Notch Regulates Fibrocartilage Stem Cell Fate and Is Upregulated in Inflammatory TMJ Arthritis.

The Notch pathway is critical for the development of the extracellular matrix in cartilage by regulating both anabolic and catabolic cellular activities. Similarly, Notch signaling plays a biphasic role in adult cartilage health and osteoarthritis by maintaining homeostasis and contributing to degeneration, respectively. The temporomandibular joint (TMJ) is the synovial joint of the craniofacial complex and is subject to injury and osteoarthritis. While Notch has been studied in axial skeletal joints, little is known about the role of Notch in TMJ development and disease. We identified fibrocartilage stem cells (FCSCs) localized within the TMJ condyle superficial zone niche that regenerate cartilage and repair joint injury. Here we investigate the role of Notch in regulating TMJ development and FCSC fate. Using a Notch reporter mouse, we discovered FCSCs localized within the TMJ superficial niche exhibit Notch activity during TMJ morphogenesis. We further showed that constitutively activating Notch promotes FCSC differentiation toward both cartilage and bone lineages, but inhibits adipogenesis. Using a TNF-α-induced TMJ inflammatory arthritis mouse model, we found that the expression of Notch receptors and ligands are upregulated and coupled with cells undergoing cartilage to bone transdifferentiation, which may contribute to TMJ pathogenesis. We also discovered that global Notch inhibition reduces osteogenic and chondrogenic differentiation of FCSCs. Together, these findings suggest that Notch is critical for FCSC fate specification and TMJ homeostasis, and reveal that inhibition of the Notch pathway may be a new therapeutic target for treating TMJ osteoarthritis.

Fibrocartilage Stem Cells Engraft and Self-Organize into Vascularized Bone.

Angiogenesis is a complex, multicellular process that is critical for bone development and generation. Endochondral ossification depends on an avascular cartilage template that completely remodels into vascularized bone and involves a dynamic interplay among chondrocytes, osteoblasts, and endothelial cells. We have discovered fibrocartilage stem cells (FCSCs) derived from the temporomandibular joint (TMJ) mandibular condyle that generates cartilage anlagen, which is subsequently remodeled into vascularized bone using an ectopic transplantation model. Here we explore FCSC and endothelial cell interactions during vascularized bone formation. We found that a single FCSC colony formed transient cartilage and host endothelial cells may participate in bone angiogenesis upon subcutaneous transplantation in a nude mouse. FCSCs produced an abundance of the proangiogenic growth factor vascular endothelial growth factor A and promoted the proliferation of human umbilical vein endothelial cells (HUVECs). Using a fibrinogen gel bead angiogenesis assay experiment, FCSC cell feeder layer induced HUVECs to form significantly shorter and less sprouts than D551 fibroblast controls, suggesting that FCSCs may initially inhibit angiogenesis to allow for avascular cartilage formation. Conversely, direct FCSC-HUVEC contact significantly enhanced the osteogenic differentiation of FCSCs. To corroborate this idea, upon transplantation of FCSCs into a bone defect microenvironment, FCSCs engrafted and regenerated intramembranous bone. Taken together, we demonstrate that the interactions between FCSCs and endothelial cells are essential for FCSC-derived vascularized bone formation. A comprehensive understanding of the environmental cues that regulate FCSC fate decisions may contribute to deciphering the mechanisms underlying the role of FCSCs in regulating bone formation.