低强度脉冲超声通过CGRP增强软骨内成骨促进脊柱融合的实验研究

Spinal fusion is a major surgical practice for spine surgery, and it is necessary to establish a safe and efficacious method to assist this procedure. Studies have shown that low-intensity pulsed ultrasound (LIPUS) can promote spinal fusion, but the mechanism is yet to be clarified. It has been reported that calcitonin gene-related peptide (CGRP) may play a role in promoting ossification. Our research suggests a significant proliferation of chondrocyte in the bone graft area and a high expression of CGRP after the intervention of LIPUS. Therefore, we speculate that LIPUS may increase CGRP expression in the bone fusion area, promoting endochondral ossification and thereby enhancing fusion of bone graft. The project of our study is designed to apply the rabbit model for posterolateral lumbar fusion with LIPUS intervention, so as to observe the time and spatial distribution of CGRP plus nerve fiber in the fusion area with the two-photon laser-scanning microscopy, to clarify its relationship with endochondral ossification, investigate the change of CGRP receptor in bone mesenchymal stem cells (BMSCs) and chondrocytes under LIPUS, and to determine the effect of CGRP on BMSCs and chondrocytes in terms of proliferation and differentiation, hence verify the effect of CGRP in spinal fusion with the application of LIPUS. This study helps to clarify the mechanism of LIPUS spinal fusion, providing experimental and theoretical basis for the clinical application of this method.

脊柱融合术是治疗脊柱疾病的重要手段之一。研究表明低强度脉冲超声(LIPUS)可以促进脊柱融合，但其机制不明。课题组前期研究提示LIPUS干预后植骨融合区的软骨细胞显著增殖，降钙素基因相关肽(CGRP)呈高表达，而已有研究提示CGRP能促进骨形成。据此推测：LIPUS通过上调骨融合区CGRP表达，增强软骨内成骨效应，最终促进植骨融合。本项目拟以LIPUS干预兔腰椎后外侧融合模型，采用双光子激光扫描显微技术观察CGRP+神经纤维在骨融合区时空分布变化，分析其与软骨内成骨关系；探究LIPUS作用下骨髓间充质干细胞和软骨细胞CGRP受体变化，及CGRP对这些细胞增殖分化的影响，验证CGRP在LIPUS促进脊柱融合中的作用。本研究对于阐明LIPUS促进脊柱融合的分子机制具有重要意义，为LIPUS促进脊柱融合的临床应用提供实验和理论依据。

脊柱融合术是治疗脊柱疾病的常用手段之一，多项研究表明低强度脉冲超声（LIPUS）可以促进骨折愈合。本项目从整体、组织、细胞三个层面证实LIPUS可以促进脊柱融合。通过micro-CT分析发现低强度脉冲超声（LIPUS）作用四周后，将脊柱融骨区域相对骨体积从33.4±1.5显著提升至42.6±2.9。组织水平上，通过HE染色观察发现在LIPUS作用下，融骨区域内骨组织和成骨细胞均显著增多，且在空间上紧紧围绕在新生骨周围，因此课题组推测成骨细胞增殖是异体骨植骨融合的重要病理过程。通过对CGRP阳性感觉神经纤维进行免疫组化染色发现：观察CGRP及其受体CRLR、RAMP的时空分布发现，CGRP及其受体CRLR、RAMP密度在脊柱融合过程中随时间推移逐渐增高，三周后达到峰值，后逐渐下降；LIPUS作用后，CGRP阳性感觉神经纤维密度显著上升，但也在三周后逐渐下降，提示CGRP可能只参与成骨细胞增殖、迁移，不参与骨痂重塑阶段。在研究过程中，课题组首次发现H型微血管存在于植骨融合区域，且在脊柱融合过程中发挥重要作用。LIPUS干预可以显著提高融骨区域H型微血管新生，使H型微血管密度增加42%。血管形成实验发现LIPUS干预血管内皮细胞可以显著促进新生血管形成。将LIPUS与CGRP共同作用，不仅可以显著提高内皮细胞增殖和迁移能力，还能够促进内皮细胞分泌VEGF，且二者具有协同作用。通过上述研究，课题组证实了LIPUS能够促进脊柱融合，且H型微血管和CGRP在植骨融合过程中发挥重要作用，为阐明LIPUS促进骨再生的机制打下坚实的基础。