Evaluation of vessel injury after simulated catheter use in an endothelialized silicone model of the intracranial arteries.

PURPOSE: Neurothrombectomy catheters can disrupt or injure the vessel wall. This potential injury is often studied in animal or cadaver models, but prior work suggests that endothelialized silicone models may be an option for early in vitro assessment. The purpose of this work was to create a complex, clinically-relevant endothelialized neurovascular silicone model, and to determine the utility of the model for evaluating vessel injury due to catheter simulated use. METHODS: Models of the ICA and MCA were fabricated out of silicone, sterilized, coated with fibronectin, placed in bioreactors, and endothelialized with HUVECs. These silicone vessels were maintained under flow for 3 and 7 days, and cellular linings were assessed. Subsequently, 24 silicone vessels were created and treated with neurovascular catheters. Vessels were accessed with a guidewire, microcatheter, and/or aspiration catheter, either once (1-pass) or three times (3-pass). Vessels were then fixed, and injury was evaluated through quantitative image analysis and a visual scoring system. RESULTS: Complex silicone models were successfully endothelialized and maintained with consistent cell linings. The transparent silicone permitted catheter simulated use without fluoroscopy, and injury to the vessel wall was observed and successfully imaged and characterized. Vessels subjected to 3-passes exhibited more injury than 1-pass, and injury increased with the number and size of devices. These results illustrated expected trends and support use of these models for early assessment of vessel injury. CONCLUSION: Complex silicone neurovascular models can be endothelialized and used in vitro to assess and compare injury due to the use of neurovascular catheters.

Cellular responses to flow diverters in a tissue-engineered aneurysm model.

BACKGROUND: Notwithstanding the widespread implementation of flow diverters (FDs) in the treatment of intracranial aneurysms, the exact mechanism of action of these devices remains elusive. We aimed to advance the understanding of cellular responses to FD implantation using a 3D tissue-engineered in vitro aneurysm model. METHODS: Aneurysm-like blood vessel mimics (aBVMs) were constructed by electrospinning polycaprolactone nanofibers onto desired aneurysm-like geometries. aBVMs were seeded with human aortic smooth muscle cells (SMCs) followed by human aortic endothelial cells (ECs). FDs were then deployed in the parent vessel of aBVMs covering the aneurysm neck and were cultivated for 7, 14, or 28 days (n=3 for each time point). The EC and SMC coverage in the neck was measured semi-quantitatively. RESULTS: At day 7, the device segment in contact with the parent vessel was partially endothelialized. Also, the majority of device struts, but not pores, at the parent vessel and neck interface were partially covered with ECs and SMCs, while device struts in the middle of the neck lacked cell coverage. At 14 days, histology verified a neointimal-like lining had formed, partially covering both the struts and pores in the center of the neck. At 28 days, the majority of the neck was covered with a translucent neointimal-like layer. A higher degree of cellular coverage was seen on the struts and pores at the neck at 28 days compared with both 7 and 14 days. CONCLUSION: aBVMs can be a valuable alternative tool for evaluating the healing mechanisms of endovascular aneurysm devices.

Endothelialized silicone aneurysm models for in vitro evaluation of flow diverters.

OBJECTIVE: The goal of this work was to endothelialize silicone aneurysm tubes for use as in vitro models for evaluating endothelial cell interactions with neurovascular devices. The first objective was to establish consistent and confluent endothelial cell linings and to evaluate the silicone vessels over time. The second objective was to use these silicone vessels for flow diverter implantation and assessment. METHODS: Silicone aneurysm tubes were coated with fibronectin and placed into individual bioreactor systems. Human umbilical vein endothelial cells were deposited within tubes to create silicone vessels, then cultivated on a peristaltic pump and harvested at 2, 5, 7, or 10 days to evaluate the endothelial cell lining. A subset of silicone aneurysm vessels was used for flow diverter implantation, and evaluated for cell coverage over device struts at 3 or 7 days after deployment. RESULTS: Silicone vessels maintained confluent, PECAM-1 (platelet endothelial cell adhesion molecule 1) positive endothelial cell linings over time. These vessels facilitated and withstood flow diverter implantation, with robust cell linings disclosed after device deployment. Additionally, the endothelial cells responded to implanted devices through coverage of the flow diverter struts with increased cell coverage over the aneurysm seen at 7 days after deployment as compared with 3 days. CONCLUSIONS: Silicone aneurysm models can be endothelialized and successfully maintained in vitro over time. Furthermore, these silicone vessels can be used for flow diverter implantation and assessment.

SIMPoly: A Matlab-Based Image Analysis Tool to Measure Electrospun Polymer Scaffold Fiber Diameter.

