Three-dimensional print of a liver for preoperative planning in living donor liver transplantation.

The growing demand for liver transplantation and the concomitant scarcity of cadaveric livers have increased the need for living donor liver transplantation (LDLT). Ensuring the safety of donors and recipients is critical. The preoperative identification of the vascular and biliary tract anatomy with 3-dimensional (3D) printing may allow better preoperative surgical planning, avert unnecessary surgery in patients with potentially unsuitable anatomy, and thereby decrease the complications of liver transplant surgery. We developed a protocol and successfully 3D-printed synthetic livers (along with their complex networks of vascular and biliary structures) replicating the native livers of 6 patients: 3 living donors and 3 respective recipients who underwent LDLT. To our knowledge, these are the first complete 3D-printed livers. Using standardized preoperative, intraoperative, and postoperative assessments, we demonstrated identical anatomical and geometrical landmarks in the 3D-printed models and native livers.

In vivo assessment of endothelial function in human lower extremity arteries.

OBJECTIVE: Endothelial function has been measured in preclinical studies in human brachial and coronary arteries but not in lower extremity arteries affected by atherosclerosis. We describe a novel, first-in-man evaluation of endothelial function of the superficial femoral arteries (SFAs) in patients with peripheral arterial disease (PAD). METHODS: Enrolled were 25 patients with PAD requiring lower extremity angiography. Endothelial-dependent relaxation was measured using intravascular ultrasound (IVUS) imaging and a Doppler flow wire after the infusion of acetylcholine (Ach). IVUS-derived virtual histology of the same vessel was calculated. Endothelial-independent relaxation was measured with an infusion of nitroglycerin (200 µg). Levels of nitric oxide and serum nitric oxide metabolites were determined by laboratory analysis. RESULTS: Patients (48% male; mean age, 62 years) had a history of hypertension (80%), coronary disease (36%), and diabetes (40%). The mean SFA diameter was 5.2 ± 1 mm (range, 3.2-6.9 mm). Patients tolerated Ach infusion with no adverse events. Endothelial-dependent relaxation increased over baseline for all patients with Ach infusion of 10(-6) to 10(-4). At Ach 10(-4), diameter (0.5%) and area (1.8%) changes in the diseased SFAs were modest and insignificant; however, average peak velocity of blood flow significantly increased 26%, 46%, and 63% with an Ach 10(-6) to 10(-4) infusion. Calculations of limb volumetric flow (68% at Ach 10(-4)) were significantly increased after Ach infusion. Lower extremity nitric oxide levels were slightly lower than systemic venous levels (P = .04). Nitroglycerin infusion indicated normal smooth muscle responsiveness (3% diameter, 9% area, and 116% velocity change over baseline). IVUS-virtual histology plaque stratification indicated predominantly fibrous morphology (46%; necrotic core, 29%; calcium, 18%). Atheroma burden was 14.9 ± 5.5 mm(3)/cm and did not correlate with endothelial responsiveness. CONCLUSIONS: Endothelial function can be measured directly in human lower extremity arteries at the sites of vascular disease. Despite extensive atherosclerosis, endothelial function is still intact. These data support the application of regional endothelial-specific biologic therapies in patients with PAD.

Choosing transcatheter aortic valve replacement in porcelain aorta: outcomes versus surgical replacement.

