Machine Perfusion Deters Ischemia-Related Derangement of a Rodent Free Flap: Development of a Model.

INTRODUCTION: Machine perfusion can enable isolated support of composite tissues, such as free flaps. The goal of perfusion in this setting is to preserve tissues prior to transplantation or provide transient support at the wound bed. This study aimed to establish a rodent model of machine perfusion in a fasciocutaneous-free flap to serve as an affordable testbed and determine the potential of the developed support protocol to deter ischemia-related metabolic derangement. METHODS: Rat epigastric-free flaps were harvested and transferred to a closed circuit that provides circulatory and respiratory support. Whole rat blood was recirculated for 8 h, while adjusting the flow rate to maintain arterial-like perfusion pressures. Blood samples were collected during support. Extracellular tissue lactate and glucose levels were characterized with a microdialysis probe and compared with warm ischemic, cold ischemic, and anastomosed-free flap controls. RESULTS: Maintenance of physiologic arterial pressures (85-100 mmHg) resulted in average pump flow rates of 360-430 µL/min. Blood-based measurements showed maintained glucose and oxygen consumption throughout machine perfusion. Average normalized lactate to glucose ratio for the perfused flaps was 5-32-fold lower than that for the warm ischemic flap controls during hours 2-8 (P < 0.05). CONCLUSIONS: We developed a rat model of ex vivo machine perfusion of a fasciocutaneous-free flap with maintained stable flow and tissue metabolic activity for 8 h. This model can be used to assess critical elements of support in this setting as well as explore other novel therapies and technologies to improve free tissue transfer.

PDMS-Zwitterionic Hybrid for Facile, Antifouling Microfluidic Device Fabrication.

Poly(dimethylsiloxane) (PDMS) has been used in a wide range of biomedical devices and medical research due to its biostability, cytocompatibility, gas permeability, and optical properties. Yet, some properties of PDMS create critical limitations, particularly fouling through protein and cell adhesion. In this study, a diallyl-terminated sulfobetaine (SB-diallyl) molecule was synthesized and then directly mixed with a commercial PDMS base (Sylgard 184) and curing agent to produce a zwitterionic group-bearing PDMS (PDMS-SB) hybrid that does not require a complex or an additional surface modification process for the desired end product. In vitro examination of antifouling behavior following exposure to fresh ovine blood showed a significant reduction in platelet deposition for the PDMS-SB hybrid surface compared to that of a PDMS control (p < 0.05, n = 5). The manufacturability via soft lithography using the synthesized polymers was found to be comparable to that for unmodified PDMS. Bonding via O2 plasma treatment was confirmed, and the strength was measured and again found to be comparable to the control. PDMS-SB microfluidic devices were successfully fabricated and showed improved blood compatibility that could reduce channel occlusion due to clot formation relative to PDMS control devices. Further, gas (CO2) transfer through a PDMS-SB hybrid membrane was also tested with a proof-of-concept microchannel device and shown to be comparable to that through the PDMS control.

Epoxy silane sulfobetaine block copolymers for simple, aqueous thromboresistant coating on ambulatory assist lung devices.

Developing an ambulatory assist lung (AAL) for patients who need continuous extracorporeal membrane oxygenation has been associated with several design objectives, including the design of compact components, optimization of gas transfer efficiency, and reduced thrombogenicity. In an effort to address thrombogenicity concerns with currently utilized component biomaterials, a low molecular weight water soluble siloxane-functionalized zwitterionic sulfobetaine (SB-Si) block copolymer was coated on a full-scale AAL device set via a one pot aqueous circulation coating. All device parts including hollow fiber bundle, housing, tubing and cannular were successfully coated with increasing atomic compositions of the SB block copolymer and the coated surfaces showed a significant reduction of platelet deposition while gas exchange performance was sustained. However, water solubility of the SB-Si was unstable, and the coating method, including oxygen plasma pretreatment on the surfaces were considered inconsistent with the objective of developing a simple aqueous coating. Addressing these weaknesses, SB block copolymers were synthesized bearing epoxy or epoxy-silane groups with improved water solubility (SB-EP & SB-EP-Si) and no requirement for surface pretreatment (SB-EP-Si). An SB-EP-Si triblock copolymer showed the most robust coating capacity and stability without prior pretreatment to represent a simple aqueous circulation coating on an assembled full-scale AAL device.

