Regenerative Adaptation to Electrochemical Perturbation in Planaria: A Molecular Analysis of Physiological Plasticity.

Anatomical homeostasis results from dynamic interactions between gene expression, physiology, and the external environment. Owing to its complexity, this cellular and organism-level phenotypic plasticity is still poorly understood. We establish planarian regeneration as a model for acquired tolerance to environments that alter endogenous physiology. Exposure to barium chloride (BaCl2) results in a rapid degeneration of anterior tissue in Dugesia japonica. Remarkably, continued exposure to fresh solution of BaCl2 results in regeneration of heads that are insensitive to BaCl2. RNA-seq revealed transcriptional changes in BaCl2-adapted heads that suggests a model of adaptation to excitotoxicity. Loss-of-function experiments confirmed several predictions: blockage of chloride and calcium channels allowed heads to survive initial BaCl2 exposure, inducing adaptation without prior exposure, whereas blockade of TRPM channels reversed adaptation. Such highly adaptive plasticity may represent an attractive target for biomedical strategies in a wide range of applications beyond its immediate relevance to excitotoxicity preconditioning.

Neural control of body-plan axis in regenerating planaria.

Control of axial polarity during regeneration is a crucial open question. We developed a quantitative model of regenerating planaria, which elucidates self-assembly mechanisms of morphogen gradients required for robust body-plan control. The computational model has been developed to predict the fraction of heteromorphoses expected in a population of regenerating planaria fragments subjected to different treatments, and for fragments originating from different regions along the anterior-posterior and medio-lateral axis. This allows for a direct comparison between computational and experimental regeneration outcomes. Vector transport of morphogens was identified as a fundamental requirement to account for virtually scale-free self-assembly of the morphogen gradients observed in planarian homeostasis and regeneration. The model correctly describes altered body-plans following many known experimental manipulations, and accurately predicts outcomes of novel cutting scenarios, which we tested. We show that the vector transport field coincides with the alignment of nerve axons distributed throughout the planarian tissue, and demonstrate that the head-tail axis is controlled by the net polarity of neurons in a regenerating fragment. This model provides a comprehensive framework for mechanistically understanding fundamental aspects of body-plan regulation, and sheds new light on the role of the nervous system in directing growth and form.

Clinical Application of Mathematical Long Bone Ratios to Calculate Appropriate Donor Limb Lengths in Bilateral Upper Limb Transplantation.

Background: Limited methods exist to aid in deciding the appropriate donor limb lengths in bilateral upper limb amputees qualifying for vascularized composite allotransplantation. We hypothesized mathematical equations could be created using long bone length ratios, and applied to radiographs, to approximate the patient's limb length prior to amputation. Methods: A data set of 30 skeletons' unilateral upper limb long bones measured using osteometric board and calipers was used. Anatomic segment ratios were calculated based on humerus length after multivariate linear regression analysis. For clinical application testing, 5 cadavers' upper limbs were radiographed. Radiographic bone lengths were then measured along the long axis of each long bone. These measured radiographic lengths were then compared with the predicted bone lengths, generated from the skeleton data set ratios, for each cadaver. Results: The chi-square goodness-of-fit test showed excellent fit (P < .01) between the predicted and radiographically measured lengths for the 5 cadavers, and interobserver measurements showed no statistical difference. Depending on the cadaver, percent error in total limb length predicted to measure ranged from 0.9% to 2.7%. The variables to multiply an individual humerus length to calculate a given anatomic segment thus proved to be effective. Conclusions: If a bilateral upper limb amputee has 1 intact humerus, ratios to the humerus length can be reliably applied to calculate the preamputation limb length based on the patient's radiographic humerus length. These formulas are indicated for finding the appropriate limb lengths, and smaller anatomic segments, for donor-recipient matching in upper limb transplantation.

Planarian regeneration as a model of anatomical homeostasis: Recent progress in biophysical and computational approaches.

Planarian behavior, physiology, and pattern control offer profound lessons for regenerative medicine, evolutionary biology, morphogenetic engineering, robotics, and unconventional computation. Despite recent advances in the molecular genetics of stem cell differentiation, this model organism's remarkable anatomical homeostasis provokes us with truly fundamental puzzles about the origin of large-scale shape and its relationship to the genome. In this review article, we first highlight several deep mysteries about planarian regeneration in the context of the current paradigm in this field. We then review recent progress in understanding of the physiological control of an endogenous, bioelectric pattern memory that guides regeneration, and how modulating this memory can permanently alter the flatworm's target morphology. Finally, we focus on computational approaches that complement reductive pathway analysis with synthetic, systems-level understanding of morphological decision-making. We analyze existing models of planarian pattern control and highlight recent successes and remaining knowledge gaps in this interdisciplinary frontier field.

