Tan S Djien, de Vries Teun J, Kuijpers-Jagtman Anne Marie, Semeins Cornelis M, Everts Vincent, Klein-Nulend Jenneke
Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam-Universiteit van Amsterdam and Vrije Universiteit, Research Institute MOVE, Amsterdam, The Netherlands.
Bone. 2007 Nov;41(5):745-51. doi: 10.1016/j.bone.2007.07.019. Epub 2007 Aug 10.
Bone has the capacity to alter its mass and structure to its mechanical environment. Osteocytes are the predominant bone cells and it is generally accepted that the osteocytes are the professional mechanosensors of bone. A strain-derived fluid flow through the lacuno-canalicular porosity seems to mechanically activate them, resulting in the production of signalling molecules such as nitric oxide (NO). We hypothesize that mechanically stimulated osteocytes modulate osteoclast formation and activity via soluble factors, thus affecting bone resorption. Osteocytes, osteoblasts, and periosteal fibroblasts were isolated from fetal chicken calvariae via enzymatic digestion. The periosteal fibroblasts were obtained from the periostea. Osteocytes were separated from osteoblasts by immunomagnetic separation. Cells were mechanically stimulated for 1 h with pulsating fluid flow (PFF, 0.70 +/- 0.30 Pa) at 5 Hz, or kept under static conditions. Conditioned medium was collected after 60 min. The effect of conditioned medium on osteoclastogenesis was tested on mouse bone marrow cells in the presence of macrophage colony stimulating factor and receptor activator of NF-kappaB ligand. After 6 days of culture, osteoclast formation and bone resorption was determined. Osteocytes subjected to 1 h pulsating fluid flow produced conditioned medium that inhibited the formation of osteoclasts. For osteoblast PFF-conditioned medium, such effect was, to a lesser extent, also observed, but not for periosteal fibroblast PFF-conditioned medium. Furthermore, PFF-treated osteocytes, but not osteoblast or periosteal fibroblast, produced conditioned medium that resulted in a decreased bone resorption. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester attenuated the inhibitory effects of osteocyte PFF-conditioned medium on osteoclast formation and resorption. We conclude that osteocytes subjected to PFF inhibit osteoclast formation and resorption via soluble factors, and the release of these factors was at least partially dependent on activation of an NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast regarding to the production of soluble factors affecting osteoclast formation and bone resorption.
骨骼有能力根据其力学环境改变自身的质量和结构。骨细胞是主要的骨细胞，人们普遍认为骨细胞是骨骼的专业机械传感器。通过腔隙-小管孔隙的应变衍生流体流动似乎能机械性地激活它们，从而产生诸如一氧化氮（NO）等信号分子。我们假设机械刺激的骨细胞通过可溶性因子调节破骨细胞的形成和活性，进而影响骨吸收。通过酶消化从鸡胚颅骨中分离出骨细胞、成骨细胞和骨膜成纤维细胞。骨膜成纤维细胞取自骨膜。通过免疫磁珠分离将骨细胞与成骨细胞分开。细胞在5Hz频率下用脉动流体流（PFF，0.70±0.30Pa）机械刺激1小时，或保持在静态条件下。60分钟后收集条件培养基。在巨噬细胞集落刺激因子和核因子κB受体激活剂配体存在的情况下，测试条件培养基对小鼠骨髓细胞破骨细胞生成的影响。培养6天后，测定破骨细胞形成和骨吸收情况。接受1小时脉动流体流刺激的骨细胞产生的条件培养基可抑制破骨细胞的形成。对于成骨细胞PFF条件培养基，也在较小程度上观察到了这种作用，但骨膜成纤维细胞PFF条件培养基则未观察到。此外，经PFF处理的骨细胞而非成骨细胞或骨膜成纤维细胞产生的条件培养基导致骨吸收减少。一氧化氮合酶抑制剂N(G)-硝基-L-精氨酸甲酯减弱了骨细胞PFF条件培养基对破骨细胞形成和吸收的抑制作用。我们得出结论，接受PFF刺激的骨细胞通过可溶性因子抑制破骨细胞的形成和吸收，并且这些因子的释放至少部分依赖于骨细胞中NO途径的激活以响应PFF。因此，就影响破骨细胞形成和骨吸收的可溶性因子的产生而言，骨细胞似乎比成骨细胞或骨膜成纤维细胞对PFF更敏感。
