Development of a novel low-background noise blood loop model for testing blood-contacting biomaterials and medical devices in fresh human blood.

A novel low volume blood loop model (Ension Triad System [ETS]) incorporating pulsatile flow and a proprietary low-activation blood-contacting surface (Ension bioactive surface [EBS]) enabling high signal-to-noise performance is described. The ETS system incorporates a test chamber that allows direct comparison of material samples or finished medical devices such as catheters with varying compositions and/or surface treatments. ETS performance is presented from two independent organizations (Medtronic and MLM Labs) and includes results for hemolysis (pfHgb), platelet count, platelet activation (ßTG), coagulation (TAT), inflammation (PMN Elastase, PMN CD112b, and monocyte CD112b) and immune response (SC5b-9) were made on: (1) the EBS-treated system itself without a test material (No Material, NM); (2) the EBS-treated system with an idealized untreated catheter (UC); and (3) the EBS-treated system with the prototype catheter treated with the EBS surface treatment (CC). The untreated catheter (UC) was associated with significant elevation of all activation marker levels (pfHgb excluded). The EBS-treated catheter, in direct comparison to the UC and NM catheters, appeared invisible with respect to the activation markers (all markers statistically different than the UC and equivalent to the NM control). Based on these data, we conclude that using a relatively small surface area test sample and a small volume of fresh human blood, the high signal-to-noise performance of the ETS system demonstrates comprehensive and statistically significant material differences in the major ISO 10993-4 categories of blood interaction. These data underscore the important benefit of minimal confounding of test/device responses with non-test-material/model-related responses. ETS offers a practical alternative to the common one-test-category-at-a-time approach when assessing blood/medical device interactions.

In vitro methodology for medical device material thrombogenicity assessments: A use condition and bioanalytical proof-of-concept approach.

Device manufacturers and regulatory agencies currently utilize expensive and often inconclusive in vivo vascular implant models to assess implant material thrombogenicity. We report an in vitro thrombogenicity assessment methodology where test materials (polyethylene, Elasthane™ 80A polyurethane, Pebax®), alongside positive (borosilicate glass) and negative (no material) controls, were exposed to fresh human blood, with attention to common blood-contact use conditions and the variables: material (M), material surface modification (SM) with heparin, model (Mo), time (T), blood donor (D), exposure ratio (ER; cm2 material/ml blood), heparin anticoagulation (H), and blood draw/fill technique (DT). Two models were used: (1) a gentle-agitation test tube model and (2) a pulsatile flow closed-loop model. Thrombogenicity measurements included thrombin generation (thrombin-antithrombin complex [TAT] and human prothrombin fragment F1.2), platelet activation (ß-thromboglobulin), and platelet counts. We report that: (a) thrombogenicity was strongly dependent (p < .0001) on M, H, and T, and variably dependent (p < .0001 - > .05) on Mo, SM, and D (b) differences between positive control, test, and negative control materials became less pronounced as H increased from 0.6 to 2.0 U/ml, and (c) in vitro-to-in vivo case comparisons showed consistency in thrombogenicity rankings on materials classified to be of low, moderate, and high concern. In vitro methods using fresh human blood are therefore scientifically sound and cost effective compared to in vivo methods for screening intravascular materials and devices for thrombogenicity.

Preclinical safety and efficacy evaluation of the Pipeline Vantage Embolization Device with Shield Technology.

BACKGROUND: The Pipeline Vantage Embolization Device with Shield Technology is a next generation flow diverter developed to improve aneurysm occlusion and implant endothelialization in addition to lowering thrombogenicity. We report here the in vivo biocompatibility and in vitro hemocompatibility performance of the Pipeline Vantage Embolization Device with Shield Technology (Vantage) compared with the Pipeline Flex Embolization Device (Flex). METHODS: Biocompatibility (via histology), aneurysm occlusion and vessel patency (via angiography), and endothelial coverage (via scanning electron microscopy (SEM)) for the Vantage and Flex devices were assessed in the rabbit elastase aneurysm model at 90 days (n=29) and 180 days (n=27). In vitro thrombogenicity for Flex and Vantage (n=16) was assessed using a human blood flow loop model at low heparin concentration (0.6 U/mL) with thrombin generation, platelet activation and thrombus visualization as outputs. RESULTS: Raymond Roy Occlusion Classification grade 1 was higher for Vantage (61%) compared with Flex (46%), but was not statistically significant (p>0.05). All branch vessels were patent. Histological measures for both devices were similar (p>0.05). Endothelial coverage of the implant was significantly better for Vantage compared with Flex (p<0.05). In vitro measurements of thrombin generation (thrombin-antithrombin complex (µg/mL): Vantage 0.49±0.45; Flex 10.57±9.84) and platelet activation (ß-thromboglobulin (IU/µl): Vantage 0.41±0.19; Flex 4.14±2.38) were both statistically lower (p<0.05) for Vantage compared with Flex. High resolution microscopy showed less accumulation of thrombus on Vantage as compared with Flex. CONCLUSION: Vantage improved aneurysm occlusion and implant endothelialization and had significantly lower thrombogenicity as compared with Flex, while preserving the biocompatibility safety profile of Flex.

