Nanoengineered Stent Surface to Reduce In-Stent ... - ACS Publications

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Nanoengineered Stent Surface to Reduce In-Stent Restenosis in Vivo Harald Nuhn,† Cesar E. Blanco,† and Tejal A. Desai*,‡ †

The Alfred E. Mann Institute for Biomedical Engineering at the University of Southern California, 1042 Downey Way, DRB Building, Suite 101, Los Angeles, California 90089-1112, United States ‡ Department of Bioengineering and Therapeutic Sciences and The UC Berkeley−UCSF Graduate Group in Bioengineering, University of CaliforniaSan Francisco, San Francisco, California 94158, United States S Supporting Information *

ABSTRACT: In-stent restenosis (ISR) is the leading cause of stent failure and is a direct result of a dysfunctional vascular endothelium and subsequent overgrowth of vascular smooth muscle tissue. TiO2 nanotubular (NT) arrays have been shown to affect vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) in vitro by accelerating VEC cell proliferation and migration while suppressing VSMCs. This study investigates for the first time the potentially beneficial effects of TiO2 NT arrays on vascular tissue in vivo. TiO2 NT arrays (NT diameter: 90 ± 5 nm, height: 1800 ± 300 nm) were grown on the surface of titanium stents and characterized in terms of surface morphology and stability. Stents were implanted into the iliofemoral artery using an overinflation model (rabbit). After 28 days, stenosis rates were determined. The data show a statistically significant reduction of stenosis by 30% compared to the control. Tissue in the presence of TiO2 NTs appears more mature, and less neointima is present between struts. In addition, the extra cellular matrix secreted by cells at the interface of the NT arrays shows complete integration into the nanostructured surface. These results document the accelerated restoration of a functional endothelium in the presence of TiO2 NT arrays and substantiate their beneficial impact on vascular tissue in vivo. Our findings suggest that TiO2 NT arrays can be used as a drug-free approach for keeping stents patent long-term and have the potential to address ISR. KEYWORDS: nanoengineered surface, titania nanotubes, anti-inflammatory, in-stent restenosis, pro-healing

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medically managed through blood thinner prophylaxis.11 Over the recent years it became more evident that stent design plays an important factor in patient outcome.12,13 Strut thickness in particular has been found to be a key factor in the development of ISR, with thin struts reported to reduce neointima formation (ISAR-STEREO Trial).14,15 In addition, there are indications that thinner struts create more favorable conditions for reendothelialization.14 Recent in vitro studies have shown that nanotopographies created by nanotubular (NT) arrays differentially affect VECs and VSMCs. Our group previously published several studies describing cell migration and cell proliferation in the presence of NT covered substrates.16−18 In the presence of NT arrays we found a significantly increased proliferation and motility of vascular endothelial cells (VECs).16,19 Vascular smooth muscle cells (VSMCs) showed the opposite cell response, i.e., decreased proliferation and decreased motility, when compared to control substrates. In the presence of NT arrays, VECs were found to

INTRODUCTION Vascular stenting has become one of the most common percutaneous vascular intervention procedures to restore blood flow in coronary and peripheral artery disease.1 During these procedures, intravascular injuries occur which result in in-stent restenosis (ISR), the loss of artery patency over time. Stent deployment leads to severe damage to the vascular endothelial cell (VEC) layer.2 The dysfunctional endothelium is deficient of antithrombic and antiatherogenic properties and is no longer able to suppress vascular smooth muscle cell (VSMC) proliferation. VSMCs will then grow inward the blood vessel, causing the loss of vessel patency.3,4 Despite the advancements in stent design of bare metal stents (BMSs), drug-eluting stents (DESs), and more recently bioresorbable drug-eluting stents, ISR remains a key factor in treatment failure.5−7 DESs are the most widely used stents, delivering cytotoxic drugs (e.g., paclitaxel, sirolimus, or everolimus), shown to significantly reduce ISR by inhibiting VSMC proliferation.2 However, this class of drugs is equally cytotoxic to VSMCs and VECs, leading to poor re-endothelialization of the lumen.2,8,9 Once the drug reservoir is depleted, studies show an increase in late-stage thrombosis.10 The potential risk of thrombosis is © XXXX American Chemical Society

