Abstract The Alzheimer’s Amyloid Precursor Protein (APP) has determinant roles in neuronal development and function, both in its full-length conformation and as some of its proteolytic peptides, particularly secreted (s)APPa. Given that APP phosphorylation tightly regulates its trafficking, proteolysis, and protein-protein binding, it consequently affects several APP functions. The S655 residue, located in the basolateral sorting motif YTSI at APP C-terminus has been observed to be phosphorylated in mature full-length APP and its C-terminal fragments. Previously observed to modify APP’s protein interactions, resulting in altered endolysosomal trafficking, andincreased half-life and sAPPa generation, phosphoS655 APP has potential to modulate APP-mediated neuronal differentiation. To study the phosphoS655 differential interactome relevant for neuronal differentiation, SH-SY5Y cells expressing Wt or S655 phosphomutants APP-GFP were differentiated at two time points. APP-GFP and their respective interacting partners were immunoprecipitated using GFP-trap, and interactors identified by mass spectrometry. Both dephospho and phosphoS655 interactomes were generally enriched in similar processes, primarily RNA processing and translation, as well as signal transduction, metabolism, and cytoskeleton remodeling. The smaller phosphoS655 interactome contributes for functional specialization via binding to e.g. FUBP3, ELAVL4, ATXN2, Tubulin, INA. Several of these specific binding partners are known to promote neurite outgrowth and likely underlie our experimental observation that phosphoS655 APP promotes neuritogenesis, particularly the formation of longer neuritic extensions. These results are not only important for the body of knowledge on this Alzheimer’s disease core protein, but may also aid in future therapies against this disease.

Notwithstanding the demonstrated benefits of electrical stimulation in enhancing tissue functionality, existing state-of-the-art electrostimulation systems often depend on invasive electrodes or planar designs. This work exploits the versatility of graphene to fabricate biocompatible electrodes for the three-dimensional in vitro electrical stimulation of neural stem cells. A conductive green graphene-based ink was formulated and screen-printed as the bottom and top electrodes in a bottom-less standard culture well plate. Upon exposure to macrophages, although some oxidative stress was observed, this graphene-based ink did not elicit an increase in the pro-inflammatory cytokine IL-6. An analysis of the electrode impedance as a function of time and frequency was performed to optimize the 3D electrical stimulation. The efficacy of these graphene electrodes for electrically stimulating cells across 3D environments was investigated in scaffolds composed of a decellularized extracellular matrix and reduced graphene oxide, which had previously shown the capability to facilitate neuronal differentiation in vitro and to create a pro-regenerative microenvironment in vivo. Neural stem cells were seeded on these scaffolds and electrically stimulated with a 10 Hz bidirectional current signal of 200 μA for 1 hour daily. At the target frequency of 10 Hz, deemed advantageous for neural regeneration, a scaffold impedance below 800 Ω was ensured. The low-frequency 3D stimulation proved to enhance cellular mechanisms essential for the development of neuronal networks, including neuronal differentiation, neuritogenesis and neurite growth.

In recent years, microneedles (MNs) have shown high potential as drug delivery devices capable of administering different drugs in a simple, fast, and minimally invasive manner. Their ability to pierce the stratum corneum barrier heavily outweighs the inconveniences posed by conventional administration methods such as hypodermal injections, creams, or ointments. In this work, high-performance, single-component polysaccharide-based microneedles, viz. pullulan MNs, were produced by micromolding, aimed for transdermal delivery of lidocaine. These dissolvable MNs were able to sustain forces up to 1.5 N per needle without breakage. Their ability to overcome the stratum corneum was validated using excised human abdominal skin, with the MNs successfully reaching the dermis. The MN tips completely dissolved after 10 min in an agarose gel skin model and porcine ear skin, with 26 and 32% of lidocaine being retained in the patch after 10 min of insertion in the agarose gel and porcine skin, respectively. Cytocompatibility against multiple cell lines (HaCaT, 3T3, and RAW 264.7) was demonstrated by the resazurin metabolic assay, with over 80% cell viability in all cases. Time-lapse fluorescence microscopy confirmed that the activity of the MN-released lidocaine on sensory neurons was comparable to that of a pure lidocaine solution at a similar concentration. In sum, given its low onset time (<10 min), swift dissolution, and effective drug release, this pullulan-based system constitutes a simple, safe, and efficient delivery device, contrasting conventional methods of local anesthetic administration.

