The developing tendon and enthesis are hypoxic and rely on hypoxia-inducible factor 1a during postnatal development

The enthesis is a specialized tissue crucial for transmission of muscle force from tendon to bone, yet its postnatal development is not fully understood. In this study, we investigated if and how hypoxia inducible factor 1 alpha (HIF-1α) regulates enthesis cell adaptation, e.g. cell survival and extracellular matrix (ECM) deposition. We investigated the spatial and temporal dynamics of hypoxia in the murine Achilles tendon enthesis and found that, whereas neonatal tendons rapidly resolve hypoxia after birth, the enthesis maintains a hypoxic niche until postnatal day 5, mirroring a hypoxic gradient observed in growth plates. Genetic disruption of Hif1a in mouse tendon/enthesis-resident cells (cKO) resulted in pronounced deficits in grip strength, abnormal tendon–bone attachment morphology, disrupted calcaneal architecture, impaired mineralization and significant ECM disorganization. We found persistent cell death and loss of collagen organization in cKO entheses. In vitro, Hif1a-deficient tendon fibroblasts exhibited blunted transcriptional responses to hypoxia, altered metabolic gene expression and disrupted ECM deposition. Collectively, our findings illuminate hypoxia as a sustained niche in the postnatal enthesis, with HIF-1α required for cell survival, ECM organization and structural integrity of entheses.

After euthanasia, intact left limbs from Ctrl (Cre-negative, n=15; Cre-positive, n=3 males) and cKO mice (n=15) at 8 weeks of age were frozen at −20°C. Achilles tendons and calcaneus were dissected and kept hydrated (with removal of the plantaris tendon and surrounding muscles). Tendon volume was imaged using OCT with polarization (TEL221PSC1, OCT-LK4 lens, ThorLabs Inc.). Intensity and polarization images were acquired in 3D (A-scan averaging=4; spacing=10 μm in x and y, 2.5 μm in z; sampling rate=28 kHz) and intensity and retardation images were exported (32-bit TIFF). The full 3D OCT image stacks were analyzed on a slice-level using a 2D FFT method to measure frequency of retardation, which describes collagen organization (MATLAB). For each slice, we extracted the distance from the center to the maximum peak in frequency space by summing the pixel values in 2-pixel-wide concentric rings radiating from the center. The AUC of dominant frequency versus slice-depth curve is reported per sample as collagen organization metric where a higher retardation frequency (i.e. more tightly packed retardation patterns) results in a higher AUC after FFT, implying increased organization. Slice analysis was used to determine tendon CSA and bifurcation distance (Dragonfly, Comet Technologies Canada Inc.). After imaging, the proximal Achilles tendon was clamped in a thin film grip (Imada) with the calcaneus secured in a custom 3D-printed fixture (FormLabs 3B, Somerville, MA, USA) prior to uniaxial tensile testing. The tendon and grip were placed in a room temperature PBS bath and a pin was used to secure the tendon-grip unit. Uniaxial tensile tests were conducted with load measured using a multi-axis load cell (±70 N; Mach-1 VS500CST, Biomomentum). Samples were preloaded to 0.2 N and preconditioned for ten cycles (±0.025 mm at 0.1 Hz). Grip-to-grip gauge length (L0) was measured at the start of each test after preload and was consistent between tests (3.6±0.5 mm). Load and torque data were collected while applying displacement to failure at 0.01 mm/min to assess axial and off-axis loading. Mechanical properties (e.g. maximum load, maximum strain, linear stiffness; linear modulus and maximum stress calculated using load and CSA) of the Achilles tendon were calculated from force-displacement data using a piecewise linear segmentation by dynamic reprogramming recursion package (dpseg) in R (v4.2.2 or later; The R Project for Statistical Computing, Vienna, Austria). Data are presented as mean±s.d. and compared using a one-way ANOVA and corrected for multiple comparisons using Tukey’s multiple comparisons tests (Prism v9.0+; GraphPad).