OR29-02 Energy Deficient Osteoblasts Inefficiently Mineralize Developing Bone In Sheep With Hypophosphatasia
Authors: Clarence Bertels Joshua , McCord Morgan , White Lauren G , Huggins Shannon , Landrock Danilo , Giles Erin D , Suva Larry J , Gaddy Dana 
Published: October 2023

Disclosure: J.C. Bertels: None. M. McCord: None. L.G. White: None. S. Huggins: None. D. Landrock: None. E.D. Giles: None. L.J. Suva: None. D. Gaddy: None.

Hypophosphatasia (HPP) is a rare genetic disorder characterized by low enzymatic activity of tissue nonspecific alkaline phosphatase (TNSALP), resulting in decreased bone mineralization, muscle weakness, and premature loss of deciduous teeth. All HPP phenotypes have been characterized in our CRISPR/Cas9 gene-edited sheep model of HPP. Previous studies identified small, abnormal mitochondria in HPP sheep cells, leading to the hypothesis that reduced bone mineralization may result from decreased osteoblast (OB) differentiation and cellular energetics. To test this idea, iliac crest bone biopsies and sternal bone marrow stromal cells (BMSCs) were harvested from 1 yr old WT and HPP sheep (n=3 per genotype). Histomorphometric analysis of bone biopsies from HPP sheep revealed increased unmineralized osteoid matrix compared to WT, indicative of decreased OB activity, whereas osteoblast and osteoclast number were unchanged. To determine whether BMSCs from HPP sheep were altered from WT in regard to differentiation capacity toward the adipocyte (AD) or OB lineage, 21 day cultures of BMSCs in OB or AD differentiation medium were performed. HPP BMSCs had significantly increased AD differentiation and similar OB differentiation compared to WT. Next, high resolution respirometry of undifferentiated and differentiating cells toward either the OB or AD lineage using Seahorse XF analysis to measure oxidative respiration and glycolysis on days 0, 7, 14 and 21 of both OB and AD differentiation was performed. Results demonstrated that both undifferentiated and differentiating HPP cells (to OB and AD) had significantly decreased oxygen consumption rates (OCR) and extracellular acidification rates (ECAR), reflective of diminished maximal and ATP-linked oxidative respiration and glycolysis. When measured, intracellular ATP levels in differentiating BMSCs, both undergoing osteoblastogenic and adiopogenic differentiation, showed a higher accumulation of ATP. Collectively, these data provide the first evidence that decreased bone mineralization in HPP sheep results from decreased BMSC and OB energetics through both oxidative respiration and glycolysis. In addition, the data demonstrate that reduced TNSALP activity also leads to enhanced bone marrow adipogenesis. Coupled with our previous results demonstrating reduced oxidative respiration and glycolysis in sheep skeletal muscle, these findings demonstrate a fundamental requirement for TNSALP activity in the maintenance of normal cellular respiration and purinergic metabolism in cells of the musculoskeletal system. Moreover, they provide important insight into the potential for targeting increased cellular respiration to alleviate the deleterious effects of HPP.

Presentation: Sunday, June 18, 2023