This project focuses on discerning the differences between human bone precursor cells (osteoprogenitor cells, preosteoblasts, and their differentiated progeny, the osteoblasts) in young (age 18-25 years) and older (ages greater than 50 years) individuals. To this end we have developed a means to purify human trabecular bone precursor cells and proliferate them in chemically-defined serum-free media. We also can differentiate these cells into osteoblast-like cells. Finally, each of these cell populations is defined by a number of parameters (cell size, cell complexity, antigenic content as defined by multiparameter flow cytometry), as well as by responsiveness to osteogenic growth factors. Therefore, we have a system in which each component of the osteogenic microenvironment (purified cells, recombinant growth factors, and purified extracellular matrix molecules; ECM) can be evaluated, in a systematic fashion, for age-related differences. The specific goals of this proposal are to first evaluate the responsiveness of bone precursor cells to osteogenic growth factors both alone and in combination as well as the responsiveness of these cells to cytokine:ECM complexes. As well, purified cells are used to examine the expression of integrins on developing bone cells, and their role in mediating bone cell:cell and cell:extracellular matrix (ECM) interactions. A related series of studies utilizes highly-sensitive fluorescent-based in vitro assays to investigate the capacity of human bone precursor cells to both migrate and within tissue; i.e., to localize within an appropriate osteogenic microenvironment. As well, age-related differences in the ability of precursor cells to differentiate into osteoblast-like cells will be assessed. These studies will determine antigenic and cell characteristic changes (by flow cytometry), and the capacity of these differentiated cells to elaborate bone ECM proteins, as well as to calcify the surrounding ECM. The final series of investigations will examine the molecular events associated with aging in human bone precursor cells. These studies first will evaluate the expression of (known) bone protein mRNA. A second series of studies seeks to isolate cDNA clones of genes which are differentially regulated during the aging process. These investigations are designed to reveal the level and nature of any age- related bone cell defect underlying the osteoporotic events occurring in elderly individuals. As such, the study will include as many women and minorities as is demographically feasible.
