Dissertations / Theses on the topic 'Neural scaffold'

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002.
Includes bibliographical references (leaves 78-91). Also available in electronic version. Access restricted to campus users.

Transplantation of olfactory ensheathing cells (OEC) is one of the most promising current approaches to repair spinal cord injury. The encouraging results from transplantation of OECs in animal models have led to several clinical applications of these cells in spinal cord injury. The first controlled clinical trial was carried out by Mackay-Sim, Féron and colleagues (Mackay-Sim et al., 2008). A number of neurosurgical teams have also implanted foetal OECs (Huang et al., 2003) or minced whole mucosal tissue (Lima et al., 2006) into spinal injuries. So far the reported functional benefits are only moderate. The Mackay-Sim team reported no improvements while others reported minor improvements (including an ongoing trial by Pawel Tabakow’s team in Poland; personal communication). The basic conclusion is that OEC transplantation is feasible and safe. However, in the studies where suspensions of OECs were used there were not enough cells to fill the lesion, and no materials were used to bridge the gaps. In order to progress to more effective transplants the two areas addressed in this thesis will be important – what is the best source of adequate numbers of cells, and what biomaterials can be used to bridge the gaps. In addressing the twin necessities of (a) identifying the tissue source needed to provide sufficient cells for transplantation and (b) the problem of bridging the large gaps present in spinal cord injuries, the results of this study were directed towards two issues. (a) The questions of cell source and proliferation were addressed by establishing the quantitative baseline for the yield of primary cultures from the olfactory bulb, and the whole and split olfactory mucosa and characterising the heterogeneity of these cultures in search for any difference between bulbar and mucosal OECs. The study of flow cytometric simultaneous antigenic bivariate cell cycle of purified OECs and ONFs from these four sources revealed the evolution of population heterogeneity and its strikingly differences between these four sources of primary tissue with additional populations that were not previously described. An unexpected highly proliferative p75+ population in the stripped mucosal epithelium was also characterised. Correlation study of the cell proliferation and population evolution revealed cell autonomous among the difference sources. (b) The feasibility of a synthesis biomaterial for the deployment of OECs and olfactory nerve fibroblasts (ONFs) as a transplant was addressed by designing and developing an electrospun PLGA nanocomposite nanofibre construct with a myriad of microfabrication techniques, focusing on how OECs and ONFs can be deployed during tissue culture and transplantation. The techniques included nanocomposite electrospinning, replica moulding from photolithographed silicon mould, design of tissue-culture membrane insert, and laser ablation. The biocompatibility study showed that when grown on a fibre mesh structured at the nano-scale, OECs responded by adopting the elongated form comparable to that which occurs when the convey regenerating fibres cross small lesions in in vivo transplants. Preliminary functional studies of using the nanocomposite nanofibers as a neural scaffold in the organotypic entorhino-hippocampus slice co-culture data provide an indication that the nanofibres are compatible with tissue and allow migration of astrocytes and growth of nerve fibres. These observations will be important in future attempts to derive larger cell populations for transplantation. The anticipated use of the OEC nanofibre prosthesis would be in the application of autologous human OECs for bridging the gap in spinal cord lesions.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2005.
Includes bibliographical references (leaves 33-35).
The performance of a biological scaffold formed by the self-assembling peptide RADA16 is comparable to the most commonly used synthetic materials employed in the culture of neural stem cells. Furthermore, improvements in the performance of RADA16 have recently been made by appending the self-assembling peptide sequence with various functional motifs from naturally occurring proteins. The focus of this work is to further analyze the performance of these functionalized self-assembling peptide scaffolds when used for the culture of neural stem cells, and to characterize these newly developed materials for comparison with RADA16. The effect of the functional motifs on the structure of the peptide scaffold was evaluated with circular dichroism and scanning electron microscopy, and the mechanical properties of the peptide scaffolds were examined through theological analysis. The functionalized peptides were found to have lower percentages of beta-sheet structure as well as reduced storage moduli in comparison with RADA16. SEM images confirmed the ability of the functionalized peptides to form three-dimensional nanofiber scaffolds capable of encompassing, neural stem cells. Three-dimensional cell culture techniques were used to evaluate the ability of the functionalized peptide scaffolds to promote neural stem cell proliferation, and a scaffold formed by the combination of different functionalized peptides was found to increase the proliferation of neural stem cells in comparison to non-functionalized RADA 16.
by Angus M. Hucknall.
S.M.

This thesis describes a highly interdisciplinary approach to discern the differing impact of scaffold dimensionality and physical space restrictions on the behavior of single cells. Rolled-up nanotechnology is employed to fabricate three-dimensional (3D) SiO/SiO2 microtube geometries of varied diameter, that after a biofunctionalization step are shown to support the growth of U2OS and six different types of stem cells. Cell confinement quantifiable through the given microtube diameter is tolerated by U2OS cells through a remarkable elongation of the cell body and nucleus down to a certain threshold, while the integrity of the DNA is maintained. This confinement for NSPCs also leads to the approaching of the in vivo morphology, underlining the space-restrictive property of live tissue. The dimensionality of the cell culture scaffold however is identified as the major determiner of NSPC migration characteristics and leads to a morphologically distinct mesenchymal to amoeboid migration mode transition. The 3D microtube migration is characterized by exclusively filopodia protrusion formation, a higher dependence on actin polymerization and adopts aspects of in vivo-reported saltatory movement. The reported findings contribute to the determination of biomaterial scaffold design principles and advance our current understanding of how physical properties of the extracellular environment affect cell migration characteristics.