Bioelectrical coupling in multicellular domains regulated by gap junctions: A conceptual approach.

We review the basic concepts involved in bioelectrically-coupled multicellular domains, focusing on the role of membrane potentials (Vmem). In the first model, single-cell Vmem is modulated by two generic polarizing and depolarizing ion channels, while intercellular coupling is implemented via voltage-gated gap junctions. Biochemical and bioelectrical signals are integrated via a feedback loop between Vmem and the transcription and translation of a protein forming an ion channel. The effective rate constants depend on the single-cell Vmem because these potentials modulate the local concentrations of signaling molecules and ions. This electrochemically-based idealization of the complex biophysical problem suggests that the spatio-temporal map of single-cell potentials can influence downstream patterning processes by means of the voltage-gated gap junction interconnectivity, much as in the case of electronic devices where the control of electric potentials and currents allows the local modulation of the circuitry to achieve full functionality. An alternative theoretical approach, the BioElectrical Tissue Simulation Engine (BETSE), is also presented. The BETSE modeling environment utilizes finite volume techniques to simulate bioelectric states from the perspective of ion concentrations and fluxes. This model has been successfully applied to make predictions and explain experimental observations in a variety of embryonic, regenerative, and oncogenic contexts.

Bioelectrical control of positional information in development and regeneration: A review of conceptual and computational advances.

Positional information describes pre-patterns of morphogenetic substances that alter spatio-temporal gene expression to instruct development of growth and form. A wealth of recent data indicate bioelectrical properties, such as the transmembrane potential (Vmem), are involved as instructive signals in the spatiotemporal regulation of morphogenesis. However, the mechanistic relationships between Vmem and molecular positional information are only beginning to be understood. Recent advances in computational modeling are assisting in the development of comprehensive frameworks for mechanistically understanding how endogenous bioelectricity can guide anatomy in a broad range of systems. Vmem represents an extraordinarily strong electric field (∼1.0 × 106 V/m) active over the thin expanse of the plasma membrane, with the capacity to influence a variety of downstream molecular signaling cascades. Moreover, in multicellular networks, intercellular coupling facilitated by gap junction channels may induce directed, electrodiffusive transport of charged molecules between cells of the network to generate new positional information patterning possibilities and characteristics. Given the demonstrated role of Vmem in morphogenesis, here we review current understanding of how Vmem can integrate with molecular regulatory networks to control single cell state, and the unique properties bioelectricity adds to transport phenomena in gap junction-coupled cell networks to facilitate self-assembly of morphogen gradients and other patterns. Understanding how Vmem integrates with biochemical regulatory networks at the level of a single cell, and mechanisms through which Vmem shapes molecular positional information in multicellular networks, are essential for a deep understanding of body plan control in development, regeneration and disease.

HCN2 Rescues brain defects by enforcing endogenous voltage pre-patterns.

Endogenous bioelectrical signaling coordinates cell behaviors toward correct anatomical outcomes. Lack of a model explaining spatialized dynamics of bioelectric states has hindered the understanding of the etiology of some birth defects and the development of predictive interventions. Nicotine, a known neuroteratogen, induces serious defects in brain patterning and learning. Our bio-realistic computational model explains nicotine's effects via the disruption of endogenous bioelectrical gradients and predicts that exogenous HCN2 ion channels would restore the endogenous bioelectric prepatterns necessary for brain patterning. Voltage mapping in vivo confirms these predictions, and exogenous expression of the HCN2 ion channel rescues nicotine-exposed embryos, resulting in normal brain morphology and molecular marker expression, with near-normal learning capacity. By combining molecular embryology, electrophysiology, and computational modeling, we delineate a biophysical mechanism of developmental brain damage and its functional rescue.

Bioelectric gene and reaction networks: computational modelling of genetic, biochemical and bioelectrical dynamics in pattern regulation.

