title: Co-culture of osteocytes and neurons on a unique patterned surface.
abstract: Neural and skeletal communication is essential for the maintenance of bone mass and transmission of pain, yet the mechanism(s) of signal transduction between these tissues is unknown. The authors established a novel system to co-culture murine long bone osteocyte-like cells (MLO-Y4) and primary murine dorsal root ganglia (DRG) neurons. Assessment of morphology and maturation marker expression on perlecan domain IV peptide (PlnDIV) and collagen type-1 (Col1) demonstrated that PlnDIV was an optimal matrix for MLO-Y4 culture. A novel matrix-specificity competition assay was developed to expose these cells to several extracellular matrix proteins such as PlnDIV, Col1, and laminin (Ln). The competition assay showed that approximately 70% of MLO-Y4 cells preferred either PlnDIV or Col1 to Ln. To co-culture MLO-Y4 and DRG, we developed patterned surfaces using micro-contact printing to create 40 μm × 1 cm alternating stripes of PlnDIV and Ln or PlnDIV and Col1. Co-culture on PlnDIV/Ln surfaces demonstrated that these matrix molecules provided unique cues for each cell type, with MLO-Y4 preferentially attaching to the PlnDIV lanes and DRG neurons to the Ln lanes. Approximately 80% of DRG were localized to Ln. Cellular processes from MLO-Y4 were closely associated with axonal extensions of DRG neurons. Approximately 57% of neuronal processes were in close proximity to nearby MLO-Y4 cells at the PlnDIV-Ln interface. The surfaces in this new assay provided a unique model system with which to study the communication between osteocyte-like cells and neurons in an in vitro environment.
authors.affiliation: University of Delaware, Department of Biological Science, Newark, 19716, USA.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Boggs
authors.forename: William R
authors.initials: WR
authors.lastname: Thompson
authors.forename: Mary C
authors.initials: MC
authors.lastname: Farach-Carson
authors.forename: Randall L
authors.initials: RL
authors.lastname: Duncan
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Beebe
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods*; D024042:Collagen Type I / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology; D005786:Gene Expression Regulation / Q000187:drug effects; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry; D007797:Laminin / Q000494:pharmacology; D051379:Mice; D018613:Microscopy, Confocal; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010011:Osteocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D056951:Photoelectron Spectroscopy; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013499:Surface Properties / Q000187:drug effects
chemical_list: D015415:Biomarkers; D024042:Collagen Type I; D003432:Cross-Linking Reagents; D019812:Heparan Sulfate Proteoglycans; D007797:Laminin; D010455:Peptides; D012333:RNA, Messenger; C071927:perlecan