ABSTRACT Peripheral nerve injury is a common clinical problem significantly affecting the patients’ quality of life. In case of severe transections, the bridging of the gap between the proximal and distal nerve stumps is required and autologous nerve grafts using sensory nerves (i.e. the sural nerve or antebrachial cutaneous nerve) are the current criterion standard. Nevertheless, donor-site morbidities, permanent loss of function, size mismatch between the donor nerve and the injured nerve and poor functional recovery rates have prompted the interest towards the identification of an alternative to this technique. To date, surgeons and researchers are turning their attention towards different grafts made of biological or artificial polymers. In fact, the development of hollow nerve guide conduits a) creating an adequate microenvironment for nutritional support/axons regeneration; b) acting as a barrier against the surrounding tissue infiltration; c) matching the effectiveness of the autologous nerve graft, would be beneficial to the field of peripheral nerve surgery. Over the years, many biomaterials of natural or synthetic origin and with different characteristics in terms of biodegradability have been studied. However, it has not been identified yet a prosthesis able to guarantee a better regenerated tissue than the others.
The aim of the present study was to manufacture and investigate in vitro and in vivo the characteristics and the regenerative potential of three different nerve conduits made up of polyvinyl alcohol (PVA); 1% Oxidized PVA (1% Ox PVA) and Silk-Fibroin (SF). While the use of PVA and SF for the realization of neuro-guides has already been studied in the past, oxidized PVA (recently patented by our research group) is a new material for this purpose. In parallel, this study also allowed to assess the quality of axonal regeneration guaranteed by neuro-guides with different origin (synthetic vs. natural) and biodegradation properties (vs non-biodegradable biodegradable). After preparing the three different polymer solutions, disk-shaped and tubular supports were manufactured. These were employed for in vitro and in vivo studies respectively. Considering in vitro analysis, a morphological characterization of supports was performed by Scanning Electron Microscopy (SEM). Thereafter, the biocompatibility and the biological activity of the three different scaffolds was assessed using a Schwann-cell line (SH-SY5Y). Cells were seeded on supports and their adhesion and proliferation was evaluated by SEM and MTT assay at two different end-points (3 and 7 days from seeding). Regarding in vivo tests, nerve conduits were implanted in animal models (Sprague-Dawley rats) of peripheral nerve injury with loss of substance (nerve gap: 5 mm). At 12-weeks from surgery, the functional recovery of the sciatic nerve was assessed; thereafter, the animals were euthanized and the dissection occurred. Prior to explant, the gross appearance of grafts was carefully observed in situ. Specimens were than processed for histological (hematoxylin and eosin staining) and immunohistochemical analysis (anti-CD3; anti-S100) as well as for further Transmission Electron Microscopy (TEM) analysis. The objective was to assess the quality of the regenerated nerve-tissue highlighting any differences in efficacy between the three types of nerve-conduits; to this end, the histomorphological analysis has been fundamental allowing to quantify the axons (myelinated vs unmyelinated nerve fibers) at different levels of the explant (proximal vs central vs distal portion); the controlateral sciatic nerve was used as control. Considering the in vitro results, SEM micrographs showed that PVA and SF supports have a smooth and regular surface; conversely, a certain roughness was noticed observing the ultrastructure of 1% Ox PVA disk-shaped scaffolds. Despite the superficial appearance of supports, it does not seem to affect the interaction with the cells. In fact, PVA-based scaffolds do not support cell adhesion and proliferation; SEM analysis and the MTT assay do not identified the presence of SH-SY5Y cells after 3 and 7 days from seeding. This result can be attributed to the high hydrophilic nature of the hydrogels. Conversely, SF scaffolds are adequate to promote SH-SY5Y cells growth. Regarding the in vivo study, all nerve conduits showed good characteristics in terms of handiness, being easy-suturing and demonstrating also an adequate tear-resistance feature; PVA-based scaffolds appear more flexible than SF guides. After 12 weeks from surgery, all animals showed a sciatic nerve functional recovery; in particular, all of them supported their body weight on the hind leg even though animals implanted with PVA and SF nerve conduits sometimes showed spasms during the walk while not limping. On the contrary, animals implanted with 1% Ox PVA nerve conduits exhibited a normal movement. At the time of dissection, the three scaffolds were still clearly identifiable. Any dislocation of the grafts or neuroma formation at the stumps was observed; moreover, the transparency of the three scaffolds allowed to identify the presence of a regenerated tissue inside. Thereafter, histological and immunohistochemical analysis were performed to evaluate the quality of axonal regeneration. Preliminarily, the haematoxylin and eosin staining of the specimens (cross-section of the central portion) highlighted the morphological integrity of the structure. In fact, three layers are recognizable proceeding from the periphery to the inside of the sections: an external fibrous capsule; a layer corresponding to the nerve conduit; a homogeneous and dense regenerated tissue in the middle. The biocompatibility of the grafts was verified by immunohistochemical analysis; anti-CD3 immunohistochemistry demonstrated the absence of severe inflammatory reactions. At the same time, several S100+ cells were identified suggesting the extensive presence of Schwann-cells. In parallel, the typical peripheral nerve morphology was highlighted also by Toluidine Blue staining by means of was considered also the appearance of the proximal and distal stumps. Although all samples support the recovery of the lesion, some differences can be found between the three experimental groups; these results were confirmed also by TEM micrographs. The histomorphometric analysis of samples evaluated the total axons number per nerve and axon density (axons/μm2); for each graft were considered the proximal, the central and the distal section. The collected data showed that 1% Ox PVA conduits assure a better outcome in nerve regeneration than the non-biodegradable PVA grafts which among the three groups proved to be the ones with the lower outcomes.
The results of this study showed that all nerve conduits considered (PVA; 1% Ox PVA and SF) promote peripheral nerve regeneration in case of neurotmesis with loss of substance. Considering the quality of regenerates, better outcomes were observed analyzing the 1% Ox PVA explants compared to PVA and Silk-Fibroin ones.
RIASSUNTO Le lesioni nervose periferiche costituiscono un problema clinico piuttosto comune, il quale inficia in modo significativo la qualità della vita dei pazienti. In caso di lesioni gravi con perdita di sostanza, al fine di colmare il gap tra il moncone prossimale ed il distale, il gold- standard prevede l’impianto di innesti nervosi autologhi utilizzando nervi sensoriali (ad es., nervo surale o nervo cutaneo antibrachiale). Tuttavia, criticità quali la morbidità del sito donatore, la perdita in funzionalità, la mancata corrispondenza dimensionale tra il nervo donatore ed il nervo lesionato oltre ad uno scarso recupero funzionale hanno spinto l'interesse verso l'identificazione di un approccio alternativo. Allo stato dell’arte, chirurghi e ricercatori stanno volgendo la loro attenzione verso innesti polimerici diversi (grafts) di natura sia biologica che artificiale. Infatti, lo sviluppo di neuroguide capaci di: a) creare un microambiente ideale per la rigenerazione assonale; b) fornire una protezione dall'infiltrazione di tessuto circostante; c) possedere un’efficacia analoga a quella garantita dall’innesto nervoso autologo; costituirebbe un vantaggio significativo nell’ambito della chirurgia del nervo periferico. Nel corso degli anni, sono stati studiati molti biomateriali di origine sia naturale che sintetica aventi caratteristiche differenti in termini di biodegradabilità. Tuttavia, considerando la qualità del tessuto rigenerato, non è ancora stata individuata una protesi più performante rispetto alle altre. L’obiettivo di questo studio è stato quello di allestire e studiare, sia in vitro che in vivo, le caratteristiche ed il potenziale rigenerativo di tre diverse neuroguide rispettivamente costituite da: alcool polivinilico (PVA); PVA ossidato 1% (PVA Ox 1%) e Fibroina della Seta (FS). Mentre l’impiego di PVA e FS per la realizzazione di grafts è già stato investigato in passato, il PVA Ox 1% (recentemente brevettato dal nostro gruppo di ricerca) costituisce un nuovo materiale per questo scopo. In parallelo, questo studio ha anche consentito di confrontare la qualità della rigenerazione assonale sostenuta da neuroguide diverse sia per origine (sintetica vs naturale) che per proprietà biodegradative (biodegradabili vs nonbiodegradabili).
Dopo aver allestito le tre diverse soluzioni polimeriche, sono stati quindi preparati scaffolds sia discoidali che in forma di graft tubulare, utilizzati rispettivamente per i successivi studi in vitro e in vivo. Nell’ambito degli studi in vitro, è stata effettuata una caratterizzazione morfologica dei supporti mediante microscopia elettronica a scansione (SEM). Successivamente, la biocompatibilità e l'attività biologica dei tre differenti scaffolds è stata valutata utilizzando una linea di cellule di Schwann (SH-SY5Y). Le cellule sono state seminate sui supporti e la loro adesione e la proliferazione è stata valutata mediante saggio MTT oltre che SEM a due differenti end-point (3 e 7 giorni dalla semina). Per quanto riguarda lo studio in vivo, i graft tubulari sono stati impiantati in modelli animali (ratti Sprague- Dawley) di lesione nervosa periferica con perdita di sostanza (gap tra moncone prossimale e distale: 5 mm). A 12 settimane dalla chirurgia, è stato valutato il recupero funzionale del nervo sciatico; successivamente, gli animali sono stati sacrificati. Dopo dissezione, prima di procedere all’espianto, l'aspetto macroscopico degli innesti è stato attentamente osservato in situ. I campioni sono stati quindi prelevati e trattati per le successive analisi istologiche (ematossilina ed eosina) ed immunoistochimiche (anti-CD3; anti-S100) nonché per ulteriori analisi di microscopia elettronica a scansione (TEM). L'obiettivo è stato quello di valutare la qualità del tessuto rigenerato evidenziando eventuali differenze di efficacia tra i tre tipi di grafts; a tal fine, anche l'analisi istomorfologica si è rivelata fondamentale, permettendo essa di quantificare gli assoni (mielinici vs amielinici) in diverse porzioni del campione (porzione prossimale vs centrale vs distale). Il nervo sciatico controlaterale è stato usato come controllo. Considerando i risultati degli studi in vitro, le immagini al SEM hanno mostrato come i supporti in PVA e FS mostrino una superficie liscia e regolare; al contrario, una certa ruvidità è stata notata osservando l’ultrastruttura degli scaffold discoidali in PVA Ox 1%. Nonostante il diverso aspetto ultrastrutturale dei supporti, esso non sembra influenzare l'interazione con le cellule. Il PVA (sia nativo che ossidato) non sostiene l'adesione e la proliferazione cellulare; infatti, sia le analisi al SEM che il saggio MTT non hanno identificato la presenza di cellule SH-SY5Y dopo 3 e 7 giorni dalla semina. Questo risultato può essere attribuito alla elevata idrofilia degli idrogeli Al contrario, gli scaffold in FS sono adeguati per promuovere la crescita delle SH-SY5Y. Per quanto riguarda lo studio in vivo, tutti i graft mostrato buone caratteristiche in termini di manipolabilità, essendo facilmente suturabili e dimostrando anche una adeguata resistenza allo strappo; gli scaffold in PVA appaiono più flessibile rispetto alle guide in FS. Dopo 12 settimane dalla chirurgia, tutti gli animali hanno mostrato un certo recupero funzionale dell’arto operato; in particolare, tutti distribuivano il proprio peso corporeo anche sulla zampa posteriore. Pur non zoppicando, gli animali impiantati con PVA e SF mostravano talvolta degli spasmi durante la deambulazione, al contrario, gli animali impiantati con graft in PVA Ox 1% esibivano un movimento normale. Al momento della dissezione, i tre graft erano ancora chiaramente identificabili. Non è stata riscontrata alcuna dislocazione degli innesti o formazione di neuroma in corrispondenza dei monconi; inoltre, la trasparenza delle tre neuroguide ha permesso di identificare la presenza di un tessuto rigenerato al loro interno. Successivamente, sono state effettuate analisi istologiche ed immunoistochimiche per valutare la qualità della rigenerazione assonale. Preliminarmente, mediante colorazione con ematossilina ed eosina (sezione trasversale della porzione centrale) è stato possibile mettere in evidenza l'integrità morfologica della struttura. Procedendo dalla periferia della sezione verso l'interno sono riconoscibili: una capsula fibrosa esterna; il graft; ed il tessuto neo-rigenerato, omogeneo e denso, nel mezzo. La biocompatibilità degli innesti è stata verificata mediante analisi immunoistochimica; la scarsa presenza di cellule CD3+ ha dimostrato l'assenza di reazioni infiammatorie gravi riconducibili all’impianto. Contestualmente, l’elevata presenza di elementi S100+ riscontrata in tutti i campioni ha comprovato una evidente rigenerazione assonale. In parallelo, la morfologia tipica del tessuto nervoso periferico è stata altresì evidenziata mediante colorazione con Blu di Toluidina mediante la quale è stato considerato anche l'aspetto dei monconi prossimale e distale.
Sebbene tutti i campioni supportino il recupero della lesione, alcune differenze possono essere riscontrate tra i tre gruppi sperimentali; questi risultati sono stati confermati anche dalle micrografie al TEM. L'analisi morfometrica dei campioni ha valutato il numero totale di assoni/nervo e la loro densità (assoni / μm2); per ogni innesto sono state considerate le sezioni prossimale, centrale e distale. I dati raccolti hanno dimostrato che il PVA Ox 1% assicura un risultato migliore nella rigenerazione assonale rispetto agli innesti non biodegradabili in PVA, il quale tra i tre gruppi è risultato essere quello con l’outcome inferiore. I risultati di questo studio hanno mostrato che, in caso di neurotmesi con perdita di sostanza, tutti i graft allestiti (PVA; PVA Ox 1% e FS) promuovono la rigenerazione del nervo. Considerando la qualità del tessuto rigenerato, sono stati osservati dei risultati migliori con graft in PVA Ox 1% rispetto a quelli ottenuti da neuroguide in PVA e FS.

