Is the a_vß₃ Integrin Necessary for Bone Mineralization?

This summer I have worked in the field of bone biology with Dr. Wozniak and Dr. Hruska in the Renal Division at Barnes-Jewish Hospital. The goal of my research had been to participate in the examination of the mechanisms of bone formation. It is known that osteoblasts are essential in carrying out mineralization which leads to the formation of new bone. Previous research by Dr. Wozniak has implicated integrins, a family of adhesion receptors that direct cell adhesion, in the formation of new bone. Using this information, I was able to hypothesize that integrins, specifically the a_vß₃ integrin, are necessary for bone mineralization.

The role of the a_vß₃ integrin in bone mineralization was examined using human osteoblasts. Human stromal cells were obtained from donors and differentiated in the laboratory along the osetoblast lineage into pre-osteoblasts, and then into mineralizing osteoblasts using a cell culture medium supplemented with osteogenic protein-1 (OP-1), also known as morphogenetic protein BMP-7. In order to determine if the a_vß₃ integrin is involved in the process of bone mineralization, specific anti-a_vß₃ inhibitors were added to the cultures. These inhibitors- labeled as inhibitor A and inhibitor B- were provided by a research company for testing. Inhibitor A was used at 1 nM and 10 nM concentrations while inhibitor B was added at concentrations of 100 nM and 1 µM. As a control, two wells in each 6-well culture dish were incubated in the absence of any a_vß₃ integrin inhibitor. Cells were incubated at 37°C (5% CO₂) for 3 weeks, changing medium every 3-5 days. The level of calcium deposition was measured using Alizarine Red S, a calcium binding dye commonly used to measure calcium deposition in the laboratory. Cell culture dishes were rinsed 3 times with phosphate buffered saline (PBS) and fixed in ice-cold 70% ethanol for 1 hour. The cells were then rinsed 6 times with millipore H₂O and stained with Alizarine Red S (0.05g/ml H₂O) for 10 minutes at room temperature. Cells were again rinsed 6 times with Millipore H₂O and developed with PBS for 15 minutes. Finally, the cell layer was dehydrated with 100% ethanol and allowed to air dry.

Results showed a significant decrease in the mineralization of cells that were treated with either A or B a_vß₃ integrin inhibitor. To determine precise amounts of Alizarine Red S in the absence and presence of inhibitors, Alizarine Red S was eluted from the cell wells using a 10% cetylpyridinium chloride solution. Absorption at 562 nm indicated that control cells (not treated with inhibitor) bound 160 µg of Alizarine Red S, while cells treated with inhibitor A at levels of 1 and 10 nM bound only 28 and 26 µg of Alizarine Red S, respectively. Cells treated with inhibitor B at 100 nM bound an average of 23 µg of Alizarine Red S, while those treated with 1 µM bound only 16 µg. Therefore, the conclusion is that the a_vß₃ integrin plays a key role in the process of bone mineralization.