Carbon nanotube-polymer scaffolds and biomimetic peptides as a system to promote human cell differentiation toward the neuronal phenotype: analysis of a model cell line and circulating multipotent cells

Carbon nanotubes (CNTs) are attractive candidates for the development of scaffolds for neural regeneration thanks to their ability to conduct electrical stimuli, to interface with cells and to mimic the neural environment. This thesis work concerns the development of a freestanding nanocomposite scaffold composed of multi-walled CNTs in a poly-L-lactic (PLLA) matrix that combines the conductive, mechanical and topographical features of CNTs with the biocompatibility of PLLA. Such CNT-PLLA scaffold resulted to support growth and differentiation of neuronal SH-SY5Y cells better than PLLA alone. In order to mimic guidance cues from the neural environment, biomimetic peptides were designed to reproduce regulatory motifs from L1CAM and LINGO1 proteins, that are involved in neurite outgrowth control. Both peptides - which neither alter cell proliferation nor induce cell death - could specifically and positively modulate neuronal differentiation when either used to coat well bottoms or added to the culture medium (with highest efficiency at 1 uM concentration). Furthermore, cell differentiation resulted to be synergistically improved by the combination of the nanocomposite scaffold and the peptides, thus suggesting a prototype for the development of implants for long-term neuronal growth and differentiation. Then, the CNT-PLLA matrix was electrospun into fibres of submicrometric size in order to better mimic the neural environment, i.e. neuronal processes and collagenous components of the extracellular matrix. These scaffolds were shown to be biocompatible and to promote the formation of new neurites that extend along the scaffold fibres. Since cells are influenced by the scaffold topography, the orientation of the scaffold fibres opens up the perspective to promote a polarized neurite outgrowth. Moreover, the neuritogenic properties of the scaffolds are further enhanced when LINGO1 derivative peptide is added to culture medium; this represents a good starting point for developing next generation scaffolds upon peptide functionalization. Moreover, human circulating multipotent cells (hCMCs) were grown onto the scaffolds and treated with peptides in order to asses if this autologous and accessible source of stem cells is capable of neuronal differentiation thanks to the scaffold and peptide characteristics. The CNT-PLLA scaffolds and its respective electrospun version resulted to be suitable for hCMCs adhesion and growth, showing a very good level of biocompatibility, and the hCMCs growing onto the scaffolds showed typical features of cells from the neuronal lineage, such as long neuritic protrusions that are tipped with fan-shaped structures resembling growth cones. Moreover, soon after cell seeding, the scaffolds were shown to promote the upregulation of markers typical of the neuronal lineage.The biomimetic peptides were also shown to influence cell morphology and to upregulate neuronal markers. These results suggest that hCMCs can acquire neuronal commitment thanks to scaffold/peptide properties per se, i.e. even in the absence of those typical growth factors that are normally used to promote the neuronal differentiation of stem cells. Further improvements in the scaffold geometry and composition, functionalization with peptides and culture conditions are necessary to achieve the complete neuronal differentiation of cells and to control the neuron subtype obtained, but our system resulted to be a good starting point for setting up implantable scaffolds for autologous neuronal differentiation. Future functional assessment of synaptic transmission and electrophysiological properties of cells onto the scaffolds will be of great interest. Moreover, coupling such scaffolds with electrical stimulation (which is readily achievable using CNT based materials) can boost further analyses aimed at studying neuronal differentiation and has great potential in nerve injury repair as well as neuron prosthesis

Carbon nanotube-polymer scaffolds and biomimetic peptides as a system to promote human cell differentiation toward the neuronal phenotype: analysis of a model cell line and circulating multipotent cells

