Biophysical control of plasticity and patterning in regeneration and cancer.

Cells and tissues display a remarkable range of plasticity and tissue-patterning activities that are emergent of complex signaling dynamics within their microenvironments. These properties, which when operating normally guide embryogenesis and regeneration, become highly disordered in diseases such as cancer. While morphogens and other molecular factors help determine the shapes of tissues and their patterned cellular organization, the parallel contributions of biophysical control mechanisms must be considered to accurately predict and model important processes such as growth, maturation, injury, repair, and senescence. We now know that mechanical, optical, electric, and electromagnetic signals are integral to cellular plasticity and tissue patterning. Because biophysical modalities underly interactions between cells and their extracellular matrices, including cell cycle, metabolism, migration, and differentiation, their applications as tuning dials for regenerative and anti-cancer therapies are being rapidly exploited. Despite this, the importance of cellular communication through biophysical signaling remains disproportionately underrepresented in the literature. Here, we provide a review of biophysical signaling modalities and known mechanisms that initiate, modulate, or inhibit plasticity and tissue patterning in models of regeneration and cancer. We also discuss current approaches in biomedical engineering that harness biophysical control mechanisms to model, characterize, diagnose, and treat disease states.

3D bioengineered neural tissue generated from patient-derived iPSCs mimics time-dependent phenotypes and transcriptional features of Alzheimer's disease.

Several iPSC-derived three-dimensional (3D) cultures have been generated to model Alzheimer's disease (AD). While some AD-related phenotypes have been identified across these cultures, none of them could recapitulate multiple AD-related hallmarks in one model. To date, the transcriptomic features of these 3D models have not been compared with those of human AD brains. However, these data are crucial to understanding the pertinency of these models for studying AD-related pathomechanisms over time. We developed a 3D bioengineered model of iPSC-derived neural tissue that combines a porous scaffold composed of silk fibroin protein with an intercalated collagen hydrogel to support the growth of neurons and glial cells into complex and functional networks for an extended time, a fundamental requisite for aging studies. Cultures were generated from iPSC lines obtained from two subjects carrying the familial AD (FAD) APP London mutation, two well-studied control lines, and an isogenic control. Cultures were analyzed at 2 and 4.5 months. At both time points, an elevated Aß42/40 ratio was detected in conditioned media from FAD cultures. However, extracellular Aß42 deposition and enhanced neuronal excitability were observed in FAD culture only at 4.5 months, suggesting that extracellular Aß deposition may trigger enhanced network activity. Remarkably, neuronal hyperexcitability has been described in AD patients early in the disease. Transcriptomic analysis revealed the deregulation of multiple gene sets in FAD samples. Such alterations were strikingly similar to those observed in human AD brains. These data provide evidence that our patient-derived FAD model develops time-dependent AD-related phenotypes and establishes a temporal relation among them. Furthermore, FAD iPSC-derived cultures recapitulate transcriptomic features of AD patients. Thus, our bioengineered neural tissue represents a unique tool to model AD in vitro over time.

Functional bioengineered models of the central nervous system.

The functional complexity of the central nervous system (CNS) is unparalleled in living organisms. Its nested cells, circuits and networks encode memories, move bodies and generate experiences. Neural tissues can be engineered to assemble model systems that recapitulate essential features of the CNS and to investigate neurodevelopment, delineate pathophysiology, improve regeneration and accelerate drug discovery. In this Review, we discuss essential structure-function relationships of the CNS and examine materials and design considerations, including composition, scale, complexity and maturation, of cell biology-based and engineering-based CNS models. We highlight region-specific CNS models that can emulate functions of the cerebral cortex, hippocampus, spinal cord, neural-X interfaces and other regions, and investigate a range of applications for CNS models, including fundamental and clinical research. We conclude with an outlook to future possibilities of CNS models, highlighting the engineering challenges that remain to be overcome.

Learning and synaptic plasticity in 3D bioengineered neural tissues.

