title: 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.
authors.affiliation: INEB - Instituto de Engenharia Biomédica, Rua do Campo Alegre, 823 4150-180 Porto, Portugal. lamghari@ineb.up.pt.
authors.forename: Estrela
authors.initials: E
authors.lastname: Neto
authors.forename: Cecília J
authors.initials: CJ
authors.lastname: Alves
authors.forename: Daniela M
authors.initials: DM
authors.lastname: Sousa
authors.forename: Inês S
authors.initials: IS
authors.lastname: Alencastre
authors.forename: Ana H
authors.initials: AH
authors.lastname: Lourenço
authors.forename: Luís
authors.initials: L
authors.lastname: Leitão
authors.forename: Hyun R
authors.initials: HR
authors.lastname: Ryu
authors.forename: Noo L
authors.initials: NL
authors.lastname: Jeon
authors.forename: Rui
authors.initials: R
authors.lastname: Fernandes
authors.forename: Paulo
authors.initials: P
authors.lastname: Aguiar
authors.forename: Ramiro D
authors.initials: RD
authors.lastname: Almeida
authors.forename: Meriem
authors.initials: M
authors.lastname: Lamghari
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000294:innervation*; D002460:Cell Line; D018920:Coculture Techniques; D005727:Ganglia, Spinal / Q000166:cytology / Q000378:metabolism*; D007150:Immunohistochemistry; D044085:Microfluidics / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D016501:Neurites / Q000378:metabolism* / Q000648:ultrastructure; D010006:Osteoblasts / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011984:Sensory Receptor Cells / Q000378:metabolism*