- Email: [email protected]
The modification of microstructure to improve the biodegradation and mechanical properties of a biodegradable Mg alloy
journal of the mechanical behavior of biomedical materials 20 (2013) 54 –60
Available online at www.sciencedirect.com
www.elsevier.com/locate/jmbbm
Research Paper
The modification of microstructure to improve the biodegradation and mechanical properties of a biodegradable Mg alloy Hyung-Seop Hana, Yin Minghuia, Hyun-Kwang Seoka, Ji-Young Byunb, Pil-Ryung Chac, Seok-Jo Yangd, Yu Chan Kima,n a
Center for Biomaterials, Korea Institute of Science & Technology, Seoul 136-650, South Korea Materials/Devices Division, Korea Institute of Science & Technology, Seoul 136-650, South Korea c School of Advanced Materials Engineering, Kookmin University, Seoul 136-702, South Korea d Department of Mechatronics Engineering, Chungnam National University, Deajeon 305-764, South Korea b
ar t ic l e in f o
abs tra ct
Article history:
The effect of microstructural modification on the degradation behavior and mechanical
Received 15 October 2012
properties of Mg–5 wt%Ca alloy was investigated to tailor the load bearing orthopedic
Received in revised form
biodegradable implant material. The eutectic Mg/Mg2Ca phase precipitated in the as-cast
5 December 2012
Mg–5 wt%Ca alloy generated a well-connected network of Mg2Ca, which caused drastic
Accepted 17 December 2012
corrosion due to a micro galvanic cell formed by its low corrosion potential. Breaking the
Available online 28 December 2012
network structure using an extrusion process remarkably retarded the degradation rate of
Keywords:
the extruded Mg–5 wt%Ca alloy, which demonstrates that the biocompatibility and
Magnesium
mechanical properties of Mg alloys can be enhanced through modification of their
Extrusion
microstructure. The results from the in vitro and in vivo study suggest that the tailored
Microstructure
microstructure by extrusion impede the deterioration in strength that arises due to the
Biodegradation
dynamic degradation behavior in body solution.
Mechanical properties
1.
Introduction
Over the past two decades, bioabsorbable polymeric devices have been utilized in many aspects of orthopaedic surgery. Since bioabsorbable implants have the advantage of dissolving in a biological environment after a certain period of functional use, these represent an appropriate solution by means of their cost, convenience, and aesthetic reasons, all of which are favorable to patients. However, these biodegradable polymers have relatively low strength and an n
Corresponding author. Tel.: þ82 29585457; fax: þ82 29585449. E-mail address: [email protected] (Y.C. Kim).
1751-6161/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jmbbm.2012.12.007
& 2012 Elsevier Ltd. All rights reserved.
unpredictable degradation rate, resulting in sudden collapse during their degradation in a living body (Hofmann, 1995; Claes and Ignatius, 2002). Moreover, biodegradable polymers decrease the pH value of body fluid upon dissolution, which is not desirable for the promotion of bone growth (Bo¨stman and Pihlajama¨ki, 2000). Recently, the use of magnesium as a biodegradable metal has been considered as a promising biomedical material due to its unique properties, namely: a larger fracture toughness than that of ceramics and an elastic modulus closer to that of
journal of the mechanical behavior of biomedical materials 20 (2013) 54 –60
natural bone compared with other commonly used metallic implants (Staiger et al., 2006). Therefore, Mg alloys can minimize or bypass the ‘‘stress-shield’’ phenomenon which exists in the current metallic implants made of stainless steel or titanium alloys (Staiger et al., 2006).Moreover, as the fourth most abundant cation, magnesium is essential to human metabolism, and is an essential nutrient in the human body. In particular, it has important influences on nerve, muscle, bone and heart function (Saris et al., 2000). Magnesium is a degradable (or bioabsorbable) metal in the environment of the human body because its highly negative standard electrode potential induces much faster corrosion than is the case for other metallic materials, thus rendering additional surgical operations