Implantation of an Impedance Sensor for Early Detection of Gastrointestinal Anastomotic Leaks.

INTRODUCTION: Accurate early diagnosis of a gastrointestinal anastomotic leak remains a challenge. When an anastomotic leak develops, the electrical properties of the tissue undergoing inflammatory processes change, resulting from the extravasation of inflammatory fluid and cellular infiltration. The method described here intends to provide a novel early anastomotic leak warning system based upon measurable changes in tissue impedance nearby an acute inflammatory process. METHODS: A biodegradable Mg-alloy was compared with a nonabsorbable stainless steel (STS) electrode connected to a wireless recording system for impedance measurement. In vitro measurements were made in physiological solutions and small animal (eight mice) and large animal (eight pigs) models with an anastomotic leak simulated by an open colotomy. Measurements were made at 10 mm intervals from the open colon at baseline and up to 120 min comparing these with a sutured colonic wound and normal tissue. RESULTS: In-vitro biodegradable magnesium electrode impedance evaluation showed good sensitivity to different media due to its environmental corrosion properties. The impedance of an acidic environment (1.06 ± 0.02 kΩ for citric acid) was twice that of phosphate-buffered saline (PBS) (0.64 ± 0.008 kΩ) with a distinction between Normal Saline (0.42 ± 0.013 kΩ) and PBS (0.64 ± 0.008 kΩ). This was in contrast to the performance characteristics of the control STS electrodes, where impedance in an acidic environment was lower than saline or PBS (citric acid:0.76 ± 0.01 kΩ versus PBS: 1.32 ± 0.014 kΩ). In a mouse model simulating an anastomotic leak, there was a significant increase in impedance after 120 min when compared with controls (99.7% increase versus 9.6% increase, respectively; P < 0.02). This effect was confirmed in a pig model when relative impedance measurements of the leak and control groups were compared (1.86 ± 0.46 versus 1.07 ± 0.02, respectively; P < 0.027). CONCLUSIONS: Electrophysiological measurement shows diagnostic sensitivity for a gastrointestinal leak with potential clinical utility in the postoperative detection of early intra-abdominal sepsis. Further investigation of biodegradable tissue sensors capable of monitoring an early anastomotic leak is required.

Sensitive and robust chemical detection using an olfactory brain-computer interface.

When it comes to detecting volatile chemicals, biological olfactory systems far outperform all artificial chemical detection devices in their versatility, speed, and specificity. Consequently, the use of trained animals for chemical detection in security, defense, healthcare, agriculture, and other applications has grown astronomically. However, the use of animals in this capacity requires extensive training and behavior-based communication. Here we propose an alternative strategy, a bio-electronic nose, that capitalizes on the superior capability of the mammalian olfactory system, but bypasses behavioral output by reading olfactory information directly from the brain. We engineered a brain-computer interface that captures neuronal signals from an early stage of olfactory processing in awake mice combined with machine learning techniques to form a sensitive and selective chemical detector. We chronically implanted a grid electrode array on the surface of the mouse olfactory bulb and systematically recorded responses to a large battery of odorants and odorant mixtures across a wide range of concentrations. The bio-electronic nose has a comparable sensitivity to the trained animal and can detect odors on a variable background. We also introduce a novel genetic engineering approach that modifies the relative abundance of particular olfactory receptors in order to improve the sensitivity of our bio-electronic nose for specific chemical targets. Our recordings were stable over months, providing evidence for robust and stable decoding over time. The system also works in freely moving animals, allowing chemical detection to occur in real-world environments. Our bio-electronic nose outperforms current methods in terms of its stability, specificity, and versatility, setting a new standard for chemical detection.

Induced neuro-vascular interactions robustly enhance functional attributes of engineered neural implants.

Engineered neural implants have a myriad of potential basic science and clinical neural repair applications. Although there are implants that are currently undergoing their first clinical investigations, optimizing their long-term viability and efficacy remain an open challenge. Functional implants with pre-vascularization of various engineered tissues have proven to enhance post-implantation host integration, and well-known synergistic neural-vascular interplays suggest that this strategy could also be promising for neural tissue engineering. Here, we report the development of a novel bio-engineered neuro-vascular co-culture construct, and demonstrate that it exhibits enhanced neurotrophic factor expression, and more complex neuronal morphology. Crucially, by introducing genetically encoded calcium indicators (GECIs) into the co-culture, we are able to monitor functional activity of the neural network, and demonstrate greater activity levels and complexity as a result of the introduction of endothelial cells in the construct. The presence of this enhanced activity could putatively lead to superior integration outcomes. Indeed, leveraging on the ability to monitor the construct's development post-implantation with GECIs, we observe improved integration phenotypes in the spinal cord of mice relative to non-vascularized controls. Our approach provides a new experimental system with functional neural feedback for studying the interplay between vascular and neural development while advancing the optimization of neural implants towards potential clinical applications.