OBJECTIVES: Porcelain aorta complicates aortic valve replacement and is an indication for transcatheter approaches. No study has compared surgical and transcatheter valve replacement in the setting of porcelain aorta. We characterize porcelain aorta patients undergoing aortic valve replacement and the association of aortic calcification and outcomes. METHODS: Patients undergoing aortic valve replacement with porcelain aorta were identified. Aortic calcium volume was determined using 3D computed tomography thresholding techniques. Propensity scoring was performed to assess the effect of surgical versus transcatheter approaches. Risk factors for composite major hospital complications (death, stroke and dialysis) were identified using random forest machine learning. RESULTS: From January 2006 to January 2015, 164 patients with porcelain aorta underwent aortic valve replacement [105 (64%) surgical replacement, 59 (36%) transcatheter replacement]. Propensity scoring matched 29 pairs (49% of transcatheter patients). Before matching, 5-year survival was 41% [(43% surgical, 35% transcatheter, P(log-rank) = 0.9]. After matching, mortality for surgical versus transcatheter replacement was 3.4% (n = 1) vs 10% (n = 3), stroke 14% (n = 4) vs 3.4% (n = 1) and dialysis 6.9% (n = 2) versus 11% (n = 3). Matched 5-year survival was 40% after surgical replacement and 29% after transcatheter replacement [P(log-rank) = 0.4]. Total aortic calcium volume was greater in transcatheter than surgical patients [18 (8.0) vs 17 (7.7) ml] and was associated with more major hospital complications after either approach. CONCLUSIONS: Surgical and transcatheter approaches are complementary options for aortic stenosis with porcelain aorta. Surgical valve replacement remains an effective treatment for patients requiring concomitant procedures. Quantifying aortic calcium volume is a helpful risk predictor in all patients with porcelain aorta.

A pilot, prospective evaluation of a direct thrombin inhibitor, bivalirudin (Angiomax), in patients undergoing lower extremity bypass.

OBJECTIVE: Replacing heparin with bivalirudin has been beneficial in patients undergoing coronary intervention and coronary artery bypass. The use of this alternative anticoagulant during peripheral bypass operations has not been studied. Concerns over distal thrombosis using this direct thrombin inhibitor (DTI) prompted a single-arm, open-label, pilot prospective trial of bivalirudin in patients undergoing lower extremity bypass to assess perioperative safety and efficacy. METHODS: Between 2006 and 2007, 18 patients met criteria for enrollment and underwent primary lower extremity bypass using bivalirudin. All patients had severe symptomatic atherosclerotic disease requiring lower extremity bypass. Bivalirudin at a bolus dose of 0.75 mg/kg and continuous infusion of 1.75 mg/kg/hr was used as the sole anticoagulant. RESULTS: Patients (mean age, 67 years) underwent femoral-popliteal (n = 14) or femoral-tibial (n = 4) bypass preferentially using saphenous vein (83%). Mean operative time was 261 minutes, with bivalirudin infusion time of 95 +/- 26 minutes (mean +/- standard deviation). Reliable anticoagulation was achieved with weight-based dosing with activated clotting time values at baseline (systemic) of 131 +/- 92 seconds, during infusion (systemic) of 347 +/- 36 seconds, and from the distal vasculature (limb) of 345 +/- 66 seconds. Distal limb bivalirudin levels were stable at 9755 +/- 3860 ng/mL during clamp occlusion. Mean estimated blood loss was 332 +/- 191 mL with four patients (22%) requiring blood products. One patient required revision of the proximal anastomosis during the initial hospitalization. At 30 days, all bypass operations were patent with improvement of mean ankle-brachial index from 0.57 to 0.81. There were no deaths, myocardial infarctions, or amputations in the 30-day postoperative period. Based on the Thrombolysis in Myocardial Infarction classification for bleeding, one patient had major bleeding (>2 units of packed red blood cells), and three patients had minor bleeding within the first 30 days. CONCLUSIONS: Bivalirudin is a safe and effective anticoagulant for lower extremity bypass operations. Thrombosis beyond the distal clamp was not seen. A comparative trial to standard anticoagulation is warranted.

Evaluation of peripheral atherosclerosis: a comparative analysis of angiography and intravascular ultrasound imaging.

