Local FK506 Dose-Dependent Study Using a Novel Three-Dimensional Organotypic Assay
Kasra Tajdaran, MASc, PhD1,2; Katelyn Chan, B.Eng BioSci1,2; Jennifer J Zhang, MD, PhD3; Tessa Gordon, PhD, DSc3; Gregory H. Borschel, MD, FAAP, FACS1,2
1Division of Plastic and Reconstructive Surgery, The Hospital for Sick Children, Toronto, ON, Canada, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada, 3The Hospital for Sick Children, Toronto, ON, Canada
Purpose: Local delivery of FK506 directly to injured nerves enhances nerve regeneration and avoids the toxicity of systemic administration of FK506. However, the optimal dose of local FK506 for enhancement of axon regeneration has not yet been established. Therefore, we sought to determine the optimal local FK506 dose in a novel in vitro model of nerve regeneration.
Methods: We devised a novel three-dimensional (3D) organotypic assay modeling the peripheral nerve. This assay consisted of a neonatal rat dorsal root ganglion (DRG) extending its neurites into a native peripheral nerve scaffold provided by an acellular nerve allograft (ANA). In the first series of experiments, the entire DRG-ANA constructs were cultured in media containing FK506 at concentrations ranging from 1 pg/mL to 200 ng/mL. In a second experiment, a novel 3D compartmented cell culture system was adapted from the 3D organotypic system to achieve local delivery of FK506 exclusively to the growing neurites in vitro. In this system, the culture media surrounding the growing neurites was isolated from the media of the DRG using a silicone sheet. FK506 with the mentioned concentrations was added to the media surrounding the growing neurites. The DRG-ANA constructs in the negative control groups of both experiments were not cultured with FK506. Following 72 hours of incubation at 37oC, the length and density of the extended neurites in both experiments were measured in longitudinal histological sections of the DRG-ANA constructs. Doses leading to the maximum neurite extension was identified.
Results: A bimodal dose response was observed by culturing the entire DRG-ANA construct with media containing different concentrations of FK506. A low drug concentration of 1 pg/mL and a high drug concentration of 100 ng/mL promoted the longest neurite extension in vitro. Furthermore, regardless of the FK506 concentration, local delivery of the drug to only the growing neurites resulted in significant increase in both neurite extension and neurite density, an effect that was not observed with the FK506 delivery to both the neurites and the neural cell bodies within DRG.
Conclusion: The findings in this study provide valuable insight into the optimal local concentration(s) of FK506 for peripheral nerve regeneration. Furthermore, for the first time, this study indicates a potential interaction of FK506 with axons at the level of the growth cone.
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