Dynamic Tensile Loading Improves Engineered Tendon Formation

Introduction: Tendon tissue engineering seeks to aid and augment surgical repair by using scaffolds, biomimetic factors, and cells to promote tendon regeneration. Synthetic polymer scaffolds with aligned fibers promote tendon-like gene expression and the formation of an aligned, collagen rich matrix. However, many tissue engineered constructs cannot withstand physiologically relevant loads. Both tendon development and normal homeostasis require loading, and tendons adapt to loading: for example, fetal movement is necessary for tendon development while immobilization impairs normal tendon formation. In vitro loading of tissue engineered constructs also promotes tendon-like gene expression and matrix synthesis, but results vary by cell type, strain applied, and frequency of the loading. However, overloading of tendon is detrimental, and high fatigue disrupts collagen organization and stiffness. The objective of this study was to determine the optimal number of daily cycles of dynamic tensile loading for in vitro human adipose stem cell (hASC) engineered tendon development.

Materials and

Methods: hASCs (IRB exempt) were seeded at 5 x 105 hASCs/cm2 on both sides of poly(ε-caprolactone) 3D meltblown scaffolds and pre-cultured for 10 days, followed by a 28-day culture period where they were loaded to 6% stain as determined by the linear region of the stress-strain curve for these biomaterials at 1Hz on a custom tensile bioreactor at 0 (control), 1000 (late development fetal kicks/day), 5000 (moderate exercise), 10000 (used previously, analogous to target steps/day) cycles 3X a week (Fig. 1A). The effect of dynamic, tensile loading was assessed via collagen organization, tendon-like matrix production, and mechanical properties. After harvest, frozen sections were stained with Picrosirius red to quantify collagen alignment. dsDNA, sulfated glycosaminoglycan (sGAG), and collagen content were quantified in scaffolds digested in papain. To determine mechanical properties, scaffolds were cut into dog bone shapes and marked with India ink for regional strain analysis. Dynamic mechanical analysis measured hysteresis, stress relaxation, and frequency sweeps at 1%, 3%, 5%, and 7% strain, before resting followed by a ramp to failure to determine linear region properties. Data are reported as mean±SD and analyzed by ANOVA followed by Tukey post-hoc test (n=3-8).

Results, Conclusions, and Discussions: Loading with 5000 cycles led to a 1.4-fold increase in dsDNA (Fig.1B) and a 2.1-fold increase for 10000 cycles compared to unloaded (0 cycle) controls. Collagen increased 2.3-fold in the 5000-cycle loaded group compared to all groups (Fig. 1C), but there was no difference in collagen content between any other groups. sGAG content was unaffected by loading. Picrosirius red stain revealed collagen matrix throughout the thickness of the scaffold (Fig. 1D) but unexpectedly, loading during culture had no effect on collagen alignment (p=0.6090) (Fig. 1E). The 5000 cycle groups had the greatest linear region modulus (Fig. 1F) and yield stress (Fig. 1G); yield stretch was unaffected by loading (p=0.3038). Stress relaxation was not different between treatment groups and decreased with increased strain, although the scaffolds exhibit lower stress relaxation than native tendons. Energy stored in the hysteresis loop increased with strain and was greatest in the 5000-cycle group at 7% strain. Secant stiffness increased with strain – the 5000-cycle group had greater stiffness at 5% and 7% strain than other groups (p < 0.05), while the 10000-cycle group had lower stiffness at 1%, 3%, and 5% strain (p < 0.05). At 0.1 Hz and 1 Hz, scaffolds in the 5000-cycle group exhibited a larger phase shift than other groups (p < 0.05), but not at 5 Hz. Loading engineered tendon at 5000-cycles 3X a week increased hASC proliferation, collagen synthesis, and mechanical function without affecting collagen alignment. These data suggest that the effect may be due to an increase in collagen fibers. Increasing the loading to 10000-cycles appears to promote an overuse phenotype where cell proliferation was increased, but cells synthesized less collagen and mechanical function showed no improvement versus unloaded control. While loading did improve engineered tendon matrix and function in a manner dependent on cycle number, it required over 5 weeks of culture (pre-culture + loading period) to achieve this benefit, without yet reaching physiologically relevant strength, suggesting that further refinement of the biomaterial and provision of additional biomimetic cues may be needed. Alternatively, the genes driving this response could be targeted therapeutically to improve engineered tendon maturation without loading in future studies.

Acknowledgements (Optional): NIH grants AR073882 (DL) and AR079844 (TJ). The authors acknowledge Matthew Fisher PhD and Paul Warren PhD for their assistance.