University of Groningen
Magnesium-based supports for stem cell therapy of vascular disease
Echeverry Rendon, Monica
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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 8
General discussion and future
perspectives
126 CHAPTER 8
General discussion
Throughout this thesis, the problem of providing an alternative solution for the treatment of cardiovascular dis-eases (CVD) such as stenosis has been approached from different perspectives. The urgency to count with a new therapy is closely linked with the high impact that this project could have in society.
The WHO (World Health Organization), expects a 15% global increase of the prevalence of CVD 2010 and 2020. This is due to adverse lifestyle including smoking, high caloric nutrition, stress, physical inactivity and excessive alcohol intake to mention a few [1]. In contrast, global life expectancy has increased and thus also the possibility to contract CVD. Furthermore CVDs are more associated with aging with a higher frequency in people aged over sixty [2][3]. In addition, CVD is a co-morbidity from other diseases in particular type 2 diabetes (T2D) which currently is considered an ‘epidemic’ problem and which is expanding fast too [4]. The costs associated with prevention, diagnosis and treatment of CVD increase steadily. According to the CDC, by 2030 in the USA alone, more than $818 billion per year are spent on medical costs associated with CVD and around $275 billion on lost productivity [5]. Taking these facts into count, it is urgent to search for suitable therapeutic alternatives to impact this field and help to dampen the impact of CVD despite a series of pharmaceutical agents that at least partially, or better: no more than partially, alleviate the burden of CVD.
Currently, tissue engineering and regenerative medicine are disciplines that gain increasing recognition by indus-try, due to significant scientific and clinical progress and success. In the past decades, novel concepts and tech-nologies emerged while scientific research expands in this direction to develop new treatment modalities for CVD patients in need of reliable and fast solutions [6]. In historical perspective, implantable medical devices have been frequently used to functionally repair or replace damaged tissues and organs. In this case the interaction of bio-materials with the implant tissue microenvironment will induce specific biological responses, preferably with a sizeable therapeutic benefit [7]. In this context, bioactive interfaces play a crucial role: this is sensed from the first moment of implantation till integration or degradation. An appropriate bioactive surface or interface is critical for the regeneration or functional recovery of damaged tissue in terms of time, quality and cost [7].
For a long time, the use of magnesium (Mg) as implant material in the body was restricted due its high reactiv-ity, which produces a high alkalization of the surrounding environment accompanied by hydrogen accumulation, which was verified through all of our experiments from the point of view of the material as well as biologically [8]. Currently, those problems are more controlled due to the use of alloys or by surface modifications of the implant [9][10]. PEO is a simple, versatile, and low-cost technique in which the material is oxidized in a controlled way generating a protective layer that decrease the degradation rate of the material. The advantage of using pure Mg implants compared to Mg-based alloys, is that elements released from alloys such as aluminum and chromium might exert local cytotoxicity, while magnesium is not. Some elements in alloys are also nephrotoxic yet difficult to excrete from the body [11][12].
This thesis was established on the hypothesis that a degradable magnesium-based (Mg) vascular implant modified by plasma electrolytic oxidation (PEO) and combined with cell therapy promotes the regeneration of atheroscle-rotic vascular lesions. The process to obtain an implant with final use in humans is time-consuming and requires profound scientific knowledge. In this process, initially a study from a material perspective is required where me-chanical and physicochemical properties should be defined. Subsequently, biological characterization involving both,in vitro and in vivo experiments is needed to validate the hypothesis. Finally, it is important to investigate aspects related with the design of the device as well to approach to strategies for delivering the combination of cells and material in a successful way without compromising the therapeutic function at the place of interest. Our project was an important first approach to the idea while several new scientific questions emerged. A major result is that our research provides a better overview on the options of surface modification of c. p Mg for biological
8
applications with the prospect of clinical applications to treat CVD.
