Elsevier

Acta Biomaterialia

Volume 48, 15 January 2017, Pages 499-520
Acta Biomaterialia

Full length article
Cytocompatibility and early inflammatory response of human endothelial cells in direct culture with Mg-Zn-Sr alloys

https://doi.org/10.1016/j.actbio.2016.10.020Get rights and content

Abstract

Crystalline Mg-Zinc (Zn)-Strontium (Sr) ternary alloys consist of elements naturally present in the human body and provide attractive mechanical and biodegradable properties for a variety of biomedical applications. The first objective of this study was to investigate the degradation and cytocompatibility of four Mg-4Zn-xSr alloys (x = 0.15, 0.5, 1.0, 1.5 wt%; designated as ZSr41A, B, C, and D respectively) in the direct culture with human umbilical vein endothelial cells (HUVEC) in vitro. The second objective was to investigate, for the first time, the early-stage inflammatory response in cultured HUVECs as indicated by the induction of vascular cellular adhesion molecule-1 (VCAM-1). The results showed that the 24-h in vitro degradation of the ZSr41 alloys containing a β-phase with a Zn/Sr at% ratio ∼1.5 was significantly faster than the ZSr41 alloys with Zn/Sr at% ∼1. Additionally, the adhesion density of HUVECs in the direct culture but not in direct contact with the ZSr41 alloys for up to 24 h was not adversely affected by the degradation of the alloys. Importantly, neither culture media supplemented with up to 27.6 mM Mg2+ ions nor media intentionally adjusted up to alkaline pH 9 induced any detectable adverse effects on HUVEC responses. In contrast, the significantly higher, yet non-cytotoxic, Zn2+ ion concentration from the degradation of ZSr41D alloy was likely the cause for the initially higher VCAM-1 expression on cultured HUVECs. Lastly, analysis of the HUVEC-ZSr41 interface showed near-complete absence of cell adhesion directly on the sample surface, most likely caused by either a high local alkalinity, change in surface topography, and/or surface composition. The direct culture method used in this study was proposed as a valuable tool for studying the design aspects of Zn-containing Mg-based biomaterials in vitro, in order to engineer solutions to address current shortcomings of Mg alloys for vascular device applications.

Statement of Significance

Magnesium (Mg) alloys specifically designed for biodegradable implant applications have been the focus of biomedical research since the early 2000s. Physicochemical properties of Mg alloys make these metallic biomaterials excellent candidates for temporary biodegradable implants in orthopedic and cardiovascular applications. As Mg alloys continue to be investigated for biomedical applications, it is necessary to understand whether Mg-based materials or the alloying elements have the intrinsic ability to direct an immune response to improve implant integration while avoiding cell-biomaterial interactions leading to chronic inflammation and/or foreign body reactions. The present study utilized the direct culture method to investigate for the first time the in vitro transient inflammatory activation of endothelial cells induced by the degradation products of Zn-containing Mg alloys.

Introduction

Magnesium (Mg) alloys specifically designed for biodegradable implant applications have been the focus of biomedical research since the early 2000s [1], [2], [3]. Physicochemical properties of Mg alloys make these metallic biomaterials excellent candidates for temporary biodegradable implants in orthopedic and cardiovascular applications [1], [2], [3], [4]. Most notable is the fact that the human body contains a large amount of Mg ions and can effectively metabolize the degradation products of Mg [1], [2], [3]. Therefore, temporary biodegradable metallic implants are idealized to be superior alternatives to permanent implants in that they would eliminate the need for implant removal surgeries following healing of the damaged tissue. By doing so, Mg-based biodegradable implants could reduce the burden on the healthcare system by mitigating risks and costs [5], [6]. Furthermore, as Mg alloys continue to be investigated for biomedical applications, it is necessary to understand whether Mg-based materials or the alloying elements have the intrinsic ability to direct an immune response to improve implant integration while avoiding cell-biomaterial interactions leading to chronic inflammation and/or foreign body reactions [7], [8]. In contrast, previous studies have shown that conventional permanent metallic implants, and associated wear debris, can trigger chronic inflammatory responses, result in tissue loss, and are prone to infection [1], [6], [9].

In general, the initial (acute) inflammatory response (i.e. innate immunity) to biomaterials is activated by the reaction of vascularized connective tissue to injury caused either by trauma or implantation [10], [11]. The physiological response of inflammation consists of a complex series of meticulously controlled responses which cannot possibly be summarized in a few sentences; however, the informed reader is referred to excellent reviews on the inflammatory response to biomaterials [8], [11]. Endothelial cells (EC) play an important role in the regulation of immune and inflammatory local responses by expressing, among other things, cell adhesion molecules (CAM) [12], [13]. CAM expression in activated ECs (type II activation) can be induced by pro-inflammatory cytokines, e.g. tumor necrosis factor α (TNFα) [12], [13], released by inflammatory cells activated on contact with adsorbed proteins on the implanted biomaterial [8], [11], [14]. In turn, these adhesion molecules help recruit leukocytes from circulating blood and facilitate transendothelial migration to the site of injury to initiate the acute inflammatory response [8], [11], [12], [13], [14]. Additionally, previous in vitro studies showed that CAM expression in ECs was activated by elevated concentrations of metallic ions typically found in permanent metallic implants [7], [15], [16], [17], [18], [19], [20], [21], [22], [23]. Vascular cell adhesion molecule-1 (VCAM-1) is an immunoglobulin superfamily-specific receptor that provides high-affinity interactions between ECs and integrins on the leukocyte surface and facilitates transendothelial migration [10], [13], [14]. Moreover, VCAM-1 binds with monocytes, but not neutrophils, and it is the first CAM expressed in chronic inflammation such as atherosclerosis (before atherosclerotic plaque development) [13], [14], [24] and restenosis following coronary stent implantation [25]. Thus, VCAM-1 can be used as an indicator of in vitro EC activation during the early stages of inflammation. Furthermore, previous studies supported the applicability of human umbilical vein endothelial cells (HUVEC) to model and investigate components of the inflammatory response, such as CAM expression [7], [17].