Received: April 7, 2017 Accepted: May 23, 2017

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DOI: 10.1021/acsami.7b04626 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

a 10% micro-90 solution (International Products Corporation), acetone (Fluka), and pure ethanol (Fluka) and sonicated at each step for 5 min. Stents were dried under a nitrogen gas stream and used for anodization. Nanotubes were introduced using a customized setup in which the stent (working electrode) is surrounded by five platinum electrodes (counter electrodes; wire OD: 0.4 mm, Alfa Aesar), placed in a circular arrangement (radius 12.5 mm). Stents were held in place by a corrosion resistant, stainless steel screw, acting as the connection between the power source and the stent. Stents were inserted into the custom built nanotube synthesis device, and the whole setup was lowered into the electrolyte until the stent was fully submerged. The electrolyte was composed from ammonium fluoride (SigmaAldrich), ethylene glycol (Fluka), and water (3 g/L; ratio 9:1). A temperature-controlled circulator (HAAKE DC10/K10) was used to control the electrolyte temperature and set to 20 °C. The power supply (Agilent E3612A) was set to constant voltage (15−90 V). Nanotube synthesis was performed over a time of 5−90 min and a voltage between 15 and 90 V. After synthesis, stents were rinsed using ethanol, and potential debris on top of the surface was removed using an ultrasound while immersed in 70% ethanol and then dried under vacuum. Field-Emission Scanning Electron Microscopy Characterization. NT arrays on stents were characterized using a Carl Zeiss Ultra 55 field emission scanning electron microscope (FE-SEM). Samples were fixated on aluminums stubs (Carl Zeiss) using silver paint (Pelco, Inc.). Prior to imaging, a 3−5 nm layer of iridium was deposited (Cressington-HR sputter coater). Assembly of Stent Delivery System. Custom-made balloon catheters (balloon length: 15 mm; expanded diameter 2.75 mm; overall length: 75 cm, monorail) for the in vivo study were fabricated and ordered from Duke Empirical. Additional balloons with diameters of 2.5, 2.75, 3.0, and 3.5 and a length of 15 mm were ordered from Creganna-Tactx Medical. Stents were crimped onto the balloon using a manual benchtop crimper, equipped with polymeric brackets (Machine Solutions, Inc.). In brief, balloons were evacuated (14 kPa) using a manual vacuum pump. Stents were crimped onto balloons, closing the crimp head for 30 s. After releasing the vacuum, balloons were pressurized for 15 s at 300 kPa, while the crimp head remained closed. Pressure was released, and systems were visually inspected. Assembled systems were disinfected using 70% ethanol and vacuum sealed for storage. Mechanical Testing: Hoop Force Measurement. The impact of NT synthesis on the mechanical properties of the stents was tested by determining the hoop force for nanostructured and control stents. Stents were compressed from an outer diameter of 2.3 mm down to a diameter of 0.81 mm. Compression speed was set to 94.34 μm/s at room temperature (RX550; Machine Solutions, Inc.). Research Objective and Study Design. The purpose of this study was to determine the impact of titanium stents with a nanoengineered surface on stenosis and re-endothelialization of stented arteries. Nanoengineered ceramic (titanium oxide) textured surfaces have previously been shown to promote the growth and terminal differentiation of vascular endothelial cells and inhibit vascular smooth muscle proliferation in vitro.18 Endothelial cells have the ability to control the proliferation of smooth muscle cells and fibroblasts through cell−cell contact inhibition.2 Animal procedures were approved by the Institutional Animal Care and Use Committees (IACUCs). The rabbit restenosis model has been studied extensively to test restenosis therapies and to understand cellular and molecular mechanisms. The rabbit model has been known to reliably reflect human restenosis histopathologically and therefore is an appropriate model for assessing and comparing the efficacy of new stents with conventional stent systems.31−34 The feasibility of the test stents were assessed with regard to these end points: clinical observations, adverse events, gross pathology, and histopathology. Test group size was determined using an unpaired t test, with values of alpha = 0.05, difference = 40%, standard deviation = 20%, and a power of 0.80. The power calculation yields a necessary per-group sample size of 4. Therefore, 5 animals per group were required to reach statistical significance in each experimental group.