In the first part of this thesis, new chemical formulations to produce surface-coatings on commercial pure magne-sium (c.p Mg) by PEO were studied. One of the novelties of this project was the use of two organic additives in the modification process of the Mg to improve the corrosion resistance of the material. In chapter 3, the effect to add hexamethylenetetramine (HMT) and mannitol (MAN) to the electrolytic solution of the anodizing was studied. It is important to clarify that although it is well known that the use of organic compounds during the anodization process can result in coatings that are more resistant to corrosion, HMT and MAN were not used before for similar applications or in c.p Mg [13]. In addition, the chemical composition of the obtained coatings was not affected by the addition of extra elements different from those used in the base solution (i.e silicate) and magnesium oxide (MgO) and magnesium hydroxide (Mg(OH)2) which are normally generated as the result of oxidation of the material. These
coatings were compared with others obtained from a studied formula where sodium fluoride (NAF) was used as an additive to the electrolytic solution, where the fluoride (F), which is an element that can be highly toxic at certain concentrations, was incorporated into the obtained anodic film [14][15]. . Additionally, an optimization of the process was carried out after the modification of the parameters such as current density (potentiostaic mode) and voltage (galvanostatic mode).during the anodizing. This experiments allowed us to conclude that the role of NAF, HMT and MAN as additives directly influenced the formation mechanisms of the coatings affecting the thickness, surface mor-phology and structural organization [16].
Once coatings with different characteristics were obtained, one per group was selected taking into account the uni-formity and morphology of the anodic films and their possible contribution to decrease the corrosion rate of the material. More detailed studies were performed with those samples and were described in chapter 4. The chosen samples were for NAF coating obtained by treatment at 104.16 mA.cm-2 for 600s, for HMT samples treated at 320 V for 600s and for MAN treatment at 380V for 600s. In addition to the samples mentioned above, two-step anodized samples were prepared. This combinations were considered in the study due to previous investigations in materials such as titanium (Ti), aluminum (Al) and zirconium (Zr) has been shown an effective increase in the ordering of the morphology after the anodization of samples with a pretreatment with a determined pattern serving as guide for self-organization[17][18][19]. The two-step anodized treatment consisted of a first anodizing with NAF solution to generate a compact layer which should be more protective and resistant to corrosion[16]. Consequently, samples were anodized for second time with either HMT or MAN in order to create a porous surface which is more attractive for biological interactions like cell adhesion [20][21].. Our results show that during the second step of anodization a competition between the old film dissolution and new film formation occurred. This influenced the morphology of the coatings and the corrosion resistance was higher compared to samples that were anodized only once. This cor-roborates findings by other authors [22][23][24]. For all the coatings, in particular for two-step anodized samples, the highest release of hydrogen production was observed during the first 48h of exposure to an aqueous solution. This was expected because when samples were in contact with the NaCl solution, the initialized degradation process first increased and then attenuated by the re-passivation of the samples, confirming literature [25]. As stated, this effect was less pronounced in samples with a single anodization step. The behavior of the samples treated with MAN solution differs from the others with a more gradual degradation profile. That means that Mg2+ was released at a
constant rate during the first month. Additionally, coated samples showed an improvement in the wettability of the material converting hydrophobic surfaces of Mg to hydrophilic features. This is crucial because the first reaction that occurs on the surface of the implant after an implantation, is the deposition of proteins. This deposition promotes attachment of cells and possibly also their proliferation and differentiation andor maturation [26]. Our results also showed that surface-coated materials were hemocompatible i.e. did not increase coagulation of blood, nor caused hemolysis in contrast to c.p Mg which was coherent with studies reported previously in literature [27][28].
