Bone Cell Culture - Perfusion Effects on Osteogenesis

Dillaman, Richard

Abstract

The objective of the proposed research is to test the hypothesis that a culture system in which osteogenic cells are exposed to a constant, unidirectional flow of nutrient medium more closely approximates the conditions cells encounter in vivo than a system where cells are grown in petri dishes or on membranes in which medium is changed every two days. Evidence is presented to indicate that such a perfusion culture system would provide two important features that would improve the growth and differentiation of embryonic chick calvaria cells in culture. The first would be that a unidirectional flow of nutrients would mimic the flow of extracellular fluid in normal bone. Such a flow might establish the polarity observed in osteoblastic cells in vivo. The second advantage of the system would be that the rate of flow of culture medium could be regulated so that concentrations of certain constituents of the medium would not fall to such low levels that the phenotypic expression of the osteogenic cells might be altered. The hypothesis will be tested using three culture systems: 1) 35 ml plastic culture dishes with medium changed daily, 2) Costar culture chambers with cells grown on Nucleopore membranes and media changed every two days, and 3) Costar culture chamber inserts placed in a perfusion chamber where medium will be pumped through the Nucleopore membrane by a precision peristaltic pump. Characters used to score the three systems will include: growth rate; histological and ultrastructural characteristics of the cells and extracellular matrix; time to mineralize and degree of mineralization; and responsiveness of the cells to parathyroid hormone. Should the proposed perfusion system lead to primary cultures that more closely represent the osteogenic phenotype, the system would allow more precise testing of physiological and biochemical factors influencing bone.