OBJECTIVE: Angiography remains a critical component for diagnostic imaging and therapeutic intervention in peripheral arterial disease (PAD). The goal of this study was to compare angiography with corresponding intravascular ultrasound (IVUS) imaging of the same vessels in patients with PAD. METHODS: From 2004 to 2008, 93 patients undergoing angiography for PAD were recruited in a prospective observational analysis. At the time of angiography, diseased lower extremities were interrogated using a 10-cm IVUS pullback with registration points. IVUS data were analyzed with radiofrequency techniques for vessel and lumen diameter, plaque volume, plaque composition, and cross-sectional area. Similarly, three vascular surgeons blinded to the IVUS data graded corresponding angiographic images according to vessel diameter, degree of stenosis, degree of calcification, and extent of eccentricity. Statistical analyses of matched IVUS images and angiograms were performed. RESULTS: The distribution of demographic and risk variables were typical for PAD: 54% male, 96% hypertension, 78% hyperlipidemia, 44% diabetic, 87% tobacco history, 65% coronary artery disease, and 10% end-stage renal disease. Symptoms precipitating the angiographic evaluation included claudication (53%), rest pain (18%), and tissue loss (29%). Angiographic and IVUS interpretation were similar for luminal diameters, but external vessel diameter was greater by IVUS imaging (7.0 +/- 0.7 vs 5.2 +/- 0.8 mm, P < .05). The two-dimensional diameter method resulted in a significant correlation for stenosis determination (r = 0.84); however, IVUS determination of vessel area stenosis was greater by 10% (95% confidence interval, 0.3%-21%, P < .05). IVUS imaging indicated that a higher proportion of plaques were concentric. Grading of calcification was moderate to severe in 40% by angiography but in only 7% by IVUS (P < .05). CONCLUSIONS: In the evaluation of PAD, angiography and IVUS imaging provide similar luminal diameters and diameter-reducing stenosis measurements. Determination of overall vessel diameter and interpretation of plaque morphology by angiography are discordant from IVUS-derived data.

Arterial calcification increases in distal arteries in patients with peripheral arterial disease.

The aim of this study was to determine if significant differences in plaque composition exist between the popliteal and tibial vessels in patients with severe peripheral arterial disease (PAD). Forty-four patients with PAD required either above-knee (n = 38), below-knee (n = 5), or through-knee (n = 1) amputation for pedal sepsis/gangrene. The 51 vessels (anterior tibial, n = 9; posterior tibial, n = 10; peroneal, n = 3; popliteal, n = 29) were obtained and underwent intravascular ultrasound (IVUS) evaluation ex vivo within 24 hr of amputation. Sequential IVUS data were obtained at known intervals throughout the vessel length and then analyzed with radiofrequency techniques for quantification of plaque composition, plaque volume, and total vessel volume. Plaque composition was categorized as fibrous, fibro-fatty, necrotic core, and dense calcium. Clinical data were obtained via review of electronic records at the time of amputation. Two-sided t-tests were performed to compare components within each plaque. Results are expressed as mean percentage +/- standard error of the mean. Tibial vessels had more dense calcium within these plaques than popliteal arteries (33.8 +/- 5.6% vs. 10.6 +/- 1.9%, p < 0.001). Consequently, distal vessels had less fibro-fatty and fibrous plaque than popliteal arteries (7.7 +/- 1.4% vs. 13.1 +/- 1.2%, p < 0.005; 42.4 +/- 4.7% vs. 61.4 +/- 2.2%, p < 0.001, respectively). Necrotic core plaque composition was found to be similar when comparing tibial versus popliteal arteries (16.1% vs. 14.9%, p = nonsignificant). Clinical factors including diabetes, hyperlipidemia, and chronic renal insufficiency were not associated with plaque composition differences using a univariate analysis. As we progress distally in the arterial tree of patients with PAD, calcium plaque content increases with decreasing burden of fibro-fatty plaque. Clinical and demographic factors, with the exception of smoking, were not found to be associated with atherosclerotic plaque composition.