Activity-induced immediate-early gene (IEG) transcription foci can be labelled with fluorescent probes, permitting high temporal and spatial resolution in mapping neuronal circuits. Previous quantification approaches have assumed a Boolean function of transcription foci, assuming that cells are either active or inactive. Due to multiple amplification steps in the in situ hybridization process, it was thought that information relating to magnitudes of firing rates was lost. However, the current data suggest that transcription foci actually exhibit non-Boolean intensity and size values which vary according to behavioural condition. Systematic characterization of transcription foci intensity and size revealed incremental variations such that: home-cage < one-environment exposure < five-environment exposure < maximal electroconvulsive shock. Visual differences in transcription foci may result from a quantifiable relationship between spiking patterns and transcription rates. The exact stoichiometry between neuronal spiking and transcription is not yet clear, but these results suggest that Boolean applications of IEG imaging may neglect accurate neuronal activation properties.
xvi, 125 leaves : ill. ; 29 cm

Pre regeneráciu a rast poranených nervových vlákien bolo preskúmaných mnoho postupov, no výsledný rast axónov je často náhodný až dezorganizovaný a odráža sa na zložitejšom zotavovaní pacienta. V tejto práci boli vyrobené nové skafoldy s mikroštruktúrnymi a mechanickými vlastnosťami nervového skafoldu pomocou metódy freeze-casting. Konkrétne boli vyrobené biokeramické skafoldy na báze fosforečnanov vápenatých, oxidu titaničitého alebo oxidu zirkoničitého. Pomocou kontrolovaného rastu ľadu v jednom smere bola pripravená orientovaná mikroštruktúra. Pozorovanie pomocou skenovacej elektrónovej mikroskopie potvrdilo lineárne orientované póry (lamelárny systém), v ktorých priemerná veľkosť pórov klesala so zvyšujúcou sa rýchlosťou mrazenia. Skafoldy pripravené pomocou mrazenia v tekutom dusíku vykazovali vynikajúce mechanické vlastnosti, kde pevnosť v ohybe bola získaná v rozmedzí 10–17 MPa. Tie isté skafoldy mali vzdialenosť medzilamelamelárnych priestorov 10–30 µm, ktorých parametre sú vhodné pre nervové skafoldy. Biokompatibilita bola vyhodnotená pomocou Schwannových buniek in vitro, kde bola pozorovaná adhézia a rast v lamelárnom smere. Cytotoxické testy odhalili negatívny vplyv vyššej koncentrácie vápnika na prežitie Schwannových buniek. Pripravené skafoldy mali schopnosť tvorby apatitu na povrchu v podobe embryonálnych a nukleačných centier a apatitu samotného. Skafoldy na báze fosforečnanov vápenatých a oxidu titaničitého vykazovali sľubné regeneračné vlastnosti, konkrétne adhéziu a rast prostredníctvom pórovitej štruktúry a taktiež vynikajúce mechanické vlastnosti.

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.
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.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.
Includes bibliographical references (p. 123-125).
Rat neural stem cells (NSCs) were cultured in monolayer or in porous collagen scaffolds and exposed to neurogenic or non-neurogenic medium to determine the effects on neural differentiation and neurite growth. Nestin, [beta]III-tubulin, and GFAP expression were determined using immunofluorescent techniques, and the neurite length was measured. NSCs differentiated into neurons, with actively growing neurites, and astrocytes when cultured in differentiation medium (DM) or neurogenic medium (NM). NSCs cultured in monolayer expressed more nestin and III-tubulin and had significantly longer neurite extensions than NSCs cultured in collagen scaffolds. Laminin coated scaffolds promoted the attachment of NSCs to the scaffold struts and resulted in a more even distribution of nestin and [beta]III-tubulin positive cells throughout the scaffold. Overall, NSCs cultured in DM for at least 14 days resulted in the most neuronal differentiation and neurite growth.
by Erica Ueda.
S.M.

Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.

The JNKs have been shown to regulate cellular processes ranging from apoptosis to differentiation and survival, depending on cell-tissue type and cell context. JIP3 was identified as a putative scaffold protein functioning in the JNK signalling pathway. JIP3 is predominantly expressed in neurons, and is thought to have a role in neuronal vesicular transport. The function of JIP3 in the context of JNK signalling has, however, not been established.;A polyclonal JIP3 antiserum was generated in the course of this study, which was suitable for western blotting, immunprecipitation and indirect immunofluorescence microscopy experiments. This antiserum showed greater efficacy than any other available antisera, and was essential in subsequent experiments.;Biochemical fractionation and immunofluorescence microscopy experiments identified the association of endogenous JIP3 with an unconventional vesicular species. The majority of JIP3 was found to localise predominantly in growth cone regions of differentiated N1E-115 and PC-12 cells. In these growth cone regions, significant co-localisation was identified between JIP3 and JNK pathway components, but was not apparent in other subcellular compartments. Immunoprecipitation experiments showed increased association of JIP3 with JNK pathway components upon differentiation of PC-12 cells.;The proposed function of the JNK pathway in neurite outgrowth led to the investigation of the potential for JIP3 regulation of this neuronal differentiation process. Overexpression of JIP3, which acts as a partial inhibitor of JNK pathway signalling, was found to significantly suppress neurite outgrowth in N1E-115 and PC-12 cells. Moreover, re-constitution of a JIP3 mediated JNK signalling module was sufficient to induce neurite outgrowth in PC-12 cells.;Finally, JIP3 co-localised with the cytoskeletal regulator paxillin specifically within growth cone regions of differentiated PC-12 cells. This suggests that JIP3 may direct JNK pathway signalling towards a growth cone associated substrate, paxillin, and thus regulate neurite outgrowth.

