Polycaprolactone-Based 3D-Printed Scaffolds as Potential Implant Materials for Tendon-Defect Repair

Chronic tendon ruptures are common disorders in orthopedics. The conventional surgical methods used to treat them often require the support of implants. Due to the non-availability of suitable materials, 3D-printed polycaprolactone (PCL) scaffolds were designed from two different starting materials as suitable candidates for tendon-implant applications. For the characterization, mechanical testing was performed. To increase their biocompatibility, the PCL-scaffolds were plasma-treated and coated with fibronectin and collagen I. Cytocompatibility testing was performed using L929 mouse fibroblasts and human-bone-marrow-derived mesenchymal stem cells. The mechanical testing showed that the design adaptions enhanced the mechanical stability. Cell attachment was increased in the plasma-treated specimens compared to the control specimens, although not significantly, in the viability tests. Coating with fibronectin significantly increased the cellular viability compared to the untreated controls. Collagen I treatment showed an increasing trend. The desired cell alignment and spread between the pores of the construct was most prominent on the collagen-I-coated specimens. In conclusion, 3D-printed scaffolds are possible candidates for the development of tendon implants. Enhanced cytocompatibility was achieved through surface modifications. Although adaptions in mechanical strength still require alterations in order to be applied to human-tendon ruptures, we are optimistic that a suitable implant can be designed

Space Odyssey: An Experimental Software Security Analysis of Satellites

Satellites are an essential aspect of our modern society and have contributed significantly to the way we live today, most notable through modern telecommunications, global positioning, and Earth observation. In recent years, and especially in the wake of the New Space Era, the number of satellite deployments has seen explosive growth. Despite its critical importance, little academic research has been con- ducted on satellite security and, in particular, on the security of onboard firmware. This lack likely stems from by now outdated assumptions on achieving security by obscurity, effectively preventing meaningful research on satellite firmware. In this paper, we first provide a taxonomy of threats against satellite firmware. We then conduct an experimental security analysis of three real-world satellite firmware images. We base our analysis on a set of real-world attacker models and find several security-critical vulnerabilities in all analyzed firmware images. The results of our experimental security assessment show that modern in-orbit satellites suffer from different software security vulnerabilities and often a lack of proper access protection mechanisms. They also underline the need to overcome prevailing but obsolete assumptions. To substantiate our observations, we also performed a survey of 19 professional satellite developers to obtain a comprehensive picture of the satellite security landscape

SGXFuzz: Efficiently Synthesizing Nested Structures for SGX Enclave Fuzzing

Intel's Software Guard Extensions (SGX) provide a non-introspectable trusted execution environment (TEE) to protect security-critical code from a potentially malicious OS. This protection can only be effective if the individual enclaves are secure, which is already challenging in regular software, and this becomes even more difficult for enclaves as the entire environment is potentially malicious. As such, many enclaves expose common vulnerabilities, e.g., memory corruption and SGX-specific vulnerabilities like null-pointer dereferences. While fuzzing is a popular technique to assess the security of software, dynamically analyzing enclaves is challenging, as enclaves are meant to be non-introspectable. Further, they expect an allocated multi-pointer structure as input instead of a plain buffer. In this paper, we present SGXFuzz, a coverage-guided fuzzer that introduces a novel binary input structure synthesis method to expose enclave vulnerabilities even without source-code access. To obtain code coverage feedback from enclaves, we show how to extract enclave code from distribution formats. We also present an enclave runner that allows execution of the extracted enclave code as a user-space application at native speed, while emulating all relevant environment interactions of the enclave. We use this setup to fuzz enclaves using a state-of-the-art snapshot fuzzing engine that deploys our novel structure synthesis stage. This stage synthesizes multi-layer pointer structures and size fields incrementally on-the-fly based on fault signals. Furthermore, it matches the expected input format of the enclave without any prior knowledge. We evaluate our approach on 30 open and closed-source enclaves and found a total of 79 new bugs and vulnerabilities