Gene regulatory networks (GRNs) describe interactions between gene products and transcription factors that control gene expression. In combination with reaction-diffusion models, GRNs have enhanced comprehension of biological pattern formation. However, although it is well known that biological systems exploit an interplay of genetic and physical mechanisms, instructive factors such as transmembrane potential (Vmem) have not been integrated into full GRN models. Here we extend regulatory networks to include bioelectric signalling, developing a novel synthesis: the bioelectricity-integrated gene and reaction (BIGR) network. Using in silico simulations, we highlight the capacity for Vmem to alter steady-state concentrations of key signalling molecules inside and out of cells. We characterize fundamental feedbacks where Vmem both controls, and is in turn regulated by, biochemical signals and thereby demonstrate Vmem homeostatic control, Vmem memory and Vmem controlled state switching. BIGR networks demonstrating hysteresis are identified as a mechanisms through which more complex patterns of stable Vmem spots and stripes, along with correlated concentration patterns, can spontaneously emerge. As further proof of principle, we present and analyse a BIGR network model that mechanistically explains key aspects of the remarkable regenerative powers of creatures such as planarian flatworms. The functional properties of BIGR networks generate the first testable, quantitative hypotheses for biophysical mechanisms underlying the stability and adaptive regulation of anatomical bioelectric pattern.

Exploring Instructive Physiological Signaling with the Bioelectric Tissue Simulation Engine.

Bioelectric cell properties have been revealed as powerful targets for modulating stem cell function, regenerative response, developmental patterning, and tumor reprograming. Spatio-temporal distributions of endogenous resting potential, ion flows, and electric fields are influenced not only by the genome and external signals but also by their own intrinsic dynamics. Ion channels and electrical synapses (gap junctions) both determine, and are themselves gated by, cellular resting potential. Thus, the origin and progression of bioelectric patterns in multicellular tissues is complex, which hampers the rational control of voltage distributions for biomedical interventions. To improve understanding of these dynamics and facilitate the development of bioelectric pattern control strategies, we developed the BioElectric Tissue Simulation Engine (BETSE), a finite volume method multiphysics simulator, which predicts bioelectric patterns and their spatio-temporal dynamics by modeling ion channel and gap junction activity and tracking changes to the fundamental property of ion concentration. We validate performance of the simulator by matching experimentally obtained data on membrane permeability, ion concentration and resting potential to simulated values, and by demonstrating the expected outcomes for a range of well-known cases, such as predicting the correct transmembrane voltage changes for perturbation of single cell membrane states and environmental ion concentrations, in addition to the development of realistic transepithelial potentials and bioelectric wounding signals. In silico experiments reveal factors influencing transmembrane potential are significantly different in gap junction-networked cell clusters with tight junctions, and identify non-linear feedback mechanisms capable of generating strong, emergent, cluster-wide resting potential gradients. The BETSE platform will enable a deep understanding of local and long-range bioelectrical dynamics in tissues, and assist the development of specific interventions to achieve greater control of pattern during morphogenesis and remodeling.

Gap Junctional Blockade Stochastically Induces Different Species-Specific Head Anatomies in Genetically Wild-Type Girardia dorotocephala Flatworms.

The shape of an animal body plan is constructed from protein components encoded by the genome. However, bioelectric networks composed of many cell types have their own intrinsic dynamics, and can drive distinct morphological outcomes during embryogenesis and regeneration. Planarian flatworms are a popular system for exploring body plan patterning due to their regenerative capacity, but despite considerable molecular information regarding stem cell differentiation and basic axial patterning, very little is known about how distinct head shapes are produced. Here, we show that after decapitation in G. dorotocephala, a transient perturbation of physiological connectivity among cells (using the gap junction blocker octanol) can result in regenerated heads with quite different shapes, stochastically matching other known species of planaria (S. mediterranea, D. japonica, and P. felina). We use morphometric analysis to quantify the ability of physiological network perturbations to induce different species-specific head shapes from the same genome. Moreover, we present a computational agent-based model of cell and physical dynamics during regeneration that quantitatively reproduces the observed shape changes. Morphological alterations induced in a genomically wild-type G. dorotocephala during regeneration include not only the shape of the head but also the morphology of the brain, the characteristic distribution of adult stem cells (neoblasts), and the bioelectric gradients of resting potential within the anterior tissues. Interestingly, the shape change is not permanent; after regeneration is complete, intact animals remodel back to G. dorotocephala-appropriate head shape within several weeks in a secondary phase of remodeling following initial complete regeneration. We present a conceptual model to guide future work to delineate the molecular mechanisms by which bioelectric networks stochastically select among a small set of discrete head morphologies. Taken together, these data and analyses shed light on important physiological modifiers of morphological information in dictating species-specific shape, and reveal them to be a novel instructive input into head patterning in regenerating planaria.

Fundamental ratios and logarithmic periodicity in human limb bones.

