Enhancing Tendon Repair Through Non-Invasive Hyperbaric Oxygen Therapy

Introduction: : Each year, approximately 7 million people worldwide experience a tendon rupture. For many, the injured tendon never fully regains its original strength or function due to the formation of scar tissue during healing. This fibrotic repair significantly weakens the tissue compared to healthy tendon and can impair daily activities. Hyperbaric oxygen therapy (HBOT), which delivers 100% oxygen at elevated atmospheric pressures, is a potential strategy to enhance tendon healing by limiting scar formation. Clinically, HBOT is currently used to treat conditions such as decompression sickness, radiation injury, and non-healing wounds. Its therapeutic effects stem from pulsed cellular hyperoxygenation, which promotes angiogenesis and reduced inflammation. In orthopaedics, HBOT has recently been investigated for improving anterior cruciate ligament graft integration, facilitating fracture healing, and treating avascular necrosis (Leite et al. JOR 2023, Yeh et al. J Orthop Res. 2007, Jeyaraman et al. Indian J Orthop. 2023). Given the limited vascularity and poor intrinsic regenerative capacity of tendons, targeted modulation of the inflammatory response may be key to improving healing. However, the dose and timing of HBOT for tendon repair is not known. Low doses may not be effective while high doses may produce toxicity. We examined the effects of HBOT at various durations on tenocyte behavior using an in vitro wound healing model. We hypothesize that HBOT would be well tolerated and have effects on collagen deposition, proliferation, and gene expression for both oxygen and tendon specific genes.

Materials and

Methods: : Tenocytes were harvested from six C57BL6 (14-16-week-old) murine Achilles tendons and digested using an established protocol (Dudakovic et al. J Cell Physiol. 2023). All murine tendon tissue extraction was exempt from Dartmouth IACUC requirements. Following expansion, tenocytes at passages 2-4 were plated in 12-well plates at a density of approximately 10,000 cells/mL. Once cells reached ~80-90% confluence, a 0.50mm scratch assay was performed on half of the wells to simulate in vitro wound healing. Plates were then placed in an HBOT chamber (Reimers Systems Inc.) at 2.5 atmosphere absolute for 30, 60, or 90 minutes, while control plates were positioned on the lab bench adjacent to the chamber to ensure equal environmental exposure outside the incubator. Following HBOT treatment, all well plates (with and without the scratch) were returned to the incubator, and the scratch assay wells were imaged every 6 hours for up to 24 hours. After 24 hours, cell viability and proliferation were analyzed across all wells via an MTS assay, picrosirius red staining, live/dead staining, and tendon (COL1A1, SCX, TNMD) and oxygen specific (HIF-1A) gene expression with RT-qPCR on all well plates. Statistical significance was determined using unpaired t-tests with a p-value threshold of 0.05.

Results, Conclusions, and Discussions:: Cell proliferation did not differ between control and HBOT-treated tenocytes after 30-minute exposures (p > 0.05). However, proliferation increased significantly following 60- and 90-minute HBOT treatments (Figure 1A). Scratch assay analysis revealed no significant differences in percent wound closure between control and HBOT groups at any exposure timepoint (Figure 1B). Similarly, picrosirius red staining showed comparable collagen deposition across all groups (Figure 1C, 1D). Live/dead staining demonstrated increased cell death in the 60- and 90-minute HBOT groups compared to both the control and 30-minute HBOT groups (Figure 1C). RT-qPCR analysis for the 60-minute exposure group indicated a trend toward decreased expression of collagen type I (COL1A1) in HBOT-treated cells compared to controls. This implies that we may see differences in collagen deposition on the picrosirius red staining at later time points due to the slower dynamics of matrix synthesis. Expression of the tendon-specific gene TNMD was not significantly affected by HBOT, while differences in SCX expression appeared to be associated with scratch injury rather than treatment. Notably, the hypoxia-responsive gene HIF-1A was not elevated in HBOT-treated cells at 24 hours post-treatment (Figure 2). These initial findings suggest that HBOT enhances tenocyte metabolic activity at longer exposure durations, potentially due to increased oxygen availability supporting ATP production and cell proliferation. However, increased cell death at 60 and 90 minutes of exposure may indicate a threshold beyond which oxygen and pressure induce oxidative stress. Ongoing analyses aim to further characterize gene and protein expression in all experimental groups. Additionally, a preliminary in vivo mouse model is being developed under an approved IACUC protocol to investigate the effects of HBOT on tendon healing in a physiological context. Initial proof-of-concept histology demonstrates a healthy Achilles tendon prior to injury and HBOT (Figure 3). In conclusion, tendon healing continues to pose a clinical challenge, often complicated by excessive scar tissue formation and limited regenerative capacity. These early in vitro results suggest that HBOT may modulate tenocyte behavior in ways that could influence healing outcomes, but determining time and dose are important. Future work will help clarify its therapeutic potential and optimal treatment parameters.

Acknowledgements and/or References (Optional):: The authors would like to acknowledge Victoria Ruiz and Lily Giurleo for their valuable assistance with data collection. We also thank Sherri Geimer and Monika Adhikari for their support in operating the HBOT chamber.