Roberto Plasenzotti

Interspecies differences in coagulation profile

Many animals are used in research on blood coagulation and fibrinolysis, but the relevance of animal models to human health is often questioned because of differences between species. The objective was to find an appropriate animal species, which mimics the coagulation profile in humans most adequately. Species differences in the coagulation profile with and without thrombin stimulation in vitro were assessed in whole blood by Rotation Thromboelastometry (ROTEM). Endogenous thrombin generation was measured in platelet-poor plasma. Measurements were performed in blood from five different species: humans, rats, pigs, sheep and rabbits. In humans and sheep, the clotting time (ROTEM) was in the same range with or without thrombin stimulation and a 100-fold lower dose of thrombin (0.002 IU) was required to cause a shortening in the clotting time as compared to rats, pigs and rabbits (0.2 IU) (p<0.05). Similarly, the endogenous thrombin potential (ETP) was in the same range in humans and sheep. The maximum clot firmness with or without thrombin stimulation was similar in rabbits and humans. The maximum lysis with or without thrombin stimulation was similar in humans and pigs. Significant species differences exist in the coagulation profile with or without thrombin stimulation. Most importantly, sheep had a clotting time most similar to humans and could thus be a suitable species for translational coagulation studies. Moreover, our findings confirm the potential usefulness of pigs as an experimental species to study fibrinolytic pathway and support the usefulness of rabbits as a species for examining platelets.

Healing characteristics of electrospun polyurethane grafts with various porosities.

Pore size and porosity control the rate and depth of cellular migration in electrospun vascular fabrics and thus have a strong impact on long-term graft success. In this study we investigated the effect of graft porosity on cell migration in vitro and in vivo. Polyurethane (PU) grafts were fabricated by electrospinning as fine-mesh, low-porosity grafts (void fraction (VF) 53%) and coarse-mesh, high-porosity grafts (VF 80%). The fabricated grafts were evaluated in vitro for endothelial cell attachment and proliferation. Prostheses were investigated in a rat model for either 7 days, 1, 3 or 6 months (n=7 per time point) and analyzed after retrieval by biomechanical analysis and various histological techniques. Cell migration was calculated by computer-assisted morphometry. In vitro, fine-pore mesh favored early cell attachment. In vivo, coarse mesh grafts revealed significantly higher cell populations at all time points in all areas of the conduit wall. Biomechanical tests indicated sufficient compliance, tensile and suture retention strength before and after implantation. Increased porosity improves host cell ingrowth and survival in electrospun conduits. These conduits show successful natural host vessel reconstitution without limitation of biomechanical properties.

Electrospun Small‐Diameter Polyurethane Vascular Grafts: Ingrowth and Differentiation of Vascular‐Specific Host Cells

No small-diameter synthetic graft has yet shown comparable performance to autologous vessels. Synthetic conduits fail due to their inherent surface thrombogenicity and the development of intimal hyperplasia. In addressing these shortcomings, electrospinning offers an interesting alternative to other nanostructured, cardiovascular substitutes because of the close match of electrospun materials to the biomechanical and structural properties of native vessels. In this study, we investigated the in vivo behavior of electrospun, small-diameter conduits in a rat model. Vascular grafts composed of polyurethane were fabricated by electrospinning. Prostheses were implanted into the abdominal aorta in 40 rats for either 7 days, 4 weeks, 3 months, or 6 months. Retrieved specimens were evaluated by histology, immunohistochemical staining, confocal laser scanning microscopy, and scanning electron microscopy. At all time points, we found no evidence of foreign body reaction or graft degradation. The overall patency rate of the intravascular implants was 95%. Within 7 days, grafts revealed ingrowth of host cells. CD34+ cells increased significantly from 7 days up to 6 months of implantation (P < 0.05). Myofibroblasts and myocytes showed increasing cell numbers up to 3 months (P < 0.05). Ki67 staining indicated unaltered cell proliferation during the whole follow-up period. Besides biomechanical benefits, electrospun polyurethane grafts exhibit excellent biocompatibility in vivo. Cell immigration and differentiation seems to be promoted by the nanostructured artificial matrix.