Month-long Respiratory Support by a Wearable Pumping Artificial Lung in an Ovine Model.

BACKGROUND: A wearable artificial lung could improve lung transplantation outcomes by easing implementation of physical rehabilitation during long-term pretransplant respiratory support. The Modular Extracorporeal Lung Assist System (ModELAS) is a compact pumping artificial lung currently under development. This study evaluated the long-term in vivo performance of the ModELAS during venovenous support in awake sheep. Feedback from early trials and computational fluid dynamic analysis guided device design optimization along the way. METHODS: The ModELAS was connected to healthy sheep via a dual-lumen cannula in the jugular vein. Sheep were housed in a fixed-tether pen while wearing the device in a holster during support. Targeted blood flow rate and support duration were 2-2.5 L/min and 28-30 days, respectively. Anticoagulation was maintained via systemic heparin. Device pumping and gas exchange performance and hematologic indicators of sheep physiology were measured throughout support. RESULTS: Computational fluid dynamic-guided design modifications successfully decreased pump thrombogenicity from initial designs. For the optimized design, 4 of 5 trials advancing past early perioperative and cannula-related complications lasted the full month of support. Blood flow rate and CO2 removal in these trials were 2.1 ± 0.3 L/min and 139 ± 15 mL/min, respectively, and were stable during support. One trial ended after 22 days of support due to intradevice thrombosis. Support was well tolerated by the sheep with no signs of hemolysis or device-related organ impairment. CONCLUSIONS: These results demonstrate the ability of the ModELAS to provide safe month-long support without consistent deterioration of pumping or gas exchange capabilities.

A biostable, anti-fouling zwitterionic polyurethane-urea based on PDMS for use in blood-contacting medical devices.

Polydimethylsiloxane (PDMS) is commonly used in medical devices because it is non-toxic and stable against oxidative stress. Relatively high blood platelet adhesion and the need for chemical crosslinking through curing, however, limit its utility. In this research, a biostable PDMS-based polyurethane-urea bearing zwitterion sulfobetaine (PDMS-SB-UU) was synthesized for potential use in the fabrication or coating of blood-contacting devices, such as a conduits, artificial lungs, and microfluidic devices. The chemical structure and physical properties of synthesized PDMS-SB-UU were confirmed by 1H-nuclear magnetic resonance (1H-NMR), X-ray diffraction (XRD), and uniaxial stress-strain curve. In vitro stability of PDMS-SB-UU was confirmed against lipase and 30% H2O2 for 8 weeks, and PDMS-SB-UU demonstrated significantly higher resistance to fibrinogen adsorption and platelet deposition compared to control PDMS. Moreover, PDMS-SB-UU showed a lack of hemolysis and cytotoxicity with whole ovine blood and rat vascular smooth muscle cells (rSMCs), respectively. The PDMS-SB-UU was successfully processed into small-diameter (0.80 ± 0.05 mm) conduits by electrospinning and coated onto PDMS- and polypropylene-based blood-contacting biomaterials due to its unique physicochemical characteristics from its soft- and hard- segments.

In vivo testing of the low-flow CO2 removal application of a compact, platform respiratory device.

BACKGROUND: Non-invasive and lung-protective ventilation techniques may improve outcomes for patients with an acute exacerbation of chronic obstructive pulmonary disease or moderate acute respiratory distress syndrome by reducing airway pressures. These less invasive techniques can fail due to hypercapnia and require transitioning patients to invasive mechanical ventilation. Extracorporeal CO2 removal devices remove CO2 independent of the lungs thereby controlling the hypercapnia and permitting non-invasive or lung-protective ventilation techniques. We are developing the Modular Extracorporeal Lung Assist System as a platform technology capable of providing three levels of respiratory assist: adult and pediatric full respiratory support and adult low-flow CO2 removal. The objective of this study was to evaluate the in vivo performance of our device to achieve low-flow CO2 removal. METHODS: The Modular Extracorporeal Lung Assist System was connected to 6 healthy sheep via a 15.5 Fr dual-lumen catheter placed in the external jugular vein. The animals were recovered and tethered within a pen while supported by the device for 7 days. The pump speed was set to achieve a targeted blood flow of 500 mL/min. The extracorporeal CO2 removal rate was measured daily at a sweep gas independent regime. Hematological parameters were measured pre-operatively and regularly throughout the study. Histopathological samples of the end organs were taken at the end of each study. RESULTS: All animals survived the surgery and generally tolerated the device well. One animal required early termination due to a pulmonary embolism. Intra-device thrombus formation occurred in a single animal due to improper anticoagulation. The average CO2 removal rate (normalized to an inlet pCO2 of 45 mmHg) was 75.6 ± 4.7 mL/min and did not significantly change over the course of the study (p > 0.05). No signs of consistent hemolysis or end organ damage were observed. CONCLUSION: These in vivo results indicate positive performance of the Modular Extracorporeal Lung Assist System as a low-flow CO2 removal device.