A eletrofiação é uma celebrada técnica de processamento de polímeros, capaz de produzir fibras de diâmetro nanométrico. A montagem comum do sistema de eletrofiação permite a captação de fibras aleatórias sob a forma de um não-tecido. Diversas modificações nessa montagem permitem a obtenção de diferentes morfologias de fibras. Tais modificações são revisadas e discutidas neste trabalho. Na produção de suportes de crescimento de células neurais, é interessante que seja incorporada alguma anisotropia no meio. Assim, um aparato de eletrofiação, capaz de produzir fibras alinhadas, foi construído e a variação dos parâmetros de seu processamento permitiu a obtenção de diferentes qualidades de alinhamento das fibras para dois polímeros biodegradáveis. Diversos parâmetros influenciaram a qualidade desse alinhamento, porém a velocidade de captação das fibras mostrou ser o mais impactante, em acordo com dados reportados na literatura. A morfologia das fibras foi avaliada quanto ao seu diâmetro, com o auxílio de micrografias de MEV e do software de edição de imagens ImageJ. Adicionalmente buscou-se avaliar a qualidade do alinhamento de tais fibras. Para tanto, foi desenvolvida uma metodologia de quantificação de qualidade de alinhamento de fibras, baseado nas micrografias e na ferramenta de FFT do ImageJ. A metodologia proposta foi capaz de ordenar de maneira objetiva e consistente a qualidade do alinhamento das fibras obtidas, mesmo quando a análise visual (usada como referência) se provava ineficiente. A metodologia proposta foi incorporada num plugin para ImageJ, via algoritmo computacional escrito em Java. Com o uso do plugin, foi possível processar diversas micrografias, obtidas em diferentes pontos das malhas eletrofiadas e com variadas magnificações, a fim de se criar uma estatística dos resultados obtidos para qualidade de alinhamento das fibras, algo inédito na literatura. Malhas eletrofiadas com diferentes qualidades de alinhamento de suas fibras foram utilizadas como substrato na cultura de células precursoras neurais, provenientes de neuroesferas. Foi feita a cultura de células progenitoras neurais, provenientes de neuroesferas, tendo como substrato malhas eletrofiadas com diferentes qualidades de alinhamento, a fim de se avaliar o impacto dos contatos físicos das fibras sobre a migração e diferenciação de tais células.
Electrospinning is a celebrated technique of polymer processing, able to produce fibers with nanometric diameter. Common assembly of electrospinning apparatus allows collection of random fibers in a non-woven matt. Several modifications on this assembly enable different fiber morphologies to be obtained. Such modifications are revised and discussed in this work. In the production of cell growth scaffolds, its interesting that some anisotropy is incorporated in the medium. Therefore, an electrospinning apparatus capable of producing aligned fibers was constructed. Variation of processing parameters of said apparatus enabled different alignment qualities of fibers to be attained for two biodegradable polymers. Many parameters influenced on the quality of said alignment; fiber collection speed, however, proved more impacting, in accordance with literature data. Fiber morphology was assessed in regard to its diameter with the aid of MEV micrographs and ImageJ software. Furthermore, assessment of fiber alignment quality was sought. For this matter, it has been developed a quantification methodology for fiber alignment quality, based on micrographs and ImageJ\'s FFT tool. The proposed methodology was able to objectively and consistently rank fiber alignment quality, even when visual analysis (used as reference) failed to do so. This methodology was incorporated in a plugin for ImageJ, via Java script algorithm. With the aid of this plugin it was feasible to process several micrographs, taken from electrospun mats at different spots and magnifications. This helped create statistics about obtained results of fiber alignment quality, on an unprecedented approach in written literature. Electrospun mats with varying quality in fiber alignment were used as substrate in the culture of neural precursor cells from neurospheres to assess the influence of contact guidance on migration and differentiation of such cells

The use of nanofibrous scaffolds for tissue engineering applications is emerging as an important area of research in the field of regenerative medicine. Due to their close morphological similarity to the extracellular matrix (ECM) these types of scaffolds could be ideal candidates for synthetic, ECM-like substitutes. The main aim of this thesis is to understand and tailor the physical and morphological properties of nanofibrous scaffolds through control of the processing conditions and the incorporation of multi-walled carbon nanotubes and finally their application as substrates for neuronal cell growth. Nanofibrous scaffolds from polycaprolactone, poly(methyl methacrylate) and gelatin were produced using a self-made electrospinning kit. Advanced experimental design was employed to understand the impact of the processing parameters on the morphology of these scaffolds. The crystalline structure of polycaprolactone scaffolds was measured as a function of the fibre morphology and the processing conditions. It was found that the mechanical properties were strongly dependant on both of these factors which allows for the production of scaffolds with similar fibre structures but markedly different mechanical properties. Carbon nanotubes were successfully incorporated into polycaprolactone and gelatin nanofibres to form composite scaffolds. The targeted functionalization – polycaprolactone was chemically grafted onto carbon nanotubes to macht the polycaprolactone matrix – of carbon nanotubes to the polymer matrix was found to be superior to a more common approach to nanotube functionalization in its reinforcement properties on the electrospun scaffolds. Finally the performance of polycaprolactone scaffolds with bespoke fibre morphologies and different levels of carbon nanotube reinforcements as substrates for human neuronal cell growth was explored in a pilot study. Initial results indicate that the addition of carbon nanotubes greatly enhance neuronal cell growth on scaffolds as determined by an increased cell proliferation and neuronal cell cell differentiation.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2010.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 79-82).
The organization and intricacy of the central and peripheral nervous systems pose special criteria for the selection of a suitable scaffold to aid in regeneration. The scaffold must have sufficient mechanical strength while providing an intricate network of passageways for axons, Schwann cells, oligodendrocytes, and other neuroglia to populate. If neural regeneration is to occur, these intricate passageways must not be impeded by macrophages, neutrophils, or other inflammatory cells. Therefore it is imperative that the scaffold does not illicit a severe immune response. Biodegradable electrospun fibers are an appealing material for tissue engineering scaffolds, as they strongly resemble the morphology of extracellular matrix. In this study, electrospun fibers composed of poly(L-lactic acid) (PLLA) and polycaprolactone (PCL) were prepared with and without the steroid anti-inflammatory drug, dexamethasone, encapsulated. Histological analysis of harvested subcutaneous implants demonstrated the PLLA fibers encapsulating dexamethasone (PLLA/dex fibers) evoked a much less severe immune response than any other fiber. These findings were supported by in vitro drug release data showing a controlled release of dexamethasone from the PLLA/dex fibers and a burst release from the PCL/dex fibers. The ability of the PLLA/dex fibers to evade an immune response provides a very powerful tool for fabricating tissue engineering scaffolds, especially when the stringent demands of a neural tissue engineering scaffold are considered. Structural support and contact guidance are crucial for promoting peripheral nerve regeneration. A method to fabricate peripheral nerve guide conduits with luminal, axially aligned, electrospun fibers is described and implemented in this study. The method includes the functionalization of the fibers with the axonal outgrowth promoting protein, laminin, to further enhance regeneration. The implantation of stem cells at the. site of a spinal cord or peripheral nerve lesion has been shown to promote nerve regeneration. Preliminary work to isolate and culture pluripotent, adult neural stem cells for seeding on the above mentioned scaffold is also described here.
by Nathaniel Vacanti.
S.M.

