Anodization of Magnesium for Biomedical Applications – Processing, Characterization, Degradation and Cytocompatibility

posted Sep 28, 2017, 3:35 PM by Huinan Liu   [ updated Sep 28, 2017, 4:06 PM ]

Cipriano AF*, Lin J*, Miller C*, Lin A*, Cortez Alcaraz MC*, Soria P*, Liu H. Anodization of Magnesium for Biomedical Applications – Processing, Characterization, Degradation and Cytocompatibility. Acta Biomaterialia. S1742-7061(17)30516-0. DOI: 10.1016/j.actbio.2017.08.017. (PMID: 28818688)

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 2.0 V showed that the sample anodized at 1.9 V 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.

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

posted Nov 17, 2016, 9:22 PM by Huinan Liu   [ updated Sep 28, 2017, 3:57 PM ]

Cipriano AF*, Sallee A*, Tayoba M*, Cortez Alcaraz MC*, Lin A*, Guan RG, Zhao ZY*, Tayoba M*, Sanchez J*, and Liu HCytocompatibility and Early Inflammatory Response of Human Endothelial Cells in Direct Culture with Mg-Zn-Sr AlloysActa Biomaterialia. 16: 30547-5, 2016. Epub ahead of print 10/13/2016. DOI: 10.1016/j.actbio.2016.10.020. (PMID: 27746360)

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.

Cytocompatibility of Magnesium Alloys with Human Urothelial Cells: A Comparison of Three Culture Methodologies

posted Sep 29, 2016, 9:34 AM by Huinan Liu   [ updated Sep 29, 2016, 9:48 AM ]

Tian Q*, Deo M*, Rivera-Castaneda L*, Liu H. "Cytocompatibility of Magnesium Alloys with Human Urothelial Cells: A Comparison of Three Culture Methodologies." ACS Biomaterials Science and Engineering. 2016. DOI: 10.1021/acsbiomaterials.6b00325.

Magnesium (Mg) is a biodegradable metallic material, which has shown great potential for medical device applications. In this study, human urothelial cells (HUCs) were cultured in vitro with Mg-based substrates to investigate their cytocompatibility for potential urological device applications. Three different in vitro culture methodologies were explored to mimic different in vivo conditions, in an attempt to establish standard methods of evaluating cytocompatibility of Mg-based biomaterials for urological device applications. Direct culture is a suitable in vitro method when it is important to evaluate direct cell attachment on the biomaterial surfaces. Direct exposure culture is a desirablein vitro method for investigating the response of well-established cells in the body with newly implanted biomaterials. The exposure culture method is appropriate for evaluating cell–biomaterial interactions in the same environment, where they are not in direct contact with each other. The results showed differences in HUC behaviors with the same Mg-based substrates when different culture methods were used. The Mg-based substrates inhibited the HUC viability with direct contact at the cell–material interface in direct culture and direct exposure culture. The faster degrading Mg alloys containing yttrium reduced HUC density in direct culture, direct exposure culture, and exposure culture. The major soluble degradation products of Mg-based materials reduced HUC density significantly when the pH increased to 8.6 and above or the Mg2+ ion concentration reached 10 mM and above. Mg-based biomaterials, especially the slower degrading alloys such as AZ31, should be further studied to determine their potential to be used for bioresorbable urological devices.

Concentration-dependent behaviors of bone marrow derived mesenchymal stem cells and infectious bacteria toward magnesium oxide nanoparticles

posted May 18, 2016, 3:31 PM by Huinan Liu   [ updated Sep 29, 2016, 9:42 AM ]

Wetteland CL*, Nguyen NYT*, and Liu H. "Concentration-dependent behaviors of bone marrow derived mesenchymal stem cells and infectious bacteria toward magnesium oxide nanoparticles."Acta biomaterialia 35 (2016): 341-356.

This article reports the quantitative relationship between the concentration of magnesium oxide (MgO) nanoparticles and its distinct biological activities towards mammalian cells and infectious bacteria for the first time. The effects of MgO nanoparticles on the viability of bone marrow derived mesenchymal stem cells (BMSCs) and infectious bacteria (both gram-negative Escherichia coli and gram-positive Staphylococcus epidermidis) showed a concentration-dependent behavior in vitro. The critical concentrations of MgO nanoparticles identified in this study provided valuable guidelines for biomaterial design toward potential clinical translation. BMSCs density increased significantly when cultured in 200 μg/mL of MgO in comparison to the Cells Only control without MgO. The density of BMSCs decreased significantly after culture in the media with 500 μg/mL or more of MgO. Concentrations at or above 1000 μg/mL of MgO resulted in complete BMSCs death. Quantification of colony forming units (CFU) revealed that the minimum bactericidal concentration (MBC) of MgO for E. coli and S. epidermidis was 1200 μg/mL. The addition of MgO nanoparticles into the cultures increased the pH and Mg2+ ion concentration in the respective culture media, which might have played a role in the observed cell responses but not the main factors. E. coli and S. epidermidis still proliferated significantly at alkaline pH up to 10 or with supplemental Mg2+ dosages up to 50 mM, indicating bactericidal properties of MgO are beyond the effects of increased media pH and Mg2+ ion concentrations. MgO nanoparticles at a concentration of 200 μg/mL provided dual benefits of promoting BMSC proliferation while reducing bacterial adhesion, which should be further studied for potential medical implant applications. The use of free MgO nanoparticles yielded detrimental effects to BMSCs in concentrations above 300 μg/mL. We recommend further study into MgO nanoparticle as a coating material or as a part of a composite.

