The Influence of Vitamin D Metabolism on Gene Expression, Matrix Production and Mineralization During Osteoprecursor Cell-Based Bone Development.

BACKGROUND: Multipotential precursor cell lines derived from human bone marrow, capable of differentiating into cartilage or bone, may provide a useful tissue development model for studying the regulation and metabolism of putative growth and differentiation factors necessary for tissue regeneration. In mammals, the process of bone development depends on the proliferation and differentiation of osteoblast lineage cells, and the subsequent synthesis and mineralization of bone extracellular matrix (ECM). Vitamin D metabolites play a pivotal role in bone and mineral homeostasis, and are positive factors on bone development. Recently, it was demonstrated that a human-derived engineered osteoblast precursor cell line (OPC1), derived from human bone marrow, can metabolize the parental precursor vitamin D3 (vitaD3) to the active steroid 1α,25-dihydroxyvitamin D3 (1,25OH2D3), and elicit an osteogenic response that results in the decrease in proliferation and increase in ECM synthesis during early bone development. The aim in this study is to characterize gene expression, matrix production and mineralization within a bone development model. METHODS: We investigated whether vitaD3 influences bone ECM mineralization in the same manner as 1,25OH2D3 in confluent cultures of OPC1s. In addition, we explored the influence of vitamin D metabolites, in combination with other commonly used osteogenic factors, ascorbic acid, ß-glycerophosphate, dexamethasone (dex) and recombinant human bone morphogenetic protein-2 (rhBMP-2) on the osteoinduction of OPC1. RESULTS: It was demonstrated that OPC1 expresses the mRNA for the enzymatic equipment necessary to convert vitaD3 to 1,25OH2D3, as well as the mRNA expression of the catabolic enzyme known to regulate the concentration of active 1,25OH2D3. It was also demonstrated that mRNA expression for the vitamin D receptor (VDR) was influenced by both vitaD3 and 1,25OH2D3. Differential results using vitamin D metabolites in combination with ascorbic acid, ß-glycerophosphate, dex and/or rhBMP-2 were observed in alkaline phosphatase (ALP) activity and calcium deposition, and mRNA expression of procollagen type I (proColI), osteocalcin (OC) and osteopontin (OP). CONCLUSIONS: Overall it was demonstrated that vitamin D in combination with osteogenic factors influences the temporal bone development sequence in a positive manner.

Extrahepatic 25-Hydroxylation of Vitamin D3 in an Engineered Osteoblast Precursor Cell Line Exploring the Influence on Cellular Proliferation and Matrix Maturation during Bone Development.

Osteoblastic precursors experience distinct stages during differentiation and bone development, which include proliferation, extracellular matrix (ECM) maturation, and ECM mineralization. It is well known that vitamin D plays a large role in the regulation of bone mineralization and homeostasis via the endocrine system. The activation of vitamin D requires two sequential hydroxylation steps, first in the kidney and then in the liver, in order to carry out its role in calcium homeostasis. Recent research has demonstrated that human-derived mesenchymal stem cells (MSCs) and osteoblasts can metabolize the immediate vitamin D precursor 25-dihydroxyvitamin D3 (25OHD3) to the active steroid lα,25-dihydroxyvitamin D3 (1,25OH2D3) and elicit an osteogenic response. However, reports of extrahepatic metabolism of vitamin D3, the parental vitamin D precursor, have been limited. In this study, we investigated whether osteoblast precursors have the capacity to convert vitamin D3 to 1,25OH2D3 and examined the potential of vitamin D3 to induce 1,25OH2D3 associated biological activities in osteoblast precursors. It was demonstrated that the engineered osteoblast precursor derived from human marrow (OPC1) is capable of metabolizing vitamin D3 to 1,25OH2D3 in a dose-dependent manner. It was also demonstrated that administration of vitamin D3 leads to the increase in alkaline phosphatase (ALP) activity associated with osteoblast ECM maturation and calcium deposits and a decrease in cellular proliferation in both osteoblast precursor cell lines 0PC1 andOMC3T3-E1. These findings provide a two-dimensional culture foundation for future three-dimensional engineered tissue studies using the OPC1 cell line.

Three-Dimensional Culture of Cells and Matrix Biomolecules for Engineered Tissue Development and Biokinetics Model Validation.

There has been considerable progress in cellular and molecular engineering due to recent advances in multiscale technology. Such technologies allow controlled manipulation of physiochemical interactions among cells in tissue culture. In particular, a novel chemomechanical bioreactor has recently been designed for the study of bone and cartilage tissue development, with particular focus on extracellular matrix formation. The bioreactor is equally significant as a tool for validation of mathematical models that explore biokinetic regulatory thresholds (Saha, A. K., and Kohles, S. S., 2010, "A Distinct Catabolic to Anabolic Threshold Due to Single-Cell Nanomechanical Stimulation in a Cartilage Biokinetics Model," J. Nanotechnol. Eng. Med., 1(3), p. 031005; 2010, "Periodic Nanomechanical Stimulation in a Biokinetics Model Identifying Anabolic and Catabolic Pathways Associated With Cartilage Matrix Homeostasis," J. Nanotechnol. Eng. Med., 1(4), p. 041001). In the current study, three-dimensional culture protocols are described for maintaining the cellular and biomolecular constituents within defined parameters. Preliminary validation of the bioreactor's form and function, expected bioassays of the resulting matrix components, and application to biokinetic models are described. This approach provides a framework for future detailed explorations combining multiscale experimental and mathematical analyses, at nanoscale sensitivity, to describe cell and biomolecule dynamics in different environmental regimes.