On the longevity of flexible neural interfaces: Establishing biostability of polyimide-based intracortical implants

Flexible neural implants are extremely favored, as the most successful strategy to promote probe-tissue integration and avoid severe gliosis relies on reducing the mechanical mismatch between probe and brain tissue. But what are the realistic requirements for achieving chronic recording stability? What are the critical dimensions and main factors determining glial scar-free device integration? To answer these questions, two types of hair sized polyimide-based flexible intracortical (PIXI) arrays were fabricated, differing only in their cross-sectional area. Chronic tissue reaction to both types was evaluated in rats, and in different implantation setups. Interfacial stresses were found to play a critical role in long-term tissue integration. Still, all the devices provided high quality chronic recordings of single units and inflammatory gene expression was not significantly upregulated for larger devices. Our study points out that the most relevant factor in eliciting FBR is played by mechanical probe tissue interactions, that polyimide is well tolerated by the tissue, and that a holistic design considering material properties, geometrical dimensions and assembling techniques is the key towards longevity and long-term performance of intracortical probes. The optimization of only one parameter did not yet lead to the successful translation of research accomplishments into chronic preclinical and clinical applications

Generation of mouse hippocampal brain organoids from primary embryonic neural stem cells

: Here we present a protocol to generate standardized cerebral organoids with hippocampal regional specification using morphogen WNT3a. We describe steps for isolating mouse embryonic (E14.5) neural stem cells from the brain subgranular zone, preparing organoids samples for immunofluorescence, calcium imaging, and metabolic profiling. This protocol can be used to generate mouse brain organoids for developmental studies, modeling disease, and drug screening. Organoids can be obtained in one month, thus providing a rapid tool for high-throughput data validation. For complete details on the use and execution of this protocol, please refer to Ciarpella et al. "Murine cerebral organoids develop network of functional neurons and hippocampal brain region identity".1

Single walled carbon nanohorns composite for neural sensing and stimulation

reserved9noOxidized single walled carbon nanohorns (ox-SWCNH) were electrodeposited onto gold microelectrode arrays in conjunction with poly(3,4-ethylenedioxythiophene) (PEDOT) and polystirenesulfonate (PSS), and the properties of the new composite material for neural recording and stimulation were assessed. PEDOT/ox-SWCNH composites were compared with films prepared with one of the most notorious carbonaceous material in this field, the oxidized multi-walled Carbon Nanotubes (ox-MWCNT). The PEDOT/ox-SWCNH exhibited superior charge transfer capability, reflecting greater electroactive surface, as confirmed by SEM and EIS characterizations. As a consequence, a charge injection limit of 11.6 mC/cm(2) was observed for the new composite, which is higher than the one of PEDOT/ox-MWCNT (8.7 mC/cm(2)). Having confirmed comparable neural recording performance, the PEDOT/ox-SWCNH composite results very promising for improving therapeutic electrical stimulation in the central and peripheral nervous systems.mixedCarli, S ; Lambertini, L ; Zucchini, E ; Ciarpella, F ; Scarpellini, A ; Prato, M ; Castagnola, E ; Fadiga, L ; Ricci, DCarli, S; Lambertini, L; Zucchini, E; Ciarpella, F; Scarpellini, A; Prato, M; Castagnola, E; Fadiga, L; Ricci,

Establishment of a 3D-dynamic osteoblasts-osteoclasts co-culture model to simulate the jawbone microenvironment in vitro

Aims We aimed to establish a 3D osteoblasts/osteoclasts co-culture system requiring limited amounts of human primary cells and useful as platform to 1. recapitulate an "oral bone microenvironment" in healthy or pathological condition, and 2. produce potential implantable cell constructs for regeneration of jawbone which can be negatively affected by bisphosphonates (BPs). Main methods Osteoblasts from normal bone chips (hOBs) or from jawbone of patients taking BPs (hnOBs) were co-cultured with monocytes (hMCs) either in static (3D-C) or dynamic (3D-DyC) condition using the RCCS-4™ bioreactor for 3 weeks. Cell aggregates were characterized for viability, histological features and specific osteoclastic and osteogenic markers. Key findings In all tested conditions hOBs supported the formation of mature osteoclasts (hOCs), without differentiating agents or exogenous scaffolds. 3D-DyC condition associated with a ground based condition (Xg) rather than modeled microgravity (μXg) produced aggregates with high level of osteogenic markers including Osteopontin (OPN), Osterix (OSX), Runx2 and appreciable bone mineral matrix. hnOBs co-cultured with hMCs in 3D-Dyc/Xg condition generated OPN and mineral matrix positive aggregates. Significance We optimized a 3D co-culture system with a limited amount of cells preserving viability and functionality of bone cellular components and generating bone-like aggregates also by using cells from jawbone necrotic tissue. The feasibility to obtain from poor-quality bone sites viable osteoblasts able to form aggregates when co-cultured with hMCs, allows to study the development of autologous implantable constructs to overcome jawbone deficiency in patients affected by MRONJ (Medication-Related Osteonecrosis of the Jaws)

