The assessment of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) for use as an intraluminal guidance scaffold for improving peripheral nerve regeneration using a developed 3D in vitro testing model

Abstract:

Introduction

Peripheral nerve injuries affect 300,000 people in Europe every year. Nerve guide conduits (NGCs) are currently used to treat injuries but have limited success in large gaps. Providing internal guidance cues, such as electrospun fibres, have been used to improve current designs. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a biodegradable and biocompatible polymer used in many different areas of tissue engineering. We previously developed a 3D in vitro model that allows the evaluation of different intraluminal fibre scaffolds inside a complete NGC. Therefore, the suitability of PHBV as an intraluminal guidance scaffold for NGCs was assessed using this model.

 

Materials and Methods

Electrospinning was used to fabricate 3 different PHBV fibre diameter sizes (2, 5 and 8µm). In vitro analysis was performed using 40,000 NG108-15 neuronal cells and 60,000 rat primary Schwann cells using live/dead analysis and immunolabelling cells against β-III tubulin and S100. PHBV fibres were then threaded into polyethylene glycol conduits fabricated from microstereolithography. Dorsal root ganglia were extracted and explanted onto fibres in PEG conduits. After 21 days in culture, fibres were removed from conduits, labelled for DAPI, S100 and β-III tubulin and imaged using confocal microscopy.

 

Results and Discussion

All fibre diameter sizes supported the adherence, proliferation and maturation of NG108-15 neuronal cells and primary Schwann cells. 8µm fibres supported the highest number of live cells (564.55 ± 215.69) and the longest neurites were recorded on the 8μm fibres (133.33 ±17.38μm). Primary Schwann cell migration and axon outgrowth length from DRGs, was significantly higher on 5µm fibres compared to 8µm fibres, 2µm fibres and a hollow conduit.

 

Conclusions

Overall, 8µm fibres supported the largest neurite outgrowth lengths from NG108-15 neuronal cells. However, 5µm fibres, supported the largest primary Schwann cell migration length and axon outgrowth length from DRGs, when using the 3D in vitro testing model. Future work would involve in vivo testing.

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