An Introduction to the Benchmarking and Publications for Non-Targeted Analysis Working Group.

Non-targeted analysis (NTA) encompasses a rapidly evolving set of mass spectrometry techniques aimed at characterizing the chemical composition of complex samples, identifying unknown compounds, and/or classifying samples, without prior knowledge regarding the chemical content of the samples. Recent advances in NTA are the result of improved and more accessible instrumentation for data generation and analysis tools for data evaluation and interpretation. As researchers continue to develop NTA approaches in various scientific fields, there is a growing need to identify, disseminate, and adopt community-wide method reporting guidelines. In 2018, NTA researchers formed the Benchmarking and Publications for Non-Targeted Analysis Working Group (BP4NTA) to address this need. Consisting of participants from around the world and representing fields ranging from environmental science and food chemistry to 'omics and toxicology, BP4NTA provides resources addressing a variety of challenges associated with NTA. Thus far, BP4NTA group members have aimed to establish a consensus on NTA-related terms and concepts and to create consistency in reporting practices by providing resources on a public Web site, including consensus definitions, reference content, and lists of available tools. Moving forward, BP4NTA will provide a setting for NTA researchers to continue discussing emerging challenges and contribute to additional harmonization efforts.

Cytotoxicity, cellular uptake and apoptotic responses in human coronary artery endothelial cells exposed to ultrasmall superparamagnetic iron oxide nanoparticles.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPION) possess reactive surfaces, are metabolized and exhibit unique magnetic properties. These properties are desirable for designing novel theranostic biomedical products; however, toxicity mechanisms of USPION are not completely elucidated. The goal of this study was to investigate cell interactions (uptake and cytotoxicity) of USPION using human coronary artery endothelial cells as a vascular cell model. Polyvinylpirrolidone-coated USPION were characterized: average diameter 17 nm (transmission electron microscopy [TEM]), average hydrodynamic diameter 44 nm (dynamic light scattering) and zeta potential -38.75 mV. Cells were exposed to 0 (control), 25, 50, 100 or 200 µg/mL USPION. Concentration- and time-dependent cytotoxicity were observed after 3-6 hours through 24 hours of exposure using Alamar Blue and Real-Time Cell Electronic Sensing assays. Cell uptake was evaluated by imaging using live-dead confocal microscopy, actin and nuclear fluorescent staining, and TEM. Phase-contrast, confocal microscopy, and TEM imaging showed significant USPION internalization as early as 3 hours after exposure to 25 µg/mL. TEM imaging demonstrated particle internalization in secondary lysosomes with perinuclear localization. Three orthogonal assays were conducted to assess apoptosis. TUNEL staining demonstrated a marked increase in fragmented DNA, a response pathognomonic of apoptosis, after a 4-hour exposure. Cells subjected to agarose gel electrophoresis exhibited degraded DNA 3 hours after exposure. Caspase-3/7 activity increased after a 3-hour exposure. USPION uptake resulted in cytotoxicity involving apoptosis and these results contribute to further mechanistic understanding of the USPION toxicity in vitro in cardiovascular endothelial cells.

Different cytotoxicity responses to antimicrobial nanosilver coatings when comparing extract-based and direct-contact assays.

This study was performed to understand how the choice of cytotoxicity assay format affects the observed biocompatibility of nanosilver (nAg). nAg coatings are physical coatings containing silver (Ag) that have feature sizes of 100 nm or less, often in the form of nanoparticles or grains. They are used on medical devices to prevent infection, but in spite of this intended benefit, observations of potential cytotoxicity from nAg have been reported in numerous published studies. For medical device regulation, cytotoxicity testing is part of a biocompatibility evaluation, in which specific test methods are chosen based on the technological characteristics and intended use of a device. For this study, nAg-coated tissue culture polystyrene surfaces were prepared using magnetron sputter coating, resulting in nAg films of 0.2 to 311 µg cm(-2) Ag. These coatings exhibited nanometer-scale morphologies and demonstrated a > 4log10 reduction in Escherichia coli viability. It was observed that extracts of nAg caused no cytotoxicity to L929 mouse fibroblasts, but cells cultured directly on nAg coatings (direct-contact assay format) showed a dose-dependent reduction in viability by up to 100% (P < 0.001). Results using inductively coupled plasma mass spectrometry to measure Ag release suggested that extracts of nAg are not toxic because the dissolved Ag in those samples becomes less cytotoxic over time, probably owing to the reaction with cell culture media and serum (six-fold cytotoxicity reductions observed over a 24-h period). These findings highlight the potential value of direct-contact cytotoxicity testing for nAg in predicting biological interactions with cells or tissue in vivo.

