Department of Materials Science and Engineering, University of Sheffield, Sheffield, UK.
INSIGNEO Institute for In Silico Medicine, University of Sheffield, Sheffield, UK.
Connect Tissue Res. 2024 Jul;65(4):313-329. doi: 10.1080/03008207.2024.2375065. Epub 2024 Jul 10.
As osteoblasts deposit a mineralized collagen network, a subpopulation of these cells differentiates into osteocytes. Biochemical and mechanical stimuli, particularly fluid shear stress (FSS), are thought to regulate this, but their relative influence remains unclear. Here, we assess both biochemical and mechanical stimuli on long-term bone formation and osteocytogenesis using the osteoblast-osteocyte cell line IDG-SW3.
Due to the relative novelty and uncommon culture conditions of IDG-SW3 versus other osteoblast-lineage cell lines, effects of temperature and media formulation on matrix deposition and osteocytogenesis were initially characterized. Subsequently, the relative influence of biochemical (β-glycerophosphate (βGP) and ascorbic acid 2-phosphate (AA2P)) and mechanical stimulation on osteocytogenesis was compared, with intermittent application of low magnitude FSS generated by see-saw rocker.
βGP and AA2P supplementation were required for mineralization and osteocytogenesis, with 33°C cultures retaining a more osteoblastic phenotype and 37°C cultures undergoing significantly higher osteocytogenesis. βGP concentration positively correlated with calcium deposition, whilst AA2P stimulated alkaline phosphatase (ALP) activity and collagen deposition. We demonstrate that increasing βGP concentration also significantly enhances osteocytogenesis as quantified by the expression of green fluorescent protein linked to Dmp1. Intermittent FSS (~0.06 Pa) rocker had no effect on osteocytogenesis and matrix deposition.
This work demonstrates the suitability and ease with which IDG-SW3 can be utilized in osteocytogenesis studies. IDG-SW3 mineralization was only mediated through biochemical stimuli with no detectable effect of low magnitude FSS. Osteocytogenesis of IDG-SW3 primarily occurred in mineralized areas, further demonstrating the role mineralization of the bone extracellular matrix has in osteocyte differentiation.
随着成骨细胞沉积矿化胶原网络，这些细胞的一个亚群分化为骨细胞。人们认为生化和机械刺激，特别是流体切应力（FSS），可以调节这一过程，但它们的相对影响尚不清楚。在这里，我们使用成骨细胞-骨细胞细胞系 IDG-SW3 评估生化和机械刺激对长期骨形成和骨细胞生成的影响。
由于 IDG-SW3 相对于其他成骨细胞系细胞具有相对新颖性和不常见的培养条件，因此首先对温度和培养基配方对基质沉积和骨细胞生成的影响进行了特征描述。随后，比较了生化（β-甘油磷酸（βGP）和抗坏血酸 2-磷酸（AA2P））和机械刺激对骨细胞生成的相对影响，采用跷跷板摇床间歇性施加低幅度的 FSS。
βGP 和 AA2P 的补充是矿化和骨细胞生成所必需的，33°C 培养物保留了更成骨细胞表型，而 37°C 培养物经历了更高的骨细胞生成。βGP 浓度与钙沉积呈正相关，而 AA2P 刺激碱性磷酸酶（ALP）活性和胶原蛋白沉积。我们证明，增加βGP 浓度也显著增强了骨细胞生成，这可以通过与 Dmp1 连接的绿色荧光蛋白的表达来定量。间歇性 FSS（~0.06 Pa）摇床对骨细胞生成和基质沉积没有影响。
这项工作证明了 IDG-SW3 在骨细胞生成研究中具有适用性和易用性。IDG-SW3 的矿化仅通过生化刺激介导，没有检测到低幅度 FSS 的影响。IDG-SW3 的骨细胞生成主要发生在矿化区域，进一步证明了骨细胞外基质矿化在骨细胞分化中的作用。
