Electrophoretic Deposition of Magnesium Oxide Nanoparticles on Magnesium: Processing Parameters, Microstructures, Degradation, and Cytocompatibility.

Magnesium (Mg) and its alloys are a class of promising materials for biodegradable orthopedic and craniomaxillofacial implants; however, rapid release of hydrogen gas remains a key challenge for clinical translation. This study reported the optimal parameters of electrophoretic deposition (EPD), at which magnesium oxide nanoparticles (nMgO) could be deposited onto Mg substrates with homogeneous surface morphology and elemental distribution. The results showed that the distribution and uniformity of the nMgO coatings on Mg improved when the nMgO concentration in ethanol increased and the time of applied voltage decreased. The nMgO-coated Mg showed a homogeneous surface and distinct degradation mode during the 9-day immersion studies in revised simulated body fluid (r-SBF) and Dulbecco's modified Eagle's medium (DMEM), when compared with the noncoated Mg controls. The nMgO coating initially mitigated hydrogen gas formation. The degradation layer on nMgO-coated Mg was thicker than the noncoated Mg and enriched with Ca and P that are favorable for skeletal implant applications. In the direct culture study with bone marrow derived mesenchymal stem cells (BMSCs) in vitro, the cell adhesion density and morphology were not affected by the solubilized degradation products released by the nMgO-coated Mg under indirect contact. However, at the cell-biomaterial interface, the cell spreading decreased under direct contact, possibly because of the continuous dynamic degradation of the samples. The electrophoretically deposited nMgO coatings on Mg-based medical implants should be further studied to improve the coating-substrate and cell-material interfaces for clinical applications.

In vitro evaluation of MgSr and MgCaSr alloys via direct culture with bone marrow derived mesenchymal stem cells.

Magnesium (Mg) and its alloys have been widely investigated as the most promising biodegradable metals to replace conventional non-degradable metals for temporary medical implant applications. New Mg alloys have been developed for medical applications in recent years; and the concept of alloying Mg with less-toxic elements have aroused tremendous interests due to the promise to address the problems associated with rapid degradation of Mg without compromising its cytocompatibility and biocompatibility. Of particular interests for orthopedic/spinal implant applications are the additions of calcium (Ca) and strontium (Sr) into Mg matrix because of their beneficial properties for bone regeneration. In this study, degradation and cytocompatibility of four binary MgSr alloys (Mg-xSr, x = 0.2, 0.5, 1 and 2 wt%) and four ternary MgCaSr alloys (Mg-1Ca-xSr, x = 0.2, 0.5, 1 and 2 wt%) were investigated and compared via direct culture with bone marrow-derived mesenchymal stem cells (BMSCs). The influence of the alloy composition on the degradation rates were studied and compared. Moreover, the cellular responses to the binary MgSr alloys and the ternary MgCaSr alloys were comparatively evaluated; and the critical factors influencing BMSC behaviors were discussed. This study screened the degradability and in vitro cytocompatibility of the binary MgSr alloys and the ternary MgCaSr alloys. Mg-1Sr, Mg-1Ca-0.5Sr and Mg-1Ca-1Sr alloys are recommended for further in vivo studies toward clinical translation due to their best overall performances in terms of degradation and cytocompatibility among all the alloys studied in the present work. STATEMENT OF SIGNIFICANCE: Traditional Mg alloys with slower degradation often contain aluminum or rare earth elements as alloying components, which raised safety and regulatory concerns. To circumvent unsafe elements, nutrient elements such as calcium (Ca) and strontium (Sr) were selected to create Mg-Sr binary alloys and Mg-Ca-Sr ternary alloys to improve the safety and biocompatibility of bioresorbable Mg alloys for medical implant applications. In this study, in vitro degradation and cellular responses to four binary Mg-xSr alloys and four ternary Mg-1Ca-xSr alloys with increasing Sr content (up to 2 wt%) were evaluated in direct culture with bone marrow derived mesenchymal stem cells (BMSCs). The roles of Sr and Ca in tuning the alloy microstructure, degradation behaviors, and BMSC responses were collectively compared in the BMSC direct culture system for the first time. The most promising alloys were identified and recommended for further in vivo studies toward clinical translation.

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.

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.

A Comparison Study on the Degradation and Cytocompatibility of Mg-4Zn-xSr Alloys in Direct Culture.

This article reports the behaviors of bone-marrow-derived mesenchymal stem cells (BMSCs) in the direct culture with four Mg-4Zn-xSr alloys (x = 0.15, 0.5, 1.0, 1.5 wt %), designated as ZSr41A, B, C, and D, respectively; and a systematic comparison on the degradation of the ZSr41 alloys and their biological impact in the direct culture with different cell types in their respective media. The direct culture method, in which cells are seeded directly onto the surface of the sample, was used to investigate cellular responses at the cell-biomaterial interface in vitro. The results showed that BMSCs adhered and remained viable on the surfaces of all ZSr41 alloys, but the faster degrading ZSr41A and ZSr41B alloys showed a significantly lower amount of viable BMSCs adhered to their surfaces. Moreover, BMSCs adhered to the culture plate surrounding the samples were unaffected by the solubilized degradation products from the ZSr41 alloys. The results from the comparison study showed that the in vitro degradation rates of Mg-based biomaterials in different culture systems might be mostly affected by media buffer capacity (i.e., HCO3- concentration), and to a lesser extent, d-glucose concentration. The comparison study also indicated that BMSCs were more robust than H9 human embryonic stem cells and human umbilical vein endothelial cells for screening the cytocompatibility of Mg-based biomaterials. In general, the adhesion and viability of BMSCs at the cell-material interface were inversely proportional to the alloy degradation rates. This study presented a clinically relevant in vitro culture system for screening bioresorbable alloys in direct culture, and provided valuable guidelines for determining the degradation rates of Mg-based biomaterials.