Carbon nanotubes (CNTs) are attractive candidates for the development of scaffolds for neural regeneration thanks to their ability to conduct electrical stimuli, to interface with cells and to mimic the neural environment. This thesis work concerns the development of a freestanding nanocomposite scaffold composed of multi-walled CNTs in a poly-L-lactic (PLLA) matrix that combines the conductive, mechanical and topographical features of CNTs with the biocompatibility of PLLA. Such CNT-PLLA scaffold resulted to support growth and differentiation of neuronal SH-SY5Y cells better than PLLA alone. In order to mimic guidance cues from the neural environment, biomimetic peptides were designed to reproduce regulatory motifs from L1CAM and LINGO1 proteins, that are involved in neurite outgrowth control. Both peptides - which neither alter cell proliferation nor induce cell death - could specifically and positively modulate neuronal differentiation when either used to coat well bottoms or added to the culture medium (with highest efficiency at 1 uM concentration). Furthermore, cell differentiation resulted to be synergistically improved by the combination of the nanocomposite scaffold and the peptides, thus suggesting a prototype for the development of implants for long-term neuronal growth and differentiation. Then, the CNT-PLLA matrix was electrospun into fibres of submicrometric size in order to better mimic the neural environment, i.e. neuronal processes and collagenous components of the extracellular matrix. These scaffolds were shown to be biocompatible and to promote the formation of new neurites that extend along the scaffold fibres. Since cells are influenced by the scaffold topography, the orientation of the scaffold fibres opens up the perspective to promote a polarized neurite outgrowth. Moreover, the neuritogenic properties of the scaffolds are further enhanced when LINGO1 derivative peptide is added to culture medium; this represents a good starting point for developing next generation scaffolds upon peptide functionalization. Moreover, human circulating multipotent cells (hCMCs) were grown onto the scaffolds and treated with peptides in order to asses if this autologous and accessible source of stem cells is capable of neuronal differentiation thanks to the scaffold and peptide characteristics. The CNT-PLLA scaffolds and its respective electrospun version resulted to be suitable for hCMCs adhesion and growth, showing a very good level of biocompatibility, and the hCMCs growing onto the scaffolds showed typical features of cells from the neuronal lineage, such as long neuritic protrusions that are tipped with fan-shaped structures resembling growth cones. Moreover, soon after cell seeding, the scaffolds were shown to promote the upregulation of markers typical of the neuronal lineage.The biomimetic peptides were also shown to influence cell morphology and to upregulate neuronal markers. These results suggest that hCMCs can acquire neuronal commitment thanks to scaffold/peptide properties per se, i.e. even in the absence of those typical growth factors that are normally used to promote the neuronal differentiation of stem cells. Further improvements in the scaffold geometry and composition, functionalization with peptides and culture conditions are necessary to achieve the complete neuronal differentiation of cells and to control the neuron subtype obtained, but our system resulted to be a good starting point for setting up implantable scaffolds for autologous neuronal differentiation. Future functional assessment of synaptic transmission and electrophysiological properties of cells onto the scaffolds will be of great interest. Moreover, coupling such scaffolds with electrical stimulation (which is readily achievable using CNT based materials) can boost further analyses aimed at studying neuronal differentiation and has great potential in nerve injury repair as well as neuron prosthesis.I nanotubi di carbonio (CNTs) sono i candidati ideali per lo sviluppo di supporti volti a promuovere la rigenerazione neurale grazie alla loro abilità di condurre gli stimoli elettrici e alla loro nanotopografia in grado di mimare l'ambiente neurale. Questo lavoro riguarda lo sviluppo di supporti nanocompositi costituiti da CNTs dispersi in una matrice di acido polilattico (PLLA) e quindi in grado di combinare le caratteristiche nanotopografiche e di conduttività dei CNTs con la biocompatibilità del PLLA. Tali supporti, sono risultati essere in grado di supportare la crescita e il differenziamento delle cellule neuronali SH-SY5Y in modo migliore rispetto al solo PLLA. Al fine di mimare gli stimoli guida dell'ambiente neurale, sono stati sintetizzati anche dei peptidi biomimetici ricavati da specifici motivi regolativi delle proteine L1CAM e LINGO1, le quali sono coinvolte nel controllo dell'accrescimento neuritico. Entrambi i peptidi non hanno dimostrato effetti negativi sulla vitalità e la proliferazione cellulare, promuovendo invece il differenziamento neuronale in modo sequenza specifico e con i maggiori effetti quando utilizzati a concentrazione 1 uM. Inoltre, quando usati in combinazione, supporti e peptidi sono in grado di agire in modo sinergico e di aumentare ulteriormente il differenziamento cellulare. Successivamente, al fine mimare al meglio l'ambiente neurale, la matrice CNT-PLLA è stata elettrospinnata in fibre di dimensione submicrometrica con lo scopo di rappresentare i processi neuronali e la componente collagenosa della matrice extracellulare. Tali supporti si sono rivelati essere biocompatibili e in grado di promuovere la formazione di nuovi neuriti che si allungano seguendo l'orientamento delle fibre del supporto. Dal momento che le cellule sono influenzate dalla topografia del supporto, l'allineamento delle fibre suggerisce la possibilità di poter ottenere una crescita neuritica polarizzata. Inoltre, le proprietà neuritogeniche del supporto aumentano quando il peptide derivato da LINGO1 viene aggiunto al terreno di coltura; questi risultati rappresentano un buon punto di partenza per sviluppare supporti più avanzati a seguito della funzionalizzazione con tale peptide. In aggiunta, cellule circolanti multipotenti umane (hCMCs) sono state coltivate sui supporti e trattate con i peptidi al fine di determinare se tale fonte di cellule staminali autologa ed accessibile sia capace di differenziazione neuronale grazie soltanto alle caratteristiche dei supporti e dei peptidi. I supporti CNT-PLLA e la rispettiva versione elettrospinnata sono risultati essere adatti all'adesione e alla crescita delle hCMCs, mostrando buoni livelli di biocompatibilità; inoltre, le hCMCs coltivate sui supporti hanno mostrato caratteristiche tipiche delle cellule neuronali come lunghe protrusioni neuritiche terminanti con strutture a forma di ventaglio simili ai coni di crescita. I supporti inoltre promuovono l'espressione di marcatori tipici del lignaggio neuronale. Anche i peptidi si sono rivelati essere in grado di influenzare la morfologia cellulare e di upregolare marcatori neuronali. Questi risultati suggeriscono che le hCMCs sono capaci di acquisire un commitment neuronale solo grazie alle caratteristiche dei supporti e dei peptidi e senza l'ausilio dei fattori di crescita che sono tradizionalmente usati per promuovere il differenziamento neuronale di cellule staminali. Sono necessari ulteriori studi riguardanti la composizione e geometria dei supporti, funzionalizzazione con i peptidi e condizioni di coltura per acquisire una completa differenziazione neuronale e controllare il tipo neuronale ottenuto; ma tale sistema sembra essere un buon punto di partenza per progettare supporti trapiantabili per promuovere la rigenerazione neurale. Sarebbe interessante poter valutare la trasmissione sinaptica e le proprietà fisiologiche delle cellule cresciute sui supporti così come utilizzare tali supporti per stimolare elettricamente le cellule e valutare un eventuale miglioramento nel differenziamento

