cells to migrate through the endothelial barrier into the blood vessels. The irreversible degradation of S1P is solely caused by the sphingosine-1-phosphate lyase (SGPL1). SGPL1 overexpression reduces cancer cell migration and therefore silences the endogenous S1P siren, which promotes cancer cell attraction-the main reason for metastasis. Since our previous metabolomics studies revealed an increased SGPL1 activity in association with successful breast cancer cell treatment in vitro, we further investigated expression and localization of SGPL1. Expression analyses confirmed a very low SGPL1 expression in all breast cancer samples, regardless of their subtype. Additionally, we were able to prove a novel SGPL expression in the cytoplasm membrane of non-tumorigenic breast cells by fusing three independent methods. The general SGPL1 downregulation and the loss of the plasma membrane expression resulted in S1P dependent stimulation of migration in the breast cancer cell lines MCF-7 and BT-20. Not only S1P stimulated migration could be repressed by overexpressing the natural SGPL1 variant not but also more general migratory activity was significantly reduced. Here, for the first time, we report on the SGPL1 plasma membrane location in human, non-malignant breast epithelial cell lines silencing the extracellular S1P siren in vitro, and thereby regulating pivotal cellular functions. Loss of this plasma membrane distribution as well as low SGPL1 expression levels could be a potential prognostic marker and a viable target for therapy. Therefore, the precise role of SGPL1 for cancer treatment should be evaluated.

Synergistic Action of Genistein and Calcitriol in Immature Osteosarcoma MG-63 Cells by SGPL1 Up-Regulation.

BACKGROUND: Phytoestrogens such as genistein, the most prominent isoflavone from soy, show concentration-dependent anti-estrogenic or estrogenic effects. High genistein concentrations (>10 µM) also promote proliferation of bone cancer cells in vitro. On the other hand, the most active component of the vitamin D family, calcitriol, has been shown to be tumor protective in vitro and in vivo. The purpose of this study was to examine a putative synergism of genistein and calcitriol in two osteosarcoma cell lines MG-63 (early osteoblast), Saos-2 (mature osteoblast) and primary osteoblasts. METHODS: Thus, an initial screening based on cell cycle phase alterations, estrogen (ER) and vitamin D receptor (VDR) expression, live cell metabolic monitoring, and metabolomics were performed. RESULTS: Exposure to the combination of 100 µM genistein and 10 nM calcitriol reduced the number of proliferative cells to control levels, increased ERß and VDR expression, and reduced extracellular acidification (40%) as well as respiratory activity (70%), primarily in MG-63 cells. In order to identify the underlying cellular mechanisms in the MG-63 cell line, metabolic profiling via GC/MS technology was conducted. Combined treatment significantly influenced lipids and amino acids preferably, whereas metabolites of the energy metabolism were not altered. The comparative analysis of the log2-ratios revealed that after combined treatment only the metabolite ethanolamine was highly up-regulated. This is the result: a strong overexpression (350%) of the enzyme sphingosine-1-phosphate lyase (SGPL1), which irreversibly degrades sphingosine-1-phosphate (S1P), thereby, generating ethanolamine. S1P production and secretion is associated with an increased capability of migration and invasion of cancer cells. CONCLUSION: From these results can be concluded that the tumor promoting effect of high concentrations of genistein in immature osteosarcoma cells is reduced by the co-administration of calcitriol, primarily by the breakdown of S1P. It should be tested whether this anti-metastatic pathway can be stimulated by combined treatment also in metastatic xenograft mice models.

Data supporting attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts.

The provided data contains the phagocytic interaction of human MG-63 osteoblasts with micro-particles 6 µm in size as well as geometric micro-pillared topography with micro-pillar sizes 5 µm of length, width, height and spacing respectively related to the research article entitled "Attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts" in the Biomaterials journal. [1] Micro-particle treatment was used as positive control triggering phagocytosis by the osteoblasts. Caveolin-1 (Cav-1) as major structural component of caveolae [2] plays an important role in the phagocytic process of micro-particles and -pillars. Data related to the experiments in [1] with siRNA-mediated knockdown are presented here as well as micro-particle control experiments, tubulin analysis on the micro-pillared topography and initial cell interaction with the micro-pillars.

Antitumor evaluation of two selected Pakistani plant extracts on human bone and breast cancer cell lines.

BACKGROUND: The medicinal plants Vincetoxicum arnottianum (VSM), Berberis orthobotrys (BORM), Onosma hispida (OHRM and OHAM) and Caccinia macranthera (CMM) are used traditionally in Pakistan and around the world for the treatment of various diseases including cancer, dermal infections, uterine tumor, wounds etc. The present study focuses on the investigation of the selected Pakistani plants for their potential as anticancer agents on human bone and breast cancer cell lines in comparison with non-tumorigenic control cells. METHODS: The antitumor evaluation was carried out on human bone (MG-63, Saos-2) and breast cancer cell lines (MCF-7, BT-20) in contrast to non-tumorigenic control cells (POB, MCF-12A) via cell viability measurements, cell cycle analysis, Annexin V/PI staining, microscopy based methods as well as migration/invasion determination, metabolic live cell monitoring and western blotting. RESULTS: After the first initial screening of the plant extracts, two extracts (BORM, VSM) revealed the highest potential with regard to its antitumor activity. Both extracts caused a significant reduction of cell viability in the breast and bone cancer cells in a concentration dependent manner. The effect of VSM is achieved primarily by inducing a G2/M arrest in the cell cycle and the stabilization of the actin stress fibers leading to reduced cell motility. By contrast BORM's cytotoxic properties were caused through the lysosomal-mediated cell death pathway indicated by an upregulation of Bcl-2 expression. CONCLUSIONS: The antitumor evaluation of certain medicinal plants presented in this study identified the methanolic root extract of Berberis orthobotrys and the methanolic extract of Vincetoxicum arnottianum as promising sources for exhibiting the antitumor activity. Therefore, the indigenous use of the herbal remedies for the treatment of cancer and cancer-related diseases has a scientific basis. Moreover, the present study provides a base for phytochemical investigation of the plant extracts.