Previously, we reported the development of Mg-Zinc-Strontium (Mg-Zn-Sr) ternary alloys and the evaluation of their biological performance for biomedical applications [26], [27], [28]. Furthermore, we reported the in vitro direct culture method to mimic in vivo physiological conditions and evaluate cell responses at the cell-biomaterial interface (direct contact) and on the culture plate (indirect contact; exposure to solubilized degradation products) surrounding the Mg-based biomaterial [29]. The direct culture method was introduced to provide a more comprehensive in vitro method, as compared with ISO 10993-based methods, for the initial rapid screening of cytocompatibility and degradation of Mg-based biomaterials [29]. The direct culture method was introduced as part of a field-wide effort to improve and standardize the in vitro testing of Mg-based biomaterials [29], [30], [31], [32]. Thus, the first objective of this study was to investigate the degradation and cytocompatibility of four Mg-4Zn-xSr alloys (x = 0.15, 0.5, 1.0, 1.5 wt%; designated as ZSr41A, B, C, and D respectively) in the direct culture with HUVECs in vitro. The second objective was to investigate the induction of an inflammatory response in HUVECs as indicated by the expression of VCAM-1 activated by the degradation products of the ZSr41 alloys. While several recent in vivo studies reported adequate immunological response during the foreign body reaction or fibrosis stages following implantation of Mg-based materials [33], [34], [35], [36], [37], sparse literature is found on the early-stage inflammatory response. Specifically, to the authors’ knowledge, early-stage inflammatory induction by the degradation of Mg-based materials has only been investigated in vitro with primary murine and human macrophages [38] and with dendritic cells [39]. In both cases, the Mg-based materials and the respective degradation products were not found to have detrimental immunomodulatory effects. This study reported for the first time on the in vitro transient inflammatory activation of ECs induced by the degradation products of Zn-containing Mg alloys.

Section snippets

Preparation of ZSr41 alloys, Mg control, and reference materials

The ZSr41 alloys in this study had a nominal composition of 4 wt% Zn with 0.15, 0.5, 1.0, or 1.5 wt% Sr; these alloys were designated as ZSr41A, ZSr41B, ZSr41C, and ZSr41D accordingly with increasing Sr content. Details pertaining to the metallurgical process and heat treatment used for alloy preparation are described elsewhere [26], [27]. The heat-treated 1.0 mm thick sheets of ZSr41 alloys were cut into 5 × 5 mm squares. Likewise, commercially pure Mg sheets (99.9%, As-rolled, 1.0 mm thick, Cat#

Microstructure of ZSr41 alloys

Fig. 1 shows the surface microstructures and elemental compositions of the ZSr41 alloys, pure Mg control, and AZ31 reference. SEM images at a low magnification of 600× (Fig. 1a) confirmed an increase in intermetallic β-phase with increasing Sr content in the ZSr41A-D alloys, respectively. As expected, the pure Mg control showed a surface free of secondary phases, and the AZ31 reference showed the presence of an intermetallic β-phase. SEM images at a high magnification of 5000× (Fig. 1a, insets)

Discussion

The degradation of four heat-treated ZSr41 crystalline Mg alloys and their interaction with HUVECs was investigated using the direct culture method to model and study possible cellular modulatory effects at the HUVEC/ZSr41 alloy interface in vitro. We also examined HUVECs on the culture plate surrounding the ZSr41 alloy samples to understand modulatory effects of solubilized degradation products. Our results showed for the first time that the degradation products of Zn-containing Mg-alloys

Conclusions

This article reported the cytocompatibility and early-stage inflammatory induction of human umbilical vein endothelial cells in response to the degradation of four Mg-4Zn-xSr alloys (x = 0.15, 0.5, 1.0, 1.5 wt%; designated as ZSr41A, B, C, and D respectively) in the direct culture in vitro. Possible factors and mechanisms that affect cellular responses were also investigated. The following major conclusions were drawn from the present study:

  • 1.

    Addition of 0.5 or 1.5 wt% Sr to the heat-treated

Acknowledgements

The authors would like to thank the American Heart Association (12SDG12220014), Hellman Faculty Fellowship (Huinan Liu), and the University of California Regents Faculty Fellowship (Huinan Liu) for financial support. Aaron F. Cipriano would like to thank the Graduate Division at UC Riverside for the Dissertation Year Program fellowship. The authors thank the Central Facility for Advanced Microscopy and Microanalysis (CFAMM) at the University of California, Riverside.

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