for its removal unnecessary. However, the intense chemical activity of magnesium would lead to an upsurge of corrosion in corrosive media containing Cl (Inoue et al., 2002; Lee et al., 2009), which can cause the plethoric production of hydrogen gas during in vivo degradation (Staiger et al., 2006; Levesque et al., 2003). Recently, magnesium alloys, such as AZ31, AZ91, WE43, and LAE442 have been investigated for the purpose of improving the corrosion resistance of magnesium for bone implant applications (Witte et al., 2006, 2005; Duygulu et al., 2007). However, the above magnesium–aluminum or magnesiumrare-earth alloys are potentially toxic because aluminum has been reported to be related to brain disease and Alzheimer’s disease (Saris et al., 2000), and rare-earth elements, such as yttrium, may cause liver toxicity. Therefore, it is necessary to develop a new magnesium alloy for biomedical application which has good biocompatibility, good corrosion resistance and good mechanical properties. In particular, it is desirable that such beneficial properties are achieved with the addition of the fewest trace elements possible. Calcium may be one of the best alloying elements to develop a Mg based biodegradable alloy for orthopaedic applications (Kim et al., 2008; Wan et al., 2008; Li et al., 2008) because it is a major component of human bone and essential in chemical signaling between cells. Nie and Muddle (1997) suggested that the Mg–Ca system has good potential in the sense that it has a useful combination of strength, creep resistance, castability, corrosion resistance, and cost because the equilibrium inter metallic phase (Mg2Ca) is structurally analogous with the magnesium matrix. Recently, Li et al. (2008) reported that only Mg alloy with Ca content less than 3 wt% was available for bioabsorbable implants because Mg alloys with larger Ca content were very corrosive and brittle, in spite of their relatively high strength. Previously, most research has involved changing the alloying elements or adjusting the amount of alloying elements to improve the mechanical and corrosion properties of Mg alloys. In this study, we propose a new route to improve both the mechanical properties and biodegradability of a Mg–Ca alloy; i.e., tailoring the microstructure. We achieved good corrosion resistance in a Mg–Ca alloy by tailoring the microstructure, even for a Ca content of up to 5 wt%. We revealed the relationship between the rapid corrosion of Mg–Ca alloys in a simulated body fluid (SBF) and its microstructure, and modified the microstructure to improve the corrosion, biocompatibility, and mechanical properties based on the identified relationship.
2.
Materials and methods
2.1.
Material preparation
55
A binary Mg–5 wt%Ca alloy was prepared by melting and casting under an Ar atmosphere in a vacuum/inert gas atmosphere furnace. Before the Ar gas was introduced to the furnace, the oxygen and moisture in the furnace were removed by applying a vacuum of 103 torr. A STS430 crucible filled with high purity Mg (99.98%) and Ca (99.95%) was kept at 850 1C for 120 min in order to melt the alloy completely. The molten alloy was cast into a STS430 steel mold after stirring for 5 min. The as-cast ingots were preheated for some hours before extrusion, and then extruded into a rod at about 350 1C with an extrusion ratio of 25:1, and cooled in air.
2.2.
Mechanical test
Mechanical properties were evaluated by compression testing. Casting samples were machined according to a f 3 6 mm cylinder shape compression system with a strain rate of 0.6 103 s1. In order to deteriorate strength as arises through dynamic degradation behavior, compression tests were performed on the sample after adequate immersion testing. At least four samples were tested for each condition.
2.3.
Immersion test
For the immersion tests, the specimens were put in a beaker containing SBF. Before the test, the pH was adjusted to 7.4. The degradation rate of the samples was estimated by measuring the amount of generated hydrogen with a funnel. After the immersion tests, the corrosion morphology was analyzed with a Scanning Electron Microscopy (SEM, HITACHI S4200) and X-ray diffractometry (XRD, Bruker D8).
2.4.
