Slow Stretch Elongation of Engineered Ligaments Drives Cellular Formation of Hierarchical Collagen Fibers Independent of Collagen Stretch

Introduction:: Hierarchically organized collagen fibers are the primary source of strength in tissues throughout the body, particularly menisci, tendons, and ligaments. After injury, these fibers do not regenerate, resulting in a loss of function and over 1.4 million surgeries a year in the US.^1,2 Engineered replacements are a promising solution, but it remains a challenge to form the large hierarchical collagen fibers essential to long-term mechanical success.^1,2 Previously, we developed a static clamping culture system which guides anterior cruciate ligament (ACL) fibroblasts in high-density collagen gels to produce aligned collagen fibrils by two weeks, which develop into native-sized fibers and fascicles by 6 weeks.² However, further maturation is required for these constructs to be clinically relevant.² Mechanical cues, such as slow stretch elongation and rapid cyclic muscle loading, are critical for fiber maturation during development; however the effect of each type of load on hierarchical fiber formation is largely unknown.^3,4 Further, growth rates of the ACL can vary by more than 10-fold between embryonic and early postnatal stages, during which time there are significant changes in hierarchical collagen organization.⁵ The objective of this study is to investigate whether slow stretch elongation at rates similar to neonatal or postnatal ACL growth rates differentially drive hierarchical collagen fiber formation in our system, and to evaluate whether these changes in maturation are cell-driven or a side-effect of stretching the collagen gel. We hypothesize that slow stretch elongations will improve hierarchical organization, composition, and tissue mechanics in a dose-dependent fashion and via cell-driven remodeling.



Materials and Methods:: Constructs were formed by mixing neonatal bovine ACL fibroblasts with rat tail type I collagen to form 20 mg/mL sheet gels at 5x10⁶ cells/mL.² Rectangular constructs were cut from sheet gels and cultured in our static clamping device or in a modified CellScale tensile bioreactor for up to 3 weeks (Fig 1A). Loaded constructs were continuously stretched for up to 3 weeks at 1 mm/day to simulate neonatal growth rates³ or at 0.1 mm/day to simulate early postnatal growth rates (Fig 1A).⁵ Acellular constructs were also cultured for up to 3 weeks, with and without load, to evaluate whether changes in construct maturation were cell-driven or from stretching the construct alone. Neonatal growth rates of 1 mm/day, in both cell-seeded and acellular constructs, resulted in early rupture of constructs, so these constructs were only cultured to 1 week. Post culture, confocal reflectance was performed to analyze collagen organization (Fig 1B). Mechanical properties were analyzed by tensile tests at 0.75% strain/sec to failure (Fig 1C). DNA, glycosaminoglycans (GAGs), LOX activity, and collagen content were measured using picogreen, DMMB, lysyl oxidase activity, and hydroxyproline (hypro) assays (Fig 1D). DNA, GAG and collagen are reported per construct to account for differences in construct size due to stretch. Statistical analysis was performed by 2-way ANOVA with Tukey’s post-hoc (p< 0.05).

Results, Conclusions, and Discussions:: Cell-seeded constructs stretched at rates similar to neonatal ACL growth rates (1 mm/day) had significantly increased collagen organization (Fig 1B), tissue tensile mechanics (Fig 1C) and increased DNA, GAG, and LOX activity by 1 week (Fig 1D), compared to static and postnatal growth-rate constructs. Acellular constructs stretched at 1 mm/day had some alignment of collagen by 1 week, however not to the same extent as cell-seeded constructs, and this alignment did not produce improved tissue mechanics, demonstrating maturation is cell driven. Cell-seeded constructs stretched at postnatal rates (0.1 mm/day) had significantly improved collagen organization, tissue mechanics, and increased DNA, GAG, and LOX activity compared to static controls also, however these increases mainly occurred at 3 weeks rather than 1 week (Fig 1B-D). In particular, the postnatal growth-rate drove cells to develop aligned fibrils by 2 weeks, and larger fibers and fascicles by 3 weeks, resulting in significantly improved mechanics. Again, acellular postnatal stretch constructs had little-to-no change in collagen organization and tissue mechanics by 3 weeks (Fig 1B-C). Interestingly, both stretch rates resulted in significantly decreased collagen content compared to static controls (Fig 1D). In our culture system, the largest turnover of collagen occurs in the first 2 weeks when cells are forming aligned collagen fibrils.² During this period, slow stretch may increase cellular turnover of collagen, resulting in lower collagen concentrations and more aligned fibrils. Once on aligned fibril, cells most likely sense the stretch differentially, resulting in a shift to increased collagen accumulation by 3 weeks. In fact, on-going work evaluating the effect of these stretch-rates at different levels of organization has found further increases in collagen when stretch is applied once fibers have formed. Further, despite the lower collagen concentration, stretched constructs had significantly higher mechanics, demonstrating the importance of collagen organization. Overall, this study found that slow stretch applied at rates similar to neonatal and postnatal ACL growth rates differentially drives increases in collagen organization and tissue mechanics, and that these changes are cell-driven. This study provides new insights vital to developing better rehabilitation protocols and engineered replacements for tendons and ligaments throughout the body.


Acknowledgements (Optional): : This work is supported by NSF CAREER #2045995 (CMMI).

References (Optional): : 1.Patel+JOR 2018; 2.Puetzer+Biomat 2021; 3.Kalson+Dev Dyn 2011; 4.Connizzo+Matrix 2013; 5.Cone+J Bone Jt Surg 2019.