Though neuroscientists have historically relied upon measurement of established nervous systems, contemporary advances in bioengineering have made it possible to design and build artificial neural tissues with which to investigate normative and diseased states [1-5] however, their potential to display features of learning and memory remains unexplored. Here, we demonstrate response patterns characteristic of habituation, a form of non-associative learning, in 3D bioengineered neural tissues exposed to repetitive injections of current to elicit evoked-potentials (EPs). A return of the evoked response following rest indicated learning was transient and partially reversible. Applying patterned current as massed or distributed pulse trains induced differential expression of immediate early genes (IEG) that are known to facilitate synaptic plasticity and participate in memory formation [6,7]. Our findings represent the first demonstration of a learning response in a bioengineered neural tissue in vitro.

Toward Studying Cognition in a Dish.

Bioengineered neural tissues help advance our understanding of neurodevelopment, regeneration, and neural disease; however, it remains unclear whether they can replicate higher-order functions including cognition. Building upon technical achievements in the fields of biomaterials, tissue engineering, and cell biology, investigators have generated an assortment of artificial brain structures and cocultured circuits. Though they have displayed basic electrochemical signaling, their capacities to generate minimal patterns of information processing suggestive of high-order cognitive analogues have not yet been explored. Here, we review the current state of neural tissue engineering and consider the possibility of a study of cognition in vitro. We adopt a practical definition of minimal cognition, anticipate problems of measurement, and discuss solutions toward a study of cognition in a dish.

A Novel Outbred Mouse Model to Study Lung Memory Immunological Response Induced by Pertussis Vaccines.

Lung tissue resident memory (TRM) T cells can provide rapid and effective protective immunity against respiratory pathogens such as Bordetella pertussis We assessed an outbred CD1 mouse model and i.m. immunization to study vaccine-induced immune memory, using pertussis vaccines as an example. The phenotypes of cells from the lungs of CD1 mice that had been primed with either i.m. whole-cell B. pertussis (wP), acellular B. pertussis (aP) vaccines or buffer (unvaccinated) and challenged with B. pertussis were determined using flow cytometry and immunohistology. We observed a rapid and high increase of CD4+T cells expressing TRM markers by flow cytometry, supported by immunohistology observations, in lungs from wP-immunized mice. Priming mice with wP vaccine induced a more potent CD4+ response in lungs following B. pertussis challenge than priming with aP vaccine, although both were less potent than that observed in primoinfected mice. We also observed for the first time, to our knowledge, that CD8+ and γδ+ TRM-like T cell responses were induced in lungs of wP-primed mice postinfection. This novel outbred CD1 mouse model with i.m. immunization that enabled us to study vaccine-induced B. pertussis-specific memory T cells in lungs could be useful for evaluating candidate parenteral vaccines against B. pertussis or others pulmonary pathogens.

A 3D Tissue Model of Traumatic Brain Injury with Excitotoxicity That Is Inhibited by Chronic Exposure to Gabapentinoids.

Injury progression associated with cerebral laceration is insidious. Following the initial trauma, brain tissues become hyperexcitable, begetting further damage that compounds the initial impact over time. Clinicians have adopted several strategies to mitigate the effects of secondary brain injury; however, higher throughput screening tools with modular flexibility are needed to expedite mechanistic studies and drug discovery that will contribute to the enhanced protection, repair, and even the regeneration of neural tissues. Here we present a novel bioengineered cortical brain model of traumatic brain injury (TBI) that displays characteristics of primary and secondary injury, including an outwardly radiating cell death phenotype and increased glutamate release with excitotoxic features. DNA content and tissue function were normalized by high-concentration, chronic administrations of gabapentinoids. Additional experiments suggested that the treatment effects were likely neuroprotective rather than regenerative, as evidenced by the drug-mediated decreases in cell excitability and an absence of drug-induced proliferation. We conclude that the present model of traumatic brain injury demonstrates validity and can serve as a customizable experimental platform to assess the individual contribution of cell types on TBI progression, as well as to screen anti-excitotoxic and pro-regenerative compounds.

