Macromolecular crowding optimisation and application in co-culture setting for tendon regeneration

Tendon is a connective tissue that links bone to muscle, allowing for maintenance of skeleton posture, joint movement, energy storage and transmission of muscle force to bone. Tendon is a hypocellular and hypovascular tissue of poor self-regeneration capacity. Current surgical treatments are of limited success, frequently resulting in reinjury. Upcoming cell therapies are primarily based on tenocytes, a cell population of limited self-renewal capacity in vitro or mesenchymal stromal cells, a cell population prone to ectopic bone formation in vivo. Over the years mono- or multi- factorial cell culture technologies have failed to effectively maintain tenocyte phenotype in culture during expansion or to prime mesenchymal stromal cells towards tenogenic lineage prior to implantation. Upon these limitations the concept of co-culture was conceived. The utilisation of co-culture techniques to induce mesenchymal stromal cell lineage-specific differentiation is a compelling area of research within the field of tissue engineering. Through co-culture, functional tissue-specific cell populations can be developed for tissues of low cellularity and cells of reduced function and / or limited expansion capacity ex vivo, such as tendon. Providing cells with relevant stimuli to support their phenotype and function through in vitro recapitulation of the in situ microenvironment with biophysical, biological and biochemical cues is of paramount importance. A strategy that has yet to be investigated in co-culture setting is macromolecular crowding, the addition of macromolecules in cell culture media that by decreasing diffusion via restriction of molecular motion, they favour protein-substrate interactions and result in enhanced and accelerated extracellular matrix deposition. In native tissue context, a continuous dynamic reciprocity between cells and their surrounding extracellular matrix maintains tissue-specific cell phenotype and/or differentiates mesenchymal stromal cells towards a tissue-specific lineage, via a finely-tuned spatiotemporal sequence of biophysical, biochemical and biological signalling cascades. This work puts forward the notion that co-culture can be significantly improved in a macromolecular crowding milieu due to the enhanced extracellular matrix present. To validate this, first an optimal (with respect to highest extracellular matrix deposition in the shortest period of time) macromolecular crowding agent that enhances and accelerates extracellular matrix deposition should be identified (objective 1, chapter 2) and then its potential in direct co-culture of mesenchymal stromal cells / tenocytes should be assessed (objective 2, chapter 3). For objective 1, the potential of gum Arabic, gum gellan, gum karaya and gum xanthan as macromolecular crowding agents in WS1 skin fibroblast cultures (no macromolecular crowding and a ready-to-use powder consisting in the combination of kappa and lambda carrageenan isoforms were used as control) were assessed. WS1 skin fibroblasts were chosen for simplicity purposes and as there was limited supply of tenocytes and mesenchymal stromal cells. Dynamic light scattering analysis revealed that all macromolecules had negative charge and were polydispersed. None of the macromolecules affected basic cellular function. At day 7 (longest time point assessed), gel electrophoresis analysis revealed that all macromolecules significantly increased collagen type I deposition in comparison to the non-macromolecular crowding group. Also at day 7, immunofluorescence analysis revealed that carrageenan; the 50 µg/ml, 75 µg/ml and 100 µg/ml gum gellan; and the 500 µg/ml and 1,000 µg/ml gum xanthan significantly increased both collagen type I and collagen type III deposition and only carrageenan significantly increased collagen type V deposition, all in comparison to the non-macromolecular crowding group at the respective time point. This preliminary study demonstrated the potential of gums as macromolecular crowding agents, but more detailed biological studies are needed to fully exploit their potential. Considering that carrageenan outperformed the assessed gums in extracellular matrix deposition, this macromolecular crowding agent, along with a Ficoll cocktail that has been used in other macromolecular crowding studies, were progressed to the second objective. In objective 2, carrageenan and Ficoll were assessed in bone marrow mesenchymal stromal cell alone, tenocyte alone and bone marrow mesenchymal stromal cell / tenocyte direct co-cultures (no macromolecular crowding cultures were used as control). These two macromolecular crowding agents were selected to also assess whether the chemistry of the macromolecular crowding agents can affect cellular phenotype. Carrageenan increased extracellular matrix deposition without altering cell metabolic activity, proliferation and morphology. Ficoll, on the other hand, did not notably enhance extracellular matrix deposition. Subsequent biological analysis via Western blotting and lectin microarray revealed chondrogenic and osteogenic lineage commitment, but not enhanced tenogenic lineage commitment and no cell-type-specific carbohydrate, respectively. Collectively, these data demonstrate that there are more than the physical properties of a macromolecular crowding agent to determine the rate of extracellular matrix deposition (e.g. negatively charged and polydispersed macromolecules enhance and accelerate extracellular matrix deposition but other factors intervene as size and shape of the crowder), whilst the chemical properties of a macromolecular crowding agent determine cell fate (e.g. sulphated and neutral polysaccharides induce osteogenic and/or chondrogenic lineage commitment).

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University of Galway

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University of Galway Theses (PhD Theses)