Topical recombinant thrombin at a concentration of 1000 IU/mL reliably shortens in vivo TTH and delivers durable hemostasis in the presence of heparin anticoagulation and clopidogrel platelet inhibition in a rabbit model of vascular bleeding.

BACKGROUND: This study was designed to evaluate the effect of recombinant human thrombin (rThrombin) concentration on time to hemostasis (TTH), clot durability, and clot strength in settings that replicate the heparinization and platelet inhibition often found in surgical populations. METHODS: A modified, anticoagulated rabbit arteriovenous shunt preparation was selected to model vascular anastomotic bleeding. Rabbits were treated with heparin or heparin + clopidogrel and TTH was measured after applying a range of topical rThrombin concentrations or placebo, in combination with absorbable gelatin sponge, USP. Treatments (placebo, rThrombin) were randomly assigned and the investigator was blinded to treatment. TTH was evaluated with the Kaplan-Meier method. After hemostasis was achieved, clot burst assessment was performed for heparin + clopidogrel treated animals. Clot viscoelastic strength and kinetics were measured in ex-vivo samples using thromboelastography (TEG) methods. RESULTS: TTH decreased with increasing concentrations of rThrombin in heparin-treated animals and was shorter after treatment with 1000 IU/mL rThrombin (73 seconds) than with 125 IU/mL rThrombin (78 seconds; p = 0.007). TTH also decreased with increasing concentrations of rThrombin in heparin + clopidogrel treated animals; again it was significantly shorter after treatment with 1000 IU/mL rThrombin (71 seconds) than with 125 IU/mL rThrombin (177 seconds; p < 0.001). Variability in TTH was significantly smaller after treatment with 1000 IU/mL rThrombin than after 125 IU/mL rThrombin, indicating greater reliability of clot formation (p < 0.001 for heparin or heparin + clopidogrel treatments). Clot durability was examined in heparin + clopidogrel treated animals. Clots formed in the presence of 1000 IU/mL rThrombin were significantly less likely to rupture during clot burst assessment than those formed in the presence of 125 IU/mL rThrombin (0% versus 79%, p < 0.001). In vitro clot strength and clot kinetics, as determined by TEG in heparin + clopidogrel samples, were positively associated with the amount of rThrombin activity added for clot initiation. CONCLUSION: In an animal model designed to replicate the anti-coagulation regimens encountered in clinical settings, topical rThrombin at 1000 IU/mL more reliably controlled the pharmacological effects of heparin or heparin + clopidogrel on hemostasis than rThrombin at 125 IU/mL. Results from in vitro assessments confirmed a positive relationship between the amount of rThrombin activity and both the rate of clot formation and clot strength.

Delta 2-specific opioid receptor agonist and hibernating woodchuck plasma fraction provide ischemic neuroprotection.

OBJECTIVES: The authors present evidence that the delta opioid receptor agonist Deltorphin-D(variant) (Delt-D(var)) and hibernating woodchuck plasma (HWP), but not summer-active woodchuck plasma (SAWP), can provide significant neuroprotection from focal ischemia in mice by a mechanism that relies in part on reducing nitric oxide (NO) release in ischemic tissue. METHODS: Cerebral ischemia was produced in wild-type and NO synthase-deficient (NOS(-/-)) mice by transient, 1-hour middle cerebral artery occlusion (MCAO). Behavioral deficits were determined at 22 hours and infarct volume was assessed at 24 hours after MCAO. Mice were treated with saline or Delt-D(var) at 2.0 and 4.0 mg/kg, or 200 microL of HWP or SAWP. NOS(-/-) mice were treated with either saline or Delt-D(var) at 4.0 mg/kg. NO release was determined using an N9 microglial cell line pretreated with delta- or mu-specific opioids and HWP or SAWP prior to activation with lipopolysaccharide and interferon-gamma. Nitrate in the medium was measured as an indicator of NO production. RESULTS: Infusion of Delt-D(var) or HWP (but not SAWP) decreased infarct volume and improved behavioral deficits following 1 hour of MCAO and 24 hours of reperfusion. In NOS(-/-) mice, endothelial NOS(+/+) is required to provide Delt-D(var)-induced neuroprotection. Delt-D(var) and HWP dose-dependently decreased NO release in cell culture, while SAWP and other delta- and mu-specific opioids did not. CONCLUSIONS: Delt-D(var) and HWP, but not SAWP, are effective neuroprotectant agents in a mouse model of transient MCAO. In cell culture, the mechanism of this ischemic neuroprotection may rely in part on their ability to block NO release.