Thrombogenicity assessment of Pipeline, Pipeline Shield, Derivo and P64 flow diverters in an in vitro pulsatile flow human blood loop model.

Flow diversion is a disruptive technology for the treatment of intracranial aneurysms. However, these intraluminal devices pose a risk for thromboembolic complications despite dual antiplatelet therapy. We report the thrombogenic potential of the following flow diversion devices measured experimentally in a novel human blood in-vitro pulsatile flow loop model: Pipeline™ Flex Embolization Device (Pipeline), Pipeline™ Flex Embolization Device with Shield Technology™ (Pipeline Shield), Derivo Embolization Device (Derivo), and P64 Flow Modulation Device (P64). Thrombin generation (Mean ±â€¯SD; µg/mL) was measured as: Derivo (28 ±â€¯11), P64 (21 ±â€¯4.5), Pipeline (21 ±â€¯6.2), Pipeline Shield (0.6 ±â€¯0.1) and Negative Control (1.5 ±â€¯1.1). Platelet activation (IU/µL) was measured as: Derivo (4.9 ±â€¯0.7), P64 (5.2 ±â€¯0.7), Pipeline (5.5 ±â€¯0.4), Pipeline Shield (0.3 ±â€¯0.1), and Negative Control (0.9 ±â€¯0.7). We found that Pipeline Shield had significantly lower platelet activation and thrombin generation than the other devices tested (p < .05) and this was comparable to the Negative Control (no device, p > .05). High resolution scanning electron microscopy performed on the intraluminal and cross-sectional surfaces of each device showed the lowest accumulation of platelets and fibrin on Pipeline Shield relative to Derivo, P64, and Pipeline. Derivo and P64 also had higher thrombus accumulation at the flared ends. Pipeline device with Phosphorylcholine surface treatment (Pipeline Shield) could mitigate device material related thromboembolic complications.

Thrombogenicity assessment of Pipeline Flex, Pipeline Shield, and FRED flow diverters in an in vitro human blood physiological flow loop model.

Endovascular treatment of intracranial aneurysms with endoluminal flow diverters (single or multiple) has proven to be clinically safe and effective, but is associated with a risk of thromboembolic complications. Recently, a novel biomimetic surface modification with covalently bound phosphorylcholine (Shield Technology™) has shown to reduce the material thrombogenicity of the Pipeline flow diverter. Thrombogenicity of Pipeline Flex, Pipeline Shield, and Flow Redirection Endoluminal Device (FRED) in the presence of human blood under physiological flow conditions-in addition to relative increase in thrombogenicity with multiple devices-remains unknown and was investigated here. Thrombin generation (mean ± SD; µg/mL; thrombin-antithrombin complex or TAT) was measured as FRED (30.3 ± 2.9), Pipeline (13.9 ± 4.4), Pipeline Shield (0.4 ± 0.3), and negative control (no device; 0.1 ± 0.0). Platelet activation (mean ± SD; IU/µL; beta-thromboglobulin or ßTG) was measured as FRED (148 ± 45), Pipeline (92.8 ± 41), Pipeline Shield (16.2 ± 3.5), and negative control (2.70 ± 0.16). FRED was significantly more thrombogenic than Pipeline and Pipeline Shield (p < 0.05) for TAT. Additionally, Pipeline Shield had significantly lower TAT and ßTG than the other devices tested (p < 0.05) and these were comparable to the negative control (p > 0.05). TAT and ßTG scaled proportionately with multiple Pipeline devices (N = 6) but was unaffected by multiple Pipeline Shield (N = 6) devices-the latter being statistically similar to negative control (p > 0.05). © 2018 The Authors. Journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3195-3202, 2018.

From in vivo to in vitro: The medical device testing paradigm shift.

Amid growing efforts to advance the replacement, reduction, and refinement of the use of animals in research, there is a growing recognition that in vitro testing of medical devices can be more effective, both in terms of cost and time, and also more reliable than in vivo testing. Although the technological landscape has evolved rapidly in support of these concepts, regulatory acceptance of alternative testing methods has not kept pace. Despite the acceptance by regulators of some in vitro tests (cytotoxicity, gene toxicity, and some hemocompatibility assays), many toxicity tests still rely on animals (irritation, sensitization, acute toxicity, reproductive/developmental toxicity), even where other industrial sectors have already abandoned them. Bringing about change will require a paradigm shift in current approaches to testing - and a concerted effort to generate better data on risks to human health from exposure to leachable chemicals from medical devices, and to boost confidence in the use of alternative methods to test devices. To help advance these ideas, stir debate about best practices, and coalesce around a roadmap forward, the JHU-Center for Alternatives to Animal Testing (CAAT) hosted a symposium believed to be the first gathering dedicated to the topic of in vitro testing of medical devices. Industry representatives, academics, and regulators in attendance presented evidence to support the unique strengths and challenges associated with the approaches currently in use as well as new methods under development, and drew next steps to push the field forward from their presentations and discussion.

