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• 1. [Article] 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 ...

  Citation × Citation Citation Abstract Copy Title: The Influence of Vitamin D Metabolism on Gene Expression, Matrix Production and Mineralization During Osteoprecursor Cell-Based Bone Development Author: Mason, Shelley S., Kohles, Sean S., Winn, Shelley R., Zelick, Randy D. Year: 2014 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 1alpha,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, beta-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, beta-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. Title: The Influence of Vitamin D Metabolism on Gene Expression, Matrix Production and Mineralization During Osteoprecursor Cell-Based Bone Development Author: Mason, Shelley S., Kohles, Sean S., Winn, Shelley R., Zelick, Randy D. Year: 2014 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 1alpha,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, beta-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, beta-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. Close

• 2. [Article] 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 ...

  Citation × Citation Citation Abstract Copy Title: 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 Author: Mason, Shelley S., Kohles, Sean S., Winn, Shelley R., Zelick, Randy D. Year: 2013 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(25OH₂D₃) to the active steroid 1α,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 D₃to 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 OPC1 and MC3T3-E1. These findings provide a two-dimensional culture foundation for future three- dimensional engineered tissue studies using the OPC1 cell line. Title: 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 Author: Mason, Shelley S., Kohles, Sean S., Winn, Shelley R., Zelick, Randy D. Year: 2013 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(25OH₂D₃) to the active steroid 1α,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 D₃to 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 OPC1 and MC3T3-E1. These findings provide a two-dimensional culture foundation for future three- dimensional engineered tissue studies using the OPC1 cell line. Close

• 3. [Article] An Inverse Method for Predicting Tissue-Level Mechanics from Cellular Mechanical Input

  Extracellular matrix (ECM) provides a dynamic three-dimensional structure which translates mechanical stimuli to cells. This local mechanical stimulation may direct biological function including tissue ...

  Citation × Citation Citation Abstract Copy Title: An Inverse Method for Predicting Tissue-Level Mechanics from Cellular Mechanical Input Author: Kim, Wangdo, Tretheway, Derek C., Kohles, Sean S. Year: 2009 Extracellular matrix (ECM) provides a dynamic three-dimensional structure which translates mechanical stimuli to cells. This local mechanical stimulation may direct biological function including tissue development. Theories describing the role of mechanical regulators hypothesize the cellular response to variations in the external mechanical forces on the ECM. The exact ECM mechanical stimulation required to generate a specific pattern of localized cellular displacement is still unknown. The cell to tissue inverse problem offers an alternative approach to clarify this relationship. Developed for structural dynamics, the inverse dynamics problem translates measurements of local state variables (at the cell level) into an unknown or desired forcing function (at the tissue or ECM level). This paper describes the use of eigenvalues (resonant frequencies), eigenvectors (mode shapes), and dynamic programming to reduce the mathematical order of a simplified cell–tissue system and estimate the ECM mechanical stimulation required for a specified cellular mechanical environment. Finite element and inverse numerical analyses were performed on a simple two-dimensional model to ascertain the effects of weighting parameters and a reduction of analytical modes leading toward a solution. Simulation results indicate that the reduced number of mechanical modes (from 30 to 14 to 7) can adequately reproduce an unknown force time history on an ECM boundary. A representative comparison between cell to tissue (inverse) and tissue to cell (boundary value) modeling illustrates the multiscale applicability of the inverse model. Title: An Inverse Method for Predicting Tissue-Level Mechanics from Cellular Mechanical Input Author: Kim, Wangdo, Tretheway, Derek C., Kohles, Sean S. Year: 2009 Extracellular matrix (ECM) provides a dynamic three-dimensional structure which translates mechanical stimuli to cells. This local mechanical stimulation may direct biological function including tissue development. Theories describing the role of mechanical regulators hypothesize the cellular response to variations in the external mechanical forces on the ECM. The exact ECM mechanical stimulation required to generate a specific pattern of localized cellular displacement is still unknown. The cell to tissue inverse problem offers an alternative approach to clarify this relationship. Developed for structural dynamics, the inverse dynamics problem translates measurements of local state variables (at the cell level) into an unknown or desired forcing function (at the tissue or ECM level). This paper describes the use of eigenvalues (resonant frequencies), eigenvectors (mode shapes), and dynamic programming to reduce the mathematical order of a simplified cell–tissue system and estimate the ECM mechanical stimulation required for a specified cellular mechanical environment. Finite element and inverse numerical analyses were performed on a simple two-dimensional model to ascertain the effects of weighting parameters and a reduction of analytical modes leading toward a solution. Simulation results indicate that the reduced number of mechanical modes (from 30 to 14 to 7) can adequately reproduce an unknown force time history on an ECM boundary. A representative comparison between cell to tissue (inverse) and tissue to cell (boundary value) modeling illustrates the multiscale applicability of the inverse model. Close