be individually larger and more spread out and showed a more elongated and extended morphology.16 A whole genome microarray study conducted on human primary ECs and VSMCs grown on NTs revealed a decreased expression of molecules involved in inflammation and coagulation in both cell types.17 ECs showed upregulated expression of prostaglandin I2, an antithrombogenic protein and antiproliferative agent targeting VSMCs.17,18 In the same study, VSMCs showed a 3-fold increase in expression of smooth muscle α-actin (SMA), a marker of cell differentiation and proliferation. In general, the expression of SMA is reduced upon vessel wall injury.20 Lowered levels of SMA are connected with VSMC differentiation and proliferation. Thus, a substrate capable of upregulating SMA expression has the potential to reduce VSMC activation, an important finding with regard to the progression of ISR. Brammer et al. showed a reduced inflammatory response for cells cultured on nanotubular arrays.21 Primary bovine aortic endothelial cells cultured on NTs demonstrated an upregulated antithrombic cellular state necessary for maintaining vascular tone and crucial toward the formation of a functional endothelium.21−23 Work by Arnold et al. and Park et al. investigated mechanotransduction and reported different cellular response of osteoblasts, fibroblasts, mesenchymal stem cellsproliferation, differentiation, and migrationas a result of variations in lateral spacing of micro- or nanosized features, including TiO2 nanotubes of various diameter.24−26 Flasker et al. recently reported a similar effect for various NT diameters for human coronary artery endothelial cells.22 Cell viability was linked to the cell’s ability of integrin clustering/focal contact formation. While both VECs and VSMCs are more viable on small diameter nanotubes (95% survival rate and zero complications. There was randomization, and the contract research organization that performed the surgeries and the pathologist were blinded. Stent Deployment. Upon transfer to the procedure room, the animal was placed in a supine position. The vascular access site was aseptically cleansed and the animal then draped in a sterile manner. Vascular access of the left carotid artery was performed through either a surgical cut-down or Seldinger technique (at the surgeon’s discretion). An appropriately sized introducer sheath was placed. The animal was heparinized to maintain an activated clotting time (ACT) of over 250 s. ACT testing was used to confirm an ACT of >250 s before performing device implantation. Animals were treated with an IV lidocaine bolus and/or a drip to prevent ventricular ectopy or ventricular tachycardia. Additionally, amiodarone was administered at the start of the procedure to prevent arrhythmias. Other intraoperative medications (epinephrine, nitroglycerine, or atropine) were administered as needed; treatment and dose were at the discretion of the certified surgical research specialist or qualified veterinary personnel. Under fluoroscopic guidance, an appropriately sized guide catheter (5−6 Fr) was advanced up to the level of the iliofemoral arteries. An angiography was performed and a target vessel identified, sized, and documented. The target vessels were the common iliofemoral arteries. Stents were delivered and placed using percutaneous coronary invention. Stents were mounted on custom-made balloon catheters. The balloon catheter OD prior to inflation was ∼1.3 mm with the stent mounted, and the OD after inflation was 2.75 mm. The balloon catheters had marker bands. The balloon length is 15 mm, and the catheter length is 75 cm. At the completion of the procedure, a postdeployment angiography was performed, and the catheter(s) and introducers were removed. The arterial access site was ligated and hemostasis verified, and the incision was closed. The animals recovered from anesthesia and were transferred to postoperative care. Histopathology. All animals were first anesthetized for angiography and then euthanized according to the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2013 Edition for necropsy examination and tissue harvest. All animals were judged to be in good body condition. Iliac arteries in each rabbit were first flushed with 0.9% saline (Hospira, Lake Forest, IL) via the descending aorta at approximately 1 psi for approximately 5 min or until the affluent was clear. They were then perfused with 10% buffered formalin (Neogen Corp., Lexington, KY) at the same pressure for approximately 15 min. The distal ends of the iliac arteries were tagged with sutures for directional indication. The vessels were removed, photographed, and immersed in the same fixative (formalin). Following fixation, the iliac artery specimens with the stents in place were forwarded to Alizee Pathology, 20 Frederick Road, Thurmont, Maryland 21788, for processing in Spurr’s resin, cutting and staining the slides with hematoxylin and eosin and Movat’s pentachrome (Electron Microscopy Sciences, 1560 Industry Road, Hatfield, PA 19440). The Spurr-embedded Iliac arteries were cut and examined at the following levels: (1) distal stent, (2) middle stent, and (3) proximal stent. The distal and proximal native control sections were embedded in paraffin for H&E stained slide preparation. In total there were 10 slides per stent. Stents of all three groups were sectioned in a comparable fashion. All sections were examined with a light microscope in a blinded manner. After the blinded evaluation, the study pathologist was unblinded and then summarized the histopathologic findings as they relate to the control and the test articles. Determination of Inflammation Score. The iliofemoral artery sections were evaluated based on the FDA recommendations contained in the “Guidance for General Considerations for Animal Studies for Cardiovascular Devices” document issued on July 29, 2010.35 When appropriate, an injury score was calculated based on Schwartz et al.’s recommendations:36

1 = Internal elastic lamina lacerated; media typically compressed but not lacerated 2 = Internal elastic lamina lacerated; media visibly lacerated; external elastic lamina intact but may be compressed 3 = External elastic lamina lacerated; typically, large lacerations of media extending through the external elastic lamina Each strut in the section was scored, and the mean injury score for each section was calculated and reported. The means of the section were calculated and reported, providing a mean injury score per stent. The predominant inflammatory cell type(s) were described, per stent section and for the entire stent. Inflammation was calculated on a strut-by-strut base, as: 0 = 0−3 inflammatory cells around the strut 1 = 4−10 inflammatory cells around the strut 2 ≥ 10 inflammatory cells around the strut area can extend into neointima but do not efface the surrounding tissue 2 ≥ 10 cells, efface the surrounding tissue Statistical Analysis. All objectives were evaluated qualitatively and, where appropriate, summarized using basic descriptive techniques. f-tests and t-tests were performed. Reported P-values are two-sided. All analyses were done with Excel (Microsoft Office Professional Plus; 2016). A P-value of less than 0.05 was considered statistically significant.

