Sensory neurons and osteoblasts: close partners in a microfluidic platform

Abstract

Innervation has proven to be critical in bone homeostasis/regeneration due to the effect of soluble factors, produced by nerve fibers, associated with changes in the activity of bone cells. Thus, in this study, we have established and characterized a coculture system comprising sensory neurons and osteoblasts to mimic the in vivo scenario where nerve fibers can be found in a bone microenvironment. Embryonic or adult primary dorsal root ganglion (DRG) and MC3T3-E1 osteoblastic cells were cocultured in compartmentalized microfluidic platforms and morphological and functional tests were performed. The time of adhesion and readout of axonal outgrowth were improved by the alignment of DRG with the axis of microgrooves, which showed to be a crucial step for the designed experiments. Cocultures of entire DRG from adult origin with osteoblasts were performed, showing extended DRG projections towards the axonal compartment, reaching osteoblastic cells. Immunocytochemistry showed that the neurites present within the osteoblastic compartment were immunoreactive to synapsin and calcitonin gene-related peptide suggesting the presence of specialized structures involved in this crosstalk. This evidence was further confirmed by electron microscopy where varicosities were detected as well as electron dense structures in neurite membranes. Aiming to mimic the properties of tissue extracellular matrices, MC3T3-E1 cells were seeded in the axonal side upon laminin, collagen or within 3D functionalized alginate matrices and axonal outgrowth was clearly observed. In order to analyze and quantify data with reproducible image analysis, a semi-automated algorithm was also developed. The collagen and laminin substrates displayed a higher amount of axons reaching the axonal side. Overall, the established method revealed to be a suitable tool to study the interaction between the peripheral nervous system and bone cells in different contexts mimicking the in vivo scenario.

Mesenchymal stem cells secretome-induced axonal outgrowth is mediated by BDNF

“…Quantification of the axonal outgrowth was performed using a MATLAB (The MathWorks Inc., Natick, MA) script named AxoFluidic 58 . This computational tool calculates the axonal networks density as a function of the distance (x-axis) to the microgrooves termination in the microfluidic chamber.…”

Bidirectional communication between sensory neurons and osteoblasts in an in vitro coculture system

“…Preceding studies have demonstrated close membrane contact and synaptic structure in cocultures comprising DRG‐derived sensory neurons and osteoblasts, by electron microscopy analysis . Immunoreactivity to synapsin, a synaptic‐related protein, has also been observed in nerve fibers attached to osteoblasts . These results suggest that direct signal transduction from sensory neurons to osteoblasts occurs via exocytosis of glutamate or substance P. In most peptidergic sensory neurons, substance P is coexpressed with CGRP .…”

“…2A,B). Preceding studies have demonstrated close membrane contact and synaptic structure in cocultures comprising DRG-derived sensory neurons and osteoblasts, by electron microscopy analysis [32,40]. Immunoreactivity to synapsin, a synaptic-related protein, has also been observed in nerve fibers attached to osteoblasts [40].…”

Sensory neurons sprouting is dependent on osteoclast-derived extracellular vesicles involving the activation of epidermal growth factor receptors

“…Microfluidic chambers had an appropriate microchannel spacing for compartmentalization and probing of axonal activity. Microfluidic chambers were also adapted by adding an extra smaller reservoir (Ø 3 mm), which allowed the seeding of the DRG in a central position to the electrode matrix (Neto et al, 2014). μEF devices were composed of two separate compartments connected by 16 microchannels of 700 μm length × 9.6 μm height × 10 μm width.…”