Knee tactile afferents act as synovial joint limit detectors, eliciting signalling upon excessive fibrous tissue strain but play little role in joint function as disruption of their activity does not induce impairments in movement or sensation. In contrast, knee nociceptive afferents gain activity upon inflammation producing painful sensation in pathology such as osteoarthritis. We hypothesize that similar in origin, fast-conducting tactile afferents become sensitized by inflammatory mediators and gain activity causing proprioceptive sensation impairment in patients with knee pathology, driving gait abnormalities and osteoarthritis progression. To investigate the activity of these neurons, we will produce a co-culture model using our existing 3D bone mimetic and iPSC derived tactile sensory neurons by utilizing the NGN2-BRN3A plasmid produced by Nickolls et al producing a model of these tactile neurons at their position within the joint at the fibrous/bony interface.
Human Y201 MSC cells embedded in type I collagen gels (0.05 × 106 cell/gel) were differentiated to osteocytes andmechanically loaded in silicone plates (5000 µstrain, 10Hz, 3000 cycles) (n=5). RNA quantified by RNAseq analysis (NovaSeq S1) and neuronal communication pathways identified using DEseq2 analysis.
Over 20 genes involved in neural communication were expressed in 100% of bone cultures, and most of these showed regulation under mechanical strain including receptors for Substance P (p= 0.91), CGRP (p=0.05), Norepinepherin (p=0.002), NPY (p=0.0002), Sema3A (p=0.01), Leptin (p=0.00005), Neutrophin3A (p=0.23), BDNF (p=0.5), GDNF (p=0.02), and glutamate(p=0.024) and signalling molecules Neutrophin3 (p=0.73), NGF (p=0.02), Sema3A (p=0.003), BDNF (p=0.02) and GDNF (p=0.006).
The production of this 3D neural co-culture model is still in its infancy. However, preliminary RNAseq data has shown our Y201 bone model expresses all the signalling pathways known to exert neural regulatory responses and therefore is now ready to move forward to neural inclusion.