Adams Mary, Cottrell Jessica
Department of Biological Sciences, Seton Hall University, South Orange, NJ, United States.
Immunology Translational Research, Translational Early Development, Bristol Myers Squibb, Summit, NJ, United States.
Front Endocrinol (Lausanne). 2024 Nov 12;15:1484912. doi: 10.3389/fendo.2024.1484912. eCollection 2024.
Three-dimensional cultures are widely used to study bone and cartilage. These models often focus on the interaction between osteoblasts and osteoclasts or osteoblasts and chondrocytes. A culture of osteoblasts, osteoclasts and chondrocytes would represent the cells that interact in the joint and a model with these cells could be used to study many diseases that affect the joints. The goal of this study was to develop 3D bone-cartilage interface (3D-BCI) that included osteoblasts, osteocytes, osteoclasts, and cartilage. Fluorescently tagged cell lines were developed to assess the interactions as cells differentiate to form bone and cartilage. Mouse cell line, MC3T3, was labeled with a nuclear GFP tag and differentiated into osteoblasts and osteocytes in Matrigel. Raw264.7 cells transfected with a red cytoplasmic tag were added to the system and differentiated with the MC3T3 cells to form osteoclasts. A new method was developed to differentiate chondrocyte cell line ATDC5 in a cartilage spheroid, and the ATDC5 spheroid was added to the MC3T3 and Raw264.7 cell model. We used an Incucyte and functional analysis to assess the cells throughout the differentiation process. The 3D-BCI model was found to be positive for TRAP, ALP, Alizarin red and Alcian blue staining to confirm osteoblastogenesis, osteoclastogenesis, and cartilage formation. Gene expression confirmed differentiation of cells based on increased expression of osteoblast markers: , , , and Runx, cartilage markers: , , , and osteoclast markers: , and . Based on staining, protein expression and gene expression results, we conclude that we successfully developed a mouse model with a 3D bone-cartilage interface.
三维培养广泛应用于骨和软骨的研究。这些模型通常聚焦于成骨细胞与破骨细胞或成骨细胞与软骨细胞之间的相互作用。成骨细胞、破骨细胞和软骨细胞的培养将代表在关节中相互作用的细胞，并且包含这些细胞的模型可用于研究许多影响关节的疾病。本研究的目的是开发包含成骨细胞、骨细胞、破骨细胞和软骨的三维骨 - 软骨界面（3D - BCI）。开发了荧光标记的细胞系以评估细胞分化形成骨和软骨时的相互作用。小鼠细胞系MC3T3用核绿色荧光蛋白标签标记，并在基质胶中分化为成骨细胞和骨细胞。将转染了红色细胞质标签的Raw264.7细胞添加到该系统中，并与MC3T3细胞一起分化形成破骨细胞。开发了一种新方法来使软骨细胞系ATDC5在软骨球状体中分化，并将ATDC5球状体添加到MC3T3和Raw264.7细胞模型中。我们使用Incucyte和功能分析来评估整个分化过程中的细胞。发现3D - BCI模型对TRAP、ALP、茜素红和阿尔新蓝染色呈阳性，以确认成骨细胞生成、破骨细胞生成和软骨形成。基因表达基于成骨细胞标志物（ 、 、 、Runx）、软骨标志物（ 、 、 ）和破骨细胞标志物（ 、 ）表达增加来确认细胞分化。基于染色、蛋白质表达和基因表达结果，我们得出结论，我们成功开发了一种具有三维骨 - 软骨界面的小鼠模型。
