Abstract
Summary Statement
The aim of this study was to compare patterns (aligned, random and grid) of electrospun polydioxanone scaffolds for tendon repair. The aligned design was optimal, directing cell shape, orientation and protein expression. Moreover, it naturally crimped, presenting tendon-like morphology.
Introduction
Nanofibrous electrospun materials have been previously proposed as potential scaffolds for tendon repair, with emphasis on biomimetic design, postulated to encourage tissue regeneration. In this study, we characterised the interaction of primary tendon-derived cells with polydioxanone (PDO) scaffolds. PDO is a polymer with an excellent in vitro and in vivo biocompatibility, and is specifically compatible with tendon-derived cells. Here, we designed electrospun PDO scaffolds with different fibre orientations, namely aligned, random and grid-like patterns. To evaluate their potential as patches for tendon repair, we grew primary tendon derived cells on these scaffolds, and tested different aspects of cell behavior, including cell shape, proliferation and protein expression.
Methods
Scaffolds with different orientations were produced using a single nozzle electrospinning set-up. Human tendon cells were extracted from rotator cuff tissue resected during surgical repair, with appropriate ethical approval. Cells were grown on different scaffolds for at least 14 days. Multiphoton microscopy (MPM) was used to image cells (green, calcein AM, and red, actin-phalloidin). Cell growth was monitored using AlamarBlue assay. Cell length, width and orientation were manually measured from images acquired by fluorescence microscopy. RNA was extracted by Trizol homogenisation in a GentleMACS (Miltenyi Biotec). RNA samples were reversibly transcribed to cDNA and RT QPCR were performed using a ViiA7 (Life Technologies) with QuantiTect primer assays (QIAGEN). Results are in relative expression to GAPDH.
Results
MPM enabled the visualization of the scaffolds and viable cells grown for at least 14 days. Images demonstrate the distinct appearance of cells grown on highly aligned scaffold compared to random, grid-orientation and glass control. They also show the crimp-like appearance of the oriented scaffold as well as the cells on these crimped fibres. Interestingly, proliferation was not significantly effected by scaffold pattern. However, cell shape was clearly affected, and cells grown on oriented scaffolds showed higher anisotropy and elongated, narrower cell shape compared to all other groups, presenting a more tendon-like phenotype. This was further supported by a strong increase in the expression of β-actin on the aligned compared to the randomly oriented scaffold, correlating with observed changes in cell length and shape.
Conclusion
The aim of this study was to compare patterns (aligned, random and grid) of electrospun polydioxanone scaffolds as templates for tendon repair. Aligned PDO scaffolds significantly affected cell length, width, and orientation. We also found that β-actin expression was significantly increased on aligned polydioxanone scaffolds. Moreover, PDO scaffolds fabricated as aligned/oriented were observed to present crimp-like morphology, similar to native tendon. Taken together, these findings suggest an aligned morphology of polydioxanone may hold the potential to improve tendon healing.
