Medical Device Industry Approaches for Addressing Sources of Failing Cytotoxicity Scores

To ensure patient safety, medical device manufacturers are required by the Food and Drug Administration and other regulatory bodies to perform biocompatibility evaluations on their devices per standards, such as the AAMI-approved ISO 10993-1:2018 (ANSI/AAMI/ISO 10993-1:2018).However, some of these biological tests (e.g., systemic toxicity studies) have long lead times and are costly, which may hinder the release of new medical devices. In recent years, an alternative method using a risk-based approach for evaluating the toxicity (or biocompatibility) profile of chemicals and materials used in medical devices has become more mainstream. This approach is used as a complement to or substitute for traditional testing methods (e.g., systemic toxicity endpoints). Regardless of the approach, the one test still used routinely in initial screening is the cytotoxicity test, which is based on an in vitro cell culture system to evaluate potential biocompatibility effects of the final finished form of a medical device. However, it is known that this sensitive test is not always compatible with specific materials and can lead to failing cytotoxicity scores and an incorrect assumption of potential biological or toxicological adverse effects. This article discusses the common culprits of in vitro cytotoxicity failures, as well as describes the regulatory-approved methodology for cytotoxicity testing and the approach of using toxicological risk assessment to address clinical relevance of cytotoxicity failures for medical devices. Further, discrepancies among test results from in vitro tests, use of published half-maximal inhibitory concentration data, and the derivation of their relationship to tolerable exposure limits, reference doses, or no observed adverse effect levels are highlighted to demonstrate that although cytotoxicity tests in general are regarded as a useful sensitive screening assays, specific medical device materials are not compatible with these cellular/in vitro systems. For these cases, the results should be analyzed using more clinically relevant approaches (e.g., through chemical analysis or written risk assessment).

