DataSheet1_Triple-gene deletion for osteocalcin significantly impairs the alignment of hydroxyapatite crystals and collagen in mice.PDF

Osteocalcin (Ocn), also known as bone Gla protein, is synthesized by osteoblasts and thought to regulate energy metabolism, testosterone synthesis and brain development. However, its function in bone is not fully understood. Mice have three Ocn genes: Bglap, Bglap2 and Bglap3. Due to the long span of these genes in the mouse genome and the low expression of Bglap3 in bone, researchers commonly use Bglap and Bglap2 knockout mice to investigate the function of Ocn. However, it is unclear whether Bglap3 has any compensatory mechanisms when Bglap and Bglap2 are knocked out. Considering the controversy surrounding the role of Ocn in bone, we constructed an Ocn-deficient mouse model by knocking out all three genes (Ocn−/−) and analyzed bone quality by Raman spectroscopy (RS), Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and MicroCT (μCT). The RS test showed that the alignment of hydroxyapatite crystals and collagen fibers was significantly poorer in Ocn−/− mice than in wild-type (WT) mice. Ocn deficiency resulted in a looser surface structure of bone particles and a larger gap area proportion. FTIR analysis showed few differences in bone mineral index between WT and Ocn−/− mice, while μCT analysis showed no significant difference in cortical and trabecular regions. However, under tail-suspension simulating bone loss condition, the disorder of hydroxyapatite and collagen fiber alignment in Ocn−/− mice led to more obvious changes in bone mineral composition. Collectively, our results revealed that Ocn is necessary for regulating the alignment of minerals parallel to collagen fibrils.

骨钙蛋白（Osteocalcin, 简称 Ocn），亦称骨型谷光甘肽蛋白，由成骨细胞合成，并被认为调控能量代谢、睾酮合成及大脑发育。然而，其在骨骼中的作用尚不明确。小鼠具有三种 Ocn 基因：Bglap、Bglap2 和 Bglap3。鉴于这些基因在小鼠基因组中的漫长序列及其在骨骼中 Bglap3 的低表达，研究人员通常采用 Bglap 和 Bglap2 敲除小鼠来探究 Ocn 的功能。然而，当 Bglap 和 Bglap2 被敲除时，Bglap3 是否存在补偿机制尚不明确。鉴于 Ocn 在骨骼中作用之争议，我们通过敲除全部三个基因（Ocn−/−）构建了 Ocn 缺陷型小鼠模型，并利用拉曼光谱（Raman spectroscopy, RS）、扫描电子显微镜（Scanning electron microscopy, SEM）、傅里叶变换红外光谱（Fourier transform infrared spectroscopy, FTIR）及微CT（MicroCT, μCT）对骨骼质量进行了分析。RS 检测显示，与野生型（wild-type, WT）小鼠相比，Ocn−/− 小鼠中羟基磷灰石晶体与胶原纤维的排列显著较差。Ocn 缺失导致骨骼颗粒表面结构松弛，间隙面积比例增大。FTIR 分析显示，在野生型和 Ocn−/− 小鼠之间骨矿物质指数差异不大，而 μCT 分析显示皮质和松质区域之间无显著差异。然而，在模拟骨丢失的尾部悬挂条件下，Ocn−/− 小鼠中羟基磷灰石与胶原纤维排列的紊乱导致骨矿物质成分发生更明显的改变。综合以上结果，我们揭示了 Ocn 对于调控矿物质与胶原纤维平行排列的必要性。