Polyisocyanopeptide hydrogels as effective tissue engineering scaffolds

Kargaard, Annette (2014-12)

Thesis (MSc)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: The extracellular matrix (ECM) provides the perfect environment for cells, with regard to mechanical strength, delivery of nutrients, facilitation of cell to cell communication, and more. The most challenging aspects of tissue engineering, the artificial construction of living tissue and organs, is to find a scaffold that is able to create an environment that mimics that of the ECM. In the search of such a scaffold, polyisocyanopeptide hydrogels, functionalised with oligo(ethylene glycol) side chains, have found to be the closest synthetic mimic of the ECM. They mimic, in almost in every way, the microenvironment of the cells. The primary aim of the current study was to decorate these thermo-responsive hydrogels with CIKVAV and cyclo(RGDfC) epitopes, in order to establish whether they can act as scaffolds in the promotion of neurite outgrowth in neuronal progenitor cells. The polyisocyanopeptides were prepared by a Ni(II)-catalysed copolymerisation of a spacer monomer and an ‘azide monomer’ with pendant azide functionality, which was used as a reactive handle to click the CIKVAV and cyclo(RGDfC) epitopes. Three copolymers were synthesised in two different monomer feed ratios of the ‘azide monomer’ and ‘spacer monomer’. The polymers were then characterised using FT-IR, SEC, AF4, CD and UV-vis analysis. The mechanism of gelation of the polymer was investigated using super-resolution fluorescence microscopy, in an effort to visualise the gelation behaviour in the solution state. It was observed that the polymers accumulate into concentrated clusters of bundles during the transition from solution to gel state. The polymers were decorated with the epitopes using copper-free click chemistry. This was achieved by clicking alkyne functionalised epitopes to the pendant azide functional groups onto the polymer. The decoration was then verified using the Kaiser test. The functionalised polymers were found to be non-cytotoxic. Thereafter, the decorated polymers were seeded with neuronal GT1-7 progenitor cells, in order to test the process formation of the cells in the gel environment. However, the cell differentiation studies were not very conclusive, the materials do induce cell differentiation, but it is not very extensive. It is necessary to optimise the system, in the future.

AFRIKAANSE OPSOMMING: Die ekstrasellulêre matriks (ESM) verskaf die perfekte omgewing vir selle met betrekkeing tot hul meganiese krag, aflewering van voedingstowwe, fasiliteite vir sel tot sel kommunikasie en meer. Die mees uitdagende aspek van weefsel manipulasie, die kunsmatige konstruksie van lewende weefsel en organe, is om ‘n steier te vind wat die omgewing van die ESM kan naboots. In die soektog tot so ‘n steier is poliisosiaanpeptied hidrojel gefunksionaliseer met oligo(etileenglikol) sykettings identifiseer as die naaste sintetiese nabootsing van die ESM. Hulle boots na, in byna elke opsig, die mikro-omgewing van die selle. Die primêre doel van die huidige studie was om hierdie termo-responsiewe hidrojel met CIKVAV en siklo(RGDfC) epitope te versier ten einde vas te stel of hulle kan dien as steier in die promosie van die uitbreiding van neurone voorloper selle tot neuriete. Die poliisosiaanpeptied was voorberei deur ‘n Ni(II) gekataliseerde ko-polimerisasie van ‘n spasiêre monomer en ‘n asied monomeer met hanger asied funktionaliteit wat kon dien as reaktiewe handvatsels om die CIKVAV en siklo(RGDfC) epitope te kliek. Drie ko-polimere is gesintetiseer deur gebruik te maak van twee verskillende invoer verhoudings van die asied monomeer en spasiêre monomeer. Die polimere is gekarakteriseer deur gebruik te maak van FT-IR, SEC, AF4, CD en UV-vis analise. Die meganisme van jelvorming van die polimeer is ondersoek deur gebruik te maak van super-resolusie fluoressensie mikroskopie, in ‘n poging om die jelvorming gedrag in oplossing te visualiseer. Daar is opgelet dat die polimeer akkumileer in gekonsentreerde groepe bondels tydens die transformasie van oplossing to jel toestand. Die polimeer is versier met die epitoop deur gebruik te maak van koper-vrye kliek chemie. Dit is bereik deur alkyn gefunktionaliseerde epitope te kliek aan die hanger asied gefunktionaliseerde groepe op die polimeer. Die versiering is geverifieer deur die Kaiser toets. Daar is gevind dat die gefunksionaliseerde polimere nie-sitotoksies is nie. Neurone GT1-7 voorloper selle was gekweek in die versierde polimeer ten einde die formasie proses van die selle in die jel omgewing te toets. Die sel differensiasie studies was egter nie baie oortuigend nie. Die material veroorsaak sel differensiasie, maar dit is nie baie ekstensief nie. Dit is nodig om in die toekoms die sisteem te verbeter.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/95787
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