(516e) Influence of Polymeric Substrate Topography On the Orientation, Proliferation and Transdifferentiation of Mesenchymal Stem Cells for Neuroregeneration Strategies

Nerve regeneration is a complex biological process. Currently, autologous nerve grafts are considered the “gold standard” for treatment of severe peripheral nerve injuries (PNI). Autologous grafting involves transplanting part of a healthy nerve from the same individual to the site of injury. Consequently, ethical issues and probability of tissue rejection are minimized. However, donor site morbidity and requirement of multiple surgeries are some of the serious concerns in utilizing this technique. As such, research into alternative routes is essential for development of experimental strategies to facilitate nerve regeneration.

A promising alternative, which is the main objective of this work, is to enhance  neuronal regeneration following PNI by incorporating interdisciplinary approaches at the intersection of neuroscience, biomaterials, tissue engineering and stem cell therapies.  Our previous work has shown that micropatterned biodegradable polymeric conduits preseeded with Schwann cells and sutured to the ends of transected sciatic nerves promote nerve regeneration and functional recovery in rats. Biocompatible and biodegradable polymeric materials were used for engineering conduits to provide mechanical support and orientation to the regenerating axons, and to facilitate localized delivery of neurotrophic factors at the site of injury. Micropatterns on the inner lumen of the conduits provide guidance to the regenerating axons. However, there is no readily available and relatively non-invasive source of Schwann cells.

Therefore, our current focus is on the transdifferentiation of bone marrow derived mesenchymal stem cells (MSCs) into a Schwann cell-like phenotype, as a substitute for myelinating Schwann cells for promoting regeneration in a PNI model. Bone marrow-derived MSCs can be extracted easily using less invasive surgical procedures in comparison to autologous nerve grafts. Also, MSCs have been suggested as an attractive cell type for neural regeneration purposes owing to their high proliferation rate, self-renewing ability and differentiation capabilities into several lineages.

MSCs isolated from Brown Norway rat were characterized for multi-potency and rat MSC markers. These isolated MSCs were grown on micropatterned Polystyrene (PS) and Poly(lactic acid) (PLA) films.  A comparative study of morphological and molecular differences was carried out by growing the MSCs on half micropatterned-half smooth (non-patterned) polymeric films.More than 90% of cells were found to be oriented (0^o-20^o) in the direction of micropatterns on both PS and PLA patterned substrates. No specific orientation was observed for cells on the smooth side of the substrate. Elongation of cells on the patterned polymeric substrates was found to be approximately 3 times that of the cells grown on smooth substrates. MSCs were treated with bromodeoxyuridine (BrDU) for 12 hours to check their proliferation. Immunolabeling of these cells with an anti-BrDU antibody revealed that 30-40% of the cells proliferated on both PS and PLA substrates. Proliferation of cells on PLA substrates was observed to be higher than PS substrates. No significant effect due to micropatterned or smooth substrates was observed, although, the smooth PLA substrates resulted in maximum proliferation.

The impact of micropatterned polymeric films on transdifferentiation of MSCs towards SC-like phenotypes was also tested. A comparison of transdifferentiated MSCs (tMSCs) and undifferentiated MSCs (uMSCs) using molecular markers such as S100β and p75^NTR was carried out. Significantly higher numbers of tMSCs expressed S100β and p75^NTR as compared to uMSCs, and the percentage of cells expressing these markers were quantified on all four substrate types. No appreciable variation was found in the degree of transdifferentiation for any substrate type. However, PLA substrates had slightly higher number of transdifferentiated cells as compared to PS, and non-patterned PLA caused maximum transdifferentiation compared to other substrate types. Results revealed that micropatterns strongly influence orientation and morphology of MSCs but have negligible effect on growth and transdifferentiation.

Fabrication and transplantation of tMSCs seeded on micropatterned PLA conduits in a rat peripheral nerve injury model will be conducted for investigating the combined ability of micropatterns and tMSCs in promoting nerve regeneration.