UC Berkeley

Articular cartilage is the highly specialized tissue found in diarthrodial joints that provides frictionless movement between bone, acting as a shock absorber and facilitating the transmission of loads. Due to the low cellularity and avascular nature of articular cartilage, the tissue has a limited ability to self-heal following trauma or degenerative diseases, such as osteoarthritis (OA). The aging population and a rise in younger patients (< 60 years old) developing cartilage defects has led to a growing interest in developing biological approaches that aim to repair early-stage cartilage lesions, reduce patient pain, and prolong the need for total joint replacements.

Cartilage tissue engineering strategies that use autologous chondrocytes require in vitro expansion of cells to obtain enough cells to produce functional engineered tissue. However, chondrocytes dedifferentiate during expansion culture, limiting their ability to produce chondrogenic tissue and their utility for cell-based cartilage repair strategies. Adding growth factors, known as “priming”, to the culture media during expansion culture has been shown to alter cell properties during two-dimensional (2D) culture and alter tissue production once the cells are returned to a three-dimensional (3D) environment. Specifically, the use of a growth factor cocktail consisting of transforming growth factor beta-1 (TGF-β1), fibroblast growth factor two (FGF2), and platelet-derived growth factor-ββ (PDGF-ββ) has demonstrated potential in enhancing chondrocyte redifferentiation and promoting the production of chondrogenic tissue. Previous studies demonstrate that alterations in expansion culture conditions have a significant and longterm impact on 3D tissue production; however, the mechanisms underlying this effect are not well understood. Further, it is not known whether specific biomarkers exist during 2D culture that could predict successful development of cartilage-like tissue in 3D culture. Thus, the overall objective of this dissertation was to establish a relationship between chondrocyte properties during 2D expansion culture and 3D matrix production.

Differences in chondrocyte transcriptional, biomechanical, and morphological properties during monolayer expansion culture and how these relate to differences in chondrogenic tissue production in bovine chondrocytes primed and unprimed during expansion culture were investigated. Unbiased analysis, supported by experimental studies, identified conditions that favor cartilage production and the mechanobiological mechanisms responsible for these benefits. Specifically, growth factor priming was shown to override mechanobiological pathways to prevent 2 chondrocytes from remodeling their cytoskeleton in response to the stiff, monolayer microenvironment. Differences in cell adhesion, morphology, and cell mechanics between primed and unprimed cells were mitigated by passage 5, suggesting that priming may have a diminished effect on cells with extended culture time. Importantly, primed cells had increased ability to redifferentiate upon transfer to 3D culture to produce engineered tissue with greater tissue modulus, glycosaminoglycan (GAG) content, and total collagen content than engineered cartilage secreted from unprimed cells. Furthermore, whether the positive effects of growth factor priming observed in healthy juvenile animal donor cells could be replicated in chondrocytes obtained from adult human donors was examined. Consistent with findings using juvenile bovine chondrocytes, cells primed during expansion culture produced extracellular matrix with greater modulus, GAG, and collagen content than cells unprimed. Taken together, these findings show promise for growth factor priming as a method for reducing variability in cartilage tissue engineering outcomes.

To investigate whether juvenile bovine chondrocytes expanded in monolayer culture maintain their ability to redifferentiate to produce chondrogenic tissue, differences in unprimed cell phenotype through multiple passages and their 3D tissue production were compared. Genomewide transcriptional differences in chondrocyte phenotype between passage 1, 3, and 5 were examined and cells from passage 3 and 5 were seeded in hydrogel scaffolds for 3D tissue culture. Furthermore, an unbiased computational approach to prioritize genes that were differentially expressed with passaging and enriched in human genome-wide association studies (GWAS) was used to identified genes differentially expressed during dedifferentiation that may also play a role in OA development. This analysis identified 24 genes differentially expressed in monolayer expansion and associated with OA, including novel genes, such as TMEM190 and RAB11FIP4. Many known genes associated with chondrocyte dedifferentiation and osteoarthritic development were identified to be enriched during passaging; however, chondrocytes from both passages were still able to redifferentiate in 3D culture. These findings highlight a shortcoming of using juvenile animal chondrocytes, rather than adult human chondrocytes, which may already be expressing some of the genes this work identified to increase in expression with passaging. These differences in gene expression may contribute to the inability of human chondrocytes to redifferentiate at higher passages.

These findings collectively provide significant insights into autologous chondrocyte dedifferentiation and hold great promise for regenerative medicine approaches that require a large number of cells for de novo tissue development. Specifically, this work enhanced our understanding of how growth factor priming and passage number lead to variations in chondrogenic tissue production. By evaluating differences in cell properties during both 2D expansion and 3D tissue production, this dissertation provides valuable insights into which cells can produce functional engineered tissue, a finding which will enable scientists to overcome a significant obstacle in translating cell-based biological repair strategies into clinical applications.