Impact of Phosphate Supplementation and PPi Reduction on Osteocyte Lacunar-Canalicular Network in XLH

Proper phosphate homeostasis is critical for bone mineralization, with disruptions leading to skeletal abnormalities affecting growth and load-bearing capacities. X-linked hypophosphatemia (XLH), caused by mutations in PHEX gene, results in elevated fibroblast growth factor 23 (FGF23), a phosphaturic hormone that can cause impaired renal phosphate reabsorption and osteomalacia. High-phosphate diets improve mineralization in Hyp mice carrying inactivating Phex mutation but fail to restore osteocyte connectivity, possibly due to persistent mineralization defects around osteocyte lacunae. Excess inorganic pyrophosphate (PPi), a mineralization inhibitor, may contribute to these defects. This study investigates whether reducing PPi via the Enpp1asj/asj mutation, which impairs ENPP1-mediated PPi production, while supplementing phosphate in Hyp;Enpp1asj/asj mice, enhances osteocyte numbers and restores lacunar-canalicular connectivity. Hyp, Enpp1asj/asj, Hyp;Enpp1asj/asj, and wild-type (WT) mice (n=16, two per group) were assigned to either a standard chow or high-phosphate diet (2%P) for two weeks starting at six weeks of age. Dissected humeri were stained with rhodamine and embedded in epoxy for confocal microscopy to examine lacunar-canalicular connectivity, with 3D reconstruction performed using Dragonfly software. Osteocyte networks were severely disrupted in Hyp mice, with larger lacunae and reduced connectivity, showing minimal improvement with phosphate supplementation. Enpp1asj/asj mice had fewer osteocytes and reduced connectivity regardless of diet. Hyp;Enpp1asj/asj mice exhibited increased osteocyte numbers on a high-phosphate diet but retained irregular lacunae and disrupted connectivity, suggesting PPi reduction increases cell number but not full network restoration. Small sample sizes limit statistical significance; future work will expand sample sizes (e.g., n=80 mice, ten per group) and refine imaging analysis to quantify osteocyte morphology and connectivity.