Influences of anandamide on the composition and microanatomy of bone formed<i>in vitro</i>by osteoblast-like Saos-2 cells

The objective of our study was to investigate the influence of an endocannabinoid, anandamide (AEA), on bone formation in vitro by cultured osteosarcoma Saos-2 cells (HTB-85). Osteogenesis requires differentiation of cells to mature osteoblasts, followed by secretion of collagen fibers and hydroxyapatite mineral composed of calcium and phosphorus. We hypothesized that Saos-2 cells chronically treated with AEA would produce bone tissue with altered compositional and microanatomical properties compared to bone produced by untreated cells. To test this hypothesis, Saos-2 cells were cultured for up to 21 days in the presence of a physiological (0.25µM) or supraphysiological (25µM) AEA concentration, which were determined to be non-cytotoxic. Osteoblast-like Saos-2 cells are a common osteogenic model as they express alkaline phosphatase (ALP), secrete Type I collagen, and produce bone nodules structurally similar to hydroxyapatite nucleation sites in vivo. ALP activity assessed cell differentiation and osteogenic ability on Days 1 and 7. Extracellular matrix composition and microanatomy were evaluated on Days 1, 7, 14, and 21. Alizarin Red S (ARS) staining of calcium was imaged to determine AEA-induced microanatomical changes (total area of mineralization, nodule count, nodule size). ARS extraction quantified calcium and Sircol assays quantified collagen. Treatment groups were compared to vehicle control dimethyl sulfoxide (DMSO≤0.1% in all groups). ALP activity (µmol PNP/min/µg protein) increased slightly but significantly from Day 1 (0.02) to Day 21 (0.021) (p=0.03), displaying culture preparedness to produce mineralized bone tissue. Physiological AEA (0.25µM) increased ALP activity above vehicle control values on Days 1 and 7, although only Day 7 reached statistical significance (p=0.025). Supraphysiological AEA (25µM) greatly enhanced ALP activity on Day 1 (p&lt;0.001), but decreased ALP activity over the 7-day period until values were comparable to vehicle control. Bone nodule formation was observed in all treatment groups and increased with time as expected from Day 1 to Day 21. Independent nodules were visible and countable in all groups on Day 7, with slight aggregation occurring. By Day 21, few single nodules were visible, and nodule aggregation predominated. Collagen quantity (µg) increased in all groups with time (Day 1, 5.99 vs. Day 21, 13.79). Analysis of AEA treatment effects on microanatomy and composition are in progress. In conclusion, the results suggest the physiological concentration of AEA enhances Saos-2 cell differentiation, as determined by ALP activity. Understanding the influence of AEA and endocannabinoids on osteogenesis has potential as a treatment for bone pathologies, including osteoporosis, and may improve bioengineering of bone graft implants. Mentoring Access &amp; Platforms in STEM (MAPS)- Colorado State University Pueblo This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.