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RESULTS TiO2 Nanotube Synthesis on Titanium Stents. We have developed a robust method to introduce TiO2 NT arrays to titanium stents with full surface coverage. Using a custom fixture in which a central stent is surrounded by five electrodes and by using the appropriate synthesis conditions our process yielded complete TiO2 NT coverage over all stent surfaces (see Experimental Section for details). Analogous to our previous in vitro work, we introduced NTs with an average diameter of 90 nm ±5 nm, determined via SEM and software-assisted size measurement (ImageJ). Nanotube length was in the range of 1800 nm ±300 nm. To demonstrate the ability to control the process, additional synthesis parameters were investigated including synthesis time, temperature of the electrolyte, and voltage. NTs grown over a constant time (15 min) at 30, 45, and 60 V display a decrease in NT length, most likely a result of accelerated dissolution of the distal ends of the NTs due to local heating and accelerated reaction kinetics.23 An increase in reaction time at constant voltage (60 V) revealed increased tube length. Increased reaction temperature revealed a honeycomb-like structure (30 min, 60 V, 50 °C) (Figure 1, Table 1, Table S1). Initially nonelectropolished stents were used to introduce NTs. The formed NT arrays revealed a crack along all edges of the struts. This is due to the growth mechanism of the nanotubes (Figure S1). NTs grow perpendicular from the surface, and above a critical tube length, the tubes start separating, resulting in the observed crack. In contrast, the electropolished stents used for this in vivo study have rounded edges. As a result, the introduced NT arrays reveal minor lateral displacement along the edges, keeping the NTs connected toward the substrate, providing additional lateral strength to the NT array (Figure S1D). Radial Strength. Since during anodization a portion of the substrate is etched off and converted into nanotubes, the risk arises that the mechanical stability of treated stents is reduced. Berger et al. reported that ∼1/3 of the tube length roughly corresponds to the etching depth into the substrate.37 With an average tube length of 1800 nm, according to Berger et al. 600 nm of material from all surfaces has been removed. With a turn

0 = Internal elastic lamina intact; endothelium typically denuded; media may be compressed but not lacerated C


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Figure 1. Examples of the different dimensions that have been obtained by the given parameter. The nanotube diameter and length change as a function of time, temperature, and voltage (Table 1). Scale bar: 400 nm.

Figure 2. Hoop force for a treated (red) and untreated (blue) stent. No significant difference in the compression force over the analyzed diameter (2.2−1.2 mm) was detected. This indicates that the mechanical properties of the stents were not altered during anodization.

Table 1. Tube Dimensions As Function of Voltage and Reaction Timea

a

reaction time [min]

voltage [V]

15 15 15 30 60 20 20 20

30 45 60 60 60 30 60 80

length [μm]

diameter [nm]

