Co-culture of osteocytes and neurons on a unique patterned surface

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.