Bone pain is a devastating complication in patients with advanced cancer with bone metastases that dramatically impacts the quality of life of the patient. Pain sensation in bone, particularly in bone cancer, is not well understood and treatment is ineffective. Osteocytes are likely candidates for bone-to-neuron communication because these cells are found throughout bone. Their location in bone provides many sites for osteocytes to associate with neuronal axons found in bone. To better understand the communication between osteoctyes and neurons, we sought to develop methods to coculture murine long-bone osteocyte-like cells (MLO-Y4) with dorsal root ganglia neurons (DRG). Patterned surfaces were prepared using micro-contact printing with alternating extracellular matrix molecules (ECM) to support different cell type attachment. Appropriate ECM molecules for surface patterning for osteocyte growth were determined by expression of maturation markers and morphology of MLO-Y4 cells. Optimal ECM protein for neuronal culturing was determined by dendrite process length and neurofilament staining. I found that MLO-Y4 cells optimally express differentiation markers, E11, dentin-matrix protein-1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE), and exhibit numerous dendritic-like processes when cultured on the novel peptide, perlecan domain IV peptide (PlnDIV) in comparison to Collagen type 1. Neuron process length was greater on laminin in comparison to fibronectin. Surfaces patterned with alternating 40µm x 40µm stripes of laminin and PlnDIV demonstrate that these cells can be grown in close proximity for communication studies.

Before cell-to-cell communication studies could be performed, the effect of ATP and ATP agonists and antagonists on DRG neurons was determined. I found that addition of ATP on DRG neurons at a concentration of 5mM resulted in depolarization of 20mV and multiple action potentials. Lower concentrations of ATP such as 100, 250 and 500 µM also resulted in depolarization, but only a single action potential was produced.

I also performed calcium imaging studies to determine the effect of ATP on intracellular calcium. Addition of ATP at concentrations of 10, 5, 1, and 0.1 µM resulted in an increase in intracellular calcium greater than the control HBSS condition. Approximately 60 to 80% of cells responded to the various concentrations of ATP. Addition of TNP-ATP, a P2X3 inhibitor, resulted in reduced number of cells responding to ATP.

The main objective of this work is to determine if mechanical stimulation of osteocytes results in soluble signal release that can activate neurons. I hypothesized that a potential soluble signal is ATP. To determine the effect of mechanical stimulation on MLO-Y4 cells, I hypotonically challenged the cells and tested the resultant media for ATP. I determined that ATP release was significantly greater in comparison to control. I then applied this conditioned media at a concentration of 0.1 µM ATP to determine its affect on DRG neuron intracellular calcium. I found that MLO-Y4 cell conditioned media resulted in an increase in intracellular calcium in DRG neurons, and that this was significantly reduced with the addition of TNP-ATP. These data indicate that ATP released by MLO-Y4 cells activates P2X3 receptors in DRG neurons. These indicate that purinergic signaling is a potential mechanism of communication between osteocytes and neuronal axons in bone.

Lastly, I aimed to utilize the co-culture system to study single cell-to-cell communication between MLO-Y4 cells and DRG neurons. I developed a system to mechanically stimulate MLO-Y4 cells in the presence of DRG neurons while monitoring intracellular calcium on a confocal microscope. Initial data shows that application of fluid flow results in increase in intracellular calcium in some MLO-Y4 cells (~9%) when the pipette was located at 50 m above the cell. Addition of fluid in the same manner did not result in increase in intracellular calcium in DRG neurons. However, addition of ATP in a similar manner did increase DRG neuron intracellular calcium. This set-up provides a unique system for studying single cell-to-cell communication between cell types.

Overall, the work presented here provides a unique system for studying single cell-to-cell communication between osteocytes and neurons with an implication in understanding pain sensation in bone. I established here that ATP released from osteocyte-like cells results in a cellular response in DRG neurons. This indicates a potential mechanism for the transmission of pain sensation in bone, and thus presents a possible therapeutic target for the treatment of bone pain.