title: Engineering surface mobility to direct stem cell fate
Creators: name: Bathawab, Fatma Mirfat
Publisher: name: University of Glasgow
Year: 2017
Subjects: subject: Polymer mobility, stem cell differentiation, BMP-2, interfacial protein layer, extracellular matrix.

description: Since the first contact and fusion of an egg and sperm and throughout development, a cell lives a life of constant communication with its environment. Cells interact with the external environment via a layer of proteins and respond to not only biochemical cues but also physical properties including stiffness and topography of adjacent surfaces. However, even though polymeric biomaterials have been described as one of the cornerstones of tissue engineering, the effect of an intrinsic polymer property known as mobility on cell behaviour is poorly characterised. Mobility is a physical property of polymers inversely proportional to the glass transition temperature (Tg); the temperature at which polymers undergo a transition between a rubbery viscous state to a glassy brittle solid. Therefore, films of four poly(alkyl acrylates) with similar surface chemistry but different glass transition temperatures achieved by varying branch chain lengths (1, 2, 4 or 6 methyl groups) were used in this work to investigate the role of polymer mobility on cell behaviour. I verified using atomic force microscopy the similarity in topography and stiffness between the four substrate surfaces and ascertained that fibronectin molecules adsorb in a globular conformation on the polymer with the shortest side chain (1 methyl group) compared with a more extended conformation on the rest of the polymers. My study of the fibronectin coatings using fluorescence recovery after photobleaching (FRAP) on the different polymer surfaces suggested that the mobility of the polymer substrate is translated to the interfacial protein layer. This interesting finding highlighted a possible pathway for cells cultured on fibronectin coated polymer surfaces to detect the underlying polymer mobility via the fibronectin coating. The interaction of cells with surfaces occurs via membrane proteins which interact with specific structural sites within extracellular matrix proteins; these include the cell binding site (RGD: Arginine Glycine Aspartic acid amino acid motif) and the Synergy site (PHSRN: Proline Histidine Serine Asparagine amino acid motif). My ELISA analyses indicated a higher exposure of these important cell-binding sites on the more extended fibronectin compared with the globular one however, this did not correlate to the mobility of polymers or the mobility of the fibronectin layer. This was also the case for myogenic cell differentiation, which was indiscriminately higher on polymers with extended fibronectin, however, cytoskeletal contractility was found to play an essential role in the myogenic differentiation of cells on these polymers in a mobility dependent manner. We then sought to understand the role of
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mobility in modulating osteogenic differentiation of human MSCs in the presence and absence of stimulation with BMP-2. The Fibronectin network-forming polymer with the lowest mobility (side chain of 2 methyl groups) induced the highest expression of osteogenic markers in the absence of BMP-2 stimulation. My mechanistic studies using specific inhibitors also revealed that the Erk1/2 pathway was required for this increase in osteogenic markers, while contractility, unlike in myogenesis produced only minimal effects on osteogenic differentiation. In this set of polymers, mobility increases with side chain length, while all the polymers with more than one methyl group in their side chain induced the independent formation fibronectin networks upon adsorption. The polymer with two methyl groups in its side chain is characterised with the lowest mobility among the three fibrillogenesis - inducing polymers, and the highest expression of osteogenic markers in the absence of BMP-2. In the presence of BMP-2, smad phosphorylation was also higher on this polymer suggesting a combined synergistic effect towards osteogenic differentiation provided by the simultaneous activation of the Erk1/2 pathway and high phosphorylation of smad1/5/8. My observations suggest that fibronectin fibrils coating a polymer with low mobility may be most suited for osteogenic differentiation of hMSCs by simultaneously exposing cell-binding sites to a higher degree. Thus, inducing Erk1/2 signalling and presenting BMP-2 in a manner that stimulates the highest phosphorylation of smad1/5/8 hence achieving a stronger synergistic effect on the overall expression of osteogenic markers. The findings from this work strongly support previous studies suggesting that polymer mobility is a subtle change in the substrate with significant downstream biological significance and is crucial to understand to improve the application of polymeric biomaterials.