Abstract
Chondrocytes, derived from the mesodermal lineage, form a cartilage template that serves as the foundation of the developing skeleton during embryogenesis, and are responsible for maintaining the tissue's homeostasis following post-natal growth. In the process of mechanotransduction, chondrocytes convert mechanical signals into a biophysical, cellular response. Work in this dissertation uses in vitro modeling with human induced pluripotent stem cells (hiPSCs) and transcriptomic analysis to investigate the mechanisms through which cartilage disease-causing mutations influence chondrocyte mechanotransduction, with a focus on physiologic joint loading conditions that activate the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4). TRPV4 plays a crucial role in cartilage development as its mutation is associated with a spectrum of pediatric musculoskeletal diseases. In particular, the V620I mutation leads to brachyolmia, a dysplasia characterized by short stature and skeletal abnormalities, while the T89I mutation is associated with a more severe phenotype of metatropic dysplasia. Both mutations have been linked to disrupting chondrocyte hypertrophy during endochondral ossification, the process of long bone growth. Using in vitro disease modeling facilitated by mechanical loading of tissue-engineered cartilage derived from iPSCs gene-edited with these mutations, we investigated their impact on chondrocyte mechanotransduction. RNA-sequencing analysis identified V620I and T89I mutant tissue-engineered cartilage were both associated with a reduced mechanoresponse. In addition, each mutation was linked to a unique transcriptomic signature, with T89I influencing the immediate, transiently activated loading response, while V620I regulated genes activated after 24 hours. A thorough understanding of the pathways through which the chondrocyte’s mechanoresponse is altered due to these mutations enables the identification of tailored targets for the treatment and prevention of skeletal dysplasias caused by gain-of-function mutations in TRPV4. Osteoarthritis (OA) is a complex, multifactorial disease characterized by the progressive degeneration of articular cartilage primarily due to an imbalance of chondrocyte catabolic and anabolic activities. Exome sequencing studies identified a variant in collagen type VI alpha three (COL6A3), one of the monomeric units forming collagen VI, in a patient with familial OA. COL6A3 encodes for one of the monomeric units forming collagen type VI, a differentiating component of the chondrocyte’s pericellular matrix (PCM). Collagen type VI maintains the structural and mechanical integrity of the PCM, acting as a key regulator for the transduction of chemical and mechanical cues to the chondrocyte. Using gene-edited iPSCs harboring the identified variant, we focused on characterizing alterations in mechanotransduction, in addition to the intrinsic and extrinsic factors regulating chondrocyte physiology, including circadian regulation and sensitivity to proinflammatory cytokines. COL6A3 mutant chondrocytes exhibited impaired TRP4-mediated mechanotransduction, characterized by an elevated osmotically-induced Ca2+ signaling response, and a lower anabolic response to channel activation reflected in gene expression and matrix production. RNA-sequencing identified that COL6A3 mutant chondrocytes dysregulated multiple pathways in response to mechanical loading, and exhibited a TRPV4-dependent difference in their mechanosensitive transcriptional profile. In addition, the variant was associated with disruptions in circadian rhythms, marked by the overexpression and shift in phase of the core clock gene BMAL1. Modeling the inflammatory osteoarthritic joint environment revealed increased chondrocyte catabolism and reduced COL6A3 synthesis in mutant chondrocytes, after challenge with the proinflammatory cytokine interleukin-1 (IL-1). Mutation-mediated effects were likely driven by an altered PCM structural and mechanical composition, reflected by the reduced expression of proteins involved in collagen type VI interactions and matrix organization, in addition to a lower PCM elastic modulus measured by immunofluorescence-guided atomic force microscopy. Finally, we developed a targeted knock-in mouse model harboring the COL6A3 mutation for in vivo modeling of the variant, with the induction of OA to be investigated through surgical, injury-induced, and spontaneous age-related models. Overall, understanding the mechanisms underlying altered mechanobiology due to mutations in TRPV4 and type VI collagen will aid in the development of drug targets for therapeutic strategies and disease prevention.
Degree
Doctor of Philosophy (PhD)
Author's Department
Biomedical Engineering
Document Type
Dissertation
Date of Award
5-9-2025
Language
English (en)
DOI
https://doi.org/10.7936/5t9w-8371
Recommended Citation
Harissa, Zainab, "Impact of Disease-Causing Mutations in TRPV4 and Collagen VI on the Mechanobiology of iPSC-Derived Tissue-engineered Cartilage" (2025). McKelvey School of Engineering Theses & Dissertations. 1238.
The definitive version is available at https://doi.org/10.7936/5t9w-8371