± ± ± ± ± ± ± ±

50 55 40 110 110 50 80 110

2.0 0.9 1.2 2.5 6.5 1.8 7.5 8.0

0.3 0.1 0.2 0.3 0.3 0.3 0.3 0.4

equipped with a digital manometer. Stability of the nanotubular arrays was analyzed by means of optical and scanning electron microscopy. Upon balloon inflation, no visible delamination of the NT arrays has been observed at and below a diameter of 2.75 mm, whereas with increased balloon diameter larger NT areas started to flake off at turns and the center of the struts (3.5 mm balloons), due to an increase in shear forces and twisting of struts, respectively (Figure S2). In summary, the NT arrays largely remain intact upon compression and inflation. For the in vivo study we selected balloons of 2.75 mm diameter. The stent geometry used in this study was optimized to further improve NT array stability upon crimping and balloon expansion. To achieve maximum NT array stability, the width of the turns and bridges was reduced from 120 μm down to 90 μm. This allowed the stent to homogeneously fold and expand, reducing the risk of NT array delamination. Figure 3A shows the NT arrays on a stent. Grooves along edges are the result of the NT growth mechanism since NTs grow perpendicular to the substrate surface, and at rounded edges the NTs start to separate from each other.23 However, no detrimental effect on the NT array stability was observed. Figure 3D shows an SEM image for a stent where one-half was inflated to 3.5 mm ID, while the other half was kept at 1.8 mm ID. No critical damage or larger areas of delamination of the NT arrays were found, preventing us from performing the in vivo study without putting the animal heath at risk. After crimping and inflation visual inspection of previously manufactured stents showed the NT arrays remained intact. Animal Model and Stent Placement. A three-arm arterial wall injury model was used to investigate the impact of unmodified vs TiO2-NT-covered stents on stenosis and reendothelialization after 27−28 days. For this study we employed the overinflation injury model. Stent oversizing/ balloon overinflation has been reported to significantly increase the damage introduced to the vascular endothelium, leading to accelerated neointimal cell proliferation and increased ISR.38 The three arms were (i) unmodified bare metal stainless steel (SS) stents, (ii) unmodified bare metal titanium (Ti) stent, and (iii) titanium oxide nanotube covered titanium (TiNT) stents. A single stent was deployed into the iliofemoral artery of each rabbit. A total of 14 rabbits were stented, with four animals

All reactions performed at 20 °C.

and strut width of 90 and 120 um, this converts to 1.3% and 1% of substrate removal. Hoop-force measurements were performed to determine a potential change in the stent’s mechanical properties. Stents were compressed from a diameter of 2.3 mm down to a diameter of 0.81 mm. Compression speed was set to 94.34 μm/s. For nanostructured and control stents, the hoop force was identical over a diameter of 2.3 (0 N) to 1.2 mm (∼4.65 N). Below a diameter of 1.2 mm the hoop force increased again, indicating that the struts are in contact (Figure 2). Therefore, for NT arrays, the compression range was set to 2.3−1.2 mm. No statistically significant difference was found between nanostructured and control stents, indicating that the anodization process does not alter the hoop force of the NT stents used in this study. NT Array Stability upon Compression and Inflation. In interventional cardiology, stents are commonly deployed using balloon catheters. Upon deployment, balloons are inflated to a given diameter, resulting in stent expansion from diameters of, e.g., 1.0 mm (compressed) to 3.5 mm (inflated). In addition, stents are mounted on the balloon by radial compression (crimping). At both events, crimping and inflating, stents are exposed to mechanical stresses, which might shear off or destabilize any given coating, rendering it unavailable for usage in medical devices. To investigate how the nanotubular arrays respond to this mechanical load, NT stents were crimped onto balloons of various inflation diameter (2.5, 2.75, 3.0, and 3.5 mm). Stent crimping diameter was set to 1.2 mm. Afterward balloons were inflated for 30 s at a pressure of 10 atm, as indicated by the manufacturer, using a 20 mL inflation syringe, D


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Figure 3. (A) Complete strut coverage by TiO2 NTs of a crimped stent onto a balloon. Inset: Nanotubes, average nanotube diameter is 90 nm. (Magnification 250.000×.) (B) TiO2 NT array covered stent. SEM revealed full NT coverage. The right half of the stent was inflated to 3.5 mm ID, and the left was kept at an ID of 1.8 mm. No critical damage at bridges, struts, or turns was observed.

Figure 4. Fluoroscopy image taken immediately after stent deployment (left) and after 27 days (right). At both time points, patency was found. Yellow frame highlights stent location (same animal).

receiving SS stents, five receiving Ti stents, and five receiving TiNT stents. Stents were deployed into the iliofemoral artery using angioplasty. Vascular access was achieved via the left carotid artery through the Seldinger technique. Under fluoroscopic guidance the left or right iliofemoral artery was selected for stent deployment, based on the lack of branching and the targeted vessel diameter of 1.5−2.5 mm. Arteries were overinflated upon stent deployment by on average 1.8 times. In all cases, immediately after stent deployment none of the fluoroscopy images revealed stent failure or vessel wall rupture, and patency was observed (Figure 4). After 27−28 days, fluoroscopic images were taken to confirm that stents had not migrated and that the arteries remained patent. Animals were then sacrificed and stents harvested and prepared for histopathology (see Experimental Section for details). In one animal, the stented ileofemoral artery (TiNT stent) was fully occluded after 28 days. The licensed veterinary pathologist who reviewed the histological slide specimens concluded that the arterial occlusion was caused by faulty stent deployment rupturing the external elastic lamina that led to aggressive formation of neointima.31 All data from this specific animal were excluded from the statistical analysis since the

