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Autologous Cell-based Human Meniscus Tissue Engineering

  • Author / Creator
    Yan Liang
  • The knee menisci are a pair of weight-bearing fibrocartilaginous tissues between the femoral condyles and tibial plateau. They are essential for mechanical load distribution and transmission, lubrication, and stability of the knee joint. Meniscus injury is a risk factor for the onset of knee osteoarthritis (OA), which is characterized as a progressive degenerative disease resulting in cartilage breakdown. The avascular nature of the inner meniscus regions limits their intrinsic healing capacity and early intervention is required after injury to prevent OA. The long-term clinical outcomes after partial meniscectomy for inner meniscus injuries are poor. Cell-based meniscus tissue engineering by (re)-differentiating autologous cells towards an inner meniscus-like extracellular matrix (IM-ECM)-forming phenotype is an emerging strategy to repair or replace damaged tissues. Human meniscus fibrochondrocytes (MFCs) and mesenchymal stem cells (MSCs) can be isolated from surgical debris and through synovial fluid using minimally invasive techniques, respectively. Even though studies using these cell sources showed promising results regarding meniscus-like ECM formation in vitro and in animal studies, tissue engineering variables are being investigated to optimize the outcomes prior to clinical implantation. The studies in this thesis focused on investigating cell isolation and expansion conditions, inductive stimuli for IM-ECM formation, and phenotype stability of in vitro preformed IM-ECM in human MFC and synovial fluid-derived MSC (SF-MSC)-based meniscus tissue engineering. For human MFCs, the first experiment aimed to identify the population doublings (PDs) of TGF-β1 and FGF-2 (T1F2)-expanded MFCs to retain their chondrogenic redifferentiation capacity under normoxia (21% O2) and hypoxia (3% O2) using an in vitro cell pellet model. The data demonstrated that MFCs with PDs of up to 10 can undergo chondrogenic redifferentiation. The IM-ECM formation was most pronounced in MFCs with PDs ranging from 2.5 to 3.3 under hypoxia. Moreover, the MFCs did not undergo osteogenic differentiation. A subsequent study was performed to investigate whether human meniscus-derived decellularized matrix (DCM) can re-differentiate T1F2-expanded MFCs to form IM-ECM under hypoxia (3% O2). The DCM supported IM-ECM formation only with an exogenous chondrogenic factor. A third experiment was performed to investigate the effects of oxygen levels, transient TGF-β3 supplementation (3 weeks), and long-term culture with TGF-β3 (8 weeks) on IM-ECM production by MFCs within a type I/III collagen scaffold. The study also assessed the behavior of the engineered IM-ECM after subcutaneous implantation in nude mice. The results showed that the constructs expressed genes and proteins associated with IM-ECM, especially in hypoxia. Long-term culture (8 weeks) led to superior IM-ECM compared to 3 weeks, but only with TGF-β3 in hypoxia. The IM-ECM formed after 3 weeks’ hypoxic chondrogenic culture was better retained compared to normoxia and became vascularized without calcification after 5 weeks’ implantation in nude mice. For human SF-MSCs, the first experiment was to investigate whether TGF-β3, insulin-like growth factor 1 (IGF-1), and human meniscus-derived (DCM) can induce differentiation of SFMSCs towards an MFC phenotype under hypoxia (3% O2). In pellets, combined TGFβ3 and IGF-1 synergistically enhanced IM-ECM formation compared to growth factors alone. In DCM, the combination of TGF-β3 and IGF1 induced IM-ECM production and upregulated aggrecan, collagens I and II expression compared to DCM alone. The differentiated SF-MSCs also showed little expression of hypertrophic differentiation marker type X collagen. A subsequent experiment was performed to assess whether hypoxic (2% O2) compared to normoxic (21% O2) chondrogenic culture can drive SF-MSCs to produce meniscus-like ECM rich with angiogenesis-promoting factors VEGF and SDF-1 within a type I collagen scaffold in vitro. The angiogenic potential of SF-MSCs and the stability of the generated ECM regarding calcification were tested using a subcutaneous nude mouse model. IM-ECM formation and production of VEGF by SF-MSCs was enhanced by HYP in vitro. IM-ECM from both oxygen tensions underwent vascularization and did not calcify in the nude mice. In summary, the experiments in this thesis showed that enough MFCs with the capacity to form IM-ECM can be obtained through growth factor supplementation. Hypoxic preculture of both cell sources promote a chondrogenic phenotype to form IM-ECM rather than osteogenic differentiation both in vitro and in vivo. Moreover, this work showed that precultured ECM allowed vascularization at the ectopic sites, which showed promise to enhance the repairing potential of avascular meniscus injuries.

  • Subjects / Keywords
  • Graduation date
    Spring 2020
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-bm4w-df11
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.