128 CHAPTER 8
Other important contribution of this work is showed in Chapter 5 where for first time different methods to sterilize the surface-coated Mg-based materials were studied. Samples sterilized by steam autoclaving did not change their coating characteristics in terms of composition, morphology and thickness however the contact angle increased affecting its wettability. For samples sterilized with UV light, morphology of the coatings were conserved but an increase in the oxygen quantified was observed indicating changes in the crystallinity of the coatings by the action of ionizing radiation [29]. Samples treated with heat dry method presented cracks on the surfaces which can act as a points of failure and fatigue of the material which negatively affects it (rate of) degradation. Finally, surface morphology of the coatings was affected by the chemical action of the formaldehyde. Even though UV sterilization showed promising results, the mechanism of killing microorganism by UV is not enough considering that this does not penetrate the porous structure of the coatings [30]. After this evaluation it was decided that all the tested sam-ples in vitro were sterilized by autoclaving because to this showed the least complications. In addition, this method is recommended because the material was partially passivated due to the water/steam contact during autoclaving. This also suppressed the high degradation rates registered in the first 48 hours, occur in the presence of cells [31]. Once the coatings were physicochemically characterized, their biological evaluation was carried out. Biological as-says were initially performed with the most characteristic cell types of blood vessels in order to investigate the influence of the Mg degradation. In the body Mg has an important role in multiple processes. For instance, Mg2+ acts
as calcium antagonist and participates in the regulation of energy metabolism, synthesis of proteins such as the DNA and it is responsible of the activation or inactivation of some enzymatic reactions. Additionally, Mg interacts with phospholipids, nucleic acids, and proteins. In the circulatory system, Mg also participates in the regulation of blood pressure and in the control of the blood glucose [28]. Excess of Mg ions, aka hypermagnesemia, affects the osmolal-ity and the general homeostasis of the body [32]. This complication is also associated with renal failure, irregular heartbeat, low blood pressure and muscle fatigue [32].
To understand how changes in Mg concentration affects the biological environment of the implant, cell behavior was evaluated by using direct contact with the samples or indirect by using leachables (extracts) obtained after incuba-tion in medium for 48 h. Chapter 6, shows results of the response of different cell types to Mg extracts. Endothe-lial cells (HUVEC) and smooth muscle cells (SMC) were more sensitive to changes in leachables’ concentration than fibroblasst (PK84), cell adipose tissue-derived stromal cells (ASC) and macrophages (THP-1). Because endothelial cells (EC) and SMCs were compromised by Mg and because these play an important role in cardiovascular tissue, in Chapter 7 the effect of Mg on ECs and SMCs was studied. First, the main question was which factor caused apoptosis: Mg concentration or changes in pH. In order to answer this question, a simple experiment were a wide range of pH (7.4 - 9.4) and Mg concentration was evaluated in HUVECs and SMCs. Results showed that both factors affected the cells: Mg2+ at concentrations above 50mM and pH above 9. Thus in subsequent experiments pH was of extracts was
neutralized to purely investigate the effect of Mg.
The corrosion of Mg in a biological environment is mechanistically hard to predict because it is affected by multiple variable factors. One is the composition of the medium to which the material is exposed. Depending on the medium, the material forms phases with different degradation rates. This is challenging to mimic or simulate in vitro. Howev-er, previous studies of Mg by using DMEM as culture medium and supplemented with FBS, showed similarities with the performance of the material in vivo [33]. But the extra components added by the medium, that are not present in the blood plasma, may generate extra phases that change the general behavior of the material in other direction far from which is really happening in the body [34]. [34]. According to Kieke et al [35], who characterized the corrosion of pure Mg after its immersion in culture medium, the main products obtained are MgO which is insoluble in water, brucite (Mg(OH)2) and magnesite (MgCO3) with a solubility in water of 12mg/L and 5.51mg/L respectively [36]. Other
8
(Mg3(PO4)2·8H2O), portlandite (Ca(OH)2), calcite(CaCO3) and hydroxyapatite (Ca5(PO4) 3(OH)) with a solubility of
4.49, 27.62, 5.38, 8.44 and 58.77mg/L respectively [35]. Some of these compounds form particulate material or precipitates that can stick close to the implant or moved through the blood system, the dissolution of these ele-ments will depends of the time and general conditions of the environment about composition and pH. Additionally, organic components from the culture medium and the FBS such as vitamins, proteins (amino acids) e.g. are able to form complexes with Mg ions inhibiting the formation of insoluble salts and coating the surfaces of the implant reducing the interaction of the material with the medium retarding its degradation [34].
