Bone Abstracts

The musculoskeletal system experiences severe mechanical strain, with repetitive or extreme strains causing significant trauma; the result being an increase in mechanobiological studies evaluating mechanical strain on musculoskeletal cells. Currently, most stretching studies utilise fibronectin-coated cultures, as these enhance cell attachment. However, recent studies suggest that fibronectin increases cell turnover and DNA damage and affects cell differentiation. Furthermore, fibronectin fragments cause extracellular matrix degradation. All indicative of diseased states, such as tendinopathy.

We employed a novel cell stretching device, where clamp-to-clamp strain is evenly distributed across the culture surface, to determine how coatings affect cell behaviour. We cultured primary rat tenocytes on 0.15 μg/ml collagen type 1 or 10 μg/ml fibronectin-coated, micro-grooved silicone and exposed them to 2 and 4% strain at 0.5 Hz for 12 h. Calcein staining and alamarBlue was used to evaluate cell morphology and viability, while differential gene expression of musculoskeletal cell-specific and inflammatory markers was measured with real-time PCR.

Fibronectin-coated cultures demonstrated greater attachment and growth compared to collagen-coated cultures; while calcein staining suggested cells cultured on fibronectin had a more tenocytic morphology. However, with both coatings, expression of tenocyte markers tenascin-C, tenomodullin, and scleraxis decreased two- to threefold compared to non-coated cultures, with little difference between coatings, non-stretched and 2% stretched cultures. Interestingly, expression of biglycan and fibromodullin, both important in maintaining a tendon stem cell niche, were significantly upregulated in collagen-coated cultures, with biglycan upregulated threefold in non-stretched and 2% stretched cultures, and eightfold in 4% stretched cultures. Neither osteoblast- or chondrocyte-specific markers were altered, while expression of MMP-3 was significantly upregulated in both coated cultures, approximately fivefold in fibronectin-coated and 15–20-fold in collagen-coated cultures.

Overall, we have demonstrated that cells respond differently to different substrates, particularly under higher levels of stretch. Notably, collagen coating may provide a more tenocytic environment for in vitro tendon mechanobiology.