OBJECTIVE:Periodontitis is a widespread and challenging oral disease, which often involves recurring infections that complicate treatment. This study explores a novel tissue engineering approach to address such an issue, involving the design of a nanofibers-based composite material containing silver, a natural antibacterial agent. METHODS:Pure β-Ag(VO3) nanorods were synthesized via microwave irradiation and loaded into electrospun polycaprolactone (PCL) membranes. Integration was confirmed through FTIR, EDS, and XRD analyses and the release profile of Ag and V from the fibers was evaluated by ICP-OES. Biocompatibility towards mammalian cells recurred to resazurin metabolic, cell scoring and microscopy assays in cultured fibroblasts. Bioactivity against bacteria was evaluated in agar diffusion susceptibility tests using E. coli (Gram-negative) and S. aureus (Gram-positive). RESULTS:A PCL-based electrospun membrane, loaded with β-Ag(VO3) nanorods, was successfully developed and characterized. Incorporation of the nanorods improved the nanofibers' morphology, and the membranes showed a high cytocompatibility with fibroblasts. Importantly, a 0.5 % β-Ag(VO3) concentration in the composite demonstrated antibacterial efficacy against Gram-negative and Gram-positive bacteria. This antibacterial effect is most likely due to a kill-on-contact mechanism rather than from the released of Ag ions, that was < 1 ppm over the 24 h of exposure. SIGNIFICANCE:The ease of incorporating silver vanadates into polymeric membranes using the methodology here described, combined with the antibacterial effects and cytocompatibility presented by the composite membranes, suggests that these materials may represent a promising avenue for tackling recurring infections in the oral cavity and become valuable options in periodontitis treatment.

Bioelectronic bone implants are being widely recognized as a promising technology for highly personalized bone/implant interface sensing and biophysical therapeutic stimulation. Such bioelectronic devices are based on an innovative concept with the ability to be applied to a wide range of implants, including in fixation and prosthetic systems. Recently, biointerface sensing using capacitive patterns was proposed to overcome the limitations of standard imaging technologies and other non-imaging technologies; moreover, electric stimulation using capacitive patterns was proposed to overcome the limitations of non-instrumented implants. We here provide an innovative low-power miniaturized electronic system with ability to provide both therapeutic stimulation and bone/implant interface monitoring using network-architectured capacitive interdigitated patterns. It comprises five modules: sensing, electric stimulation, processing, communication and power management. This technology was validated using in vitro tests: concerning the sensing system, its ability to detect biointerface changes ranging from tiny to severe bone-implant interface changes in target regions was validated; concerning the stimulation system, its ability to significantly enhance bone cells’ full differentiation, including matrix maturation and mineralization, was also confirmed. This work provides an impactful contribution and paves the way for the development of the new generation of orthopaedic biodevices.

The present work consisted of an exploratory study aiming to evaluate in vitro the potential of AuNPs during Radiation Therapy (RT) in human pancreatic adenocarcinoma cells. AuNPs coated with hyaluronic and oleic acids (HAOA-AuNPs) or with bombesin peptides (BBN-AuNPs) were used. AuNPs were characterized by Atomic Force Microscopy (AFM) and Dynamic Light Scattering. BxPC-3 tumor cells were irradiated with a 6 MV X-rays beam, in the absence or presence of AuNPs. AFM showed that HAOA-AuNPs and BBN-AuNPs are spherical with a mean size of 83 ± 20 nm and 49 ± 12 nm, respectively. For RT alone, a reduction in cell viability of up to 33 ± 12% was obtained compared to the control (p ≤ 0.0001). HAOA-AuNPs alone at 200 and 400 μM showed a reduction in cell viability of 20 ± 4% and 35 ± 4%, respectively, while for BBN-AuNPs, at 50 and 200 μM, a reduction in cell viability of 25 ± 3% and 37 ± 3% was obtained, respectively, compared to the control (p < 0.0001). At 72 h post-irradiation, a decrease in cell viability of 26 ± 3% and 22 ± 2% between RT + HAOA-AuNPs at 400 μM and RT + BBN-AuNPs at 50 μM, compared to RT alone, was obtained (p < 0.004). The combination of RT with AuNPs led to a significant decrease in cell viability compared to the control, or RT alone, thus representing an improved effect.

The present work consisted of an exploratory study aiming to evaluate in vitro the potential of AuNPs during Radiation Therapy (RT) in human pancreatic adenocarcinoma cells. AuNPs coated with hyaluronic and oleic acids (HAOA-AuNPs) or with bombesin peptides (BBN-AuNPs) were used. AuNPs were characterized by Atomic Force Microscopy (AFM) and Dynamic Light Scattering. BxPC-3 tumour cells were irradiated with a 6 MV X-rays beam, in the absence or presence of AuNPs. AFM showed that HAOA-AuNPs and BBN-AuNPs are spherical with a mean size of 83+-20 nm and 49+-12 nm, respectively. For RT alone, a reduction in cell viability of up to 33+-12% was obtained compared to the control (p ≤ 0.0001). HAOA-AuNPs alone at 200 and 400 μM showed a reduction in cell viability of 20+-4% and 35+-4%, respectively, while for BBN-AuNPs, at 50 and 200 μM, a reduction in cell viability of 25+-3% and 37+-3% was obtained, respectively, compared to the control (p < 0.0001). At 72 h post-irradiation, a decrease in cell viability of 26+-3% and 22+-2% between RT+HAOA-AuNPs at 400 μM and RT+BBN-AuNPs at 50 μM, compared to RT alone, was obtained (p < 0.004). The combination of RT with AuNPs led to a significant decrease in cell viability compared to the control, or RT alone, thus representing an improved effect.