We consider that the static model was imprecise to study the Mg degradation. The volume/area (V/A) ratio for im-mersion testing was chosen in agreement with the ISO 10993-12(2007) in which the evaluated ranges should range from 0.17 to 0.8 mL/cm2 [37]. However, these values are not suitable to study Mg because in static conditions the
buffer capacity of the medium is too low to maintain a stable pH [33]. Additionally, during the first 48h the material had the highest corrosion rate and thus increased the pH concomitantly. This caused passivation of the material and consequently a reduced solubility due to the hydroxide layer that had formed. This is, due to its hydroxide nature, thermodynamically stable at high(er) pH-values (>8.3) [38].
Discrepancies in the models used in vitro has been discussed before. For instance, Fischer et al [39], suggested that indirect evaluations of cytotoxicity of Mg sample extracts from Mg samples should be diluted at least ten fold for reliable and predictable results. Besides Mg concentration, changes in pH and osmolality strongly affect the biological evaluation of Mg as a biomaterial [40]l. In this study four cell types were evaluated with extracts from c.p Mg: two primary cultures of bone marrow-derived mesenchymal stromal cells (BMSCs) and osteoblasts and two commercial cell lines, MC3T3-E1 (pre-osteoblasts) and L929 (fibroblasts). Their results show primary cell types had an IC50 of about 35 mM, while the cells lines had an IC50 of 15 mM, below 10 mM Mg2+ was non-cytotoxic.
This is corroborated by our results, albeit that in our hands the IC50 was slightly higher (~40 mM Mg2+). Also we
corroborate their results that cells have a reduced proliferation at pH > 8.5.
During the static evaluation the mass transfer is irrelevant because all material is dissolved and accumulated in the medium. The periodic replenishment of part of the medium, which mimics fluid exchange, circumvents this accumulation [41][42][43].
In contrast, the influence of mass transfer, which continuously removes corrosion products, is possible under liquid flow. Under liquid flow will also affect the speed of the degradation because the chemical balance between cor-rosion and degradation is kept out of equilibrium [44]Additionally, the flow helps to avoid galvanostatic corcor-rosion in which anodic and cathodic point are creating on the surfaces by the non-uniform presence of species. Similar results were found for Grogan et al [37] and Wang et al [45] who investigated the influence of pulsatile flow on a magnesium alloy (AZ31) compared tos static condition. As expected, flow increased the corrosion rate which means that Mg for applications in the circulatory system should be tested under flow.
We conclude that c.p Mg modified by PEO improves its corrosion resistance and with this its biological performance. Our study also provided directions for use of surface-modified Mg in cardiovascular stents. Investigations of the morphological and physicochemical properties of the surface-modified Mg showed that these surfaces may serve as a platform to deliver therapeutic cells to lesions with the benefit that the material will gradually disappear. Coat-ing controls the degradation of the material (reducCoat-ing the fast reactivity and possible toxic direct effect of the Mg) without altering the balance of magnesium in the body. In general, a significant difference at the level of cytotoxic-ity and biological behavior was found between all the coatings and the bare material. However, after comparing the coatings obtained under different treatments and conditions, no significant changes were evident between them. Which makes it difficult to select one specific coating. Finally, although in this project different approaches to the real application were evaluated, in vivo studies are warranted to validate this information.
130 CHAPTER 8
Future perspectives
Our results provide a preliminary view on the future use and safety of the material in vivo which warrants valida-tion in animal models. We anticipate, based on published literature that in vivo results will be better than our in vitro data, due to the severe measurement conditions and other arguments that were mentioned previously. The results in this thesis open the door to different routes. The surface modification of the Mg not only can be used in cardiovascular applications as described in this thesis. Yet, also other applications such as bone implants, surgical staples, wires, and other kinds of temporal and degradable implants are worth to consider. The challenge with this material lies in improvement of it mechanical properties and the design of the implant.
Additionally, this project allowed to explore the use of Mg in other presentations such as nanoparticles of Mg. Which can be used as a nanocarriers or deliver molecules or drugs to specific cells in order to kill or treat then selectively.
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