Objective

Disuse osteoporosis is a major type of bone loss disease characterized by regional bone loss and microstructure alterations. The condition is induced by a marked decrease in weight bearing over time, which usually occurs due to limb immobilization, therapeutic bed rest or space flight. To date, the most commonly used mouse model of disuse osteoporosis is constructed using the classical tail suspension method, which causes tail injury, movement inconvenience and mental stress. This study aimed to propose a noninvasive and effective method for the establishment of a mouse model of disuse osteoporosis and compared this method with the tail suspension method.

Methods

3D printing technology was applied to construct a movable unloading device. A movable noninvasive 3D-printed unloading device (3D-ULD) was used to unload the hindlimbs of the mice. The bone microstructure and bone volume of unloaded femurs were analysed through micro-CT and H&E staining, and von Kossa staining was performed for the detection of bone mineralization in the femurs. TRAP staining, IHC-CTSK and Q-PCR were performed for evaluation of the bone resorption ability, and double labelling, IHC-DMP1, ALP staining and Q-PCR assays were conducted to assess the osteogenic ability. The mechanical properties of disused bone were detected using the three-point bending test. The body, thymus and spleen weights of the mice were recorded, and the serum corticosterone level of the mice was assayed by enzyme-linked immunosorbent assay (ELISA).

Results

The micro-CT results showed significant trabecular bone loss, and 3D-ULD induced cortical bone loss in disused femurs as well as a decrease in the bone mineral density in the unloaded mice. TRAP staining and IHC-CTSK staining results indicated increases in the osteoclast number per bone perimeter (Oc.N/B.Pm) and the osteoclast surface per bone surface (Oc.S/BS) in the unloaded mice. The Ctsk, Trap and Mmp9 expression levels were significantly increased in the unloaded mice. Decreases in the ratio of the mineral surface to bone surface (MS/BS), mineral apposition rate (MAR) and bone formation rate per bone surface (BFR/BS) were found in unloaded mice in the 3D-ULD by double labelling. The IHC-DMP1 and ALP staining results showed decreases in the osteoblast number per bone perimeter (Ob.N/B.Pm) and osteoblast surface per bone surface (Ob. S/BS) in the mice unloaded in the 3D-ULD, and these mice also showed decreased Runx2, Alp and Dmp1 expression levels. Three-point bending test results showed that the mechanical properties were attenuated in the disused femurs of the unloaded mice. Less skin rupture and rare alterations in the thymus and spleen weights were found in the unloaded mice in the 3D-ULD. The ELISA results indicated the serum corticosterone level of the mice unloaded in the 3D-ULD was significantly lower than that of mice suspended by their tail.

Conclusion

This new disuse osteoporosis mouse model based on 3D-ULD could induce effective disuse bone loss with significantly alleviated side effects.

Translational potential of this article

This study proposes a new disuse osteoporosis mouse model based on 3D-ULD that can be used to better understand disuse bone loss in the future.

中文翻译：

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基于可移动无创3D打印卸载装置的废用性骨质疏松症小鼠模型

客观的

废用性骨质疏松症是一种主要类型的骨质流失疾病，其特征是局部骨质流失和微结构改变。这种情况是由随着时间的推移负重显着减少引起的，这通常是由于肢体固定、治疗性卧床休息或太空飞行而发生的。迄今为止，最常用的废用性骨质疏松小鼠模型是采用经典的悬尾法构建的，该方法会导致尾部损伤、运动不便和精神压力。本研究旨在提出一种无创有效的废用性骨质疏松小鼠模型建立方法，并将该方法与悬尾法进行比较。

方法

应用3D打印技术构建移动式卸料装置。使用可移动的无创 3D 打印卸载装置 (3D-ULD) 卸载小鼠的后肢。通过显微CT和H&E染色分析空载股骨的骨显微结构和骨体积，以及von Kossa进行染色以检测股骨中的骨矿化。TRAP染色、IHC-CTSK和Q-PCR用于评估骨吸收能力，并进行双标记、IHC-DMP1、ALP染色和Q-PCR测定以评估成骨能力。采用三点弯曲试验检测废弃骨的力学性能。记录小鼠的体重、胸腺和脾脏重量，采用酶联免疫吸附试验（ELISA）检测小鼠血清皮质酮水平。

结果

微 CT 结果显示显着的骨小梁丢失，3D-ULD 诱导废弃股骨的皮质骨丢失以及空载小鼠的骨矿物质密度降低。TRAP 染色和 IHC-CTSK 染色结果表明，空载小鼠中每骨周长的破骨细胞数量 (Oc.N/B.Pm) 和每骨表面的破骨细胞表面 (Oc.S/BS) 增加。Ctsk , Trap和Mmp9 _空腹小鼠的表达水平显着增加。通过双重标记在 3D-ULD 中的空载小鼠中发现矿物质表面与骨表面的比率 (MS/BS)、矿物质沉积率 (MAR) 和每个骨表面的骨形成率 (BFR/BS) 降低。IHC-DMP1 和 ALP 染色结果显示，在 3D-ULD 中卸载的小鼠中，每骨周长的成骨细胞数量 (Ob.N/B.Pm) 和每骨表面的成骨细胞表面 (Ob. S/BS) 减少，并且这些小鼠还表现出Runx2、Alp和Dmp1降低表达水平。三点弯曲试验结果表明，空载小鼠的废弃股骨的力学性能有所减弱。在 3D-ULD 中空载的小鼠中发现较少的皮肤破裂和胸腺和脾脏重量的罕见变化。ELISA结果表明，在3D-ULD中空载小鼠的血清皮质酮水平明显低于悬尾小鼠。

结论

这种基于 3D-ULD 的新型废用性骨质疏松症小鼠模型可以诱导有效的废用性骨质流失，并显着减轻副作用。

本文的转化潜力

本研究提出了一种新的基于 3D-ULD 的废用性骨质疏松症小鼠模型，可用于更好地了解未来的废用性骨质流失。