ENGINEERING A 3D HUMAN BONE-CARTILAGE INTERFACE

In vitro models are critical for understanding the pathophysiology of the cartilage-bone interface. We are developing a human 3D cartilage-bone interface (3D-hBCI) model to mimic cytokine-induced cellular and matrix changes that lead to cartilage degradation and bone destruction, as seen in diseases like rheumatoid arthritis. Our aim is to create a preclinical drug screening tool, support personalized medicine development, and provide insights into underlying disease mechanisms. To achieve this, we engineered human MSC-derived cells to form both bone and cartilage tissues, establishing a system that replicates the human bone-cartilage interface found in joints. The model develops over 30 days (Fig 1). Initially, human primary bone marrow mononuclear cells (hPBMCs) are plated in Matrigel and differentiated into osteoblasts for 21 days. Then, hPBMCs are added to differentiate into osteoclasts over 7 days. Meanwhile, Chond2 cells differentiate into 3D cartilage spheroids for 14 days. On Day 28, the cartilage spheroids are combined with the 3D bone. After 7 more days, the 3D-hBCI samples are fixed and stained. Our data demonstrates that the 3D-hBCI model exhibits both bone and cartilage tissue characteristics, confirmed by tissue staining, gene expression, and protein expression. Specifically, 3D-hBCIs stain positive for Alkaline Phosphatase (ALP, osteoblast marker), Tartrate-Resistant Acid Phosphatase (TRAP, osteoclast marker), Alizarin Red (calcium deposition by bone cells), and Alcian blue (cartilage tissue). Gene expression analysis on Day 36 shows markers for bone (Alpl, Bglap, Col2a1, Mmp2, Runx2, Sparc2, Spp1), osteoclasts (Acp5, Ctsk, Dcstamp, Ocstanp, Rank), and cartilage (Acan, Col2a1, Crtap, Plod2, Sox9). Protein expression, assessed by Luminex, also shows characteristic levels of bone and cartilage markers (OPG, SOST, DKK1). We are further characterizing the model using microCT and histology/immunohistochemistry. Once validated, this model will serve as a platform to explore questions about the joint bone-cartilage interface, providing valuable insights into treatment efficacy, biological principles, and personalized medicine applications.