3D Printed Graphene-PLA Scaffolds Promote Cell Alignment and Differentiation

Traumas and chronic damages can hamper the regenerative power of nervous, muscle, and connective tissues. Tissue engineering approaches are promising therapeutic tools, aiming to develop reliable, reproducible, and economically affordable synthetic scaffolds which could provide sufficient biomimetic cues to promote the desired cell behaviour without triggering graft rejection and transplant failure. Here, we used 3D-printing to develop 3D-printed scaffolds based on either PLA or graphene@PLA with a defined pattern. Multiple regeneration strategies require a specific orientation of implanted and recruited cells to perform their function correctly. We tested our scaffolds with induced pluripotent stem cells (iPSC), neuronal-like cells, immortalised fibroblasts and myoblasts. Our results demonstrated that the specific “lines and ridges” 100 µm-scaffold topography is sufficient to promote myoblast and fibroblast cell alignment and orient neurites along with the scaffolds line pattern. Conversely, graphene is critical to promote cells differentiation, as seen by the iPSC commitment to neuroectoderm, and myoblast fusions into multinuclear myotubes achieved by the 100 µm scaffolds containing graphene. This work shows the development of a reliable and economical 3D-printed scaffold with the potential of being used in multiple tissue engineering applications and elucidates how scaffold micro-topography and graphene properties synergistically control cell differentiation

Enhanced neuronal cell differentiation combining biomimetic peptides and a carbon nanotube-polymer scaffold

Carbon nanotubes are attractive candidates for the development of scaffolds able to support neuronal growth and differentiation thanks to their ability to conduct electrical stimuli, to interface with cells and to mimic the neural environment. We developed a biocompatible composite scaffold, consisting of multi-walled carbon nanotubes dispersed in a poly-L-lactic acid matrix able to support growth and differentiation of human neuronal cells. Moreover, to mimic guidance cues from the neural environment, we also designed synthetic peptides, derived from L1 and LINGO1 proteins. Such peptides could positively modulate neuronal differentiation, which is synergistically improved by the combination of the nanocomposite scaffold and the peptides, thus suggesting a prototype for the development of implants for long-term neuronal growth and differentiation. The study describes the design and preparation of nanocomposite scaffolds with multi-walled carbon nanotubes in a poly-L-lactic acid matrix. This compound used in combination with peptides leads to synergistic effects in supporting neuronal cell growth and differentiation

