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Title: An ex vivo model of peripheral nerve regeneration
Author: Behbehani, Mehri
ISNI:       0000 0004 7964 529X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2019
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Nerve guidance conduits (NGCs) fail to promote peripheral nerve regeneration at gap lengths >3 cm in humans. Internal NGC scaffolds through microfibres could provide additional outgrowth guidance to enhance regeneration outcome. No ideal NGC scaffold has yet been identified, and no study has systematically attempted to evaluate different scaffold variables (such as fibre diameter, fibre density, or surface modification) directly inside an NGC. Therefore, this thesis reports on a study to develop a 3D ex vivo model of peripheral nerve regeneration. The overall goal was a platform that permitted the reproducible and reliable comparison of NGC scaffolds made directly inside an NGC using a single setup, that also enables the reduction of more traditional in vivo nerve injury models. The response of NG108-15 neuronal cells and primary rat Schwann cells to polycaprolactone (PCL) fibres with 1, 5, 8, 10 and 13 µm diameter was explored, finding that fibre diameter and fibre surface topography influenced axon and Schwann cell growth. Fibres with 8 and 10 µm diameter supported optimal neurite alignment, incidence and length. In contrast, Schwann cell metabolic activity and attachment was limited on 1 µm fibres. Topographies on fibre surface were deep and elongated on fibres 5 – 13 µm and shallow, round-shaped and more frequent on 1 µm fibres. A method based on calculated fibre weight was developed to reproducibly fill NGCs with different fibre densities. From this, a 3D ex vivo model was developed placing dorsal root ganglia (DRGs) on scaffold-filled NGCs to mirror the proximal nerve stump after injury. Chick embryo DRGs and light-sheet microscopy imaging were of particular value and enhanced model reliability, accuracy and ethical acceptance. DRG outgrowth incidence and length was initially poor on PCL fibre scaffolds, but improved significantly (by more than five-fold) after air plasma modification. XPS confirmed that a 60 second air plasma treatment was sufficient to increase the surface polarity of fibre scaffolds inside conduits along their entire length. The model proved valuable for evaluating and comparing scaffolds with different fibre diameters, densities (10, 20 and 40 %), fibre materials, and for studying potential cell therapy approaches to aid peripheral nerve repair. For this, Schwann cells from chick embryo sciatic nerves were successfully isolated, purified and cultured inside NGCs using a selective DMEM D-valine method. Finally, across all variables investigated, it was concluded that conduits containing a 20 % density of air plasma-modified 10 µm diameter fibres supported optimal chick DRG outgrowth, regenerating more than 85 % of the conduits in 21 days.
Supervisor: Haycock, John W. ; Claeyssens, Frederik Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available