Mechanosensitivity of Tendon Using a Novel In Vitro Microtissue Platform

Introduction: The elucidation of isolated cellular pathways is a complex matrix of local and systemic signals. To isolate specific pathways, there exists a need to create accurate models of specific organs and tissues. In tendon, current models are optimal for quantifying protein expression and use for tensile testing but require high cell numbers and long periods to contract1; conversely, other organs have highly developed organ-on-a-chip models, such as the heart. Here, we hypothesize that using previously established load-bearing models, we can create a high throughput, translatable model for tendon using a modular chip platform. We find that primary tendon cells isolated from ScxGFP transgenic mice can spontaneously form 3D “tendon tissue” and undergo contraction within 3 hours and increase ScxGFP reporter activity. Furthermore, tissues grown on stiff cantilevers generate highly aligned collagen fibers in just 24 hours. Lastly, we show that modulating the stiffness of the modular chip alters biomechanical properties of the in vitro tissue, with the ability to introduce chemical dosing and genetic knockdowns to further determine in vivo translatability. Future work on this model revolves around drug dosing dynamically modulating cantilever stiffness after a tissue forms.

Materials and

Methods: Silicon master molds of cantilever devices were fabricated in nanofabrication facilities using SU-8 negative photoresists. 10:1 184 PDMS (base:curing agent) was applied to the silicon wafer to generate negative cantilever stamps. Positive molds in culture dishes were generated using 5:1 184 PDMS (“stiff”) or 15:1 184 PDMS (“soft”). Culture dishes were then sterilized before application of 0.2% (w/v) Pluronic F-127 solution for tissue culture.
ScxGFP tenocytes were isolated from 3-week-old healthy mouse tails. Tails were digested in 2 mg/ml collagenase I at 37 °C with shaking at 400 rpm for 45 minutes before using a 40 µm nylon mesh for cell filtration. We expanded the tenocytes in hypoxia before the first passage. Tenocytes were suspended in a rat tail collagen type I solution for microtissue seeding before being randomly seeded within the cantilever wells as illustrated in Fig. 1. The solution was polymerized for 10 minutes before a media exchange. Each microtissue well was imaged in brightfield and epifluorescence every 15 minutes for 24 hours.
We used MicroBundleCompute (MBC) to generate tissue masks and track pillar deflection2. Briefly, binary cantilever masks were created in ImageJ before MBC processing, with cantilever spring constant approximations being used from previously described methods3. For ScxGFP expression, MBC masks were applied to the epifluorescent image to remove the background signal. Custom Matlab code was used to determine tissue architectural metrics, average fluorescent signal, and integrated density. The interaction of force generation and integrated density was determined via 2-way ANOVA with Tukey’s Honest Significant Difference test.

Results, Conclusions, and Discussions: Here, we’ve demonstrated baseline research into the development of a novel tendon microtissue model. Tendon microtissues force generation was tracked over 24 hours (Fig. 2A). Through examination of cantilever deflection, we found that the microtissues seeded on stiff cantilevers generated significantly more force over 24 hours compared to the tendon microtissues on the soft cantilevers (Fig. 2B). Moreso, microtissues seeded onto the stiff cantilevers displayed increased ScxGFP over the 24 hours, as measured by integrated density (Fig. 2C). When binning for integrated density against force generation, 2-way ANOVA results deemed an interaction between ScxGFP reporter activity and force generation (Fig. 2D). All data shown has a sample size of 8 per condition.
By utilizing a uniaxial cantilever platform, we allow tendon microtissues to form in a manner aligned to the direction of the applied load. This is corroborated through the formation of highly aligned collagen fibers in tissues formed on stiff cantilevers (the soft cantilevers did not generate aligned fibers), as shown in Fig. 2E. This result is in agreement with ScxGFP reporter activity, as Scx induces matrix production.
Thus far, we have demonstrated translatability through altered Scx reporter activity. In conjunction with increased Scx reporter activity, the tendon microtissues cultured on stiff cantilevers generated highly aligned collagen fibers in just 24 hours; this is expected, as Scx regulates extracellular matrix deposition. Our model provides a unique platform to investigate molecular pathways in primary tenocytes using genetic and pharmacological approaches. Future work aims to determine how increasing dosages of TGF-β affects tenocyte phenotype and genotype on this 3D load-bearing model. Long-term, we aim to prove that this system can act as a platform for future genotypic disease studies and as a drug modeling preclinical platform.

Acknowledgements (Optional): We thank Dr. Lim for the idea for the collaboration and providing ScxGFP mouse tenocytes and Inyoung Kang for performing the cell isolation and initial expansion.

References:
1. Kalson NS, Holmes DF, Kapacee Z, et al. An experimental model for studying the biomechanics of embryonic tendon: Evidence that the development of mechanical properties depends on the actinomyosin machinery. Matrix Biol. 2010;29(8):678-689. doi:10.1016/j.matbio.2010.08.009
2. Kobeissi H, Jilberto J, Karakan MC, Gao X, Depalma SJ. MICROBUNDLECOMPUTE: AUTOMATED SEGMENTATION, TRACKING, AND ANALYSIS OF SUBDOMAIN DEFORMATION IN CARDIAC MICROBUNDLES. arXiv. 2023.
3. Legant WR, Pathak A, Yang MT, Deshpande VS, McMeeking RM, Chen CS. Microfabricated tissue gauges to measure and manipulate forces from 3D microtissues. Proc Natl Acad Sci. 2009;106(25):10097-10102. doi:10.1016/0002-9394(83)90280-5