Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation.

Microfluidic technology has become a valuable tool to the scientific community, allowing researchers to study fine cellular mechanisms with higher variable control compared with conventional systems. It has evolved tremendously, and its applicability and flexibility made its usage grow exponentially and transversely to several research fields. This has been particularly noticeable in neuroscience research, where microfluidic platforms made it possible to address specific questions extending from axonal guidance, synapse formation, or axonal transport to the development of 3D models of the CNS to allow pharmacological testing and drug screening. Furthermore, the continuous upgrade of microfluidic platforms has allowed a deeper study of the communication occurring between different neuronal and glial cells or between neurons and other peripheral tissues, both in physiological and pathological conditions. Importantly, the evolution of microfluidic technology has always been accompanied by the development of new computational tools addressing data acquisition, analysis, and modeling.

Innervation has proven to be critical in bone homeostasis/regeneration due to the effect of soluble factors, produced by nerve fibers, associated with changes in the activity of bone cells. Thus, in this study, we have established and characterized a coculture system comprising sensory neurons and osteoblasts to mimic the in vivo scenario where nerve fibers can be found in a bone microenvironment. Embryonic or adult primary dorsal root ganglion (DRG) and MC3T3-E1 osteoblastic cells were cocultured in compartmentalized microfluidic platforms and morphological and functional tests were performed. The time of adhesion and readout of axonal outgrowth were improved by the alignment of DRG with the axis of microgrooves, which showed to be a crucial step for the designed experiments. Cocultures of entire DRG from adult origin with osteoblasts were performed, showing extended DRG projections towards the axonal compartment, reaching osteoblastic cells. Immunocytochemistry showed that the neurites present within the osteoblastic compartment were immunoreactive to synapsin and calcitonin gene-related peptide suggesting the presence of specialized structures involved in this crosstalk. This evidence was further confirmed by electron microscopy where varicosities were detected as well as electron dense structures in neurite membranes. Aiming to mimic the properties of tissue extracellular matrices, MC3T3-E1 cells were seeded in the axonal side upon laminin, collagen or within 3D functionalized alginate matrices and axonal outgrowth was clearly observed. In order to analyze and quantify data with reproducible image analysis, a semi-automated algorithm was also developed. The collagen and laminin substrates displayed a higher amount of axons reaching the axonal side. Overall, the established method revealed to be a suitable tool to study the interaction between the peripheral nervous system and bone cells in different contexts mimicking the in vivo scenario.