Protective constriction of coronary vein grafts with knitted nitinol.

OBJECTIVES: Different flow patterns and shear forces were shown to cause significantly more luminal narrowing and neointimal tissue proliferation in coronary than in infrainguinal vein grafts. As constrictive external mesh support of vein grafts led to the complete suppression of intimal hyperplasia (IH) in infrainguinal grafts, we investigated whether mesh constriction is equally effective in the coronary position. METHODS: Eighteen senescent Chacma baboons (28.8 ± 3.6 kg) received aorto-coronary bypass grafts to the left anterior descending artery (LAD). Three groups of saphenous vein grafts were compared: untreated controls (CO); fibrin sealant-sprayed controls (CO + FS) and nitinol mesh-constricted grafts (ME + FS). Meshes consisted of pulse-compliant, knitted nitinol (eight needles; 50 µm wire thickness; 3.4 mm resting inner diameter, ID) spray attached to the vein grafts with FS. After 180 days of implantation, luminal dimensions and IH were analysed using post-explant angiography and macroscopic and histological image analysis. RESULTS: At implantation, the calibre mismatch between control grafts and the LAD expressed as cross-sectional quotient (Qc) was pronounced [Qc = 0.21 ± 0.07 (CO) and 0.18 ± 0.05 (CO + FS)]. Mesh constriction resulted in a 29 ± 7% reduction of the outer diameter of the vein grafts from 5.23 ± 0.51 to 3.68 ± 0 mm, significantly reducing the calibre discrepancy to a Qc of 0.41 ± 0.17 (P < 0.02). After 6 months of implantation, explant angiography showed distinct luminal irregularities in control grafts (ID difference between widest and narrowest segment 74 ± 45%), while diameter variations were mild in mesh-constricted grafts. In all control grafts, thick neointimal tissue was present [600 ± 63 µm (CO); 627 ± 204 µm (CO + FS)] as opposed to thin, eccentric layers of 249 ± 83 µm in mesh-constricted grafts (ME + FS; P < 0.002). The total wall thickness had increased by 363 ± 39% (P < 0.00001) in CO and 312 ± 61% (P < 0.00001) in CO + FS vs 82 ± 61% in ME + FS (P < 0.007). CONCLUSIONS: In a senescent non-human primate model for coronary artery bypass grafts, constrictive, external mesh support of saphenous veins with knitted nitinol prevented focal, irregular graft narrowing and suppressed neointimal tissue proliferation by a factor of 2.5. The lower degree of suppression of IH compared with previous infrainguinal grafts coincided with a lesser reduction of calibre mismatch in the coronary grafts.

Remodeling leads to distinctly more intimal hyperplasia in coronary than in infrainguinal vein grafts.

BACKGROUND: Flow patterns and shear forces in native coronary arteries are more protective against neointimal hyperplasia than those in femoral arteries. Yet, the caliber mismatch with their target arteries makes coronary artery bypass grafts more likely to encounter intimal hyperplasia than their infrainguinal counterparts due to the resultant slow flow velocity and decreased wall stress. To allow a site-specific, flow-related comparison of remodeling behavior, saphenous vein bypass grafts were simultaneously implanted in femoral and coronary positions. METHODS: Saphenous vein grafts were concomitantly implanted as coronary and femoral bypass grafts using a senescent nonhuman primate model. Duplex ultrasound-based blood flow velocity profiles and vein graft and target artery dimensions were correlated with dimensional and histomorphologic graft remodeling in large, senescent Chacma baboons (n = 8; 28.1 ± 4.9 kg) during a 24-week period. RESULTS: At implantation, the cross-sectional quotient (Q(c)) between target arteries and vein grafts was 0.62 ± 0.10 for femoral grafts vs 0.17 ± 0.06 for coronary grafts, resulting in a dimensional graft-to-artery mismatch 3.6 times higher (P < .0001) in coronary grafts. Together with different velocity profiles, these site-specific dimensional discrepancies resulted in a 57.9% ± 19.4% lower maximum flow velocity (P = .0048), 48.1% ± 23.6% lower maximal cycling wall shear stress (P = .012), and 62.2% ± 21.2% lower mean velocity (P = .007) in coronary grafts. After 24 weeks, the luminal diameter of all coronary grafts had contracted by 63%, from an inner diameter of 4.49 ± 0.60 to 1.68 ± 0.63 mm (P < .0001; subintimal diameter: -41.5%; P = .002), whereas 57% of the femoral interposition grafts had dilated by 31%, from 4.21 ± 0.25 to 5.53 ± 1.30 mm (P = .020). Neointimal tissue was 2.3 times thicker in coronary than in femoral grafts (561 ± 73 vs 240 ± 149 µm; P = .001). Overall, the luminal area of coronary grafts was an average of 4.1 times smaller than that of femoral grafts. CONCLUSIONS: Although coronary and infrainguinal bypass surgery uses saphenous veins as conduits, they undergo significantly different remodeling processes in these two anatomic positions.