Matrix Deformation with Ectopic Cells Induced by Rotational Motion in Bioengineered Neural Tissues.

The brain's extracellular matrix (ECM) is a dynamic protein-based scaffold within which neural networks can form, self-maintain, and re-model. When the brain incurs injuries, microscopic tissue tears and active ECM re-modelling give way to abnormal brain structure and function including the presence of ectopic cells. Post-mortem and neuroimaging data suggest that the brains of jet pilots and astronauts, who are exposed to rotational forces, accelerations, and microgravity, display brain anomalies which could be indicative of a mechanodisruptive pathology. Here we present a model of non-impact-based brain injury induced by matrix deformation following mechanical shaking. Using a bioengineered 3D neural tissue platform, we designed a repetitive shaking paradigm to simulate subtle rotational acceleration. Our results indicate shaking induced ectopic cell clustering that could be inhibited by physically restraining tissue movement. Imaging revealed that the collagen substrate surrounding cells was deformed following shaking. Applied to neonatal rat brains, shaking induced deformation of extracellular spaces within the cerebral cortices and reduced the number of cell bodies at higher accelerations. We hypothesize that ECM deformation may represent a more significant role in brain injury progression than previously assumed and that the present model system contributes to its understanding as a phenomenon.

A 3D human brain-like tissue model of herpes-induced Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder that causes cognitive decline, memory loss, and inability to perform everyday functions. Hallmark features of AD-including generation of amyloid plaques, neurofibrillary tangles, gliosis, and inflammation in the brain-are well defined; however, the cause of the disease remains elusive. Growing evidence implicates pathogens in AD development, with herpes simplex virus type I (HSV-1) gaining increasing attention as a potential causative agent. Here, we describe a multidisciplinary approach to produce physiologically relevant human tissues to study AD using human-induced neural stem cells (hiNSCs) and HSV-1 infection in a 3D bioengineered brain model. We report a herpes-induced tissue model of AD that mimics human disease with multicellular amyloid plaque-like formations, gliosis, neuroinflammation, and decreased functionality, completely in the absence of any exogenous mediators of AD. This model will allow for future studies to identify potential downstream drug targets for treating this devastating disease.

Modeling Controlled Cortical Impact Injury in 3D Brain-Like Tissue Cultures.

Traumatic brain injury (TBI) survivors suffer long term from mental illness, neurodegeneration, and neuroinflammation. Studies of 3D tissue models have provided new insights into the pathobiology of many brain diseases. Here, a 3D in vitro contusion model is developed consisting of mouse cortical neurons grown on a silk scaffold embedded in collagen and used outcomes from an in vivo model for benchmarking. Molecular, cellular, and network events are characterized in response to controlled cortical impact (CCI). In this model, CCI induces degradation of neural network structure and function and release of glutamate, which are associated with the expression of programmed necrosis marker phosphorylated Mixed Lineage Kinase Domain Like Pseudokinase (pMLKL). Neurodegeneration is observed first in the directly impacted area and it subsequently spreads over time in 3D space. CCI reduces phosphorylated protein kinase B (pAKT) and Glycogen synthase kinase 3 beta (GSK3ß) in neurons in vitro and in vivo, but discordant responses are observed in phosphprylated ribosomal S6 kinase (pS6) and phosphorylated Tau (pTau) expression. In summary, the 3D brain-like culture system mimicked many aspects of in vivo responses to CCI, providing evidence that the model can be used to study the molecular, cellular, and functional sequelae of TBI, opening up new possibilities for discovery of therapeutics.

A Long-Living Bioengineered Neural Tissue Platform to Study Neurodegeneration.