Magnetic Nanocomposite Hydrogel for Potential Cartilage Tissue Engineering: Synthesis, Characterization, and Cytocompatibility with Bone Marrow Derived Mesenchymal Stem Cells

posted Nov 6, 2015, 5:15 PM by Huinan Liu   [ updated Nov 6, 2015, 5:16 PM ]

Zhang N*, Lock J*, Sallee A*, and Liu H. Magnetic Nanocomposite Hydrogel for Potential Cartilage Tissue Engineering: Synthesis, Characterization, and Cytocompatibility with Bone Marrow Derived Mesenchymal Stem Cells. ACS Applied Materials and Interfaces. 7(37): pp 20987–20998, 2015.

Hydrogels possess high water content and closely mimic the microenvironment of extracellular matrix. In this study, we created a hybrid hydrogel containing type II collagen, hyaluronic acid (HA), and polyethylene glycol (PEG) and incorporated magnetic nanoparticles into the hybrid hydrogels of type II collagen-HA-PEG to produce a magnetic nanocomposite hydrogel (MagGel) for cartilage tissue engineering. The results showed that both the MagGel and hybrid gel (Gel) were successfully cross-linked and the MagGel responded to an external magnet while maintaining structural integrity. That is, the MagGel could travel to the tissue defect sites in physiological fluids under remote magnetic guidance. The adhesion density of bone marrow derived mesenchymal stem cells (BMSCs) on the MagGel group in vitro was similar to the control group and greater than the Gel group. The morphology of BMSCs was normal and consistent in all groups. We also found that BMSCs engulfed magnetic nanoparticles in culture and the presence of magnetic nanoparticles did not affect BMSC adhesion and morphology. We hypothesized that the ingested nanoparticles may be eventually broken down by lysosome and excreted through exocytosis; further studies are necessary to confirm this. This study reports a promising magnetic responsive nanocomposite hydrogel for potential cartilage tissue engineering applications, which should be further studied for its effects on cell functions when combined with electromagnetic stimulation.

Nanostructured calcium phosphate coatings on magnesium alloys: characterization and cytocompatibility with mesenchymal stem cells

posted Jun 9, 2015, 10:45 AM by Huinan Liu   [ updated Jun 9, 2015, 10:47 AM ]

Iskandar ME, Aslani A, Tian Q*, and Liu H. Nanostructured calcium phosphate coatings on magnesium alloys: characterization and cytocompatibility with mesenchymal stem cells. Journal of Materials Science: Materials in Medicine 26(5): 1-18, 2015.

This article reports the deposition and characterization of nanostructured calcium phosphate (nCaP) on magnesium–yttrium alloy substrates and their cytocompatibility with bone marrow derived mesenchymal stem cells (BMSCs). The nCaP coatings were deposited on magnesium and magnesium–yttrium alloy substrates using proprietary transonic particle acceleration process for the dual purposes of modulating substrate degradation and BMSC adhesion. Surface morphology and feature size were analyzed using scanning electron microscopy and quantitative image analysis tools. Surface elemental compositions and phases were analyzed using energy dispersive X-ray spectroscopy and X-ray diffraction, respectively. The deposited nCaP coatings showed a homogeneous particulate surface with the dominant feature size of 200–500 nm in the long axis and 100–300 nm in the short axis, and a Ca/P atomic ratio of 1.5–1.6. Hydroxyapatite was the major phase identified in the nCaP coatings. The modulatory effects of nCaP coatings on the sample degradation and BMSC behaviors were dependent on the substrate composition and surface conditions. The direct culture of BMSCs in vitro indicated that multiple factors, including surface composition and topography, and the degradation-induced changes in media composition, influenced cell adhesion directly on the sample surface, and indirect adhesion surrounding the sample in the same culture. The alkaline pH, the indicator of Mg degradation, played a role in BMSC adhesion and morphology, but not the sole factor. Additional studies are necessary to elucidate BMSC responses to each contributing factor.

Electrophoretic deposition and characterization of nanocomposites and nanoparticles on magnesium substrates

posted Jun 9, 2015, 10:29 AM by Huinan Liu   [ updated Jun 9, 2015, 11:06 AM ]

This study introduces a triphasic design of biodegradable materials composed of nanophase hydroxyapatite (nHA), poly(lactic-co-glycolic acid) (PLGA), and magnesium (Mg) substrates for musculoskeletal implant applications. Specifically, nHA_PLGA composites and nHA nanoparticles were synthesized, deposited on three-dimensional Mg substrates using electrophoretic deposition (EPD), and characterized. The three components involved, that is, nHA, PLGA, and Mg are all biodegradable in the human body, thus promising for biodegradable implant and device applications. Mg and its alloys are attractive for musculoskeletal implant applications due to their comparable modulus and strength to cortical bone. Controlling the interface of Mg with the biological environment, however, is the key challenge that currently limits this biodegradable metal for broad applications in medical implants. This article particularly focuses on creating nanostructured interface between the biodegradable Mg and surrounding tissue for the dual purposes of (1) mediating the degradation of the Mg-based substrates and (2) potentially enhancing osteointegration. Nanophase hydroxyapatite (nHA) is an excellent candidate as a coating material due to its osteoconductivity, while the polymer phase promotes interfacial adhesion between the nHA and Mg. Moreover, the degradation products of PLGA and Mg neutralize each other. Surface characterization showed successful deposition of nHA_PLGA composite microspheres and nHA nanoparticles on Mg substrates using EPD. Mg substrates coated with nHA_PLGA composites showed greater adhesion strength when compared with nHA coating, and slower corrosion rate than nHA coated Mg and non-coated Mg. The triphasic composites of nHA, PLGA and Mg are promising as the next-generation biodegradable materials for medical applications.

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

posted Mar 26, 2015, 11:00 AM by Huinan Liu   [ updated Mar 26, 2015, 11:00 AM ]

Nguyen TY*, Cipriano AF*, Guan R, Zhao Z, and Liu H. In Vitro Interactions of Blood, Platelet, and Fibroblast with Biodegradable Magnesium-Zinc-Strontium Alloys. Journal of Biomedical Materials Research Part A. Epub ahead of print.

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 Mg2+ 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 Mg2+ 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.

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

posted Dec 19, 2014, 11:42 AM by Huinan Liu   [ updated Dec 19, 2014, 11:44 AM ]

Cipriano AF*, Sallee A*, Guan RG, Zhao ZY, Tayoba M*, Sanchez J*, Liu H. Investigation on Magnesium-Zinc-Calcium Alloys and Bone Marrow Derived Mesenchymal Stem Cell Responses in Direct Culture. Acta Biomaterialia. 12(1): 298-321, 2015.

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, 4.0 wt.%) 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 27.6 mM Mg2+ 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.

Anodic Growth and Biomedical Applications of TiO2 Nanotubes

posted Jul 10, 2014, 8:42 PM by Huinan Liu   [ updated Aug 17, 2014, 7:29 PM ]

Cipriano AF*, Miller CT*, Liu H. Anodic Growth and Biomedical Applications of TiO2 Nanotubes. Journal of Biomedical Nanotechnology. 10(10): 2977-3003, 2014.

Over the past decades, self-assembled, vertically-aligned nanotubes have been generated on metallic substrates via anodization, which attracted significant scientific interest for a broad range of applications. These nano-tubular structures integrate highly controllable geometry at the nano-scale with fascinating chemical and biological properties. In this review, we first discussed mechanistic aspects of nanotube growth primarily on titanium (Ti) substrates by controlled anodization, a relatively inexpensive and scalable electrochemical process. We thoroughly reviewed electrochemical conditions that led to formation of self-assembled, vertically- aligned nano-tubular layers as they apply primarily to Ti substrates; we also reviewed anodization conditions that have led to formation of nanotubes on zirconium and various Ti alloys. We discussed how to adjust a set of anodization parameters to fine-tune the geometry of vertically oriented titania (TiO2) nanotubes, such as nanotube diameter, wall thickness, and length. We critically analyzed the key anodization parameters in the literature, including applied voltage, anodization duration, voltage ramp, electrolyte composition and concentration, electrolyte pH, electrolyte temperature, and electrolyte fluoride and water concentrations. Lastly, we discussed the promising properties of anodically grown TiO2 nano-tubular arrays for a wide range of biomedical applications including: directing cell bioactivity, anti-bacterial efficacy, modulating deposition of hydroxyapatite, drug delivery, biosensors, and orthopedic implants (in vivo). We highlighted ongoing in vitro and in vivo studies on the effects of nanotube geometry and aspect ratio on their hydrophilicity and interactions with biological entities at the protein, cellular and tissue level.

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