Acute In Vivo Evaluation of the Pittsburgh Pediatric Ambulatory Lung.

Respiratory failure is a significant problem within the pediatric population. A means of respiratory support that readily allows ambulation could improve treatment. The Pittsburgh Pediatric Ambulatory Lung (P-PAL) is being developed as a wearable pediatric pump-lung for long-term respiratory support and has previously demonstrated positive benchtop results. This study aimed to evaluate acute (4-6 hours) in vivo P-PAL performance, as well as develop an optimal implant strategy for future long-term studies. The P-PAL was connected to healthy sheep (n = 6, 23-32 kg) via cannulation of the right atrium and pulmonary artery. Plasma-free hemoglobin (PfHb) and animal hemodynamics were measured throughout the study. Oxygen transfer rates were measured at blood flows of 1-2.5 L/min. All animals survived the complete study duration with no device exchanges. Flow limitation because of venous cannula occlusion occurred in trial 2 and was remedied via an altered cannulation approach. Blood exiting the P-PAL had 100% oxygen saturation with the exception of trial 4 during which inadequate device priming led to intrabundle clot formation. Plasma-free hemoglobin remained low (<20 mg/dl) for all trials. In conclusion, this study demonstrated successful performance of the P-PAL in an acute setting and established the necessary methods for future long-term evaluation.

Effects of Hollow Fiber Membrane Oscillation on an Artificial Lung.

Gas transfer through hollow fiber membranes (HFMs) can be increased via fiber oscillation. Prior work, however, does not directly translate to present-day, full-scale artificial lungs. This in vitro study characterized the effects of HFM oscillations on oxygenation and hemolysis for a pediatric-sized HFM bundle. Effects of oscillation stroke length (2-10 mm) and frequency (1-25 Hz) on oxygen transfer were measured according to established standards. The normalized index of hemolysis was measured for select conditions. All measurements were performed at a 2.5 L min-1 blood flow rate. A lumped parameter model was used to predict oscillation-induced blood flow and elucidate the effects of system parameters on oxygenation. Oxygen transfer increased during oscillations, reaching a maximum oxygenation efficiency of 510 mL min-1 m-2 (97% enhancement relative to no oscillation). Enhancement magnitudes matched well with model-predicted trends and were dependent on stroke length, frequency, and physical system parameters. A 40% oxygenation enhancement was achieved without significant hemolysis increase. At a constant enhancement magnitude, a larger oscillation frequency resulted in increased hemolysis. In conclusion, HFM oscillation is a feasible approach to increasing artificial lung gas transfer efficiency. The optimal design for maximizing efficiency at small fiber displacements should minimize bundle resistance and housing compliance.

In Vitro Characterization of the Pittsburgh Pediatric Ambulatory Lung.

