Endothelialization and drug release kinetics of roughness induced polymer-free drug eluting stents
Abstract: Drug-eluting stents (DES) are usually created by coating bare-metal stents with polymer film and loading it with drugs. This causes adverse effects like late stent thrombosis. Polymer-free DES may help prevent this problem. In this study, polymer-free DES were created by applying three different surface treatments to three different stents to test their effectiveness in inducing different surface roughness. The stents were then seeded with endothelial cells to test the effect of roughness on endothelialization. Another fresh batch of surface-treated stents was coated with Biolimus to study the effect of roughness on drug release kinetics. It was observed that i.) surface treatments were successful in inducing different levels of roughness on stent surfaces; ii.) roughness has a significant impact on cell proliferation; iii.)The stent surface with maximum roughness showed maximum endothelial cell growth; iv.) roughness has no significant impact on drug release kinetics.

1. Introduction
 
1.1. Background
Coronary stents are hollow devices, usually bare-metal stents (BMS) inserted into an obstructed natural passage to open and eliminate the block. Although BMS work in this sense, they have been closely associated with risks such as neointimal hyperplasia which subsequently causes in-stent restenosis [1][2]. DES are metal stents coated with a polymer film which is loaded with antiproliferative drugs that are released gradually (usually over a period of 28 days) to prevent inflammation response [1]. Drug-eluting stents (DES) are associated with a major milestone in coronary heart disease treatment. It causes a lower rate of in-stent restenosis compared to the metal stents that were used initially, meaning, fewer patients need to return to the hospital for repeat treatments [1] [3].

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Though the loaded drugs have been proven to offer favourable results, the polymer coatings of the first generation DES like Cypher and Taxus were found to cause negative effects such as late stent thrombosis (LST) [1][3]. Clinical and experimental evidence added to the existing literature all of which pointed to the negative effects of permanent and biodegradable polymer coating such as extensive inflammatory response and neointimal thickening within pig coronary arteries [3]. Elimination of usage of polymer coatings may hence be beneficial in the creation of a safer DES.
Drug release kinetics
Several technologies have been employed to develop polymer-free DES (PDES). NEVO (Cordis Corp.) and Janus Carbostent (Sorin Group) are macroporous PDES developed using platform modification. Reservoirs were embedded on to the stent struts which were then loaded with drugs for sustained release over a longer period of time. While both of the stents were effective in gradual drug release, they were withdrawn due to different reasons. Cre8 (Alvimedica) and Polymer-free drug-filled stent (DFS) (Medtronic Inc.) are two other macroporous stents with innovative features that showed promising results [3].
Yukon stent (Translumina GmbH) and BioFreedom stent (Biosensors Int) are platform modified microporous PDES. The burst release of drugs within the first few days by the Yukon stent point to the fact it is not superior to the Taxus stent. The BioFreedom stent showed comparable long-term efficacy as the Taxus stent [3]. Since the molecular dimensions of drug molecules are usually of the order of nanometers, micro and macroporous surfaces may not significantly affect the drug release kinetics of such molecules. However, if the roughness of the surfaces is on submicron levels, forces such as Van der Waals interactions will come to play and significantly retard the release of drugs from the surface [2]. Hence, in this research, nanometerlevel roughness was introduced on the stent surfaces through various surface treatments to have a beneficial contribution to drug release kinetics as well as reendothelialization.
Nanoporous stents are a type of platform modified PDES where the pore size is reduced to nanoscale dimensions. Electrochemical approaches were used to develop such stents but due to safety considerations caused by particle debris, this approach is not used anymore. As an alternative, sputter coating techniques are used. The Setagon stent is an example of PDES made with this approach [3].
Endothelialization Reendothelialization occurs after vascular injury and also after stent placement. Hence, delayed healing of endothelium after stent placement leaves the patient at risk of LST [3][4]. The firstgeneration DES inhibited restenosis at the cost of impaired recovery of the endothelium [3]. A morphological autopsy study comparing coronary segments from patients after DES and BMS found delayed arterial healing and reendothelialization after DES compared to BMS [4]. Hence, there is a need to optimize DES to improve this aspect while maintaining all its existing benefits.
The stent surface hugely impacts endothelialization. Cells respond differently to variations in surface topography. Studies show that microscale grooves significantly increases the mitigation rate of endothelial cells on stent surfaces. After drug release, the rough surface left behind was claimed to enhance reendothelialization. Micro – and nanoporous surfaces were found to induce endothelialization and hence, reduce neointima formation [1][3].
Hence, PDES can be designed with specific topographical features to aid vascular healing. Ongoing research implies that optimization of surface groove dimensions can encourage targeted endothelial cell migration. Various studies have also indicated that stent surfaces need not be perfectly smooth to be safe and effective [3]. These factors are the basis for the research described in this paper which investigates the effectiveness of surface treatments in inducing roughness on the stent surfaces and the impact of stentsurface roughness on reendothelialization and drug release kinetics.
1.2. Outline
In this paper, an experiment modelled to test the effect of three different surface treatment on inducing different levels of roughness on three different stents is described. The same surface-treated stents were further seeded with endothelial cells and antiproliferative drugs in separate trials to study their reendothelialization abilities and drug release kinetics respectively. Promising results were obtained with the surface treatments successfully induced varying degrees of roughness on the stent surfaces. It was observed that roughness has a significant impact on reendothelization. It was also observed that roughness did not make any significant contribution to drug release kinetics with all the surface treated stents exhibiting similar drug release characteristics.
2. Methodology
2.1. Experiment
2.1.1. To study the effect of surface treatments on stent surface roughness
Three different stent types A, B and C were subjected to electropolishing, coarse grain sandblasting and fine grain sandblasting respectively. Atomic Force Microscopy (AFM) was used to estimate the root mean square (RMS) values of 5 samples which provided the measure of surface roughness for each stent. One-way ANOVA with a confidence interval of 95% followed by Tukey’s post-hoc was used to analyse the effectiveness of the surface treatment at inducing varying levels of roughness on the stent surfaces.
2.1.2. To study the effect of roughness on reendothelialization
The same stent surfaces were further seeded with endothelial cells and incubated under standard cell culture conditions with the number of cells present on the surface being counted after 7 days. One-way ANOVA with a confidence interval of 95% followed by Tukey’s post-hoc test was used to analyse the impact of roughness on endothelial cell proliferation.
2.1.3. To study the effect of roughness on drug release kinetics
A fresh batch of 3 samples of the same surfacetreated stents was coated with 100 µg of the antiproliferative drug, Biolimus. The coated stents were then incubated in a physiological solution which was maintained at 37°C. The in vitro drug release from the surface was quantified at specific time points up to 28 days. One-way ANOVA was used to analyse the impact of surface roughness on drug release kinetics. Drug release profiles were also created for each stent from the experimentally obtained data (Figure 1).

