IN VITRO ANALYSIS OF HUMAN FEMORAL CANCELLOUS BONE AS A VIABLE SOURCE OF OSTEOPROGENITOR CELLS AND THE EFFECTS OF BONE MORPHOGENETIC PROTEIN 2 AND DOXYCYCLINE ON THEIR DIFFERENTIATION INTO BONE FORMING COLONIES

Abstract

<p>The purpose of this investigation was 1) to establish an optimal technique for isolating osteoprogenitor cells in vitro using human femoral cancellous bone as a donor site, and 2) to evaluate the effects of various factors on differentiation of osteoprogenitor cultures. Two isolation techniques evaluated were enzyme digestion and primary explant technique. Furthermore, bone morphogenetic protein 2 (BMP-2) and doxycycline were supplemented in a dose dependent manner into various osteoprogenitor cell containing culture dishes. To compare isolation techniques and the effect of supplemented factors, we isolated cell populations from cancellous bone of the femoral neck from 7 and 6 patients, respectively, with osteoarthritis undergoing total hip replacement surgery. Bone derived osteoblasts and their bone nodules were identified using Von Kossa stain. The cell yield of the two isolation techniques was quantified by hemocytometer counts. The ability of the cells to differentiate into bone forming osteoblasts was evaluated by comparing numbers of Von Kossa positive nodule counts. Using alkaline phosphatase (ALP) staining, ALP quantitative assay and osteocalcin polymerase chain reaction, our study is the first attempt to determine the effects of doxycycline, along with the more commonly used BMP-2, on the differentiation of osteoblasts into bone forming colonies.</p>