Title: Cartilage Development and Maturation In Vitro and In Vivo
Description: The articular cartilage has a limited capacity to regenerate. Cartilage lesions often result in degeneration, leading to osteoarthritis. Current treatments are mostly palliative and reparative, and fail to restore cartilage function in the long term due to the replacement of hyaline cartilage with fibrocartilage. Although a stem-cell based approach towards regenerating the articular cartilage is attractive, cartilage generated from human mesenchymal stem cells (hMSCs) often lack the function, organization and stability of the native cartilage. Thus, there is a need to develop effective methods to engineer physiologic cartilage tissues from hMSCs in vitro and assess their outcomes in vivo. This dissertation focused on three coordinated aims: establish a simple in vivo model for studying the maturation of osteochondral tissues by showing that subcutaneous implantation in a mouse recapitulates native endochondral ossification (Aim 1), (ii) develop a robust method for engineering physiologic cartilage discs from self-assembling hMSCs (Aim 2), and (iii) improve the organization and stability of cartilage discs by implementing spatiotemporal control during induction in vitro (Aim 3).

First, the usefulness of subcutaneous implantation in mice for studying the development and maintenance of osteochondral tissues in vivo was determined. By studying juvenile bovine osteochondral tissues, similarities in the profiles of endochondral ossification between the native and ectopic processes were observed. Next, the effects of extracellular matrix (ECM) coating and culture regimen on cartilage formation from self-assembling hMSCs were investigated. Membrane ECM coating and seeding density were important determinants of cartilage disc formation. Cartilage discs were functional and stratified, resembling the native articular cartilage. Comparing cartilage discs and pellets, compositional and organizational differences were identified in vitro and in vivo. Prolonged chondrogenic induction in vitro did not prevent, but expedited endochondral ossification of the discs in vivo. Finally, spatiotemporal regulation during induction of self-assembling hMSCs promoted the formation of functional, organized and stable hyaline cartilage discs.

Selective induction regimens in dual compartment culture enabled the maintenance of hyaline cartilage and potentiated deep zone mineralization. Cartilage grown under spatiotemporal regulation retained zonal organization without loss of cartilage markers expression in vivo. Instead, cartilage discs grown under isotropic induction underwent extensive endochondral ossification. Together, the methods established in this dissertation for investigating cartilage maturation in vivo and directing hMSCs towards generating physiologic cartilage in vitro form a basis for guiding the development of new treatment modalities for osteochondral defects.