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hypothesis has been supported by a variety of co-culture experiments in vitro.^14-17 Structurally, the niche is formed by supporting cells that provide a microenvironment for stem cells and the signals emanating from the supporting cells.^18-20 However, it still remains a major challenge to accurately define the precise cellular components and anatomical structure of the niche.

The ECM comprises a scaffold of collagens and other structural proteins that are interlaced with proteoglycans which, together, control the local mechanical environment and contribute to the

“stem cell niche” microenvironment through their own signaling moieties and their ability to bind growth factors, cytokines, enzymes, and other diffusible molecules. Compared to traditional 2D culture, culturing stem cells in 3D environments provides another dimension for external mechanical inputs and for cell adhesion, which dramatically affects integrin ligation, cell contraction, and associated intracellular signaling.^21;22 Furthermore, the 3D environment might be necessary to model morphogenetic and remodeling events that occur over larger-length scales.

The mechanisms by which nanotopographic cues derived from ECM influence stem cell proliferation and differentiation are not well investigated yet but appear to involve changes in cytoskeletal organization and structure, potentially in response to the geometry and size of the underlying features of the ECM. That is, changes in the feature size of the substrate may influence the clustering of integrins and other cell adhesion molecules, thus altering the number and distribution of focal adhesions. The composition and function of adhesions characterized in 3D matrices derived from tissues or cell culture were demonstrated to be different from focal and fibrillar adhesions characterized on 2D substrates in their content of α5β1 and α^νβ3 integrins, paxillin, other cytoskeletal components, and tyrosine phosphorylation of FAK.²³ Relative to 2D substrates, 3D matrix interactions also display enhanced cell biological activities and narrowed integrin usage. Similarly, in recent years, the importance of ECM has been recognized due to the application of decellularized tissue matrix in organ or tissue regeneration.^24-30 Partially, it is because of the highly conservative nature of ECM components between species. Different from the above studies, we deposited ECM using stem cells and decellularized it for reconstructing this ex vivo microenvironment for cartilage engineering due to its demand of a large quantity of high quality cells. Our first success came from the porcine SDSC deposited DECM, which enhanced the proliferation and chondrogenic potential when applied as an expansion system compared to traditional expansion on plastic flask for porcine SDSC.¹¹ Human SDSC (both fetal and adult SDSC) deposited DECM also exhibited the same effect as shown in Chapter 4, Chapter 5 and Chapter 6. The investigation of the DECMs deposited by adipose and urine derived stem cells and dermal fibroblasts in Chapter 4 also showed that, despite the differences in these DECMs, all were able to enhance the self-renewal and chondrogenic potential compared to the 2D culture. It not only demonstrated the advantage of 3D DECMs over 2D plastic culture in facilitating ex vivo expansion but also suggested that there are common core components among all these DECMs.

The exploration of these similarities in future studies would benefit the development of an ex vivo expansion system for cartilage engineering.

Communication within the niche is essential for the maintenance of proper stem cell function and for determining the rate of stem cell self-renewal. Soluble factors may act locally or may diffuse

throughout the niche to direct stem cell fate decisions. Studies indicate that supporting cells, which are located adjacent to stem cells, secrete soluble factors that are required for maintaining stem cell identity and for specifying stem cell self-renewal.^31-33 Soluble factors, such as growth factors and cytokines, are important for the initiation and control of stem cell differentiation. A wide variety of soluble growth factors, such as FGF-2,³⁴ TGF-β,³⁵ vascular endothelial growth factor, and hepatocyte growth factor,³⁶ bind to a component of ECM, which greatly slows their diffusion and therefore serves to fine-tune their local concentrations and gradients.^35;36 Matrix binding can create locally higher concentrations of autocrine growth factors,³⁷ allowing smaller amounts of the factor to signal more effectively.³⁸ In this dissertation, we also investigated the influence of soluble growth factor, FGF-2, on SDSC stemness in Chapter 3. We showed that addition of FGF-2 during cell expansion significantly increased SDSC proliferation and chondrogenic potential. The combination of DECM with FGF-2 further enhanced the proliferation and chondrogenic potential in recellularized SDSCs, suggesting a synergistic effect between them. However, unlike DECM, the addition of FGF-2 also significantly increased the expression of hypertrophic markers (type X collagen).

The physiological condition, including oxygen tension, is an important component of the stem cell microenvironment and has been shown to play a role in regulating both embryonic and adult stem cells. Low oxygen tensions (hypoxia) maintain undifferentiated states of embryonic, hematopoietic, mesenchymal, and neural stem cell phenotypes and also influence proliferation and cell-fate commitment. Despite the presence of a decreased osteogenic and chondrogenic potential when induced to differentiate in hypoxic conditions,³⁹ adipose stem cells exhibited increased chondrocytic markers when expanded in hypoxic conditions and differentiated in normoxic cultures.⁴⁰ The effect of hypoxia in committing adipose stem cells to chondrocytes is thought to be mediated by hypoxia-inducible transcription factor (HIF)-1α. Inhibition of HIF-1α leads to decreased chondrogenic potential, normal osteogenic potential, and enhanced adipogenic potential.⁴¹ The role of hypoxia and HIF-1α in cell differentiation is tissue-specific, because HIF-1α maintains the stem cells in an undifferentiated state, inhibits differentiation of mesenchymal cells into osteoblasts, adipocytes, and myocytes, and stimulates differentiation into chondrocytes.^42-45 These data support the role of oxygen tension as an important factor in the determination of cell fate and maintenance of stemness in adipose and bone marrow derived MSCs. In Chapter 3, application of low oxygen level (5%) in combination with FGF-2 or DECM during expansion significantly promoted proliferation with strong chondrogenic potential, suggesting the maintenance of stemness by hypoxia. One limitation of this study is that expression of HIF-1α was not analyzed. In Chapter 5, we also investigated the influence of hypoxia during pellet culture for chondrogenesis. Our data showed that low oxygen level

increased the pellet size, GAG content, and chondrogenic marker expression after a 14 day induction. Thus, the low oxygen level in pellet culture for chondrogenesis is established.