Method development and characterization of chick embryo tendon mechanical properties.

Tendons are involved in multiple disorders and injuries, ranging from birth deformities to tendinopathies to acute ruptures. The ability to characterize embryonic tendon mechanical properties will enable elucidation of mechanisms responsible for functional tendon formation. In turn, an understanding of tendon development could inform approaches for adult and embryonic tendon tissue engineering and regenerative medicine. The chick embryo is a scientifically relevant model that we have been using to study Achilles (calcaneal) tendon development. Chick embryo calcaneal tendons are challenging to mechanically test due to small size and delicate nature, and difficulty distinguishing embryonic tendons from muscle and fibrocartilage using the naked eye. Here, we developed and implemented a "marking protocol" to identify and isolate calcaneal tendons at different stages of chick embryonic development. Mechanical testing of tendons isolated using the marking protocol revealed trends in mechanical property development that were not observed with tendons isolated by naked eye (eyeballing). Marked tendons exhibited non-linear increases in tensile modulus and ultimate tensile strength, whereas eyeballed tendons exhibited linear increases in the same properties, reflecting a need for the marking protocol. Furthermore, the tensile mechanical properties characterized for marked tendons are consistent with previously reported trends in cell length-scale mechanical properties measured using atomic force microscopy. This report establishes new methodology to enable tensile testing of chick embryo tendons and provides new information about embryonic tendon mechanical property development.

Phenotype stability, expansion potential, and senescence of embryonic tendon cells in vitro.

Embryonic tendon cells have been studied in vitro to better understand mechanisms of tendon development. Outcomes of in vitro cell culture studies are easily affected by phenotype instability of embryonic tendon cells during expansion in vitro to achieve desired cell numbers, yet this has not been characterized. In the present study, we characterized phenotype stability, expansion potential, and onset of senescence in chick embryo tendon cells from low to high cell doublings. We focused on cells of Hamburger-Hamilton stages (HH) 40 and HH42, where HH40 is the earliest stage associated with substantial increases in extracellular matrix and mechanical properties during embryonic tendon development. HH40 and HH42 cells both downregulated expression levels of tendon phenotype markers, scleraxis and tenomodulin, and exhibited onset of senescence, based on p16 and p21 expression levels, cell surface area, and percentage of ß-galactosidase positive cells, before significant decreases in proliferation rates were detected. These findings showed that embryonic tendon cells destabilize phenotype and become senescent earlier than they begin to decline in proliferation rates in vitro. Additionally, embryonic stage of isolation appears to have no effect on proliferation rates, whereas later stage HH42 cells downregulate phenotype and become susceptible to senescence sooner than earlier stage HH40 cells. Based on our data, we recommend chick embryo tendon cells be used before a maximum cumulative doubling level of 12 (passage 4 in this study) to avoid phenotype destabilization and onset of senescence.