Dorsal root ganglion neurons regulate the transcriptional and translational programs of osteoblast differentiation in a microfluidic platform

Innervation by the sensory nervous system plays a key role in skeletal development and in orchestration of bone remodeling and regeneration. However, it is unclear how and in which bone cells can sensory nerves act to control these processes. Here, we show a microfluidic coculture system comprising dorsal root ganglion (DRG) neurons and mesenchymal stem cells (MSCs) that more faithfully represents the in vivo scenario of bone sensory innervation. We report that DRG neurons promote the osteogenic differentiation capacity of MSCs, by mediating the increase of alkaline phosphatase activity and the upregulation of osteoblast-specific genes. Furthermore, we show that DRG neurons have a positive impact on Cx43 levels in MSCs during osteoblastogenesis, especially at an early stage of this process. Conversely, we described a negative impact of DRG neurons on MSCs N-cadherin expression at a later stage. Finally, we demonstrate a cytoplasmic accumulation of β-catenin translocation into the nucleus, and subsequently Lymphoid Enhancer Binding Factor 1—responsive transcriptional activation of downstream genes in cocultured MSCs. Together, our study provides a robust body of evidence that the direct interaction of DRG neurons with MSCs in a bone-like microenvironment leads to an enhancement of osteoblast differentiation potential of MSCs. The osteogenic effect of DRG neurons on MSCs is mediated through the regulation of Cx43 and N-cadherin expression and activation of the canonical/β-catenin Wnt signaling pathway.


Microfluidic devices fabrication
Microfluidic devices were obtained using standard photolithography and soft lithography procedures. The first step comprised the fabrication of a master mold, which consists of two layers of photoresist structures on a flat silicon wafer substrate. Afterward, poly (dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning, Midland, MI, USA) was mixed with a curing agent at a w/w of 10:1 and poured onto the silicon wafer. Master mold with PDMS was then placed in a vacuum desiccator for 15 min to remove air bubbles from the PDMS. Subsequently, PDMS was cured at 60 ºC for 2 h, and microfluidic chambers were cut and separated from the master mold. Reservoirs were punched out using an 8 mm tissue biopsy punch. Finally, the microfluidic devices were sterilized in 70 % (v/v) ethanol and allowed to dry in a laminar flow hood.
Before use, microfluidic devices were irradiated with UV light for 20 min to create reactive species on the surface, which when placed together with glass coverslips to form an electrostatic bond. UV irradiation also makes the surface hydrophilic helping the addition of liquids.

Rat bone marrow mesenchymal stem cells isolation
Primary bone marrow MSCs were obtained from a healthy male 6-10 week-old Wistar rat. Briefly, after sacrificing the rat by CO 2 , back limbs were harvested and soft tissue attached to the skeleton was removed. Femora and tibia were washing with ice-cold hank's balanced salt solution (HBSS, Gibco ® ), supplemented with 10 % (v/v) Pen/Strep, and the extremities were clipped to expose the marrow. Bones were then transferred to a 1.5 mL microcentrifuge tube, which was subsequently inserted into a 15 mL centrifuge tube and centrifuged for 1 minute at 3000 rpm to collect the marrow. The obtained pellet was resuspended in standard culture medium, which consisted of DMEM -low glucose with 10 % (v/v) FBS and 1 % (v/v) Pen/Strep. Cells were dispersed using a 21G needle for 4-6 times and filtered through a 100 µm cell strainer to remove bone fragments and cell clumps. The cell suspension was then centrifuged at 1200 rpm for 5 min, resuspended in standard culture medium, and maintained in a humidified incubator (37 °C and 5 % CO 2 ). The medium was changed after 3 days to remove non-adherent cells, and subsequently, the adherent cells were cultured to confluence (the medium was renewed every 3/4 days). Cells from the first passage were used in all studies.

Rat dorsal root ganglion neurons isolation
Primary DRG neurons were obtained from healthy male 6-10 week-old Wistar rats.
Briefly, after sacrificing the rats by CO 2 , spinal columns were removed and placed in HBSS with 10 % (v/v) Pen/Strep. Columns were opened from the caudal to the rostral end with scissors to reveal the DRG. Then, DRG were individually recovered, placed in DMEM and the nerve trunks removed with a scalpel blade. Subsequently, DRG were digested with 10 mg/mL Collagenase, Type IV (Gibco ® ) for 2 h at 37 ºC. After centrifugation at 1200 rpm for 5 min, the pellet was resuspended in DMEM. Afterward, DRG were mechanically dissociated using fire-polished glass Pasteur pipettes (full diameter and ½ diameter). The cell suspension was then centrifuged at 1200 rpm for 5 min 3 times and resuspended in growing medium composed of DMEM with 2 % (v/v) B-27, 1 µM cytosine arabinofuranoside (AraC, also known as cytarabine), and 1 % (v/v) Pen/Strep.