Establishment of a 3D-dynamic osteoblasts-osteoclasts co-culture model to simulate the jawbone microenvironment in vitro

The research on bone diseases highlights the need for reliable experimental models that may faithfully recapitulate in vitro the pathological bone microenvironment, and therefore provide a valuable tool for the development of novel strategies for bone regeneration. Taking into account that, at its simplest level, the production of bone mineralized tissue requires the presence of osteoblasts (hOBs) and osteoclasts (hOCs) enclosed in a structured matrix, nowadays much effort is focused on the set-up of specific in vitro hOBs/hOCs co-culture systems. Such simplified experimental models allow a functional characterization of the single cell types, while preserving the intimate crosstalk that naturally occurs in vivo between bone-forming and bone-resorbing cells. Our work was aimed at establishing a 3D hOBs/hOCs co-culture model to simulate the microenvironment present either in healthy or pathological jawbone, specifically for osteopenic or osteonecrotic diseases. In particular, we focused on bisphosphonate-related osteonecrosis of the jaw (BRONJ), a clinical complication found in patients treated with bisphosphonates leading to impaired jawbone turnover and tissue necrosis. We first confirmed the possibility to isolate hOBs from samples of necrotic bone (hnOB) obtained by BRONJ patients, despite the poor quality of the biological specimens. hnOB cultured in vitro maintained the typical features of osteoblastic cells, such as expression of osteogenic markers (OPN, Runx2) and mineralization capacity after culture in osteogenic medium. For the set-up of the co-colture model, our choice fell on mature and osteoprogenitor cells namely hOBs from nasal septum and MSCs from periodontal ligament (hPDLMSCs), respectively. By co-culturing hOBs or hPDLMSCs with monocytes from peripheral blood (hMCs) in transwell plates, we first demonstrated their ability to induce osteoclasts (hOCs) maturation in the absence of inducers after 7 days of co-culture. Culture in osteogenic medium for further 14 days induced the expression of OPN and deposition of mineral matrix, confirming that the co-culture system preserved the functional activity of the hOBs. We then established and compared two 3D co-culture systems, carried out in static condition using agarose-coated wells (3D-C) or dynamic condition using the Rotary Cell Culture System (3D-Dyc). After optimization of the culture parameters, we determined the lowest cell numbers that could allow the formation of viable hOBs/hOCs constructs, as the low amount of cells is a major issue when working with osteonecrotic samples. We demonstrated the presence of mature hOCs in the constructs cultured in 3D already after 7 days, in the absence of osteoclastogenic inducers. After further 14 days of culture in osteogenic medium, constructs derived from both 3D-C and 3D-Dyc culture systems were highly viable and showed the presence of mature hOCs and bone mineral matrix within the aggregates. In addition, the constructs stained positive for OPN and Col1A1. However, constructs cultured in 3D-C condition were poorly structured and showed areas with a disorganized matrix, while constructs cultured in 3D-Dyc had a very well definite structure with a uniform distribution of the mineral matrix. In conclusion, the formation of viable constructs is possible also co-culturing limited amounts of hOBs and hMCs in 3D-Dyc condition in the absence of osteoclastogenic inducers and avoiding the use of exogenous scaffolds. The finding that it is possible i. to obtain functional cells from anatomic locations with less than adequate bone quality and volume or a compromised area lacking sufficient stem and progenitor cells due to disabling conditions such as BRONJ, and ii.to create reliable 3D combinations with a few cells opens new scenarios to achieve autologous implantable constructs

Conformable polyimide-based μECoGs: Bringing the electrodes closer to the signal source

