Biomimetic Scaffolds and Mechano-Hypoxia Conditioning for Meniscus Tissue Engineering

  • Author / Creator
    Szojka, Alexander R.A.
  • The menisci are a pair of knee joint structures that are wedge-shaped in cross-section and semi-lunar in superior view. They play roles in force distribution that help maintain the health of the articular cartilage. In adults, the menisci can be divided into a fibrous, vascularized outer region and a fibrocartilaginous, avascular inner region. Being avascular, the inner regions cannot heal upon injury, which predisposes the knee to early osteoarthritis development. The principal biochemical constituents of meniscus fibrocartilage include type I collagen and hyaline cartilage-associated biochemicals type II collagen and aggrecan. Meniscus fibrocartilage is synthesized and maintained by cells known as meniscus fibrochondrocytes (MFCs), which exist natively in a hypoxic and mechanically-loaded environment. Meniscus tissue engineering (MTE) aims to generate cell-based meniscus replacements in vitro that slow or prevent osteoarthritis development after incidence of non-healing meniscus injuries. This is accomplished using a suitable combination of biomaterial scaffolds, cells, and growth signals. To this end, this thesis focuses on: i) development of biomimetic meniscus scaffolds; ii) investigating the MFC response to combined hypoxia and mechanical loading; and iii) identifying how this knowledge can be used to enhance fibrocartilage histogenesis in vitro.

    Chapters 1 and 2 introduced the research topic with a review of meniscus development, comparative anatomy, and hypoxia and mechanical loading in MTE. This review suggested that the fibrocartilaginous phenotype of the adult human inner meniscus is likely an adaptation to its hypoxic and mechanically-loaded environment. The first project (Chapter 3) described design and production of 3D printed biomimetic meniscus scaffolds. The second project (Chapter 4) presented an investigation of the simultaneous response of MFCs to combined hypoxia and supplementation of the growth factor TGF-β3 in high-density cell aggregates (pellets). These treatments had synergistic interactions that potently promoted fibrocartilage histogenesis. Chapter 5 extended this study to a larger-scale model with a porous type I collagen scaffold. Hypoxia had few effects in this scaffold, even when combined with mechanical loading (dynamic compression (DC)). Chapter 6 investigated the factorial effects of cell expansion, hypoxia, and culture time to identify more suitable static pre-culture conditions on the scaffold before treatment with hypoxia and mechanical loading. Cell expansion had few effects, but mechanical properties and the hypertrophic phenotype increased continually up to the latest measured time point (9 weeks). Hypoxia suppressed both the hypertrophic phenotype and mechanical property development. Based on these outcomes, Chapter 7 investigated an intermediate pre-culture period in normal oxygen levels prior to a short-term treatment with hypoxia and mechanical loading (DC) to identify their effects on transcriptome-wide gene expression. The immediate MFC response to the combined treatment supported fibrocartilage histogenesis in an additive rather than synergistic manner. Chapter 8 described a series of four follow-up studies that used a shortened pre-culture period of 3 weeks based on experiences in Chapter 7 prior to hypoxia and mechanical loading treatments. First, the treatments were extended to five days and to include additional types of daily mechanical loading: i) a novel DC regime combining displacement and load control, and ii) cyclic hydrostatic pressure. The original DC regime, used in Chapter 7, was most effective in gene regulation. RNA sequencing showed that the longer-term combined treatment also supported fibrocartilage histogenesis, especially in promoting the hyaline cartilage-like aspect of the inner meniscus phenotype. The second and third studies of Chapter 8 showed that the gene expression response to DC saturated after 5 minutes and increased in magnitude with higher applied strain. The last study considered a long-term treatment of mechano-hypoxia conditioning, applying continuous hypoxia with four daily incidents of DC. The outcomes included increased equilibrium dynamic modulus to levels that rival the native meniscus, although further experimentation is required. Chapter 9 summarized the work and suggested future research directions.

    This thesis has three main contributions: i) the design of biomimetic meniscus scaffolds that can be used for complete meniscus tissue engineering; ii) the knowledge that the gene expression response of MFCs to combined hypoxia and mechanical loading is, after up to five days, primarily additive; and iii) the early evidence that combined hypoxia and mechanical loading, “mechano-hypoxia conditioning,” can be a useful strategy to promote fibrocartilage histogenesis in vitro for MTE.

  • Subjects / Keywords
  • Graduation date
    Fall 2021
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.