The prevalence of dementia and other neurodegenerative diseases continues to rise as age demographics in the population shift, inspiring the development of long-term tissue culture systems with which to study chronic brain disease. Here, it is investigated whether a 3D bioengineered neural tissue model derived from human induced pluripotent stem cells (hiPSCs) can remain stable and functional for multiple years in culture. Silk-based scaffolds are seeded with neurons and glial cells derived from hiPSCs supplied by human donors who are either healthy or have been diagnosed with Alzheimer's disease. Cell retention and markers of stress remain stable for over 2 years. Diseased samples display decreased spontaneous electrical activity and a subset displays sporadic-like indicators of increased pathological ß-amyloid and tau markers characteristic of Alzheimer's disease with concomitant increases in oxidative stress. It can be concluded that the long-term stability of the platform is suited to study chronic brain disease including neurodegeneration.

Optogenetically induced cellular habituation in non-neuronal cells.

Habituation, defined as the reversible decrement of a response during repetitive stimulation, is widely established as a form of non-associative learning. Though more commonly ascribed to neural cells and systems, habituation has also been observed in single aneural cells, although evidence is limited. Considering the generalizability of the habituation process, we tested the degree to which organism-level behavioral and single cell manifestations were similar. Human embryonic kidney (HEK) cells that overexpressed an optogenetic actuator were photostimulated to test the effect of different stimulation protocols on cell responses. Depolarization induced by the photocurrent decreased successively over the stimulation protocol and the effect was reversible upon withdrawal of the stimulus. In addition to frequency- and intensity-dependent effects, the history of stimulations on the cells impacted subsequent depolarization in response to further stimulation. We identified tetraethylammonium (TEA)-sensitive native K+ channels as one of the mediators of this habituation phenotype. Finally, we used a theoretical model of habituation to elucidate some mechanistic aspects of the habituation response. In conclusion, we affirm that habituation is a time- and state-dependent biological strategy that can be adopted also by individual non-neuronal cells in response to repetitive stimuli.

On the Generalization of Habituation: How Discrete Biological Systems Respond to Repetitive Stimuli: A Novel Model of Habituation That Is Independent of Any Biological System.

Habituation, a form of non-associative learning, isno longer studied exclusively within the fields of psychology and neuroscience. Indeed, the same stimulus-response pattern is observed at the molecular, cellular, and organismal scales and is not dependent upon the presence of neurons. Hence, a more inclusive theory is required to accommodate aneural forms of habituation. Here an abstraction of the habituation process that does not rely upon particular biological pathways or substrates is presented. Instead, five generalizable elements that define the habituation process are operationalized. The formulation can be applied to interrogate systems as they respond to several stimulation paradigms, providing new insights and supporting existing behavioral data. The model can be used to deduce the relative contribution of elements that contribute to the measurable output of the system. The results suggest that habituation serves as a general biological strategy that any system can implement to adaptively respond to harmless, repetitive stimuli.

Functional Effects of a Neuromelanin Analogue on Dopaminergic Neurons in 3D Cell Culture.

The substantia nigra pars compacta (SNpc) is a discrete region of the brain that exhibits a dark pigment, neuromelanin (NM), a biomaterial with unique properties and the subject of ongoing research pertaining to neurodegenerative conditions like Parkinson's disease (PD). Obtaining human tissue to isolate this pigment is costly and labor intensive, making it necessary to find alternatives to model the biochemical interaction of NM and its implications on PD. To address this limitation, we modified our established silk 3D brain tissue model to emulate key characteristics of the SNpc by using a structural analogue of NM to examine the effects of the material on dopaminergic neurons using Lund's human mesencephalon (LUHMES) cells. We utilized a sepia-melanin, squid ink, derived NM analogue (NM-sim) to chelate ferric iron, and this iron-neuromelanin precipitate (Fe-NM) was purified and characterized. We then exposed LUHMES dopaminergic cells to the NM-sim, Fe-NM-sim, and control vehicle within 3D silk protein scaffolds. The presence of both NM-sim and Fe-NM-sim in the scaffolds negatively impacted spontaneous electrical activity from the LUMES networks, as evidenced by changes in local field potential (LFP) electrophysiological recordings. Furthermore, the Fe-NM-sim precipitate generated peroxides, depleted nutrients/antioxidants, and increased protein oxidation by carbonylation in sustained (>2 weeks) 3D cultures, thereby contributing to cell dysfunction. The results suggest that this 3D tissue engineered brain-like model may provide useful readouts related to PD neuro-toxicology research.