Proangiogenic scaffolds as functional templates for cardiac tissue engineering.

We demonstrate here a cardiac tissue-engineering strategy addressing multicellular organization, integration into host myocardium, and directional cues to reconstruct the functional architecture of heart muscle. Microtemplating is used to shape poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogel into a tissue-engineering scaffold with architectures driving heart tissue integration. The construct contains parallel channels to organize cardiomyocyte bundles, supported by micrometer-sized, spherical, interconnected pores that enhance angiogenesis while reducing scarring. Surface-modified scaffolds were seeded with human ES cell-derived cardiomyocytes and cultured in vitro. Cardiomyocytes survived and proliferated for 2 wk in scaffolds, reaching adult heart densities. Cardiac implantation of acellular scaffolds with pore diameters of 30-40 microm showed angiogenesis and reduced fibrotic response, coinciding with a shift in macrophage phenotype toward the M2 state. This work establishes a foundation for spatially controlled cardiac tissue engineering by providing discrete compartments for cardiomyocytes and stroma in a scaffold that enhances vascularization and integration while controlling the inflammatory response.

Amphiphilic self-assembled "polymeric drugs": morphology, properties, and biological behavior of nanoparticles.

This article reports the fabrication and characterization of NPs based on the self-assembling of polymeric drugs with amphiphilic character synthetized from oleyl 2-acetamido-2-deoxy-α-d-glucopyranoside methacrylate and vinyl pyrrolidone (OAGMA-VP). NPs were spherical, with an apparent hydrodynamic diameter between 91 and 226 nm and with zeta potential values that ensure stability. Atomic concentrations of C, O, and N, determined by X-ray photoelectron spectroscopy (XPS) of NPs, compared well with the corresponding theoretical values. High resolution XPS C1s spectra suggest that the carbons bound to heteroatoms or carbonyl groups are preferentially situated on the surface of the NP samples. ToF-SIMS spectra analyzed by principal component analysis (PCA) indicated that ions coming from acetyl and oleyl groups of OAGMA play important roles in the outer structure of NPs. Water contact angle and surface tension values of NPs were characteristic of hydrophilic surfaces, confirming the location of VP sequences on the surface. Cell culture assays showed that copolymeric NPs did not compromise biocompatibility of human fibroblasts according to ISO standard, but they were cytotoxic on a human glioblastoma cell line (A-172).

Chemical Characterization and Non-targeted Analysis of Medical Device Extracts: A Review of Current Approaches, Gaps, and Emerging Practices.

The developers of medical devices evaluate the biocompatibility of their device prior to FDA's review and subsequent introduction to the market. Chemical characterization, described in ISO 10993-18:2020, can generate information for toxicological risk assessment and is an alternative approach for addressing some biocompatibility end points (e.g., systemic toxicity, genotoxicity, carcinogenicity, reproductive/developmental toxicity) that can reduce the time and cost of testing and the need for animal testing. Additionally, chemical characterization can be used to determine whether modifications to the materials and manufacturing processes alter the chemistry of a patient-contacting device to an extent that could impact device safety. Extractables testing is one approach to chemical characterization that employs combinations of non-targeted analysis, non-targeted screening, and/or targeted analysis to establish the identities and quantities of the various chemical constituents that can be released from a device. Due to the difficulty in obtaining a priori information on all the constituents in finished devices, information generation strategies in the form of analytical chemistry testing are often used. Identified and quantified extractables are then assessed using toxicological risk assessment approaches to determine if reported quantities are sufficiently low to overcome the need for further chemical analysis, biological evaluation of select end points, or risk control. For extractables studies to be useful as a screening tool, comprehensive and reliable non-targeted methods are needed. Although non-targeted methods have been adopted by many laboratories, they are laboratory-specific and require expensive analytical instruments and advanced technical expertise to perform. In this Perspective, we describe the elements of extractables studies and provide an overview of the current practices, identified gaps, and emerging practices that may be adopted on a wider scale in the future. This Perspective is outlined according to the steps of an extractables study: information gathering, extraction, extract sample processing, system selection, qualification, quantification, and identification.

