Regeneration of Articular Cartilage using Mesenchymal Stem Cells

Articular cartilage injury is a major risk factor for the development of osteoarthritis, a condition that results in significant patient morbidity and substantial cost to the healthcare system. Due to the limited capacity of articular cartilage to regenerate, early intervention is required to prevent the progression of focal chondral and osteochondral defects to advanced disease and joint degeneration. Effective management options are limited at present. Mesenchymal stem cell (MSC) transplantation is a promising treatment strategy given the high proliferative capacity of MSCs and their potential to differentiate into cartilage-producing cells. MSCs may be harvested through minimally invasive techniques, such as bone marrow aspiration, which do not require joint surgery or extraction of cells from healthy cartilage as required by other cell-based strategies. Although preclinical and clinical studies have demonstrated that MSCs are capable of producing hyaline-like repair tissue and improving functional outcome following transplantation into articular cartilage defects, tissue engineering and transplantation variables are actively being studied with the goal of optimizing this treatment modality. The experiments of this thesis focused on the investigation of bone marrow-derived MSC (BMSC) isolation and expansion environment, biomaterial matrix composition and seeding density, and chondrogenic differentiation conditions in BMSC-based cartilage engineering using in vitro and in vivo models. The first study involved an assessment of incubator oxygen tension during ovine BMSC isolation, expansion and differentiation on in vitro BMSC chondrogenesis within porous scaffolds composed of type I collagen or esterified hyaluronic acid. Hypoxic culture was shown to augment chondrogenesis. Differences in gene expression and construct size were noted between scaffolds, although extracellular matrix formation consistent with hyaline-like cartilaginous tissue was noted within each scaffold. This was followed by a second study that characterized contraction of collagen I scaffolds seeded with human BMSCs that were isolated and expanded under different oxygen tensions. During chondrogenesis, BMSC-seeded scaffolds increased in size initially and then progressively contracted to the end of the 30-day culture period. Scaffold-specific and cell-mediated diameter changes were elucidated. Hypoxic isolation and expansion significantly reduced scaffold contraction. Thereafter, a third study investigated in vitro chondrogenesis of ovine BMSCs within a collagen I scaffold following isolation and expansion in either two-dimensional or three-dimensional environments. Both protocols yielded cells with hyaline-associated gene expression and extracellular matrix molecule production. Optimal scaffold seeding densities for chondrogenesis were established. The final study utilized an in vivo sheep model with full-thickness articular cartilage defects to assess a novel protocol for BMSC transplantation that involved BMSCs that were isolated, expanded, seeded within an esterified hyaluronic acid scaffold, and chondrogenically primed for a short duration in chondrogenic medium prior to implantation. The impact of oxygen tension during pre-implantation ex vivo culture on cartilaginous tissue formation was investigated. Implantation of BMSC-seeded scaffolds yielded repair tissues that varied in quality between hyaline-like cartilage and fibrocartilage. Defects implanted with cell-seeded scaffolds had significantly higher histological scores and repair tissue areas than cell-free controls. A consistent effect of oxygen tension was not established across animals. The studies described in this thesis demonstrate methods of optimization of BMSC chondrogenesis within in vitro and in vivo models through modulation of tissue engineering variables. Although the outcomes of this work are promising, further investigation is required to establish techniques that may be used in BMSC transplantation protocols to promote the reproducible creation of tissue that resembles articular cartilage.