Karp Jeffrey M, Tanaka Tetsuya S, Zohar Ron, Sodek Jaro, Shoichet Molly S, Davies John E, Stanford William L
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, Toronto, Ontario, Canada.
Bone. 2005 Sep;37(3):337-48. doi: 10.1016/j.bone.2005.04.022.
Given that thrombin is ubiquitously expressed at sites of vascular injury, and that osteogenic cells express receptors for thrombin, we questioned whether thrombin could attract osteogenic cells to a bony wound. Using a scratch wound assay, thrombin stimulated a significant increase in migration of osteogenic cultures of primary marrow cells. This effect was dependent on thrombin proteolytic activity; however, thrombin was unable to stimulate the migration of a more differentiated marrow-derived osteogenic cell line. To better understand the role of thrombin in osteoprogenitor migration, we developed an osteoprogenitor migration assay that combines a modified Boyden chamber with a bone nodule assay. Primary cells that migrated through the transwell filter in the presence of thrombin formed significantly more bone nodules compared to the condition without thrombin. This was not due to proliferation or differentiation effects of thrombin. In contrast, thrombin was unable to stimulate an increase in the number of nodules for the more differentiated osteogenic cell line. Thus, our results suggest that thrombin exhibits differential motogenic effects on osteogenic cells depending on their differentiation state. The cell migration/bone nodule assay described here is the first assay that can be specifically used to examine the effects of factors on the migration of osteoprogenitor cells, particularly those derived from primary populations.
鉴于凝血酶在血管损伤部位普遍表达，且成骨细胞表达凝血酶受体，我们不禁要问，凝血酶是否能将成骨细胞吸引至骨损伤处。通过划痕实验，凝血酶刺激原代骨髓细胞成骨培养物的迁移显著增加。这种效应依赖于凝血酶的蛋白水解活性；然而，凝血酶无法刺激更分化的骨髓来源成骨细胞系的迁移。为了更好地理解凝血酶在骨祖细胞迁移中的作用，我们开发了一种骨祖细胞迁移实验，该实验将改良的博伊登小室与骨结节实验相结合。与不存在凝血酶的情况相比，在凝血酶存在下穿过Transwell滤膜迁移的原代细胞形成的骨结节明显更多。这并非由于凝血酶的增殖或分化作用。相反，凝血酶无法刺激更分化的成骨细胞系的结节数量增加。因此，我们的结果表明，凝血酶对成骨细胞的促迁移作用因其分化状态而异。本文所述的细胞迁移/骨结节实验是首个可专门用于检测因子对骨祖细胞迁移影响的实验，尤其是对源自原代细胞群体的骨祖细胞的影响。
