During germ-cell migration in the mouse, the dynamics of embryo growth cause many germ cells to be left outside the range of chemoattractive signals from the gonad. At E10.5, movie analysis has shown that germ cells remaining in the midline no longer migrate directionally towards the genital ridges, but instead rapidly fragment and disappear. Extragonadal germ cell tumors of infancy, one of the most common neonatal tumors, are thought to arise from midline germ cells that failed to die. This paper addresses the mechanism of midline germ cell death in the mouse. We show that at E10.5, the rate of apoptosis is nearly four-times higher in midline germ cells than those more laterally. Gene expression profiling of purified germ cells suggests this is caused by activation of the intrinsic apoptotic pathway. We then show that germ cell apoptosis in the midline is activated by down-regulation of Steel factor (kit ligand) expression in the midline between E9.5 and E10.5. This is confirmed by the fact that removal of the intrinsic pro-apoptotic protein Bax rescues the germ-cell apoptosis seen in Steel null embryos. Two interesting things are revealed by this: first, germ-cell proliferation does not take place in these embryos after E9.0; second, migration of germ cells is highly abnormal. These data show first that changing expression of Steel factor is required for normal midline germ cell death, and second, that Steel factor is required for normal proliferation and migration of germ cells.

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Dipyridamole significantly improves the calvarial bone regeneration capacity of 3DPBC scaffolds. The most significant difference in bone regeneration was observed centrally within the interface between the 3DPBC scaffold and the dura mater.

The hypothesis that ultrasound increases antibiotic transport through biofilms of Escherichia coli and Pseudomonas aeruginosa was investigated using colony biofilms. Biofilms grown on membrane filters were transferred to nutrient agar containing 50 μg/mL gentamicin. A smaller filter was placed on top of the biofilm and a blank concentration disk was situated atop the filter. Diffusion of antibiotic through the biofilms was allowed for 15, 30, or 45 min at 37°C. Some of these biofilms were exposed to 70 kHz ultrasound and others were not. Each concentration disk was then placed on a nutrient agar plate spread with a lawn of E. coli. The resulting zone of inhibition was used to calculate the amount of gentamicin that was transported through the biofilm into the disk. The E. coli and P. aeruginosa biofilms grown for 13 and 24 h were exposed to two different ultrasonic power densities. Ultrasonication significantly increased the transport of gentamicin through the biofilm. Insonation of biofilms of E. coli for 45 minutes more than doubled the amount of gentamicin compared to their non-insonated counterparts. For P. aeruginosa biofilms, no detectable gentamicin penetrated the biofilm within 45 min without ultrasound; however, when insonated (1.5 W/cm 2 ) for 45 min, the disks collected more than 0.45 μg of antibiotic. Ultrasonication significantly increased transport of gentamicin across biofilms that normally blocked or slowed gentamicin transport when not exposed to ultrasound. This enhanced transport may be partially responsible for the increased killing of biofilm bacteria exposed to combinations of antibiotic and ultrasound.

SUMMARYInfection of implanted medical devices by Gram-positive organisms such as Staphylococcus ssp. is a serious concern in the biomaterial community. In this research the application of low frequency ultrasound to enhance the activity of vancomycin against implanted Staphylococcus epidermidis biofilms was examined. Polyethylene disks covered with a biofilm of S. epidermidis were implanted subcutaneously in rabbits on both sides of their spine. The rabbits received systemic vancomycin for the duration of the experiment. Following 24 h of recovery, one disk was insonated for 24 or 48 h while the other was a control. Disks were removed and viable bacteria counted. At 24 h of insonation, there was no difference in viable counts between control and insonated biofilms, while at 48 h of insonation there were statistically fewer viable bacteria in the insonated biofilm. The S. epidermidis biofilms responded favorably to combinations of ultrasound and vancomycin, but longer treatment times are required for this Gram-positive organism than was observed previously for a Gram-negative species.

Distraction osteogenesis is a bone-regenerative process in which an osteotomy is followed by distraction of the surrounding vascularized bone segments, with formation of new bone within the distraction gap. Distraction osteogenesis is efficacious for reconstructing critical sized bony defects in the appendicular and craniofacial skeleton. To provide opportunity to expand applications of distraction osteogenesis, it is important to have a thorough understanding of the underlying molecular biology and physiology of bone development and fracture healing. To accomplish these objectives a review of the literature was performed using search terms "endochondral ossification, intramembranous ossification, craniofacial skeleton, appendicular skeleton, fracture healing, bone development, and distraction osteogenesis." Bones of the craniofacial and appendicular skeleton have distinct mechanisms of embryonic development. The former develops from growth centers of mesenchymal precursors through intramembranous ossification. The latter forms though endochondral ossification in growth plates. However, both endochondral and intramembranous bone share similar master regulatory transcription factors and downstream growth factors. Fracture healing mirrors the pathway by which these bones developed embryonically. In contrast, bone formed by distraction osteogenesis does so by intramembranous ossification, regardless of whether it occurs within the appendicular or craniofacial skeleton. Understanding molecular pathway differences between bone formation by these mechanisms may allow for optimization and expansion of skeletal reconstruction by distraction osteogenesis.

Background: This paper examines the mechanism of ultrasonic enhanced drug delivery from Pluronic micelles. In previous publications by our group, fluorescently labeled Pluronic was shown to penetrate HL-60 cells with and without the action of ultrasound, while drug uptake was increased with the application of ultrasound.

Low-frequency ultrasound has been investigated as an adjuvant to antimicrobial therapy, targeted at both planktonic and biofilm (sessile) organisms. Our previous work showed that ultrasound (US) effectively enhances the bactericidal activity of certain antibiotics against planktonic cultures (Pitt et al., 1994;Rediske et al., 1999) and in vitro biofilms (Johnson et al., 1998;Qian et al., 1999) and in vivo biofilms (Carmen et al., 2004b(Carmen et al., , 2005Rediske et al., 2000) of gram-positive and gram-negative bacteria. Ultrasound was shown to increase the transport of antibiotics through biofilms (Carmen et al., 2004a) which could account for some (or all) of the enhanced antibiotic activity against insonated biofilms; but such a mechanism could not account for US-enhanced antibiotic activity in planktonic cultures which have no extensive exopolymer matrix to retard antibiotic transport.Because this ultrasonic enhancement of antibiotic activity operates on both planktonic and sessile bacteria, we posit that US does more than simply increase the transport of antibiotic to the cells; ultrasound is postulated to increase uptake of antibiotic into the cells by rendering the cell membrane more permeable to the antibiotic. To examine this postulate, we must first review how ultrasound interacts with cells.Bacterial cells are fairly transparent to ultrasound; that is, ultrasonic waves go right through cells with little absorption, scattering or other interaction. However, the pressure oscillations of ultrasound produce size oscillations in any gas bubbles in the liquid (Brennen, 1995). These bubbles range in size from approximately 1 mm to 100 mm in diameter (Brennen, 1995). The oscillations of bubbles, called cavitation, are generally divided into "stable" and "collapse" types of cavitation. Stable cavitation is the low intensity oscillation of the bubbles without complete collapse of the bubble, while collapse cavitation occurs at higher intensity levels and lower frequencies wherein these bubbles collapse and violently accelerate the fluid around them. During bubble collapse, adiabatic heating of the gas produces very high temperature, produces free radicals, generates very high liquid shear force, and generates a shock wave as the collapsing spherical wall slams into itself (Brennen, 1995). With a sufficient number of collapse cavitation events, cell membranes