Magnesium biomaterials for orthopedic ... - Wiley Online Library

Review Article Magnesium biomaterials for orthopedic application: A review from a biological perspective Jemimah Walker,1* Shaylin Shadanbaz,1 Timothy B. F. Woodfield,2 Mark P. Staiger,3 George J. Dias1 1

Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand Department of Orthopaedic Surgery, University of Otago, Christchurch, New Zealand 3 Department of Mechanical Engineering, University of Canterbury, Christchurch, New Zealand 2

Received 11 August 2013; revised 22 November 2013; accepted 7 January 2014 Published online 24 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33113 Abstract: Magnesium (Mg) has a long history of investigation as a degradable biomaterial. Physicians first began using Mg for biomedical applications in the late 19th century. Experimentation continued with varying levels of success until the mid-20th century when interest in the metal waned. In recent years the field of Mg-based biomaterials has once again become popular, likely due to advancements in technology allowing improved control of corrosion. Although this has led to success in vascular applications, continued difficulties in predicting and controlling the corrosion rate of Mg in an intraosseous environment has impeded the development of Mg-based biomaterials for orthopedic applications. In this review, an initial summary of the basic properties and the physiological role of Mg are followed by a discussion of the

physical characteristics of the metal which lend it to use as a degradable biomaterial. A description of the historical and modern applications for Mg in the medical field is followed by a discussion of the methods used to control and assess Mg corrosion, with an emphasis on alloying. The second part of this review concentrates on the methods used to assess the corrosion and biocompatibility of Mg-based orthopedic biomaterials. This review provides a summary of Mg as a C 2014 Wiley Periodibiomaterial from a biological perspective. V cals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 1316–1331, 2014.

Key Words: biodegradation, biocompatibility/hard tissue, biocompatibility/soft tissue, orthopaedic, corrosion

How to cite this article: Walker J, Shadanbaz S, Woodfield TBF, Staiger MP, Dias GJ. 2014. Magnesium biomaterials for orthopedic application: A review from a biological perspective. J Biomed Mater Res Part B 2014:102B:1316–1331.

Magnesium (Mg) alloys were first developed as degradable metallic biomaterials for vascular and orthopedic applications in the late 1800s. However, a propensity towards rapid corrosion led to excessive hydrogen production and a premature loss of mechanical strength, resulting in the near abandonment of Mg for biomedical applications until the late 20th century. At this time advancements in alloying, surface treatments, and coating technologies provided the ability to control the corrosion behavior and improve the mechanical properties of Mg, reigniting the field of Mg-based biomaterials. However, despite the successful clinical application of Mg alloys as degradable vascular stents, the development of Mg biomaterials as orthopedic implants is still hampered by unpredictable corrosion behavior and a limited understanding of the tissue response to Mg implants. The following seeks to provide an overview of the fundamentals within the field, and to discuss the various methods adopted to control and assess

both the corrosion and the biocompatibility of Mg-based biomaterials. INTRODUCTION TO MAGNESIUM

As the second most abundant element in the hydrosphere, and the eighth most abundant in the lithosphere, magnesium (Mg) is an element widely distributed throughout the natural world.1,2 Due to the high level of reactivity of the free element, Mg is found throughout the biosphere only as a divalent cation (Mg21), or in salt or mineral form.3 Owing to its relative ubiquity, Mg has been incorporated into a wide range of biological functions in both plants and animals.4 Of particular note is the essential role Mg plays in both the chlorophyll molecule, and in any reaction requiring adenosine triphosphate (ATP), which indicates its particular importance in energy utilization.5,6 As the fourth most abundant element found within the human body, and the most abundant intracellular divalent cation, it is unsurprising that

Correspondence to: J. Walker (e-mail: [email protected])

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Mg is involved in more than 300 known enzymatic reactions.3,7 In addition to its importance in reactions involving ATP, Mg has roles in protein and nucleic acid synthesis, mitochondrial activity and integrity, ion channel modulation, plasma membrane stabilisation and translational processes, as well as many other cellular functions.6–9 Due to the importance of the element, the homeostasis and potential pathological effects of disrupting Mg balance must be understood before exposure of the human body to a potential excess of Mg due to its use as an orthopedic biomaterial.

hypomagnesemia.6 There is an equally broad range of effects of magnesium deficiency, including hypocalcaemia, hypokalaemia, neuromuscular hyperexcitability and cardiovascular dysfunction including hypertension, arrhythmia and myocardial infarction.1,9,12 Long-term Mg deficiency has also been associated with conditions such as atherosclerosis, preeclampsia and osteoporosis.1,6 Fortunately, hypomagnesemia is unlikely to be associated with the implantation of Mgbased biomaterials. However, the effects of Mg deficiency do highlight the importance of the ion in a vast range of functions.

MAGNESIUM PHYSIOLOGY

Hypermagnesemia The use of Mg-based biomaterials is much more likely to result in an excess of stored and circulating Mg which could potentially manifest clinically as hypermagnesemia. The most common cause of hypermagnesemia is impaired renal function resulting in a reduction in the amount of Mg excreted in the urine.4 The acute breakdown of muscle tissue and subsequent renal failure, as is seen with rhabdomyolysis, can also result in an excess of Mg being released into the circulation, which is then not effectively excreted.15 Other causes can be iatrogenic, with inappropriate administration of Mg therapy for conditions such as preeclampsia or drug overdose, although this is a relatively rare occurrence requiring some negligence in the monitoring of serum Mg concentration.1,4 As would be expected, the symptoms of severe hypermagnesemia are often the opposite of those associated with hypomagnesemia. Early symptoms can involve a fall in blood pressure, nausea and mental impairment.1,15 Higher concentrations of circulating Mg are associated with the impairment of the neuromuscular system, with progressive muscle weakness that can result in respiratory failure.4 Additionally, cardiovascular function is impaired with high serum Mg concentrations resulting in further hypotension, conduction failure and bradycardia, which in severe cases can result in cardiac arrest.6,12 The primary treatments for hypermagnesemia involve reducing the intake of Mg, promoting kidney function or using dialysis in cases involving renal inadequacy.1 In the case of corrosion of a Mg-based implant resulting in an excess release of Mg, these treatments may not be entirely appropriate. In mild cases, ensuring a diet low in Mg may improve any appearance of symptoms, however, avoiding the situation would be the ideal solution. Although the body is capable of coping with a relatively wide range of Mg concentrations with normal renal capability, ensuring the material corrodes slowly is the optimal way to ensure hypermagnesemia does not occur. Accordingly, corrosion analysis and the monitoring of serum Mg needs to be an important aspect of assessing Mg-based biomaterials, with more thorough assessment of total Mg necessary at clinically applicable stages of investigation.

