Engineering Articular Cartilage with Bone Morphogenetic Proteins

Abstract

Osteoarthritis, the progressive degeneration of articular cartilage, affects more than 200 million people worldwide and represents a major health problem. Current therapeutic strategies to restore the joint function are based on engineering functional articular cartilage for transplantation into chondral lesions. We previously found that BMP9 is a remarkable inducer for joint regeneration in a mouse digit amputation model, suggesting that chondrocyte progenitor cells are present in the amputation wound. In Chapter 2, the in vivo findings of BMP9 induced chondrogenesis was replicated in vitro, demonstrating that amputation wound fibroblasts are BMP9 responsive, and a mouse digit fibroblast cell line (P3) displayed a parallel chondrogenic response. For chondrogenic differentiation, a self-aggregation protocol with BMP9 treatment was developed to differentiate P3 fibroblasts into transplantable hyaline cartilage. In Chapter 3, clones of P3 fibroblasts were established and screened for BMP9 induced chondrogenesis. This screen identified clonal lines that differentiated distinct cartilage phenotypes and identified a chondroprogenitor line for articular cartilage (P3_D8 clone) and one for hypertrophic cartilage (P3_E3 clone). These clones were used to enhance the self-aggregation protocol by improving self-aggregation with growth factor treatment. Using this modified protocol, the P3_D8 clonal cells differentiate stable articular cartilage based on in vivo transplantation into an acute joint defect. Detailed studies with the P3_D8 clonal line found that articular cartilage differentiates with an encapsulating fibrous connective tissue layer that contains sequestered progenitor cells that can be rederived following multiple rounds of differentiation. P3_D8 clonal cells as well as rederived cells display a cell surface marker profile similar to skeletal stem cells and can differentiate into osteoblasts indicating that they are multipotent progenitor cells. P3_E3 clonal cells display similar multipotent characteristics. Since osteoarthritis progression is associated with the transition of articular chondrocytes to hypertrophic chondrocytes, we used RNAseq to establish transcriptional signatures that distinguish the P3_D8 and P3_E3 clonal cells during cartilage differentiation. This dissertation bridges in vivo regeneration with in vitro tissue engineering strategies, identifies articular and hypertrophic chondrocyte progenitor cell lines, established a novel cartilage differentiation protocol, and identifies a transcriptional signature that distinguishes articular and hypertrophic chondrocyte differentiation.