Daptomycin, Fosfomycin, or Both for Treatment of Methicillin-Resistant Staphylococcus aureus Osteomyelitis in an Experimental Rat Model

ABSTRACT The in vivo activities of daptomycin, fosfomycin, and a combination of both antibiotics against a clinical isolate of methicillin-resistant Staphylococcus aureus (daptomycin MIC, 0.25 μg/ml; fosfomycin MIC, 0.5 μg/ml) were evaluated in a rat model of osteomyelitis. A total of 37 rats with experimental osteomyelitis were treated for 4 weeks with either 60 mg/kg of body weight of daptomycin subcutaneously once daily, 75 mg/kg fosfomycin intraperitoneally once daily, a combination of both drugs, or a saline placebo. After the completion of treatment, animals were euthanized, and the infected tibiae were processed for quantitative bacterial culture. Bone cultures were found to be positive for methicillin-resistant S. aureus in 9 of 9 (100%) animals of the placebo group, in 9 of 9 (100%) animals treated with daptomycin, in 1 of 10 (10%) fosfomycin-treated rats, and in 1 of 9 (22.2%) rats comprising the combination group. Results of bacterial counts in the bone samples were expressed as log10 CFU/g of bone and analyzed by using the Mann-Whitney U test followed by Bonferronis multiple-comparison test. Based on bacterial counts, treatment with daptomycin was significantly superior to placebo, although it remained inferior to treatment with fosfomycin. No synergistic or antagonistic effect was observed for the combination therapy. No development of resistance against daptomycin or fosfomycin was observed after the 4-week treatment period.

Decellularized, xenogeneic small-diameter arteries: Transition from a muscular to an elastic phenotype in vivo

Reports regarding the biocompatibility of xenogeneic, decellularized bioprosthetic implants differ between bioinertness and complete graft degradation. We investigated heparin-crosslinked and nonheparinized, xenogeneic vascular substitutes in a rat model. Porcine arteries (15 x 1.5 mm) were decellularized by multistep detergent and enzymatic techniques, which were followed by heparin-crosslinking in 50% of the implants. Prostheses were implanted into the abdominal aorta of 76 rats for 1 day and up to 6 months. Retrieved specimens were evaluated by histology, immunohistochemistry, laser scanning, and scanning electron microscopy. Graft patency did not differ between groups (97.3%). Heparinized grafts showed a statistically significant lower rate of aneurysm formation (p = 0.04 %). Implants revealed infiltration with granulocytes and macrophages up to 3 months. Recellularization with endothelial cells and myofibroblasts was detectable within 1 month. After 6 months elastin biosynthesis and complete graft remodeling toward an elastic vessel was evident. These results indicate that temporary inflammation does not interfere with long-term vascular remodeling.

Efficacy of Fosfomycin in Experimental Osteomyelitis Due to Methicillin-Resistant Staphylococcus aureus

ABSTRACT The activity of fosfomycin was evaluated in an experimental methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis model. Eighteen rats were treated for 4 weeks with 150 mg of fosfomycin/kg of body weight intraperitoneally once daily or with saline placebo. After treatment, animals were euthanized and the infected tibiae were processed for quantitative bacterial culture. Bone cultures were positive for methicillin-resistant S. aureus in all 9 (100%) untreated controls and in 2 of 9 (22.2%) fosfomycin-treated rats. Thus, fosfomycin treatment was significantly more efficacious than placebo. No development of resistance was observed after the 4-week treatment period.

Multi-Functional OCT Enables Longitudinal Study of Retinal Changes in a VLDLR Knockout Mouse Model

We present a multi-functional optical coherence tomography (OCT) imaging approach to study retinal changes in the very-low-density-lipoprotein-receptor (VLDLR) knockout mouse model with a threefold contrast. In the retinas of VLDLR knockout mice spontaneous retinal-chorodoidal neovascularizations form, having an appearance similar to choroidal and retinal neovascularizations (CNV and RNV) in neovascular age-related macular degeneration (AMD) or retinal angiomatous proliferation (RAP). For this longitudinal study, the mice were imaged every 4 to 6 weeks starting with an age of 4 weeks and following up to the age of 11 months. Significant retinal changes were identified by the multi-functional imaging approach offering a threefold contrast: reflectivity, polarization sensitivity (PS) and motion contrast based OCT angiography (OCTA). By use of this intrinsic contrast, the long-term development of neovascularizations was studied and associated processes, such as the migration of melanin pigments or retinal-choroidal anastomosis, were assessed in vivo. Furthermore, the in vivo imaging results were validated with histological sections at the endpoint of the experiment. Multi-functional OCT proves as a powerful tool for longitudinal retinal studies in preclinical research of ophthalmic diseases. Intrinsic contrast offered by the functional extensions of OCT might help to describe regulative processes in genetic animal models and potentially deepen the understanding of the pathogenesis of retinal diseases such as wet AMD.