Homeostasis An average adult contains approximately 1 mole of Mg (between 21 and 28 g).7,8 Over half of this is sequestered in bone, with approximately 35 to 40% found in soft tissue, and less than 1% in serum.3,6,10 Generally, sufficient Mg is provided by a balanced diet, with both passive and active absorption occurring primarily in the jejunum and ileum of the gastrointestinal system.5,6 Once absorbed, Mg is transported by the circulatory system and taken up as needed by tissues. The influx and efflux of Mg from cells is then strictly regulated to prevent fluctuations in the intracellular concentration that could affect a wide range of reactions.10 An additional homeostatic control is the large concentration of Mg in bone which provides a reservoir of the element for buffering acute changes in serum Mg levels.6,11 However, it is the kidney that is primarily involved in maintaining Mg homeostasis.8,12,13 Approximately 80% of circulating Mg is filtered through the glomerular membrane, with the majority being reabsorbed through the thick ascending loop of Henle.6,13 Only 3 to 5% of the filtered Mg is actually excreted in urine, providing Mg intake is within the normal range.7 If serum Mg concentrations increase, or the kidney is functioning at a reduced level, the amount of Mg excreted can reach almost 100%.12 A number of factors can influence this resorption and excretion, including specific divalent cation sensing receptors in the distal tubule in nephrons that respond to changes in serum Mg concentrations,10 and a range of hormones including parathyroid hormone, calcitonin, vitamin D, and glucagon.7,10 The combination of the regulated intestinal absorption of Mg, use of bone as an exchangeable store of Mg, and the resorption and excretion of Mg by the kidney, allows the effective maintenance of Mg homeostasis over a relatively wide range of exposure to the element.12,14 However, in circumstances where perturbations to these systems cause disruption of this homeostasis, pathological conditions can occur. Hypomagnesemia Mg deficiency can occur for a wide range of reasons. Primary causes are associated with intestinal malabsorption of Mg, or renal disorders resulting in increased excretion of Mg.14 Secondary factors can include poor diet, drug induced renal loss of Mg, alcoholism, or pathological conditions such as diabetes mellitus.6,9,14 However, this is not an exhaustive list, with a large number of other conditions also associated with

THE PHYSICAL PROPERTIES AND APPLICATIONS OF MAGNESIUM

Although Joseph Black discovered elemental Mg in 1755, it was not until 1808 that it was isolated.16 Over the following

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century, Mg was used for a variety of purposes including pyrotechnics, as a reducing agent for the production of aluminum (Al), and in photography.16,17 The metal itself is lightweight with a density of 1.74 g/cm3, has a high strength to weight ratio, is thermally conductive, and can be easily cast.18,19 These characteristics made Mg an appropriate metal for use in a range of applications including the automotive and aeromotive industries.20 Unfortunately, the propensity of Mg to corrode and its low elastic modulus prevented its continued use in a wide range of applications.19 However, it is precisely these characteristics, together with low relative density, which suggest Mg for application as a degradable metallic biomaterial for orthopedic applications. The use of Mg would allow the production of lightweight implants with an elastic modulus much more comparable to bone, than commonly used metallic materials such as titanium alloys and stainless steels (Bone: 3–20 GPa, Mg: 41–45 GPa, Ti alloy: 110–117 GPa, Stainless Steels: 189–205 GPa).21 This, along with the corrosion of the implant, would reduce some of the pathological issues associated with the implantation of permanent metallic materials, such as the production of inflammatory wear particles and osteopaenia.22–25 However, of the features mentioned above, the corrosion of Mg is of primary importance, as the function of a biomaterial is reliant on maintaining appropriate mechanical stability for specific periods of time. It is therefore necessary to understand the mechanisms associated with the corrosion of Mg, and the potential byproducts of this corrosion, particularly in a physiological environment. THE GENERAL CORROSION BEHAVIOR OF MAGNESIUM

The rapid corrosion of Mg and Mg alloys has been the primary limitation in the use of these materials for a range of applications in which there is exposure to a corrosive environment.26 The following reactions encompass the corrosion mechanisms that occur when Mg is exposed to an aqueous environment.27–29 Mg ! Mg21 12e2 2

2H2 O12e ! H2 12OH

ðanodic reaction Þ 2

Mg12H2 O ! MgðOHÞ2 1H2

ðcathodic reaction Þ ðoverall reaction Þ

As can be identified by the overall reaction, the result of Mg corrosion is the production of magnesium hydroxide and hydrogen gas. Under standard environmental conditions, the hydroxide layer that forms on the surface of the material is somewhat protective of corrosion.26,30 However, with exposure to high chloride concentrations such as those seen in a physiological environment, Mg(OH)2 reacts with chloride ions to produce MgCl2, which is highly soluble.21 This promotes the rapid dissolution of the Mg substrate, with the subsequent production of hydrogen gas and hydroxide ions.21 There are two primary types of corrosion that can affect Mg and Mg alloys in a physiological environment. For single-phase materials the corrosion is typically localized,

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which results in the formation of pits on the surface of the material.26 The presence of a secondary phase due to impurities or alloying components results in galvanic corrosion due to the secondary phase acting as a local cathode also resulting in localized corrosion.26 The lack of generalized corrosion is an important factor in the use of Mg as a biomaterial, as extensive areas of local corrosion are likely to result in the mechanical failure of an implant at specific points. This must be taken into account in the investigation of Mg materials for surgical applications. Additionally, due to the corrosion mechanisms occurring, the use of a Mg biomaterial that corrodes too quickly could result in the production of hydrogen gas within the implant environment, and an increase in the local pH, which could both have significant effects on the surrounding tissues. It is these issues that are likely to make the corrosion behavior of Mg-based materials one of the primary influencing factors in their success as orthopedic biomaterials.

HISTORICAL USE OF MAGNESIUM AS A BIOMATERIAL

It was only a short time after the commercialization of Mg production in the mid-19th century that the metal was first used as a biomaterial. Edward C. Huse is credited with the initial use of Mg as a wire ligature to stop bleeding during surgery in three human patients in 1878.31 For the ensuing 50 years numerous physicians would apply Mg and Mg alloy devices to applications in vascular, orthopedic, and general surgery. Of the early pioneers, Erwin Payr was primarily responsible for promoting Mg as a biomaterial with a series of investigations around the turn of the 20th century.32 With varying levels of success he applied Mg to a range of surgical uses including thin tubes for anastomosis, plates for joint arthroplasty, pins and nails for fracture fixation, pegs for intramedullary stabilisation of long bones, plates and sheets for suturing organs after partial resection, and arrow shaped implants for the treatment of hemangiomas.32 Of these, the most effective applications were arguably nonorthopedic, with particular success seen in the treatment of hemangioma and the suturing of organs (particularly liver).32 Other relatively successful vascular and general surgery applications of Mg by several physicians included the use of tubes and ring plates, clips for vessel anastomosis,32 clips for gastrointestinal anastomosis,31 and tubes for ureterorectostomy.33 However, these applications were all suited to quickly degrading devices, for which mechanical integrity was required for minimal time periods, and where the production of hydrogen gas would cause minimal tissue disruption. The application of Mg to orthopedic use had slightly more varied results. Of the earliest uses of Mg in musculoskeletal applications, Albin Lambotte’s were perhaps the most successful. However, his earliest attempt in 1907 to use a Mg plate with steel screws to fix fractures of the tibia and fibula of a 17-year-old boy, failed catastrophically due to galvanic corrosion resulting from the use of the two different metals.34 With the realization that two metallic materials could not be used together, Lambotte undertook a series of animal

