Development and characterization of an in vitro fluorescently tagged 3D bone-cartilage interface model.

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: Alpl, Bglap, Col1A2, and Runx2, cartilage markers: Acan, Col2A1, Plod2, and osteoclast markers: Acp5, Rank and Ctsk. Based on staining, protein expression and gene expression results, we conclude that we successfully developed a mouse model with a 3D bone-cartilage interface.