Abstract
The increasing investigation of cellular mechanotransduction mechanisms requires biomaterials combining biofunctionality and suitable mechanical properties. Agarose is a standard biomaterial for cartilage and intervertebral disc mechanobiology studies, but lacks adhesion motifs and the necessary cell-matrix interaction for mechanotransduction. Here, collagen type I was blended at two concentrations (2 and 4.5 mg/mL) with agarose 2% wt/vol. The composite hydrogels were characterized in terms of structural homogeneity, rheological properties and size stability. Nucleus pulposus (NP) cell viability, proliferation, morphology, gene expression, GAG production, adhesion and mechanotransduction ability were further tested. Blended hydrogels presented a homogenous network of the two polymers. While the addition of 4.5 mg/mL collagen significantly decreased the storage modulus and increased the loss modulus of the gels, blended gels containing 2 mg/mL collagen displayed similar mechanical properties to agarose. Hydrogel size was conserved over 21 days for all agarose-based gels. Embedded cells were viable (>80%) and presented reduced proliferation and a round morphology typical of NP cells in vivo. Gene expression of collagen types I and II and aggrecan significantly increased in blended hydrogels from day 1 to 7, further resulting in a significantly superior GAG/DNA ratio compared to agarose gels at day 7. Agarose-collagen hydrogels not only promoted cell adhesion, contrary to agarose gels, but also showed a 5.36-fold higher focal adhesion kinase phosphorylation (pFAK/β-tubulin) when not compressed, and increased pFAK/FAK values 10 min after compression. Agarose-collagen thus outperforms agarose, mimics native tissues constituted of non-fibrillar matrix and collagens, and allows exploring complex loading in a highly reproducible system.
Highlights
As many degenerative and regenerative processes are related to mechanical loading, the field of mechanobiology is widely expanding
Reflection microscopy of collagen I revealed no signal in agarose hydrogels (Figure 1A), while a strong signal was detected in collagen hydrogels (Figure 1B)
Both the 2 and 4.5 mg/mL agarose-collagen hydrogels displayed an intermediate signal, with collagen I fibrillar structures appearing homogenously distributed throughout the agarose matrix (Figures 1C,D)
Summary
As many degenerative and regenerative processes are related to mechanical loading, the field of mechanobiology is widely expanding. Mechanical loading is a known contributor to intervertebral disc (IVD) degeneration, which when associated with pain and inflammation, is defined as degenerative disc disease (Adams and Roughley, 2006). Degenerative disc disease is a leading source of low back pain, which is itself the first cause of disability worldwide with a lifetime prevalence of 84% (Balague et al, 2012). Due to poor knowledge about the underlying molecular mechanisms, current treatments, such as antiinflammatory drugs, are not targeted and have low effect sizes (Balague et al, 2012). Studies aiming at unlocking mechanotransduction mechanisms in the IVD are needed
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