Restricted cell functions on micropillars are alleviated by surface-nanocoating with amino groups

The topographical and chemical surface features of biomaterials are sensed by the cells, affecting their physiology at the interface. When placed on titanium, we recently discovered osteoblasts attempted caveolae-mediated phagocytosis of the sharp-edged microstructures. This active, energy-consuming process resulted in decreased osteoblastic cell functions (e.g. secretion of extracellular matrix proteins). However, chemical modification with plasma polymerized allylamine (PPAAm) was able to amplify osteoblast adhesion and spreading, resulting in better implant osseointegration in vivo. In the present in vitro study, we analyzed whether this plasma polymer nanocoating is able to attenuate the microtopography-induced changes of osteoblast physiology. On PPAAm, we found cells showed a higher cell interaction with the geometrical micropillars by 30 min, and a less distinct reduction in the mRNA expression of collagen type I, osteocalcin and fibronectin after 24 h of cell growth. Interestingly, the cells were more active and sensitive on PPAAm-coated micropillars, and react with a substantial Ca2+ ion mobilization after stimulation with ATP. These results highlight that it is important for osteoblasts to establish cell surface contact for them to perform their functions

Combined Knockout of RIPK3 and MLKL Reveals Unexpected Outcome in Tissue Injury and Inflammation

Necroptosis, initially identified as a backup cell death program when apoptosis is hindered, is a prominent feature in the etiology and progression of many human diseases, such as ischemic injury and sepsis. Receptor-interacting protein kinase 3 (RIPK3) is the cardinal regulator of this cell death modality, recruiting and phosphorylating the executioner mixed lineage kinase domain-like protein (MLKL) to signal necroptosis, which is terminated by a cellular plasma membrane rupture and the leakage of intracellular contents from dying cells. Experimental data to date indicate that RIPK3 and MLKL is the core machinery essential for all necroptotic cell death responses. By using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) technology, we showed that Ripk3 and Mlkl knockout and Ripk3/Mlkl double-knockout in necroptosis-sensitive cell lines extensively block susceptibility to necroptosis, in each case to an indistinguishable degree. In vivo studies using Ripk3- or Mlkl-deficient mice validated kidney ischemia reperfusion injury and high-dose tumor necrosis factor (TNF) availability, as druggable targets in necroptotic-mediated pathologies. Here, we demonstrated that Ripk3 or Mlkl-deficient mice are protected to a similar extent from kidney ischemia reperfusion injury and TNF-induced toxicity. Remarkably, in contrast to each single knockout, Ripk3/Mlkl double-deficient mice did not have appreciable protection from either of the above necroptotic-mediated pathologies. Paradoxically, the double-knockout mice resembled, in each case, the vulnerable wild-type mice, revealing novel complexities in the mechanisms of inflammation-driven diseases, due to aberrant cell death

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

A High-Throughput, Cell-Based Screening Method for siRNA and Small Molecule Inhibitors of mTORC1 Signaling Using the In Cell Western Technique

The mTORC1 pathway is a central regulator of cell growth, and defective mTORC1 regulation plays a causative role in a variety of human diseases, including cancer, tumor syndromes such as the tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), and metabolic diseases such as diabetes and obesity. Given the importance of mTORC1 signaling in these diseases, there has been significant interest in developing screening methods suitable for identifying inhibitors of mTORC1 activation. To this end, we have developed a high-throughput, cell-based assay for the detection of rpS6-phosphorylation as a measure of mTORC1 signaling. This assay takes advantage of the “In Cell Western” (ICW) technique using the Aerius infrared imaging system (LI-COR® Biosciences). The ICW procedure involves fixation and immunostaining of cells in a manner similar to standard immunofluorescence methods but takes advantage of secondary antibodies conjugated to infrared-excitable fluorophores for quantitative detection by the Aerius® scanner. In addition, the cells are stained with an infrared-excitable succinimidyl ester dye, which covalently modifies free amine groups in fixed cells and provides a quantitative measure of cell number. We present validation data and pilot screens in a 384-well format demonstrating that this assay provides a statistically robust method for both small molecule and siRNA screening approaches designed to identify inhibitors of mTORC1 signaling

