A 3D fibre composite material to study integrin-mediated cellular behaviour in response to shear and tensile mechanical cues

摘要

Introduction: The hierarchically organised tendon structure leads to complex extensibility mechanisms in which the collagen fibres both stretch and slide as the tendon is loaded. Tenocytes are located on collagen fibres and between fascicles, consequently experiencing both shear and tension during physiological loading. Tendon pathology is poorly understood, and exacerbated by limited in vitro systems available to study physiologically representative tenocyte strain conditions, consisting of both tension and shear. To understand the effects of physiologically relevant shear on cell behaviour, a fibre composite system incorporating cell attachment pepticles (CAPs) was developed to expose cells to controllable levels of shear and tension. Different CAPs engage different integrins, potentially altering how cells perceive mechanical cues. A collagen type Ⅰ mimetic peptide, DGEA, and fibronectin associated peptide, YRGDS, were compared to assess their effects on tenocyte response to shear-tension (S-T) ratios. Methods: Fibre composites were made with UV polymerised polyethylene glycol dimethacrylate (PEGDM) fibres (300 μm diameter, 4 mm length) containing either DGEA or YRGDS for cell attachment. They were seeded with primary bovine tenocytes, aligned in a mould and encapsulated in 20% PEGDM. The composite S-T ratio was controlled by changing the fibre stiffness, or by altering the fibre soak time before UV polymerisation. Composites were made with 4 different S-T ratios (4 composites/group; 2 strained, 2 non-strained controls), and the experiment performed with 3 biological repeats. Strained samples were exposed to 5% cyclic strain (1 Hz) for 24 hours. The expression of 14 matrix related genes were analysed, using L30 as the reference gene. Cells prior to seeding were first compared with non-strained control composites to assess the effects of CAPs, after which the effects of strain were investigated. Results: Tenocytes in non-strained DGEA composites exhibited basal gene expression profiles closer to those of the pre-seeded tenocytes when compared with YRGDS composites. Tenocytes in DGEA composites were also more mechano-sensitive than those in YRGDS composites; tenocytes in DGEA composites exhibited upregulation of COL-3, MMP-3 and IL-6, and downregulation of SCX with shear, while tenocytes in YRGDS composites downregulated TIMP-3 with shear. Discussion: The main integrin involved in DGEA binding is α2β1 while those associated with YRGDS attachment include α5β1, αVβ3 and αllbβ3. The data from this study emphasise the importance of integrins in the role of mechanotransduction of mechanical cues containing shear and tension, as integrins involved in collagen type Ⅰ binding induce functionally different responses in tenocytes to those not involved in collagen type Ⅰ binding. This information is critical in future studies of cell behaviour and tissue engineering approaches as shear can regulate cell behaviour, and its mechanotransduction important for tissue homeostasis.