Medical devices are engineered to be of durable construction and to accommodate the functionality needed for proper device application. The biocompatibility of the materials, as well as their processing, is also important to ensure that the patients are not negatively affected by the devices when they enter the clinical setting. Certain materials of constructions used for medical devices (and manufacturing processes or processing aids) may contain chemicals that can lead to failing cytotoxicity scores using traditional, regulatory-mandated methodologies. Examples of common materials include plastics (e.g., polyethylene or polypropylene [co]polymers, polyvinyl chloride [PVC]) and metals (e.g., nitinol, copper [Cu]-containing alloys). Although providing stable and reliable materials for use in relation to performance parameters, various metals/alloys and plastics may evoke undesired cytotoxic effects. These effects might be observed as reduced cellular activity or decay in the in vitro assay, especially when standard methods and test parameters (e.g., extraction ratios) are used.1,2To prevent adverse effects (e.g., toxicity, or other types of biocompatibility-related issues) from occurring among patients and clinical end users, manufacturers are required to perform biocompatibility evaluations per guidance provided in e.g., ANSI/AAMI/ISO 10993-1:2018.3 This standard provides an overall framework for the biological evaluation, emphasizing a risk-based approach, as well as general guidance on relevant tests for specific types of contact to patients or users. Of note, traditional biocompatibility tests, within the battery of both in vivo and in vitro methods, could take up to 6 months (or take years, in the case of long-term systemic toxicity testing). Lengthy turnaround times stem from in vivo test methods, which are performed on animal models and include irritation, sensitization, systemic toxicity, genotoxicity, and carcinogenicity studies. Traditional in vitro tests involve exposure of cells or cellular material to device extracts in order to characterize toxicity in terms of cytotoxicity, genotoxicity, cellular metabolic activity, and aspects of hemocompatibility.3In recent years, as a complement to or a substitute for traditional testing methods, a risk-based approach using a chemical and materials characterization for evaluation of patient safety has become mainstream. The framework for this approach is provided in ISO 10993-18:2020.4 Moreover, the Association for the Advancement of Medical Instrumentation (AAMI) and, by extension, regulatory bodies (including the Food and Drug Administration [FDA] and International Organization for Standardization [ISO]) have driven the use of chemical and material characterization. Particularly for medical devices in long-term contact with patient (e.g., implantable devices), use of chemical and material characterization can reduce unnecessary animal testing and provide results that are scientifically sound and detailed, while being more cost and time efficient. For example, ISO 10993-13 highlights that a correctly conducted risk assessment can provide justification to exclude long-term biological testing, where the nature and extent of exposure confirms that the patient is being exposed to very low levels of chemicals that are below relevant toxicological thresholds.3Throughout the ISO 10993 series, it also is emphasized that conducting animal testing for biological risk evaluation should only be considered after all alternative courses of action (review of prior knowledge, chemical or physical characterization, in vitro evaluations, or alternative means of mitigation) have been exhausted. In addition, analytical chemistry used for chemical characterization can be used as a means for investigating possible culprits when traditional biocompatibility tests, such as cytotoxicity tests, fail, especially in cases where a known substance(s) in the material has cytotoxic potential (e.g., silver-infused wound dressing that provides antibacterial properties).However, it should be kept in mind that although chemistry can be a powerful tool in many cases, not all medical devices extracts are compatible with the analytical methods and instruments used, and these studies may not provide the full understanding of the toxicity profile of the device. In those cases, animal testing or further justification may still be needed to demonstrate a safe biocompatibility profile for the device.Cytotoxicity testing per AAMI/ISO 10993-5:2009/(R)20145 has historically been one of the most used (and is considered the most reactive) of the biocompatibility tests6,7 and can be efficiently used to detect abnormal effects to cells that may arise if harmful chemicals are present in device extracts. However, it also is recognized that cell-based test methods do not necessarily correlate to in vivo toxicological effects and actual clinical patient safety, often showing a reaction when no clinical adverse effects are known or expected to occur. For instance, some soluble metal ions (e.g., Cu, nickel [Ni]) are known to exert toxic effects on cells in an in vitro setting; however, their presence in surgical instruments and implants has demonstrated high patient tolerance and negligible effects upon clinical use.This article provides a brief evaluation of the clinical impact of metals and plasticizers commonly used in medical device materials that may lead to patient exposure during the use of devices, with emphasis given to those that may result in cytotoxicity failures in an in vitro setting. In addition, an approach to evaluating valid clinical risks using a toxicological risk assessment is discussed.     

The specificity of the malarial VAR2CSA protein for chondroitin sulfate depends on 4-O-sulfation and ligand accessibility

Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant subfragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (∼80–85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection.     

Reversal of response to artificial selection on body size in a wild passerine

A general assumption in quantitative genetics is the existence of an intermediate phenotype with higher mean individual fitness in the average environment than more extreme phenotypes. Here, we investigate the evolvability and presence of such a phenotype in wild bird populations from an eleven‐year experiment with four years of artificial selection for long and short tarsus length, a proxy for body size. The experiment resulted in strong selection in the imposed directions. However, artificial selection was counteracted by reduced production of recruits in offspring of artificially selected parents. This resulted in weak natural selection against extreme trait values. Significant responses to artificial selection were observed at both the phenotypic and genetic level, followed by a significant return toward preexperimental means. During artificial selection, the annual observed phenotypic response closely followed the predicted response from quantitative genetic theory ( = 0.96, = 0.56). The rapid return to preexperimental means was induced by three interacting mechanisms: selection for an intermediate phenotype, immigration, and recombination between selected and unselected individuals. The results of this study demonstrates the evolvability of phenotypes and that selection may favor an intermediate phenotype in wild populations.     

Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion

Recent studies have suggested that regeneration of non-haematopoietic cell lineages can occur through heterotypic cell fusion with haematopoietic cells of the myeloid lineage. Here we show that lymphocytes also form heterotypic-fusion hybrids with cardiomyocytes, skeletal muscle, hepatocytes and Purkinje neurons. However, through lineage fate-mapping we demonstrate that such in vivo fusion of lymphoid and myeloid blood cells does not occur to an appreciable extent in steady-state adult tissues or during normal development. Rather, fusion of blood cells with different non-haematopoietic cell types is induced by organ-specific injuries or whole-body irradiation, which has been used in previous studies to condition recipients of bone marrow transplants. Our findings demonstrate that blood cells of the lymphoid and myeloid lineages contribute to various non-haematopoietic tissues by forming rare fusion hybrids, but almost exclusively in response to injuries or inflammation.     

Bulk Flow Revisited: Transport of a Soluble Protein in the Secretory Pathway

The C-terminal domain, Cp, of the Semliki Forest virus capsid protein, known for its rapid, efficient and chaperone-independent folding, was used to measure bulk fluid flow in the secretory pathway of Chinese hamster ovary cells. Being small, nonglycosylated, soluble and cytoplasmic in origin, Cp was not likely to interact with lectins, cargo receptors and retention factors. Using pulse-chase analysis, we observed that translocation into the endoplasmic reticulum resulted in rapid and efficient folding and transport of the newly synthesized Cp protein to the extracellular medium. The first Cp molecules were secreted 15 min after synthesis, which is the fastest transport of a protein so far recorded in mammalian cells. The rate constant of secretion was 1.2% per min, which amounts to an estimated bulk flow rate of about 155 coat protein II (COPII) vesicles per second. Transport was independent of expression level, and blocked by CI-976, brefeldin A and ATP depletion indicating that it depended on COPII vesicle formation, and followed the classical secretory pathway. In polarized Madin-Darby canine kidney cells, the secretion rate was similar but occurred mainly apically. The results demonstrated that fluid flow in the secretory pathway is fast, and can therefore play a significant role in the secretion of soluble secretory products.     

Introduction to "In Focus: Global Change and Adaptation in Local Places"

ABSTRACT   In recognition of unavoidable changes that human actions are producing in our environment, the term adaptation has become ubiquitous in the environmental and climate-change literature. Human adaptation is a field with a significant history in anthropology, yet anthropological contributions to the burgeoning field of climate change remain limited. This "In Focus" section presents studies of local adaptations to climate variation and change. Each is concerned with current environmental challenges and future environmental change, and each study is placed within a wider context that includes processes of globalization and integration into market economies, formal and informal institutions, and disasters. These studies highlight the challenges involved in understanding complex adaptations under conditions of stress. They also illustrate how anthropologists engage the larger climate-change and human-adaptation discussions and enhance our ability to respond to the challenges of a changing environment.     

Complete genome sequence of the hyperthermophilic, piezophilic, heterotrophic, and carboxydotrophic archaeon Thermococcus barophilus MP

Thermococcus barophilus is a hyperthermophilic, anaerobic, mixed heterotrophic, and carboxydotrophic euryarchaeon isolated from the deep sea hydrothermal vent Snakepit site on the mid-Atlantic ridge at a depth of 3,550 m. T. barophilus is the first true piezophilic, hyperthermophilic archaeon isolated, having an optimal growth at 40 MPa. Here we report the complete genome sequence of strain MP, the type strain of T. barophilus. The genome data reveal a close proximity with Thermococcus sibiricus, another Thermococcus isolated from the deep biosphere and a possible connection to life in the depths.     