Nitinol Release of Nickel under Physiological Conditions: Effects of Surface Oxide, pH, Hydrogen Peroxide, and Sodium Hypochlorite.

Nitinol is a nickel-titanium alloy widely used in medical devices for its unique pseudoelastic and shape-memory properties. However, nitinol can release potentially hazardous amounts of nickel, depending on surface manufacturing yielding different oxide thicknesses and compositions. Furthermore, nitinol medical devices can be implanted throughout the body and exposed to extremes in pH and reactive oxygen species (ROS), but few tools exist for evaluating nickel release under such physiological conditions. Even in cardiovascular applications, where nitinol medical devices are relatively common and the blood environment is well understood, there is a lack of information on how local inflammatory conditions after implantation might affect nickel ion release. For this study, nickel release from nitinol wires of different finishes was measured in pH conditions and at ROS concentrations selected to encompass and exceed literature reports of extracellular pH and ROS. Results showed increased nickel release at levels of pH and ROS reported to be physiological, with decreasing pH and increasing concentrations of hydrogen peroxide and NaOCl/HOCl having the greatest effects. The results support the importance of considering the implantation site when designing studies to predict nickel release from nitinol and underscore the value of understanding the chemical milieu at the device-tissue interface.

Aseptic and septic prosthetic joint loosening: Impact of biomaterial wear on immune cell function, inflammation, and infection.

The success of total joint replacements has led to consistent growth in the use of arthroplasty in progressively younger patients. However, more than 10 percent of patients require revision surgeries due to implant failure caused by osteolytic loosening. These failures are classified as either aseptic or septic and are associated with the presence of particulate wear debris generated by mechanical action between implant components. Aseptic loosening results from chronic inflammation caused by activation of resident immune cells in contact with implant wear debris. In contrast, septic loosening is defined by the presence of chronic infection at the implant site. However, recent findings suggest that subclinical biofilms may be overlooked when evaluating the cause of implant failure, leading to a misdiagnosis of aseptic loosening. Many of the inflammatory pathways contributing to periprosthetic joint infections are also involved in bone remodeling and resorption. In particular, wear debris is increasingly implicated in the inhibition of the innate and adaptive immune response to resolve an infection or prevent hematogenous spread. This review examines the interconnectivity of wear particle- and infection-associated mechanisms of implant loosening, as well as biomaterials-based strategies to combat infection-related osteolysis.

Temperature dependence of nickel ion release from nitinol medical devices.

Nitinol exhibits unique (thermo)mechanical properties that make it central to the design of many medical devices. However, nitinol nominally contains 50 atomic percent nickel, which if released in sufficient quantities, can lead to adverse health effects. While nickel release from nitinol devices is typically characterized using in vitro immersion tests, these evaluations require lengthy time periods. We have explored elevated temperature as a potential method to expedite this testing. Nickel release was characterized in nitinol materials with surface oxide thickness ranging from 12 to 1564 nm at four different temperatures from 310 to 360 K. We found that for three of the materials with relatively thin oxide layers, ≤ 87 nm nickel release exhibited Arrhenius behavior over the entire temperature range with activation energies of 80 to 85 kJ/mol. Conversely, the fourth ''black-oxide'' material, with a much thicker, complex oxide layer, was not well characterized by an Arrhenius relationship. Power law release profiles were observed in all four materials; however, the exponent from the thin oxide materials was approximately 1/4 compared with 3/4 for the black-oxide material. To illustrate the potential benefit of using elevated temperature to abbreviate nickel release testing, we demonstrated that a > 50 day 310 K release profile could be accurately recovered by testing for less than 1 week at 340 K. However, because the materials explored in this study were limited, additional testing and mechanistic insight are needed to establish a protective temperature scaling that can be applied to all nitinol medical device components.

Screening for extractables in additive-manufactured acrylonitrile butadiene styrene orthopedic cast.

