Multiscale, Converging Defects of Macro-Porosity, Microstructure and Matrix Mineralization Impact Long Bone Fragility in NF1

Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.因骨质减少、骨质疏松或致残性局灶性骨骼发育不良引发的骨脆性，在孟德尔遗传病1型神经纤维瘤病（Neurofibromatosis type 1, NF1）中较为常见。为明确NF1患者骨脆性的潜在发病机制，本研究分析了两种条件性小鼠模型——Nf1Prx1（肢体敲除模型）与Nf1Col1（成骨细胞特异性敲除模型），同时纳入NF1患者的皮质骨样本开展研究。研究人员通过显微计算机断层扫描、定性与定量组织学检测、力学拉伸分析、小角度X射线散射（small-angle X-ray scattering, SAXS）、能量色散X射线光谱（energy dispersive X-ray spectroscopy, EDX）以及扫描声学显微镜（scanning acoustic microscopy, SAM）对小鼠骨组织进行了系统性检测。在Nf1Prx1小鼠的皮质骨中，研究人员检测到与骨干矿化缺陷相关的异位血管；血管周边区域的矿质结合能力受损，这极有可能源于骨胶原形成障碍——该区域完全缺乏酸性基质蛋白，且仅含有纤细的胶原纤维。此外，研究团队还发现骨材料的力学强度显著降低，该现象部分由骨细胞体积增大所介导。与上述观察结果一致，合并胫骨发育不良的NF1患者骨样本中，骨细胞陷窝体积出现明显增加。通过SAM检测可知，力学性能下降与基质刚度降低显著相关。与之相符的是，合并胫骨发育不良的NF1患者骨组织呈现矿化异质性，且胶原纤维厚度降低、排列紊乱。综合以上实验数据，本研究表明NF1相关胫骨发育不良患者的骨脆性，部分源于骨细胞相关微孔性增加、矿化不足，以及有机基质形成的全身性缺陷；该缺陷在张力与弯曲应力整合区域更为严重，最终还会出现与局部大孔隙性骨病变相关的异位血管残留。