Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Bioengineering

First Advisor

Robert L. Mauck

Second Advisor

Nathaniel A. Dyment

Abstract

The meniscus is an integral load bearing fibrous tissue of the knee joint that derives its mechanical function from the unique geometry and precise organization and composition of its extracellular matrix (ECM). While the importance of the highly specialized ECM is well appreciated in the mature meniscus, how this structural complexity is achieved during development remains poorly understood, and in particular, what interplay exists between the cells that build the matrix and their rapidly evolving microenvironment is unclear. To address these knowledge gaps, we begin by establishing a detailed timeline of the concurrent spatiotemporal changes that occur at both the cellular and matrix level during murine meniscus maturation, through use of Col1-YFP, Col2-CFP, Col10-mCherry fluorescent reporter mice, as well as histological analysis, and region specific high-throughput qPCR. We report that distinct cellular and matrix features defining specific meniscus tissue zones are present at birth, and that regional specialization continues during postnatal growth and maturation, possibly due to onset of load bearing use. Importantly, we define a framework for investigating the reciprocal feedback between cells and their evolving microenvironment—thus laying the foundation for future mechanistic work. Informed by the finding that key structural features of the meniscus matrix are established at birth, the remainder of this thesis addresses how this nascent organization is established. By analyzing key timepoints in knee joint development, we show that the genesis of ordered meniscus matrix is downstream of early cellular patterning characterized by marked fibrillation of the actin cytoskeleton. This suggests that cells and subcellular structures act as a physical template that directs alignment of the deposited fibrous matrix. Through the use of muscular dysgenesis (mdg) and splotch-delayed (Spd) mouse mutants that lack skeletal muscle contraction and joint motion, we further show that this critical cellular re-arrangement prior to meniscus formation does not fully occur without muscle contraction and leads to tissue dissociation—demonstrating that extrinsic forces play an instructive role in the tissue’s formation. Finally, we probe the impact of embryonic cell-mediated physical cues (adhesion, cytoskeletal arrangement) on subsequent meniscus assembly by generating targeted deletion of non-muscle myosin isoforms NM-IIA and NM-IIB (Myh dKO) in meniscus precursor cells during knee development. We demonstrate that cells of Myh dKO animals have defective cellular connectivity and so assemble a disorganized fibrillar matrix at birth, but these deficiencies in matrix alignment are somewhat corrected with postnatal maturation. Together, this work establishes that both cell-generated and extrinsic physical cues are imperative in the establishment of the initial meniscus structure that is built upon and further refined during postnatal growth.

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