In vitro biocompatibility test
Biocompatibility for cytotoxicity was evaluated by in vitro cell experiments. An MEM serum free solution was used as the extraction medium and the extracts were prepared with a surface area to extraction medium ratio of 0.7 cm2/ml under the conditions of 37 1C and 5 vol% CO2 in humidified air for 1–7 days, respectively. The extracts were collected, centrifuged, and filtered before storing at 4 1C prior to the cytotoxicity test. Mouse fibrosarcoma L929 cells and Human osteoblast-like HOS cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin at 37 1C in a humidified atmosphere of 5% CO2. In vitro cytotoxicity tests were carried out by indirect contact as suggested by the ISO 10993-5 standards. A MEM medium, and MEM medium containing 0.64% phenol, acted as negative and positive controls, respectively. Cells were incubated in 96-well flatbottomed cell culture plates with 5 103 cells/100 ml medium in each well, and were incubated for 12 h to allow attachment. All wells were aspirated and 100 ml of extracts and controls (positive and negative) were added; thus 3 replicates for each sample. All wells were then incubated for 24 h. After
journal of the mechanical behavior of biomedical materials 20 (2013) 54 –60
incubation, XTT assay was carried out by using CCK-8 kit (Dojindo/Japan) according to the manufacturer’s directions.
2.5.
In vivo biocompatibility test
This study was approved by the Animal Care and Use Committee of Chungnam National University and eight adult male Sprague Dawley rats weighing approximately400 g were used (Orient Bio, Gyunggi, South Korea). Rats were anesthetized with 0.8 ml ketamine with 0.01 ml lumpun and the implantation was performed in the dorsal abdominal region to evaluate hydrogen evolution in vivo. Bar shaped specimens (20 mm (length) 5 mm (width) 1 mm (height)) of as-cast Mg–5 wt%Ca alloy and extruded Mg–5 wt%Ca were implanted in the rats and analyzed after 12 weeks.
3.
Results and discussion
3.1.
Mechanical properties
Extrusion was adopted in order to control microstructural parameters without changing the composition. Fig. 1(a) and (b) show the typical microstructures of as-cast and extruded Mg–5 wt%Ca. As-cast Mg–5 wt%Ca is composed of the primary a-Mg and eutectic Mg/Mg2Ca phases. High contrast represents the Mg2Ca phase in the eutectic phase which constructs a well-connected network in the whole specimen. On the other hand, the secondary phase with fine lamellar contrasts was observed in the extruded Mg–5 wt%Ca alloy and refinement of the microstructure and dispersion of the lamellar phase were observed after extrusion. Fig. 2 shows the XRD patterns obtained from as-cast and extruded Mg–5 wt%Ca alloys. The X-ray diffraction spectrums of both the as-cast and extruded Mg–5 wt%Ca alloy include the diffraction peaks corresponding to the Mg2Ca phase as well as strong a-Mg peaks, which implies that the phase of the extruded sample is the same as that of the as-cast alloy. During the extrusion process, the phase compositions of the alloy did not change. The effect of different microstructures on mechanical properties and biodegradable behavior before and after extrusion was investigated with compression tests and hydrogen evolution tests, respectively. Fig. 3 shows the yield strength (YS) and ultimate compression strength (UCS) of as-cast and
extruded Mg–5 wt%Ca alloys. For comparison, pure Mg data have also been included. The YS and UCS increased from 1570.75 MPa and 3771.85 MPa to 15077.50 MPa and 202710.0 MPa with the addition of 5 wt% Ca, respectively. Also, the extrusion was enhanced by up to 18079.00 MPa and 393718.25 MPa, respectively. From the viewpoint of mechanical strength, the extruded Mg–5 wt%Ca alloy may be considered as a load bearing implant, but the biodegradation rate in body solution should be also considered for its use as a biomaterial.
3.2.
Biodegradation behavior
Fig. 4 shows the results of the hydrogen evolution test for the as-cast and extruded Mg–5 wt%Ca alloys. As the dissolution of one Mg atom produces one hydrogen molecule, the measured hydrogen volume can be converted into the amount of dissolved Mg. As shown in Fig. 4, the hydrogen evolution rate of the as-cast sample was much higher than that of the extruded sample in both longitudinal and radial directions, i.e., the degradation rate of the as-cast sample was higher than that of the extruded sample. The degradation rate of as-cast Mg–5 wt%Ca alloy was much higher than that of the as-cast and extruded pure magnesium. However, A
As-cast Mg-5wt%Ca
A: Mg B: Mg2Ca
A B A
Relative Intensity (a.u.)
56
A BB
A
B
A
A B
A
As-extruded Mg-5wt%Ca A A
B
10
20
A BB
30
B A
40
A
50 2-Theta
B A
BB
60
70
A
80
90
Fig. 2 – XRD pattern obtained from as-cast and extruded Mg–5 wt%Ca alloy.
