Extracellular matrix protein-modified hydrogels enhance adipose-stem cell therapies for peripheral nerve regeneration

Abstract

Current clinical treatments of severe peripheral nerve injuries fail to support complete regeneration and function. Tissue engineering strategies offer alternative approaches, but their translation is not yet a reality. Schwann cell transplantation has improved tissue regeneration and functional outcomes in experimental models. However, translation into novel cell therapies is hindered due to complex and costly harvest and maintenance procedures. Adipose stem cells (ASCs), on the contrary, are relatively easy to harvest in abundance and have a high proliferation rate without the need for specialised media. ASCs exert their regenerative ability mostly through paracrine effects, secreting neurotrophic and angiogenic growth factors to improve nerve regeneration. However, important drawbacks such as cell death under stress conditions, poor retention, and unstable populations after transdifferentiation into the glial phenotypes should be taken into consideration. Polymers synthesised from 2-hydroxyethyl methacrylate (HEMA) and glycerol methacrylate (GMA) have been previously used in the biomedical field. However, they lack biorecognisable cues and have antifouling properties, which limit their use for cell culture applications. Regardless, these materials are easy to manufacture, and their physical and biochemical properties can be easily tuned. This thesis demonstrates that ASCs with increased neurotrophic potential can be easily obtained using laminin stimulation, without the need for expensive transdifferentiation protocols. Gene expression analysis corroborated the upregulation of genes associated with key neurotrophic factors; and the presence of secreted NGF, GDNF, and BDNF suggests that paracrine signalling might be a potential mechanism for these cells to aid in nerve regeneration. Additionally, the redox-initiated free radical polymerisation of HEMA and GMA rendered hydrophilic poly(HEMA-co-GMA) hydrogels in a cost-effective and reproducible manner. These hydrogels were homogenous, soft, mechanically compliant with an approximate storage modulus of 16 kPa, highly swellable, and transparent. Additionally, due to their exceptional tunability, poly(HEMA-co-GMA) hydrogels can be modified using ECM-derived proteins to improve cell adhesion, copolymerised with enzymatically degradable crosslinkers to improve biodegradation, or modified with photolabile molecules to increase their functionalities. Finally, this work demonstrates that protein-modified poly(HEMA-co-GMA) hydrogels can be used as alternative 3D-like cell culture platforms. In combination with ASCs, protein-functionalised poly(HEMA-co-GMA) hydrogels were able to support cell survival and proliferation for up to 7 days in vitro. Furthermore, these hydrogels have been shown to protect ASCs from detachment and can be used to study nerve regeneration events in a more physiologically-relevant manner in vitro.

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Adam Reid (Supervisor) & Lee Fielding (Supervisor)