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Ultrasound contrast agents are clinically used for diagnosis of internal organs, but ultrasound contrast agents are rarely applied clinically in musculoskeletal disorders. Our study aims to comparatively analyze the differences between ultrasonographic images through peri-injury injection of the clinically used microbubble and researched nanoparticle contrast agents in various muscular injury models. To compare contrast-enhanced images in different muscle injury models, we prepared groups of rats with sham, laceration, punch, contusion, and toxin injection injuries. We measured H 2 O 2 levels using the Amplex Red assay by extracting tissue from the damaged area. As comparative contrast agents, SonoVue ® , a commercially available microbubble contrast agent, and poly(vanillinoxalte) (PVO) nanoparticles, which are H 2 O 2 -responsive nanoparticles, were used. The difference in contrast between the two contrast agents was recorded as an ultrasound movie, and J-image software 1.53p was used to quantify and analyze the maximum and minimum echogenicity values of the images after contrast enhancement. In the Amplex red assay for the highest H 2 O 2 level in each muscle injury model, the maximum level showed 24 h after the modeling. In the sham rats, PVO injection showed no increased echogenicity except at the needle insertion site, but SonoVue ® injection showed increased echo signal throughout the injected muscle immediately after injection. One day after the preparation of the lesion, PVO and SonoVue ® were injected into the lesion site and ultrasound was performed on the lesion site. After the injection of PVO nanoparticles, contrast enhancement was observed at the lesion site immediately. SonoVue ® injections, on the other hand, showed a widespread pattern of echo signals and an increase in echo retention only at the lesion site over time, but this was not clear. There were statistically significant differences between the highest and lowest echogenicity in PVO and SonoVue ® contrast-enhanced images in all models. Contrast enhancement lasted more than 3 h in the PVO injection, but disappeared within 3 h in the SonoVue ® injection. PVO nanoparticles showed the possibility of physiologic contrast by CO 2 generated by conjugation with H 2 O 2 generated by muscle injuries, and SonoVue ® injection observed the possibility of microbubble contrast as a contrast agent with a pooling effect that lasts longer on the lesion. Further research is needed to investigate the use of various ultrasound contrast agents, including nanoparticles, in musculoskeletal disorders, as well as the potential for further utilities of microbubble contrast agents.

Ultrasound is clinically used for diagnosis and interventions for musculoskeletal injuries like muscle contusion, but contrast of ultrasonography still remains a challenge in the field of the musculoskeletal system. A level of hydrogen peroxide (H 2 O 2 ) is known to be elevated during mechanical tissue damage and therefore H 2 O 2 can be exploited as a diagnostic and therapeutic marker for mechanical injuries in the musculoskeletal system. We previously developed poly(vanillin-oxalate) (PVO) as an inflammation-responsive polymeric prodrug of vanillin, which is designed to rapidly respond to H 2 O 2 and exert antioxidant and anti-inflammatory activities. The primary aim of this study is to verify whether PVO nanoparticles could serve as contrast agents as well as therapeutic agents for musculoskeletal injuries simultaneously. In a rat model of contusion-induced muscle injury, PVO nanoparticles generated CO 2 bubbles to enhance the ultrasound contrast in the injury site. A single intramuscular injection of PVO nanoparticles also suppressed contusion-induced muscle damages by inhibiting the expression of pro-inflammatory cytokines and inflammatory cell infiltration. We, therefore, anticipate that PVO nanoparticles have great translational potential as not only ultrasound imaging agents but also therapeutic agents for the musculoskeletal disorders such as contusion.