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studies which indicated Mg implants alone partially corroded after 3 months, and completely corroded after 7 to 10 months.34 With these experiments indicating appropriate corrosion rates, Lambotte revisited the idea of using Mg for fracture fixation in humans. He used Mg nails and studs for the fixation of supracondylar humeral fractures in several children, and successfully showed total resorption of implants, and healing of the bone 1 year after surgery.32,34 Lambotte’s assistant Jean Verbrugge continued on with this work, investigating Mg implants in both animals and humans. Verbrugge reported the successful use of the material with no adverse reactions with the exception of small volumes of harmless gas.32 Conversely, Ernest Hey Groves investigated the use of Mg intramedullary pegs in animal models in 1913, and did not achieve successful results. He observed excessive callus formation and the rapid corrosion and subsequent disintegration of the implants before adequate healing could occur.35,36 However, these negative results were not enough to dissuade other physicians from applying Mg to orthopedic applications. In the late 1930s, Earl McBride used a Mg alloy containing Al and manganese (Mn) for screws and nails for successful fracture fixation in humans.37 He made several pertinent observations during his studies including that pure Mg did not provide the strength required, and that alloys were preferable in this manner, and that in general Mg-based implants should not be used in areas of high mechanical load.37 He also identified that Mg alloy plates and intramedullary pegs corroded faster than intraosseous implants.32 Additionally, he suggested that while the corrosion of Mg-based materials did result in a tissue reaction, this should not discourage the use of the material.38 However, he did identify that the use of Mg had to be specific to applications in which the advantages of a corrodible material would outweigh the affect on the surrounding tissue.37 Further successful uses of Mg for orthopedic implants in the 1940s included Maier’s use of sheets for humeral fracture fixation, and Troitskii’s use of Mg-cadmium (Cd) alloy plates and screws to treat pseudarthrosis.32 The reason for the waning interest in Mg over the following decades is not explicitly clear. However, it is likely to be due to the variable results observed in the above experiments, which were unquestionably associated with the rapid corrosion of the implant materials utilized. Perhaps, without the ability to accurately alloy Mg, and before the common application of coatings on implants, the corrosion could not be controlled sufficiently to promote its use as a biomaterial. With the exception of a few studies, the field of Mg biomaterials did not regain popularity until the late 1990s. Since then it has been growing exponentially.

applications, and the field devoted to identifying appropriate Mg-based materials for orthopedic application. Of these, the use of Mg alloys as materials for vascular implants has been the most successful. After extensive testing, and with successful use in large animal investigations,39 a Mg alloy stent was used effectively to correct a ligation of the left pulmonary artery in a preterm baby, allowing reperfusion of the lung.40 Subsequent clinical studies have shown further successful use of Mg alloy stents in both babies and adults, indicating complete corrosion of the implants within 4 months.41–44 The development of Mg-based materials for orthopedic use is somewhat further behind. Current research is at a relatively early stage, with large amounts of in vitro work being performed on a variety of materials, with some in vivo preliminary investigations being carried out. However, it is relatively rare for Mg materials to be investigated in either an in vitro or in vivo situation as clinically relevant implants. Of the few who have investigated Mg for such applications, the majority have implanted screws into the long bones of rabbits,45,46 with one study implanting screws into the sheep pelvis.47 In one case, the investigation of the mechanical performance of the implant was relatively well assessed.45 However, in general, analyses of the implant behavior and tissue response have been relatively rudimentary.46,47 Additionally, these implants were not assessed in a functional sense, with no surgical trauma being applied to the bones before their implantation. The likely cause of this fairly minimal investigation of clinically applicable Mg-based implants, is the continuing issues associated with the unpredictable corrosion of the implant materials. In particular, corrosion will compromise the mechanical integrity of the implant, requiring extensive knowledge of the behavior of any material before the implant can be ethically applied to a clinically applicable situation such as fracture fixation. However, once this knowledge is gained there is the potential for Mg materials to not only be applied to screws, but also plates and wires for fracture fixation, as well as the production of Mg scaffolds for applications in which bone grafts have traditionally been used.48,49 Of these, the latter is of particular importance, as the current standard for the replacement of diseased or excised bone is to use autografts.50,51 However, these require a secondary surgical site, and are often associated with significant morbidity at the site of extraction.52,53 Other options such as allografts or xenografts are associated with immunological reactions and pathogen transfer.50,53,54 Therefore, the ability to produce an organized, degradable, porous device with appropriate mechanical properties would provide an excellent alternative. As a candidate material, Mg delivers many of the desired requirements, with initial research assessing the manufacturing processes and initial corrosion rates indicating excellent potential for such materials.55–59

MODERN APPLICATIONS FOR MAGNESIUM AS A BIOMATERIAL

The suggested modern applications for Mg-based biomaterials are not too dissimilar to those that were attempted historically. The two primary fields of investigation are associated with the development of Mg alloys for vascular

IMPROVING THE PERFORMANCE OF MAGNESIUM FOR BIOMEDICAL APPLICATION

Despite significant improvements in the production of Mg since its historical application as a biomaterial, the


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commercially available pure metal cannot provide either the appropriate mechanical properties or corrosion resistance for application in a load bearing orthopedic environment.60– 62 Various techniques are currently being investigated to improve both these features, together with the biocompatibility and osseointegrative potential of Mg as a biomaterial. These primarily include alloying Mg, surface modification of the substrate, or various coating techniques.63–65 The following is an overview of these techniques and the mechanisms by which they improve these features, with particular emphasis on alloying. Alloying The specific composition of an alloy has the ability to affect the ductility, strength, and corrosion properties of the material.66,67 For Mg alloys, improvements in the latter two of these features are primarily associated with changes to microstructural characteristics, in particular a reduction in grain size when compared to pure Mg.63,68 However, the majority of research investigating Mg alloys has been directed towards improving all of these characteristics for industrial purposes.69 Subsequently, a substantial amount of the research into the development of Mg alloys for biomaterials has been conducted on alloys that were originally developed for use in the automotive industry.70 Generally, these alloys contain aluminum (Al) or rare earth elements (REEs).69,71 Whilst these alloys are still being widely investigated for application to the biomedical field, another branch of Mg research has concentrated on the production and assessment of Mg alloys containing arguably non-toxic elements such as calcium (Ca), manganese (Mn), zinc (Zn), and zirconium (Zr). Other alloying components that are being investigated that do not fit in either of these categories include lithium (Li), cadmium (Cd), tin (Sn), strontium (Sr), silicon (Si), silver (Ag), and bismuth (Bi). These alloys can be binary, ternary or more, with both the components and the composition of the alloys contributing significantly to different mechanical properties and corrosion behaviors. The following is a description of the most commonly utilized alloying components and their properties as part of a Mg alloy system. Aluminum. Aluminum (Al) is a common addition to Mg alloys as it is generally accepted to improve both the strength and the corrosion resistance of the metal.61 Whilst the former is largely due to both solid solution and precipitation strengthening,70 the mechanism associated with the improved corrosion resistance is not entirely understood.72,73 However, it does seem to be established that increasing concentrations of Al up to a point reduces the corrosion rate, but too high a concentration reduces the corrosion resistance due to the increased presence of the Mg17Al12 phase, which then promotes galvanic corrosion.73,74 One of the postulated mechanisms for the enhanced corrosion resistance of Al containing Mg alloys, is the production of an Al2O3 layer on the surface of the material, which unlike Mg(OH)2, is insoluble in chloride containing solutions.74 However, regardless of the improvements in the mechanical properties and corrosion resistance of Al