Fig. 1 – SEM image of Mg–5 wt%Ca alloys: (a) as-cast and (b), (c) extruded sample (longitudinal and radial direction, respectively).
57
journal of the mechanical behavior of biomedical materials 20 (2013) 54 –60
extruded Mg–5 wt%Ca alloy showed similar degradation rate to the extruded pure magnesium. The detailed microstructures of the as-cast and extruded Mg–5 wt%Ca samples after immersion test were investigated to clarify why extrusion caused such a big change in the degradation rate. Fig. 5(a) shows the SEM image of the as-cast Mg–5 wt%Ca sample after immersion testing. The interconnected Mg2Ca dissolved drastically due to the formation of a micro galvanic cell between Mg and Mg2Ca. Kim et al. (2008) reported that the corrosion potential of Mg2Ca (1.54 VSCE) is higher than that of Mg (2.37 VSHE), which can cause a micro-
450
Mg (casting)
400
Mg (extrusion)
200 150 100 50 0 YS
UCS
Fig. 3 – Yield strength (YS) and ultimate compression strength (UCS) of as-cast and extruded pure Mg and Mg–5 wt%Ca alloys.
H2 Evolution [ml/cm2]
2.0
As cast Pure Mg Extruded Pure Mg As cast Mg- 5wt % Ca Extruded Mg- 5wt % Ca - radial direction Extruded Mg- 5wt % Ca - longitudinal direction
1.5
1.0
0.5
3 Compression stress H2 evolution
200
2
150
1.5 100
1
50
0.5 0
0 0
0
20
40 60 Immersion time [hr]
Fig. 4 – Hydrogen evolution rates of specimens.
80
0.5
1
1.5 Time (hr)
2
2.5
3
450
3 Compression stress
400
H2 evolution
350 300
2.5 2
250
1.5
200 150
1
100
0.5
50 0 0
0.0
2.5
H2 evolution (ml/cm2)
250
250
20
40 60 Time (hr)
80
H2 evolution (ml/cm2)
Mg5wt%Ca (extrusion)
300
Compression strength (MPa)
Mg5wt%Ca (casting)
350
Comrpession strength (MPa)
Strength (MPa)
500
galvanic circuit between the Mg2Ca phase (cathode) and Mg matrix (anode). In our current work (Yang, 2011), however, the open circuit potential (OCP) of Mg2Ca in Hanks’ solution with a pH of 7.4 and at a temperature of 37 1C is lower by 360 mV than that of the Mg matrix, which explains the reason for the drastic corrosion along well-connected Mg2Ca in SBF as shown in Fig. 5(a). In the extruded sample, however, the well-connected network structure of Mg2Ca was broken during deformation as shown in Fig. 1(a) and (b), which explains why the degradation rate was remarkably retarded as shown in Fig. 4(b). The anisotropic degradable behavior was also investigated. Corrosion in the longitudinal direction was faster than that in the radial direction because the inter metallic compound Mg2Ca was connected along the
0 100
Fig. 6 – Variation of compression strength by immersion time for the (a) as-cast and (b) extruded Mg–5wt%Ca specimens.
Fig. 5 – Microstructure after immersion tests: (a) as-cast and (b) extruded Mg–5 wt%Ca alloy.