Knitted nitinol represents a new generation of constrictive external vein graft meshes.

OBJECTIVE: Constriction of vein grafts with braided external nitinol meshes had previously led to the successful elimination of neointimal tissue formation. We investigated whether pulse compliance, smaller kink-free bending radius, and milder medial atrophy can be achieved by knitting the meshes rather than braiding, without losing the suppressive effect on intimal hyperplasia. METHODS: Pulse compliance, bending stiffness, and bending radius, as well as longitudinal-radial deformation-coupling and radial compression, were compared in braided and knitted nitinol meshes. Identical to previous studies with braided mesh grafts, a senescent nonhuman primate model (Chacma baboons; bilateral femoral interposition grafts/6 months) mimicking the clinical size mismatch between vein grafts and runoff arteries was used to examine the effect of knitted external meshes on vein grafts: nitinol mesh-constricted (group 1); nitinol mesh-constricted and fibrin sealant (FS) spray-coated for mesh attachment (group 2); untreated control veins (group 3), and FS spray-coated control veins (group 4). RESULTS: Compared with braided meshes, knitted meshes had 3.8-times higher pulse compliance (3.43 ± 0.53 vs 0.94 ± 0.12%/100 mm Hg; P = .00002); 30-times lower bending stiffness (0.015 ± 0.002 vs 0.462 ± 0.077 Nmm(2); P = .0006); 9.2-times narrower kink-free bending radius (15.3 ± 0.4 vs 140.8 ± 22.4 mm; P = .0006), and 4.3-times lower radial narrowing caused by axial distension (18.0% ± 1.0% vs 77.0% ± 3.7%; P = .00001). Compared with mesh-supported grafts, neointimal tissue was 8.5-times thicker in group I (195 ± 45 µm) vs group III (23.0 ± 21.0 µm; P < .001) corresponding with a 14.3-times larger neointimal area in group I (4330 ± 957 × 103 µm(2)) vs group III (303 ± 221× 103 µm(2); P < .00004). FS had no significant influence. Medial muscle mass remained at 43.4% in knitted meshes vs the 28.1% previously observed in braided meshes. CONCLUSION: Combining the suppression of intimal hyperplasia with a more physiologic remodeling process of the media, manifold higher kink-resistance, and lower fraying than in braided meshes makes knitted nitinol an attractive concept in external vein graft protection.

Tailored sizes of constrictive external vein meshes for coronary artery bypass surgery.

External mesh constriction of vein grafts was shown to mitigate intimal hyperplasia by lowering circumferential wall stress and increasing fluid shear stress. As under-constriction leaves vein segments unsupported and thus prone to neointimal proliferation while over-constriction may cause wall folding optimal mesh sizing holds a key to clinical success. Diameter fluctuations and the occurrence of wall folding as a consequence of external constriction with knitted Nitinol meshes were assessed in saphenous vein grafts from 100 consecutive coronary artery bypass (CABG) patients. Subsequently, mesh dimensions were identified that resulted in the lowest number of mesh sizes for all patients either guaranteeing tight continual mesh contact along the entire graft length (stipulation A) or preventing wall folding (stipulation B). A mathematical data classification analysis and a statistical single-stage partitioning approach were independently applied alternatively prioritizing stipulation A or B. Although the risk of folding linearly increased when constriction exceeded 24.6% (Chi squared test p = 0.0004) the actual incidence of folding (8.6% of veins) as well as the degree of lumenal encroachment (6.2 ± 2.1%) were low. Folds were always single, narrow longitudinal formations (height: 23.3 ± 4.0% of inner diameter/base: 16.6 ± 18.1% of luminal circumference). Both analytical methods provided an optimum number of 4 mesh sizes beyond which no further advantage was seen. While the size ranges recommended by both methods assured continual tight mesh contact with the vein the narrower range suggested by the mathematical data classification analysis (3.0-3.7 mm) put 20.6 ± 12.5% of length in 69% of veins at risk of folding as opposed to 21.3 ± 25.9% being at risk in the wider size range (3.0-4.2 mm) suggested by the statistical partitioning approach. Four mesh sizes would provide uninterrupted mesh contact in 98% of vein grafts in CABG procedures with only 26% of their length being at risk of relatively mild wall folding.