Covalent functionalization enables good dispersion and anisotropic orientation of multi-walled carbon nanotubes in a poly(l-lactic acid) electrospun nanofibrous matrix boosting neuronal differentiation

A biocompatible porous scaffold obtained via electrospinning a nanocomposite solution of poly(l-lactic acid) and 4-methoxyphenyl functionalized multi-walled carbon nanotubes is presented here for the first time for the enhancement of neurite outgrowth. Optimization of blend preparation and deposition parameters paves the way to the obtainment of defect-free random networks of nanofibers with homogeneous diameters in the hundreds of nanometers length scale. The tailored covalent functionalization of nanotube surfaces allows a homogeneous dispersion of the nanofillers within the polymer matrix, diminishing their natural tendency to aggregate and form bundles. This results in a remarkable effect on the crystallization temperature, as evidenced through differential scanning calorimetry. Furthermore, transmission electron microscopy shows carbon nanotubes anisotropically aligned along the fiber axes, a feature believed to enhance neurite adhesion and growth. Indeed, microscopy images show neurites extension along the direction of nanofibers, highlighting the extreme relevance of scaffold morphology in engineering complex tissue environments. Furthermore, a remarkable effect on increasing the neurite outgrowth results when using the fibrous scaffold containing dispersed carbon nanotubes in comparison with an analogous one made of only polymer, providing further evidence of the key role played by carbon nanostructures in inducing neuronal differentiation

Carbon Nanotubes-Polymer Electrospun Nanofibers as Biocompatible Scaffold for Neuronal Cells Differentiation

none7noneN. Vicentini; T. Gatti; P. Salice; G. Scapin; C. Marega; F. Filippini; E. MennaVicentini, Nicola; Gatti, Teresa; Salice, Patrizio; Scapin, Giorgia; Marega, Carla; Filippini, Francesco; Menna, Enz

NOG-Derived Peptides Can Restore Neuritogenesis on a CRASH Syndrome Cell Model

Homo- and heterophilic binding mediated by the immunoglobulin (Ig)-like repeats of cell adhesion molecules play a pivotal role in cell-cell and cell-extracellular matrix interactions. L1CAM is crucial to neuronal differentiation, in both mature and developing nervous systems, and several studies suggest that its functional interactions are mainly mediated by Ig2–Ig2 binding. X-linked mutations in the human L1CAM gene are summarized as L1 diseases, including the most diagnosed CRASH neurodevelopmental syndrome. In silico simulations provided a molecular rationale for CRASH phenotypes resulting from mutations I179S and R184Q in the homophilic binding region of Ig2. A synthetic peptide reproducing such region could both mimic the neuritogenic capacity of L1CAM and rescue neuritogenesis in a cellular model of the CRASH syndrome, where the full L1CAM ectodomain proved ineffective. Presented functional evidence opens the route to the use of L1CAM-derived peptides as biotechnological and therapeutic tools

A conserved Neurite Outgrowth and Guidance motif with biomimetic potential in neuronal Cell Adhesion Molecules

none10noThe discovery of conserved protein motifs can, in turn, unveil important regulatory signals, and when properly designed, synthetic peptides derived from such motifs can be used as biomimetics for biotechnological and therapeutic purposes. We report here that specific Ig-like repeats from the extracellular domains of neuronal Cell Adhesion Molecules share a highly conserved Neurite Outgrowth and Guidance (NOG) motif, which mediates homo- and heterophilic interactions crucial in neural development and repair. Synthetic peptides derived from the NOG motif of such proteins can boost neuritogenesis, and this potential is also retained by peptides with recombinant sequences, when fitting the NOG sequence pattern. The NOG motif discovery not only provides one more tile to the complex puzzle of neuritogenesis, but also opens the route to new neural regeneration strategies via a tunable biomimetic toolbox.openScapin, Giorgia; Gasparotto, Matteo; Peterle, Daniele; Tescari, Simone; Porcellato, Elena; Piovesan, Alberto; Righetto, Irene; Acquasaliente, Laura; De Filippis, Vincenzo; Filippini, FrancescoScapin, Giorgia; Gasparotto, Matteo; Peterle, Daniele; Tescari, Simone; Porcellato, Elena; Piovesan, Alberto; Righetto, Irene; Acquasaliente, Laura; De Filippis, Vincenzo; Filippini, Francesc