具CGRP缓释功能的PLGA电纺纤维在腱骨愈合中的作用及其分子机制研究

ACL reconstruction has been widely considered as the main therapeutic option to restore the knee function and attenuate OA progression of patients after their ACL injuries. However, poor tendon-bone healing may easily cause unsatisfactory clinical outcomes after reconstruction. The improvement of the new bone formation towards the tendon graft at the tendon-bone interface can enhance the graft healing. Our previous work showed that a neurotransmitter, called calcitonin gene-related peptide (CGRP), can statistically enhance phosphorylation of CREB and promote the osteogenic differentiation ability of mesenchymal stem cells. Besides, our work also validated that the increase of CGRP levels in the periosteum can better stimulate the periosteum wrapped tendon graft-bone healing, which may be ascribed to the osseous ingrowth into the tendon graft for improved graft healing. However, the underlying mechanism behind the role of CGRP in new bone formation still keeps unclear. As CGRP can also stimulate cAMP production, which subsequently activates PKA, we hypothesize that CGRP can upregulate the expression levels of important transcription factors involved in the osteogenic differentiation of BMSCs via modulating cAMP-PKA-CREB signaling pathway, which may potentially improve tendon-bone healing. Therefore, we will modulate the expression levels of the elements of cAMP-PKA-CREB signaling pathway by using inhibitors and activators, aiming to elucidate the mechanism of cAMP-PKA-CREB signaling pathway mediating CGRP regulating osteogenic differentiation ability of BMSCs. Meanwhile, we will prepare biodegradable PLGA electrospun fibers for CGRP delivery in rats with ACL reconstruction and testify the bio-efficacy of such novel complex in tendon-bone healing. The in vivo findings can also help us to validate the proposed mechanism and provide scientific proof for the use strategy of CGRP in future clinical applications.

ACL受损后，手术重建是恢复膝关节运动功能并延缓OA发展的主要治疗手段。腱骨愈合不佳易导致ACL重建失败，促进新骨向肌腱长入利于腱骨愈合。我们前期研究发现一种神经递质，CGRP（降钙素基因相关肽），能显著上调间充质干细胞CREB磷酸化水平和增强其成骨分化能力，且证实CGRP高表达骨膜包裹肌腱更有利于界面新骨长入促进腱骨愈合。然而，CGRP促成骨分化机制尚不清楚。因CGRP能刺激胞内cAMP的产生，进而增强PKA活性，由此提出科学假设：CGRP通过调节cAMP-PKA-CREB信号通路上调间充质干细胞成骨分化相关转录因子表达水平，进而有促进腱骨愈合的应用前景。本项目拟正反调节该信号通路各元件表达水平，以阐明该通路在CGRP调控BMSCs成骨分化过程中的作用机制。制备具CGRP缓释功能的可降解PLGA电纺纤维用于大鼠ACL重建，检验腱骨愈合效果和进一步验证作用机制，为CGRP临床应用提供依据。

ACL受损后，手术重建是恢复膝关节运动功能并延缓OA发展的主要治疗手段。腱骨愈合不佳易导致ACL重建失败，促进新骨向肌腱长入利于腱骨愈合。我们前期研究发现一种神经递质，CGRP（降钙素基因相关肽），能显著上调骨膜源干细胞CREB磷酸化水平和增强其成骨分化能力，提示着CGRP可能作为潜在的生物活性因子改善腱骨界面愈合效果。然而，CGRP对骨髓间充质干细胞（BMSC）是否具有类似效应以及如何有效地利用CGRP治疗运动型损伤仍然值得关注。基于此，本研究中我们（1）探索了CGRP对骨髓间充质干细胞（BMSC）成骨分化的生物学效应及作用机制；（2）制备了PLGA电纺纤维以作为潜在缓释载体；（3）构建大鼠ACL重建模型，评估CGRP对术后腱骨愈合的影响。相应地，我们发现：（1）CGRP能通过显著上调BMSC成骨分化关键转录因子Runx2和SP7增强BMSC成骨分化能力；（2）PLGA电纺纤维形貌及表面积能有效得到调控；（3）CGRP能增加腱骨界面新骨生成与长入。本项目验证了CGRP对BMSC成骨分化的作用效果，成功制备了一系列PLGA静纺纤维，并初步确认了CGRP可促进ACL重建后腱骨界面愈合。该研究工作可为CGRP在运动医学中的临床应用提供科学依据。