LRRC8/VRAC volume-regulated anion channels are crucial for hearing.

Hearing crucially depends on cochlear ion homeostasis as evident from deafness elicited by mutations in various genes encoding cation or anion channels and transporters. Ablation of ClC­K/barttin chloride channels causes deafness by interfering with the positive electrical potential of the endolymph, but roles of other anion channels in the inner ear have not been studied. Here we report the intracochlear distribution of all five LRRC8 subunits of VRAC, a volume-regulated anion channel that transports chloride, metabolites, and drugs such as the ototoxic anti-cancer drug cisplatin, and explore its physiological role by ablating its subunits. Sensory hair cells express all LRRC8 isoforms, whereas only LRRC8A, D and E were found in the potassium-secreting epithelium of the stria vascularis. Cochlear disruption of the essential LRRC8A subunit, or combined ablation of LRRC8D and E, resulted in cochlear degeneration and congenital deafness of Lrrc8a-/- mice. It was associated with a progressive degeneration of the organ of Corti and its innervating spiral ganglion. Like disruption of ClC-K/barttin, loss of VRAC severely reduced the endocochlear potential. However, the mechanism underlying this reduction seems different. Disruption of VRAC, but not ClC-K/barttin, led to an almost complete loss of Kir4.1 (KCNJ10), a strial K+ channel crucial for the generation of the endocochlear potential. The strong downregulation of Kir4.1 might be secondary to a loss of VRAC-mediated transport of metabolites regulating inner ear redox potential such as glutathione. Our study extends the knowledge of the role of cochlear ion transport in hearing and ototoxicity.

Bone Fracture Sensing Using Ultrasound Pitch-Catch Measurements: A Proof-of-Principle Study.

The most common imaging method used to diagnose and monitor bone fractures and healing is multiple radiographic images performed by highly trained professionals with expensive equipment that can expose patients to high levels of ionizing radiation. Here we introduce and illustrate proof-of-concept of a potential alternative method for measuring bone fractures: ultrasound pitch-catch measurement technique. Measurements are performed with two piezoelectric transducers, housed in standard orthopedic screws and fixed on either side of simulated fractures, with and without an orthopedic plate, in ex vivo pig limb bones. Using this measurement method, we were able to detect significant differences between uncut and 2-, 5- and 10-mm-deep bone cuts using a two-sided t-test with an α level of 5%.

Effects of Electrical Stimulation on Stem Cells.

Recent interest in developing new regenerative medicine- and tissue engineering-based treatments has motivated researchers to develop strategies for manipulating stem cells to optimize outcomes in these potentially, game-changing treatments. Cells communicate with each other, and with their surrounding tissues and organs via electrochemical signals. These signals originate from ions passing back and forth through cell membranes and play a key role in regulating cell function during embryonic development, healing, and regeneration. To study the effects of electrical signals on cell function, investigators have exposed cells to exogenous electrical stimulation and have been able to increase, decrease and entirely block cell proliferation, differentiation, migration, alignment, and adherence to scaffold materials. In this review, we discuss research focused on the use of electrical stimulation to manipulate stem cell function with a focus on its incorporation in tissue engineering-based treatments.

Role of Bioelectricity During Cell Proliferation in Different Cell Types.

Most living organisms possess varying degrees of regenerative capabilities but how these regenerative processes are controlled is still poorly understood. Naturally occurring bioelectric voltages (like Vmem) are thought to be playing instructive role in tissue regeneration, as well as embryonic development. The different distribution of ions on the either side of the cell membrane results in intra- and extra-cellular voltage differences, known as membrane potential or Vmem. The relationship between Vmem and cell physiology is conserved in a wide range of cell types and suggests that Vmem regulation is a fundamental control mechanism for regeneration related processes e.g., proliferation and differentiation. In the present study we measured Vmem in three different cell types (human osteogenic sarcoma cell line (OSC), rat bone marrow derived mesenchymal stem cells (BM-MSC), and rat dermal fibroblasts) and characterized the relationship between their Vmem and proliferation. In order to find out if Vmem controls proliferation, or visa-versa, we blocked and then unblocked Na+/K+-exchanging ATPase using ouabain and measured the proliferation. Our results demonstrate that Vmem can be pharmacologically manipulated to control proliferation in certain cell types like BM-MSC. Taken together, it is clear that control of bioelectrical properties in non-excitable cells could prove to be potentially a useful tool in regenerative medicine efforts.

Electrical stimulation in bone tissue engineering treatments.

