Local FK506 dose‐dependent study using a novel three‐dimensional organotypic assay

Abstract

Local administration of FK506, an FDA approved immunosuppressant with neuroregenerative properties, is a promising technique to achieve improved peripheral nerve regeneration while preventing the side effects associated with the systemic administration of this drug. Although considerable research has been devoted to the development of clinically suitable systems for local delivery of FK506 to the site of nerve injury and repair, the optimal dose of FK506 for enhancement of axon regeneration in the peripheral nerve has not yet been established. To this end, we devised a threedimensional (3D) organotypic assay capable of mimicking the peripheral nerve. This assay consisted of a neonatal rat dorsal root ganglion (DRG) extending its neurites into the native peripheral nerve scaffold provided by an acellular nerve allograft (ANA). A novel 3D compartmented cell culture system was adapted from the 3D organotypic assay to achieve local delivery of FK506 just to the growing neurites in vitro and establish the required local dose of FK506 for peripheral nerve regeneration. A bimodal dose response was observed by culturing the entire DRG-ANA construct with media containing different concentrations of FK506. Low drug concentration of 1 pg/ml and high drug concentration of 100 ng/ml lead to the longest neurite extension in vitro. Furthermore, regardless of the FK506 concentration, concentrating the drug to 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 neurites and neural cell bodies within DRG. The findings in this study provide valuable insight into the optimal local dose of FK506 for peripheral nerve regeneration. Furthermore, for the first time, this study suggests the potential interaction of FK506 with axons at the level of the growth cone. K E Y W O R D S biomaterial, dose-dependent study, FK506, local drug delivery, organotypic assay, peripheral nerve regeneration, rat model, regenerative medicine 1 | INTRODUCTION After peripheral nerve injury, the expression of intrinsic growth factors and neurotrophic factors necessary for axon regeneration reduces with time and regeneration distances (Brushart et al.injured peripheral nerve with proteins and molecules that have neurotrophic and neuroregenerative properties such as brain-derived neurotrophic factor, glial-derived neurotrophic factor, and FK506 to compensate for reduced expression of the intrinsic factors, holds promise for enhancing Biotechnology and Bioengineering. 2019;116:405-414.wileyonlinelibrary.com/journal/bit

Advances in the repair of segmental nerve injuries and trends in reconstruction

“…FK‐506, an immunosuppressive drug, enhances nerve regeneration, as it increases the axonal growth rate in animal models . Now, approaches have been developed to provide FK506 locally to enhance axon regeneration given its abilities to stimulate more rapid axon outgrowth . This strategy is quite intriguing given that it reduces the potential for any systemic toxicity from its immunosuppression.…”

Local FK506 delivery at the direct nerve repair site improves nerve regeneration

“…11,39 Recently, Tajdaran et al studied the in vitro neurotrophic effects of FK506 when delivered globally to the cell body and axon growth cones and just locally to the axon growth cones. 34 They reported that global FK506 increased axon extension and density in a dose dependent manner, while local FK506 delivered to the axon growth cones similarly increased axon extension and density but not in a dose dependent manner. 40 has been shown to increase axon density by promoting collateral axon sprouting/branching, but a secondary inhibitory effect at higher concentrations that reduces axon elongation may explain the differences in gastrocnemius muscle mass and EMG amplitude.…”

“…34 They reported that global FK506 increased axon extension and density in a dose dependent manner, while local FK506 delivered to the axon growth cones similarly increased axon extension and density but not in a dose dependent manner. 40 has been shown to increase axon density by promoting collateral axon sprouting/branching, but a secondary inhibitory effect at higher concentrations that reduces axon elongation may explain the differences in gastrocnemius muscle mass and EMG amplitude. 25,40 In a previous in vitro study, we determined a dose-dependent effect that FK506 concentration had on neurite extension and reported a diminished effect at higher concentrations, with the highest used concentrations having no significant differences than no-drug controls.…”

“…Numerous in vivo studies have demonstrated that systemic FK506 delivery is neurotrophic, but less is known about localized delivery of FK506 to nerve injuries . In recent years, a few studies have been published investigating the neurotrophic effects of locally administered FK506 using in vitro and in vivo models . Tajdaran et al created a polymeric microparticle drug delivery system that shows promise with in vitro FK506 release duration lasting approximately 30 days; however, an in vivo study was not performed so its nerve regeneration capabilities are at this time are still unknown .…”

“…[8][9][10][11][12][24][25][26][28][29][30] In recent years, a few studies have been published investigating the neurotrophic effects of locally administered FK506 using in vitro and in vivo models. [31][32][33][34] Tajdaran et al created a polymeric microparticle drug delivery system that shows promise with in vitro FK506 release duration lasting approximately 30 days; however, an in vivo study was not performed so its nerve regeneration capabilities are at this time are still unknown. 31 Azizi et al…”