Calcif Tissue Int (1995) 56:123-129 Calcified Tiss1]e International 9 1995 Springer-Verlag New York Inc. Laboratory Investigations Bone Progenitor Cell Deficits and the Age-Associated Decline in Bone Repair Capacity R. Quarto,* D. Thomas, C. T. Liang Laboratory of Biological Chemistry, Gerontology Research Center, National Institute on Aging, NIH, 4940 Eastern Avenue, Baltimore, Maryland 21224, USA Received: 4 April 1994/Accepted: 8 September 1994 Abstract. Aging bone shows a progressive decline in mass and strength. Previous studies have suggested that bone marrow stem cells are reduced with aging and that this could be responsible, in part, for age-associated bone deficits. We measured the number of osteoprogenitor cells present in the bone marrow from adult and aged rats as well as their ability to differentiate in vitro and to form bone in vivo. We found that the number of adherent colony-forming cells was signif- icantly lower (65%) in marrow cells isolated from aged com- pared with adult rats. Furthermore, 88% of the colonies ob- tained from aged rats were alkaline phosphatase (AP) posi- tive, whereas virtually all the colonies from adult rats were positive. The addition of dexamethasone to the culture me- dium decreased the proliferation of the adherent cells and reduced the number of colonies obtained from both adult and aged bone marrow, all of which were AP positive. No sig- nificant differences were found in the expression of certain major bone cell marker genes as a function of donor age. However, dexamethasone treatment increased expression of osteopontin (OP) by fivefold. Adult stromal cells not treated with dexamethasone and implanted subcutaneously in recip- ient rats exhibited about 10-fold greater formation of bone compared with cells from aged rats. In contrast, dexameth- asone-treated cells exhibited high levels of bone formation, irregardless of donor age or the age of the recipient into which the cells were grafted. These studies are consistent with a deficit of osteoprogenitor cells in the bone marrow site as a contributing, perhaps correctable factor in the de- cline in bone repair and bone mass with age. Bone mass decreases and bone fragility increases with age in both men and women, although an accelerated bone loss occurs at menopause which increases the risk of osteoporo- sis. The causes of bone loss with age are not well understood but probably include both increased resorption and de- creased deposition of bone [1-3]. In part, these changes may be due to declines in the production of sex and trophic hor- mones [4-8] which function in the maintenance of bone. It has been established that the rate of fracture repair is re- duced with age [9]. Biomechanical analysis also shows that *On leave from the Laboratory of Cell Differentiation, National Institute for Cancer Research, Viale Benedetto XV n. 10 16132 Gen- ova Italy Correspondence to: C.T. Liang the capacity of load bearing in the healing fractures is much lower in old animals [9]. Bone formation induced by implants of demineralized bone powder is also delayed in old com- pared with young hosts [10-12]. Finally, rats show a much reduced ability with age to produce bone induced by the experimental removal of marrow [13]. Such findings of age- associated declines in bone formation are consistent with a decrease in the number of the progenitor cells in bone or their inability to differentiate. It is now well established that bone marrow contains a small proportion of cells (about 1 in 106) which adhere to plastic substrate, produce alkaline phosphatase, differentiate into bone cells, and produce bone when implanted subcuta- neously into a syngeneic host [14-16]. Current concepts pro- pose that these adherent cells, although not homogeneous, contain osteoprogenitor cells involved in the regeneration and repair of bone. Importantly, decreased numbers of these adherent progenitor cells are obtained from the bone marrow of old animals, suggesting that this deficit could be related to the decline in bone mass and bone repair with age [17, 18]. In this study, we have examined the loss of adherent cells in the marrow of young adult and old rats. Similar decreases in this population of ceils were observed as reported previ- ously [17, 18]. However, similar amounts of bone were pro- duced by dexamethasone-treated cells from old and young marrow after reimplantation, and this was largely indepen- dent of the age of the donor or recipient. It is likely that the factor(s) that sustains stem cell levels in the bone marrow decline with age, suggesting that this deficit might be cor- rected and, therefore, slow the age-associated loss of bone. Materials and Methods Reagents and Animals F344 Fisher male rats (6, 12, and 24 months old) were purchased from Harlan Sprague Dawley, Inc (Indianapolis, IN) under NIH contract. The animals were housed with a cycle of 12 hours of light and 12 hours of dark, and fed ad libitum standard National Institute of Health rat chow consisting of 23.5% protein, 1.2% calcium, and 1.0% phosphate. The animal protocol used in this study was ap- proved by the Animal Care and Use Committee at the Gerontology Research Center, National Institute on Aging. Dexamethasone was purchased from Sigma Chemical Co. (St. Louis, MO). Cell culture medium was Coon's modified Ham's F-12 medium (NIH Media Unit, Bethesda, MD) supplemented with 10% fetal calf serum (200, Biofluids Inc. Rockville, Md), 100 IU/ml penicillin, and 100 txg/ml streptomycin (NIH Media Unit, Bethesda, MD).