Developing an electrospun membrane to promote regeneration of periodontal ligament

Periodontal disease is an inflammatory condition, which can lead to tooth loss. The Clinical treatment and management of periodontal disease is focussed on the regeneration of the periodontal tissues to achieve a good alveolar bone mass and to maximise re-attachment of the periodontal ligament. Guided tissue regeneration (GTR) using a biomaterial membrane is currently the method of choice in more severe tissue loss. The clinical regeneration treatments can give varied results in patients, however, the reasons for this are still unknown. The overall aim of this study was to use electrospinning and surface modification techniques to fabricate a biodegradable GTR membrane/scaffold composed of aligned fibres with enhanced regenerative and biomimetic (biologically responsive) properties. Model systems using random-fibre and aligned-fibre Poly-L-lactic acid (PLLA) and poly (Ɛ-caprolactone) (PCL) scaffolds were prepared via electrospinning. However, commercial aligned-fibre PCL scaffolds had to be ordered. The morphology and the size of the fibres was determined by scanning electron microscopy (SEM). The surfaces of the scaffold fibres were modified by the addition of amine groups by using cold plasma deposition of allylamine. 6mm diameter scaffolds were then incubated with heparin. The heparin-bound scaffolds were then incubated for 24 hours with growth factors (10 μg of TGF-β1, TGF-β3 or FGF2 in PBS/ 1 mg/ml BSA). Human periodontal ligament cells were seeded onto the functionalised and control membranes and incubated at 37oC. Cell viability was checked using PrestoBlue™ on days 7, 14 and 21 and investigated using Live-Dead staining using 5-chloromethylflourescein diacetate (CMFDA) and Propidium iodide (PI) dyes. On day 35, the cell/scaffold constructs were taken to measure the amount of DNA using PicoGreen, total protein using bicinchoninic acid (BCA) and total collagen by measuring the amount of hydroxyproline using a Total Collagen assay kit. Random-fibre scaffolds of PCL or PLLA were successfully fabricated by electrospinning and commercial aligned fibre PCL scaffolds were used. A smaller range of fibre diameters was observed by SEM in the PCL scaffolds compared to PLLA scaffolds. On day 7, 14 and 21 - the cells were fully viable as determined using PrestoBlue™. Moreover, live-dead staining showed mainly green (live) cells on both PLLA and PCL membranes with few dead cells (stained red). DNA determination indicated a lower cell number on the PLLA random membrane than PCL random membrane. Cold plasma functionalisation of the random and aligned fibre scaffolds with allylamine was carried out and XPS analysis of the amine-functionalised scaffolds showed the presence of nitrogen groups on the surface. All the growth factors tested (TGF β1, TGF β3 and FGF-2) bound to the heparin-functionalised membranes and eluted from the scaffolds for a minimum period of 14 days. On biofunctionalization with the growth factors, the aligned TGF-β3 bio-functionalised scaffolds constructs produced significantly more DNA, total protein and collagen (P≤0.01) than control and plasma treated cell/scaffolds constructs.