Acute and chronic respiratory failure are a significant source of pediatric morbidity and mortality. Current respiratory support options used to bridge children to lung recovery or transplantation typically render them bedridden and can worsen long-term patient outcomes. The Pittsburgh Pediatric Ambulatory Lung (P-PAL) is a wearable pediatric blood pump and oxygenator (0.3 m surface area) integrated into a single compact unit that enables patient ambulation. The P-PAL is intended for long-term use and designed to provide up to 90% of respiratory support in children weighing 5-25 kg. Computational fluid dynamics and numerical gas exchange modeling were used to design the P-PAL and predict its performance. A P-PAL prototype was then used to obtain pressure versus flow curves at various impeller rotation rates using a blood analog fluid. In vitro oxygen exchange rates were obtained in blood in accordance with ISO standard 7199. The normalized index of hemolysis (NIH) was measured over a 6 hour period at blood flow rates of 1 and 2.5 L/min. The P-PAL provided blood flows of 1-2.5 L/min against the pressure drop associated with its intended-use pediatric cannulas. The oxygen exchange rate reached a maximum of 108 ml/min at a blood flow rate of 2.5 L/min and met our respiratory support design target. Device-induced hemolysis was low with NIH values of 0.022-0.027 g/100 L in the intended blood flow rate range. In conclusion, the current P-PAL design met our pumping, oxygenation, and hemolysis specifications and has the potential to improve treatment for pediatric respiratory failure.

Effects of Fluorosurfactant Structure and Concentration on Drug Availability and Biocompatibility in Water-in-Perfluorocarbon Emulsions for Pulmonary Drug Delivery.

The efficacy of inhaled antibiotics is often impaired by insufficient drug penetration into plugged and poorly ventilated airways. Liquid ventilation with perfluorocarbon (PFC) containing emulsified aqueous antibiotics, or antibacterial perfluorocarbon ventilation, could potentially improve treatment of respiratory infections when used as an adjunct therapy to inhaled antibiotics. The molecular structure and concentration of the fluorosurfactant used to stabilize such water-in-PFC emulsions will have significant effects on the efficacy and safety of the resulting treatment. In the present study, emulsions are formulated with tobramycin in the aqueous phase using two different fluorosurfactants (termed FSL-PEG+FSL and FSH-PEG) at varying concentrations (Cfs ). An aqueous gel is used to evaluate the availability of emulsified drug to diffuse into an aqueous interface (such as mucus or biofilm) for varying emulsion formulations. Lastly, the cytotoxicity of the fluorosurfactants is characterized using human alveolar basal epithelial cells. Results showed that tobramycin delivery is reduced at low Cfs due to inadequate drug emulsification and at large Cfs due to hindered drug availability. Thus, maximal delivery occurs at intermediate values of Cfs equal to 2 and 10 mg mL-1 for the FSH-PEG and FSL-PEG+FSL fluorosurfactants, respectively. The optimal emulsion formulation utilized FSH-PEG and demonstrated improved drug delivery relative to previously used formulations while exhibiting no cytotoxic effect. This work increases understanding of the physical means of pulmonary drug delivery via a water-in-PFC emulsion and represents a critical step in optimizing emulsion formulation for safe and effective treatment.

Effects of Emulsion Composition on Pulmonary Tobramycin Delivery During Antibacterial Perfluorocarbon Ventilation.

BACKGROUND: The effectiveness of inhaled aerosolized antibiotics is limited by poor ventilation of infected airways. Pulmonary delivery of antibiotics emulsified within liquid perfluorocarbon [antibacterial perfluorocarbon ventilation (APV)] may solve this problem through better airway penetration and improved spatial uniformity. However, little work has been done to explore emulsion formulation and the corresponding effects on drug delivery during APV. This study investigated the effects of emulsion formulation on emulsion stability and the pharmacokinetics of antibiotic delivery via APV. METHODS: Gravity-driven phase separation was examined in vitro by measuring emulsion tobramycin concentrations at varying heights within a column of emulsion over 4 hours for varying values of fluorosurfactant concentration (Cfs = 5-48 mg/mL H2O). Serum and pulmonary tobramycin concentrations in rats were then evaluated following pulmonary tobramycin delivery via aerosol or APV utilizing sufficiently stable emulsions of varying aqueous volume percentage (Vaq = 1%-5%), aqueous tobramycin concentration (Ct = 20-100 mg/mL), and Cfs (15 and 48 mg/mL H2O). RESULTS: In vitro assessment showed sufficient spatial and temporal uniformity of tobramycin dispersion within emulsion for Cfs ≥15 mg/mL H2O, while lower Cfs values showed insufficient emulsification even immediately following preparation. APV with stable emulsion formulations resulted in 5-22 times greater pulmonary tobramycin concentrations at 4 hours post-delivery relative to aerosolized delivery. Concentrations increased with emulsion formulations utilizing increased Vaq (with decreased Ct) and, to a lesser extent, increased Cfs. CONCLUSIONS: The emulsion stability necessary for effective delivery is retained at Cfs values as low as 15 mg/mL H2O. Additionally, the pulmonary retention of antibiotic delivered via APV is significantly greater than that of aerosolized delivery and can be most effectively increased by increasing Vaq and decreasing Ct. APV has been further proven as an effective means of pulmonary drug delivery with the potential to significantly improve antibiotic therapy for lung disease patients.