Methods for the Analysis of Polyphosphate in the Life Sciences

Inorganic polyphosphate (polyP) is the polymer of orthophosphate and can be found in all living organisms. For polyP characterization, one or more of six parameters are of interest: the molecular structure (linear, cyclic, or branched), the concentration, the average chain length, the chain length distribution, the cellular localization, and the cation composition. Here, the merits, limitations, and critical parameters of the state-of-the-art methods for the analysis of the six parameters from the life sciences are discussed. With this contribution, we aim to lower the entry barrier into the analytics of polyP, a molecule with prominent, yet often incompletely understood, contributions to cellular function

Polyphosphate Chain Length Determination in the Range of Two to Several Hundred P-Subunits with a New Enzyme Assay and 31 P NMR

Currently, 31P NMR is the only analytical method that quantitatively determines the average chain length of long inorganic polyphosphate (>80 P-subunits). In this study, an enzyme assay is presented that determines the average chain length of polyphosphate in the range of two to several hundred P-subunits. In the enzyme assay, the average polyP chain length is calculated by dividing the total polyphosphate concentration by the concentration of the polyphosphate chains. The total polyphosphate is determined by enzymatic polyphosphate hydrolysis with Saccharomyces cerevisiae exopolyphosphatase 1 and S. cerevisiae inorganic pyrophosphatase 1, followed by colorimetric orthophosphate detection. Because the exopolyphosphatase leaves one pyrophosphate per polyphosphate chain, the polyphosphate chain concentration is assayed by coupling the enzymes exopolyphosphatase (polyP into pyrophosphate), ATP sulfurylase (pyrophosphate into ATP), hexokinase (ATP into glucose 6-phosphate), and glucose 6-phosphate dehydrogenase (glucose 6-phosphate into NADPH), followed by fluorometric NADPH detection. The ability of 31P NMR and the enzyme assay to size polyP was demonstrated with polyP lengths in the range from 2 to ca. 280 P-subunits (no polyP with a longer chain length was available). The small deviation between methods (−4 ± 4%) indicated that the new enzyme assay performed accurately. The limitations of 31P NMR (i.e., low throughput, high sample concentration, expensive instrument) are overcome by the enzyme assay that is presented here, which allows for high sample throughput and requires only a commonly available plate reader and micromole per liter concentrations of polyphosphate

Biofunctionalized Silica Nanoparticles: Standards in Amyloid-β Oligomer-Based Diagnosis of Alzheimer’s Disease

Amyloid-β (Aβ) oligomers represent a promising biomarker for the early diagnosis of Alzheimer’s disease (AD). However, state-of-the-art methods for immunodetection of Aβ oligomers in body fluids show a large variability and lack a reliable and stable standard that enables the reproducible quantitation of Aβ oligomers. At present, the only available standard applied in these assays is based on a random aggregation process of synthetic Aβ and has neither a defined size nor a known number of epitopes. In this report, we generated a highly stable standard in the size range of native Aβ oligomers that exposes a defined number of epitopes. The standard consists of a silica nanoparticle (SiNaP), which is functionalized with Aβ peptides on its surface (Aβ-SiNaP). The different steps of Aβ-SiNaP synthesis were followed by microscopic, spectroscopic and biochemical analyses. To investigate the performance of Aβ-SiNaPs as an appropriate standard in Aβ oligomer immunodetection, Aβ-SiNaPs were diluted in cerebrospinal fluid and quantified down to a concentration of 10 fM in the sFIDA (surface-based fluorescence intensity distribution analysis) assay. This detection limit corresponds to an Aβ concentration of 1.9 ng l–1 and lies in the sensitivity range of currently applied diagnostic tools based on Aβ oligomer quantitation. Thus, we developed a highly stable and well-characterized standard for the application in Aβ oligomer immunodetection assays that finally allows the reproducible quantitation of Aβ oligomers down to single molecule level and provides a fundamental improvement for the worldwide standardization process of diagnostic methods in AD research

Nanoparticle standards for immuno-based quantitation of α-synuclein oligomers in diagnostics of Parkinson's disease and other synucleopathies