Angiography underestimates peripheral atherosclerosis: lumenography revisited.

PURPOSE: To compare angiograms, considered the gold standard for diagnostic imaging of peripheral arterial disease (PAD), to the corresponding histological sections of popliteal and tibial vessels obtained after amputation to determine if angiography fails to define atheroma burden in "normal appearing" arteries in patients with PAD. METHODS: Between 2004 and 2006, 69 patients underwent amputation of a lower extremity for severe tissue loss, gangrene, or pedal sepsis precluding limb salvage. Popliteal and tibial vessels were harvested, perfusion-fixed, and analyzed histologically. Thirty-four of these patients had pre-amputation angiography during attempted salvage procedures. Angiograms with patent or minimally diseased vessel segments (n = 19) were assessed for stenoses, diameter, and calcification by 3 vascular surgeons (n = 72 evaluations). These results were compared to corresponding cross-sectional histological slides (n = 66) in a blinded manner. RESULTS: Angiograms performed prior to above-knee (n = 9) or below-knee (n = 10) amputation revealed 24 stenoses with a mean (+/-SD) diameter-reducing stenosis of 19.5%+/-15.2%. Corresponding histological cross sections revealed greater linear stenoses measured via boundaries of the internal elastic lamina (IEL stenosis, 28.9%+/-20.2%, p = 0.003 versus angiography) or via boundaries of the external elastic membrane (vessel stenosis, 43.1%+/-15.2%, p<0.0001). Stenosis calculated by area methods (IEL area) were greater and measured 39.2%+/-24.2% (p<0.0001) and 60.9%+/-15.2% (vessel area, p<0.0001). Popliteal arteries had greater discrepancy in stenosis measurement than tibial arteries (18.5%+/-14.6% versus 34.9%+/-21.0%, p = 0.0005). However, evaluations of tibial arteries for concentricity of plaque (44% versus 69%, p = 0.08) and calcification grade (1.6 versus 2.2, p = 0.002) by angiography were discordant with histological analyses. Measurement of arterial diameter by histology for popliteal arteries (6.2+/-0.9 mm) and tibial arteries (3.1+/-0.7 mm) was greater than angiographic diameter determination (p<0.001). CONCLUSION: Angiography provides information on luminal characteristics of peripheral arteries but severely underestimates the extent of atherosclerosis in patients with PAD even in "normal appearing" vessels.

Calcium channel blockers and angiotensin-converting enzyme inhibitors may be associated with altered atherosclerotic plaque size and morphology.