Contrary to the adult central nervous system, the peripheral nervous system has an intrinsic ability to regenerate that relies on the expression of regenerationassociated genes, such as some kinesin family members. Kinesins contribute to nerve regeneration through the transport of specific cargo, such as proteins and membrane components, from the cell body towards the axon periphery. We show here that KIF4A, associated with neurodevelopmental disorders and previously believed to be only expressed during development, is also expressed in the adult vertebrate nervous system and up-regulated in injured peripheral nervous system cells. KIF4A is detected both in the cell bodies and regrowing axons of injured neurons, consistent with its function as an axonal transporter of cargoes such as β1-integrin and L1CAM. Our study further demonstrates that KIF4A levels are greatly increased in Schwann cells from injured distal nerve stumps, particularly at a time when they are reprogrammed into an essential proliferative repair phenotype. Moreover, Kif4a m RNA levels were approximately ～6-fold higher in proliferative cultured Schwann cells compared with non-proliferative ones. A hypothesized function for Kif4a in Schwann cell proliferation was further confirmed by Kif4a knockdown, as this significantly reduced Schwann cell proliferation in vitro. Our findings show that KIF4A is expressed in adult vertebrate nervous systems and is up-regulated following peripheral injury. The timing of KIF4A up-regulation, its location during regeneration, and its proliferative role, all suggest a dual role for this protein in neuroregeneration that is worth exploring in the future.

Musculoskeletal disorders are among the main causes of disease‐associated disability. Moreover, the incidence and prevalence of osteoporosis and osteoarthritis, as well as the risk of bone fractures and the need for joint replacements, are expected to increase with longer life expectancy. New approaches based on electromagnetic stimulation have been developed, aiming to shorten bone healing time, attenuate osteoporosis and osteoarthritis, and increase implants' osseointegration. Inductive coupling (IC), a non‐invasive methodology to deliver magnetic stimuli, has reached clinical trials and some clinical practices but is not yet considered a standard procedure. Indeed, its feasibility in clinical use is still under discussion, and optimal stimulation parameters are fairly undefined. This comprehensive review describes the research trends and applicability of IC‐based therapeutics for musculoskeletal disorders, and starts identifying top‐performing magnetic stimulation parameters. Insights into the magnetic stimuli setups that promote osteogenesis are provided, based on pre‐clinical and clinical evidence from 117 in vivo studies in animal models and human patients. Potential cellular and molecular biomechanisms mediating IC‐induced effects on osteoblasts and osteoclasts are also explored. The transversal knowledge herein delivered will hopefully support innovative designs and medical devices that will implement IC stimulation as a clinical standard and effective therapeutic for musculoskeletal disorders.

Replacement orthopedic surgeries are among the most common surgeries worldwide, but clinically used passive implants cannot prevent failure rates and inherent revision arthroplasties. Optimized non-instrumented implants, resorting to preclinically tested bioactive coatings, improve initial osseointegration but lack long-term personalized actuation on the bone–implant interface. Novel bioelectronic devices comprising biophysical stimulators and sensing systems are thus emerging, aiming for long-term control of peri-implant bone growth through biointerface monitoring. These acting-sensing dual systems require high frequency (HF) operations able to stimulate osteoinduction/osteoconduction, including matrix maturation and mineralization. A sensing-compatible capacitive stimulator of thin interdigitated electrodes and delivering an electrical 60 kHz HF stimulation, 30 min/day, is here shown to promote osteoconduction in pre-osteoblasts and osteoinduction in human adipose-derived mesenchymal stem cells (hASCs). HF stimulation through this capacitive interdigitated system had significant effects on osteoblasts’ collagen-I synthesis, matrix, and mineral deposition. A proteomic analysis of microvesicles released from electrically-stimulated osteoblasts revealed regulation of osteodifferentiation and mineralization-related proteins (e.g. Tgfb3, Ttyh3, Itih1, Aldh1a1). Proteomics data are available via ProteomeXchange with the identifier PXD028551. Further, under HF stimulation, hASCs exhibited higher osteogenic commitment and enhanced hydroxyapatite deposition. These promising osteoinductive/conductive capacitive stimulators will integrate novel bioelectronic implants able to monitor the bone–implant interface and deliver personalized stimulation to peri-implant tissues.

Neural tissue-related illnesses have a high incidence and prevalence in society. Despite intensive research efforts to enhance the regeneration of neural cells into functional tissue, effective treatments are still unavailable. Here, a novel therapeutic approach based on vertically aligned carbon nanotube forests (VA-CNT forests) and periodic VA-CNT micropillars produced by thermal chemical vapor deposition is explored. In addition, honeycomb-like and flower-like morphologies are created. Initial viability testing reveals that NE-4C neural stem cells seeded on all morphologies survive and proliferate. In addition, free-standing VA-CNT forests and capillary-driven VA-CNT forests are created, with the latter demonstrating enhanced capacity to stimulate neuritogenesis and network formation under minimal differentiation medium conditions. This is attributed to the interaction between surface roughness and 3D-like morphology that mimics the native extracellular matrix, thus enhancing cellular attachment and communication. These findings provide a new avenue for the construction of electroresponsive scaffolds based on CNTs for neural tissue engineering.