Structural biocompatibility is a fundamental requirement for chronically stable bioelectronic devices. Newest neurotechnologies are increasingly focused on minimizing the foreign body response through the development of devices that match the mechanical properties of the implanted tissue and mimic its surface composition, often compromising on their robustness. In this study, an analytical approach is proposed to determine the threshold of conformability for polyimide-based electrocorticography devices. A finite element model was used to quantify the depression of the cortex following the application of devices mechanically above or below conformability threshold. Findings were validated in vivo on rat animal models. Impedance measurements were performed for 40 days after implantation to monitor the status of the biotic/abiotic interface with both conformable and nonconformable implants. Multi-unit activity was then recorded for 12 weeks after implantation using the most compliant device type. It can therefore be concluded that conformability is an essential prerequisite for steady and reliable implants which does not only depend on the Young's modulus of the device material: it strongly relies on the relation between tissue curvature at the implantation site and corresponding device's thickness and geometry, which eventually define the moment of inertia and the interactions at the material-tissue interface

Environmental Enrichment Induces Meningeal Niche Remodeling through TrkB-Mediated Signaling

Neural precursors (NPs) present in the hippocampus can be modulated by several neurogenic stimuli, including environmental enrichment (EE) acting through BDNF-TrkB signaling. We have recently identified NPs in meninges; however, the meningeal niche response to pro-neurogenic stimuli has never been investigated. To this aim, we analyzed the effects of EE exposure on NP distribution in mouse brain meninges. Following neurogenic stimuli, although we did not detect modification of the meningeal cell number and proliferation, we observed an increased number of neural precursors in the meninges. A lineage tracing experiment suggested that EE-induced \u3b23-Tubulin+ immature neuronal cells present in the meninges originated, at least in part, from GLAST+ radial glia cells. To investigate the molecular mechanism responsible for meningeal reaction to EE exposure, we studied the BDNF-TrkB interaction. Treatment with ANA-12, a TrkB non-competitive inhibitor, abolished the EE-induced meningeal niche changes. Overall, these data showed, for the first time, that EE exposure induced meningeal niche remodeling through TrkB-mediated signaling. Fluoxetine treatment further confirmed the meningeal niche response, suggesting it may also respond to other pharmacological neurogenic stimuli. A better understanding of the neurogenic stimuli modulation for meninges may be useful to improve the effectiveness of neurodegenerative and neuropsychiatric treatments

Incorporation of Silicon Carbide and Diamond-Like Carbon as Adhesion Promoters Improves In Vitro and In Vivo Stability of Thin-Film Glassy Carbon Electrocorticography Arrays

Thin-film neural devices are an appealing alternative to traditional implants, although their chronic stability remains matter of investigation. In this study, a chronically stable class of thin-film devices for electrocorticography is manufactured incorporating silicon carbide and diamond-like carbon as adhesion promoters between glassy carbon (GC) electrodes and polyimide and between GC and platinum traces. The devices are aged in three solutions-phosphate-buffered saline (PBS), 30 x 10(-3) and 150 x10(-3) M H2O2/PBS-and stressed using cyclic voltammetry (2500 cycles) and 20 million biphasic pulses. Electrochemical impedance spectroscopy (EIS) and image analysis are performed to detect eventual changes of the electrodes morphology. Results demonstrate that the devices are able to undergo chemically induced oxidative stress and electrical stimulation without failing but actually improving their electrical performance until a steady state is reached. Additionally, cell viability tests are carried out to verify the noncytotoxicity of the materials, before chronically implanting them into rat models. The behavior of the GC electrodes in vivo is monitored through EIS and sensorimotor evoked potential recordings which confirm that, with GC being activated, impedance lowers and quality of recorded signal improves. Histological analysis of the brain tissue is performed and shows no sign of severe immune reaction to the implant

Murine cerebral organoids develop network of functional neurons and hippocampal brain region identity

Brain organoids are in vitro three-dimensional (3D) self-organized neural structures, which can enable disease modeling and drug screening. However, their use for standardized large-scale drug screening studies is limited by their high batch-to-batch variability, long differentiation time (10–20 weeks), and high production costs. This is particularly relevant when brain organoids are obtained from human induced pluripotent stem cells (iPSCs). Here, we developed, for the first time, a highly standardized, reproducible, and fast (5 weeks) murine brain organoid model starting from embryonic neural stem cells. We obtained brain organoids, which progressively differentiated and self-organized into 3D networks of functional neurons with dorsal forebrain phenotype. Furthermore, by adding the morphogen WNT3a, we generated brain organoids with specific hippocampal region identity. Overall, our results showed the establishment of a fast, robust and reproducible murine 3D in vitro brain model that may represent a useful tool for high-throughput drug screening and disease modeling.publishedVersionPeer reviewe