Single-sided deaf tactical unit officer treated with cochlear implantation.

OBJECTIVES: To demonstrate the efficacy of cochlear implantation for management of a severe single-sided deaf tactical unit officer. Showing that hearing improvement provided by cochlear implantation was sufficient for him to return to work as a police tactical officer. METHODS: Case report. RESULTS: A 43 years-old man working as a tactical unit officer suffered from a work-related severe single-sided hearing loss. He tried unsuccessfully many types of hearing aids including a contralateral routing of sound (CROS) system and a bone conduction hearing aid with a headband. He was finally treated with cochlear implantation which provided enough localization and hearing abilities for a complete return to work as a tactical unit officer. DISCUSSION: This case report demonstrates that cochlear implantation was very successful for a patient suffering from severe single-sided hearing loss. The current literature demonstrates benefits from cochlear implant over CROS and osseointegrated devices in unilateral hearing loss. Nevertheless, literature does not report any case of patient suffering from single-sided deafness that received a cochlear implant and a complete bilateral hearing rehabilitation that allowed him to return to work as a police tactical officer. CONCLUSION: Cochlear implantation was the only effective treatment to provide our patient enough localization and hearing abilities for a complete return to work as a police tactical officer. As no consensus has been established about selection criteria for cochlear implantation in single-sided deafness, we suggest that the patient's occupation should be considered when reviewing his candidacy for cochlear implantation.

Biophotonic markers of malignancy: Discriminating cancers using wavelength-specific biophotons.

Early detection is a critically important factor when successfully diagnosing and treating cancer. Whereas contemporary molecular techniques are capable of identifying biomarkers associated with cancer, surgical interventions are required to biopsy tissue. The common imaging alternative, positron-emission tomography (PET), involves the use of nuclear material which poses some risks. Novel, non-invasive techniques to assess the degree to which tissues express malignant properties are now needed. Recent developments in biophoton research have made it possible to discriminate cancerous cells from normal cells both in vitro and in vivo. The current study expands upon a growing body of literature where we classified and characterized malignant and non-malignant cell types according to their biophotonic activity. Using wavelength-exclusion filters, we demonstrate that ratios between infrared and ultraviolet photon emissions differentiate cancer and non-cancer cell types. Further, we identified photon sources associated with three filters (420-nm, 620-nm., and 950-nm) which classified cancer and non-cancer cell types. The temporal increases in biophoton emission within these wavelength bandwidths is shown to be coupled with intrisitic biomolecular events using Cosic's resonant recognition model. Together, the findings suggest that the use of wavelength-exclusion filters in biophotonic measurement can be employed to detect cancer in vitro.

Antibodies to PcpA and PhtD protect mice against Streptococcus pneumoniae by a macrophage- and complement-dependent mechanism.

