Establishment of a resazurin-based aortic valve tissue viability assay for dynamic culture in a microphysiological system.

BACKGROUND/AIM: Tissue pathogenesis of aortic valve (AV) stenosis is research focus in cardiac surgery. Model limitations of conventional 2D culture of human or porcine valvular interstitial/endothelial cells (VIC/VECs) isolated from aortic valve tissues but also limited ability of (small) animal models to reflect human (patho)physiological situation in AV position raise the need to establish an in vitro setup using AV tissues. Resulting aim is to approximate (patho)physiological conditions in a dynamic pulsatile Microphysiological System (MPS) to culture human and porcine AV tissue with preservation of tissue viability but also defined ECM composition. MATERIALS/METHODS: A tissue incubation chamber (TIC) was designed to implement human or porcine tissues (3×5 mm2) in a dynamic pulsatile culture in conventional cell culture ambience in a MPS. Cell viability assays based on lactate dehydrogenase (LDH)-release or resazurin-conversion were tested for applicability in the system and applied for a culture period of 14 days with interval evaluation of tissue viability on every other day. Resazurin-assay setup was compared in static vs. dynamic culture using varying substance saturation settings (50-300µM), incubation times and tissue masses and was consequently adapted. RESULTS: Sterile dynamic culture of human and porcine AV tissue segments was established at a pulsatile flow rate range of 0.9-13.4µl/s. Implementation of tissues was realized by stitching the material in a thermoplastic polyurethane (TPU)-ring and insertion in the TIC-MPS-system. Culture volume of 2 ml caused LDH dilution not detectable in standard membrane integrity assay setup. Therefore, detection of resazurin-conversion of viable tissue was investigated. Optimal incubation time for viability conversion was determined at two hours at a saturated concentration of 300µM resazurin. Measurement in static conditions was shown to offer comparable results as dynamic condition but allowing optimal handling and TIC sterilization protocols for long term culture. Preliminary results revealed favourable porcine AV tissue viability over a 14 day period confirmed via resazurin-assay comparing statically cultured tissue counterparts. CONCLUSIONS: Human and porcine AV tissue can be dynamically cultured in a TIC-MPS with monitoring of tissue viability using an adapted resazurin-assay setup. Preliminary results reveal advantageous viability of porcine AV tissues after dynamic TIC-MPS culture compared to static control.

Intermittent claudication: magnitude of the problem, patient evaluation, and therapeutic strategies.

Intermittent claudication (IC), the symptom of exercise-induced muscle ischemia of peripheral arterial disease (PAD), afflicts and limits the activities of a significant number of patients. Incidence and prevalence of IC depends on the population studied and the diagnostic instruments used. In large studies, prevalence has ranged from 3% to 10%, with a sharp increase in those aged > or =70 years. Over the next 20 years, the total number of patients affected is expected to increase significantly due to anticipated demographic changes. Analysis of the natural history of IC demonstrates that the risk of cardiovascular morbidity and mortality far exceeds that of severe limb ischemia or limb loss. In fact, only 2% to 4% of all patients with IC will require a major amputation in their lifetime. However, life expectancy is approximately 10 years less than that of an age-matched cohort. By now, PAD is well recognized as a marker of systemic atherosclerosis. The cornerstone of patient evaluation is a history and physical examination, including a detailed atherosclerotic risk-factor assessment. In the differential diagnosis of IC, clinicians should consider etiologies such as arthritis, spinal stenosis, radiculopathy, venous claudication, or inflammatory processes. In >80% of all patients, it is possible to locate the responsible arterial segment by combining the location and severity of pain with a pulse examination. Noninvasive diagnostic studies help determine the level of disease, may unmask a hemodynamically significant stenosis, and are useful in follow-up. Arteriography is reserved for patients in whom the decision for revascularization has been made. Knowing the anatomic detail of a lesion allows the clinician to determine whether and what type of intervention is feasible. Standard therapy for all patients should be directed at both peripheral and systemic atherosclerosis, beginning with risk-factor modification in the form of smoking cessation, optimal diabetes control, and lipid normalization. The benefits of supervised exercise rehabilitation include significantly increased walking distance and enhanced quality of life. Platelet inhibition has been shown to reduce the risk of ischemic stroke, myocardial infarction, and vascular death and should be prescribed for all but those in whom it is medically contraindicated. Symptom-specific pharmacotherapy with a broad range of medications has yielded disappointing results in the past. However, recent studies have demonstrated that patients receiving the novel agent cilostazol experienced increases in walking distance and improvements in quality of life.

Intraoperative lytic therapy: agents and methods of administration.

Intraoperative intraarterial thrombolysis is a valuable adjunct for the removal of residual arterial thrombi after mechanical thromboembolectomy. Early reports by some investigators indicated high rates of bleeding complications, most likely caused by inappropriately high doses of plasminogen activators infused over long periods of time, which led to systemic lytic effects. Animal models and controlled human experiments subsequently have shown the potential efficacy and safety of intraarterial thrombolysis. Since urokinase (UK) has been withdrawn from the market, recombinant tissue-type plasminogen activator (rt-PA) has become the plasminogen activator of choice. Reteplase and other plasminogen activators may be beneficial (and safe); however, data on intraoperative use currently are not available. The most common methods of delivery into the distal arterial tree are bolus infusion with inflow occlusion, drip infusion after restoration of arterial inflow, and the isolated limb perfusion technique. The number of distal vessels involved, the amount of residual thrombus, and severity of ischemia guide the dose of plasminogen activator, volume of perfusate, and the technique and duration of infusion.