Department of Orthopedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.
Department of Orthopedics, Shanghai Key Laboratory for Prevention and Treatment of Bone and Joint Diseases, Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine;
J Vis Exp. 2023 Nov 13(201). doi: 10.3791/64507.
Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular matrix (osteoid) express the Pdpn gene to form cellular dendrites and transform into preosteocytes. Later, preosteocytes express the Dmp1 gene to promote matrix mineralization and thereby transform into mature osteocytes.This process is called osteocytogenesis. IDG-SW3 is a well-known cell line for in vitro studies of osteocytogenesis. Many previous methods have used collagen I as the main or the only component of the culturing matrix. However, in addition to collagen I, the osteoid also contains a ground substance, which is an important component in promoting cellular growth, adhesion, and migration. In addition, the matrix substance is transparent, which increases the transparency of the collagen I-formed gel and, thus, aids the exploration of dendrite formation through imaging techniques. Thus, this paper details a protocol to establish a 3D gel using an extracellular matrix along with collagen I for IDG-SW3 survival. In this work, dendrite formation and gene expression were analyzed during osteocytogenesis. After 7 days of osteogenic culture, an extensive dendrite network was clearly observed under a fluorescence confocal microscope. Real-time PCR showed that the mRNA levels of Pdpn and Dmp1 continually increased for 3 weeks. At week 4, the stereomicroscope revealed an opaque gel filled with mineral particles, consistent with the X-ray fluorescence (XRF) assay. These results indicate that this culture matrix successfully facilitates the transition from osteoblasts to mature osteocytes.
成骨细胞被认为是终末分化自成骨细胞的非增殖细胞。嵌入骨细胞外基质（类骨质）中的成骨细胞表达 Pdpn 基因，形成细胞树突，并转化为前成骨细胞。随后，前成骨细胞表达 Dmp1 基因，促进基质矿化，从而转化为成熟的成骨细胞。这个过程称为成骨细胞发生。IDG-SW3 是成骨细胞发生体外研究的知名细胞系。许多先前的方法都使用 I 型胶原作为培养基质的主要或唯一成分。然而，除了 I 型胶原，类骨质还含有一种基质，它是促进细胞生长、黏附和迁移的重要组成部分。此外，基质物质是透明的，这增加了 I 型胶原形成凝胶的透明度，从而有助于通过成像技术探索树突形成。因此，本文详细介绍了一种使用细胞外基质和 I 型胶原建立 IDG-SW3 存活的 3D 凝胶的方案。在这项工作中，分析了成骨细胞发生过程中的树突形成和基因表达。在成骨培养 7 天后，荧光共焦显微镜下清楚地观察到广泛的树突网络。实时 PCR 显示，Pdpn 和 Dmp1 的 mRNA 水平在 3 周内持续增加。第 4 周，立体显微镜显示充满矿化颗粒的不透明凝胶，与 X 射线荧光（XRF）分析一致。这些结果表明，这种培养基质成功地促进了成骨细胞向成熟成骨细胞的转变。
