The Chronic Challenge: Strategies to Improve Biocompatibility and Performance of Implanted Neural Devices.

Neural interfaces are direct connections that enable bidirectional exchange of information with the nervous system. These connections can have various degrees of invasiveness and several types of recording electrode devices have been developed to access different forms of neural information. Among those, intracortical electrodes linked to prostheses have the potential to make positive impacts on patients affected by neurological disorders or trauma. Such arrays are implanted into the patient’s cortical tissue and record extracellular potentials from nearby neurons, allowing to control external devices through information encoded by the neuronal discharges. Unfortunately, a widespread use of this kind of devices is hindered by the limited chronic reliability of current neural probe technology. Available evidence suggests that a major failure mode of electrode arrays is the brain tissue reaction against these implants over time, making the biocompatibility of implanted electrodes a primary concern in device design. To overcome the neuro-inflammatory reaction and to improve recording quality, different strategies have been adopted, spanning from materials innovation to bioactive approaches. On one hand, research in material science aims either at minimizing the microelectrode footprint or at incorporating compliant materials, while bioactive methods directly target the changes that occur at the electrode/tissue interface. This thesis is structured as follows: at first, one finds an overview (Introduction) on present day traditional electrodes and a detailed description of the underlying mechanism of the glial response that leads to electrode encapsulation and failure over time, followed by a description of the approaches that we have adopted to improve intracortical electrode implants, both from the materials and biological point of view, focusing mainly on the analysis of the reduced foreign body reaction elicited by different innovative intracortical implants. Chapter 1 details the design and manufacturing of functional bio-hybrid electrodes, discussing their improved biocompatibility with respect to traditional commercial microelectrodes. Chapter 2 describes the foreign body reaction elicited by new ultra-small and flexible devices that we made in collaboration with the Institute of Microsystem Technology in Freiburg and reports the surprising integration within the brain tissue reached by probes having cell-like size. Chapter 3 is a collection of papers published during my PhD course in which I was involved mainly in the assessment of electrodes and materials biocompatibility. Collectively this thesis presents the research path followed during all these years in which I addressed the intracortical chronic challenge investigating different approaches both to minimize the tissue reaction and to improve the electrodes performance.Il termine inglese neural device o interface si riferisce ad un dispositivo capace di creare una connessione diretta e bidirezionale con il sistema nervoso centrale. Tale dispositivo è generalmente rappresentato da uno o più elettrodi, in grado dunque di trasmettere e/o ricevere specifici segnali neuronali, posti a diretto contatto con il tessuto nervoso. In ambito clinico, l’utilizzo di elettrodi intracorticali connessi a neuroprotesi potrebbe avere un forte impatto positivo sulla qualità della vita di numerose persone colpite da neuropatologie, fornendo ad esempio un importante supporto a soggetti con disabilità. Tuttavia, l’utilizzo di tali dispositivi che permettono la stimolazione o registrazione cronica in pazienti umani è attualmente limitata da una serie di problematiche che causano nel tempo il danneggiamento sia del tessuto neuronale sia del dispositivo stesso. In particolare, la reazione infiammatoria evocata dalla presenza di un corpo estraneo all’interno del tessuto cerebrale viene considerata una delle principali cause del fallimento nell’utilizzo di elettrodi intracorticali. Negli ultimi anni, diversi approcci sono stati adottati per fronteggiare l’instaurarsi della risposta infiammatoria cronica e per migliorare la qualità del segnale acquisito. Da un lato, la ricerca nell’ambito della scienza dei materiali mira allo sviluppo di microelettrodi sempre più conformi alla natura stessa del tessuto neuronale, con un ridotto grado di invasività sia dal punto di vista geometrico che dimensionale. D’altro lato, l’utilizzo di strategie “bioattive” si propone di limitare direttamente i cambiamenti che si verificano all'interfaccia elettrodo/tessuto. Un continuo studio volto a migliorare la stabilità e la biocompatibilià dei materiali utilizzati per la creazione di interfacce neurali è dunque di fondamentale importanza. La seguente tesi è strutturata come segue: l’Introduzione fornisce una panoramica dei tradizionali dispositivi neurali attualmente in uso così come una descrizione dettagliata del meccanismo alla base della risposta infiammatoria e dei fenomeni responsabili del fallimento nell’utilizzo cronico di elettrodi intracorticali. A seguire, sono riportati i diversi approcci sperimentali innovativi che ho perseguito, sia dal punto di vista dei materiali sia dal punto di vista biologico, al fine di migliorare la biocompatibilità e dunque le performance di elettrodi intracorticali, concentrandomi principalmente sull'analisi della reazione infiammatoria. Il Capitolo 1 descrive l’ideazione e sviluppo di elettrodi funzionali bio-ibridi, sottolineando la loro migliore biocompatibilità rispetto ai tradizionali microelettrodi commerciali. Il Capitolo 2 descrive la reazione immunitaria provocata dall’impiego di nuovi dispositivi ultra-flessibili e di ridotte dimensioni, realizzati in collaborazione con l'Institute of Microsystem Technology di Friburgo, riportando la sorprendente integrazione nel tessuto cerebrale raggiunta da sonde con dimensioni simili a quelle delle cellule. Infine, il Capitolo 3 si presenta come una raccolta degli articoli pubblicati durante questo corso di dottorato, in cui sono stata coinvolta principalmente nello studio della biocompatibilità di nuovi materiali utilizzati per lo sviluppo di dispositivi neurali di nuova generazione. Collettivamente questa tesi presenta il percorso di ricerca seguito in questi anni, accumunato dal tentativo di affrontare la sfida cronica intracorticale indagando diversi approcci sia per minimizzare la reazione tissutale sia per migliorare le prestazioni degli elettrodi

