Publication date: 25th July 2016
The protein kinase A (PKA) signalling pathway is key in both mediating osteogenic differentiation and transducing mechanical signalling. Carney complex (CNC) is a multiple endocrine neoplasia disease caused by mutations in PKA subunits. Mouse models of CNC, which harbour mutations of PKA subunits, develop vertebral bone tumours exhibiting abnormal bone structures. These phenotypes were attributed to a population of adult bone marrow stromal cells with aberrant PKA expression. PKA functions in mechanotransduction pathways, and subunit-specific expression has a critical role in altering bone phenotype. This research aimed to elucidate the PKA subunit-specific osteogenic differentiation response of MC3T3-E1 murine pre-osteoblasts to substrate mechanical properties. The effect of substrate mechanical properties on PKA subunit expression has not previously been observed.
Hydrogels are three-dimensional, water-insoluble polymer networks, used here as synthetic ECMs. Hydrogel mechanical properties were manipulated by altering fabrication parameters. Single-component and composite hydrogels derived from poly(ethylene glycol) dimethacrylate (PEGDMA) and alginate (ALG) were fabricated with a range of total polymer concentrations (%T) to create systems with varying structural and mechanical properties. Spherical indentation was determined to be an effective method to measure the mechanical properties of the gels, including their time-dependent behaviour. All systems demonstrated an increase in shear modulus with increasing %T. The time-dependent behaviours of the system were found dependent upon the polymer constituents rather than %T, with ALG exhibiting more relaxation over time than PEGDMA. Composite hydrogels were constructed that had similar stiffnesses as the single-component gels, but different time-dependent mechanical properties.
Wild-type, empty vector, and Cα-knockdown MC3T3s were seeded onto PEGDMA and PEGDMA-ALG composite gels. The addition of ALG to the PEGDMA formed a gel with greater time-dependent relaxation, but similar stiffness to the pure PEGDMA gel, as measured by spherical indentation. All cell lines demonstrated reduced expression of both regulatory and catalytic PKA subunits when seeded on composite gels as compared to those on PEGDMA. Osteogenic differentiation appeared to be delayed as a result of the hydrogel substrates, when compared to expression of osteogenic genes of cells seeded on tissue culture plastic. Changes in PKA expression due to mechanical properties of the substrate take place prior to the transcription of the subunits. Ultimately, understanding how substrate properties affect internal signalling mechanisms will help inform design of tissue engineering constructs for regenerative medicine.
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