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containing alloys, there can be little doubt that Al is indicated in several pathological conditions in the human.75 Of these the most commonly associated include dementia and Alzheimer’s disease.76–78 Considering these conditions are often associated with environmental exposure to Al, and that the long-term effects of implanting Al containing materials have not been elucidated, it may be unwise to investigate these alloys further as biomaterials before more significant longitudinal studies are carried out.62 Rare earth elements. The rare earth elements (REEs) include the 15 elements in the periodic table between lanthanum (La) and lutetium (Lu), as well as scandium (Sc) and yttrium (Y).79 These are generally added to Mg alloys by way of hardeners or master alloys that are used to increase the strength, ductility, corrosion resistance, and creep resistance of the metal.60,70 Like Al, they can increase the strength by both solid solution and precipitation hardening, and improve corrosion resistance in high chloride environments due to the formation of an oxide rich passivation layer.80,81 However, the issue that arises with the use of the REEs as part of Mg alloys for biomedical applications is their relatively unknown effects on the physiological system.82 Historically, they were identified as having both anticarcinogenic and anticoagulant properties,83 and more recently have been included in a Mg alloy stent used to treat the ligation of a pulmonary artery in a preterm baby without observed side effects.40 At a cellular level, low exposures to a range of the elements do not appear to cause cytotoxicity, with the exception of La and cerium (Ce), with some of the elements even producing a positive effect on cellular viability.79,84 However, higher concentrations do appear to upregulate inflammatory genes, and dosage studies carried out in rats indicate at least some of the REEs are highly toxic.79,85 These mixed results indicate that more research needs to be carried out regarding the biocompatibility of these alloying elements. As has been pointed out by Kirkland (2012), much of the research on Mg alloys containing REEs and Al being carried out currently may be of limited value if these elements are found in the future to be too toxic for use as biomaterials.62 Calcium. Although Ca does not have the strengthening properties of Al or REEs, its inclusion as part of a Mg alloy confers several benefits. It has the ability to reduce the grain size of the alloy, therefore improving the mechanical properties when compared to pure Mg. Ca has also been shown to improve corrosion resistance when included in low quantities (0.6–0.8% being optimal).74,82,86,87 Additionally, Ca is an essential element in the human body, involved in innumerable reactions including an important role in cell signaling, and is a primary component of bone.88,89 It is therefore perhaps unsurprising that Ca is widely investigated as part of binary Mg alloys as well as in combination with other elements for application as orthopedic biomaterials. Manganese. Manganese (Mn) is an essential macronutrient that is only toxic when there are high levels of exposure to


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the element, either through occupation or in patients receiving long-term parenteral nutrition.90,91 Its use as a component of Mg alloys is primarily to improve corrosion resistance by reducing the detrimental effects of impurities such as iron (Fe).26,92 Mn addition can also enhance the ductility of a Mg alloy, and somewhat improve yield strength.70,93 It is often included as part of other alloy systems, particularly those containing Al and Zn.26 Zinc. Of the commonly used alloying components, Zinc (Zn) is second only to Al in its ability to improve the strength of Mg alloys.92,93 Similarly to Mn it also has the ability to reduce the corrosion enhancing effects of all of the common impurities including Fe, nickel (Ni), and copper (Cu).26 Zn is also an important macronutrient for humans, and is involved in a wide range of physiological functions including protein synthesis, immune system regulation and many enzymatic reactions.69,75 However, cytotoxicity has been identified in vitro with exposure of cells to high concentrations of Zn,94,95 and a rare human fatality due to Zn toxicity has been recorded after a patient ingested a large number of coins.96 Although it is unlikely that exposure will reach these levels with the use of a Mg-Zn alloy implant, the potential toxicity should not be completely dismissed. Zirconium. Zirconium (Zr) is primarily added to Mg alloy systems due to its highly effective grain refining ability.61,70 This improves the strength of the alloy, and the corrosion resistance is improved by the precipitation of combined ZrFe particles before an alloy is cast.26,97 Zr is already used in a wide range of medical implants including dental alloys and relatively inert orthopedic implants, and is widely accepted as biocompatible.98,99 Surface treatments and coatings In the biomaterials field, surface treatments or coatings are primarily applied to substrates to improve the biocompatibility of the underlying material.100 However, in the development of Mg as a biomaterial such surface modifications have been investigated primarily to improve the corrosion resistance of the substrate, whether it be pure Mg or Mg alloys.101 As defined by Wang et al. (2012), these modifications can be classified as either chemical, physical or a combination of the two.102 Chemical surface modifications include: acid etching, alkaline treatment, fluoride treatment, anodization, and ion implantation, all of which are associated with replacement of the natively forming, and not particularly corrosion resistant oxide layer on the surface of the Mg substrate.102 Physical, or deposition coatings utilize various techniques to create protective coatings on the Mg substrate that can be organic, inorganic, or metallic in nature and generally provide a physical barrier between the metal and the corrosive environment.20,101,103 Frequently, a combination of both chemical and physical surface modification techniques are investigated, with the initial chemical pre-treatment used to improve the adhesion of the secondary physical coating.102 Arguably, the most commonly researched surface modification technique in the field of Mg biomaterials is the

application of calcium phosphate (CaP) coatings with or without pre-treatment steps.62,101 As well as convincing evidence that CaP coatings can improve the corrosion resistance of Mg-based materials,46,104–107 CaPs are highly biocompatible, are an important precursor for bone growth, and have already been applied to a variety of orthopedic applications.108–113 However, it has been reported that the inadequate control of phase formation, cracking, and poor adherence remain issues within the field.101 Despite these issues with CaP coatings, this technique, and many of the other briefly mentioned surface modifications show promise as a means to control the corrosion of Mg and Mg alloys for application as orthopedic biomaterials. ASSESSING THE CORROSION OF MAGNESIUM-BASED BIOMATERIALS

