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Hernandez Gomez, Yuriko Suemi (2018) Nanocomposite scaffolds and biomimetic peptides in neural regenerative medicine. [Ph.D. thesis]

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Abstract (italian or english)

The incapacity of injured adult central nervous system to restore damaged neuronal circuitry and the large peripheral nervous system nerve defect inability to be naturally regenerated are a critical medical and social issue.
An emerging approach in neuronal regenerative medicine is the use of native extracellular stimuli at nano-scale level influencing cell growth, differentiation and regeneration. Our biomimetic nanosystems mimic as much as possible the nanotopographic, conductive features and guidance cues of the neuronal extracellular environment. They are made of a freestanding and biocompatible nanocomposite scaffold, combining conductive, mechanical and topographical feature of carbon-based nanomaterials with the biocompatible properties of the poly-L-lactic acid (PLLA) matrix. Moreover, biomimetic peptides have been developed deriving them from neuronal proteins involved in the control of neurite outgrowth and axon pathfinding. In recent work from our team, the combination of the nanocomposite scaffold and the peptides proved to enhance neuronal differentiation of a human neuroblastoma cell line and to promote per se neural differentiation of human multipotent stem cells, even in the absence of exogenously added neurotrophins.
In my PhD project I further developed such biomimetic nanosystems.
About the scaffold, we checked the biocompatibility and effect on neuronal differentiation of varying types and concentration of nanofiller. We increased from 0.25 to 5% CNTs dispersed in the PLLA-matrix to improve electrical conductivity and nanoroughness of our nanocomposite scaffold. The enhanced CNTs concentration doesn’t affect cell proliferation, viability and adhesion while promoting neurite elongation. Moreover, we tested the same range of carbon nanohorns (CNHs) and reduced graphene oxide (RGO) dispersed in the PLLA matrix and proved they are as biocompatible as CNTs. Interestingly, 5%RGO has an inductive effect on neuronal differentiation. In last months, 3D printing has been used for patterned scaffold that allow to control the cell growth direction.
About biomimetic peptides, we focused on the characterization of novel peptides sharing a conserved motif to better reproduce neuronal biochemical cues. These peptides are derived from the Ig-like domain of a number of proteins playing important roles in neuronal differentiation and axon elongation: CHL1, Neurofascin, NrCAM, DCC, ROBO2 and 3, Contactin 1, 2 and 5. All such peptides were able to promote neuritogenesis and neuronal differentiation of SH-SY5Y cells, with efficacy similar to previously tested peptides.
In order to shed light on the mechanism by which our peptides act, we studied L1-A peptide in comparison to L1CAM extracellular domain it is derived from. As negative controls we used a scrambled and mutant version of the L1-A peptide. In silico simulations and in vitro evidence suggest an agonist-antagonist mechanism for our peptides: L1-A peptide binds L1CAM and exerts the same neuritogenic effect of the protein acting as L1CAM’s agonist; scrambled and mutant peptides bind the protein and inhibit the L1CAM homophilic binding, but they are not able to activate the signalling intracellular pathway leading to neuronal differentiation, acting as antagonists of L1CAM.
In conclusion, our new nanocomposite scaffold and biomimetic peptides are potential tools for neuronal regenerative medicine, even if further investigations are needed to check their effect in combination.


Abstract (a different language)

The incapacity of injured adult central nervous system to restore damaged neuronal circuitry and the large peripheral nervous system nerve defect inability to be naturally regenerated are a critical medical and social issue.
An emerging approach in neuronal regenerative medicine is the use of native extracellular stimuli at nano-scale level influencing cell growth, differentiation and regeneration. Our biomimetic nanosystems mimic as much as possible the nanotopographic, conductive features and guidance cues of the neuronal extracellular environment. They are made of a freestanding and biocompatible nanocomposite scaffold, combining conductive, mechanical and topographical feature of carbon-based nanomaterials with the biocompatible properties of the poly-L-lactic acid (PLLA) matrix. Moreover, biomimetic peptides have been developed deriving them from neuronal proteins involved in the control of neurite outgrowth and axon pathfinding. In recent work from our team, the combination of the nanocomposite scaffold and the peptides proved to enhance neuronal differentiation of a human neuroblastoma cell line and to promote per se neural differentiation of human multipotent stem cells, even in the absence of exogenously added neurotrophins.
In my PhD project I further developed such biomimetic nanosystems.
About the scaffold, we checked the biocompatibility and effect on neuronal differentiation of varying types and concentration of nanofiller. We increased from 0.25 to 5% CNTs dispersed in the PLLA-matrix to improve electrical conductivity and nanoroughness of our nanocomposite scaffold. The enhanced CNTs concentration doesn’t affect cell proliferation, viability and adhesion while promoting neurite elongation. Moreover, we tested the same range of carbon nanohorns (CNHs) and reduced graphene oxide (RGO) dispersed in the PLLA matrix and proved they are as biocompatible as CNTs. Interestingly, 5%RGO has an inductive effect on neuronal differentiation. In last months, 3D printing has been used for patterned scaffold that allow to control the cell growth direction.
About biomimetic peptides, we focused on the characterization of novel peptides sharing a conserved motif to better reproduce neuronal biochemical cues. These peptides are derived from the Ig-like domain of a number of proteins playing important roles in neuronal differentiation and axon elongation: CHL1, Neurofascin, NrCAM, DCC, ROBO2 and 3, Contactin 1, 2 and 5. All such peptides were able to promote neuritogenesis and neuronal differentiation of SH-SY5Y cells, with efficacy similar to previously tested peptides.
In order to shed light on the mechanism by which our peptides act, we studied L1-A peptide in comparison to L1CAM extracellular domain it is derived from. As negative controls we used a scrambled and mutant version of the L1-A peptide. In silico simulations and in vitro evidence suggest an agonist-antagonist mechanism for our peptides: L1-A peptide binds L1CAM and exerts the same neuritogenic effect of the protein acting as L1CAM’s agonist; scrambled and mutant peptides bind the protein and inhibit the L1CAM homophilic binding, but they are not able to activate the signalling intracellular pathway leading to neuronal differentiation, acting as antagonists of L1CAM.
In conclusion, our new nanocomposite scaffold and biomimetic peptides are potential tools for neuronal regenerative medicine, even if further investigations are needed to check their effect in combination.


EPrint type:Ph.D. thesis
Tutor:Filippini, Francesco
Ph.D. course:Ciclo 31 > Corsi 31 > BIOSCIENZE
Data di deposito della tesi:30 November 2018
Anno di Pubblicazione:30 November 2018
Key Words:regenerative medicine
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/10 Biochimica
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:11518
Depositato il:06 Nov 2019 11:13
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