ROS Dependent Wnt/β-Catenin Pathway and Its Regulation on Defined Micro-Pillars—A Combined In Vitro and In Silico Study

The physico-chemical surface design of implants influences the surrounding cells. Osteoblasts on sharp-edged micro-topographies revealed an impaired cell phenotype, function and Ca2+ mobilization. The influence of edges and ridges on the Wnt/β-catenin pathway in combination with the cells’ stress response has not been clear. Therefore, MG-63 osteoblasts were studied on defined titanium-coated micro-pillars (5 × 5 × 5 µm) in vitro and in silico. MG-63s on micro-pillars indicated an activated state of the Wnt/β-catenin pathway. The β-catenin protein accumulated in the cytosol and translocated into the nucleus. Gene profiling indicated an antagonism mechanism of the transcriptional activity of β-catenin due to an increased expression of inhibitors like ICAT (inhibitor of β-catenin and transcription factor-4). Cells on pillars produced a significant reactive oxygen species (ROS) amount after 1 and 24 h. In silico analyses provided a detailed view on how transcriptional activity of Wnt signaling is coordinated in response to the oxidative stress induced by the micro-topography. Based on a coordinated expression of regulatory elements of the Wnt/β-catenin pathway, MG-63s are able to cope with an increased accumulation of β-catenin on micro-pillars and suppress an unintended target gene expression. Further, β-catenin may be diverted into other signaling pathways to support defense mechanisms against ROS

Novel dalbavancin-PLLA implant coating prevents hematogenous Staphylococcus aureus infection in a minimally invasive mouse tail vein model

Infective/bacterial endocarditis is a rare but life-threatening disease with a hospital mortality rate of 22.7% and a 1-year mortality rate of 40%. Therefore, continued research efforts to develop efficient anti-infective implant materials are of the utmost importance. Equally important is the development of test systems that allow the performance of new materials to be comprehensively evaluated. In this study, a novel antibacterial coating based on dalbavancin was tested in comparison to rifampicin/minocycline, and the suitability of a recently developed mouse tail vein model for testing the implant coatings was validated. Small polymeric stent grafts coated with a poly-L-lactic acid (PLLA) layer and incorporated antibiotics were colonized with Staphylococcus (S.) aureus before implantation into the tail vein of mice. The main assessment criteria were the hematogenous spread of the bacteria and the local tissue reaction to the contaminated implant. For this purpose, colony-forming units (CFU) in the blood, spleen and kidneys were determined. Tail cross sections were prepared for histological analysis, and plasma cytokine levels and expression values of inflammation-associated genes were examined. Both antibiotic coatings performed excellently, preventing the onset of infection. The present study expands the range of available methods for testing the anti-infectivity of cardiovascular implants, and the spectrum of agents for effective surface coating

The anticonvulsive Phenhydan® suppresses extrinsic cell death

Different forms of regulated cell death-like apoptosis and necroptosis contribute to the pathophysiology of clinical conditions including ischemia-reperfusion injury, myocardial infarction, sepsis, and multiple sclerosis. In particular, the kinase activity of the receptor-interacting serine/threonine protein kinase 1 (RIPK1) is crucial for cell fate in inflammation and cell death. However, despite its involvement in pathological conditions, no pharmacologic inhibitor of RIPK1-mediated cell death is currently in clinical use. Herein, we screened a collection of clinical compounds to assess their ability to modulate RIPK1-mediated cell death. Our small-scale screen identified the anti-epilepsy drug Phenhydan (R) as a potent inhibitor of death receptor-induced necroptosis and apoptosis. Accordingly, Phenhydan (R) blocked activation of necrosome formation/activation as well as death receptor-induced NF-kappa B signaling by influencing the membrane function of cells, such as lipid raft formation, thus exerting an inhibitory effect on pathophysiologic cell death processes. By targeting death receptor signaling, the already FDA-approved Phenhydan (R) may provide new therapeutic strategies for inflammation-driven diseases caused by aberrant cell death