Characterization of Electrospun Substrates for Ligament Regeneration using Bone Marrow Stromal Cells

Challenges persist in the tissue engineering and regeneration of ligaments. Of the various aspects contributing to the success of ligament tissue engineering (such as the scaffold, cell source and stimulatory biochemical and mechanical cues), architecture and material involved in scaffold design remain as a significant area of study. Essentially, the scaffold for ligament regeneration, especially the cell-seed substrate, should be viable for cell attachment, promotes nutrient and waste transfer, be mechanically viable, and stimulates initial cell proliferation and ECM production. In this study, electrospun substrates using the silk fibroin (SF) and PLGA material, with different electrospun fiber arrangements (aligned (AL) and random (RD)) were compared for their ability to promote MSC attachment and subsequent differentiation down the ligament fibroblast cell lineage. The rationale for such characterizations lies in the hypothesis that SF, being a natural protein, provides significantly more favorable surface chemistry for cell attachment and differentiation than synthetic polymers such as PLGA. On the other hand, the aligned electrospun fiber arrangement will mimic the native ligament ECM environment, to guide MSCs down the fibroblast lineage. Electrospun PLGA and SF substrates were fabricated by first dissolving the respective material in HFIP (7-10% w/v). Following which, aligned PLGA and SF substrates were collected from a rotational electrospin setup, while the random types were collected from a grounded plate. These four groups of substrates (SF-AL, SF-RD, PLGA-AL, PLGA-RD) were then characterized in terms of cell adhesion, proliferation, viability and function via post-seed cell counting, AlamarBlue (TM) assay, Fluorescence Microscopy, Sircol (R) Collagen Assay and SEM. Results from this characterization show that cells remained viable when cultured on these substrates, with the AL types being mechanically stronger and promoted increased proliferation and collagen production as compared to the RD types. SF substrates promoted cell attachment and differentiation, as inferred from the increased collagen production.