脉冲电磁场和周期载荷协同刺激的抗骨丢失效应及其对骨中细胞钙信号的调控机制研究

Osteoporosis has exerted increasingly tremendous economic and social burdens to our country, as the issue of aging populations is becoming even more serious. The high cost or potential side effects of traditional anti-osteoporotic drugs might become a nonnegligible limitation. Substantial evidence has proved that both cyclic loading (CL) and pulsed electromagnetic fields (PEMF) have the capacity of partially inhibiting osteoporosis; nevertheless the efficiency of CL and PEMF was modest and the overall process of bone deterioration could not be completely reversed. Previous studies have shown that the therapeutic target and mechanism between CL and PEMF were totally different. It is believed that osteocytes behave as the central sensory system for mechanical signals in bone, whereas PEMF might modulate bone remodeling by directly affecting bone effector cells (i.e., osteoblasts and osteoclasts) on the bone surface. Our previous studies have demonstrated that osteocytes in bone exhibited unique calcium oscillation with multiple robust calcium spikes under CL, whereas osteoblasts showed negligible changes in calcium signaling. Moreover, PEMF stimulation caused significantly increased calcium spikes in osteoblasts. Thus, in this proposal, we aim to verify our novel hypothesis whether the combined application of CL and PEMF is able to produce more significant inhibition for osteoporosis. First, we compare the effects of CL, PEMF and CL+PEMF on the cellular activities and functions of in vitro bone cells. Then, the effects of combined application of CL and PEMF on bone quality and bone metabolism in osteoporotic mice with distinct bone turnover characteristics will be systematically evaluated. Finally, the intracellular calcium oscillations in bone cells under the combined stimulation of CL and PEMF will be investigated for deciphering the related mechanism. Together, the present proposal expects to provide a novel therapeutic scheme and strategy for osteoporosis in clinics, and also shed light on the mechanism how the skeleton processes the mechanical and electromagnetic signals.

随着我国人口老龄化问题的加剧，骨质疏松（OP）带来的经济社会负担逐年加重。常见抗OP药物或价格高昂或副作用明显。大量研究证实周期载荷（CL）和脉冲电磁场（PEMF）能一定程度抑制骨丢失，但二者均无法完全逆转OP发生和发展。既往研究表明CL和PEMF对骨的作用靶点和机制完全不同，CL主要靶细胞是骨细胞，PEMF则直接作用于骨表面的效应细胞。申请人研究表明，CL能诱发骨细胞产生独特的多钙尖峰钙振荡，而对成骨细胞影响不明显，而PEMF诱发成骨细胞产生更多钙尖峰。因此本课题我们提出了CL和PEMF协同作用的新理念，体外实验比较CL和PEMF协同与单一作用对骨骼细胞功能的影响，体内实验评估二者协同作用对不同骨代谢OP小鼠骨质量和骨代谢的影响，并从骨中细胞钙振荡这一崭新角度入手系统分析协同作用的机制。本研究旨在为OP临床治疗提供新思路和新策略，并为深入了解骨骼应力和电磁信号转导机制提供崭新实验依据。

该项目旨在通过体外和在体实验系统阐明脉冲电磁场和周期载荷协同作用对于骨及骨中细胞的作用效果，并从胞浆钙信号入手分析骨中细胞对于力-电-磁偶联信号的响应过程及机制。主要完成内容包括：（1）研制了新型的反馈程控式脉冲电磁场协同周期机械应力刺激装置，能够实现力-电-磁偶联的多物理场信号的智能化的精准、同步输出。（2）使用微接触压印与自组装单分子膜技术构建了新颖的体外图案化骨组织细胞网络，发现了脉冲电磁场与周期机械力对于骨组织作用靶细胞的差异性，应力能诱发骨细胞产生独特的多钙尖峰钙振荡，而脉冲电磁场诱发成骨细胞产生更多钙尖峰，而协同作用能够诱导骨组织细胞网络产生更充分的钙信号激活。（3）以体外研究为基础，开发了小动物长骨原位细胞钙成像技术，并进一步证实了脉冲电磁场与周期机械力对于小鼠胫骨原位骨组织细胞网络的协同激活效应。（4）发现了协同刺激能够在改善体外成骨细胞活性和成骨分化能力的基础上，进一步提升体外骨细胞的生物活性与功能，并间接实现对于破骨细胞的功能抑制。（5）通过动物实验发现协同刺激能够在正常和老年大鼠骨组织中产生对于成骨细胞和骨细胞网络更充分的功能激活效应，从而实现了对于正常和骨质疏松骨组织骨代谢更充分的正向调控和骨质量的高效提升。