Characterization of a reverse-phase perfluorocarbon emulsion for the pulmonary delivery of tobramycin.

BACKGROUND: Aerosolized delivery of antibiotics is hindered by poor penetration within distal and plugged airways. Antibacterial perfluorocarbon ventilation (APV) is a proposed solution in which the lungs are partially or totally filled with perfluorocarbon (PFC) containing emulsified antibiotics. The purpose of this study was to evaluate emulsion stability and rheological, antibacterial, and pharmacokinetic characteristics. METHODS: This study examined emulsion aqueous droplet diameter and number density over 24 hr and emulsion and neat PFC viscosity and surface tension. Additionally, Pseudomonas aeruginosa biofilm growth was measured after 2-hr exposure to emulsion with variable aqueous volume percentages (0.25, 1, and 2.5%) and aqueous tobramycin concentrations (Ca=0.4, 4, and 40 mg/mL). Lastly, the time course of serum and pulmonary tobramycin concentrations was evaluated following APV and conventional aerosolized delivery of tobramycin in rats. RESULTS: The initial aqueous droplet diameter averaged 1.9±0.2 µm with little change over time. Initial aqueous droplet number density averaged 3.5±1.7×10(9) droplets/mL with a significant (p<0.01) decrease over time. Emulsion and PFC viscosity were not significantly different, averaging 1.22±0.03×10(-3) Pa·sec. The surface tensions of PFC and emulsion were 15.0±0.1×10(-3) and 14.6±0.6×10(-3) N/m, respectively, and the aqueous interfacial tensions were 46.7±0.3×10(-3) and 26.9±11.0×10(-3) N/m (p<0.01), respectively. Biofilm growth decreased markedly with increasing Ca and, to a lesser extent, aqueous volume percentage. Tobramycin delivered via APV yielded 2.5 and 10 times larger pulmonary concentrations at 1 and 4 hr post delivery, respectively, and significantly (p<0.05) lower serum concentrations compared with aerosolized delivery. CONCLUSIONS: The emulsion is bactericidal, retains the rheology necessary for pulmonary delivery, is sufficiently stable for this application, and results in increased pulmonary retention of the antibiotic.

Thoracic artificial lung impedance studies using computational fluid dynamics and in vitro models.

Current thoracic artificial lungs (TALs) possess blood flow impedances greater than the natural lungs, resulting in abnormal pulmonary hemodynamics when implanted. This study sought to reduce TAL impedance using computational fluid dynamics (CFD). CFD was performed on TAL models with inlet and outlet expansion and contraction angles, θ, of 15°, 45°, and 90°. Pulsatile blood flow was simulated for flow rates of 2-6 L/min, heart rates of 80 and 100 beats/min, and inlet pulsatilities of 3.75 and 2. Pressure and flow data were used to calculate the zeroth and first harmonic impedance moduli, Z(0) and Z(1), respectively. The 45° and 90° models were also tested in vitro under similar conditions. CFD results indicate Z(0) increases as stroke volume and θ increase. At 4 L/min, 100 beats/min, and a pulsatility of 3.75, Z(0) was 0.47, 0.61, and 0.79 mmHg/(L/min) for the 15°, 45°, and 90° devices, respectively. Velocity band and vector plots also indicate better flow patterns in the 45° device. At the same conditions, in vitro Z (0) were 0.69 ± 0.13 and 0.79 ± 0.10 mmHg/(L/min), respectively, for the 45° and 90° models. These Z(0) are 65% smaller than previous TAL designs. In vitro, Z(1) increased with flow rate but was small and unlikely to significantly affect hemodynamics. The optimal design for flow patterns and low impedance was the 45° model.