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

Impatto emotivo dell’alopecia indotta da chemioterapia: studio qualitativo fenomenologico in pazienti oncologici adulti.

Introduction chemotherapy-induced alopecia (CIA) has a negative emotional impact on patients as it is experienced as a deterioration of one's body image and a clear sign of the disease in progress. Objectives this study aims to understand the experiences and emotions felt by people in oncological follow-up who have suffered from chemotherapy-induced alopecia. Materials and methods a qualitative phenomenological study was conducted through semi-structured interviews on a convenience sampling of adult cancer patients from June to December 2022. Analysis was carried out by two experts using the Colaizzi method. Results 12 interviews were carried out from which 4 main themes emerged: (I) emotions experienced by patients after hair loss; (II) alteration of self-perception regarding physical appearance; (III) relationship with others and interpersonal dynamics; (IV) the closest people during changes in personal identity and body image where multidisciplinary educational and emotional support are highlighted. Discussion and Conclusions results are consistent with scientific literature and highlight a discomfort due to CIA negatively associated with body image and self psycho-social well-being. Health workers must acquire awareness of the extent of the distress for educational interventions aimed at developing effective coping strategies.Introduzione l’alopecia da chemioterapia (CIA) ha un impatto emotivo negativo sugli assistiti in quanto vissuta come compromissione della propria immagine corporea e segno evidente della malattia in atto. Obiettivo comprendere il vissuto e le emozioni provate da persone in follow-up oncologico che hanno sofferto di alopecia indotta da chemioterapia. Materiali e metodi studio qualitativo fenomenologico condotto attraverso interviste semi-strutturate su un campione di convenienza di pazienti adulti oncologici tra giugno e dicembre 2022. Analisi effettuata attraverso il metodo Colaizzi da due esperti. Risultati sono state effettuate 12 interviste da cui sono emerse 4 tematiche principali: (I) emozioni vissute dagli assistiti dopo la perdita dei capelli, cioè come gli assistiti hanno vissuto l’esperienza relativa alla perdita dei capelli. (II) Alterazione della percezione del proprio aspetto fisico; la percezione di sé e le annesse reazioni emotive. (III) Relazione con gli altri ossia i cambiamenti del rapporto con gli altri. (IV) Infine, le persone più vicine durante i cambiamenti dell’identità personale e dell’immagine corporea dove si evidenzia il supporto educativo ed emotivo multidisciplinare. Discussione e Conclusioni i risultati sono in linea con la letteratura scientifica ed evidenziano un disagio dovuto alla CIA associato negativamente all’immagine corporea ed al benessere psico-sociale. Gli operatori sanitari devono acquisire consapevolezza dell’entità del disagio per interventi educativi mirati a sviluppare strategie di coping efficaci

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

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

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

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