58
journal of the mechanical behavior of biomedical materials 20 (2013) 54 –60
longitudinal direction while the connection along the radial direction was broken, as shown in Fig. 1(a) and (b). Another important point obtained from the results of this study is that the anisotropic microstructure with broken lamellar distribution of Mg2Ca formed by extrusion can also reduce the deterioration in strength caused by the dynamic degradation in human body. To assess the degree of deterioration in strength by immersion in SBF, compression tests were performed for the samples with various immersion times. Fig. 6 shows the variation of compression strength with immersion time for the as-cast and extruded Mg–5 wt%Ca specimens. The deterioration in strength with immersion time remarkably decreased for the extruded specimens as compared with the as-cast specimens. For example, the compression test results obtained at the same amount of hydrogen evolution (1 ml/cm2) for the as-cast and extruded Mg–5 wt%Ca specimens, which corresponds to 1.5 h for the as-cast sample and 20 h for the as-extruded sample, respectively, show that the UCS decreased from 202710.0 MPa to 12977.75 MPa for the as-cast sample and from 393718.25 MPa to 360712.5 MPa for the as-extruded sample, respectively. The ratio of UTS before and after immersion testing is about 60% for the as-cast sample and
about 90% for the as-extruded sample, respectively. As the above comparison is based on the same amount of corrosion, the reduced deterioration in strength may originate from the anisotropic and broken lamellar microstructure of Mg2Ca in the extruded sample rather than retarded degradation rate. It can be explained that the changes in microstructure by extrusion transmutes the degradation behavior into a uniform mode. Fig. 7 shows the scanning electron microscopy (SEM) micrographs recorded from the side surfaces of the fractured samples before and after the immersion tests for the as-cast and extruded Mg–5 wt%Ca specimens. All specimens (both as-cast and extruded) failed in shear mode, which means that the microstructure change and corrosion do not affect the deformation mode. Therefore, the results displayed in Figs. 6 and 7 demonstrate that the anisotropic and broken lamellar microstructure formed by extrusion can effectively prevent the deterioration in strength caused by the dynamic degradation in a human body.
3.3.
Biocompatibility
Since excessive ion leaching from biodegradable implants may produce undesirable effects, biocompatibility is another
Fig. 7 – Scanning electron microscopy (SEM) micrographs recorded from the side surfaces of the fractured samples before and after immersion testing of the as-cast and extruded Mg–5 wt%Ca specimens: (a) as-cast Mg–5 wt%Ca alloy, (b) as-cast Mg–5 wt%Ca alloy after immersion tested for 1.5 h, (c) as-extruded Mg–5 wt%Ca alloy and (d) as-extruded Mg–5 wt%Ca alloy after immersion tested for 20 h.
journal of the mechanical behavior of biomedical materials 20 (2013) 54 –60
Fig. 8 – Cell viability expressed as a percentage of the viability of cells in the control after up to 7 days of culture in as-cast and extruded Mg–5 wt%Ca alloys: (a) Mouse fibrosarcoma L929 cells and (b) Human osteoblastlike HOS.
59
crucial parameter for the use of medical devices and their component materials. In order to identify the level of biocompatibility, in vitro cytotoxicity tests were carried out by indirect contact as suggested by the ISO 10993-5 standards. Fig. 8 shows the viability of mouse fibrosarcoma L-929(Fig. 8(a)), and human osteoblast-like HOS (Fig. 8(b)) expressed as a percentage of the viability of cells cultured after being cultured in as-cast and extruded Mg–5 wt%Ca alloy extraction medium solutions for up to 7 days. It can be seen that for both types of cells, the extract of as-cast Mg5 wt%Ca leads to significantly reduced cell viability compared to the pure magnesium. This is due to the faster corrosion rate of the as-cast Mg–5 wt%Ca alloy. However, the extract of extruded Mg–5 wt%Ca alloy shows no toxicity in the 7-day culture. This result indicates that biocompatibility can be also enhanced through retardation of the degradation rate by changing the microstructure. Fig. 9 shows the X -ray images of biodegraded as-casted and extruded Mg–Ca implants four weeks post-implantation. The size of implanted samples used in this preliminary test was relatively large to show the clear difference in corrosion rate and hydrogen evolution. The trend of in vivo corrosion was similar to that of in vitro result and the images clearly showed the difference in amount of corroded samples and accumulated hydrogen gas within the tissue. Although it was smaller for the extruded Mg–Ca alloys, rats implanted with both cast and extruded Mg–Ca showed clinically visible gas bubbles. The smaller gas bubbles observed for extruded Mg–Ca started to disappear after two weeks and rats implanted with extruded Mg–Ca alloy did not show visible gas bubbles clinically after 12 weeks. The gas bubbles formed within the first week after surgery and persisted for the entire 12 weeks period for as-cast Mg–Ca alloy. All animals survived until the expected sacrifice date and there were no adverse effects caused by the gas bubbles. Typical methods of modifying the mechanical and corrosion properties of alloys have been via alloy design, but the toxicity of adding elements, which imposes much difficulty on alloy design, should be essentially considered for their use as biomaterials. In this
Fig. 9 – Biodegraded samples implanted on dorsal abdominal region of Sprague-Dawley rats for 4 weeks: (a) as-cast Mg–5 wt%Ca alloy and (b) extruded Mg–5 wt%Ca alloy.