Parkinson's disease is a neurodegenerative disorder that is characterized by symptoms such as rigor, tremor and bradykinesia. A reliable and early diagnosis could improve the development of early therapeutic strategies before death of dopaminergic neurons leads to the first clinical symptoms.The sFIDA (surface-based fluorescence intensity distribution analysis) assay is a highly sensitive method to determine the concentration of α-synuclein (α-syn) oligomers, which are presumably the major toxic isoform of α-syn and potentially the most direct biomarker for PD.Oligomer-based diagnostic tests require standard molecules that closely mimic the native oligomer. This is particularly important for calibration and assessment of inter-assay variation. In this study, we generated a standard in form of α-syn coated silica nanoparticles (α-syn-SiNaPs) that are in the size range of α-syn oligomers and provide a defined number of α-syn epitopes.The preparation of the sFIDA assay was realized on an automated platform to allow handling of high number of samples and reduce the effects of human error. The assay outcome was analyzed by determination of coefficient of variation and linearity for the applied α-syn-SiNaPs concentrations. Additionally, the limit of detection and lower limit of quantification were determined yielding concentrations in the lower femtomolar range

sFIDA automation yields sub-femtomolar limit of detection for Aβ aggregates in body fluids

Objectives: Alzheimer's disease (AD) is a neurodegenerative disorder with yet non-existent therapeutic and limited diagnostic options. Reliable biomarker-based AD diagnostics are of utmost importance for the development and application of therapeutic substances. Wehave previously introduced a platformtechnology designated ‘sFIDA’ for the quantitation of amyloid β peptide (Aβ) aggregates as AD biomarker. In this study we implemented the sFIDA assay on an automated platform to enhance robustness and performance of the assay.Design and methods: In sFIDA (surface-based fluorescence intensity distribution analysis) Aβ species are immobilized by a capture antibody to a glass surface. Aβ aggregates are then multiply loaded with fluorescent antibodies and quantitated by high resolution fluorescence microscopy. As a model system for Aβ aggregates, weused Aβ-conjugated silica nanoparticles (Aβ-SiNaPs) diluted in PBS buffer and cerebrospinal fluid, respectively. Automation of the assay was realized on a liquid handling system in combination with a microplate washer.Results: The automation of the sFIDA assay results in improved intra-assay precision, linearity and sensitivity in comparison to the manual application, and achieved a limit of detection in the sub-femtomolar range.Conclusions: Automation improves the precision and sensitivity of the sFIDA assay, which is a prerequisite for high-throughput measurements and future application of the technology in routine AD diagnostics

Analysis of anticoagulants for blood-based quantitation of amyloid β oligomers in the sFIDA assay

Early diagnostics at the preclinical stage of Alzheimer’s disease is of utmost importance for drug development in clinical trials and prognostic guidance. Since soluble Aβ oligomers are considered to play a crucial role in the disease pathogenesis, several methods aim to quantify Aβ oligomers in body fluids such as cerebrospinal fluid (CSF) and blood plasma. The highly specific and sensitive method surface-based fluorescence intensity distribution analysis (sFIDA) has successfully been established for oligomer quantitation in CSF samples. In our study, we explored the sFIDA method for quantitative measurements of synthetic Aβ particles in blood plasma. For this purpose, EDTA, citrate and heparin treated blood plasma samples from five individual donors were spiked with Aβ coated silica nanoparticles (Aβ-SiNaPs) and were applied to the sFIDA assay. Based on the assay parameters linearity, coefficient of variation and limit of detection, we found that EDTA plasma yields the most suitable parameter values for quantitation of Aβ oligomers in sFIDA assay with a limit of detection of 16 fM

Biotechnological synthesis of water-soluble food-grade polyphosphate with Saccharomyces cerevisiae

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2 to 40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally-friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, Generally-Regarded-As-Safe (GRAS) wild-type baker’s yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5 % polyP (as KPO(3), in cell dry weight). The cells were lysed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11 P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, (31)P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP