Aaron F. Cipriano

Development and evaluation of a magnesium–zinc–strontium alloy for biomedical applications — Alloy processing, microstructure, mechanical properties, and biodegradation

A new biodegradable magnesium-zinc-strontium (Mg-Zn-Sr) alloy was developed and studied for medical implant applications. This first study investigated the alloy processing (casting, rolling, and heat treatment), microstructures, mechanical properties, and degradation properties in simulated body fluid (SBF). Aging treatment of the ZSr41 alloy at 175 °C for 8h improved the mechanical properties when compared to those of the as-cast alloy. Specifically, the aged ZSr41 alloy had an ultimate tensile strength of 270 MPa, Vickers hardness of 71.5 HV, and elongation at failure of 12.8%. The mechanical properties of the ZSr41 alloy were superior as compared with those of pure magnesium and met the requirements for load-bearing medical implants. Furthermore, the immersion of the ZSr41 alloy in SBF showed a degradation mode that progressed cyclically, alternating between pitting and localized corrosion. The steady-state average degradation rate of the aged ZSr41 alloy in SBF was 0.96 g/(m(2)·hr), while the pH of SBF immersion solution increased. The corrosion current density of the ZSr41 alloy in SBF solution was 0.41 mA/mm(2), which was much lower than 1.67 mA/mm(2) for pure Mg under the same conditions. In summary, compared to pure Mg, the mechanical properties of the new ZSr41 alloy improved while the degradation rate decreased due to the addition of Zn and Sr alloying elements and specific processing conditions. The superior mechanical properties and corrosion resistance of the new ZSr41 alloy make it a promising alloy for next-generation implant applications.

An in vivo study on the metabolism and osteogenic activity of bioabsorbable Mg–1Sr alloy

UNLABELLED Previous studies indicated that local delivery of strontium effectively increased bone quality and formation around osseointegrating implants. Therefore, implant materials with long-lasting and controllable strontium release are avidly pursued. The central objective of the present study was to investigate the in vivo biocompatibility, metabolism and osteogenic activity of the bioabsorbable Mg-1Sr (wt.%, nominal composition) alloy for bone regeneration. The general corrosion rate of the alloy implant as a femoral fracture fixation device was 0.55±0.03mm·y(-1) (mean value±standard deviation) in New Zealand White rabbits which meet the bone implantation requirements and can be adjusted by material processing methods. All rabbits survived and the histological evaluation showed no abnormal physiology or diseases 16 weeks post-implantation. The degradation process of the alloy did not significantly alter 16 primary indexes of hematology, cardiac damage, inflammation, hepatic functions and metabolic process. Significant increases in peri-implant bone volume and direct bone-to-implant contact (48.3%±15.3% and 15.9%±5.6%, respectively) as well as the expressions of four osteogenesis related genes (runt-related transcription factor 2, alkaline phosphatase, osteocalcin, and collagen, type I, alpha 1) were observed after 16 weeks implantation for the Mg-1Sr group when compared to the pure Mg group. The sound osteogenic properties of the Mg-1Sr alloy by long-lasting and controllable Sr release suggesting a very attractive clinical potential. STATEMENT OF SIGNIFICANCE Sr (strontium) has exhibited pronounced effects to reduce the bone fracture risk in osteoporotic patients. Nonetheless, long-lasting local Sr release is hardly achieved by traditional methods like surface treatment. Therefore, a more efficient Sr local delivery platform is in high clinical demand. The stable and adjustable degradation process of Mg alloy makes it an ideal Sr delivery platform. We combine the well-known osteogenic properties of strontium with magnesium to manufacture bioabsorbable Mg-1Sr alloy with stable Sr release based on our previous studies. The in vitro and in vivo results both showed the alloys suitable degradation rate and biocompatibility, and the sound osteogenic properties and stimulation effect on bone formation suggest its very attractive clinical potential.

Investigation of magnesium-zinc-calcium alloys and bone marrow derived mesenchymal stem cell response in direct culture.

Crystalline Mg-Zn-Ca ternary alloys have recently attracted significant interest for biomedical implant applications due to their promising biocompatibility, bioactivity, biodegradability and mechanical properties. The objective of this study was to characterize as-cast Mg-xZn-0.5Ca (x=0.5, 1.0, 2.0, 4.0wt.%) alloys, and determine the adhesion and morphology of bone marrow derived mesenchymal stem cells (BMSCs) at the interface with the Mg-xZn-0.5Ca alloys. The direct culture method (i.e. seeding cells directly onto the surface of the sample) was established in this study to probe the highly dynamic cell-substrate interface and thus to elucidate the mechanisms of BMSC responses to dynamic alloy degradation. The results showed that the BMSC adhesion density on these alloys was similar to the cell-only positive control and the BMSC morphology appeared more anisotropic on the rapidly degrading alloy surfaces in comparison with the cell-only positive control. Importantly, neither culture media supplemented with up to 27.6mM Mg(2+) ions nor media intentionally adjusted up to alkaline pH 9 induced any detectable adverse effects on BMSC responses. We speculated that degradation-induced dynamic surface topography played an important role in modulating cell morphology at the interface. This study presents a clinically relevant in vitro model for screening bioresorbable alloys, and provides useful design guidelines for determining the degradation rate of implants made of Mg-Zn-Ca alloys.

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

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, 1.5wt%; 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 24h was not adversely affected by the degradation of the alloys. Importantly, neither culture media supplemented with up to 27.6mM 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.

In vitro interactions of blood, platelet, and fibroblast with biodegradable magnesium-zinc-strontium alloys

Magnesium (Mg) alloy is an attractive class of metallic biomaterial for cardiovascular applications due to its biodegradability and mechanical properties. In this study, we investigated the degradation in blood, thrombogenicity, and cytocompatibility of Magnesium-Zinc-Strontium (Mg-Zn-Sr) alloys, specifically four Mg-4 wt % Zn-xSr (x = 0.15, 0.5, 1, and 1.5 wt %) alloys, together with pure Mg control and relevant reference materials for cardiovascular applications. Human whole blood and platelet rich plasma (PRP) were used as the incubation media to investigate the degradation behavior of the Mg-Zn-Sr alloys. The results showed that the PRP had a greater pH increase and greater concentration of Mg(2+) ions when compared with whole blood after 2 h of incubation with the same respective Mg alloys, suggesting that the Mg alloys degraded faster in PRP than in whole blood. The Mg alloy with 4 wt % Zn and 0.15 wt % Sr (named as ZSr41A) was identified as the most promising alloy for cardiovascular stent applications, because it showed slower degradation and less thrombogenicity, as indicated by the lower concentrations of Mg(2+) ions released and less deposition of platelets. Additionally, ZSr41 alloys were cytocompatible with fibroblasts in direct exposure culture in which the cells adhered and proliferated around the samples, with no statistical difference in cell adhesion density compared with the blank reference. Future studies on the ZSr41 alloys are necessary to investigate their direct interactions with other important cells in cardiovascular system, such as vascular endothelial cells and smooth muscle cells.

Anodization of magnesium for biomedical applications – Processing, characterization, degradation and cytocompatibility

This article reports anodization of Mg in KOH electrolyte and the associated surface, degradation, and biological properties for bioresorbable implant applications. The preparation procedures for electrodes and anodization setup significantly enhanced reproducibility of samples. The results of anodization performed at the applied potentials of 1.8, 1.9, or 2.0V showed that the sample anodized at 1.9V and annealed, referred to as the 1.9 AA sample, had homogenous surface microstructure and elemental composition, and a reduction in corrosion current density in the electrochemical testing. In comparison with Mg control, the 1.9 AA sample showed a distinct mode of degradation, e.g., continuous growth of a passivation layer enriched with Ca and P instead of typical localized pitting and undermining, and a greater release rate of Mg2+ ions when immersed in physiologically relevant media. In the direct culture with bone marrow derived mesenchymal stem cells (BMSCs) in vitro, the 1.9 AA sample did not affect BMSC adhesion and morphology under indirect contact; however, the 1.9 AA sample showed a reduction in cell spreading under direct contact. The change in surface topography/composition at the dynamic interface of the anodized-annealed Mg sample might have contributed to the change in BMSC morphology. In summary, this study demonstrated the potential of anodic oxidation to modulate the degradation behaviors of Mg-based biomaterials and BMSC responses in vitro, and confirmed the value of direct culture method for studying cytocompatibility of Mg-based biomaterials for medical implant applications. STATEMENT OF SIGNIFICANCE Magnesium (Mg)-based biomaterials have been specifically designed and actively explored for biodegradable implant applications since the early 2000s. To realize the benefits of Mg-based materials for medical implant applications, it is critical to control the rate of Mg degradation (i.e. corrosion) in the body. We investigated an environmentally friendly anodization process using KOH electrolyte for modifying the surface of Mg-based materials, and the resulted surface, degradation, and biological properties for biomedical applications. This study reported critical considerations that are important for repeatability of anodization process, homogeneity of surface microstructure and composition, and in vitro evaluations of the degradation and biological properties of surface treated Mg samples. The details in preparation of electrodes, anodization setup, annealing, and sample handling before and after surface treatment (e.g. re-embedding) reported in this article are valuable for studying a variety of electrochemical processes for surface treatment of Mg-based metals, because of enhanced reproducibility.

Degradation of Bioresorbable Mg–4Zn–1Sr Intramedullary Pins and Associated Biological Responses in Vitro and in Vivo

This article reports the degradation and biological properties of as-drawn Mg-4Zn-1Sr (designated as ZSr41) and pure Mg (P-Mg) wires as bioresorbable intramedullary pins for bone repair. Specifically, their cytocompatibility with bone marrow derived mesenchymal stem cells (BMSCs) and degradation in vitro, and their biological effects on peri-implant tissues and in vivo degradation in rat tibiae were studied. The as-drawn ZSr41 pins showed a significantly faster degradation than P-Mg in vitro and in vivo. The in vivo average daily degradation rates of both ZSr41 and P-Mg intramedullary pins were significantly greater than their respective in vitro degradation rates, likely because the intramedullary site of implantation is highly vascularized for removal of degradation products. Importantly, the concentrations of Mg2+, Zn2+, and Sr2+ ions in the BMSC culture in vitro and their concentrations in rat blood in vivo were all lower than their respective therapeutic dosages, i.e., in a safe range. Despite of rapid degradation with a complete resorption time of 8 weeks in vivo, the ZSr41 intramedullary pins showed a significant net bone growth because of stimulatory effects of the metallic ions released. However, proportionally released OH- ions and hydrogen gas caused adverse effects on bone marrow cells and resulted in cavities in surrounding bone. Thus, properly engineering the degradation properties of Mg-based implants is critical for harvesting the bioactivities of beneficial metallic ions, while controlling adverse reactions associated with the release of OH- ions and hydrogen gas. It is necessary to further optimize the alloy processing conditions and/or modify the surfaces, for example, applying coatings onto the surface, to reduce the degradation rate of ZSr41 wires for skeletal implant applications.

In vitro degradation and cytocompatibility of Magnesium-Zinc-Strontium alloys with human embryonic stem cells

Magnesium-based alloys have attracted great interest for medical applications due to their unique biodegradable capability and desirable mechanical properties. When considered for medical applications, the degradation rate of these alloys must be tailored so that: (i) it does not exceed the rate at which the degradation products can be excreted from the body, and (ii) it is slow enough so that the load bearing properties of the implant are not jeopardized and do not conflict prior to and during synthesis of new tissue. Implant integration with surrounding cells and tissues and mechanical stability are critical aspects for clinical success. This study investigated Magnesium-Zinc-Strontium (ZSr41) alloy degradation rates and the interaction of the degradation products with human embryonic stem cells (hESC) over a 72 hour period. An in vitro hESC model was chosen due to the higher sensitivity of ESCs to known toxicants which allows to potentially detect toxicological effects of new biomaterials at an early stage. Four distinct ZSr41 compositions (0.15 wt.%, 0.5 wt.%, 1 wt.%, and 1.5 wt.% Sr) were designed and produced through metallurgical processing. ZSr41 alloy mechanical properties, degradation, and cytocompatibility were investigated and compared to pure polished Magnesium (Mg). Mechanical properties evaluated included hardness, ultimate tensile strength, and elongation to failure. Degradation was characterized by measuring total weight loss of samples and pH change in the cell culture media. Cytocompatibility was studied by comparing fluorescence and phase contrast images of hESCs after co-culture with Mg alloys. Results indicated that the Mg-Zn-Sr alloy with 0.15 wt.% Sr improved cytocompatibility and provided slower degradation as compared with pure Mg.

Cytocompatibility of Magnesium-Zinc-Calcium Alloys with Bone Marrow Derived Mesenchymal Stem Cells

Magnesium (Mg)-based alloys have attracted great interest as metallic biomaterials for orthopedic applications due to their biocompatibility, biodegradability, and mechanical properties that resemble those of cortical bone. However, the potential toxicity of alloying elements in commercially available Mg alloys makes it critical to engineer and screen new alloys specifically for biomedical applications. The objective of this study was to evaluate and compare the in vitrodegradation and cytocompatibility of two distinct Mg - Zinc (Zn) - Calcium (Ca) alloys (Mg-4%Zn-1%Ca and Mg-9%Zn-1%Ca, wt. %; abbreviated as ZCa41 and ZCa91, respectively) using a bonemarrow derived mesenchymal stem cell (BMSC) model. Both Zn and Ca play critical roles in boneformation and growth, and have been shown to increase mechanical and corrosion properties of Mgalloys. BMSCs provide vertebrates the continuous supply of osteoblasts needed for bone remodelingand repair, and thus were selected to determine the effect of increasing Zn content on cell behavior.Surface microstructure and composition of the alloys were characterized before and after BMSC culture using field emission scanning electron microscopy (FESEM) and energy dispersive X-rayspectroscopy (EDS). Thermanox® treated glass and plasma treated tissue culture polystyrene were used as a control and reference, respectively. Results indicated that the ZCa91 alloy improved BMSC adhesion as compared with ZCa41 alloy. The formation of high-aspect ratio needle-likefeatures on the surface of ZCa41 alloy after its degradation in cell culture media was speculated tocontribute to the lower cell adhesion. This study provided an early indication on cytocompatibility of Mg-Zn-Ca alloys for biomedical applications.

Surface Characterization of Magnesium Anodized in a 10M KOH Electrolyte

Magnesium (Mg) is a promising implant material for orthopedic applications due to its biodegradability and desirable mechanical properties. However, in order for Mg to have widespread clinical applications, engineering solutions that address the rapid degradation in physiological environments and promote bone-forming activity are needed. The objective of this study was to develop an anodization process using a toxicant-free electrolyte to modulate nanoscale surface features and surface chemistry on Mg. Anodic polarization and potentiostatic anodization tests were used to evaluate the effect of applied potential on surface morphology of Mg in a 10 M KOH electrolyte. Nucleation of oxides as a function of anodization duration was also investigated in order to optimize the synthesis process. The alkaline electrolyte used for anodization of Mg offers an alternative to commercial processes that use hazardous elements. The anodized samples were annealed to investigate the effect of thermal treatments on surface morphology and chemical composition. The nanostructure and chemical composition of the anodized and annealed Mg substrates were characterized using scanning electron microscopy and energy dispersive X-ray spectroscopy. Our results showed that the nanostructures and chemical composition of anodically-generated oxide layers on Mg are specific to each oxidation process in a 10 M KOH electrolyte. Furthermore, results indicated that anodization durations of two hours generated surface oxide layers with homogeneous topography on the Mg substrates atapplied potentials of 0.5 V, 1.5 V and 2 V.. This study showed a promising approach for creating nanoscale surface features on Mg for improved bioactivity and degradation property.