Electrical stimulation (EStim) has been shown to promote bone healing and regeneration both in animal experiments and clinical treatments. Therefore, incorporating EStim into promising new bone tissue engineering (BTE) therapies is a logical next step. The goal of current BTE research is to develop combinations of cells, scaffolds, and chemical and physical stimuli that optimize treatment outcomes. Recent studies demonstrating EStim's positive osteogenic effects at the cellular and molecular level provide intriguing clues to the underlying mechanisms by which it promotes bone healing. In this review, we discuss results of recent in vitro and in vivo research focused on using EStim to promote bone healing and regeneration and consider possible strategies for its application to improve outcomes in BTE treatments. Technical aspects of exposing cells and tissues to EStim in in vitro and in vivo model systems are also discussed.

Electrical Stimulation Decreases Dental Pulp Stem Cell Osteo-/Odontogenic Differentiation.

Dental pulp stem cells (DPSCs) have great potential for use in tissue engineering (TE)-based dental treatments. Electrical stimulation (EStim) has been shown to influence cellular functions that could play an important role in the success of TE treatments. Despite many recent studies focused on DPSCs, few have investigated the effect EStim has on these cells. The aim of this research was to investigate the effects of direct current (DC) EStim on osteo-/odontogenic differentiation of DPSCs. To do so cells were isolated from male Sprague Dawley rats (7-8 weeks old), and phenotype characterization and multilineage differentiation analysis were conducted to verify their "stemness." Different voltages of DC EStim were administrated 1 h/day for 7 days, and the effect of EStim on DPSC osteo-/odontogenic differentiation was assessed by measuring calcium and collagen deposition, alkaline phosphatase (ALP) activity, and expression of osteo- and odontogenic marker genes at days 7 and 14 of culture. We found that while 10 and 50 mV/mm of EStim had no effect on cell number or metabolic activity, 100 mV/mm caused a significant reduction in cell number, and 150 mV/mm resulted in cell death. Despite increased gene expression of osteo-/odontogenic gene markers, Osteocalcin, RunX2, BSP, and DMP1, at day 7 in EStim treated cells, 50 mV/mm of EStim decreased collagen deposition and ALP activity at both time points, and calcium deposition was found to be lower at day 14. In conclusion, under the conditions tested, EStim appears to impair DPSC osteo-/odontogenic differentiation. Additional studies are needed to further characterize and understand the mechanisms involved in DPSC response to EStim, with an eye toward its potential use in TE-based dental treatments.

Electrical stimulation-based bone fracture treatment, if it works so well why do not more surgeons use it?

BACKGROUND: Electrical stimulation (EStim) has been proven to promote bone healing in experimental settings and has been used clinically for many years and yet it has not become a mainstream clinical treatment. METHODS: To better understand this discrepancy we reviewed 72 animal and 69 clinical studies published between 1978 and 2017, and separately asked 161 orthopedic surgeons worldwide about their awareness, experience, and acceptance of EStim for treating fracture patients. RESULTS: Of the 72 animal studies, 77% reported positive outcomes, and the most common model, bone, fracture type, and method of administering EStim were dog, tibia, large bone defects, and DC, respectively. Of the 69 clinical studies, 73% reported positive outcomes, and the most common bone treated, fracture type, and method of administration were tibia, delayed/non-unions, and PEMF, respectively. Of the 161 survey respondents, most (73%) were aware of the positive outcomes reported in the literature, yet only 32% used EStim in their patients. The most common fracture they treated was delayed/non-unions, and the greatest problems with EStim were high costs and inconsistent results. CONCLUSION: Despite their awareness of EStim's pro-fracture healing effects few orthopedic surgeons use it in their patients. Our review of the literature and survey indicate that this is due to confusion in the literature due to the great variation in methods reported, and the inconsistent results associated with this treatment approach. In spite of this surgeons seem to be open to using this treatment if advancements in the technology were able to provide an easy to use, cost-effective method to deliver EStim in their fracture patients.

Ultrasound Bone Fracture Sensing and Data Communication: Experimental Results in a Pig Limb Sample.

Approximately 6.3 million fractures occur each year in the United States alone. Accurately monitoring the progression of fracture healing is essential to be able to advise patients when it is safe to return to normal activity. The most common method used to confirm and monitor fracture healing is the acquisition of multiple radiographic images over the many months required for healing. This imaging method uses large expensive equipment and exposes patients to high levels of ionizing radiation. In the study described here, we tested another technology for monitoring fracture healing that could minimize the need for multiple radiographic images. We tested a piezoelectric transducer fixed to the surface of a bone that uses electromechanical impedance spectroscopy to measure simulated fractures and transmits the measurement data to an acoustic receiver located externally on the skin surface.

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro.

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Membrane potential (Vmem) measurements during mesenchymal stem cell (MSC) proliferation and osteogenic differentiation.

