Osteoarthritis is an intractable problem affecting millions of Americans every year. Cartilage damaging knee injuries greatly increase an individual’s likelihood of developing osteoarthritis. Therefore, restoring the injured cartilage and normal joint function is paramount to preventing progressive cartilage degeneration. Unfortunately, cartilage has a poor intrinsic healing capacity. Current repair techniques are limited by the formation of cartilage that is mechanically inferior to native cartilage, the limited availability of donor cells for tissue engineering efforts, donor site morbidity, and limited fixation of the graft itself within the defect. Toward engineering a cartilage repair therapy to overcome these shortcomings, the global objectives of this work were:​ 1) to establish benchmark data from native cartilage for cartilage tissue engineering efforts, 2) to use passaged cells to engineer native-like self-assembled neocartilage, 3) to explore the use of a nonchondrocyte cell alternative for cartilage tissue engineering, and 4) to engineer an osteochondral construct incorporating self-assembled neocartilage.

Toward these objectives, this dissertation 1) elucidated structure-function relationships within potential sources of chondrocytes, 2) improved the functional properties of neocartilage engineered with passage chondrocytes, 3) improved the chondrogenic potential of a chondrocyte alternative cell type, dermis-isolated adult stem (DIAS) cells, and 4) examined the suitability of self-assembling neocartilage for inclusion in an osteochondral construct and enhanced the interfacial mechanical properties between the chondral and osseous phases.

The results of this work establish a comprehensive repository of quantitative data regarding both the fetal and juvenile ovine patellofemoral articular cartilages. These data address the dearth of knowledge of the fetal joint, and through a more complete understanding of the juvenile joint, significantly enhance the translational pathway for cartilage repair technologies. Within fetal cartilage, region-based differences in histological, biochemical, and mechanical properties, as well as significant correlations between tensile properties and pyridinoline contents are illustrated. This importantly suggests that fetal cartilage is not biomechanically blank, and that functional adaptation begins in utero. A developmental “order of operations” is proposed, suggesting the following functional properties develop in a specific order:​ GAG content, compressive stiffness, collagen content, pyridinoline content, and finally tensile properties. The order of development of self-assembled neocartilage parallels this pattern, lending further credence to it as a model of in vitro cartilage formation and development.

This work also results in the significant enhancement of the functional properties of engineered neocartilage. First, it is shown that neocartilage is greatly affected by contamination via red blood cells. Treatment of cell isolates with ammonium-chloridepotassium lysing buffer not only significantly reduces the red blood cell content in cell isolates, it also eliminates apoptotic chondrocytes. Carrying this treatment forward to other studies, it is also shown that mimicking salient steps in cartilage formation and treatment of neocartilage with developmentally inspired exogenous stimuli further enhance neocartilage functional properties. Resulting neocartilage proceperties exceed those of the native cartilage from which chondrocytes were sourced and approach adult tissue levels. Furthermore, it is suggested that chondrogenically tuned expansion and aggregate redifferentiation culture together are able to recalibrate passaged/redifferentiated cells to a more immature state, allowing them to be more synthetic than primary chondrocytes. Ultimately, this work allows the use of 8,000 times fewer primary cells to engineer superior neocartilage than previous methods.

The use of DIAS cells as a non-chondrocyte cell alternative for cartilage engineering is considered to address limitations in cell sourcing. DIAS cells that undergo aggregate culture prior to self-assembly show significant increases in GAG and collagen production, as well as compressive stiffness. This work makes strides toward enhancing DIAS cell chondro-differentiation protocols. Despite these results, the use of passaged/redifferentiated chondrocytes is deemed more promising and is therefore examined in subsequent studies.

Toward engineering an osteochondral construct, it is shown that the presence of hydroxyapatite in culture does not affect neocartilage functional properties and that an early time of osteochondral assembly is beneficial. First in this work, hydroxyapatite is identified as a superior ceramic composition to incorporate into an osteochondral graft based on its ability to allow for bone ingrowth, its ability to allow for neocartilage interdigitation, and its superior mechanical strength. It is subsequently shown that the inclusion of this ceramic in culture does not affect neocartilage self-assembly. Furthermore, it is shown that assembling the chondral phase (composed of selfassembled neocartilage) and the osseous phase (composed of the hydroxyapatite ceramic) into an osteochondral culture at an earlier time point greatly enhances the interfacial properties of the construct.

Collectively, this research makes strides toward creating a biomimetic treatment to address cartilage injuries. Through the replication of cartilage developmental processes, the use an in vitro model of cartilage formation, and the application of developmentally inspired stimuli, the formation of native-like neocartilage may be achieved. Through the modulation of timing, a robust interface within a large, shapespecific osteochondral construct can be engineered. Together, these steps establish a translational direction toward treating both small and large defects in articular cartilage which continue to be the most vexing problems in musculoskeletal medicine.