Page 106 - Magnesium-based supports for stem cell therapy of vascular disease - Mónica Echeverry Rendón

CHAPTER 6
4. Discussion
The aim of our study was to develop and evaluate a temporal scaffold based on degradable anodized commercial pure magnesium (c.p Mg) loaded with regenerative cells i.e. ASC for future use in stenting of arterial lesions. The main findings of our study are that by PEO surface oxidation, the cytotoxic and bio-incompatible c.p Mg is converted into a biocompatible material that supports adhesion of therapeutically relevant ASC while it does not negatively af- fect their gene expression profile. The variation of electrolyte solution additives i.e. sodium fluoride, hexamethylene- tetramine and mannitol or their combination was of little influence to cytotoxicity or in vitro biological processes. While mesenchymal cell types (fibroblasts, ASC) and immune cells (THP-1 monocytes) were refractory to leachables from surface-coated materials, vascular cell types (EC and SMC) were more vulnerable to their cytotoxic components. This was corroborated by the adverse influence of leachables on pre-established vascular network in vitro in which the endothelial tubes disintegrated while the ASC remained viable. This was further confirmed in scratch wound clos- ing assays in which leachables only reduced, but not abolished, the closure rate of damaged fibroblast monolayers. In aqueous solutions, the adverse biocompatibility and high cytotoxicity of (metallic) Mg is caused by oxidation, i.e. formation, of Mg(OH)2 (corrosion product) that dissolves and raises the pH beyond physiologically tolerable levels. In addition, corrosion of Mg, releases cytotoxic hydrogen gas (H2). We corroborated the cytotoxicity of c.p Mg on all evaluated cell types. Therefore, modification of the surface of c.p Mg was warranted to improve its biocompat- ibility and reduce its cytotoxicity. Indeed, PEO treatment of c.p Mg reduced the release of cytotoxic components yet vascular cell types were more vulnerable than mesenchymal cell types such as fibroblasts and ASC while monocytes (THP-1) that play an important role in arterial wound healing were refractory too. Leachables from Mg-Zn-Ca alloys caused increased proliferation of ASC, while pure Mg leachables were non-cytotoxic to ASC [34] which is at least partially confirmed by our results. Differences in extraction, materials and ASC likely explain their resilience to pure Mg leachables. The Mg2+ concentration in the leachables in our leachables may have been higher than in the study of Yazdi [34]. In general, mesenchymal cell types such as human umbilical cord perivascular cells [35], osteoblasts (U2OS) [36] are refractory to Mg2+ and respond in a concentration-dependent fashion.
The degradation of Mg-based biomaterials involves release of particulate fragments that require phagocytic clear- ance by macrophages. Indeed, phagocytic uptake of corrosion products released from Mg-2.1Nd-0.2Zn-0.5Zr alloys [37], pure Mg particles [38] do not negatively affect primary human macrophages. Larger sized particles, however, reduce vitality of J774 macrophages after phagocytosis [39]. Our results confirm that leachables released from sur- face-coated c.p. Mg did not induce a cytotoxic response in THP-1. Thus, while we did not assess the presence of nano and microparticulate fragments in leachables nor the Mg2+ concentration, the surface-coating is suitable to support proper functioning of ASC and immune cells. As a matter of fact, surface-coated materials did not induce apoptosis i.e. cleavage of pro-caspase 3 nor of PARP, in adhered ASC, while proliferation (PCNA expression) was maintained. The type of surface-coated had virtually no influence on the adhered ASC, although minor differences in cellular morphology were observed that cannot be explained based on the current data. In contrast, ASC adhered to c.p Mg surface died by apoptosis, which emphasizes the protecting influence of either of our evaluated surface-coatings. The biological activity of ASC e.g. the capacity to support wound closure or angiovasculogenesis and the secretion of beneficial paracrine factors and the absence of pro-inflammatory activation was not affected by surface-coatings[40] [41][12]. Although, closure of scratch wounds in vitro was maintained, the addition of ASC conditioned medium, un- expectedly, reduced the rate of wound healing. Likely, leachables had inactivated part of the bioactive molecules in the conditioned medium, e.g. by binding and steric hindrance or by competition of Mg2+ for Zn2+ in e.g. matrix metalloproteinases which require Zn2+ for enzymatic activity. Moreover, their differentiation capacity to adipocytes, osteoblasts and, more relevant to arterial repair, SMC is reported to be unaltered upon exposure to leachables from surface-coated materials[42][43]. No data exist in literature on changes of differentiation of ASC to vascular cell types under influence of Mg compounds unfortunately. The high expression of extracellular matrix
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