An implementation for the simulation of cells on micro-post arrays.

The mechanical interaction between cells and their underlying substrates is important in understanding the processes that take place at an interface between biological tissue and the surface of implants. There have been numerous studies that examine these interactions both by experimental and numerical modeling. The bio-chemo-mechanical model for cell contractility by Deshpande et al. [1] has numerous applications and advantages. This work shows a way to implement this model in COMSOL MULTIPHYSICS® so it can be easily modified or extended. This will allow us in a next step to couple the differential system with additional external stimuli.

Attempted caveolae-mediated phagocytosis of surface-fixed micro-pillars by human osteoblasts.

Cells are sensitive to their underlying micro- and nano-topography, but the complex interplay is not completely understood especially if sharp edges and ridges of stochastically modified surfaces interfere with an attached cell body. Micro-topography offers cues that evoke a large range of cell responses e.g. altered adhesion behavior and integrin expression resulting in disturbed cell functions. In this study, we analyzed why osteoblastic cells mimic the underlying geometrical micro-pillar structure (5 × 5 × 5 µm, spacing of 5 µm) with their actin cytoskeleton. Interestingly, we discovered an attempted caveolae-mediated phagocytosis of each micro-pillar beneath the cells, which was accompanied by increased intracellular reactive oxygen species (ROS) production and reduced intracellular ATP levels. This energy consuming process hampered the cells in their function as osteoblasts at the interface. The raft-dependent/caveolae-mediated phagocytic pathway is regulated by diverse cellular components including caveolin-1 (Cav-1), cholesterol, actin cytoskeleton as well as actin-binding proteins like annexin A2 (AnxA2). Our results show a new aspect of osteoblast-material interaction and give insight into how cells behave on extraordinary micro-structures. We conclude that stochastically structured implants used in orthopedic surgery should avoid any topographical heights which induce phagocytosis to prevent their successful ingrowth.

Continuous cellularization of calcium phosphate hybrid scaffolds induced by plasma polymer activation.

The generation of hybrid materials based on ß-tricalcium phosphate (TCP) and various biodegradable polymers like poly(l-lactide-co-d,l-lactide) (PLA) represents a common approach to overcoming the disadvantages of pure TCP devices. These disadvantages lie in TCP's mechanical properties, such as brittleness. The positive characteristic of PLA - improvement of compressive strength of calcium phosphate scaffolds - is diametrically opposed to its cell attractiveness. Therefore, the objective of this work was to optimize osteoblast migration and cellularization inside a three-dimensionally (3D) printed, PLA polymer stabilized TCP hybrid scaffold by a plasma polymer process depositing amino groups via allylamine. MG-63 osteoblastic cells inside the 10mm hybrid scaffold were dynamically cultivated for 14days in a 3D model system integrated in a perfusion reactor. The whole TCP/PLA hybrid scaffold was continuously colonized due to plasma polymerized allylamine activation inducing the migration potential of osteoblasts.

Cellular Nutrition in Complex Three-Dimensional Scaffolds: A Comparison between Experiments and Computer Simulations.

Studies on bone cell ingrowth into synthetic, porous three-dimensional (3D) implants showed difficulties arising from impaired cellular proliferation and differentiation in the core region of these scaffolds with increasing scaffold volume in vitro. Therefore, we developed an in vitro perfusion cell culture module, which allows the analysis of cells in the interior of scaffolds under different medium flow rates. For each flow rate the cell viability was measured and compared with results from computer simulations that predict the local oxygen supply and shear stress inside the scaffold based on the finite element method. We found that the local cell viability correlates with the local oxygen concentration and the local shear stress. On the one hand the oxygen supply of the cells in the core becomes optimal with a higher perfusion flow. On the other hand shear stress caused by high flow rates impedes cell vitality, especially at the surface of the scaffold. Our results demonstrate that both parameters must be considered to derive an optimal nutrient flow rate.

Intracellular calcium dynamics dependent on defined microtopographical features of titanium.

Detailed insights into the complex cellular behavior at the biomaterial interface are crucial for the improvement of implant surfaces with respect to their acceptance and integration. The cells perceive microtopographical features and, in consequence, rearrange their adhesion structures like the actin cytoskeleton and adaptor proteins. But little is known about whether these altered cellular phenotypes have consequences for intracellular calcium signaling and its dynamics. To elucidate if an artificial, geometrical microtopography influences calcium ion (Ca(2+)) mobilization in osteoblasts, human MG-63 cells were stained with the calcium dye Fluo 3-acetoxymethyl ester and set on defined silicon-titanium (Ti) arrays with regular pillar structures (P5, 5 × 5 × 5 µm) and compared with planar Ti. To induce an immediate calcium signal, cells were stimulated with adenosine 5'-triphosphate (ATP). Interestingly, osteoblasts on micropillars expressing a shortened actin cytoskeleton were hampered in their calcium mobilization potential in signal height as well duration. Even the basal level of the intracellular Ca(2+) concentration was reduced, which was accompanied by a disturbed fibronectin synthesis. The expression of the voltage-sensitive calcium channels Cav1.2, Cav1.3 (L-type) and Cav3.1, Cav3.2, Cav3.3 (T-type) as well as the signaling proteins phospho-AKT and phospho-GSK3α/ß remained unaffected on pillars. The topography-dependent calcium dynamics observed here provide new insights into how topographical cues alter cell functions - via the intracellular Ca(2+) signaling.