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

Low-intensity pulsed ultrasound (LIPUS) has been shown to be effective for orthopedic fracture repair and nonunion defects, but the specific mechanism behind its efficacy is still unknown. Previously, we have shown a measurable acoustic radiation force at LIPUS intensities traditionally used for clinical treatment and have applied this force to osteoblastic cells encapsulated in type I collagen hydrogels. Our goal in this study is to provide insight and inform the appropriate design of a cell therapy approach to bone repair in which osteoblasts are embedded in collagen hydrogels, implanted into a bony defect, and then transdermally stimulated using LIPUS-derived acoustic radiation force to enhance bone formation at the earliest time points after bone defect repair. To this end, in this study, we demonstrate the ability to measure local hydrogel deformations in response to LIPUS-induced acoustic radiation force and reveal that hydrogel deformation varies with both LIPUS intensity and hydrogel stiffness. Specifically, hydrogel deformation is positively correlated with LIPUS intensity and this deformation is further increased by reducing the stiffness of the hydrogel. We have also shown that encapsulated osteoblastic cells respond to increases in LIPUS intensity by upregulating both cyclooxygenase 2 and prostaglandin E2 (PGE2), both implicated in new bone formation and well-established responses to the application of fluid forces on osteoblast cells. Finally, we demonstrate that combining an increase in LIPUS with a three-dimensional culture environment upregulates both markers beyond their expression noted from either experimental condition alone, suggesting that both LIPUS and hydrogel encapsulation, when combined and modulated appropriately, can enhance osteoblastic response considerably. These studies provide important information toward a clinically relevant cell therapy treatment for bone defects that allows the transdermal application of mechanical loading to bone defects without physically destabilizing the defect site.

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

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.