Quantifying fiber diameter is important for characterizing electrospun polymer scaffolds. Many researchers use manual measurement methods, which can be time-consuming and variable. Semi-automated tools exist, but there is room for improvement. The current work used Matlab to develop an image analysis program to quickly and consistently measure fiber diameter in scanning electron micrographs. The new Matlab method, termed "SIMPoly" (Semiautomated Image Measurements of Polymers) was validated by using synthetic images with known fiber size and was found to be accurate. The Matlab method was also applied by three different researchers to scanning electron microscopy (SEM) images of electrospun poly(lactic-co-glycolic acid) (PLGA). Results were compared with the semi-automated DiameterJ method and a manual ImageJ measurement approach, and it was found that the Matlab-based SIMPoly method provided measurements in the expected range and with the least variability between researchers. In conclusion, this work provides and describes SIMPoly, a Matlab-based image analysis method that can simply and accurately measure polymer fiber diameters in SEM images with minimal variation between users.

Custom tissue engineered aneurysm models with varying neck size and height for early stage in vitro testing of flow diverters.

Endovascular techniques for treating cerebral aneurysms are rapidly advancing and require testing to optimize device configurations. The purpose of this work was to customize tissue-engineered aneurysm "blood vessel mimics" (aBVMs) for early stage in vitro assessment of vascular cell responses to flow diverters and other devices. Aneurysm scaffolds with varying neck size and height were created through solid modeling, mold fabrication, mandrel creation, and electrospinning. Scaffold dimensions and fiber morphology were characterized. aBVMs were created by depositing human smooth muscle and endothelial cells within scaffolds, and cultivating within perfusion bioreactors. These vessels were left untreated or used for flow diverter implantation. Cellular responses to flow diverters were evaluated at 3 days. Custom scaffolds were created with aneurysm neck diameters of 2.3, 3.5, and 5.5 mm and with aneurysm heights of 2, 5, and 8 mm. A set of scaffolds with varying neck size was used for aBVM creation, and dual-sodding of endothelial and smooth muscle cells resulted in consistent and confluent cellular linings. Flow diverters were successfully implanted in a subset of aBVMs, and initial cell coverage over devices was seen in the parent vessel at 3 days. Direct visualization of the device over the neck region was feasible, supporting the future use of these models for evaluating and comparing flow diverter healing. Tissue-engineered aneurysm models can be created with custom neck sizes and heights, and used to evaluate cellular responses to flow diverters and other endovascular devices.

Tissue-engineered blood vessel mimics in complex geometries for intravascular device testing.

OBJECTIVE: Intravascular stents are commonly used to treat occluded arteries during coronary heart disease. After coronary stent implantation, endothelial cells grow over the stent, which is referred to as re-endothelialization. Re-endothelialization prevents blood from clotting on the stent surface and is a good predictor of stent success. Blood vessel mimics (BVMs) are in vitro tissue-engineered models of human blood vessels that may be used to preclinically test stents for re-endothelialization. BVMs have been developed in straight geometries. However, the United States Food and Drug Administration recommends that devices intended to treat coronary occlusions be preclinically tested in bent and bifurcated vessels due to the complex geometries of native coronary arteries. The main objectives of this study were to develop and characterize BVMs in complex geometries. DESIGN: Bioreactors were designed and constructed so that BVMs could be cultivated in bent (>45°) and bifurcated geometries. Human umbilical vein endothelial cells were sodded onto complex-shaped scaffolds, and the resulting BVMs were characterized for cell deposition. For a final proof of concept, a coronary stent was deployed in a severely angulated BVM. RESULTS: The new bioreactors were easy to use and mounting scaffolds in complex geometries in the bioreactors was successful. After sodding scaffolds with cells, there were no statistically significant differences between the cell densities along the length of the BVMs, on the top and bottom halves of the BVMs, or on the inner and outer halves of the BVMs. This suggests cells deposited evenly throughout the scaffolds, resulting in consistent complex-geometry BVMs. Also, a coronary stent was successfully deployed in a severely angulated BVM. CONCLUSIONS: Bioreactors can be constructed for housing complex-shaped vessels. BVMs can be developed in the complex geometries observed in native coronary arteries with endothelial cells evenly dispersed throughout BVM lumens.

Tissue-engineered aneurysm models for in vitro assessment of neurovascular devices.

PURPOSE: Preclinical testing of neurovascular devices is crucial for successful device design and is commonly performed using in vivo organisms such as the rabbit elastase-induced aneurysm model; however, simple in vitro models may help further refine this testing paradigm. The purpose of the current work was to evaluate, and further develop, tissue-engineered blood vessel mimics (BVMs) as simple, early-stage models to assess neurovascular devices in vitro prior to animal or clinical use. METHODS: The first part of this work used standard straight-vessel BVMs to evaluate flow diverters at 1, 3, and 5 days post-deployment. The second part developed custom aneurysm-shaped scaffolds to create aneurysm BVMs. Aneurysm scaffolds were characterized based on overall dimensions and microstructural features and then used for cell deposition and vessel cultivation. RESULTS: It was feasible to deploy flow diverters within standard BVMs and cellular linings could withstand and respond to implanted devices, with increasing cell coverage over time. This provided the motivation and foundation for the second phase of work, where methods were successfully developed to create saccular, fusiform, and blister aneurysm scaffolds using a wax molding process. Results demonstrated appropriate fiber morphology within different aneurysm shapes, consistent cell deposition, and successful cultivation of aneurysm BVMs. CONCLUSION: It is feasible to use tissue-engineered BVMs for assessing cellular responses to flow diverters, and to create custom aneurysm BVMs. This supports future use of these models for simple, early-stage in vitro testing of flow diverters and other neurovascular devices.