Engineering vascularized flaps using adipose-derived microvascular endothelial cells and mesenchymal stem cells.

Human adipose-derived microvascular endothelial cells (HAMEC) and mesenchymal stem cells (MSC) have been shown to bear angiogenic and vasculogenic capabilities. We hypothesize that co-culturing HAMEC:MSC on a porous biodegradable scaffold in vitro, later implanted as a graft around femoral blood vessels in a rat, will result in its vascularization by host vessels, creating a functional vascular flap that can effectively treat a range of large full-thickness soft tissue defects. HAMEC were co-cultured with MSC on polymeric three-dimensional porous constructs. Grafts were then implanted around the femoral vessels of a rat. To ensure vessel sprouting from the main femoral vessels, grafts were pre-isolated from the surrounding tissue. Graft vascularization was monitored to confirm full vascularization before flap transfer. Flaps were then transferred to treat both abdominal wall and exposed bone and tendon of an ankle defects. Flaps were analysed to determine vascular properties in terms of maturity, functionality and survival of implanted cells. Findings show that pre-isolated grafts bearing the HAMEC:MSC combination promoted formation of highly vascularized flaps, which were better integrated in both defect models. The results of this study show the essentiality of a specific adipose-derived cell combination in successful graft vascularization and integration, two processes crucial for flap survival. Copyright © 2017 John Wiley & Sons, Ltd.

Implantation of 3D Constructs Embedded with Oral Mucosa-Derived Cells Induces Functional Recovery in Rats with Complete Spinal Cord Transection.

Spinal cord injury (SCI), involving damaged axons and glial scar tissue, often culminates in irreversible impairments. Achieving substantial recovery following complete spinal cord transection remains an unmet challenge. Here, we report of implantation of an engineered 3D construct embedded with human oral mucosa stem cells (hOMSC) induced to secrete neuroprotective, immunomodulatory, and axonal elongation-associated factors, in a complete spinal cord transection rat model. Rats implanted with induced tissue engineering constructs regained fine motor control, coordination and walking pattern in sharp contrast to the untreated group that remained paralyzed (42 vs. 0%). Immunofluorescence, CLARITY, MRI, and electrophysiological assessments demonstrated a reconnection bridging the injured area, as well as presence of increased number of myelinated axons, neural precursors, and reduced glial scar tissue in recovered animals treated with the induced cell-embedded constructs. Finally, this construct is made of bio-compatible, clinically approved materials and utilizes a safe and easily extractable cell population. The results warrant further research with regards to the effectiveness of this treatment in addressing spinal cord injury.

Morphogenesis of 3D vascular networks is regulated by tensile forces.

Understanding the forces controlling vascular network properties and morphology can enhance in vitro tissue vascularization and graft integration prospects. This work assessed the effect of uniaxial cell-induced and externally applied tensile forces on the morphology of vascular networks formed within fibroblast and endothelial cell-embedded 3D polymeric constructs. Force intensity correlated with network quality, as verified by inhibition of force and of angiogenesis-related regulators. Tensile forces during vessel formation resulted in parallel vessel orientation under static stretching and diagonal orientation under cyclic stretching, supported by angiogenic factors secreted in response to each stretch protocol. Implantation of scaffolds bearing network orientations matching those of host abdominal muscle tissue improved graft integration and the mechanical properties of the implantation site, a critical factor in repair of defects in this area. This study demonstrates the regulatory role of forces in angiogenesis and their capacities in vessel structure manipulation, which can be exploited to improve scaffolds for tissue repair.

Adipose-derived endothelial and mesenchymal stem cells enhance vascular network formation on three-dimensional constructs in vitro.

