Understanding the Effect of Microenvironments on Extracellular Vesicle Profile using a Nanofibrous In Vitro Tendon Model

Introduction:: Introduction: Tendon pathology, macroscopically, is characterized by a lack of collagen fiber alignment, reduced fibril diameter, diminished mechanical integrity, and the presence of inflammatory cytokines¹. Studies suggest that mesenchymal stem cell derived extracellular vesicles (EVs) play a role in the cell-to-cell communication throughout the tissue healing process by exchanging information between the cells in the injured tissue microenvironment ^2,3. EVs are membrane bound vesicles secreted by cells that function in delivering proteins, metabolites and nucleic acids to recipient cells⁴. Understanding the innate role of EVs in tendon pathology, immunomodulation, and healing has yet to be fully understood, which could be key information in elucidating the unknown mechanisms that contribute to insufficient tendon healing. To understand the different roles and characteristics of EVs in various tendon healing microenvironments, we fabricated highly aligned and unaligned polycaprolactone (PCL) electrospun scaffolds to mimic healthy and pathologic tendon tissues, respectively. We aim to characterize EVs within our in vitro tendon models by observing differences in size distribution, concentration, and total protein content within two different microenvironments, which we hypothesize EV secretion profiles will depend on the tissue microenvironment.


Materials and Methods:: Methods: 50% w/v PCL scaffolds were electrospun onto a spinning mandrel at approximately 100 RPM and 800 RPM to create nanofibrous structures that replicate the disorganized pathologic and aligned healthy tendon matrices, respectively. Scanning electron microscopy (SEM) and ImageJ were performed to analyze fiber alignment, diameter, orientation, overall nanofibrous matrix morphology. Scaffolds were seeded with NIH3T3 fibroblasts at a density of 1x10⁶ cells/scaffold. Culture media was collected at days 1, 3, 5 and 7 for EV isolation. EVs were isolated from culture media using a qEV 70 nm columns (IZON Science). Nanoparticle tracking analysis (NTA) was performed to assess EV size distribution and EV concentration. Total protein content was determined through BCA (Bicinchoninic Acid) protein assay. Tensile mechanical testing was performed on un-seeded scaffolds utilizing a ramp-to-failure protocol of 18% strain/min. A two-sample t-test was performed on mechanical data to determine a statistically significant difference in the elastic moduli and ultimate tensile strength (UTS) between the aligned and unaligned scaffolds. A two-sample t-test was performed to determine significant difference in fiber diameter as well. A two-way analysis of variance (ANOVA) was performed on BSA protein quantification to determine the effect of scaffold microarchitecture on EV purity over time.


Results, Conclusions, and Discussions:: Results and Discussion: SEM analysis demonstrated PCL constructs spun at 100 RPM displayed unaligned fibers relative to the constructs spun at 800 RPM, being phenotypically conducive to pathologic tendon tissue (Fig. 1A,1B). Fiber diameter was also assessed between the scaffolds, with the 800 RPM scaffold having significantly smaller fiber diameters (0.524 ± 0.320 µm) than the 100 RPM group (0.636 ± 0.588 µm). Larger diameter fibers are more phenotypically conducive to pathologic microenvironments in the latter stages of tendon healing⁵. The scaffolds spun at 800 RPM had a significantly greater elastic modulus (38.998 ± 18.104 MPa) than the scaffolds spun at 100 RPM (12.93 ± 2.484 MPa) (Fig. 2A). Similarly, the average UTS of the 800 RPM scaffolds (2.12 ± 0.897 MPa) was significantly higher than the 100 RPM scaffolds (0.50 ± 0.087 MPa) (Fig. 2B). NTA demonstrated a higher EV concentration for the 100 RPM and 800 RPM scaffolds relative to the TCP for all timepoints except day 7, with the 100 RPM group having a lower EV concentration. (Fig 2A). This observation is consistent with literature on scaling up EV-stimulation in three-dimensional culture systems, as well as biophysical stimulation resulting from electrospun nanofibers ^6,7,8. BCA analysis demonstrated no significant difference in protein content over time amongst the microenvironments (Fig. 3B). This could be due to the alignment demonstrated in figures 1C and 1D being similar. Although the nanofibers between the environments were significantly different, further biophysical augmentation is necessary to upscale EV secretion. Additionally, future work would entail supplementing culture media with pro-inflammatory cytokines such as tumor necrosis factor-α (TNF- α), which is a marker in tendinitis, to further emulate the inflammatory microenvironment present in pathologic tendon.

Conclusions: From this study, we have demonstrated an increase in EV secretion in response to the nanofibrous microenvironment. Further understanding of EV secretion in various microenvironments can not only potentially elucidate molecules crucial for understanding the role of EVs in the progression of tendon pathologies and throughout tendon healing, but also demonstrate the use of EVs as biomarkers for tendinopathy.


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