underlying injury mechanism was due to stent deployment and not to the stent. Postmortem Observations. Fluoroscopic images revealed distal stent migration for two stents in the Ti stent control group by 10 mm and within the TiNT test article group for two stents by 5.0 mm distal from the point of deployment. Histopathology. Following fixation, the artery specimens with the stents in place were embedded in Spurr’s resin and cut, and slides were stained with hematoxylin and eosin (H&E) and Movat’s pentachrome, respectively. The Spurr-embedded Iliac arteries were cut and examined at the following levels: (1) distal stent, (2) middle stent, and (3) proximal stent. Distal and proximal native control sections were embedded in paraffin for H&E stained slide preparation. Compared to the Ti and SS stents, the TiNT stents showed a 15.6% and 5.6% thinner neointima over the struts. The neointima between the struts was thinner for both the TiNT stents (−8.75%) and Ti stents (−9.34%) compared to the SS stents. The neointima formed over TiNT stent struts consists of thin layers of cells, indicating maturing tissue. The media between struts was found to be thinner for the TiNT stents when compared to both control groups (Ti stent +18.3%; SS stent +1.23%). E


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Figure 5. Peripheral rabbit arteries after 28 days (iliofemoral artery). Top to bottom: SS stent, Ti stent, TiNT stent. Left column (Moffat trichrome stain): The nanoengineered stents show a 15.6% and 5.6% thinner neointima over the struts when compared to the Ti stent and SS stent control groups. Cell type and appearance indicate maturing tissue for the TiNT stents. A slightly pale neointima immediately surrounding the strut is supported by a delicate vascular network and a few macrophages. Central column: Dark-field representation of the images in the left column. After sample processing the nanoengineered surface remained in contact with the tissue, indicating a significantly more distinct tissue interaction and integration. Right column (H&E stain): Nanotube-covered stents show less stenosis compared to SS stent and Ti stent. The neointima formed between the struts for the TiNT stents is thinner (SS stent: +1.23%; Ti stent: +18.3%).

Figure 6. Left: Little to no tissue interaction was found between the SS stent and neointima. Right: The newly formed tissue integrated with the TiNT surface. The inset shows the strong interaction between the secreted extracellular matrix at the cell/NT array interface. SEM revealed collagen bundles. (Pig, coronary, 28 days, detailed info in Supporting Information). SS: stainless steel; TiNT: TiO2 nanotubular arrays.

Morphometric Analyses and Stenosis Rates. Slides of the proximal, middle, and distal levels from each animal were analyzed morphometrically. The following measurements were taken: lumen area, internal elastic lamina (IEL) area, external elastic lamina (EEL) Area. This measurement gives reference points to calculate the thickness of the media, maximum intimal width, and media thickness. Lumen, IEL, and EEL areas were measured directly by tracing the outline of the section. The

neointimal area was calculated by subtracting the lumenal area from the internal elastic lamina area. EEL and maximum intimal width were measured for reference purposes only. These values are not used for comparison purposes. Percent area stenosis was calculated by the following formula39 percent area stenosis = 100 ·(1 − (Lumen area/IEL area)) (1) F


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ACS Applied Materials & Interfaces Table 2. Summary of Histomorphometric Analysis of Stenosis Ratesa SS stent stenosis neointima between struts neointima over struts media between struts compressed media inflammatory score injury score a

24.76% 178.02 77.65 89.143 45.367 0.320 0.164

± ± ± ± ± ± ±

Ti stent

7.45% 30.80 μm 28.88 μm 6.96 μm 5.80 μm 0.213 0.25

20.02% 161.39 86.82 107.875 55.027 0.309 0.155

± ± ± ± ± ± ±

3.76% 25.29 μm 17.91 μm 19.21 μm 10.13 μm 0.146 0.089

TiNT stent 17.22% 162.45 73.28 88.138 39.348 0.437 0.138

± ± ± ± ± ± ±

1.11% 28.09 μm 20.01 μm 9.49 μm 6.81 μm 0.091 0.127

Significantly lower ISR for TiNT stents compared to the Ti stents was found.

