Advanced nanofabrication techniques and materials for mechanotransduction studies and tissue engineering applications

Although peripheral nerves display regenerative abilities compared to the central nervous system, regeneration after centimeters nerve loss is very limited. To date, peripheral nerve injuries represent a major cause for morbidity and disability in the affected patients. Severe nerve lesions might occur at any age and result from many different types of traumas. In particular, young males are often involved in peripheral nerve injuries after car accidents and traumatic limb amputations. The incidence of peripheral nerve injuries lies at about 300,000 cases per year in Europe: the socio-economic impact is therefore high, comparable to diseases such as diabetes (European Commission report, 2016). Moreover, peripheral nerve lesions result to be clinically relevant with an incidence of 2/100,000 persons per year, value recorded only for hand amputation traumas. Hence, there is a considerable need for innovative therapies in the area of repair and regeneration of peripheral nerve injuries. Nowadays, functional recovery after a nerve injury is achieved through the regeneration of the severed axons and the reinnervation of target tissues. In particular, nerve regeneration is fostered by a set of phenomena at the cellular level that recreate the connection from the proximal up to the distal stump. Nonetheless, a number of factors can interfere with functional recovery, hindering the complete healing of denervated target tissues. In this context, tissue engineering can substantially enhance the repair of neural tissues through the use of artificial scaffolds. Although standard micro- and nanofabrication techniques have been employed to produce biomimetic scaffolds during the last two decades, it is still difficult to recreate the physiological complexity in an in vitro system. The results reported in this thesis include novel nanofabrication techniques and materials for mechanotransduction studies and tissue engineering applications. In particular, the main topic of my Ph.D. project concerns the development and use of innovative biomaterials and nanoimprint lithography schemes for biomedical applications. The current work is organized as follows. After this introduction (Chapter 1), I will provide a comprehensive overview of the current literature on engineered polymers, identifying the key-features that make them leading candidates to regenerative medicine applications. In particular, I will focus on fabrication techniques, mechanotransduction processes, peripheral nerve regeneration and neural scaffolds technologies, and nanostructured scaffolds (Chapter 2). In Chapter 3 I will show the fabrication of novel microstructured phantoms to mimic vascularized tissues for photoacoustic system optimization. I will also investigate the possible use of novel light-responsive hydrogels for biomedical applications. Both of these studies will have the final aim of characterizing novel materials before their in vivo experimental tests. Chapter 4 will be dedicated to the development and use of high-resolution intermediate molds for nanoimprint lithography (NIL). In this section I will detail the optimization of perfluoropolyether (PFPE) as a soft mold for NIL, proving enhanced resolution and fidelity of the replica process. In Chapter 5 I will demonstrate the applicability of PFPE intermediate molds for the fabrication of transparent and biocompatible cyclic olefin copolymer (COC) and polyethylene terephthalate (PET) scaffolds, patterned with two original types of sub-100 nm topographies, named nanoripples and nanogratings. Preliminary results on topographical gradients of directionality will be reported in Chapter 6. I carried out this research activity mostly at ETH in Zurich, where I have been involved firsthand in the design of novel micropatterned structures as well as their fabrication, and in vitro cell migration assays. Finally, in Chapter 7 I will summarize my research achievements, highlighting the contribution of the present work to the complex issue of peripheral nerve regeneration, and their potential for a medium-term clinical translation.