The use of additive-manufactured components in medical applications, specifically medical devices (e.g., orthopedic casts), has increased in recent years. Such devices may be fabricated at the point of care using consumer-grade additive manufacturing. Limited studies have been conducted to evaluate the extractable substances of these devices. Chemical characterization followed by toxicological risk assessment is one means of evaluating safety of devices. This study was designed to determine the extractables profile of additive-manufactured materials according to filament grade and post-processing method. Feedstocks for additive manufacturing were tested as filament and manufactured casts, while the cast from consumer-grade filament (CGF) was post-processed. Samples were extracted using three solvents of varying polarities. Extracts were analyzed by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) techniques. In GC/MS analysis, isopropanol extracts generated fewer compound identifications for USP Class VI filament (USPF)-based casts (3) compared with the respective filament (17) while hexane generated the most compound identifications for the finished cast manufactured from CGF. CGF was found to have the highest number of nonvolatile extractables for isopropanol (15) and hexane (34) by positive ion LC/MS. Additionally, CGF produced more non-polar extractables in hexane than the USPF. A known polymer byproduct and potential genotoxicant, styrene acrylonitrile (SAN) trimer, was one of the compounds identified in both GC/MS and LC/MS at quantities ranging from 19 to 270 µg g-1. Overall these results suggested that the extractables profile was affected by the filament material, printing procedure, and post-processing method.

Leveraging Extraction Testing to Predict Patient Exposure to Polymeric Medical Device Leachables Using Physics-based Models.

Toxicological risk assessment approaches are increasingly being used in lieu of animal testing to address toxicological concerns associated with release of chemical constituents from polymeric medical device components. These approaches currently rely on in vitro extraction testing in aggressive environments to estimate patient exposure to these constituents, but the clinical relevance of the test results is often ambiguous. Physics-based mass transport models can provide a framework to interpret extraction test results to provide more clinically relevant exposure estimates. However, the models require system-specific material properties, such as diffusion (D) and partition coefficients (K), to be established a priori for the extraction conditions. Using systems comprised high-density polyethylene and 4 different additives, we demonstrate that these properties can be quantified through standard extraction testing in hexane and isopropyl alcohol. The values of D and K derived in this manner were consistent with theoretical predictions for these quantities. Based on these results, we discuss both the challenges and benefits to leveraging extraction data to parameterize physics-based exposure models. Our observations suggest that clinically relevant, yet still conservative, exposure dose estimates provided by applying this approach to a single extraction measurement can be more than 100 times lower than would be measured under typical aggressive extraction conditions. However, to apply the framework on a routine basis, limiting values of D and K must be established for device-relevant systems either through the aggregation and analysis of more extensive extraction test data and/or advancements in theoretical and computational modeling efforts to predict these quantities.

Predicting patient exposure to nickel released from cardiovascular devices using multi-scale modeling.

Many cardiovascular device alloys contain nickel, which if released in sufficient quantities, can lead to adverse health effects. However, in-vivo nickel release from implanted devices and subsequent biodistribution of nickel ions to local tissues and systemic circulation are not well understood. To address this uncertainty, we have developed a multi-scale (material, tissue, and system) biokinetic model. The model links nickel release from an implanted cardiovascular device to concentrations in peri-implant tissue, as well as in serum and urine, which can be readily monitored. The model was parameterized for a specific cardiovascular implant, nitinol septal occluders, using in-vitro nickel release test results, studies of ex-vivo uptake into heart tissue, and in-vivo and clinical measurements from the literature. Our results show that the model accurately predicts nickel concentrations in peri-implant tissue in an animal model and in serum and urine of septal occluder patients. The congruity of the model with these data suggests it may provide useful insight to establish nickel exposure limits and interpret biomonitoring data. Finally, we use the model to predict local and systemic nickel exposure due to passive release from nitinol devices produced using a wide range of manufacturing processes, as well as general relationships between release rate and exposure. These relationships suggest that peri-implant tissue and serum levels of nickel will remain below 5 µg/g and 10 µg/l, respectively, in patients who have received implanted nitinol cardiovascular devices provided the rate of nickel release per device surface area does not exceed 0.074 µg/(cm2 d) and is less than 32 µg/d in total. STATEMENT OF SIGNIFICANCE: The uncertainty in whether in-vitro tests used to evaluate metal ion release from medical products are representative of clinical environments is one of the largest roadblocks to establishing the associated patient risk. We have developed and validated a multi-scale biokinetic model linking nickel release from cardiovascular devices in-vivo to both peri-implant and systemic levels. By providing clinically relevant exposure estimates, the model vastly improves the evaluation of risk posed to patients by the nickel contained within these devices. Our model is the first to address the potential for local and systemic metal ion exposure due to a medical device and can serve as a basis for future efforts aimed at other metal ions and biomedical products.