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DISCUSSION Here we have successfully shown that we can introduce TiO2 NT arrays over the complete surface of a complex 3D structure, i.e., a medical-grade titanium stent. Varying synthesis parameters allow controlling length and diameter of the obtained nanotubes. The TiO2 NT arrays largely withstand the occurring forces when crimping modified stents onto regular delivery balloon catheters, as used in angioplasty, and they largely remain intact when inflating them to their final diameter upon stent deployment. No adverse effects on mechanical properties of NT covered stents were found by comparing their radial compression force to unmodified stents. The primary goal of this study was to investigate the ability of TiO2 NT arrays to reduce stenosis rates in vivo compared to bare metal stents with intact and unmodified native oxide surfaces. We found significantly lower stenosis rates for modified versus unmodified titanium stents. Stenosis rates were 19.14% and 30.45% lower for Ti and TiNT stents compared to SS stent controls. For the first time, this study reports the beneficial impact of TiO2 NT arrays on arterial tissue. This reduction in restenosis is in agreement with our previous in vitro work on monocultures, where VEC proliferation and migration on NT arrays was increased, whereas VSMC proliferation and migration was decreased. We are aware that our current stent and delivery system was not optimized with respect to stent geometry, strut thickness, NTarray stability, or nanotube geometry. We expect that with further optimization of the TiO2 NT topography there will be a larger difference between modified and unmodified surfaces regarding stenosis. To optimize the biological response of the stent, the nanotube diameter could be adjusted to achieve maximal VEC proliferation, migration, and function while at the same time further minimizing VSMC growth. Our group and the work of others have previously shown the relevance of NT diameter on cell fate. Different cell types respond similarly when cultured on smaller NT diameter between 10 and 30 nm,18,26,41 whereas the same cell types respond differently when cultured on top of NTs of larger diameters (>30 to 100 nm), reflected in altered proliferation, growth, and health.27 In an earlier study we have demonstrated the impact of NT array-covered Nitinol sheets on the deposition of extracellular matrix proteins.28 Primary human aortic endothelial cells were found to produce more collagen (2−3-fold increase) and elastin (5−8-fold increase per cell) compared to cells cultured on bare Nitinol sheets. For this study, light microscopy (Figure 5) and SEM data (Figure 6) show distinct tissue interactions with the nanoengineered surface supporting the hypothesis that a TiO2 nanotubular surface array might foster endothelial cell interaction. A major finding of this study is that the impact of nanotubular arrays may not be limited to cells directly

Overall, all stents maintained widely expanded within the iliofemoral arteries. The percent area stenosis for the control SS stent group was 24.76% ± 7.45%. The restenosis rate for the Ti stent group was 20.02% ± 3.76%. The TiNT stent group showed the lowest percent area stenosis of 17.22% ± 1.11%. Compared to the restenosis rates for the SS stent the restenosis rates for the Ti and TiNT stents were 19.14% and 30.45% lower. Figure 5 shows a representation of this finding. A statistically significant difference in the total percent stenosis area was found between the control Ti stents and TiNT stent group (P-value: 0.04). This finding reflects our previous in vitro work where we compared the cellular response of ECs and SMVCs when cultured on bare and on TiO2 NT covered titanium sheets, respectively.17 Due to the large variance in the SS stent data, no statistical significance was found between the SS stent and TiNT stent groups. No significant difference was found between the two control groups. Interaction between Stent Surface and Tissue. In some cases, the strut material was displaced during tissue sectioning. For the group of TiNT stents, bright-field and dark-field microscopy showed that the NT array remained in place (Figure 5). This finding is in agreement with results from an earlier study (Supporting Information), where Field emission scanning electron microscopy (FE-SEM) images show TiNTtissue integration but little to no interaction or integration between the tissue and stainless steel surface. As part of the sample preparation for the SEM analysis, stents were longitudinally cut. Due to the applied forces, NT arrays flaked off the substrate but remained connected to the ECM, indicating a strong interaction with the NT surface. SEM images show collagen bundles being integrated into the NT surface (Figure 6, inset; Figures S3 and S4). The absence of a collagen capsule in the presence of the NT arrays indicated that the surface was not recognized as a foreign body.40 Inflammatory Response. The injury scores found for all groups have been low (SS stent: 0.164 ± 0.259; Ti stents: 0.155 ± 0.089; TiNT stents: 0.138 ± 0.127) (Table 2). Although the discontinuity of the internal elastic lamina (IEL) was occasionally noted circumferentially in all test groups, no substantial injury of the medial layer immediately underneath the associated struts was found. The inflammatory reaction was minimal for all groups (SS stent: 0.320 ± 0.213; Ti stents: 0.309 ± 0.146; TiNT stent: 0.437 ± 0.091), represented by infiltrates of approximately 3−10 macrophages confined to the immediate peri-strut regions. Rare multinucleated giant cells were noted as well as minor differences in the cellular infiltrates between the groups. G


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technology may ultimately reduce in-stent restenosis rates and lead to an increased outcome for affected patients.

