Design and Characterization of Tendon-Mimetic Scaffolds Capable of Withstanding Physiological Cyclic Loading

Introduction:: INTRODUCTION: Polycaprolactone (PCL) is used in soft tissue engineering (TE) due to its ability to transfer energy and mimic viscoelastic tendon matrices ¹. The mechanical properties of electrospun PCL have been widely investigated; however, the morphological and mechanical changes of the fibrous matrix under cyclic straining have yet to be evaluated. This information is critical in designing a scaffold that can withstand physiological cyclic loads with minimal deformation. Additionally, the relationship between tendon fibroblasts and PCL matrices during mechanical stimulation has not been fully elucidated. The objective of the study was to investigate the effect of cyclically straining PCL scaffolds of varying properties and evaluate the impact of culturing cell-seeded constructs within a bioreactor. We hypothesized that higher density, aligned scaffolds would retain viscoelastic properties better than low density, unaligned scaffolds and that culturing within the bioreactor would not affect cell viability.

Materials and Methods:: MATERIALS & METHODS: 33%, 50%, and 66% w/v PCL with dimethylformamide and dichloromethane solutions were electrospun at approximately 100 and 500 RPM onto a spinning mandrel to create unaligned and aligned scaffolds. The scaffolds were strained to 5% at a rate of 1 Hz for 4 hours in a bioreactor to replicate the percent strain at which tendon microtears occur ². Scanning electron microscopy (SEM) images were taken of strained and unstrained scaffolds to assess fiber diameter and alignment. The scaffolds were then mechanically tested at a tensile straining rate of 0.3% per second until failure on an Instron to determine the ultimate tensile strengths (UTS) and elastic moduli (E). Lastly, cellular viability studies were conducted using NIH3T3 fibroblasts seeded at 120,000 cells/cm² maintained with normal culture media ³. Comparisons were made between cell-seeded scaffolds in the bioreactor and the static cell plate to evaluate the impact of culturing cells within the bioreactor. Metabolic activity of the cells was assessed over time as an indirect measure of cellular viability. Two-way ANOVA and students’ t-tests statistical analyses were conducted in R Studio with significance set at p < 0.05.


Results, Conclusions, and Discussions:: RESULTS AND DISCUSSION: SEM image analysis confirmed a positive correlation between density and fiber size (Figures 1 & 4). Qualitatively, the scaffold spun at the higher RPM had a more aligned organized matrix than the unaligned fibrous matrix of the 100 RPM scaffolds (Figure 1). Additionally, aligned 33% and 50% w/v scaffolds displayed a significantly higher ultimate tensile stress and elastic modulus than their unaligned counterparts (Figures 2 & 3). It was also shown that straining the samples generally lead to a decrease in UTS and increase in E. Further, aligned samples were shown to have significantly thinner fibers at higher fabrication densities than their unaligned counterparts (Figure 4). Lastly, culturing fibroblasts in the bioreactor did not have a detrimental effect on the cellular metabolic activity compared to the cell plate, affirming the use of the bioreactor for future cell-seeded scaffold studies (Figure 5).

CONCLUSION: The data suggests there is an optimal density and alignment that allows for viscoelastic properties of tendons to be replicated. Higher density scaffolds appear to retain plastic characteristics, as demonstrated by failure in loading, and would not be sufficient for tendinous applications. Aligned 33% w/v scaffolds performed the best in terms of retaining mechanical properties and need to be further investigated. This finding is supported by literature that demonstrates PCL fibers electrospun at lower concentrations with smaller diameters had a higher degree of crystallinity, molecular orientation, and entanglement, which all contribute to the strength and elasticity of the scaffold ^4,5. It was expected for straining to elongate fibers; however, this was not demonstrated in our study. Future studies will evaluate the impact of increasing the statistical power of the study on fiber analysis. Further, static culturing within the bioreactor did not have a significant effect on cellular viability and serves as a preliminary evaluation to investigate the influence of cyclic straining and matrix deformation on tendon cell morphology and behavior. Studies such as this will help elucidate the relationship between tendon fibroblasts and PCL nanofibers cyclic straining, which is imperative for designing scaffolds that display minimal deformation when subjected to physiologically relevant loading in vivo.

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