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 E (PGE), 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.
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.
Few gene markers selectively identify mesenchymal progenitor cells inside the bone marrow. We have investigated a cell population located in the mouse bone marrow labeled by Connective Tissue Growth Factor reporter expression (CTGF-EGFP). Bone marrow flushed from CTGF reporter mice yielded an EGFP+ stromal cell population. Interestingly, the percentage of stromal cells retaining CTGF reporter expression decreased with age in vivo and was half the frequency in females compared to males. In culture, CTGF reporter expression and endogenous CTGF expression marked the same cell types as those labeled using Twist2-Cre and Osterix-Cre fate mapping approaches, which previously has been shown to identify mesenchymal progenitors in vitro. Consistent with this past work, sorted CTGF+ cells displayed an ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and into osteoblast, adipocyte, and stromal cell lineages after transplantation into a parietal bone defect. In vivo examination of CTGF reporter expression in bone tissue sections revealed it marked cells highly localized to the trabecular bone region and was not expressed in the perichondrium or periosteum. Mesenchymal cells retaining high CTGF reporter expression were adjacent to, but distinct from mature osteoblasts lining bone surfaces and endothelial cells forming the vascular sinuses. Comparison of CTGF and Osterix reporter expression in bone tissue sections indicated an inverse correlation between the strength of CTGF expression and osteoblast maturation. Down-regulation of CTGF reporter expression also occurred during in vitro osteogenic differentiation. Collectively, our studies indicate that CTGF reporter mice selectively identify a subpopulation of bone marrow mesenchymal progenitor cells that reside in the trabecular bone region.
Cell therapy is emerging as an effective treatment strategy for many diseases. Here we describe a novel approach to bone tissue repair that combines hydrogel‐based cell therapy with low intensity pulsed ultrasound (LIPUS), an FDA approved treatment for fracture repair. Bone marrow‐derived stromal cells (BMSCs) have been encapsulated in type I collagen hydrogels and mechanically stimulated using LIPUS‐derived acoustic radiation force (ARF). We observed the expression and upward trend of load‐sensitive, osteoblast‐specific markers and determined that the extent of cell response is dependent on an optimal combination of both hydrogel stiffness and ARF intensity. Specifically, cells encapsulated in hydrogels of optimal stiffness respond at the onset of ultrasound by upregulating early bone‐sensitive markers such as calcium, cyclooxygenase‐2, and prostaglandin E2, and later by supporting mineralized tissue formation after 21 days of culture. In vivo evaluation of a critical size calvarial defect in NOD scid gamma (NSG) mice indicated that the implantation of BMSC‐laden hydrogels of optimal stiffness improved healing of calvarial defects after daily administration of ARF over 4 weeks. Collectively, these findings validate the efficacy of our system of localized cell delivery for treating bone defects where undifferentiated BMSCs are induced to the osteoblastic lineage. Further, in vivo healing may be enhanced via non‐invasive transdermal mechanical stimulation of implanted cells using ARF.
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