Poster AA6 - HYPOXIC CULTURE UPREGULATES COLLAGEN I, COLLAGEN III, AND LYSYL OXIDASE EXPRESSION AND ENHANCES BIOMECHANICAL PROPERTIES OF ENGINEERED SCAFFOLD-FREE TENDON FIBERS

Introduction: To better understand tendon development, our lab has established a scaffold-free tendon fiber engineering platform that allows us to apply controlled stimuli as fibers develop and evaluate their functional consequences (Fig. 1) [1,2]. Given the key role of hypoxia in mammalian development [3], we seek to explore the effects of hypoxic culture on tendon fiber development and functional maturation in our in vitro system. While atmospheric oxygen comprises roughly 21% of dry air, the concentration of oxygen in the developing mammalian embryo is only 1 to 5% [3]. Our prior work showed that hypoxic culture (5% O2) improved biomechanical function of engineered tendon fibers, increasing fiber peak stress and moduli compared to normoxic controls, with additional improvements observed with time in culture [4]. In this study, we evaluate the effect of hypoxic culture (5% O2) on gene expression of engineered tendon fibers over 7 days of development.

Materials and

Methods: Fibers were engineered using our previously established method [2]. Growth channels (17.5mm*150µm*300µm) were molded into agarose gels, with collagen disks (4-mm diameter) functioning as anchors for the fiber (Fig. 1). Human fibronectin (0.375 mg/mL) was wicked through the channel to promote cell adhesion, and human dermal fibroblasts (HDFs) were seeded into the fibronectin-coated growth channels at 30 million cells/mL. Seeded channels were cultured in normoxic (21% O2¬) or hypoxic (5% O2) conditions. Cells self-assembled within 18 hours to form continuous fibers and were maintained in culture for 1, 3, or 7 days (Fig. 2A). Fibers were harvested for biomechanical testing or gene expression analyses via reverse transcription quantitative real time polymerase chain reaction (RT-qPCR).
For PCR, samples were homogenized, RNA was extracted and treated, concentration and sample quality were evaluated, and low-quality RNA samples were cleaned. Extracted RNA samples were reverse transcribed, and cDNA samples were diluted. qPCR was run for 40 cycles in a thermocycler, using 2 ng cDNA/sample and iQ SYBR Green Supermix. Relative gene expression, compared to 1-day normoxic fibers, was calculated using the 2-ΔΔCT method with two replicates per sample. For biomechanical evaluation, as in our prior work, fibers were pulled to failure using a custom ADMET tensile bioreactor, with mN-force resolution [2]. Resulting force-displacement data were normalized by specimen geometry to obtain stress-strain curves, then analyzed to assess engineered fiber strength (peak stress), stiffness (elastic modulus), toughness, as well as toe-in and yield strain (not shown) (Fig. 2C). All statistical analyses were conducted using Minitab®.

Results, Conclusions, and Discussions: Hypoxic culture significantly upregulated expression of Col1a1, Col3a1, and LOX compared to 1-day normoxic fibers (Fig. 3). Expression of these genes did not change significantly with time in culture, though LOX trended downward at later timepoints (Fig. 3). Preliminary biomechanical data show a significant increase in peak stress, Young’s modulus, and toughness with hypoxic culture, compared to normoxic, and further increases with time in culture (Fig. 4).
Culture of developing engineered tendon fibers in hypoxia upregulated expression of Collagen type 1 (Col1a1) and Collagen type 3 (Col3a1), compared to normoxic controls. Hypoxic culture also significantly upregulated LOX across all timepoints. LOX trended downward with time in culture, which could potentially indicate a decreasing need for this protein as the fiber develops. Hypoxia also enhanced biomechanical properties of our fibers, with a significantly greater peak stress, elastic modulus, and toughness for fibers grown in hypoxic conditions. These functional enhancements are consistent with increased matrix production and collagen crosslinking, as supported by the observed upregulation of Col1a1, Col3a1, and LOX expression with hypoxia. Prior work in embryonic chicks showed that hypoxia contributes to biomechanical maturation of developing tendons in vivo via LOX-mediated collagen cross-linking [5], providing further support to our in vitro observations.
We are currently expanding our biomechanical characterization groups to enable additional data analyses to explore potential correlations between gene expression changes with hypoxia and the resulting biomechanical function of fibers. Additionally, our future work seeks to explore roles of matrix alignment, remodeling, and collagen crosslinking via histological analysis, as this may reveal other mechanisms by which hypoxia contributes to biomechanical maturation of these engineered fibers. Such investigations will provide structural and compositional insight to complement our genetic and biomechanical observations, thus connecting gene expression, tissue composition, and fiber function. Further, we will explore other macroenvironmental stimuli, such as macromolecular crowding, and their potential synergies with hypoxia to promote tendon development. The work herein provides novel insight into biological underpinnings for use of hypoxic culture to enhance engineered tendon maturation, with potential applications in larger-scale tendon engineering approaches.

Acknowledgements (Optional): We would like to acknowledge Dr. Connie Chamberlain (University of Wisconsin – Madison) and Dr. Elizabeth Blaber (Rensselaer Polytechnic Institute for assistance in RT-qPCR experimental design and interpretation of results.

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[4] Bramson, M.T.K, Thesis, (2024).
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