Cellular Response to Cyclic Stretch in Engineered Ligament is Dependent on Degree of Hierarchical Collagen Organization

Introduction:: The primary source of strength and function in tendons and ligaments is hierarchically organized collagen fibers. Injuries to these tissues disrupt collagen fibers resulting in loss of function, pain, and decreased mobility.¹ These collagen fibers do not regenerate after injury and whether repaired or replaced, current treatment strategies fail to restore the functional properties of native tissue.^1,2 Engineered replacements are promising; however, it remains a challenge to form the large hierarchically organized collagen fibers essential to long-term mechanical success. We previously developed a novel culture system (Fig 1A) that guides Anterior Cruciate Ligament (ACL) fibroblasts in high-density collagen gels to produce aligned collagen fibrils by 2 weeks, which mature into native-sized fibers and fascicles by 4 and 6 weeks.¹ These constructs are promising, but further maturation is needed for clinical relevancy.

Mechanical cues are critical for tissue development in vivo and have been shown to improve maturation of engineered tissues;^2,3,4 however the effect on larger fiber and fascicle formation is largely unknown. The objective of this study was to investigate whether intermittent cyclic stretch, mimicking rapid muscle activity, could drive further maturation in our system. Additionally, we were interested in exploring whether cyclic loading had differential effects on cells when applied at different degrees of collagen organization. We hypothesize that intermittent cyclic loading will improve overall collagen organization, composition, and tissue mechanics, via cell-driven collagen remodeling, resulting in significantly stronger, functional ligament replacements.


Materials and Methods:: To form constructs, rat tail type I collagen and neonatal bovine ACL fibroblasts were mixed and cast into 1.5 mm thick sheet gels at 20 mg/mL collagen and 5x10⁶ cells/mL and gelled at 37º C for 1 hour, as previously described.¹ Rectangles (8 x 30 mm) were cut from gels, divided between groups, and cultured for up to 6 weeks with timepoints at 0, 2, 4, and 6 weeks. Static constructs were clamped into our culture device to drive fiber formation (Fig 1A), while loaded constructs were clamped into a modified CellScale tensile bioreactor and loaded with an established loading regime⁴ to mimic rapid muscle movement. Specifically, constructs were loaded with either 5% or 10% strain at 1 Hz for 1 hr, twice daily, 3x a week (Fig. 1B) to evaluate dose effect of cyclic loading. Both loads are within the reported human tensile strains for ACL (2-12%).^5,6 Zero-week samples were collected 24 hours after one loading cycle. Acellular constructs, denoted (-)cell, were also cultured for up 6 weeks to evaluate whether changes in construct maturation were cell-driven or from stretching of the construct alone. Post culture, confocal reflectance and SEM were performed to analyze collagen organization. DNA, glycosaminoglycans (GAGs), Collagen content, and LOX activity were measured via Picogreen, DMMB, hydroxyproline (Hypro), and LOX activity assays. Tissue mechanics were analyzed by tensile tests at 0.75% strain/sec to failure. All data are mean ± SD. Statistical analysis was performed by 1- and 2-way ANOVA with Tukey’s post-hoc (p< 0.05 significant).


Results, Conclusions, and Discussions:: All cell-seeded constructs significantly contracted to 30-40% their original size by 6 weeks (Fig 1C), while acellular constructs ((-)cell) remained at ~100%. As expected, the static clamping device guided cells to produce aligned collagen fibrils by 2 weeks and larger fibers by 6 weeks. Cyclic loading further increased cell-driven hierarchical fiber maturation with crimp formation by 2 weeks and more consistent, uniform crimp with highly aligned fibers and fibrils by 6 weeks (Fig 1D). Acellular constructs remained largely unorganized by 6 weeks, demonstrating the increased fiber maturation was cell-driven.

DNA remained constant throughout culture (data not shown), while loading increased LOX activity, GAG, and collagen accumulation in a dose- and time-dependent manner (Fig 1E). Specifically, in unorganized gels (0-2 weeks), loading increased GAG and decreased collagen concentration. However, once cells had formed aligned fibrils, cyclic loading resulted in decreased GAG accumulation and increased collagen concentration (Fig 1E). Collectively, cellular response to cyclic loading varied depending on the degree of organization, with cyclic load producing a more catabolic response in unorganized collagen, and a more anabolic response later in culture once cells are anchored on aligned fibrils, suggesting a shift in mechanotransduction. These results mirror previous work in 2D.^7,8


Loading also significantly improved tensile mechanical properties in a dose-dependent manner. While 5% cyclic strain resulted in increased elastic modulus (Fig 1F) and collagen at 6 weeks, this was lost with 10% loading, suggesting a mechanical threshold in cellular response. Interestingly, both 5 and 10% cyclic loading improved toe mechanics compared to controls by 6 weeks (Fig 1F), suggesting the development of crimp and improved fibril alignment lead to more functional mechanics (Fig 1D). Acellular constructs had no change in mechanics, further demonstrating maturation is cell-driven.

Collectively, cyclic loading drove cells to increase hierarchical collagen organization, collagen crimping, and tissue mechanics. Further, the effect of loading on cells varied depending on the degree of organization. This study provides new insight into how cyclic loading affects cell-driven hierarchical fiber formation and maturation. A better understanding of how mechanical cues regulate fiber formation will help to develop better rehabilitation protocols and better engineer replacements.


Acknowledgements (Optional): : This work is supported by NSF CAREER #2045995 (CMMI) & the IREK12 Program sponsored by NIH K12D073945.


References (Optional): : 1. Puetzer+Biomat 2021; 2. Galloway+J Bone Surg Am 2013; 3. Connizzo+Matrix Bio 2013; 4. Puetzer+Tis Eng 2016; 5. McPherson+Orthop J. Sport. Med 2018 ; 6. Englander+J. Biomech 2020: 7. Baker+Tis Eng 2011; 8. Chen+Acta Biomat 2022