In vitro methods for the analysis of Mg corrosion As has been previously mentioned, the primary reason Mgbased biomaterials have not been successfully applied to the orthopedic field is the inability to satisfactorily control their corrosion rate. Whilst historically direct animal and human experimentation were used to assess the corrosion behavior of Mg and Mg alloys, implanting untested materials in vivo nowadays would be regarded as unethical. It is therefore important that this parameter be assessed in an in vitro environment to allow the identification of appropriately corroding materials for further analysis as potential biomaterials. Various methods can be adopted to measure the corrosion of Mg, which can be used alone or in combination to approximate the likely corrosion behavior of the material in a physiological environment. In an extensive review of these techniques, Kirkland et al. (2012) categorized these as either unpolarized or polarized methods, referring to the use of an applied voltage for the latter.114 The unpolarized methods involve immersion testing in which samples are submerged in varying volumes and types of solution. The Mg corrosion is then measured by the analysis of released Mg ions, weight loss, or hydrogen evolution. The polarized or electrochemical methods include potentiodynamic polarization, and electrochemical impedance spectroscopy. Each of these techniques is associated with both advantages and limitations for the analysis of Mg corrosion, which will be addressed below. Magnesium ion release. The analysis of released Mg ions into the immersion solution is one of the least utilized methods for measuring Mg corrosion, with only a few studies adopting the technique.64,104,115–119 The general method involves the immersion of a Mg or Mg alloy sample into a solution for extended periods of time. Samples of the immersion solution are then collected at regular intervals, and the ionic concentrations of Mg are measured either by colorimetric techniques (xylidyl blue assay),117 flame atomic absorption spectroscopy,115 or inductively coupled plasma atomic emission spectrometry (ICP-AES).64,104,116,118 With the use of the latter technique, other ions of interest are also measured including the applicable alloying components, Ca, phosphorous (P), and oxygen (O). The benefits of this


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method include the ability to analyze corrosion over time, and to measure the concentration of other ions, allowing inference into some of the corrosion mechanisms occurring. For example, Huan et al. (2010) showed an increase in Mg21 concentration, and a decrease in both Ca21 and phosphate (PO432) in the initial stages of immersion, which then normalized over time.116 They hypothesized this was due to the formation of a surface layer preventing further corrosion and Ca21 and PO432 deposition.116 Whilst observations such as these can be valuable, there are also several interfering factors that can reduce the reliability of such methods. Firstly, if the solution is not refreshed throughout the experimental time period, the pH of the solution will rise due to the production of hydroxide ions, which are associated with the corrosion of Mg. If this increase in alkalinity reaches pH 8.5 or higher, a corrosion resistant stable hydroxide film forms on the surface of the sample preventing further corrosion.18,26,120 As this pH would be unlikely to occur in a physiological environment, this would confound the effects of the corrosion measurement. If this eventuality is taken into account and the solution is at least partially refreshed on a regular basis, the concentration of ions in the solution would be affected by the removal and replenishment of the new solution. Additionally, the analysis of Mg21 in the immersion solution, does not take into account the insoluble corrosion products that may include magnesium compounds.121 Due to these inherent issues with ionic concentration as a measure of Mg corrosion, this technique is not used in isolation. Additional methods are utilized to provide complementary results by analysis of sample volume, mass loss, hydrogen evolution or electrochemical testing.64,115–118 Weight loss. Gravimetric analysis, or the measurement of weight loss, is a commonly utilized technique for the assessment of corrosion in the field of Mg biomaterials, and is used either alone or in combination with other methods.64,65,69,92,122–144 Its popularity is due to both its relative simplicity and reliability.145 The general methodology is similar to that described above for ion release analysis. A sample of known weight is immersed in a specified volume of solution, and at predetermined time points the samples are removed, the corrosion products are dissolved, and the samples are dried and weighed. The mass loss is then taken as a measure of corrosion itself, or used to calculate the corrosion rate. The benefits of the weight loss method for the analysis of Mg corrosion lie in both the minimal requirements for specialized equipment, and in the ability to gain accurate results provided good experimental techniques are adopted. This requires the inclusion of adequate replicates, the use of an accurate microbalance and the effective removal of corrosion products.114 The latter point is of particular importance, as the ineffective removal of corrosion products will lead to a reduced indication of the extent of corrosion. In contrast, the overzealous removal of corrosion products has the potential to degrade the remaining substrate, providing inaccurately high corrosion results.145 To achieve the effective removal of these corrosion products,

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the samples are generally submerged in a chromic acid solution,64,65,69,92,122,127,130–132,135–139,141 although occasionally hydrofluoric acid is utilized.45,146,147 Both these acids are known to minimally affect Mg-based biomaterials, and therefore do not corrode the remaining substrate.148,149 Occasionally, this step is omitted entirely, which generally results in a net gain in weight, or a negative corrosion rate being calculated.124–126 A further limitation to the weight loss technique is in its inability to provide information on the mechanisms of corrosion or how the corrosion behaviors alter over time.114,121,145 The results can only provide an overview of the amount of corrosion that has occurred at individual time points.145 However, it is unlikely that any corrosion mechanism occurring in an in vitro test would exactly replicate that which would occur in an in vivo environment. Therefore, it could be argued that the corrosion rate is the important factor, not the mechanism, if the aim of using the in vitro method is to allow the selection of appropriately corroding materials for further in vivo analysis. Hydrogen evolution. The analysis of hydrogen evolution as a measure of Mg corrosion is a technique that has become increasingly popular over the last four years.75,82,88,89,116,121,123,128,135,141,150–158 Although not as widely used as weight loss, the technique requires a similarly simple experimental setup. The general method utilizes an upturned funnel attached vertically to a burette, placed over a sample submerged in an immersion solution.159 As the Mg corrodes, hydrogen gas (H2) is produced and displaces the solution in the burette, allowing measurement of the volume of evolved gas.114 This technique allows the assessment of Mg corrosion, as theoretically 1 mole of H2 is produced with the corrosion of one mole of Mg metal.28,145 The benefit of this technique over the weight loss method is the ability to assess the corrosion of Mg over time.156 However, there are several experimental limitations that require consideration when utilizing this technique. First, differences in the environment including atmospheric pressure and temperature can markedly affect the volume of H2 measured.70,114 Second, an unknown portion of the produced H2 will be dissolved into the immersion solution, and therefore cannot be measured.150 Third, there is the potential for gas to escape the apparatus if not sufficiently assembled, or if plastic components are used instead of glass, as plastics can be permeable to H2.114 Despite the potential shortcomings of this technique, it can still provide results indicative of corrosion behavior, and can therefore be a valuable method when used in conjunction with others such as weight loss or electrochemical testing. Electrochemical methods. The two electrochemical techniques most commonly used in the investigation of Mg and Mg alloy corrosion are potentiodynamic polarization (PDP), and electrical impedance spectroscopy (EIS). Both involve the use of Mg as the working electrode, alongside a reference and counter electrode, which are all immersed in solution. PDP is a direct current (DC) technique which involves