BACKGROUND: Adipose-derived mesenchymal stem cells (MSCs) have been gaining fame mainly due to their vast clinical potential, simple isolation methods and minimal donor site morbidity. Adipose-derived MSCs and microvascular endothelial cells have been shown to bear angiogenic and vasculogenic capabilities. We hypothesized that co-culture of human adipose-derived MSCs with human adipose-derived microvascular endothelial cells (HAMECs) will serve as an effective cell pair to induce angiogenesis and vessel-like network formation in three-dimensional scaffolds in vitro. METHODS: HAMECs or human umbilical vein endothelial cells (HUVECs) were co-cultured on scaffolds with either MSCs or human neonatal dermal fibroblasts. Cells were immunofluorescently stained within the scaffolds at different time points post-seeding. Various analyses were performed to determine vessel length, complexity and degree of maturity. RESULTS: The HAMEC:MSC combination yielded the most organized and complex vascular elements within scaffolds, and in the shortest period of time, when compared to the other tested cell combinations. These differences were manifested by higher network complexity, more tube alignment and higher α-smooth muscle actin expression. Moreover, these generated microvessels further matured and developed during the 14-day incubation period within the three-dimensional microenvironment. CONCLUSIONS: These data demonstrate optimal vascular network formation upon co-culture of microvascular endothelial cells and adipose-derived MSCs in vitro and constitute a significant step in appreciation of the potential of microvascular endothelial cells and MSCs in different tissue engineering applications that can also be advantageous in in vivo studies.

Spontaneous Activity Characteristics of 3D "Optonets".

Sporadic spontaneous network activity emerges during early central nervous system (CNS) development and, as the number of neuronal connections rises, the maturing network displays diverse and complex activity, including various types of synchronized patterns. These activity patterns have major implications on both basic research and the study of neurological disorders, and their interplay with network morphology tightly correlates with developmental events such as neuronal differentiation, migration and establishment of neurotransmitter phenotypes. Although 2D neural cultures models have provided important insights into network activity patterns, these cultures fail to mimic the complex 3D architecture of natural CNS neural networks and its consequences on connectivity and activity. A 3D in-vitro model mimicking early network development while enabling cellular-resolution observations, could thus significantly advance our understanding of the activity characteristics in the developing CNS. Here, we longitudinally studied the spontaneous activity patterns of developing 3D in-vitro neural network "optonets," an optically-accessible bioengineered CNS model with multiple cortex-like characteristics. Optonet activity was observed using the genetically encodable calcium indicator GCaMP6m and a 3D imaging solution based on a standard epi-fluorescence microscope equipped with a piezo-electric z-stage and image processing-based deconvolution. Our results show that activity patterns become more complex as the network matures, gradually exhibiting longer-duration events. This report characterizes the patterns over time, and discusses how environmental changes affect the activity patterns. The relatively high degree of similarity between the network's spontaneously generated activity patterns and the reported characteristics of in-vivo activity, suggests that this is a compelling model system for brain-in-a chip research.

Microfluidic Chip for Site-Specific Neuropharmacological Treatment and Activity Probing of 3D Neuronal "Optonet" Cultures.

The study introduces a "brain-on-a-chip" microfluidic platform that hosts brain-like 3D cultures ("optonets") whose activity and responses to flowing drugs are recorded optically. Optonets are viable, optically accessible 3D neural networks whose characteristics approximate cortical networks. The results demonstrate the ability to monitor complex 3D activity patterns during extended site-specific, reversible neuropharmacogical exposure, suggesting an interesting potential in drug screening.

An engineered muscle flap for reconstruction of large soft tissue defects.

Large soft tissue defects involve significant tissue loss, requiring surgical reconstruction. Autologous flaps are occasionally scant, demand prolonged transfer surgery, and induce donor site morbidity. The present work set out to fabricate an engineered muscle flap bearing its own functional vascular pedicle for repair of a large soft tissue defect in mice. Full-thickness abdominal wall defect was reconstructed using this engineered vascular muscle flap. A 3D engineered tissue constructed of a porous, biodegradable polymer scaffold embedded with endothelial cells, fibroblasts, and/or myoblasts was cultured in vitro and then implanted around the femoral artery and veins before being transferred, as an axial flap, with its vascular pedicle to reconstruct a full-thickness abdominal wall defect in the same mouse. Within 1 wk of implantation, scaffolds showed extensive functional vascular density and perfusion and anastomosis with host vessels. At 1 wk posttransfer, the engineered muscle flaps were highly vascularized, were well-integrated within the surrounding tissue, and featured sufficient mechanical strength to support the abdominal viscera. Thus, the described engineered muscle flap, equipped with an autologous vascular pedicle, constitutes an effective tool for reconstruction of large defects, thereby circumventing the need for both harvesting autologous flaps and postoperative scarification.