University of Groningen
Magnesium-based supports for stem cell therapy of vascular disease
Echeverry Rendon, Monica
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Publication date: 2018
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Echeverry Rendon, M. (2018). Magnesium-based supports for stem cell therapy of vascular disease. University of Groningen.
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CHAPTER 1
INTRODUCTION AND AIMS OF THE
THESIS
CHAPTER 1
10
Metallic materials are used for fabrication of medical devices to alleviate clinical symptoms of a plethora of diseases and to improve the quality of life of patients. These diseases include cardiovascular disease (CVD), the prime global cause of death [1]. Arterial lesions and occlusions underlie a majority of CVD and have been the particular target of ‘metal-based’ interventions i.e. the use of stents to treat atherosclerotic plaque-related occlusion but also pathologi-cal dilatations i.e. aneurysms [2]. The long term persistence of undegradable materials in the body always elicits a bi-ological response called the foreign body reaction (FBR). The FBR generally causes the formation of a fibrous capsule around the implant, but may additionally cause a long term inflammation, albeit smoldering [3]. Stents implanted in arteries that are damaged by balloon catheterization, may induce a pathological medial hyperplasia that leads to a renewed occlusion of the lumen (restenosis) [4][5]. Currently, drug-eluting stents are highly fashionable to use. These elute mTor inhibitors such as analogs of FK506 and rapamycin, which inhibit proliferation of vascular smooth muscle cells (SMC) and thus prevent restenosis. However, these drugs also reduce endothelial proliferation and thus hamper re-endothelialization of stented lesions [6][7]. In other words, the drug inhibits intima formation which is essential to maintain an anti-coagulatory state. As a consequence, long term clinical studies reveal an increased risk for thrombo-sis. In addition, the elution of the drugs on obviously wears off in time after implantation and with it its therapeutic value. To address these caveats, biodegradable stents have been developed in the past decade, that are based on polymers or degradable metal alloys [8][9][10]. A favorable metal to fabricate biodegradable stents is magnesium (Mg), because magnesium ions are essential to proper functioning of several enzymes and thus essential to sustain human life [11][12]. Nevertheless, the oxidation of magnesium in aqueous solution is rather vigorous and thus Mg demands (surface) modification to control its degradation (corrosion) and resident time in the body at lesion sites.
Fig.1 General therapeutic concept of the present project
Over the past two decades an increasing number of studies reported on the development of magnesium-based de-vices with fine-tuned mechanical properties and increased corrosion resistance. On occasion, alloys of Mg and poten-tially toxic metals such as aluminum or rare earth metals, reduced the biocompatibity in a concentration-dependent fashion, albeit that alloys have a lower corrosion rate [13][14][15]. Thus novel methods to adjust corrosion while maintaining biocompatibility of Mg are warranted. A promising alternative to improve the corrosion resistance of Mg is though the surface modification of the material by using of conversion techniques that involve chemical or stry transformations (read: oxidation), in particular anodization methods [16][17][18].
1
The main aim of this project was to develop a new formulation of biodegradable stent based on c.p Mg (chemically pure Mg), surface-modified by PEO and loaded with therapeutic cells. Along this line of ideas, the material will act as temporal platform to deliver stem cells and with time the material will gradually disappear addressing the healing of damaged cardiovascular tissues (Fig.1).
In order to provide the sufficient information about the context of this topic in Chapter 2 an overview of the state of the art around this topic is summarized. The thesis consists of two parts: part one includes development and optimization of the processing and characterization of the coatings of so-called commercial pure magnesium (c.p Mg) and in the second part contains all the biological validation. The contents of this thesis are summarized in Fig. 2
Fig.2 Structure of the thesis according to the chapters and their contents
In this specific project, c.p Mg was modified by plasma electrolytic oxidation (PEO), also known as micro-arc oxida-tion (MAO). In this process, c.p Mg was used as anode in an electrolytic cell and it was immersed in a conductive solu-tion through which a current was applied during a specific time controlling the growth of a protective hybrid coating consisted of magnesium oxide (MgO) and magnesium hydroxide (Mg(OH)2). These oxidation products are poorly
soluble yet not insoluble which is a major advantage because this allows to tune the degradation rate of future stents. Details about the processing and optimization of the Mg anodization is describe in Chapter 3, where organic compounds, hexamethylenetetramine or mannitol, instead of traditional anorganic additives, where added to the electrolytic anodizing solution and compared to fluoride. In Chapter 3, we also describe the influence of voltage and current density on the characteristics of the coatings.
The purpose of the surface modifications was to generate a protective layer that decreases the rate of degradation and improve the corrosion resistance of the material and with this improve its biological performance. In Chapter
4, five formulations, three from the previous work and two new in which a two-step anodized was obtained were
characterized. All these coatings differ with respect to morphology, thickness and surface energy. A physicochemical analysis of the coating was performed in which topography, contact angle, surface energy and hydrogen evolution was assessed to determine the corrosion resistance in comparison to untreated Mg.
Additionally, in Chapter 5, the effect of different sterilization methods of coated Mg was studied including steam autoclaving, UV irradiation, dry heat treatment and steam-formaldehyde sterilization. Because Mg is a highly
reac-CHAPTER 1
12
tion. Because Mg is a highly reactive material to different environments such as chemicals or aqueous solutions, it is important to validate that the integrity of the coatings remains intact. This is relevant because the material will be implanted in vivo in the future which requires adequate sterilization.
Besides the material’s perspective, a biological characterization of surface-modified Mg on different cell types relat-ed to (repair of) cardiovascular tissue was performrelat-ed. In Chapter 6 the cytotoxic effect of surface-modifirelat-ed c.p Mg on fibroblasts, endothelial cells, smooth muscle cells, adipose tissue-derived stromal cells (ASC) and macrophages was investigated. The biological function of ASCs in direct and indirect evaluations was investigated in more detail. Assessment of proliferation, apoptosis, angiogenesis and wound healing revealed the protective effect of coatings of Mg on the survival and functionality of ASCs. In addition, different models or experimental approaches for the evaluation of Mg in vitro and ex-vivo were studied and are described in Chapter 7. There, the effect of changes in pH and Mg2+ concentration was evaluated in HUVECs and SMCs. Moreover, functional experiment to verify the effect of
corrosion products from Mg on HUVECs were performed.
This thesis concludes with a General Discussion and Perspectives Chapter (8) in which we compare and validate our overall results and generate new dogmas and directions for future research.
It is important to emphasize that our investigations were the first approach to a new concept, therefore the questions that were raised outnumber the answers obtained. Therefore, our studies are an important basis of study for future work on the application of surface-modified and cell-loaded c.p Mg for therapeutic purposes that extend beyond cardiovascular disease
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