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 ß-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.

Label-Free SERS Selective Detection of Dopamine and Serotonin Using Graphene-Au Nanopyramid Heterostructure.

Ultrasensitive detection and spatially resolved mapping of neurotransmitters, dopamine and serotonin, are critical to facilitate understanding brain functions and investigate the information processing in neural networks. In this work, we demonstrated single molecule detection of dopamine and serotonin using a graphene-Au nanopyramid heterostructure platform. The quasi-periodic Au structure boosts high-density and high-homogeneity hotspots resulting in ultrahigh sensitivity with a surface enhanced Raman spectroscopic (SERS) enhancement factor ∼10(10). A single layer graphene superimposed on a Au structure not only can locate SERS hot spots but also modify the surface chemistry to realize selective enhancement Raman yield. Dopamine and serotonin could be detected and distinguished from each other at 10(-10) M level in 1 s data acquisition time without any pretreatment and labeling process. Moreover, the heterostructure realized nanomolar detection of neurotransmitters in the presence of simulated body fluids. These findings represent a step forward in enabling in-depth studies of neurological processes including those closely related to brain activity mapping (BAM).

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.

Bone marrow stromal cell adhesion and morphology on micro- and sub-micropatterned titanium.

The objective of this study was to investigate the adhesion and morphology of bone marrow derived stromal cells (BMSCs) on bulk titanium (Ti) substrates with precisely-patterned surfaces consisting of groove-based gratings with groove widths ranging from 50 micro m down to 0.5 micro m (500 nm). Although it is well known that certain surface patterning enhances osteoblast (bone-forming cell) functions, past studies on cell-pattern interactions reported in the literature have heavily relied on surface patterning on materials with limited clinical relevance for orthopedic applications, such as polymeric substrates. The clinical need for improving osseointegration and juxtaposed bone formation around load-bearing Ti implants motivated this in vitro study. BMSCs were selected as model cells due to their important role in bone regeneration. The results showed significantly greater BMSC adhesion density and more favorable cell morphology on sub-micropatterned gratings when compared with larger micropatterned gratings and non-patterned control surfaces after both 24 hr and 72 hr cultures. We observed increasing cellular alignment and elongation with decreasing feature size. We also identified two distinctive cellular morphologies: Type I-Attached and spread cells that elongated along the pattern axes; and Type II-Superficially adhered round cells. Sub-micropatterned gratings demonstrated significantly greater Type I cell density than the non-patterned control, and lower Type II cell density than the larger micropatterned gratings. Collectively, these results suggest potential for rationally designing nano-scale surface topography on Ti implants to improve osseointegration.

Anodic growth and biomedical applications of TiO2 nanotubes.

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.

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.

In vitro degradation of four magnesium-zinc-strontium alloys and their cytocompatibility with human embryonic stem cells.

Magnesium alloys have attracted great interest for medical applications due to their unique biodegradable capability and desirable mechanical properties. When designed for medical applications, these alloys must have suitable degradation properties, i.e., their degradation rate should not exceed the rate at which the degradation products can be excreted from the body. Cellular responses and tissue integration around the Mg-based implants are critical for clinical success. Four magnesium-zinc-strontium (ZSr41) alloys were developed in this study. The degradation properties of the ZSr41 alloys and their cytocompatibility were studied using an in vitro human embryonic stem cell (hESC) model due to the greater sensitivity of hESCs to known toxicants which allows to potentially detect toxicological effects of new biomaterials at an early stage. Four distinct ZSr41 alloys with 4 wt% zinc and a series of strontium compositions (0.15, 0.5, 1, and 1.5 wt% Sr) were produced through metallurgical processing. Their degradation was characterized by measuring total mass loss of samples and pH change in the cell culture media. The concentration of Mg ions released from ZSr41 alloy into the cell culture media was analyzed using inductively coupled plasma atomic emission spectroscopy. Surface microstructure and composition before and after culturing with hESCs were characterized using field emission scanning electron microscopy and energy dispersive X-ray spectroscopy. Pure Mg was used as a control during cell culture studies. Results indicated that the Mg-Zn-Sr alloy with 0.15 wt% Sr provided slower degradation and improved cytocompatibility as compared with pure Mg control.

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.

Improved bone marrow stromal cell adhesion on micropatterned titanium surfaces.

Implant longevity is desired for all bone replacements and fixatives. Titanium (Ti) implants fail due to lack of juxtaposed bone formation, resulting in implant loosening. Implant surface modifications have shown to affect the interactions between the implant and bone. In clinical applications, it is crucial to improve osseointegration and implant fixation at the implant and bone interface. Moreover, bone marrow derived cells play a significant role for implant and tissue integration. Therefore, the objective of this study is to investigate how surface micropatterning on Ti influences its interactions with bone marrow derived cells containing mesenchymal and hematopoietic stem cells. Bone marrow derived mesenchymal stem cells (BMSC) have the capability of differentiating into osteoblasts that contribute to bone growth, and therefore implant/bone integration. Hematopoietic stem cell derivatives are precursor cells that contribute to inflammatory response. By using all three cells naturally contained within bone marrow, we mimic the physiological environment to which an implant is exposed. Primary rat bone marrow derived cells were seeded onto Ti with surfaces composed of arrays of grooves of equal width and spacing ranging from 0.5 to 50 µm, fabricated using a novel plasma-based dry etching technique. Results demonstrated enhanced total cell adhesion on smaller micrometer-scale Ti patterns compared with larger micrometer-scale Ti patterns, after 24-hr culture. Further studies are needed to determine bone marrow derived cell proliferation and osteogenic differentiation potential on micropatterned Ti, and eventually nanopatterned Ti.