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the measurement of current when voltage is applied at a steady rate, both in anodic and cathodic directions.160,161 The resultant curve can be used to calculate the corrosion current density (and therefore the corrosion rate), as well as imparting some information on the anodic and cathodic reactions of the material of interest.114 In contrast, EIS uses an alternating current (AC) to provide information on the frequency-dependant impedance at the surface of a material.162,163 This can be used to calculate corrosion resistance, analyze the formation of surface layers, and provides more detailed information on several other aspects of the corrosion mechanisms occurring that can not be revealed with PDP.163 For this reason the use of EIS has recently become increasingly popular,115,164–167 although PDP is still the most commonly utilized electrochemical method in the field of Mg biomaterials.46,60,64,69,71,75,82,88,92,93,125,126,129,131,136,137,142,151,154,168–173 However, both techniques are often used within a single study to provide complementary results regarding corrosion behavior.63,80,127,132,143,144,155,157,174–184 The primary advantage in utilizing these electrochemical techniques rather than the immersion methods described above is the ability to gain instantaneous data on the corrosion rate of the materials, while also elucidating some of the mechanisms resulting in this corrosion behavior.145,161 However, it remains unclear as to whether these mechanisms are relevant to the corrosion that occurs in an in vivo environment. There is also some disagreement within the research community as to whether these techniques can provide accurate data pertaining to the actual corrosion of Mg-based materials. This is due to an occurrence known as the negative difference effect (NDE). The NDE for Mg is associated with a tendency towards an apparent increase in both the anodic and cathodic reactions when potential is applied, whereas for most metals the anodic reaction would increase whilst the cathodic would decrease.28,185 The mechanisms postulated to explain this occurrence include: the breakdown of a protective surface film, particle undermining resulting in disintegration of the working electrode, the potential production of monovalent Mg ions, and the production of magnesium hydride (the last two of which result in the production of H2 chemically, rather than being due to an increase in the cathodic reaction).121,186,187 These can then result in mass loss from the material being investigated, which is not taken into account with the electrochemical interpretation of corrosion rate.121 It can therefore be argued that whilst the electrochemical methods can provide valuable data on corrosion mechanisms, they cannot provide a corrosion rate relevant to those that would occur with long term in vivo implantation. Overview of the in vitro techniques for the assessment of Mg corrosion. Each of the described methods can provide valuable insights into certain aspects of Mg corrosion behavior. However, there is one aspect common to all of these techniques that must be taken into account. All of the methods involve the immersion of the material of interest into a solution, whether as a sample or an electrode. It is the variables associated with the choice of this solution that can

drastically affect the results pertaining to both the corrosion rate and the corrosion mechanisms occurring. These include the control and maintenance of pH, temperature, solution composition, the time points investigated, the size of the samples, the volume of solution, and the use of agitation or flow (the latter being of importance for immersion testing, not electrochemical methods). The potential for these variables to affect the corrosion behavior of Mg and Mg alloys is now widely accepted within the research community, with a number of articles published specifically investigating these environmental effects.18,26,70,74,117,131,135,140,154,156,157,169,178,188–191 Consequently there has been a progression towards using solutions that are deemed to be more physiologically relevant, so as to be more applicable to the in vivo environment the biomaterials are intended for. This can include appropriate pH (7.4–7.6), temperatures of 37 C, physiologically applicable ionic concentrations, the inclusion of vitamins, amino acids, and proteins within the test solution, the use of a dynamic environment and justification for the sample size and solution volume used. With the additional variable of the specific Mg material being investigated, there are very few studies available for which the results of corrosion analysis in vitro can be directly compared to another. Additionally, it is not currently known whether or not these modifications to the techniques actually improve the comparability of the results to those that occur in vivo. This is primarily due to only very few investigations directly comparing the corrosion behaviors of Mg-based materials in vitro to those in vivo.88,125,126,143 Therefore, a systematic analysis of the effects of different environmental parameters on the in vitro corrosion of Mg-based materials in reference to results from in vivo analysis, would provide a basis for standardized testing parameters. In vivo methods for the analysis of Mg corrosion The techniques utilized for assessing the corrosion of Mg after in vivo implantation are somewhat less complicated than the in vitro methods described above. All of the techniques are associated with measuring the physical reduction in size of the materials due to the corrosion that has occurred over the implantation period. The most common of these methods is the analysis of the remaining volume of the implanted material using micro-computed tomography (l-CT).45,64,106,107,138,146,147,151,192,193 This is generally performed on explants of tissue containing an implant that have been fixed and embedded in resin. One primary advantage of this technique is that it is nondestructive, allowing both the implant and surrounding tissue to remain intact. The result is that a range of other analyses can be carried out on the same sample, including the assessment of new bone growth, bone-implant contact, and osseointegration with the obtained l-CT data.64,193 Additionally there is the opportunity to cut sections of the samples after l-CT imaging for further histological analysis. The only disadvantage with this technique is the specialized equipment necessary for both the l-CT, and the cutting of resin embedded samples containing metallic implants should histological analysis be carried out.


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Another method that is utilized to analyze the corrosion of Mg materials in vivo is the measurement of the weight loss of the implant.45,88,143,149,173,194 The benefit in using this technique is both the potential to compare results directly with weight loss data gathered from in vitro studies, and the minimal requirements for specialist equipment. However, the necessity to remove the implant from the surrounding tissue eliminates the opportunity to investigate the implant-tissue interface. Additionally, the shear forces required to mechanically dislodge an implant are likely to damage the tissue that had surrounded the material, removing the potential for analysis of this tissue. This is of particular importance with the intraosseous investigation of Mgbased materials where osseointegration could make implant removal difficult, but is possibly less of an issue with soft tissue or intramedullary implantation studies. The only other quantitative method used to assess Mg corrosion in vivo, is the analysis of the remaining crosssectional area of the implants in two-dimensional sections.119,195–197 These results are then compared to the original size of the implant, allowing an indication of the corrosion occurring. This technique is generally associated with transverse sections of the implant being cut with the associated surrounding tissue, allowing some analysis of biocompatibility. However, the limitation with this method is the assumption that corrosion of the implant is occurring uniformly throughout the tissue the material is implanted in, whereas realistically, an orthopedic device is likely to be exposed to multiple tissue types, which for Mg would result in different corrosion rates.151 ASSESSING THE BIOCOMPATIBILITY OF MAGNESIUMBASED BIOMATERIALS

In vitro methods for the analysis of biocompatibility The in vitro analysis of the biocompatibility of potential biomaterials is an important initial step in determining the potential safety of a material for implantation in vivo. In the Mg field, there are two primary techniques for assessing this biocompatibility. The first is to assess the compatibility of the materials with blood, most commonly with hemolytic assays,46,93,115,127,170,173 but also through the investigation of coagulation and platelet aggregation.75,198 However, these methods are often (although not exclusively) utilized in the development of Mg-based materials for cardiovascular applications such as stents. For the investigation of Mg-based materials for orthopedic applications, the in vitro analysis of biocompatibility is predominantly carried out using cell culture. The majority of cell culture studies are carried out using one of two methods adapted from international standards (ISO 10993-5 and ISO 10993-12).199,200 The first is an indirect assay in which cultured cells are exposed to an extract of the material of interest for variable lengths of time. The extracts are prepared by immersion of the material in an applicable cell culture media for between 24 and 72 h. The intent is that the viability of the cells exposed to the extracts can then be measured to assess the cytotoxic or proliferative effects of the materials. This is a popular method within the