Effects of intraoperatively applied glucocorticoid-hydrogels on residual hearing and foreign-body reaction in a guinea pig model of cochlear implantation

Abstract Conclusion: The intraoperative application of glucocorticoid-loaded hydrogels seems to cause a reduction in neutrophil infiltration. No beneficial effect on hearing thresholds was detected. Objectives: To evaluate the application of dexamethasone- and triamcinolone acetonide-loaded hydrogels for effects on hearing preservation and foreign body reaction in a guinea pig model for cochlear implantation (CI). Methods: A total of 48 guinea pigs (n = 12 per group) were implanted with a single channel electrode and intraoperatively treated with 50 μl of a 20% w/v poloxamer 407 hydrogel loaded with 6% dexamethasone or 30% triamcinolone acetonide, a control hydrogel, or physiological saline. Click- and tone burst-evoked compound action potential thresholds were determined preoperatively and directly postoperatively as well as on days 3, 7, 14, 21, and 28. At the end of the experiment, temporal bones were prepared for histological evaluation by a grinding/polishing technique with the electrode in situ. Three ears per treatment group were serially sectioned and evaluated for histological alterations. Results: The intratympanic application of glucocorticoid-loaded hydrogels did not improve the preservation of residual hearing in this cochlear implant model. The foreign body reaction to the electrode appeared reduced in the glucocorticoid-treated animals. No correlation was found between the histologically described trauma to the inner ear and the resulting hearing threshold shifts.

Extended in vivo evaluation of a miniaturized axial flow pump with a novel inflow cannula for a minimal invasive implantation procedure

BACKGROUND Minimally invasive techniques are desirable to minimize surgical trauma during left ventricular assist device (LVAD) implantation. This is particularly challenging for full-flow support. In this study, a minimally invasive implantation technique was developed for a microaxial rotary pump. The system was evaluated in a chronic sheep model. METHODS A HeartWare MVAD (HeartWare, Miami Lakes, FL) pump (length, 50 mm; diameter, 21 mm; maximum flow, 7-8 liters/min) was combined with a novel inflow cannula, including a new flow-optimized tip. The device was implanted into sheep (range, 60-80 kg, mean, 71.6 ± 6.8 kg) through a right-sided minithoracotomy. The inflow cannula was inserted through the superior pulmonary vein, passing through the left atrium into the left ventricle. Scheduled implant period was 30 days for 8 sheep and 100 days for 3 sheep. Mean support flow was set to half of the nominal cardiac output. RESULTS Six of 8 sheep finished the scheduled 30-day investigation period (one failed due to early non-pump-related post-operative bleeding and one due to prototype controller failure). The 3 sheep scheduled for 100 days reached the study end point. Peak pump flows of up to 6.9 liters/min were achieved. At necropsy, no signs of mitral valve lesions or thrombus formation around the cannula, the tip, or the insertion site were observed, except for valve leaflet erosion in 1 animal, where the cannula had been entangled in the sub-valvular chords due to lack of ultrasound monitoring. CONCLUSIONS The minimally invasive implantation technique using the HeartWare MVAD pump, together with a new cannula, provided excellent results in a chronic animal model.

Cartilage damage and bone erosion are more prominent determinants of functional impairment in longstanding experimental arthritis than synovial inflammation

ABSTRACT Chronic inflammation of articular joints causing bone and cartilage destruction consequently leads to functional impairment or loss of mobility in affected joints from individuals affected by rheumatoid arthritis (RA). Even successful treatment with complete resolution of synovial inflammatory processes does not lead to full reversal of joint functionality, pointing to the crucial contribution of irreversibly damaged structural components, such as bone and cartilage, to restricted joint mobility. In this context, we investigated the impact of the distinct components, including synovial inflammation, bone erosion or cartilage damage, as well as the effect of blocking tumor necrosis factor (TNF) on functional impairment in human-TNF transgenic (hTNFtg) mice, a chronic inflammatory erosive animal model of RA. We determined CatWalk-assisted gait profiles as objective quantitative measurements of functional impairment. We first determined body-weight-independent gait parameters, including maximum intensity, print length, print width and print area in wild-type mice. We observed early changes in those gait parameters in hTNFtg mice at week 5 – the first clinical signs of arthritis. Moreover, we found further gait changes during chronic disease development, indicating progressive functional impairment in hTNFtg mice. By investigating the association of gait parameters with inflammation-mediated joint pathologies at different time points of the disease course, we found a relationship between gait parameters and the extent of cartilage damage and bone erosions, but not with the extent of synovitis in this chronic model. Next, we observed a significant improvement of functional impairment upon blocking TNF, even at progressed stages of disease. However, blocking TNF did not restore full functionality owing to remaining subclinical inflammation and structural microdamage. In conclusion, CatWalk gait analysis provides a useful tool for quantitative assessment of functional impairment in inflammatory destructive arthritis. Our findings indicate that cartilage damage and bone erosion, but not synovial inflammation, are the most important determinants for progressive functional impairment in this chronic erosive arthritis model. Summary: Gait-profile assessment is an objective quantitative measurement to monitor functional impairment in arthritis models. Longstanding functional impairment correlates with inflammation-driven structural damage.