BACKGROUND: Electrochemical signals play an important role in cell communication and behavior. Electrically charged ions transported across cell membranes maintain an electrochemical imbalance that gives rise to bioelectric signaling, called membrane potential or Vmem. Vmem plays a key role in numerous inter- and intracellular functions that regulate cell behaviors like proliferation, differentiation and migration, all playing a critical role in embryonic development, healing, and regeneration. METHODS: With the goal of analyzing the changes in Vmem during cell proliferation and differentiation, here we used direct current electrical stimulation (EStim) to promote cell proliferation and differentiation and simultaneously tracked the corresponding changes in Vmem in adipose derived mesenchymal stem cells (AT-MSC). RESULTS: We found that EStim caused increased AT-MSC proliferation that corresponded to Vmem depolarization and increased osteogenic differentiation that corresponded to Vmem hyperpolarization. Taken together, this shows that Vmem changes associated with EStim induced cell proliferation and differentiation can be accurately tracked during these important cell functions. Using this tool to monitor Vmem changes associated with these important cell behaviors we hope to learn more about how these electrochemical cues regulate cell function with the ultimate goal of developing new EStim based treatments capable of controlling healing and regeneration.

Histological Scoring Method to Assess Bone Healing in Critical Size Bone Defect Models.

Large bone defects are a major problem in trauma and orthopedic surgery. Tissue engineering based treatments have emerged as promising alternatives to traditional bone grafting techniques. Critical size bone defect animal models have been developed and widely used to evaluate and compare therapeutic effectiveness in bone tissue engineering treatments. To measure healing in a given defect after treatment, histological assessment methods are commonly used. These histological methods are typically qualitative and only measure the amount of newly formed bone. In this study, we introduce a new histological scoring method that in addition to new bone formation also measures newly formed "cartilage," "fibrous tissue," and "remnant bone defect size." Using Kappa analysis and interclass correlation analysis, we verified the reliability of our new scoring method. These additional parameters make it possible to differentiate between the hard callus and soft callus phases of healing and, thus, derive more valuable information about the effect different tissue-engineering treatments have on the healing process.

Time course of traumatic neuroma development.

This study was designed to characterize morphologic stages during neuroma development post amputation with an eye toward developing better treatment strategies that intervene before neuromas are fully formed. Right forelimbs of 30 Sprague Dawley rats were amputated and limb stumps were collected at 3, 7, 28, 60 and 90 Days Post Amputation (DPA). Morphology of newly formed nerves and neuromas were assessed via general histology and neurofilament protein antibody staining. Analysis revealed six morphological characteristics during nerve and neuroma development; 1) normal nerve, 2) degenerating axons, 3) axonal sprouts, 4) unorganized bundles of axons, 5) unorganized axon growth into muscles, and 6) unorganized axon growth into fibrotic tissue (neuroma). At early stages (3 & 7 DPA) after amputation, normal nerves could be identified throughout the limb stump and small areas of axonal sprouts were present near the site of injury. Signs of degenerating axons were evident from 7 to 90 DPA. From day 28 on, variability of nerve characteristics with signs of unorganized axon growth into muscle and fibrotic tissue and neuroma formation became visible in multiple areas of stump tissue. These pathological features became more evident on days 60 and 90. At 90 DPA frank neuroma formation was present in all stump tissue. By following nerve regrowth and neuroma formation after amputation we were able to identify 6 separate histological stages of nerve regrowth and neuroma development. Axonal regrowth was observed as early as 3 DPA and signs of unorganized axonal growth and neuroma formation were evident by 28 DPA. Based on these observations we speculate that neuroma treatment and or prevention strategies might be more successful if targeted at the initial stages of development and not after 28 DPA.

Pretreating mesenchymal stem cells with electrical stimulation causes sustained long-lasting pro-osteogenic effects.

BACKGROUND: Electrical stimulation (ES) has a long history of successful use in the clinical treatment of refractory, non-healing bone fractures and has recently been proposed as an adjunct to bone tissue-engineering treatments to optimize their therapeutic potential. This idea emerged from ES's demonstrated positive effects on stem cell migration, proliferation, differentiation and adherence to scaffolds, all cell behaviors recognized to be advantageous in Bone Tissue Engineering (BTE). In previous in vitro experiments we demonstrated that direct current ES, administered daily, accelerates Mesenchymal Stem Cell (MSC) osteogenic differentiation. In the present study, we sought to define the optimal ES regimen for maximizing this pro-osteogenic effect. METHODS: Rat bone marrow-derived MSC were exposed to 100 mV/mm, 1 hr/day for three, seven, and 14 days, then osteogenic differentiation was assessed at Day 14 of culture by measuring collagen production, calcium deposition, alkaline phosphatase activity and osteogenic marker gene expression. RESULTS: We found that exposing MSC to ES for three days had minimal effect, while seven and 14 days resulted in increased osteogenic differentiation, as indicated by significant increases in collagen and calcium deposits, and expression of osteogenic marker genes Col1a1, Osteopontin, Osterix and Calmodulin. We also found that cells treated with ES for seven days, maintained this pro-osteogenic activity long (for at least seven days) after discontinuing ES exposure. DISCUSSION: This study showed that while three days of ES is insufficient to solicit pro-osteogenic effects, seven and 14 days significantly increases osteogenic differentiation. Importantly, we found that cells treated with ES for only seven days, maintained this pro-osteogenic activity long after discontinuing ES exposure. This sustained positive osteogenic effect is likely due to the enhanced expression of RunX2 and Calmodulin we observed. This prolonged positive osteogenic effect, long after discontinuing ES treatment, if incorporated into BTE treatment protocols, could potentially improve outcomes and in doing so help BTE achieve its full therapeutic potential.

