Characterizing the Effects of Indirect Histotripsy Treatment on Tendon Structure and Function

Introduction: : Focused ultrasound (FUS) therapies are an emerging non-invasive approach for ablating various tumors, including osteosarcoma (OS) and soft tissue sarcomas1,2. Current standards of care for these tumors consist of limb amputation, limb-salvage surgery, pre- and post-operative chemotherapy, and thermal ablation methods for both humans and dogs2. While these current standards of care are used to ablate and/or provide pain relief, there are risks and limitations associated with them, including potential deleterious effects on surrounding tissues. Histotripsy is a novel limb-salvage technique that can be used to treat these tumors. Histotripsy is a non-invasive, non-thermal, and non-ionizing focused ultrasound method that mechanically breaks down targeted tissue into subcellular components by generating acoustic cavitation bubble clouds3. In order to establish the clinical safety of histotripsy for OS and soft tissue sarcomas, it is important to ensure that there is no damage to supporting structures, which include tumor-adjacent tendons, as tendons are vital musculoskeletal structures that facilitate joint movement. Therefore, the objective of this study is to evaluate the effects of indirect histotripsy treatment on tendon structure and function via biomechanical testing, ultrasound (US) image texture analysis, and histological analysis.

Materials and

Methods: : A total of 33 canine extensor tendons were collected from medium to large breed male and female dogs and randomly assigned to one of 3 treatment conditions (n=8 tendons/group, Table 1): control (untreated), histotripsy using 1000 ppp (pulses per point, a clinical dose) and 4000 ppp (supraclinical dose). In this ex vivo model, treated tendons were fixed in 7.5% porcine gelatin and treated indirectly in the middle region of the tendon (i.e. focal point is below the tendon, Figure 1). Grayscale and shear wave elastography (SWE) ultrasound (US) images were acquired (Supersonic Aixplorer, SL18-5 Transducer) prior to and after histotripsy treatments for each dose, followed by load-to-failure mechanical testing at 20 mm/min (MTS Insight 10). Image standardization of the grayscale images was performed using a histogram specification technique to account for variability of gain. Pixelwise US texture analysis for across the three regions (distal, middle, and proximal) was performed to calculate 1st (mean, median, variance, skewness, kurtosis, and entropy) and 2nd (contrast, homogeneity, and energy) order statistics. Collagen fiber and cell structure and arrangement of Trichrome and Hematoxylin & Eosin (H&E) stained sections (n=3 tendons/group) were evaluated using a custom histologic grading scale.

Results, Conclusions, and Discussions:: Results from biomechanical testing, US image texture analysis, and histological analysis generally showed non-significant changes between untreated and treated tendons at each dose. Subtle dose-dependent, localized changes in texture parameters within the treatment region (4000 ppp) did not alter tendon mechanical properties. Statistically significant correlations were observed between several texture parameter outcomes and mechanical properties, which supports the potential use of quantitative US texture analysis as a non-invasive approach for assessing tendon mechanical integrity. This work establishes histotripsy as a safe and effective limb-salvage treatment that preserves tendon function. The data from this study can be used to refine histotripsy treatment protocols for tumor treatments and establish a safety profile for tendons. From a clinical perspective, this data can inform treatment planning by identifying cases where higher histotripsy doses are required, such as when treating a larger or denser tumor, so that appropriate approaches, including delayed return to activity or rehabilitation, can be implemented to allow for tendon recovery and reduce risk of reinjury. This study establishes an experimental platform for understanding how specific histotripsy parameters influence tendon tissue, which will facilitate the design of non-invasive, ablative treatments, such as debridement.

Acknowledgements and/or References (Optional):: Supported by a research grant from the Focused Ultrasound Foundation (FUSF).
1. Rugers L et al., Cancers (2023) 2. Rugers L et al., Int J Hyperthermia (2023) 3. Arnold L et al., Ultrasound Med Biol (2021)