interacting with the nanoengineered surface. At a stent diameter of 3.0 mm the struts cover only 14% of the arterial wall. The observation of a lower stenosis rate over the whole stented area and not only in close proximity to the struts suggests an increase in EC proliferation and migration as seen in our in vitro work but also allows the hypothesis of cell to cell signal transmission beyond the provided cell stimulus.42 Given the high number of stenting procedures performed each year it is of great interest to both health care providers and patients to prevent the onset or to reduce the progression of ISR. The current solutions, despite their benefits, are still putting patients at risk. These limitations could be resolved by controlling cell response on a cellular level using nanoengineered surfaces. Our in vivo data strongly support the use of nanoengineered TiO2 NT surfaces to lower the degree of ISR and to promote re-endothelialization. The lowered stenosis rate promotes the use of TiO2 nanotubular arrays as a drug-free approach to keep stents patent. Furthermore, NT arrays seem to promote the formation of a functional endothelium, as seen in our histology data, which potentially reduces the risk of thrombosis and lowers the need for anticoagulants. Overall, a “pro-healing” surface, such as provided by the TiO2 NT surfaces, could have a significant impact on the managing of coronary and peripheral artery diseases and could possibly improve the long-term clinical outcome. A major limitation of this study consists in the limited control and test group size, resulting in larger variance, thus affecting the statistical analyses. Additional time points would have provided additional data on re-endothelialization (14 days) and ISR progression (90 days). A longer study time frame would have allowed for tissue maturation. Since mature tissue is denser, the restenosis rates might have decreased further, most likely in favor of the nanoengineered surfaces. In order to further decrease ISR rates, the stent design could be optimized in order to reduce flow turbulences. Titanium is currently not used as stent material. However, a stent material of choice is Nitinol, and Nitinol stents are readily available clinically, primarily being used to treat peripheral artery diseases. Recently, we successfully applied NT arrays to Nitinol foils and examined cell motility and proliferation of primary ECs and SMCs.16,28 Similar to our work on titanium NTs, primary human aortic ECs grown on Nitinol NTs showed increased mobility and proliferation, whereas primary human aortic SMCs displayed decreased mobility and proliferation. These in vitro results using Nitinol nanotubes may be predictive of their impact on vascular endothelial and smooth muscle cells in vivo.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b04626. TiO2 nanotube growth model. NT array damage as a function of balloon diameter. Additional synthesis parameter and NT topographies. Overview of the unpublished performed animal study, describing the animal model (pig), stenting location, and investigated surfaces (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Harald Nuhn: 0000-0003-2490-6061 Author Contributions

H.N. led development of synthesis methods on stents, introduced and characterized NT arrays on stents, optimized stent designs, performed FE-SEM imaging, assembled the delivery stent system, and performed statistical analysis of in vivo data with input from C.E.B. and T.A.D. H.N. wrote the manuscript, with contributions from all the other authors. C.E.B. led the animal study and designed and analyzed experiments with contribution from H.N and T.A.D. T.A.D. conceived the project, gave scientific input, and reviewed the data. Funding

This work was supported in part by the Alfred E. Mann Institute for Biomedical Engineering at the University of Southern California. This work was partially supported by the U.S. National Institutes of Health Vascular Interventions/ Innovations and Therapeutic Advances Program (VITA) Award HHSN268201400005C (TAD) Notes

The authors declare the following competing financial interest(s): HN is VP of research and development of Biothelium, Inc., a medical device company in the vascular space. CEB is the CTO and a member of the Board of Directors of Biothelium, Inc. TAD is on the scientific board of Biothelium, Inc. The University of California, San Francisco (UCSF) has filed a provisional patent application on nanotechnology for vascular applications.

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CONCLUSION The reduced stenosis rates (−30% compared to the control) and accelerated tissue maturation document the promoted restoration of a functional endothelium in the presence of TiO2 NT arrays and substantiate their beneficial impact on vascular tissue in vivo. This result are in agreement with our previous in vitro work and study (Supporting Information).17,18 Our findings suggest that TiO2 NT arrays can be used as a drugfree approach for keeping stents patent and have the potential to address ISR, a major challenge in the long-term use of stents. Restoration of a functional endothelium reduces the risk of thrombosis, mitigating the need for prolonged prophylaxis with anticoagulants and antiplatelet drugs. The clinical impact of this

ACKNOWLEDGMENTS We gratefully acknowledge use of the Carl Zeiss Ultra 55 FESEM and supporting equipment at SF State. The FE-SEM and supporting facilities were obtained under NSF-MRI award #0821619 and NSF-EAR award #0949176, respectively.

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ABBREVIATIONS EEL, external elastic lamina; FE-SEM, field emission scanning electron microscopy; IEL, internal elastic lamina; ISR, in-stent restenosis; SMA, smooth-muscle α-actin; SS, stainless steel; TiNT, titania nanotubes; TiO2, titania; NT, nanotube; VECs, vascular endothelial cells; VSMCs, vascular smooth muscle cells H


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