Thinking inside the box: keeping tissue-engineered constructs in vitro for use as preclinical models.

Tissue engineers have made great strides toward the creation of living tissue replacements for a wide range of tissue types and applications, with eventual patient implantation as the primary goal. However, an alternate use of tissue-engineered constructs exists: as in vitro preclinical models for purposes such as drug screening and device testing. Tissue-engineered preclinical models have numerous potential advantages over existing models, including cultivation in three-dimensional geometries, decreased cost, increased reproducibility, precise control over cultivation conditions, and the incorporation of human cells. Over the past decade, a number of researchers have developed and used tissue-engineered constructs as preclinical models for testing pharmaceuticals, gene therapies, stents, and other technologies, with examples including blood vessels, skeletal muscle, bone, cartilage, skin, cardiac muscle, liver, cornea, reproductive tissues, adipose, small intestine, neural tissue, and kidney. The focus of this article is to review accomplishments toward the creation and use of tissue-engineered preclinical models of each of these different tissue types.

Assessment of the intimal response to a protein-modified stent in a tissue-engineered blood vessel mimic.

Protein-coated intravascular stents have emerged as potential pro-healing modifications for or alternatives to anti-proliferative drug-eluting stents. To support the development of these devices, preclinical testing is required to evaluate the intimal response to new coatings and modifications. The purpose of this work was to implement a tissue-engineered blood vessel as an in vitro testing system to evaluate extracellular matrix-modified stents with regard to endothelialization of the stent surface. Stents were modified by submersion in a protein-enriched medium and were subsequently deployed within tissue-engineered blood vessels and cultivated in vitro under flow to assess the intimal response. Scanning electron microscopy, fluorescent nuclear staining with en face imaging, and histological assessments were performed 7 or 14 days postdeployment. Results illustrated accelerated cellular regeneration over protein-modified stent strut surfaces, with increased coverage and increased tissue thickness atop protein-modified stent struts. In addition, the intimal response to modified stents differed significantly from bare metal stents. Conclusions from this work support the use of a tissue-engineered blood vessel mimic system for evaluation of modified stent surfaces. These findings are important to stent researchers as well as laboratories developing tissue-engineered constructs.

An automatic algorithm for detecting stent endothelialization from volumetric optical coherence tomography datasets.

Recent research has suggested that endothelialization of vascular stents is crucial to reducing the risk of late stent thrombosis. With a resolution of approximately 10 microm, optical coherence tomography (OCT) may be an appropriate imaging modality for visualizing the vascular response to a stent and measuring the percentage of struts covered with an anti-thrombogenic cellular lining. We developed an image analysis program to locate covered and uncovered stent struts in OCT images of tissue-engineered blood vessels. The struts were found by exploiting the highly reflective and shadowing characteristics of the metallic stent material. Coverage was evaluated by comparing the luminal surface with the depth of the strut reflection. Strut coverage calculations were compared to manual assessment of OCT images and epi-fluorescence analysis of the stented grafts. Based on the manual assessment, the strut identification algorithm operated with a sensitivity of 93% and a specificity of 99%. The strut coverage algorithm was 81% sensitive and 96% specific. The present study indicates that the program can automatically determine percent cellular coverage from volumetric OCT datasets of blood vessel mimics. The program could potentially be extended to assessments of stent endothelialization in native stented arteries.

Tissue-engineered vascular grafts as in vitro blood vessel mimics for the evaluation of endothelialization of intravascular devices.

The accelerating use of minimally invasive procedures for the treatment of cardiovascular disease, and the commensurate development of intravascular devices such as stents, has lead to a high demand for preclinical assessment techniques. A 3-dimensional in vitro blood vessel mimic (BVM) would be ideal for device testing before animal or clinical studies. This is possible based on current capabilities for the creation of tissue-engineered vascular grafts (TEVGs). Using an established method of pressure-sodding human endothelial cells onto a polymer scaffold, a BVM was created in an in vitro bioreactor system under flow. Scanning electron microscopy and immunohistochemistry verified a cellular lining and revealed a luminal monolayer of endothelial cells. After BVM development, bare metal stents were deployed. Stented and unstented BVMs were evaluated using fluorescent nuclear staining and optical coherence tomography (OCT). En face and cross-sectional evaluation of bisbenzimide-stained nuclei revealed cellular coverage of the stent surfaces. Cross-sectional evaluation using OCT also illustrated a cellular layer developing over the stent struts. These data support the use of TEVGs as in vitro BVMs for pre-clinical evaluation of the endothelial cell response to stents and endovascular devices.