Combining electrical stimulation and tissue engineering to treat large bone defects in a rat model.

Bone Tissue engineering (BTE) has recently been introduced as an alternative to conventional treatments for large non-healing bone defects. BTE approaches mimic autologous bone grafts, by combining cells, scaffold, and growth factors, and have the added benefit of being able to manipulate these constituents to optimize healing. Electrical stimulation (ES) has long been used to successfully treat non-healing fractures and has recently been shown to stimulate bone cells to migrate, proliferate, align, differentiate, and adhere to bio compatible scaffolds, all cell behaviors that could improve BTE treatment outcomes. With the above in mind we performed in vitro experiments and demonstrated that exposing Mesenchymal Stem Cells (MSC) + scaffold to ES for 3 weeks resulted in significant increases in osteogenic differentiation. Then in in vivo experiments, for the first time, we demonstrated that exposing BTE treated rat femur large defects to ES for 8 weeks, caused improved healing, as indicated by increased bone formation, strength, vessel density, and osteogenic gene expression. Our results demonstrate that ES significantly increases osteogenic differentiation in vitro and that this effect is translated into improved healing in vivo. These findings support the use of ES to help BTE treatments achieve their full therapeutic potential.

Brain regions for sound processing and song release in a small grasshopper.

We investigated brain regions - mostly neuropils - that process auditory information relevant for the initiation of response songs of female grasshoppers Chorthippus biguttulus during bidirectional intraspecific acoustic communication. Male-female acoustic duets in the species Ch. biguttulus require the perception of sounds, their recognition as a species- and gender-specific signal and the initiation of commands that activate thoracic pattern generating circuits to drive the sound-producing stridulatory movements of the hind legs. To study sensory-to-motor processing during acoustic communication we used multielectrodes that allowed simultaneous recordings of acoustically stimulated electrical activity from several ascending auditory interneurons or local brain neurons and subsequent electrical stimulation of the recording site. Auditory activity was detected in the lateral protocerebrum (where most of the described ascending auditory interneurons terminate), in the superior medial protocerebrum and in the central complex, that has previously been implicated in the control of sound production. Neural responses to behaviorally attractive sound stimuli showed no or only poor correlation with behavioral responses. Current injections into the lateral protocerebrum, the central complex and the deuto-/tritocerebrum (close to the cerebro-cervical fascicles), but not into the superior medial protocerebrum, elicited species-typical stridulation with high success rate. Latencies and numbers of phrases produced by electrical stimulation were different between these brain regions. Our results indicate three brain regions (likely neuropils) where auditory activity can be detected with two of these regions being potentially involved in song initiation.

Multielectrode recordings from auditory neurons in the brain of a small grasshopper.

BACKGROUND: Grasshoppers have been used as a model system to study the neuronal basis of insect acoustic behavior. Auditory neurons have been described from intracellular recordings. The growing interest to study population activity of neurons has been satisfied so far with artificially combining data from different individuals. NEW METHOD: We for the first time used multielectrode recordings from a small grasshopper brain. We used three 12µm tungsten wires (combined in a multielectrode) to record from local brain neurons and from a population of auditory neurons entering the brain from the thorax. Spikes of the recorded units were separated by sorting algorithms and spike collision analysis. RESULTS: The tungsten wires enabled stable recordings with high signal to noise ratio. Due to the tight temporal coupling of auditory activity to the stimulus spike collisions were frequent and collision analysis retrieved 10-15% of additional spikes. Marking the electrode position was possible using a fluorescent dye or electrocoagulation with high current. Physiological identification of units described from intracellular recordings was hard to achieve. COMPARISON WITH EXISTING METHODS: 12µm tungsten wires gave a better signal to noise ratio than 15µm copper wires previously used in recordings from bees' brains. Recording the population activity of auditory neurons in one individual prevents interindividual and trial-to-trial variability which otherwise reduce the validity of the analysis. Double intracellular recordings have quite low success rate and therefore are rarely achieved and their stability is much lower than that of multielectrode recordings which allows sampling of data for 30min or more.