Beverly L. Roeder

Pulsed Ultrasound Enhances the Killing of Escherichia coli Biofilms by Aminoglycoside Antibiotics In Vivo

ABSTRACT Escherichia coli biofilms on two polyethylene disks were implanted subcutaneously into rabbits receiving systemic gentamicin. Ultrasound was applied for 24 h to one disk. Both disks were removed, and viable bacteria were counted. Pulsed ultrasound significantly reduced bacterial viability below that of nontreated biofilms without damage to the skin.

Ultrasonically enhanced vancomycin activity against Staphylococcus epidermidis biofilms in vivo

Infection 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.

Using stable isotopes to unravel and predict the origins of great cormorants (Phalacrocorax carbo sinensis) overwintering at Kinmen

The Food and Agricultural Organization of the United Nations and the World Organization for Animal Health have called for a better understanding of the role that migrating birds may play in spreading H5N1 highly pathogenic avian influenza (HPAI). Bird banding, traditionally used in studies of migration, is limited by low recapture rates. Telemetry can only be applied to larger species and a limited number of birds. We show that analyses of multiple stable isotopes (delta(13)C, delta(15)N, delta(18)O and deltaD) can provide an understanding of the number of breeding populations represented at large congregations of wintering birds, probable locations of these breeding populations, and which breeding populations do not contribute migrants to a wintering site. As Asia is thought to be the origin of many HPAI strains and the center of their evolution, and as bird migration is poorly understood in this part of the world, we recommend that, in addition to banding, satellite, and VHF telemetry, the stable isotope analysis of migration patterns should become a part of long-term surveillance studies.

Distribution of doxorubicin in rats undergoing ultrasonic drug delivery.

Ultrasound (US) increases efficacy of drugs delivered from micelles, but the pharmacokinetics have not been studied previously. In this study, US was used to deliver doxorubicin (Dox) sequestered in micelles in an in vivo rat model with bilateral leg tumors. One of two frequencies with identical mechanical index and intensity was delivered for 15 min to one tumor immediately after systemic injection of micellar Dox. Pharmacokinetics in myocardium, liver, skeletal muscle, and tumors were measured for 1 week. When applied in combination with micellar Dox, the ultrasoincated tumor had higher Dox concentrations at 30 min, compared to bilateral noninsonated controls. Initially, concentrations were highest in heart and liver, but within 24 h they decreased significantly. From 24 h to 7 days, concentrations remained highest in tumors, regardless of whether they received US or not. Comparison of insonated and noninsonated tumors showed 50% more Dox in the insonated tumor at 30 min posttreatment. Four weekly treatment produced additional Dox accumulation in the myocardium but not in liver, skeletal leg muscle, or tumors compared to single treatment. Controls showed that neither US nor the empty carrier impacted tumor growth. This study shows that US causes more release of drug at the targeted tumor.

Automation of Pressure Control Improves Whole Porcine Heart Decellularization.

Whole heart decellularization combined with patient-specific cells may prove to be an extremely valuable approach to engineer new hearts. Mild detergents are commonly used in the decellularization process, but are known to denature and solubilize key proteins and growth factors and can therefore be destructive to the extracellular matrix (ECM) during the decellularization process. In this study, the decellularization of porcine hearts was accomplished in 24 h with only 6 h of sodium dodecyl sulfate exposure and 98% DNA removal. Automatically controlling the pressure during decellularization reduced the detergent exposure time while still completely removing immunogenic cell debris. Stimulation of macrophages was greatly reduced when comparing native tissue samples to the processed ECM. Complete cell removal was confirmed by analysis of DNA content. General collagen and elastin preservation was demonstrated. Glycosaminoglycans and collagen quantification both showed no significant differences in content after decellularization. The compression elastic modulus of the ECM after decellularization was lower than native at low strains, but there was no significant difference at high strains. Polyurethane casts of the vasculature of native and decellularized hearts demonstrated that the microvasculature network was preserved after decellularization. A static blood thrombosis assay using bovine blood was also developed. Finally, the recellularization potential of the ECM samples was demonstrated by reseeding cardiac fibroblasts and endothelial cells on the myocardium and endocardium samples.

Strategies and processes to decellularize and recellularize hearts to generate functional organs and reduce the risk of thrombosis.

Heart failure is one of the leading causes of death in the United States. Current therapies, such as heart transplants and bioartificial hearts, are helpful, but not optimal. Decellularization of porcine whole hearts followed by recellularization with patient-specific human cells may provide the ultimate solution for patients with heart failure. Great progress has been made in the development of efficient processes for decellularization, and the design of automated bioreactors. Challenges remain in selecting and culturing cells, growing the cells on the decellularized scaffolds without contamination, characterizing the regenerated organs, and preventing thrombosis. Various strategies have been proposed to prevent thrombosis of blood-contacting devices, including reendothelization and the creation of nonfouling surfaces using surface modification technologies. This review discusses the progress and remaining challenges involved with recellularizing whole hearts, focusing on the prevention of thrombosis.