Trauma or degenerative diseases such as osteonecrosis may determine bone loss whose recover is promised by a "tissue engineering“ approach. This strategy involves the use of stem cells, grown onboard of adequate biocompatible/bioreabsorbable hosting templates (usually defined as scaffolds) and cultured in specific dynamic environments afforded by differentiation-inducing actuators (usually defined as bioreactors) to produce implantable tissue constructs. The purpose of this thesis is to evaluate, by finite element modeling of flow/compression-induced deformation, alginate scaffolds intended for bone tissue engineering. This work was conducted at the Biomechanics Laboratory of the Institute of Biomedical and Neural Engineering of the Reykjavik University of Iceland. In this respect, Comsol Multiphysics 5.1 simulations were carried out to approximate the loads over alginate 3D matrices under perfusion, compression and perfusion+compression, when varyingalginate pore size and flow/compression regimen. The results of the simulations show that the shear forces in the matrix of the scaffold increase coherently with the increase in flow and load, and decrease with the increase of the pore size. Flow and load rates suggested for proper osteogenic cell differentiation are reported.

Il sistema nervoso centrale svolge un ruolo chiave nella raccolta, integrazione ed elaborazione delle informazioni provenienti dagli organi di senso e dall’ambiente interno dell’organismo. La sua struttura, formata da una complessa rete neurale finemente organizzata può essere soggetta a danni di vario tipo, che ne minano le funzionalità. Per far fronte a tale evenienza, negli ultimi anni, sta emergendo una nuova scienza dal carattere fortemente multidisciplinare, ovvero la medicina neurorigenerativa. Essa comprende l’ingegneria tissutale, che mira all’ingegnerizzazione dei materiali per la produzione di supporti per la rigenerazione (scaffold), la neurologia, e l’ingegneria biomedica. Scopo di questa tesi è stato quello di passare in rassegna le ricerche più recenti sui materiali impiegati per la medicina neurorigenerativa, così come le tecniche e le tecnologie emergenti per il loro processo. In particolare, i polimeri naturali e di sintesi rappresentano una reale potenzialità per possibili interventi terapeutici. D’altra parte, tecnologie di processo quali l’electrospinning e la stampa tridimensionale (3D BIOPRINTING) hanno consentito di progredire notevolmente nella fabbricazione di supporti finalizzati allo sviluppo di impianti neurorigenerativi eterologhi o coadiuvanti l’impianto autologo. In particolare, il 3D Bioprinting è una tecnologia che ha la potenzialità di consentire, in un prossimo futuro, di riprodurre con precisione sempre maggiore la delicata organizzazione spaziale e strutturale gerarchica della matrice extracellulare neuronale. I vantaggi e le applicazioni del Bioprinting sono potenzialmente vastissimi e ancora in fase di esplorazione. L’impatto di nuove tecnologie, nuovi materiali e tecniche bioigegneristiche d’avanguardia potrebbero rappresentare non solo un passo avanti nel campo della rigenerazione neurale, ma anche per la comprensione di processi fisiologici ed ancor di più patofisiologici del Sistema Nervoso Centrale.

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.

Il Sistema Nervoso Centrale (SNC), possiede una limitata capacità di rigenerarsi spontaneamente in seguito a traumi o malattie; le poche strategie rigenerative rivolte a pazienti con traumi o malattie al SNC oggi disponibili, sono tuttavia limitate. Strategie rigenerative dei tessuti neurali, come la somministrazione di cellule e/o farmaci, hanno condotto a risultati sperimentali promettenti nei modelli animali; l’estensione degli esiti all’ambito clinico risulta però difficoltosa. L’efficacia della terapia cellulare, che adopera il trapianto di cellule staminali nel SNC al fine di sostituire i tessuti danneggiati, si è dimostrata limitata a causa della bassa sopravvivenza cellulare e delle criticità di integrazione cellulare che si presentano durante il trapianto. La somministrazione di molecole terapeutiche al SNC tramite metodi convenzionali, come la somministrazione orale o endovenosa, viene invece ostacolata dalla difficoltosa diffusione attraverso la barriera ematoencefalica. Per favorire la sopravvivenza e l’integrazione cellulare post-trapianto, così come per somministrare localmente e in modalità sostenibile agenti biologici ai siti danneggiati del SNC, oggi si ricorre attivamente all’utilizzo di biomateriali. Poiché i trattamenti farmacologici attualmente presenti si limitano a ritardare la progressione delle malattie del SNC, risultano urgenti metodi di medicina rigenerativa che siano in grado di prevalere sul progredire della malattia, promuovendo quindi la rigenerazione tissutale. Questo lavoro di tesi vuole essere uno studio sui recenti biomateriali impiegati come veicoli per la trasmissione di cellule e farmaci per la riparazione di danni acuti e/o cronici del SNC, con particolare attenzione ai casi di lesioni traumatiche encefaliche e del midollo spinale, e a malattie degenerative come la degenerazione maculare legata all’età e la retinite pigmentosa, che determinano degenerazione dei fotorecettori e dell’epitelio pigmentato retinico.

Danos ao sistema nervoso central (SCN) resultam em perda de conexões axonais, das funções motoras e sensoriais. Uma das estratégias para seu reparo é o transplante de células-tronco mesenquimais (CTMs). Porém essa alternativa requer uma adequada via de aplicação. Nesse sentido, o uso de matrizes alinhadas pode ser usado para apoiar o crescimento e diferenciação das CTMs e, quando incorporadas com fatores de crescimento, otimizam o processo de regeneração tecidual. O objetivo desse trabalho foi avaliar a diferenciação neural das CTMs cultivadas sobre matrizes de nanofibras orientadas com o fator de crescimento epidermal (EGF) incorporado. Os scaffolds com fibras alinhadas foram produzidos por electrospinning de emulsão e avaliados conforme a sua morfologia, o diâmetro das nanofibras, a degradabilidade e a liberação do EGF. As CTMs utilizadas foram provenientes da polpa de dentes decíduos esfoliados humanos. Essas células foram cultivadas nos scaffolds e avaliadas conforme os testes biológicos: adesão, viabilidade, proliferação, citotoxicidade e diferenciação neural. Os scaffolds com fibras alinhadas controle (AC) e contendo o EGF (AE) apresentaram morfologia, diâmetro das nanofibras e tempo de degradação semelhantes. Com base no total de EGF presente na matriz AE, 90,14% foi liberado após 28 dias. O citoesqueleto e o núcleo das CTMs cultivadas nos scaffolds AC e AE estavam mais alongados e alinhados quando comparado com as CTMs cultivadas no poço de cultura (controle). As CTMs aderiram mais nas matrizes AE em relação às matrizes AC, porém a proliferação e viabilidade celular foram similares, exceto no tempo de 72 horas, o qual a viabilidade no grupo controle foi maior, em comparação aos demais grupos. Os scaffolds AC e AE não foram tóxicos para as CTMs. Em relação aos resultados da neuro-diferenciação, a expressão de nestina e neurofilamentos consideravelmente maior em todos os grupos analisados quando comparado ao grupo controle. A expressão de βIII-tubulina e GFAP foi maior em todos os grupos diferenciados quando comparada ao grupo controle. A maioria das CTMs cultivadas nas matrizes AC e AE, induzidas ou não à diferenciação neural, apresentaram correntes dependente de voltagem para sódio. O valor de condutância máxima foi maior para todos os grupos analisados quando comparado ao grupo controle onde as células não foram diferenciadas. Portanto, as matrizes com nanofibras orientadas induzem à diferenciação neural das CTMs em neurônios funcionais tanto na ausência como na presença de EGF incorporado. As matrizes AE ainda mostraram ser capazes de melhorar a adesão celular. Dessa forma, conclui-se que as matrizes de nanofibras estudadas são uma possível estratégia para otimização da regeneração de lesões neurológicas.
Damage to the central nervous system (CNS) results in loss of axonal connections and motor and sensory functions. One of the strategies for its repair is the transplantation of mesenchymal stem cells (MSCs). However, this requires a suitable application route. Accordingly, the use of scaffolds support the growth of MSCs and, when incorporated with growth factors, optimize the regeneration process. The purpose of this study was to evaluate the neural differentiation of MSCs cultured on nanofiber matrices oriented with epidermal growth factor (EGF) incorporated. Aligned scaffolds were produced by electrospinning emulsion and evaluated according to their degradation, the morphology and diameter of the nanofibers, and release of EGF from the nanofibers. MSCs used were from human exfoliated deciduous teeth (SHED). These cells were cultured on the scaffolds and evaluated according to biological tests: adhesion, viability, proliferation, cytotoxicity and neural differentiation. The aligned control scaffolds (AC) containing EGF (AE) presented similar morphology, diameter of nanofibers and degradation time. Based on the total EGF present in the scaffold AE, 90.14% was released after 28 days. The cytoskeleton and the core of the MSCs cultured on scaffolds AC and AE were more aligned and elongated when compared to the MSCs grown on plate wells (control). MSCs adhered more to matrices AE when compared to matrices AC, although proliferation and cell viability were similar, except after 72 hours. In this period, the viability of the control group was higher when compared to the rest of the groups. Scaffolds AC and AE were not toxic to MSCs. In regard to the results of neuro-differentiation, the expression of nestin and neurofilament was much higher in all groups than the control group. The expression of βIII tublin and GFAP was higher in all differentiated groups than the control group. Most of the MSCs grown in matrices AC and AE, induced or not to neural differentiation, showed voltage-dependent sodium currents. The maximum value of conductance of these groups was higher for the cells in all groupscompared to the control group, where the cells were not differentiated. Therefore, oriented nanofiber matrices induce neural differentiation of MSCs into functional neurons both in the absence and in the presence of incorporated EGF. The matrices AE also showed improved cell adhesion. Thus, these matrices are a possible strategy for optimizing the regeneration of neurologic lesions.

