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Roanne R. Jones, Shengli Mi, Bo Chen, Ian W. Hamley, Che J. Connon; Investigating The Mechanical Properties Of Collagen Substrates & Their Influence On The Ex Vivo Expansion Of Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):312.
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© ARVO (1962-2015); The Authors (2016-present)
Various biological materials have been employed for the transplantation of corneal stem cells in the treatment of limbal stem cell deficiency1. Currently amniotic membrane is the preferred substrate for the ex vivo expansion of limbal stem cells. Whilst conventional collagen gels are highly hydrated, they are also structurally week and therefore compressed collagen gels have been suggested as a suitable substrate for this purpose2,3. Previously it has been shown that substrate stiffness can influence stem cell differentiation4 therefore we aim to characterise collagen gels by assessing their mechanical properties and we also aim to measure the effects of substrate stiffness on limbal epithelial cell (LEC) growth and differentiation.
Materials examined were laminin coated compressed (CC), medium (MC) and uncompressed (UC) collagen gels. CC & UC gels were prepared as previously described2. MC gels were partially compressed under a fixed load of 64 g for 2.5 minutes. Rheology was performed to determine the stiffness our substrates. Immunohistochemistry was performed to probe the expression of CK3, CK14 and ZO1 in bovine LEC on collagen constructs.
Rheology measurements show that there is a difference between the storage moduli (stiffness) of CC (2500 Pa), MC (170 Pa) and UC (4 Pa). Stress sweeps indicate that CC can withstand shearing under increased stress up to 20 Pa whilst MC and UC can only withstand shearing up to 5 and 1 Pa respectively.Immunohistochemistry showed dense cell stratification on CC constructs with CK3 and ZO1 expression and loose cell stratification on UC constructs. CK14 and ZO1 expression was evident in MC constructs whilst UC constructs did not appear to express ZO1 markers.
We conclude that the mechanical properties of collagen gels are improved by compression load and that substrate stiffness can influence LEC differentiation. Substrates with weaker mechanical properties do not give rise to well ordered, compact cell layers typically shown by amniotic membrane and plastic compressed collagen gels3,5. By characterising the mechanical stiffness of collagen gels, we can relate measured differences in limbal epithelial growth and differentiation to substrate stiffness.References1. Levis & Daniels (2009) New Current Opinion in Biotechnology (20) 593-5972. Mi et al. (2010) Tissue Engineering Part A 16 (6) 2091-21003. Levis et al (2010) Biomaterials 31 7726-77374. Engler et al (2006) Cell 126 677-6895. Chen et al (2010) Tissue Engineering Part A 16 (9) 2721-2729
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