Freezing/Thawing without Cryoprotectant Damages Native but not Decellularized Porcine Renal Tissue

abstract Whole organ decellularization of porcine renal tissue and recellularization with a patients own cells would potentially overcome immunorejection, which is one of the most significant problems with allogeneic kidney transplantation. However, there are obstacles to achieving this goal, including preservation of the decellularized extracellular matrix (ECM), identifying the proper cell types, and repopulating the ECM before transplantation. Freezing biological tissue is the best option to avoid spoilage; however, it may damage the structure of the tissue or disrupt cellular membranes through ice crystal formation. Cryoprotectants have been used to repress ice formation during freezing, although cell toxicity can still occur. The effect of freezing/thawing on native (n = 10) and decellularized (n = 10) whole porcine kidneys was studied without using cryoprotectants. Results showed that the elastic modulus of native kidneys was reduced by a factor of 22 (P < 0.0001) by freezing/thawing or decellularization, while the elastic modulus for decellularized ECM was essentially unchanged by the freezing/thawing process (p = 0.0636). Arterial pressure, representative of structural integrity, was also reduced by a factor of 52 (P < 0.0001) after freezing/thawing for native kidneys, compared to a factor of 43 (P < 0.0001) for decellularization and a factor of 4 (P < 0.0001) for freezing/thawing decellularized structures. Both freezing/thawing and decellularization reduced stiffness, but the reductions were not additive. Investigation of the microstructure of frozen/thawed native and decellularized renal tissues showed increased porosity due to cell removal and ice crystal formation. Orcein and Sirius staining showed partial damage to elastic and collagen fibers after freezing/thawing. It was concluded that cellular damage and removal was more responsible for reducing stiffness than fibril destruction. Cell viability and growth were demonstrated on decellularized frozen/thawed and non-frozen samples using human renal cortical tubular epithelial (RCTE) cells over 12 d. No adverse effect on the ability to recellularize after freezing/thawing was observed. It is recommended that porcine kidneys be frozen prior to decellularization to prevent contamination, and after decellularization to prevent protein denaturation. Cryoprotectants may still be necessary, however, during storage and transportation after recellularization.

Rapid separation of bacteria from blood—review and outlook

The high morbidity and mortality rate of bloodstream infections involving antibiotic‐resistant bacteria necessitate a rapid identification of the infectious organism and its resistance profile. Traditional methods based on culturing the blood typically require at least 24 h, and genetic amplification by PCR in the presence of blood components has been problematic. The rapid separation of bacteria from blood would facilitate their genetic identification by PCR or other methods so that the proper antibiotic regimen can quickly be selected for the septic patient. Microfluidic systems that separate bacteria from whole blood have been developed, but these are designed to process only microliter quantities of whole blood or only highly diluted blood. However, symptoms of clinical blood infections can be manifest with bacterial burdens perhaps as low as 10 CFU/mL, and thus milliliter quantities of blood must be processed to collect enough bacteria for reliable genetic analysis. This review considers the advantages and shortcomings of various methods to separate bacteria from blood, with emphasis on techniques that can be done in less than 10 min on milliliter‐quantities of whole blood. These techniques include filtration, screening, centrifugation, sedimentation, hydrodynamic focusing, chemical capture on surfaces or beads, field‐flow fractionation, and dielectrophoresis. Techniques with the most promise include screening, sedimentation, and magnetic bead capture, as they allow large quantities of blood to be processed quickly. Some microfluidic techniques can be scaled up.

The impact of decellularization agents on renal tissue extracellular matrix

The combination of patient-specific cells with scaffolds obtained from natural sources may result in improved regeneration of human tissues. Decellularization of the native tissue is the first step in this technology. Effective decellularization uses agents that lyse cells and remove all cellular materials, leaving intact collagenous extracellular matrices (ECMs). Removing cellular remnants prevents an immune response while preserving the underlying structure. In this study, the impact of five decellularization agents (0.1 N NaOH, 1% peracetic acid, 3% Triton X-100, 1% sodium dodecyl sulfate (SDS), and 0.05% trypsin/EDTA) on renal tissue was examined using slices of porcine kidneys. The NaOH solution induced the most efficient cell removal, and resulted in the highest amount of cell viability and proliferation after recellularization, although it also produced the most significant damage to collagenous fiber networks, glycosaminoglycans (GAGs) and fibroblast growth factor (FGF). The SDS solution led to less severe damage to the ECM structure but it resulted in lower metabolic activity and less proliferation. Peracetic acid and Triton X-100 resulted in minimum disruption of ECMs and the most preserved GAGs and FGF. However, these last two agents were not as efficient in removing cellular materials as NaOH and SDS, especially peracetic acid, which left more than 80% of cellular material within the ECM. As a proof of principle, after completing the comparison studies using slices of renal ECM, the NaOH process was used to decellularize a whole kidney, with good results. The overall results demonstrate the significant effect of cell lysing agents and the importance of developing an optimized protocol to avoid extensive damage to the ECM while retaining the ability to support cell growth.

Comparison of four decontamination treatments on porcine renal decellularized extracellular matrix structure, composition, and support of human renal cortical tubular epithelium cells.

Engineering whole organs from porcine decellularized extracellular matrix and human cells may lead to a plentiful source of implantable organs. Decontaminating the porcine decellularized extracellular matrix scaffolds is an essential step prior to introducing human cells. However, decontamination of whole porcine kidneys is a major challenge because the decontamination agent or irradiation needs to diffuse deep into the structure to eliminate all microbial contamination while minimizing damage to the structure and composition of the decellularized extracellular matrix. In this study, we compared four decontamination treatments that could be applicable to whole porcine kidneys: 70% ethanol, 0.2% peracetic acid in 1 M NaCl, 0.2% peracetic acid in 4% ethanol, and gamma (γ)-irradiation. Porcine kidneys were decellularized by perfusion of 0.5% (w/v) aqueous solution of sodium dodecyl sulfate and the four decontamination treatments were optimized using segments (n = 60) of renal tissue to ensure a consistent comparison. Although all four methods were successful in decontamination, γ-irradiation was very damaging to collagen fibers and glycosaminoglycans, leading to less proliferation of human renal cortical tubular epithelium cells within the porcine decellularized extracellular matrix. The effectiveness of the other three optimized solution treatments were then all confirmed using whole decellularized porcine kidneys (n = 3). An aqueous solution of 0.2% peracetic acid in 1 M NaCl was determined to be the best method for decontamination of porcine decellularized extracellular matrix.