Embryonic chick tenocytes cultured in fixed-length three-dimensional fibrin gels synthesise a taught collagen fibril-rich extracellular matrix that closely resembles embryonic tendon (Kapacee et al., Matrix Biology, 27: 371–375, 2008). Importantly, the cells replace fibrin with parallel arrays of collagen fibrils that are able to resist pulling forces. Regenerative medicine strategies for musculoskeletal applications require the development of tissue with mechanical strength comparable to that of native tissues. How the cells generate tension is not understood and the mechanical properties and the cellular behaviour of this culture system have not been described. This project aimed to describe the morphology of cells in the tendon-like constructs and to quantitate the mechanical properties of the constructs.

Multiphoton imaging (MPI) can image deep into objects with reduced phototoxicity, allowing live-cell applications. MPI of the tendon constructs revealed that cells under tension were aligned longitudinally with the matrix. However, when tension was released the cells became rounded. The results suggested that embryonic tendon cells align along lines of force. Mechanical testing of newly-formed tendon constructs (T0), then at weekly intervals to six weeks (T7 to T42) was performed using an INSTRON^® failure-testing machine. An initial increase in ultimate tensile strength (UTS) was seen from T0 to T7 (1.023±0.031N to 1.150±1.150N, p=0.006), followed by a gradual decline at T35 to 0.350±0.043N, after which there was no further decrease. The UTS of the constructs was comparable with embryonic day 14 chick tendon. The initial increase in strength between T0 and T7 was cell dependent; constructs immersed in Triton-X 100 to remove cells were weaker than cellular constructs (1.277±0.096 versus 0.508±0.099, p< 0.001). Stress-strain plots demonstrated toe, heel, linear and failure regions that are classically observed in tendon.

The results show that embryonic tendon cells synthesise an extracellular matrix of collagen fibrils that are tensioned by the cells, and that the tendon constructs have mechanical strength comparable to in vivo generated tissue.

The research is generously supported by grants from The Wellcome Trust.