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Mg field, and is most commonly associated with the use of colorimetric viability assays in which the metabolic activity of a living cell reduces a tetrazolium salt to a colored formazan product.75,88,116,122,123,129,138,143,166,170,173,181,198,201,202 This theoretically allows the quantification of the number of living cells if appropriate controls are used, or at the very least provides the ability to compare the cytotoxic or proliferative potential of materials to one another within an experiment. Interestingly, the use of tetrazolium based assays has remained popular despite research indicating that Mg ions interfere with the assay by promoting the conversion to formazan.203 This is of particular importance when the toxicity of a Mg-based material is in question, as this would result in higher cellular viability being reported than actually occurred. Although there is some indication that thorough washing of the cells before the application of the assay can mediate this effect,202 this step is rarely mentioned. Additional issues associated with this indirect method are related to the by-products of Mg corrosion. If the extracts are prepared and used in accordance with the ISO 10993-12 standard, the resultant solutions would likely be associated with a toxic osmolality and pH increase that would result in minimal viability.202 This is often avoided within the literature by diluting the extraction media before the application to cells, or by increasing the solution volume compared to the size of the sample, effectively achieving the same result.88,129,138,143,166,198 The second method that has been adapted from the international standard (ISO 10993-5) is a technique involving direct contact between the material of interest and the cultured cells. This most commonly involves the growth of cells directly on the material, and as with the extract method, is often used to assess cellular viability. However, as colorimetric methods require the quantification of absorbance of the solution, such assays cannot be used when a sample is present within the culture environment. Whilst several studies have detached the cells before quantification,110,204 the majority use techniques that allow the visualization of the cells adhered to the Mg samples. The most popular of these techniques within the Mg field involves the use of fluorescent dyes such as calcein, ethidium homodimer, DAPI (4’,6-diamidino-2-phenylindole dilactate), alexa red phalloidin, and Hoechst 33342, in association with fluorescent microscopy.81,82,164,205,206 Another involves fixing and drying the cells and sputter coating the samples with gold (Au) or carbon (C), and visualizing them using scanning electron microscopy (SEM).104,109,124,170 The number of cells adhered can then be quantified or the morphology can be assessed giving an indication of the biocompatibility of the material. Both the indirect and direct methods described above can be used to quantify the viability of cells. These techniques are used with many cell types including both adherent primary cells (cultured directly from living tissue) and adherent cell lines (immortalized or cancerous cells grown continuously). These can be either human or animal in origin, and can be derived from various tissue types. Primary endothelial cells, or endothelial cell lines are often utilized


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for the investigation of Mg-based materials intended for cardiovascular applications.82,166,207 Fibroblastic cell lines are used as a general tool for the analysis of the biocompatibility of Mg materials for any biomedical applications.88,109,138,143,171,181 However, if the material is being investigated specifically for orthopedic use, osteoblastic cell lines or primary bone marrow stromal cells are commonly used.81,104,110,116,124,129,144,170,173,198,204,208,209 This is because they not only allow the analysis of viability, but also the investigation of specific cellular responses that could indicate the potential effects of Mg-based materials on bone tissue. Most commonly this involves the analysis of alkaline phosphatase (ALP) activity as an indicator of osteoblastic differentiation,104,110,124,209 but several studies have also used the reverse transcription-polymerase chain reaction (RT-PCR) to identify the expression of osteoblastic differentiation markers such as osteopontin, bone sialoprotein, osteocalcin and pre-procollagen type I.110,209 As with the in vitro corrosion techniques previously discussed, the difficulty with the investigation of the biocompatibility of Mg-based biomaterials in vitro is the lack of comparability between studies. A large number of variables can affect the results gathered including choice of technique, cell type, and the time points investigated. Additionally, it is likely that for many of the Mg-based materials being investigated, any cytotoxicity identified with in vitro methods is likely to be associated with the increased osmolality and pH that are associated with the corrosion of the materials. This adds further variables to the biocompatibility analysis of Mg materials in the choice of solution volume and whether the pH is adjusted. Therefore, whilst these in vitro biocompatibility methods can be used to indicate broadly whether or not a material is toxic, it is very difficult to extrapolate the data to an in vivo environment. In vivo methods for the analysis of biocompatibility The previously described in vitro methods for assessing biocompatibility are a necessary initial step in the investigation of materials for biomedical applications. These techniques provide information on general toxicity as well as the opportunity to avoid the in vivo implantation of potentially toxic materials.210 However, these in vitro techniques cannot reproduce the complex environment a biomaterial will be exposed to when implanted in a physiological environment, necessitating the use of in vivo investigations. The techniques used for assessing the in vivo response to a biomaterial are associated primarily with identifying the tissue reaction occurring in the area in direct contact with, and surrounding the implant. In the field of Mg biomaterials there are a diverse range of animal models and methods utilized to investigate this tissue reaction. A technique that is used relatively rarely within the field is the implantation of Mg-based biomaterials in a subcutaneous or intramuscular environment.126,138,151,166,194 This is primarily due to the preferred use of intramedullary or intraosseous locations when the material is being developed for orthopedic use. However, in addition to being a standard and widely accepted technique for initial in vivo

investigations,211–213 a soft tissue environment has several benefits specific to the use of Mg. Firstly, the implant can be easily removed, and therefore assessed for corrosion behavior. Secondly, soft tissue is likely to be much more forgiving with the inevitable production of hydrogen gas. This is of particular importance when the in vivo corrosion behavior of a material is yet to be determined. In the studies in which Mg-based materials have been investigated in a soft tissue environment, the biocompatibility has been investigated with the use of histological stains such as hematoxylin and eosin (H&E),126,138,166,194 Masson-Goldner trichrome,151 and pentachrome.166 These allow the identification of a range of cell types and tissue features, which are then used to provide a qualitative account of the tissue response, including any inflammatory reaction or neovascularization in response to the implantation of the material of interest. Several other investigations have used similar techniques to analyze the soft tissue reaction to Mg implants by specifically removing the muscle and connective tissue overlying an intraosseous implantation site.86,173 As has been previously mentioned, the investigation of Mg-based biomaterials in either an intramedullary or intraosseous location is a technique much more commonly utilized within the field than soft tissue implantation. As with subcutaneous and intramuscular investigations, the predominant method for analyzing the tissue reaction to the material relies on the use of histological techniques. These can be used to indicate inflammation and neovascularization, in addition to orthopedic specific responses such as new bone formation. However, the analyses of these reactions are completed to varying standards within the Mg field. The most minimal involve the qualification of the tissue response, with a description of the microscopic appearance that can range from several sentences to a very detailed account of the reaction to the implanted material.64,88,104,106,107,143,151,170,196,197,214 This is often the case when the investigation of biocompatibility is not the primary aim of the experiment, but rather the emphasis is placed on the analysis of corrosion behavior. More thorough investigations of the tissue response to Mg-based biomaterials involve the use of semiquantitative scales for the analysis of biocompatibility.193,215–218 These are often adapted from the scoring system outlined in the international standard for the assessment of local effects after implantation (ISO 10993-6).212 Unfortunately, the majority of studies within the Mg field rely on brief qualitative descriptions, with only relatively few investigators adopting the use of these semiquantitative scales. The most thorough microscopic investigations of the tissue response to implants involve the use of immunohistochemical techniques to categorically identify specific cell types or proteins within the tissue. These can include osteoclasts, bone morphogenic proteins, and growth factors.109,193 However, these techniques are time consuming, expensive, and require the use of antibodies for a wide range of antigens in order to provide a comprehensive view of the tissue reaction occurring. There are also additional methods that are used for the analysis of certain aspects of the tissue response to Mg


