Role of Mineral Nutrition in Neonatal Bone Health: Effects on Porcine Mesenchymal Stem Cells

Abstract

ABSTRACT

MAHAJAN, AVANIKA. Role of Mineral Nutrition in Neonatal Bone Health: Effects on Porcine Mesenchymal Stem Cells. (Under the direction of Chad H. Stahl).

Both calcium and phosphorus tremendously impact bone health, and their deficiencies are associated with reduced bone parameters in growing mammals due to altered levels of calcitropic hormones (Parathyroid hormone (PTH) and 1,25(OH)2D3). However, effects of mineral deficiency on bone during the neonatal period are less characterized. Mesenchymal stem cell (MSC) differentiation into osteoblasts and adipocytes is a major determinant of bone quality and quantity. The aim of this research was to examine the effects of mineral nutrition and calcitropic hormones on MSC activity, bone parameters and gene expression during the neonatal period using pig as the model system. In the first study, 1,25(OH)2D3 was found to stimulate adipocytic differentiation of MSC isolated from 2-week old piglet based on dose-dependent increase in adipogenic (PPARG, LPL and AP2) expression and lipid accumulation (Oil Red O staining) in cells treated in vitro for a period of 12 days. A trend for reduced Runx2 (osteogenic transcription factor) expression and a significant reduction in alkaline phosphatase (ALP) activity suggested inhibition of osteogenesis, however, the effects were unclear due to a dose-dependent increase in osteogenic marker osteocalcin expression with 1,25(OH)2D3. Stimulatory effects of 1,25(OH)2D3 were also found to be altered in the presence of physiologically relevant electrical stimulation. Elevated levels of 1,25(OH)2D3 during P deficiency seen in older animals could impact bone by altering MSC activity. In the second study, dietary P deficiency during the neonatal period (d0-d15) resulted in dramatically reduced in vivo MSC proliferation accompanied by increased adipogenic markers (PPARG and LPL) and reduced osteocalcin expression in the bone marrow. Although the reduction in growth performance, bone growth and reduced PTH levels associated with classical P deficiency were observed, unlike older animals, circulating levels of 1,25(OH)2D3 did not change. The reduced PTH levels seen with P deficiency could impact MSC activity. In the third study, PTH was found to alter MSC activity depending on the mode of treatment as it caused increased adipogenic expression (PPARG, LPL) and lipid accumulation (Oil Red O) with continuous treatment and increased osteogenic expression (ALP, Runx2) and ALP activity by both continuous and intermittent treatment. Also, PTH and 1,25(OH)2D3 acted antagonistically when added together as the ability of 1,25(OH)2D3 to stimulate adipogenesis was partly inhibited by PTH while the ability of PTH to cause osteogenic expression was partly inhibited by 1,25(OH)2D3. Since both calcitropic hormones affect MSC activity, dietary calcium deficiency may alter developmental programming of bone during the neonatal period. In the fourth study, dietary Ca deficiency during neonatal period (d0-d18) caused a dramatic reduction in the in vivo MSC proliferation. Although bone quality and quantity were significantly reduced, unlike older animals, the circulating levels of Ca, P and 1,25(OH)2D3 were unaltered and PTH levels were increased only by end of the study with calcium deficiency. In vitro studies utilizing homologous sera demonstrated that MSC activity was affected by both the calcium status of the animal and the sera, as well as by their interaction Altered MSC activity maybe attributed to one or more of the 22 differentially expressed serum proteins identified by proteomic analysis of homologous sera. Overall, the data from these studies indicates that neonatal mineral nutrition is crucial for bone integrity and suggests that early life Ca and P restriction may have long-term effects on bone integrity via programming of MSC. Also, the circulating levels of calcitropic hormones may affect in vivo bone development based on their ability to alter MSC differentiation potential in vitro as seen in these studies.