School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, China.
Department of Orthopedics-Spine Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Front Endocrinol (Lausanne). 2022 Feb 22;13:826660. doi: 10.3389/fendo.2022.826660. eCollection 2022.
Previous studies have revealed that melatonin could play a role in anti-osteoporosis and promoting osteogenesis. However, the effects of melatonin treatment on osteoporotic bone defect and the mechanism underlying the effects of melatonin on angiogenesis are still unclear. Our study was aimed to investigate the potential effects of melatonin on angiogenesis and osteoporotic bone defect. Bone marrow mesenchymal stem cells (BMSCs) were isolated from the femur and tibia of rats. The BMSC osteogenic ability was assessed using alkaline phosphatase (ALP) staining, alizarin red S staining, qRT-PCR, western blot, and immunofluorescence. BMSC-mediated angiogenic potentials were determined using qRT-PCR, western blot, enzyme-linked immunosorbent assay, immunofluorescence, scratch wound assay, transwell migration assay, and tube formation assay. Ovariectomized (OVX) rats with tibia defect were used to establish an osteoporotic bone defect model and then treated with melatonin. The effects of melatonin treatment on osteoporotic bone defect in OVX rats were analyzed using micro-CT, histology, sequential fluorescent labeling, and biomechanical test. Our study showed that melatonin promoted both osteogenesis and angiogenesis . BMSCs treated with melatonin indicated higher expression levels of osteogenesis-related markers [ALP, osteocalcin (OCN), runt-related transcription factor 2, and osterix] and angiogenesis-related markers [vascular endothelial growth factor (VEGF), angiopoietin-2, and angiopoietin-4] compared to the untreated group. Significantly, melatonin was not able to facilitate human umbilical vein endothelial cell angiogenesis directly, but it possessed the ability to promote BMSC-mediated angiogenesis by upregulating the VEGF levels. In addition, we further found that melatonin treatment increased bone mineralization and formation around the tibia defect in OVX rats compared with the control group. Immunohistochemical staining indicated higher expression levels of osteogenesis-related marker (OCN) and angiogenesis-related markers (VEGF and CD31) in the melatonin-treated OVX rats. Then, it showed that melatonin treatment also increased the bone strength of tibia defect in OVX rats, with increased ultimate load and stiffness, as performed by three-point bending test. In conclusion, our study demonstrated that melatonin could promote BMSC-mediated angiogenesis and promote osteogenesis-angiogenesis coupling. We further found that melatonin could accelerate osteoporotic bone repair by promoting osteogenesis and angiogenesis in OVX rats. These findings may provide evidence for the potential application of melatonin in osteoporotic bone defect.
先前的研究表明，褪黑素在抗骨质疏松和促进成骨方面可能发挥作用。然而，褪黑素治疗对骨质疏松性骨缺损的影响以及褪黑素对血管生成影响的机制尚不清楚。本研究旨在探讨褪黑素对血管生成和骨质疏松性骨缺损的潜在作用。从大鼠股骨和胫骨中分离骨髓间充质干细胞（BMSCs）。使用碱性磷酸酶（ALP）染色、茜素红 S 染色、qRT-PCR、western blot 和免疫荧光来评估 BMSC 的成骨能力。通过 qRT-PCR、western blot、酶联免疫吸附测定、免疫荧光、划痕实验、Transwell 迁移实验和管形成实验来确定 BMSC 介导的血管生成潜能。使用微 CT、组织学、连续荧光标记和生物力学测试分析褪黑素处理对去卵巢（OVX）大鼠胫骨缺损模型中骨质疏松性骨缺损的影响。研究表明，褪黑素促进成骨和血管生成。与未处理组相比，用褪黑素处理的 BMSCs 表现出更高的成骨相关标志物（ALP、骨钙素（OCN）、 runt 相关转录因子 2 和osterix）和血管生成相关标志物（血管内皮生长因子（VEGF）、血管生成素 2 和血管生成素 4）的表达水平。重要的是，褪黑素不能直接促进人脐静脉内皮细胞血管生成，但通过上调 VEGF 水平，它具有促进 BMSC 介导的血管生成的能力。此外，我们还发现褪黑素治疗组与对照组相比，OVX 大鼠胫骨缺损周围的骨矿化和形成增加。免疫组织化学染色表明，褪黑素治疗的 OVX 大鼠中骨形成相关标志物（OCN）和血管生成相关标志物（VEGF 和 CD31）的表达水平更高。然后，三点弯曲试验表明，褪黑素治疗还增加了 OVX 大鼠胫骨缺损的骨强度，增加了最大载荷和刚度。总之，本研究表明，褪黑素可以促进 BMSC 介导的血管生成并促进成骨-血管生成偶联。我们进一步发现，褪黑素可以通过促进 OVX 大鼠的成骨和血管生成来加速骨质疏松性骨修复。这些发现可能为褪黑素在骨质疏松性骨缺损中的潜在应用提供依据。