Correlations of atherosclerotic plaque attributes with clinical presentation have not been studied in peripheral arterial disease (PAD). The aim of the current study was to identify clinical variables associated with alterations in PAD plaque morphology. Thirty-one patients underwent intravascular ultrasonography (IVUS) at the time of arteriography for symptomatic PAD. IVUS data were analyzed with radiofrequency techniques for quantification of plaque composition, plaque volume, and total vessel volume. Associations between plaque characteristics and clinical variables were evaluated. Univariable and multivariable analyses were performed using t-test, Pearson correlations, F-tests, and analysis of variance. Calcium (Ca2+) channel blocker use was associated with a smaller total atherosclerotic plaque burden (44.2 +/- 2.7 vs 52.9 +/- 2.5%; p < .05), and decreased fibrous plaque content (18.2 +/- 1.8% vs 24.0 +/- 1.9%; p < .05). Angiotensin-converting enzyme (ACE) inhibitor use, however, was associated with a larger total atherosclerotic plaque burden (58.3 +/- 2.2% vs 42.9 +/- 2.1%; p < .01) and larger fibrous plaque content (27.2 +/- 2.0% vs 17.7 +/- 1.6%; p < .001). Multivariable analysis was performed to evaluate which factors may differentially impact the response variable measurements of plaque volume to vessel volume. Based on this model, those without the use of an antihyperlipidemic agent or ACE inhibitor had an average total atherosclerotic plaque burden of 47.7%. Those on an antihyperlipidemic agent had an average decrease of 7.0% (p < .05), whereas those on ACE inhibitors had an average increase of 16.2% from the baseline value (p < .001). The use of calcium channel blockers is associated with significantly decreased atherosclerotic plaque burden and decreased fibrous plaque content, whereas the use of ACE inhibitors was associated with an increase in plaque burden and an increased fibrous plaque content. The use of these medications in PAD may alter plaque morphology with the potential to affect clinical outcomes.

Comparison of recombinant human thrombin and plasma-derived human alpha-thrombin.

Bleeding can be a serious complication of surgery, and topical thrombin is widely used as an adjunct to hemostasis in diverse surgical settings. The potent hemostatic properties of thrombin derive from its ability to activate platelets directly to aggregate and adhere to damaged vessels and to catalyze the formation simultaneously of a fibrin matrix. Application of exogenous thrombin bypasses the physiological process of generating a thrombin burst by directly initiating the terminal reactions of blood clot formation. Currently, thrombin used to control surgical bleeding is primarily from bovine plasma, with a small percentage from human plasma. Human thrombin isolated from pooled plasma carries the risk of transmitting plasma-borne pathogens or prion diseases. The bovine preparations have been associated with protein and preparative contaminants that pose potential risks of developing cross-reacting antibodies. There is a need for a pure therapeutic preparation of human thrombin. Recombinant human thrombin (rhThrombin) has been efficiently produced from a prethrombin-1 precursor obtained from Chinese hamster ovary cell culture. This rhThrombin is substantially free of process-derived contaminants and has been characterized extensively in terms of composition, primary, secondary, and tertiary structure, enzymatic activity; and in vivo pharmacology. In vivo studies of topically applied rhThrombin have shown it is effective in achieving hemostasis in a rabbit liver excisional wound model. Clinical studies are ongoing to evaluate the safety and efficacy of rhThrombin as an adjunct to hemostasis in patients undergoing surgery.

Factor XIIIa incorporates thymosin beta4 preferentially into the fibrin(ogen) alphaC-domains.

It was shown recently that tissue transglutaminase and presumably plasma transglutaminase, factor XIIIa, can covalently incorporate into fibrin(ogen) a physiologically active peptide, thymosin beta(4) [(Huff et al. (2002) FASEB J. 16, 691-696]. To clarify the mechanism of this incorporation, we studied the interaction of thymosin beta(4) with fibrinogen, fibrin, and their recombinant fragments, the gamma-module (gamma-chain residues 148-411), and the alphaC-domain (Aalpha-chain residues 221-610) and its truncated variants by immunoblot and ELISA. No significant noncovalent interaction between them was detected in the absence of activated factor XIII, while in its presence thymosin beta(4) was effectively incorporated into fibrin and to a lesser extent into fibrinogen. The incorporation at physiological concentrations of fibrin(ogen) and factor XIII was significant with molar incorporation ratios of thymosin beta(4) to fibrinogen and fibrin of 0.2 and 0.4, respectively. Further experiments revealed that although activated factor XIII incorporates thymosin beta(4) into the isolated gamma-module and alphaC-domain, in fibrin the latter serves as the major incorporation site. This site was further localized to the COOH-terminal portion of the alphaC-domain including residues 392-610.