碩士
國防醫學院
生物及解剖學研究所
99
Axonal regeneration and glial remyelination are major problems of spinal cord injury (SCI) therapy in clinical. In references, glial scar produced by activated astrocyte after SCI is the primary target need to be solved. This study planned to use gelatin/collagen I (GC-I) membrane, a degradable biocomposite, as the glial scar replace strategy to provide an orderly environment for spinal cord injury repair. The aim of this study is to determine whether the neural plasticity can be manipulated by GC-I membrane in SCI rats. This experiment designed four groups included lesion SCI covered with GC-I membrane, lesion control, normal cord covered with GC-I membrane and normal control. Results of this study reveal the injured spinal cord covered with GC-I membrane group shown the increase of Glial fibrillary acidic protein (GFAP) (1 week) and Neurofilament (4 weeks) expression compared with lesion control group. According these evidences, GC-I membrane prevented the glial scar formation, and nerves seems had regrowth along the GC-I membrane at lesion site in SCI plus GC-I membrane group after 4 weeks. That provide the feasibility of the GC-I membrane using in SCI repair. Summarize of this study, the GC-I membrane is a suitable biological scaffold for axonal regeneration in spinalized rats.

Tissue engineering investigates new therapeutic approaches for spinal cord regeneration. Biodegradable scaffolds are employed aiming at creating an appropriate environment to support cell regrowth and transplantation. The transplantation of neural stem/progenitor cells (NSPCs) is a promising strategy under investigation. The main objective of this work was the synthesis of new soft materials for the production of nanostructured scaffolds able to support NSPCs transplantation and enable spinal cord regeneration. Polyurethanes (PUs) are segmented polymers, with tunable properties. PUs were synthesized using polycaprolactone-diol (PCL-diol) as soft segment, and isophorone diisocyanate and dimethylol propionic acid (DMPA) as hard segment. To introduce biological cues in the polymer backbone, chitosan (CS) and gelatin (Gel) were used to substitute DMPA as chain extender. The PUs were characterized regarding their chemical composition and thermal properties. Electrospun fibrous mats are convenient structures for cell support. In particular, aligned nanofibers provide a guidance cue to axon regrowth. Electrospinning was used to produce scaffolds of randomly oriented and aligned fibers from the different PU formulations. Scaffolds were characterized regarding their morphology, mechanical behavior, crystallinity, surface properties and hydrolytic degradation. Their impact on cells was evaluated in vitro using human fibroblasts. Cell adhesion and proliferation was highest for scaffolds produced from PUs containing CS or Gel as the only chain extender. Stem cell interaction with PU-CS and PU-Gel scaffolds was studied using human umbilical cord mesenchymal stem cells (MSCs) and human fetal spinal cord neural stem cells (NSCs). MSCs proliferated best on PU-Gel randomly oriented fibers whereas NSCs proliferated best on PU-CS with aligned fiber morphology. Neuronal differentiation of NSCs was confirmed using neuronal markers. Neurites aligned along the fibers direction. The physical, chemical and biological properties of PU-CS and PU-Gel fibrous mats make them promising substrates for NSPC in order to promote neural regeneration.

Nos últimos anos, a engenharia de tecidos (ET) surgiu como uma abordagem promissora para colmatar os processos insuficientes de regeneração do sistema nervoso (SN) e, consequentemente, atenuar algumas consequências físicas, psicológicas e sociais devastadoras para os pacientes em todo o mundo. O sucesso das estratégias de ET neural depende fortemente da combinação de biomateriais com técnicas avançadas de nanofabricação para o fabrico de scaffolds fibrosos capazes de apresentar uma topografia biomimética e uma elevada razão área superficial/volume capaz de melhorar a resposta celular. As excelentes propriedades físico-químicas dos nanomateriais à base de carbono, como o grafeno, encorajaram a sua exploração para a regeneração neural. Para além das propriedades elétricas, óticas e mecânicas inovadoras do grafeno, o óxido de grafeno (GO) apresenta uma superfície altamente funcionalizada por oxigénio, o que confere um caráter hidrofílico ao material e, consequentemente, leva a excelentes características biológicas. Assim, neste trabalho, fabricou-se uma ampla gama de scaffolds eletrofiados compósitos de policaprolactona-gelatina-GO para estudar a influência da concentração, tamanho e nível de redução do GO nas propriedades morfológicas, mecânicas, químicas e biocompatibilidade das fibras. Apesar de algumas variações, de um modo geral todos os compósitos apresentaram propriedades morfológicas, mecânicas e de molhabilidade compatíveis com células do tipo neuronal. O scaffold com nanofolhas de GO reduzidas apresentou os melhores resultados no ensaio celular com células SH-SY5Y. Os valores de viabilidade obtidos encorajam a realização de culturas celulares superiores a sete dias, ensaios com estimulação elétrica e/ou fatores de diferenciação
In recent years, tissue engineering (TE) has emerged as a promising approach to complete the deficient natural regeneration processes of the nervous systems and consequently attenuate some devastating physical, psychological and social consequences for patients worldwide. The success of neural TE strategies deeply rely on the combination of biomaterials with advanced nanofabrication techniques towards the fabrication of fibrous scaffolds able to present a biomimetic topography and a high surface area/volume ratio capable of enhancing the cell response. The excellent physicochemical properties of carbon-related nanomaterials, such as graphene, have encouraged their exploitation for neural regeneration. Complementary to the ground-breaking electrical, optical and mechanical properties of graphene, graphene oxide (GO) presents a highly oxygen functionalized surface, which impart a hydrophilic character to the material and consequently leads to excellent biologically features. Thus, in this work, we fabricated a wide range of composite electrospun polycaprolactone-gelatin-GO scaffolds to study the influence of concentration, size and reduction level of GO on the morphological, mechanical, chemical properties and biocompatibility of the fibres. In general, all composites showed morphological, mechanical and wettability properties compatible with neuronal-like cell cultures. The scaffold with reduced GO nanosheets showed the best results in the cell culture with SH-SY5Y cells. Viability results encourage cell cultures greater than seven days, electrical stimulation tests and/or addition of differentiation factors
Mestrado em Materiais e Dispositivos Biomédicos

This thesis describes a highly interdisciplinary approach to discern the differing impact of scaffold dimensionality and physical space restrictions on the behavior of single cells. Rolled-up nanotechnology is employed to fabricate three-dimensional (3D) SiO/SiO2 microtube geometries of varied diameter, that after a biofunctionalization step are shown to support the growth of U2OS and six different types of stem cells. Cell confinement quantifiable through the given microtube diameter is tolerated by U2OS cells through a remarkable elongation of the cell body and nucleus down to a certain threshold, while the integrity of the DNA is maintained. This confinement for NSPCs also leads to the approaching of the in vivo morphology, underlining the space-restrictive property of live tissue. The dimensionality of the cell culture scaffold however is identified as the major determiner of NSPC migration characteristics and leads to a morphologically distinct mesenchymal to amoeboid migration mode transition. The 3D microtube migration is characterized by exclusively filopodia protrusion formation, a higher dependence on actin polymerization and adopts aspects of in vivo-reported saltatory movement. The reported findings contribute to the determination of biomaterial scaffold design principles and advance our current understanding of how physical properties of the extracellular environment affect cell migration characteristics.