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implants including hydrogen gas production and new bone formation. Many studies use radiography throughout the experimental period and before euthanasia as a tool to identify gas pockets within the soft tissue associated with the implants.64,143,147,214,219 Visualization techniques such as lCT are used to identify the structure of the bone tissue surrounding intraosseously implanted materials, or to quantify bone-implant contact and new bone growth at the perimeter of the implantation site.64,104,147,149,193 Several studies have also injected fluorescent vital dyes such as calcein green and xylenol orange throughout the experimental period to investigate the mineral apposition rate associated with bone ingrowth as the Mg implant corrodes.195,220 These techniques can all provide additional information on the local effects of the implantation of Mg-based biomaterials. Additional methods are also used to assess the general toxicity of the implanted materials. Most commonly this involves the analysis of serum for the presence of Mg ions or alloying components,64,88,107,138,214 but can also be used to measure general markers for the health of the animal (such as serum creatinine, blood urea nitrogen, and cell counts).194,196,197 Histological samples of visceral organs have also been analyzed to identify any systemic pathology that could be due to the implantation of Mg-based biomaterials.138,143,151 As with the corrosion analysis and in vitro biocompatibility of Mg-based biomaterials, the in vivo analysis of biocompatibility is an area in which experimental variables and differing methodologies make the comparison between investigations difficult. Additionally, the importance placed on assessing corrosion behavior in the largely engineering dominated field leads to decreased emphasis on the appropriate analysis of biocompatibility. Too many investigations are carried out in vivo only to provide qualitative descriptions of the resultant tissue response. However, the increasing popularity of semiquantitative scales is a promising sign within the field. CONCLUSION

There is significant promise in the field of biomaterials for the identification of Mg alloys that could be applied as orthopedic implants. The benefit of a relatively high strength material for which the corrosion could be tailored according to the specific application is clear. Additionally, the clinical precedent for the use of such materials has already been set by the recent successful use of Mg alloys as vascular stents. However, the field is plagued by the difficulties in both controlling and assessing the corrosion and biocompatibility of Mg-based biomaterials for orthopedic applications. For there to be success, two factors are of vital importance. Firstly, the development of a set of standardized protocols for both corrosion and biocompatibility assessment would allow the comparison of materials between experimental groups. This would vastly increase the volume of comparable data, preventing excessive repetition of experimental testing. Secondly, it is imperative that the field encourages more extensive collaboration with clinicians, allowing the design and development of the materials at the

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earliest stages for specific clinical uses. Only then can the characteristics of a Mg-based biomaterial truly be tailored to a functional end point, rather than fitting an appropriate application to the characteristics of a material. REFERENCES 1. Rude RK. Magnesium. Modern Nutrition in Health and Disease, 10th ed. Baltimore: Lippincott Williams and Wilkins; 2006. pp 224–47. 2. Wolf FI, Cittadini A. Chemistry and biochemistry of magnesium. Mol Aspects Med 2003;24:3–9. 3. Maguire ME, Cowan JA. Magnesium chemistry and biochemistry. Biometals 2002;15:203–210. 4. Rude RK, Singer FR. Magnesium deficiency and excess. Annu Rev Med 1981;32:245–259. 5. Wester PO. Magnesium. Am J Clin Nutr 1987;45(5 Suppl): 1305–1312. 6. Touyz RM. Magnesium in clinical medicine. Front Biosci 2004;9: 1278–1293. 7. Saris NE, Mervaala E, Karppanen H, Khawaja JA, Lewenstam a. Magnesium. An update on physiological, clinical and analytical aspects. Clin Chim Acta 2000;294:1–26. 8. Quamme GA, De Rouffignac C. Epithelial magnesium transport and regulation by the kidney. Front Biosci 2000;5:D694–D711. 9. Gums JG. Magnesium in cardiovascular and other disorders. Am J Health Syst Pharm 2004;61:1569–1576. 10. Vormann J. Magnesium: Nutrition and metabolism. Mol Aspect Med 2003;24:27–37. 11. Spiegel DM. Magnesium in chronic kidney disease: Unanswered questions. Blood Purif 2011;31:172–176. 12. Topf JM, Murray PT. Hypomagnesemia and hypermagnesemia. Rev Endocr Metab Disord 2003;4:195–206. 13. Quamme GA. Renal magnesium handling: New insights in understanding old problems. Kidney Int 1997;52:1180–1195. 14. Laires MJ, Monteiro CP, Bicho M. Role of cellular magnesium in health and human disease. Front Biosci 2004;9:262–276. 15. Allan R, Mara N. Magnesium and the acute physician. Acute Med 2012;11:3–7. 16. Friedrich H, Mordike B, editors. Magnesium Technology Metallurgy, Design Data, Applications. Berlin: Springer-Verlag; 2006. 17. Baker H, Avedesian MM; ASM International. Handbook Committee. Magnesium and Magnesium Alloys. ASM Specialty Handbook. Materials Park, OH: ASM International; 1999. pp ix, 314. 18. Ghali E, Dietzel W, Kainer KU. General and localized corrosion of magnesium alloys: A critical review. J Mater Eng Perform 2004; 13:7–23. 19. Mordike BL, Ebert T. Magnesium—Properties, applications, potential. Mater Sci Eng 2001;302:37–45. 20. Gray JE, Luan B. Protective coatings on magnesium and its alloys—A critical review. J Alloys Comp 2002;336:88–113. 21. Staiger MP, Pietak AM, Huadmai J, Dias G. Magnesium and its alloys as orthopedic biomaterials: A review. Biomaterials 2006;27: 1728–1734. 22. Geetha M, Singh A, Asokamani R, Gogia A. Ti based biomaterials, the ultimate choice for othopaedic implants—A review. Prog Mater Sci 2009;54:397–425. 23. Gallo J, Goodman SB, Konttinen YT, Raska M. Particle disease: Biologic mechanisms of periprosthetic osteolysis in total hip arthroplasty. Innate immunity. 2013;19:213–224. 24. Wang J, Yu W, Sandhu H, Betts F, Bhuta S, Delamarter R. Metal debris from titanium spinal implants. Spine 1999;24:899. 25. St. Pierre C, Chan M, Iwakura Y, Ayers D, Kurt-Jones A, Finberg R. Periprosthetic osteollysis: Characterizing the innate immune response to titanium wear-particles. J Orthop Res 2010;28:1418–1424. 26. Song GL, Atrens A. Corrosion mechanisms of magnesium alloys. Adv Eng Mater 1999;1:11–33. 27. Atrens A, Liu M, Zainal Abidin NI. Corrosion mechanicsm applicable to biodegradable magnesium implants. Mater Sci Eng B 2011; 176:1609–1636. 28. Song G, Atrens A. Understanding magnesium corrosion: A framework for improved alloy performance. Adv Eng Mater 2003;5: 837–58.


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