Figure 1: Straight line plot of means of mass of drug released (n = 3) against time for Stents A, B and C. Stent A shows burst release of drug within 10 days while B and C show a more gradual release.
2.2. Analysis
ANOVA comparison of roughness between each different type of stents gives evidence that there is a significant difference between the mean roughness of each stent (p-value = 0.000). Tukey’s posthoc test indicates a significant difference between the mean surface roughness of the 3 stents (Figure 2). Stent C displays maximum roughness and Stent A exhibits the minimum roughness pointing to the fact that the surface treatments were effective in inducing different levels of roughness on the stents.
For endothelialization, there was a significant difference in cell growth on the stent surface of each stent

Figure 2: Grouping using the Tukey method shows that there is a significant difference between the roughness of the three stents since all of them belong to different groups.
(p-value = 0.000). Performing Tukey’s posthoc test showed that Stent C exhibits maximum cell growth while the cell growth on stents A and B were similar (Figure 3).

Figure 3: Grouping using the Tukey method shows that stent C exhibits maximum cell growth compared to A and B which shows identical cell growth (same group).
It was found that roughness did not have a significant impact on the drug release kinetics of the stents (p-values>0.005 for all the samples). Plotting the drug release data shows that stents A, B and C released almost all the drug into the solution by the 28th day with stent C faring slightly better than the other two in terms of retaining some of the drug (Figure 1). Stent A released almost all the drug within the first 10 days while stent B and C showed a more gradual release of the drug.
3. Results and Discussion
All the experiments point to the fact roughness does have an impact on endothelialization after stent implants which aligns with the results of similar research recorded in the literature. Stent C shows considerable advantages compared to stents A and B, in terms of roughness and endothelialization. Stent A shows the lowest roughness and the lowest cell growth while stent C showed the highest roughness and maximum cell growth after day 7. Hence, roughness in submicron scale might promote reendothelialization on the stent surface. The surface of stent C was fine grain sand-blasted – this method induced maximum roughness. Hence, it might be a useful technique in creating nanoporous stents and can be applied instead of electrochemical processes.
It was also found that different levels of roughness did not have an impact on drug release kinetics which may be owed to the limitations in the current research. In this study, roughness of nanometer levels were induced on all the stent surfaces. Hence, no conclusions can be made on the lack of impact of this factor on drug release kinetics. If the level of roughness were significantly different, say micron level and submicron level for different stents, the results would be more useful and comparable. The study was conducted in vitro in a biological environment which is different from the actual environment the stents are supposed to work in which is also a limitation of this study.
Overall, fine grain sandblasted stent such as stent C holds some potential for future research.
4. Conclusions
BMS stents which were used to treat coronary blocks are associated with the risk of in-stent restenosis. DES removes this risk through the sustained release of antiproliferative drugs which prevent inflammatory responses. DES are usually made by coating BMS with a polymer film which is then loaded with antiproliferative drugs. Polymer-coated DES cause detrimental effects like LST. To remove such risks, PDES can be used. In this study, three stents were surface treated to induce roughness and they were tested for endothelialization capabilities and drug release kinetics. Results showed that roughness can be induced with fine grain sand-blasting the stent and that roughness has a significant impact on endothelial cell growth while it has no significant impact on drug release kinetics.
5. References
[1] Chen, W., Habraken, T., Hennink, W. and Kok, R., 2015. Polymer-Free Drug-Eluting Stents: An
Overview of Coating Strategies and Comparison with Polymer-Coated Drug-Eluting Stents. Bioconjugate Chemistry, 26(7), pp.1277-1288.
[2] Vo, T., Morgan, S., McCormick, C., McGinty, S., McKee, S. and Meere, M., 2018. Modelling drug release from polymer-free coronary stents with microporous surfaces. International Journal of Pharmaceutics, 544(2), pp.392-401.
[3] McCormick, C., 2018. Polymer-free drug-eluting stents. Functionalised Cardiovascular Stents, pp.57-74.
[4] Luscher, T., Steffel, J., Eberli, F., Joner, M., Nakazawa, G., Tanner, F. and Virmani, R., 2007. DrugEluting Stent and Coronary Thrombosis. Circulation, 115(8), pp.1051-1058.