Modulation of Mesenchymal Stem Cell Behaviour via Integrin- Extracellular Matrix Interactions

Richard Mills (2011). Modulation of Mesenchymal Stem Cell Behaviour via Integrin- Extracellular Matrix Interactions PhD Thesis, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland.

       
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Author Richard Mills
Thesis Title Modulation of Mesenchymal Stem Cell Behaviour via Integrin- Extracellular Matrix Interactions
School, Centre or Institute Australian Institute for Bioengineering and Nanotechnology
Institution The University of Queensland
Publication date 2011-06
Thesis type PhD Thesis
Supervisor Professor Justin Cooper-White
Associate Professor Ernst Wolvetang
Total pages 248
Total colour pages 50
Total black and white pages 198
Language eng
Subjects 10 Technology
Abstract/Summary Mesenchymal stem cells (MSCs) have great potential for regenerative medicine and are an ideal cell source for tissue engineering (TE) applications. Realising the potential of MSCs within tissue engineered therapies relies on our ability to develop integrated biomaterials that combine various microenvironmental cues to guide cellular behaviour. This will require an increased understanding of basic MSC biology and instructive cues to predictably and explicitly control cell-biomaterial interactions. This thesis primarily focuses on understanding integrin-extracellular matrix (ECM) interactions to dictate MSC differentiation and migration characteristics. Integrins are the primary link between MSCs and their surrounding ECM, with different integrin pairs having specificity for different ECM molecules. It is widely acknowledged that the type of ECM present can influence MSC differentiation and migration. However, it is yet to be determined how integrin-ECM expression changes during the differentiation process or how specific integrin-ECM interactions may alter MSC differentiation or migration characteristics. Initially, a comprehensive analysis of integrin and ECM expression throughout osteo-, adipo- and myogenesis of MSCs was performed to gain a clearer understanding of the changes during differentiation. We determined that osteogenesis is characterized by a sharp increase in integrin a5 expression after seven days, adipogenesis by a significant increase in a6 expression and smooth muscle differentiation (myogenesis) by an increase in a4. In parallel to the changes of integrin expression, we observed increased collagen-I and -IV and decreased fibronectin during osteogenesis. Collagen-I and -IV were also laid down during adipo- and myogenic differentiation, however there was additional expression of laminin during adipogenesis and fibronectin during myogenesis, respectively. After identifying the key integrin and ECM changes, we investigated the premise that MSC differentiation can be optimised by activating the appropriate integrin-ECM interactions. We subsequently used a PS-PEO surface to present defined ECM motifs (RGD, RRETAWA, IKVAV and YIGSR), to activate the desired MSC integrins. By activating previously identified integrin-ECM interactions, we demonstrated that differentiation could be enhanced towards adipogenic (IKVAV), chondrogenic (YIGSR) and smooth muscle (RGD) lineages. Surprisingly, our data showed that activating a5B1 (RRETEWA), even though the a5 subunit dramatically increases during osteogenesis, did not provide the best surface for osteoblast induction and IKVAV was most conducive for osteogenic differentiation. These results demonstrate that the lineage progression of hMSCs is influenced by the presentation of integrin binding motifs, further confirming the role of integrin specificity in hMSC differentiation. Following on from this, the role of integrin-ECM interactions on MSC migration was examined. However, assessing this using traditional assays is difficult, as experimental outcomes are often compromised by the inability to maintain a defined surface prior to the initiation of migration. We therefore developed a microfluidic migration device that maintains a defined surface with no cellular damage and that has not been confounded by previous exposure to cells or secretions during commencement of the assay. This device allows the investigation into defined cell-substrate interactions and was subsequently utilised to evaluate the key integrin-ECM interactions during MSC migration over ECM molecules (fibronectin, collagen I, II and IV, laminin) and the effect of osteo- and chondrogenic differentiation on their migration potential. We determined that MSCs interactions with fibronectin are mediated through a3B1, a5B1 and aVB3, collagen I via a1B1 and a3B1, collagen II with a2B1, collagen IV through a1B1 and laminin via a2B1 and a3B1. In addition, we observed that as MSCs progressed down either lineage their ability to migrate, on any of the tested ECM substrates, decreased. These findings are of interest in the basic understanding of MSC migration but are also relevant to the colonization of scaffolds and development of TE strategies. The architectural features of a scaffold are also known to influence cellular infiltration and colonisation. To investigate the relationship between cell migration and geometrical constraints we modified the channel configuration of the migration assay to present two dimensional geometric constraints often seen within traditional scaffold architectures. Using 3T3 fibroblasts and MSCs we determined that the migration characteristics of different cell types influences their response to various geometric challenges, including channel width, channel contraction and expansion gradient and channel junction type. 3T3 fibroblasts migrated as a wave and were substantially affected by a variety of geometrical encounters, where as hMSCs migrated as single cells, and only responded to geometric constraints when they were comparable to the length scales of a single cell. Through N-cadherin blocking studies we showed that the difference in migration characteristics was a result of the cell-cell contact present whilst 3T3 fibroblasts migrated. This highlights that differences in migration characteristics of different cell types can be deterministic of how they may respond to geometric constraints within porous TE constructs. These studies illustrate the importance of understanding cell-biomaterial interactions in order to present appropriate cues to dictate MSC behaviour, in addition to presenting a number of novel microfluidic platforms to probe cell migration in response to bound, soluble and geometrical stimuli. It is hoped that the insights provided by this thesis will aide in the development of new generation, integrated biomaterials for tissue engineering therapies.
Keyword Mesenchymal stem cells (MSC)
Multipotent Mesenchymal Stromal Cells
Biomaterials
Integrin
Extracellular Matrix
Microenvironment
Differentiation
Microfluidics
Migration Assay
Migration
Additional Notes Colour Pages: 27, 29, 33, 38, 51, 61, 63, 79, 81, 83, 84, 85, 95, 105, 107, 108, 110, 112, 114, 117, 120, 123, 133, 138, 145, 151, 157, 162, 169, 174, 177, 179, 181, 182,189,195, 198 – 205, 215, 225, 234 – 236, 239 Landscape Pages: 120

 
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Created: Fri, 09 Dec 2011, 15:56:04 EST by Mr Richard Mills on behalf of Library - Information Access Service