Assessment of total silver and silver nanoparticle extraction from medical devices.

There is concern over the release of silver nanoparticles (AgNPs) from medical devices due to their potential toxicological consequences inside the body. Towards developing the exposure component of a risk assessment model, the purpose of this study was to determine the amount and physical form of silver released from medical devices. Scanning electron microscopy was used to confirm that three of five marketed medical devices contained nanosilver coatings (mean feature sizes 115-341 nm). Aqueous device extracts (water, saline and human plasma) were analyzed with inductively coupled plasma mass spectrometry, ultraviolet-visible spectroscopy, dynamic light scattering, transmission electron microscopy, and nanoparticle tracking analysis. The amount of silver extracted from the devices ranged from 1 × 10(-1) to 1 × 10(6) ng/cm(2) (conditions ranged from 37 to 50 °C, over one hour to seven days). The results further indicated that one of the five devices (labeled MD1) released significantly more AgNPs than the other devices. This data suggests that some but not all devices that are formulated with nanosilver may release detectable levels of AgNPs upon extraction. Further work is underway to quantitate the proportion of silver released as AgNPs and to incorporate this data into a risk assessment for AgNP exposure from medical devices.

Porous implants modulate healing and induce shifts in local macrophage polarization in the foreign body reaction.

The foreign body reaction (FBR) to implanted materials is of critical importance when medical devices require biological integration and vascularization to support their proper function (e.g., transcutaneous devices, implanted drug delivery systems, tissue replacements, and sensors). One class of materials that improves FBR outcomes is made by sphere-templating, resulting in porous structures with uniform, interconnected 34 µm pores. With these materials we observe reduced fibrosis and increased vascularization. We hypothesized that improved healing is a result of a shift in macrophage polarization, often measured as the ratio of M1 pro-inflammatory cells to M2 pro-healing cells. In this study, macrophage polarity of 34 µm porous implants was compared to non-porous and 160 µm porous implants in subcutaneous mouse tissue. Immunohistochemistry revealed that macrophages in implant pores displayed a shift towards an M1 phenotype compared to externalized cells. Macrophages in 34 µm porous implants had up to 63% greater expression of M1 markers and up to 85% reduction in M2 marker expression (p < 0.05). Macrophages immediately outside the porous structure, in contrast, showed a significant enrichment in M2 phenotypic cells. This study supports a role for macrophage polarization in driving the FBR to implanted materials.

Single-step process to produce surface-functionalized polymeric nanoparticles.

Nanoparticles (NPs) are a versatile medium for the localization of therapeutics to tumors and for cellular and tissue imaging. The ability to impart targeting capability or enhance cellular uptake is dependent in part on the presentation of relevant surface functionality, among other design parameters. Currently, the production of functionalized polymeric NPs requires the a priori synthesis of polymers bearing such functionality. Here we describe a process to produce functionalized polymeric NPs derived from nonfunctional polymers in a single step. This was achieved by tailoring the solvation of the polymer using a binary solvent system such that the addition of an aqueous phase rich in water-soluble polymer or polyelectrolytes results in the formation of NPs with the concomitant functionalization of NP surfaces with the polymeric moieties introduced into the aqueous phase. This strategy also allows for easy control over NP size independent of surface functionality. We have demonstrated that poly(lactic-co-glycolic acid) (PLGA) NPs bearing surface functionality as diverse as biological polysaccharides such as heparin, water-soluble ionic polymers, and poly(ethylene glycol) can be prepared under identical conditions in a single step, with surface coverage (mass %) ranging from 3 to >70%. We expect this novel process to enable complex surface engineering of NP chemistry that hitherto was impossible using existing approaches.