Harnessing microfluidic biofabrication and cell printing to develop an in vitro bone pain model

3D bone models remain of significant interest for the study of diseases associated with the skeletal system. Current approaches seek to use living organisms as bioreactors; however, these do not address the 3Rs initiative. We detail a novel animal-free three-dimensional model for drug screening against bone pain, printing human bone marrow stromal cells (hBMSCs) and induced pluripotent stem cell-derived nociceptors (iPSC-NCs) to fabricate a 3D platform to study the pathophysiological interaction of these cells. An innovative clay-based biomaterial ink (bone ink), as previously established [1], was used to print hBMSCs. A low-polymeric fraction gelatin methacryloyl–Matrigel (GelMA-Ma) ink (neuro ink) was developed and characterized for printing iPSC-derived nociceptors. Extrusion of the bone and neuro inks was tested on a polydimethylsiloxane (PDMS) flow-focusing microfluidic chip using high-speed imaging (Photron-UX100) to determine ideal extrusion parameters at 5 and 8 µl·min⁻¹, respectively. By utilizing the microfluidic chip's ability to rapidly switch between bone and neuro bioinks, hBMSCs and iPSC-NCs were printed serially, critically demonstrating viability and functionality post-printing, with positive staining for alkaline phosphatase and expression of TRKA and CGRP, and limited expression of NEFH and RET. Notably, mechanical disruption of the neuro portion impaired functional neuro-signaling, confirming the ability to mimic neural damage. In conclusion, we demonstrate the functionality of a bone-neuro model capable of recapitulating complex skeletal innervation and simulating/modulating the neural network, illustrating the potential of the model in testing novel therapeutic agents for bone pain studies.