[Show abstract][Hide abstract] ABSTRACT: Previous studies have demonstrated the beneficial effect of mechanical loading on in vitro tendon engineering. To understand the mechanism, human tenocytes and polyglycolic acid long fibers were used for in vitro tendon engineering in a bioreactor system for 12 weeks with and without dynamic loading. The engineered neo-tendons were subjected to proteomic analysis using mass spectrometry along with shotgun strategy. As expected, mechanical loading resulted in a more mature tendon tissue characterized by a firmer tissue texture and densely deposited matrices which formed longitudinally aligned collagen fibers in a highly compact fashion. In contrast, non-loaded neo-tendon revealed loosely and less deposited matrices in a relatively less organized pattern. Proteins isolated from two groups of tissues exhibited similar distribution of isoeletric point and molecular weight indicating the similarity and comparability of the tissue specimens. Further, proteomic analysis showed that total 758 proteins were identified from both groups with 194 and 177 proteins uniquely presented in loaded and non-loaded tendons, respectively. Comparison of loaded and non-loaded tendons revealed 195 significantly up-regulated proteins and 189 significantly down-regulated proteins. The differentially expressed proteins could generally be classified into the categories of extracellular matrix, intra-cellular signaling, cytoskeleton and inflammatory response. Among them, significantly up-regulated collagens I and VI, MMP-14, WNT5A, microfilament molecules and some inflammatory factors suggest that the possible mechanism for this particular biological phenomenon may involve increased production of tendon specific matrices, enhanced cross-link of collagens and other matrix molecules, proper matrix remodeling for tissue maturation and mechanotransduction (including non-canonical Wnt signal pathway) mediated other biological processes.
[Show abstract][Hide abstract] ABSTRACT: Engineering of extensor tendon complex remains an unexplored area in tendon engineering research. In addition, less is known about the mechanism of mechanical loading in human tendon development and maturation. In the current study, an ex vivo approach was developed to investigate these issues. Human fetal extensor tenocytes were isolated, expanded and seeded on polyglycolic acid (PGA) fibers that formed a scaffold with a shape mimicking human extensor tendon complex. After in vitro culture for 6 weeks, 7 cell-scaffold constructs were further in vitro cultured with dynamic mechanical loading for another 6 weeks in a bioreactor. The other 14 constructs were in vivo implanted subcutaneously to nude mice for another 14 weeks. Seven of them were implanted without loading, whereas the other 7 were sutured to mouse fascia and animal movement provided a natural dynamic loading in vivo. The results demonstrated that human fetal cells could form an extensor tendon complex structure in vitro and become further matured in vivo by mechanical stimulation. In contrast to in vitro loaded and in vivo non-loaded tendons, in vivo loaded tendons exhibited bigger tissue volume, better aligned collagen fibers, more mature collagen fibril structure with D-band periodicity, and stronger mechanical properties. These findings indicate that an extensor tendon complex like structure is possible to generate by an ex vivo approach and in vivo mechanical loading might be an optimal niche for engineering functional extensor tendon.