(A-13) Focused Ultrasound Mediated Release of Bone Morphogenetic Protein 2 for Bone Regeneration

Introduction:: Segmental bone defects impact human health at the global scale, causing disabilities in populations suffering from aging, osteomyelitis, traumatic injuries, and cancer. In 2010, 43.9% of the aging population exhibited low bone mass, a leading cause of osteoporosis and segmental bone defects.1 With the elderly population expected to multiply threefold, this translates to over 570 million individuals globally who will experience a significant loss of bone mass by 2050.² The lack of success with current methods of vascularized bone transport, bone grafting, distraction osteogenesis, cytokine therapeutics, and stem cell-based therapies present major challenges to segmental bone reconstruction.3 Current methods deliver bone morphogenetic protein 2 (BMP-2) by injection or device. One key limitation to these delivery techniques is the lack of precise control over location and time (spatiotemporal control). Due to uncontrolled diffusion into surrounding tissues, the current cytokine delivery methods often lead to inflammation, bone resorption, or abnormal bone formation.4 Furthermore, the uncontrolled diffusion presents the need for repetitive treatments or counter treatments, decreasing overall effectiveness and increasing cost. As a novel solution to this challenge, we aim to use focused ultrasound (fUS) to release growth factors from stimuli-responsive hydrogel systems. With fUS as an image-guided external stimulus, cytokine release from hydrogel systems can be precisely and spatiotemporally controlled. Through careful design of the hydrogel systems and crosslinking groups, a controlled release delivery system can be engineered to provide independent control of BMP-2 delivery at the site of defect.


Materials and Methods:: The rationally designed polyethylene glycol (PEG) hydrogel system combined with tunable ultrasound-sensitive Diels-Alder (DA) chemistry (Figure 1A) provides controlled and sustained protein delivery. The responsive 4-arm and 8-arm Diels-Alder PEG constructs were crosslinked through cycloaddition of 3,2-Furyl-propionic acid and maleimide, resulting in PEG FDA-4 and PEG FDA-8 respectively. The 4-arm PEG control group substituted responsive crosslinking with glutaraldehyde, resulting in PEG GLUT-4.

Constructs were loaded with trypsin (~24kDa) as a model payload, with similar size to the final payload BMP-2 (~26kDa). Trypsin release kinetics between groups and protein activity post-fUS exposure were measured. Each hydrogel was crosslinked within a 9mm diameter ultrasound plate. Trypsin released from ultrasound-induced hydrogel restructuring was resuspended in ultrapure water. Each sample was exposed to fUS from a 1.5 MHz transducer at 2% duty cycle with waveform amplitudes of 100mV, 300mV, and 700mV. The ultrapure water was extracted for protein quantification measurements via bicinchoninic acid (BCA) assay to compare release kinetics between constructs. Fully active trypsin was diluted to similar protein quantity through BCA assay and cell-lifting studies were completed to determine optimized fUS parameters of 300mV (p- = 15 MPa and p+ = 68 MPa). The optimized parameters were applied with single targeting at times of 0, 1, and 3 minutes, as well as double and triple targeting at 3 minutes for release studies.

Hydrogel groups were crosslinked with media and degraded via fUS, resulting in 5% w/v PEG hydrogel media to assess biocompatibility using LIVE/DEAD, PicoGreen, and CellTiter Blue assays (Figure 1E-G).


Results, Conclusions, and Discussions:: Focused ultrasound proved to be a successful image-guided external trigger (Figure 1C) for retrograde Diels-Alder reactions through local heating (Figure 1B). Exposure to fUS also induced higher levels of protein release in PEG hydrogels with DA linkers. This ultrasound-mediated release is shown through quantified trypsin BCA assay measurements (Figure 1D). Finally, the degraded PEG hydrogel constructs proved to be biocompatible with bone-marrow derived stem cells (BMSC’s). This was determined by assessing cell viability, quantification, and metabolic activity using LIVE/DEAD, PicoGreen, and CellTiter Blue respectively on BMSC’s and 5% w/v degraded PEG matrices in cell media (Figure 1E-G).


(Figure 1 insert here)

Figure 1. (A) Graphical depiction of Diels-Alder reaction to fUS and the associated BMP-2 release. (B) Thermocouple temperature measurements of PEG FDA-4 during 5 minutes of 300 mV fUS exposure and the 5 minutes following exposure. (C) Guided ultrasound targeting at 300mV and imaging capabilities of PEG FDA-4 and PEG GLUT-4 hydrogel restructuring after 3 minutes of ultrasound exposure. (D) Trypsin release from PEG GLUT-4, PEG FDA-4, and PEG FDA-8 using 1.5 MHz transducer at 300mV (n = 4, ns = no significance, *** = p < 0.001, **** = p < 0.0001). (E) Representative LIVE/DEAD staining of BMSC’s with and without exposure to 5% w/v degraded PEG hydrogel media at days 1, 3, and 5. Scale bar = 200 μm. (F) Metabolic activity relative to BMSC’s cultured without exposure to degraded PEG hydrogel (n = 3, ns = no significance). (G) Cell count of BMSC’s cultured with and without exposure to degraded PEG hydrogel matrices (n = 3, ns = no significance).


Hydrogel systems crosslinked with stimuli-responsive DA linkages were successfully restructured at a higher rate than the control crosslinking group. Protein quantification data and ultrasound imaging show highest levels of release and restructuring in PEG FDA-4. Ultrasound-mediated protein release from hydrogels is a promising approach for the future of bone regeneration. Current studies on the ultrasound-responsive PEG hydrogel constructs include BMP-2 release kinetics and differentiation of BMSC’s from hydrogel release. With promising in vitro data, future studies would include an in vivo femoral defect model.


Acknowledgements (Optional): :

References (Optional): : ¹Wright, Nicole C, et al. 2014. Journal of Bone and Mineral Research. DOI: 10.1002/jbmr.2269

²Peterson, Peter G. 1999. Foreign Affairs. DOI: 10.2307/20020238

³Mauffrey, Cyril, et al. 2015. Journal of the American Academy of Orthopaedic Surgeons. DOI:10.5435/JAAOS-D-14-00018

⁴James, Aaron W., et al. 2016. Tissue Engineering Part B: Reviews. DOI: 10.1089/ten.teb.2015.0357