Currently marketed Streptococcus pneumoniae (Spn) vaccines, which contain polysaccharide capsular antigens from the most common Spn serotypes, have substantially reduced pneumococcal disease rates but have limited coverage. A trivalent pneumococcal protein vaccine containing pneumococcal choline-binding protein A (PcpA), pneumococcal histidine triad protein D (PhtD), and detoxified pneumolysin is being developed to provide broader, cross-serotype protection. Antibodies against detoxified pneumolysin protect against bacterial pneumonia by neutralizing Spn-produced pneumolysin, but how anti-PhtD and anti-PcpA antibodies protect against Spn has not been established. Here, we used a murine passive protection sepsis model to investigate the mechanism of protection by anti-PhtD and anti-PcpA antibodies. Depleting complement using cobra venom factor eliminated protection by anti-PhtD and anti-PcpA monoclonal antibodies (mAbs). Consistent with a requirement for complement, complement C3 deposition on Spn in vitro was enhanced by anti-PhtD and anti-PcpA mAbs and by sera from PhtD- and PcpA-immunized rabbits and humans. Moreover, in the presence of complement, anti-PhtD and anti-PcpA mAbs increased uptake of Spn by human granulocytes. Depleting neutrophils using anti-Ly6G mAbs, splenectomy, or a combination of both did not affect passive protection against Spn, whereas depleting macrophages using clodronate liposomes eliminated protection. These results suggest anti-PhtD and anti-PcpA antibodies induced by pneumococcal protein vaccines protect against Spn by a complement- and macrophage-dependent opsonophagocytosis.

Right cerebral hemispheric sensitivity to pH and physiological ions in fixed post-mortem Wistar rat brains.

Post-mortem human neural tissues fixed in ethanol and aldehyde-based solutions express modulated frequency-dependent microvolt potentials when probed by chemical and electrical stimuli. These observations run contrary to the assumption that basic tissue functions are irreversibly impaired upon fixation, in the absence of nutrients and sufficient concentrations of physiological ions. The aim of the current study was to investigate the relative effects of pH and specific charged particles relevant to normal cell physiology upon electric potentials associated with fixed post-mortem rat brain tissue. We identified a positive relationship between the total time the brains had been immersed in ethanol-formalin-acetic acid and high-frequency microvolt potentials within the dorsal right hemisphere of the rat cerebrum. Measuring the pH of the fixative solution surrounding the brains indicated that as time increased, a logarithmic trend toward alkalinity could be observed. Further experiments revealed that high-frequency microvolt potentials were related to pH changes within the right hemisphere only. The right ventral cerebrum displayed a unique response to potassium chloride in ways uncounted for by pH alone. The results suggest that the fixed post-mortem right cerebrum of the rat is particularly sensitive to pH and physiological ions which explains a subset of previous findings with respect to stimulus-response patterns in human coronal brain sections. A concluding hypothesis is presented which suggests that brain tissue expresses material properties independent of metabolic activity though perhaps relevant to living brain function.

When Is the Brain Dead? Living-Like Electrophysiological Responses and Photon Emissions from Applications of Neurotransmitters in Fixed Post-Mortem Human Brains.

The structure of the post-mortem human brain can be preserved by immersing the organ within a fixative solution. Once the brain is perfused, cellular and histological features are maintained over extended periods of time. However, functions of the human brain are not assumed to be preserved beyond death and subsequent chemical fixation. Here we present a series of experiments which, together, refute this assumption. Instead, we suggest that chemical preservation of brain structure results in some retained functional capacity. Patterns similar to the living condition were elicited by chemical and electrical probes within coronal and sagittal sections of human temporal lobe structures that had been maintained in ethanol-formalin-acetic acid. This was inferred by a reliable modulation of frequency-dependent microvolt fluctuations. These weak microvolt fluctuations were enhanced by receptor-specific agonists and their precursors (i.e., nicotine, 5-HTP, and L-glutamic acid) as well as attenuated by receptor-antagonists (i.e., ketamine). Surface injections of 10 nM nicotine enhanced theta power within the right parahippocampal gyrus without any effect upon the ipsilateral hippocampus. Glutamate-induced high-frequency power densities within the left parahippocampal gyrus were correlated with increased photon counts over the surface of the tissue. Heschl's gyrus, a transverse convexity on which the primary auditory cortex is tonotopically represented, retained frequency-discrimination capacities in response to sweeps of weak (2µV) square-wave electrical pulses between 20 Hz and 20 kHz. Together, these results suggest that portions of the post-mortem human brain may retain latent capacities to respond with potential life-like and virtual properties.