Mouse Bone Marrow Mononuclear Cells

H₂ Inhibits the Osteoclastogenesis of Mouse BMMCs

Hydrogen (H₂) is one of the major biodegradation products of magnesium (Mg) alloys implanted for bony fracture healing and reconstruction; H₂ thus plays a significant role in the regulation of local microenvironment and the biology of resident cells. Liu's team investigated how H₂, with different concentrations and durations, regulates the osteoclastogenesis of mouse bone marrow mononuclear cells (BMMCs).

First, they measured the osteoclastogenesis of mouse BMMCs under five H₂ concentrations (0%, 2%, 25%, 50%, and 75%) and three durations (5, 7, and 10 days) using TRAP staining, F-actin ring formation assay, pit formation assay, and RT-qPCR analysis. The number of multinucleated giant cells was significantly higher in the control and 2% H₂ groups than in the 25%, 50%, and 75% H₂ groups. Compared to the control group, 2% H₂ did not significantly inhibit osteoclastogenesis after 5, 7, or 10 days of induction, while 25%, 50%, and 75% H₂ did (Fig. 1A). Trap+ cells appeared after one day of induction, and Trap+ multinucleated cells appeared after three days. Their number peaked after seven days. The F-actin ring formation assay showed no differences in the number and size of F-actin rings between the 2% H₂ and control groups after 5, 7, or 10 days of induction. However, these parameters were significantly reduced in the 25%, 50%, and 75% H₂ groups, indicating significant inhibition of F-actin ring formation compared to the control group (Fig. 1B).

NaHS Mediated H₂S Release, Enhances CXCR4 Expression and Chemotaxis to SDF-1Α

For successful transplantation of Hematopoietic Stem cells (HSCs), it is quite necessary that efficient homing, engraftment and retention of HSC self-renewal capacity takes place, which is often restricted due to inadequate number of adult HSCs. Here, Khanna et al. reported that short-term ex-vivo treatment of mouse bone marrow mononuclear cells (BMMNCs) to Sodium Hydrogen Sulfide (NaHS, hydrogen sulfide-H2S donor) can be used as a possible strategy to overcome such hurdle.

SDF-1α and CXCR4 guide HSPC migration. Elevated CXCR4 expression is vital for HSPC homing and engraftment. Here, CXCR4 mRNA and surface expression were measured in NaHS- and vehicle-treated cells (Fig. 2a). qRT-PCR showed maximal CXCR4 expression at 300 µM NaHS (3.4-fold). At this concentration, CXCR4 expression peaked at 24 hours (Fig. 2b, c). NaHS also increased CXCR4 surface expression, shown by higher MFI in LSK, ST-HSCs, and LT-HSCs (Fig. 2d). BAPTA-AM reduced CXCR4 expression, highlighting calcium's role. In a trans-well assay, linneg and LSK cells migrated toward SDF-1α, with NaHS-treated cells showing significantly more migration (Fig. 2e, f). Similar results were seen in ST-HSCs and LT-HSCs (Fig. 2g, h). AMD3100 blocked NaHS-induced migration, confirming CXCR4 mediation (Fig. 2g, h).