The mature central nervous system has a very limited capacity for self-renewal and repair following injury. Neural stem cells (NSCs), however, provide a promising new therapeutic option and can be readily expanded in vitro . Towards the development of an effective therapy, greater understanding and control is needed over the mechanisms regulating the differentiation of these cells into function-restoring neurons. In vivo, the neural stem cell niche plays a critical role in directing stem cell self-renewal and differentiation. By understanding and harnessing the power of this niche, a tissue engineered system with encapsulated neural stem cells could be designed to encourage neuronal differentiation and ultimately regeneration of damaged neural tissue. Poly(ethylene glycol)-based hydrogels were used here as a platform for isolating and investigating the response of neural stem cells to various matrix, soluble, and cellular components of the niche. When covalently modified with a cyclic RGD peptide, the synthetic scaffold was demonstrated to support attachment and proliferation of a human NSC line under conditions permissive to cell growth. Under differentiating conditions, the scaffold maintained appropriate lineage potential of the cells by permitting the development of both neuronal and glial populations. Expansion and differentiation of NSCs was also observed in a more biomimetic, three dimensional environment following encapsulation within a degradable hydrogel material. To simulate the soluble signals in the niche, fibroblast growth factor and nerve growth factor were tethered to the hydrogel and shown to direct NSC proliferation and neuronal differentiation respectively. Finally, as an example of the cell-cell interactions in the niche, the pro-angiogenic capacity of encapsulated neural stem cells was evaluated both in vitro and in vivo. Ideally, the optimal scaffold design will be applied to guide NSCs in a therapeutic application. Toward this goal, a novel method was developed for encapsulation of the cells within injectable hydrogel microspheres. This technique was optimized for high cell viability and microsphere yield and was demonstrated with successful microencapsulation and delivery of neural stem cells in rodent model of ischemic stroke.

碩士
中原大學
奈米科技碩士學位學程
105
PartI: Electrospun fibrous scaffolds have been widely applied in tissue engineering. The objective of this study was developing aligned and random silica nanofiber scaffolds with and without laminin to evaluate the potential of rat neural stem cells (rNSCs) for neural differentiation. Herein, we used various methods such as trypan blue exclusion test, MTS assay, real-time polymerase chain reaction, and immunocytochemistry to evaluate the effects of the scaffolds on cell adhesion, cellular viability, and neuron-specific gene expression of the cells. The results show that the rNSCs cultivated on all groups of scaffolds were able to adhere. More importantly, fluorescence microscopy images illustrated that the scaffold with aligned 2-laminin (A2/L) fibers greatly increased the average neurite length and directed neurite extension of differentiated rNSCs along the fiber. Gene expression analysis demonstrated that the highest expression of neural-related genes, tuj1 was observed in rNSCs cultured on A2/L scaffolds. Other results indicated that the modification of laminin could enhance the glial differentiation of the rNSCs and it was independent of the fiber alignment. Based on the experimental results, the aligned nanofibrous silca scaffold with laminin could be used as a are superior candidates in neural tissue engineering. PartII: Parkinson’s disease (PD) is a common neurodegenerative disorder, which is characterized by the selective and progressive death of dopaminergic (DA) neurons in the substantia nigra. Increasing evidence suggests that inhibition of microglia-mediated neuroinflammation may become a reliable protective strategy for PD. (-)-Epigallocatechin-3-gallate (EGCG) is a major polyphenol in green tea, has been known to possess antioxidant, anticancer, and anti-inflammatory properties. We Used liposome as a drug carrier, which can prolonged release of the EGCG. The aim of this study was to investigate the neuroprotective effect of liposome-VE-EGCG in a rat model of PD. Microglial activation and the injury of dopaminergic neurons were induced by LPS intranigral injection. Animal behavioral tests and biochemical assays were performed to evaluate the dopamine neuron degeneration and neuroprotective effects of liposome-VE-EGCG. Liposome-VE-EGCG significantly reduced amphetamine-induced rotational behavior in LPS-lesioned rats after 4 weeks. Furthermore, Liposome-VE-EGCG significantly decreased TNF-α levels, a marker of neuroinflammation in PD rats compared with saline group. These findings suggest that liposome-VE-EGCG exerts neuroprotection against LPS-induced dopaminergic neurodegeneration, and TNF-α.Thus EGCG represents a potent and useful neuroprotective agent for inflammation-mediated neurological disorders.

The debilitating symptoms that arise from damage to the central nervous system (CNS) have received significant attention due to the limited capacity for self-repair of both the brain and spinal cord. Cell replacement therapy (CRT) has emerged as a potential treatment alternative for CNS damage where by stem cells are implanted at the site of injury in order to replace damaged neural tissue. Clinical trials have demonstrated proof-of-concept that CRT could provide an effective alternative treatment for CNS damage. Significant issues, however, including the survival of transplanted cells and reinnervation of host tissue, must still be overcome for successful translation to the clinic. Biomaterials have been identified as potential tools that could help improve cell survival by providing a supportive environment in an otherwise hostile milieu. Furthermore, engineered biomaterials can direct cell behaviour including adhesion, migration and differentiation. Through the rational design of materials that appropriately mimic the native extracellular environment, we seek to present structural and biochemical cues to cells in an effort to enhance current CRT strategies and enhance neural tissue repair. In this thesis, the development of a novel, nanofibrous biomaterial is described. Bioactive peptide sequences were incorporated in minimalist fluorenylmethyloxycarbonyl-based self-assembling peptides (Fmoc-SAPs) to form nanofibrous hydrogel networks via non-covalent interactions known as pi-beta self-assembly. The research reported here demonstrates a materials development approach to the tissue specific biofunctionalisation of these Fmoc-SAPs inspired by extracellular matrix (ECM) proteins. Following materials development and characterisation, we assessed the biocompatibility of these Fmoc-SAP hydrogels in vivo. Initial evaluation was carried out in the intact brain of mice, showing no negative effect on the survival and fibre innervation of injected SAPs carrying primary neural progenitor cells as well as a limited inflammatory response after 28 days. This work was expanded on by investigation of the support afforded by Fmoc-SAPs to ventral mesencephalon (VM) donor cells in a Parkinson's disease model. Finally, we enhanced the biomimetic capacity of Fmoc-SAPs through investigation of these injectable hydrogels as neurotrophic growth factor delivery vehicles. As well as demonstrating the ability of Fmoc-SAPs to stabilise soluble proteins for long-term growth factor delivery, we developed a modified Fmoc-SAP with the capacity for localised delivery of genetic material via non-covalent immobilisation of viral vectors. The described evolution in this thesis of a biologically inert biomaterial to a sophisticated biomimetic highlights the research and development process required for optimisation of an adjuvant scaffold to improve CRT. Through this progression in bioactive Fmoc-SAP development, we provide a platform for further expansion in the design of enhanced biomaterials for tissue engineering, not only for CRT in the CNS, but for applications in regenerative medicine globally.

碩士
中原大學
奈米科技碩士學位學程
107
Sulfonated polyaniline was synthesized by oxidative polymerization and PC12 cell was cultured on the film of sulfonated polyaniline in this experiment, this study would observe the influences on the differentiation of PC12 cell by controlling the voltages and amount of sulfonate group in polyaniline chain Proceeding the structure identification by Fourier Transform Infrared Spectrometer and Elemental analysis, measuring the properties with Contact Angle, Cyclic Voltammetry and Electric Conductivity, this study found out sulfonated polyaniline had reversible doping and reversible reduction-oxidation which was known as a characteristic of conductive polymer. For further research about the effects of neuron regeneration, PC12 cell was cultured on material of sulfonated polyaniline, the adhesion rate and cell viability increased as sulfonation rate had increased under the experiments of adhesion and cell viability. After calculating neurite length by microscope and immunofluorescence, the best differentiation effects came from the sulfonated polyaniline cotted with laminin.

