Safety & Efficacy of LIFU for Non-Invasively Triggering In Vivo Silk Scaffold Degradation

Introduction: Limited supplies of autologous grafts, and their associated morbidity, have motivated the need for alternative sources of tissues for soft tissue grafts. Tissue engineering provides a platform for restoring or replacing tissue and organ function, where biomaterials can initially support cells to proliferate and secrete matrix before gradually degrading as de novo tissue replaces the scaffold to maintain intrinsic properties throughout the healing process. However, a major barrier to clinical translation is that patients have differing regenerative tissue capacities due to complex, interacting, and currently unpredictable factors.2-6 When biomaterials persist beyond the initial healing phase, they are plagued with poor outcomes, which account for 10% of failed implants and close to $10 billion annually. The long-term goal of our work is to develop a personalized biomaterial platform, where biomaterial degradation can be adjusted with focused ultrasound following routine monitoring of how the patient is healing. Unlike imaging ultrasound, therapeutic ultrasound often has higher intensities and lower frequencies. Therapeutic low-intensity focused ultrasound (LIFU) is considered harmless and safe for cells and has several biomedical applications involving microbubble interactions. Based on our recent work that indicated LIFU induces degradation in silk scaffolds in vitro, we hypothesized that LIFU would be both safe and effective for tailoring the degradation of silk scaffolds to match the regenerative environment in mice.

Materials and

Methods: Silk scaffolds were prepared from Bombyx mori cocoons as described previously. Scaffolds were cut to size (2mm thickness x 4mm diameter), autoclaved, and soaked in sterile PBS. Scaffolds were treated with LIFU in a dish, and gravimetric analysis was used for degradation efficacy verification. Additional scaffolds were implanted subcutaneously in both male and female C57BL/6J mice (4 months old, 20-30g each, Jackson Laboratory) at 4 marked dorsal sites per mouse via incisions in the skin, with IACUC approval (IPROTO202200000017). Incisions were treated with iodine to keep sterile and surgical glue to re-seal, with Carprofen (5mg/kg SC) for mild pain management. Four tissue-only dorsal sites were also marked. Two weeks post-operatively, LIFU was applied to two scaffold sites and two tissue-only sites per mouse using parameters described previously. After two additional weeks of monitoring, mice were euthanized and marked scaffold and tissue-only sites were resected and formalin-fixed for endpoint analysis. Scaffolds and tissue-only sites were fluorescently stained with SYTOX Deep Red (nuclei), BODIPY FL (lipids), Alexa Fluor 555 Phalloidin (F-actin), and polyclonal IL-6 antibody + Alexa Fluor 405 (inflammation). Z-stacks were captured via confocal microscopy.

Results, Conclusions, and Discussions: In preliminary testing with our silk biomaterial system, which did not involve a processing step to artificially add or produce gas microbubbles, LIFU treatment significantly decreased the mass of silk scaffolds compared to untreated scaffolds (Fig 1). The findings indicate that naturally occurring microbubbles trapped at hydrophobic sites in the silk fibroin scaffold architecture were adequate for producing effective acoustic cavitation in response to the chosen LIFU parameters, and are consistent with our hypothesis that LIFU would be effective for non-invasively triggering scaffolds to degrade. Further studies will need to be performed to assess the in vivo degradation timeline and characteristics of implanted scaffolds, such as the implementation of in vivo monitoring methods (fluorescent IVIS imaging) or volumetric analyses (μ-CT). From tissue-only samples resected from mice at the 4-week endpoint (Fig 2a-d), IL-6 staining indicated no significant increase in chronic tissue inflammation as a result of LIFU treatment (Fig 2e), and this was also found to be the case across both sexes (Fig 2f), consistent with our hypothesis that LIFU treatments would be safe in vivo as tested with mice. Further studies will need to be performed to investigate the acute inflammatory effects of LIFU immediately following treatment as an additional safety measure.

Our LIFU technique was demonstrated to be both safe and effective for degrading silk scaffolds in vivo in mice. This marks the first step in designing a personalizable system for degrading implanted biomaterials at different time points and rates on a patient-by-patient basis, which can help to mitigate current issues in the field and improve clinical outcomes for patients.

Acknowledgements (Optional): This work was supported (in part) by a Focused Ultrasound Foundation grant (FUS987R1). The authors would like to acknowledge Angela Malaney and Paul Berger at the CMU Mellon Institute Centralized Vivarium for their guidance and assistance in carrying out in vivo work, and Megan DeBari and Rachel Niu for assisting with LIFU equipment setup.