Dietary Calcium and Phosphate levels affect bone development and mesenchymal stem cell lineage allocation in neonatal pigs
Authors: Zhang Wei , Murray Robert L. , Rhoads Robert , Stahl Chad H. 
Published: June 2021

Deficits in bone deposition during early‐life have been associated with increased risk of osteoporosis later in life. Bone marrow mesenchymal stem cells (MSC) are a subset of multipotent stem cells residing within the bone which provide the life‐long supply of bone forming cells. Molecular mechanisms affecting the activity and differentiation potential of MSC are still poorly understood. Our previous work has revealed that the activity and differentiation potential of MSC are subject to alteration based on neonatal nutrition. Dietary calcium (Ca) and phosphate (PO4) levels play a critical role in regulation of bone development. With increasing interest in MSC therapies and an increasing incidence of bone diseases, we aimed to elucidate the impact of marginal dietary Ca and PO4 deficiency and excess on the activity and differentiation potential of MSC during neonatal development. During a 16‐day study, 27 neonatal pigs (n= 27, 24±6h old) were fed milk replacers that were isonitrogenous and isocaloric, but either 25% deficient (D), adequate (A), or 25% in excess (E) of Ca and PO4 requirements. Piglets were fed 8 times/d at a rate to match the growth rate of sow‐reared piglets. Body weight and feed intake were recorded daily. Blood was collected on d8 and d16 for analysis. Humeri were collected for the isolation of MSC and radial/ulna bones were collected for physical measurement and determination of bone mineral content. Serum PO4 concentrations were significantly higher in the A group compared to the D group on day 8, however at day 16 the D group had the highest serum PO4 concentrations with the E group having the lowest PO4 concentrations (P<0.05). Serum FGF23 was greater in the D group than the E group at 16d (77.21±1.90 pg/mL vs. 70.38±1.90 pg/mL, P<0.05), with values for the A group being intermediate to these groups. The D group had lower bone mineral content and bone volume as compared to the E group. Among the MSC, those isolated from D pigs had significantly lower rates of in vivo proliferation of than those isolated from A pigs, as determined by BrdU labelling (0.063±0.007 vs. 0.089±0.007), but significantly higher in vitro proliferation than those isolated from E pigs at both 12 and 24 h post‐plating (12 h: 0.40 ±0.04 vs 0.23±0.04; 24h: 0.28±0.04 vs 0.10±004). Ca and PO4 deficiency appears, based on the gene expression of key adipogenic factors as well as cellular accumulation of neutral lipids, to have predisposed MSC towards adipogenic differentiation. MSC from D pigs had increased expression of PPARγ2, FABP4, and LPL (P<0.05) and greater accumulation of neutral lipids as indicated by Oil Red O staining. Coupled with this apparent predisposition for adipocytic differentiation, a decrease in osteogenic capabilities were seen among MSC isolated from D pigs. This was determined based on reduced expression of the osteogenic regulators osteocalcin and fibronectin at d6 post differentiation. These findings demonstrated that even minor dietary deficiencies of Ca and PO4 during the neonatal period can have a profound impact on bone development. These dietary deficiencies may also program future bone health by altering the differentiation capabilities of MSC.Support or Funding InformationThis project is support by USDA grantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.