Segment-specific generation of Drosophila Capability neuropeptide neurons by multi-faceted Hox cues

In the Drosophila ventral nerve cord, the three pairs of Capability neuropeptide-expressing Va neurons are exclusively found in the second, third and fourth abdominal segments (A2–A4). To address the underlying mechanisms behind such segment-specific cell specification, we followed the developmental specification of these neurons. We find that Va neurons are initially generated in all ventral nerve cord segments and progress along a common differentiation path. However, their terminal differentiation only manifests itself in A2–A4, due to two distinct mechanisms: segment-specific programmed cell death (PCD) in posterior segments, and differentiation to an alternative identity in segments anterior to A2. Genetic analyses reveal that the Hox homeotic genes are involved in the segment-specific appearance of Va neurons. In posterior segments, the Hox gene Abdominal-B exerts a pro-apoptotic role on Va neurons, which involves the function of several RHG genes. Strikingly, this role of Abd-B is completely opposite to its role in the segment-specific apoptosis of other classes of neuropeptide neurons, the dMP2 and MP1 neurons, where Abd-B acts in an anti-apoptotic manner. In segments A2–A4 we find that abdominal A is important for the terminal differentiation of Va cell fate. In the A1 segment, Ultrabithorax acts to specify an alternate Va neuron fate. In contrast, in thoracic segments, Antennapedia suppresses the Va cell fate. Thus, Hox genes act in a multi-faceted manner to control the segment-specific appearance of the Va neuropeptide neurons in the ventral nerve cord.     

An EMG-driven musculoskeletal model to estimate muscle forces and knee joint moments in vivo

This paper examined if an electromyography (EMG) driven musculoskeletal model of the human knee could be used to predict knee moments, calculated using inverse dynamics, across a varied range of dynamic contractile conditions. Muscle-tendon lengths and moment arms of 13 muscles crossing the knee joint were determined from joint kinematics using a three-dimensional anatomical model of the lower limb. Muscle activation was determined using a second-order discrete non-linear model using rectified and low-pass filtered EMG as input. A modified Hill-type muscle model was used to calculate individual muscle forces using activation and muscle tendon lengths as inputs. The model was calibrated to six individuals by altering a set of physiologically based parameters using mathematical optimisation to match the net flexion/extension (FE) muscle moment with those measured by inverse dynamics. The model was calibrated for each subject using 5 different tasks, including passive and active FE in an isokinetic dynamometer, running, and cutting manoeuvres recorded using three-dimensional motion analysis. Once calibrated, the model was used to predict the FE moments, estimated via inverse dynamics, from over 200 isokinetic dynamometer, running and sidestepping tasks. The inverse dynamics joint moments were predicted with an average R(2) of 0.91 and mean residual error of approximately 12 Nm. A re-calibration of only the EMG-to-activation parameters revealed FE moments prediction across weeks of similar accuracy. Changing the muscle model to one that is more physiologically correct produced better predictions. The modelling method presented represents a good way to estimate in vivo muscle forces during movement tasks.     

The inter-monomer interface of the major light-harvesting chlorophyll a/b complexes of photosystem II (LHCII) influences the chlorophyll triplet distribution

Under strong light conditions, long-lived chlorophyll triplets (³Chls) are formed, which can sensitize singlet oxygen, a species harmful to the photosynthetic apparatus of plants. Plants have developed multiple photoprotective mechanisms to quench ³Chl and scavenge singlet oxygen in order to sustain the photosynthetic activities. The lumenal loop of light-harvesting chlorophyll a/b complex of photosystem II (LHCII) plays important roles in regulating the pigment conformation and energy dissipation. In this study, site-directed mutagenesis analysis was applied to investigate triplet–triplet energy transfer and quenching of ³Chl in LHCII. We mutated the amino acid at site 123 located in this region to Gly, Pro, Gln, Thr and Tyr, respectively, and recorded fluorescence excitation spectra, triplet-minus-singlet (TmS) spectra and kinetics of carotenoid triplet decay for wild type and all the mutants. A red-shift was evident in the TmS spectra of the mutants S123T and S123P, and all of the mutants except S123Y showed a decrease in the triplet energy transfer efficiency. We propose, on the basis of the available structural information, that these phenomena are related to the involvement, due to conformational changes in the lumenal region, of a long-wavelength lutein (Lut2) involved in quenching ³Chl.