Fundamental mathematical relationships are widespread in biology yet there is little information on this topic with regard to human limb bone lengths and none related to human limb bone volumes. Forty-six sets of ipsilateral upper and lower limb long bones and third digit short bones were imaged by computed tomography. Maximum bone lengths were measured manually and individual bone volumes calculated from computed tomography images using a stereologic method. Length ratios of femur : tibia and humerus : ulna were remarkably similar (1.21 and 1.22, respectively) and varied little (<7%) between individuals. The volume ratio of femur : tibia was approximately half that of humerus : ulna (1.58 and 3.28, respectively; P < 0.0001). Lower limb bone volume ratios varied much more than upper limb ratios. The relationship between bone length and volume was found to be well described by power laws, with R(2) values ranging from 0.983 to 0.995. The most striking finding was a logarithmic periodicity in bone length moving from distal to proximal up the limb (upper limb λ = 0.72, lower limb λ = 0.93). These novel data suggest that human limb bone lengths and volumes follow fundamental and highly conserved mathematical relationships, which may contribute to our understanding of normal and disordered growth, stature estimation, and biomechanics.

Growth of calcium phosphates on magnesium substrates for corrosion control in biomedical applications via immersion techniques.

Magnesium (Mg) has been suggested as a revolutionary biodegradable replacement for current permanent metals used in orthopedic applications. Current investigations concentrate on the control of the corrosion rate to match bone healing. Calcium phosphate coatings have been a recent focus of these investigations through various coating protocols. Within this investigation, an in situ crystallization technique was utilized as an inexpensive and relatively simple method to produce a brushite and monetite coating on pure Mg. Coatings were characterized using energy dispersive spectroscopy, glancing angle X-ray diffraction and field emission scanning electron microscopy. Corrosion protection properties of the coatings were assessed in physiological buffers, Earles balanced salt solution, minimum essential media, and minimum essential media containing serum albumin, over a 4-week period. Using this novel coating protocol, our findings indicate brushite and monetite coated Mg to have significant corrosive protective effects when compared with its uncoated counterpart whilst maintaining high coating substrate adhesion, homogeneity, and reproducibility.

Structural evidence for electromagnetic resonance in plant morphogenesis.

How a homogeneous collective of cells consistently and precisely establishes long-range tissue patterns remains a question of active research. This work explores the hypothesis of plant organs as resonators for electromagnetic radiation. Long-range structural patterns in the developing ovaries and male flower buds of cucurbit plants (zucchini, acorn, and butternut squash), in addition to mature cucurbit fruits (acorn, butternut, and zucchini squash; watermelon, and cucumber), were investigated. A finite element analysis (FEA) model was used to determine resonant EM modes for models with similar geometric and electrical parameters to those of developing organs. Main features of the developing ovaries (i.e. shape of placental lines, ovum location, definition of distinct tissue regions), male flower buds (i.e. early pollen tube features), and mature fruits (i.e. septa placement, seed location, endocarp and mesocarp) showed distinct correlations with electric and magnetic field components of electromagnetic resonant modes. On account of shared pattern signatures in developing organs and the EM resonant modes supported by a modelled structure with similar geometric and electrical properties to those of cucurbit organs, experimental investigations are warranted. The concept of a developing organ as an EM dielectric resonator may extend to a variety of morphogenetic phenomena in a number of living systems.

Endogenous electromagnetic fields in plant leaves: a new hypothesis for vascular pattern formation.

Electromagnetic (EM) phenomena have long been implicated in biological development, but few detailed, practical mechanisms have been put forth to connect electromagnetism with morphogenetic processes. This work describes a new hypothesis for plant leaf veination, whereby an endogenous electric field forming as a result of a coherent Frohlich process, and corresponding to an EM resonant mode of the developing leaf structure, is capable of instigating leaf vascularisation. In order to test the feasibility of this hypothesis, a three-dimensional, EM finite-element model (FEM) of a leaf primordium was constructed to determine if suitable resonant modes were physically possible for geometric and physical parameters similar to those of developing leaf tissue. Using the FEM model, resonant EM modes with patterns of relevance to developing leaf vein modalities were detected. On account of the existence of shared geometric signatures in a leaf's vascular pattern and the electric field component of EM resonant modes supported by a developing leaf structure, further theoretical and experimental investigations are warranted. Significantly, this hypothesis is not limited to leaf vascular patterning, but may be applicable to a variety of morphogenetic phenomena in a number of living systems.

Describing long-range patterns in leaf vasculature by metaphoric fields.