In recent years, significant success has been made in the field of regenerative medicine. Tissue engineering scaffolds have been developed to repair and replace different types of tissues. The overall goal of the current work was to develop scaffolds of native extracellular matrix components for soft tissue regeneration, more specifically, neural tissue engineering. To date, much research has been focused on developing a nerve guidance scaffold for its ability to fill and heal the gap between the damaged nerve ends. Such scaffolds are marked by several intrinsic properties including: (1) a biodegradable scaffold or conduit, consisting of native ECM components, with controlled internal microarchitecture; (2) support cells (such as Schwann cells) embedded in a soft support matrix; and (3) sustained release of bioactive factors. In the current dissertation, we have developed such scaffolds of native biomaterials including hyaluronic acid (HA) and collagen. HA is a nonsulphated, unbranched, high-molecular weight glycosaminoglycan which is ubiquitously secreted by cells in vivo and is a major component of extracellular matrix (ECM). High concentrations of HA are found in cartilage tissue, skin, vitreous humor, synovial fluid of joints and umbilical cord. HA is nonimmunogenic, enzymatically degradable, non-cell adhesive which makes HA an attractive material for biomedical research. Here we developed new photopolymerizable HA based materials for soft tissue repair application. First, we developed interpenetrating polymer networks (IPN) of HA and collagen with controlled structural and mechanical properties. The IPN hydrogels were enzymatically degradable, porous, viscoelastic and cytocompatible. These properties were dependent on the presence of crosslinked networks of collagen and GMHA and can be controlled by fine tuning the polymer ratio. We further developed these hydrogel constructs as three dimensional cellular constructs by encapsulating Schwann cells in IPN hydrogels. The hydrogel constructs supported cell viability, spreading, proliferation, and growth factor release from the encapsulated cells. Finally, we fabricated scaffolds of photopolymerizable HA with controlled microarchitecture and developed designer scaffolds for neural repair using layer-by-layer fabrication technique. Lastly, we developed HA hydrogels with unique anisotropic swelling behavior. We developed a dual-crosslinking technique in which a super-swelling chemically crosslinked hydrogel is patterned with low-swelling photocrosslinked regions. When this dual-crosslinked hydrogel is swelled it contorts into a new shape because of differential swelling among photopatterned regions.
text

yes
Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50 Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50 Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.

Electrospun biomaterial scaffolds can be engineered to support the neural differentiation of induced pluripotent stem cells. As electrospinning produces scaffolds consisting of nano or microfibers, these topographical features can be used as cues to direct stem cell differentiation. These nano and microscale scaffolds can also be used to deliver chemical cues, such as small molecules and growth factors, to direct the differentiation of induced pluripotent stem cells into neural phenotypes. Induced pluripotent stem cells can become any cell type found in the body, making them a powerful tool for engineering tissues. Therefore, a combination of an engineered biomaterial scaffold with induced pluripotent stem cells is a promising approach for neural tissue engineering applications. As detailed in this thesis, electrospun scaffolds support the neuronal differentiation of induced pluripotent stem cells through delivering the appropriate chemical cues and also presenting physical cues, specifically topography to enhance neuronal regeneration. This thesis seeks to evaluate the following topics: multifunctional electrospun scaffolds for promoting neuronal differentiation of induced pluripotent stem cells, neuronal differentiation of human induced pluripotent stem cells seeded on electrospun scaffolds with varied topographies, and controlled release of glial cell-derived neurotrophic factor from random and aligned electrospun nanofibers.
Graduate
nkhadem@uvic.ca

Despite tremendous progress in medicine, injuries of the adult central neural system remain without satisfactory solution. Regenerative medicine employs tissue engineering, cellular therapies, medical devices, gene therapy, or growth factors with the aim to bridge the lesion, re-establish lost connections and enhance endogenous repair in order to restore neural function. The aim of my thesis was to evaluate therapeutic potential of two approaches, transplantation of human mesenchymal stromal cells (hMSCs) and biological scaffolds derived from extracellular matrix (ECM) for neural regeneration, particularly in models of spinal cord injury (SCI). First, hMSCs from various sources - bone marrow (BM), adipose tissue (AT) and Wharton's jelly (WJ) - were isolated and characterized in vitro. All cell types met the minimal criteria for MSC phenotype and displayed similar properties in terms of their surface marker expression, differentiation potential, migratory capacity, and secretion of cytokines and growth factors. On the other hand, the cell yield from WJ and AT was significantly higher, and MSCs isolated from these tissues proliferated better than from BM. Therapeutic effect of intrathecal application of hWJ-MSCs was then evaluated in SCI compression model in rats. The effect of low (0.5 million) and...

碩士
國立交通大學
應用化學系碩博士班
104
The communication in the neuron network is still a mystery. We expect to fabricate specific scaffold to induce PC12 growth so that we can easily survey the characteristics of neuron network. Herein, we fabricate 2D scaffold by femtosecond laser system and 3D scaffold by aligned electrospinning method. In 2D scaffold fabrication, we used glass substrate coated by cytophobic MPC polymer and utilized femto second laser system to ablate the MPC polymer in specific part. Then, poly-L-Lysine coating change the cytophilicity of glass surface. In 3D scaffold fabrication, we chose a kind of biocompatible material PLA as our model. Combining electrospinning with aluminium hollow collector and applied electron field, we can get the aligned PLA on the aluminium hollw. Then, fluorescence stain was used to observe the PC12 growth. The result showed that PC12 can well-attached on the 2D scaffold even controlled the growth direction by the 2D scaffold. Because of the laser focus limitation, we cannot improve their quality. In contrast, 3D scaffold can restrict PC12 to grow in one direction and connect to each other so that we can do the further experiment by this method. Finally, we used FITC-dopamine to observe the communication between PC12. Due to the floating scaffold, it is difficult to observe the FITC-dopamine release from cells in right focus. We need to immobilize the scaffold to assist the observation by confocal microscopy.

博士
義守大學
電機工程學系
102
The repairing process in the nervous system is complicated and brings great challenges to researchers. Tissue engineering scaffolds provide an alternative approach for neural regeneration. The collagen based scaffolds which mimic the topography of natural extracellular matrix (ECM) can be potential scaffold candidates for neural tissue engineering. In this project, we will establish a 50-m porous and bio-degradable collagen scaffold that mimic nature extracellular matrix for 3-D neuron cancer stem cell culture. The aim of this study is to investigate cell multiply and differentiation of neuroblastoma cancer stem cells (NCSCs) on 3-D collagen scaffold growth. Here we will demonstrate a new approach for instant monitor the transitions of morphologically changes and multiply on 3-D scaffold growth by introducing green fluorescence protein (GFP) transgene into NCSCs with lentiviral infection. Under a scanning electron microscopy analysis, the 50-m porous size of collagen scaffold might allow the GFP-NCSCs to adhere with high multiply and neurite out growth formation. To validate the high multiply of GFP-NCSCs growth in 3-D collagen scaffold, the living cell imaging will be taken by confocal laser microscopy and a significant cell mass growth from single cell was observed after 5 days culture. The significant neurite outgrowth of GFP- NCSCs was observed by immunohistochemistry staining with a neuron specific NeuN antibody. These results confirmed that the native conformation collagen scaffold could enhance the attachment, viability, and cell differentiation of the cultured cancer neural stem cells that could provide the application of nerve tissue engineering and nerve regeneration.

Human induced pluripotent stem cells (hiPSCs) have two main properties: pluripotency and self-renewal. Physical cues presented by biomaterial scaffolds can stimulate differentiation of hiPSCs to neurons. In this work, we develop and analyze a mathematical model of aggregate growth and neural differentiation on melt electrospun biomaterial scaffolds. An ordinary differential equation model of population size of each cell state (stem, progenitor, differentiated) was developed based on experimental results and previous literature. Analysis and numerical simulations of the model successfully capture many of the dynamics observed experimentally. Analysis of the model gives optimal parameter sets, that correspond to experimental procedures, to maximize particular populations. The model indicates that a physiologic oxygen level (~5%) increases population sizes compared to atmospheric oxygen levels (~21%). Model analysis also indicates that the optimal scaffold porosity for maximizing aggregate size is approximately 63%. This model allows for the use of mathematical analysis and numerical simulations to determine the key factors controlling cell behavior when seeded on melt electrospun scaffolds.
Graduate