The effect of acoustic radiation force on osteoblasts in cell/hydrogel constructs for bone repair

Abstract

Ultrasound, or the application of acoustic energy, is a minimally invasive technique that has been used in diagnostic, surgical, imaging, and therapeutic applications. Low-intensity pulsed ultrasound (LIPUS) has been used to accelerate bone fracture repair and to heal non-union defects. While shown to be effective the precise mechanism behind its utility is still poorly understood. In this study, we considered the possibility that LIPUS may be providing a physical stimulus to cells within bony defects. We have also evaluated ultrasound as a means of producing a transdermal physical force that could stimulate osteoblasts that had been encapsulated within collagen hydrogels and delivered to bony defects. Here we show that ultrasound does indeed produce a measurable physical force and when applied to hydrogels causes their deformation, more so as ultrasound intensity was increased or hydrogel stiffness decreased. MC3T3 mouse osteoblast cells were then encapsulated within hydrogels to measure the response to this force. Statistically significant elevated gene expression for alkaline phosphatase and osteocalcin, both well-established markers of osteoblast differentiation, was noted in encapsulated osteoblasts (p < 0.05), suggesting that the physical force provided by ultrasound may induce bone formation in part through physically stimulating cells. We have also shown that this osteoblastic response is dependent in part on the stiffness of the encapsulating hydrogel, as stiffer hydrogels resulted in reducing or reversing this response. Taken together this approach, encapsulating cells for implantation into a bony defect that can potentially be transdermally loaded using ultrasound presents a novel regenerative engineering approach to enhanced fracture repair.

Mechanically Loading Cell/Hydrogel Constructs with Low-Intensity Pulsed Ultrasound for Bone Repair

“…Toward this end, we have developed a methodology that can mechanically stimulate hydrogel-encapsulated cells using low-intensity pulsed ultrasound (LIPUS)-derived acoustic radiation force (ARF). 5 ARF is the force that is introduced when an area is irradiated with a sound field 6 and manifests itself by driving particles within the sound field to drift and cluster. Traditionally, ARF is used at very high intensities, as high as 1 MW/cm 2 , for techniques such as tissue imaging and elastography to evaluate the mechanical properties of intact human tissue, [7][8][9] but resulting thermal effects from the required high intensities limit its clinical utility.…”

Low-intensity pulsed ultrasound promotes cell motility through vinculin-controlled Rac1 GTPase activity

“…The pulse modulation of the LIPUS signal produces motion occurring at a frequency of 1 kHz, and has been shown to produce tissue movement in the nanometre range (Harrison et al, 2016); replication of this motion has been shown to generate the same phenotype in chondrocytes as stimulation with LIPUS (Argadine et al, 2005). More recently, Veronick et al (2016) showed that LIPUS stimulation induces movement of fluorescent beads encapsulated within a collagen hydrogel.…”

A Comparison of 1‐ and 3.2‐MHz Low‐Intensity Pulsed Ultrasound on Osteogenesis on Porous Titanium Alloy Scaffolds: An In Vitro and In Vivo Study

“…Numerous reports suggest that LIPUS does not affect cell proliferation . Akagi et al reported no influence of LIPUS on cell proliferation in rat bone marrow cells.…”

“…Akagi et al reported no influence of LIPUS on cell proliferation in rat bone marrow cells. Veronick et al demonstrated that LIPUS had no effect on MC3T3 mouse osteoblast cell proliferation on hydrogels. In our in vitro study, we also did not detect any effect of LIPUS on cell proliferation by CCK‐8 and PI assays.…”