Some patterns in dicotyledonous leaf vasculature depict rather precise, long-range structural features. This work identifies and quantifies these previously unrecognized features in terms of an empirically derived mathematical formalism that generates wave-like spatial patterns referred to as metaphoric fields. These patterns were used to specify regularities in the long-range structure of dicot leaf vasculature, and were found to account significantly for the predominant features of all 27 dicot species studied. The conserved features of these metaphoric fields are discussed in terms of existing models for leaf pattern formation based on efflux-protein mediated auxin transport in a developing cellular field. This work highlights the complex, regular, long-range structures existing in leaf vascular patterns, and provides a means for specifying and identifying the inherent global features of vascular patterns which must be accounted for in functional developmental models.

Seeing tissue as a 'phase of matter': exploring statistical mechanics for the cell.

The field of tissue engineering aims to produce living, biological constructs which possess the appropriate spatial ordering of cells and their extra cellular matrix products. The complexity of a single cell and its interactions in a large collective have made development of useful models to assist in tissue culture difficult, and consequentially most tissue culture endeavors are limited to trial and error approaches. Some cell types display a natural tendency to spontaneously self-assemble into large domains of parallel-oriented cells. In this work, we show that these cell culture systems can be studied in the context of continuous disorder-order phase transformations. We suggest that collective ordering of the cells is controlled by the amount of noise in the walk of the individual cells (directional persistence) because undifferentiated mesenchymal stem cells display a seven-times higher directional persistence than mature fibroblasts and have a 24-times larger final-oriented domain size, an observation that corresponds with collective ordering in self-propelled particle systems. The study of cell culture systems using analogies derived from statistical mechanics yields simple, practical models offering insight into how a long-range order can be obtained in tissue-engineered constructs, providing a new paradigm for managing operations with large collectives of living cells.

Bone-like matrix formation on magnesium and magnesium alloys.

Mg metal and its alloys have promise as a biocompatible, degradable biomaterials. This work evaluates the potential of in vitro cell culture work with osteoblast-like cells on Mg based materials, and investigates cell differentiation and growth on Mg alloyed with various non-toxic or low-toxicity elements. Mg based substrates support the adhesion, differentiation and growth of stromal cells towards an osteoblast-like phenotype with the subsequent production of a bone like matrix under in vitro conditions. No significant difference in the final tissue layer is observed on pure Mg, an AZ21 alloy or a 0.5 wt% Ca alloy. Only a 0.8 wt% Ca alloy which shows complete structural disintegration shows minimal cell growth. Due to association of non-soluble degradation products formed when Mg is incubated in physiological-like fluid, mass changes typically used to report Mg degradation are not viable estimates of degradation. Methods quantifying the time dependent change in the mechanical integrity of samples as a function of incubation time are required for a proper assessment of Mg degradation. We conclude that in vitro cell culture of bone cells on Mg substrates is expected to be a viable screening technique to assess the relative biological activity of Mg-based materials.

Silicon substitution in the calcium phosphate bioceramics.

Silicon (Si) substitution in the crystal structures of calcium phosphate (CaP) ceramics such as hydroxyapatite (HA) and tricalcium phosphate (TCP) generates materials with superior biological performance to stoichiometric counterparts. Si, an essential trace element required for healthy bone and connective tissues, influences the biological activity of CaP materials by modifying material properties and by direct effects on the physiological processes in skeletal tissue. The synthesis of Si substituted HA (Si-HA), Si substituted alpha-TCP (Si-alpha-TCP), and multiphase systems are reviewed. The biological performance of these Si substituted CaP materials in comparison to stoichiometric counterparts is discussed. Si substitution promotes biological activity by the transformation of the material surface to a biologically equivalent apatite by increasing the solubility of the material, by generating a more electronegative surface and by creating a finer microstructure. When Si is included in the TCP structure, recrystallization to a carbonated HA is mediated by serum proteins and osteoblast-like cells. Release of Si complexes to the extracellular media and the presence of Si at the material surface may induce additional dose-dependent stimulatory effects on cells of the bone and cartilage tissue systems.

Magnesium and its alloys as orthopedic biomaterials: a review.

As a lightweight metal with mechanical properties similar to natural bone, a natural ionic presence with significant functional roles in biological systems, and in vivo degradation via corrosion in the electrolytic environment of the body, magnesium-based implants have the potential to serve as biocompatible, osteoconductive, degradable implants for load-bearing applications. This review explores the properties, biological performance, challenges and future directions of magnesium-based biomaterials.