Focused Ultrasound Stimulation of Nitric Oxide-releasing Hydrogels for Tendon Healing

Introduction: : Achilles tendinopathy is a common, chronic injury that is characterized by debilitating pain and decreased mobility, particularly when left untreated. Common treatments, such as physical therapy and surgery, can be costly, time-consuming, and ineffective at preventing reinjury. The long-term success of exercise protocols (e.g., physical therapy) points to the importance of mechano-stimulation in treating tendinopathies [1]. Focused ultrasound (FUS) therapy is an emerging, non-invasive approach for treating musculoskeletal disorders via its thermal and mechanical bioeffects. Recent work from our group has demonstrated the potential of low intensity FUS to precisely deliver acoustic energy to murine Achilles tendons in vivo [2]. FUS has also been proposed as a tool for enhancing drug delivery in combination with other materials, such as hydrogels and bioscaffolds, to promote synergistic healing effects. Nitric oxide (NO) is known to modulate several factors involved in tissue regeneration and potentiate wound healing. Recently, our group developed an injectable, bioadhesive hydrogel that can be thermally stimulated by FUS to control NO release in vitro [3]. The current investigation compared the effects of FUS therapy, an injected hydrogel for NO delivery, and a dual treatment (applying both therapies) on healing of murine Achilles tendinopathy. We hypothesized that dual treatment would exhibit superior tendon biomechanical properties compared with individual effects of FUS and NO release.

Materials and

Methods: : Under IACUC approval, 11-week-old, C57BL/6J male mice were received and randomly assigned to one of 10 experimental groups (Table 1) to assess healing at either 17 or 31 days post-injury. Murine Achilles tendinopathy was induced using rHuTGF-β1 injections, as previously reported by our group [4]. Polyethylene-glycol (PEG)-fibrinogen hydrogels, containing either S-Nitroso-N-acetyl penicillamine (SNAP)-fibrin (utilized as a NO donor) microparticles or fibrin-only microparticles (as a control), were implanted in a liquid state after injury induction via subcutaneous injection, then allowed to cure. FUS-only treated mice received a hydrogel injection containing fibrin-only microparticles (without the NO donor). FUS treatments were delivered in two sessions separated by three days, with the first session taking place 10 days after the hydrogel injection. A 1.1 MHz transducer and water coupling cone were aligned directly above the center of the tendon. FUS treatment was applied continuously, inducing an approximate peak negative pressure of 0.67 MPa, for 200 seconds. Following euthanasia, limbs were assigned to either biomechanical testing (n = 8/group) or histological analysis (n = 2/group). Biomechanical testing consisted of preconditioning followed by a load-to-failure test (0.05 mm/sec). Samples assigned for histological analysis were paraffin processed and stained with Toluidine Blue and Safranin-O for assessment of collagen fiber organization, cell density, cell shape, and abundance of sulfated glycosaminoglycans (s-GAGs).

Results, Conclusions, and Discussions:: At the 17d time point, tendon cross-sectional area was significantly increased in IU (p = 0.035), hydrogel-only (p = 0.033), and dual (p = 0.045) treatment groups compared to naïve samples. Maximum stress was significantly increased in the FUS-only treatment group compared to the IU (p = 0.007) and hydrogel-only treatment group (p = 0.034) and decreased for IU versus naïve (p = 0.0457) (Figure 1). Histologic analysis of the 17d groups revealed matrix disorganization and chondroid metaplasia in the IU samples, and a pattern of increased cell density and s-GAGs along the posterior side of the tendon in all groups that received the hydrogel. Deviations from naïve tendon properties were least pronounced in the FUS only group (Figure 2), with limited fiber disorganization and decreased Saf-O staining compared to IU samples. At the 31d time point, elastic modulus was significantly elevated in the FUS-only group relative to naïve tendons (p = 0.022), but no other biomechanical properties differed between groups. The histologic appearance of tendons 31d post-injury included small areas of fiber disorganization in all groups other than naïve with s-GAG staining similar to naïve levels.
Contrary to our hypothesis, biomechanical properties of healing tendons (17d) were superior for FUS-only treatment, which restored tensile properties to naïve levels. These results provide encouraging evidence of the potential for FUS to stimulate tendon repair, particularly at early stages of healing. Biomechanical and histologic results suggest a near-complete healing response at the 31d post injury time point when few differences were observed across treatment groups. An increase in the presence of rounded cells and intensity of Saf-O staining along the posterior aspect of the tendon may indicate a slower healing response in regions adjacent to the hydrogel material at early healing stages. Surprisingly, NO did not significantly improve the healing response of the injured tendons, potentially due to the timing of hydrogel delivery and FUS treatment. The current study demonstrates the feasibility of a dual hydrogel-FUS approach for the treatment of murine tendinopathy, and this experimental platform can be used to further investigate delivery of a variety of active therapeutic agents.

Acknowledgements and/or References (Optional):: This study was funded by NIH R15 Grant GM112082. We thank Laura Lyons, Zoe Miloszewski, and Kyle Cooper for technical assistance.

References:
[1] van der Vlist AC, et al. Br J Sports Med. 2021; 55(5):249-256.
[2] Meduri C, et al. Trans Orthop Res Soc. 2024; 49:2315.
[3] Meyers KM, et al. Front Mater. 2023; 10:1159444.
[4] Bell R, et al. J Biomech. 2013; 46(3):498-505.