60
journal of the mechanical behavior of biomedical materials 20 (2013) 54 –60
sense, tailoring of the microstructure by a process of deformation is one of the most effective methods of improving the biodegradation and biomechanical properties while keeping the mechanical properties, without further considering elemental toxicity. Based on this concept, we invented a new method to improve the corrosion and mechanical properties of Mg alloys; that is, tailoring of the microstructure.
4.
Conclusions
Phases and their spatial distribution, which are the constituents of microstructure, are demonstrated to be the crucial factor in the biodegradation behavior of magnesium alloys. A well-connected network of Mg2Ca in the as-cast Mg–5 wt%Ca binary alloy drastically increased the degradation rate due to micro-galvanic cells formed by its low corrosion potential. The extrusion and deformation processes refine primary Mg grains and break the network structure of Mg2Ca, which resulted in a significant retardation of the degradation rate. The microstructure modification by extrusion improved the mechanical properties in both an ambient environment and body solution. Grain refinement and breaking the network structure of the relatively brittle and corrosive Mg2Ca phase increased mechanical strength in an ambient environment and retarded abrupt deterioration in strength during degradation in a body solution. All the above results demonstrate that tailoring of the microstructure by applying a proper deformation process can be one of the most effective methods of improving the biodegradation properties without elemental toxicity.
Acknowledgments This research was financial support by ‘‘KIST project (2E23090, 2E23720)’’ and ‘‘Seoul R&BD program, Seoul Development Institute, Republic of Korea (SS100008)’’.
references
Bo¨stman, O., Pihlajama¨ki, H., 2000. Clinical biocompatibility of biodegradable orthopaedic implants for internal fixation: a review. Biomaterials 21, 2615–2621.
Claes, L., Ignatius, A., 2002. Development of new biodegradable implants. Der Chirurg 73, 990–996. Duygulu, O., Kaya, R.A., Oktay, G., Kaya, A.A., 2007. Investigation on the potential of magnesium alloy AZ31 as a bone implant. Materials Science Forum 421–4, 546–549. Hofmann, G.O., 1995. Biodegradable implants in traumatology: a review on the state-of-the-art. Archives of Orthopaedic and Trauma Surgery114, 123–132. Inoue, H., Sugahara, K., Yamamoto, A., Tsubakino, H., 2002. Corrosion rate of magnesium and its alloys in buffered chloride solutions. Corrosion Science 44, 603–610. Kim, W.C., Kim, J.G., Lee, J.Y., Seok, H.K., 2008. Influence of Ca on the corrosion properties of magnesium for biomaterials. Materials Letters 62, 4146–4148. Lee, J.Y., Han, G., Kim, Y.C., Byun, J.Y., Jang, J.I., Seok, H.K., Seok, J.Y., 2009. Effects of impurities on the biodegradation behavior of pure magnesium. Metals and Materials International 15, 955–961. Levesque, J., Dube, D., Fiset, M., Mantovani, D., 2003. Investigation of corrosion behaviour of magnesium alloy AM60B-F under pseudo-physiological conditions. Materials Science Forum 521–6, 426–432. Li, Z., Gu, X., Lou, S., Zheng, Y., 2008. The development of binary Mg–Ca alloys for use as biodegradable materials within bone. Biomaterials 29, 1329–1344. Nie, J.F., Muddle, B.C., 1997. Precipitation hardening of Mg–Ca(–Zn) alloys. Scripta Materialia 37, 1475–1481. Staiger, M.P., Pietak, A.M., Huadmai, J., Dias, G., 2006. Magnesium and its alloys as orthopedic. Biomaterials 27, 1728–1734. Saris, N.E.L., Mervaala, E., Karppanen, H., Khawaja, J.A., Lewenstam, A., 2000. Magnesium: an update on physiological clinical and analytical aspects. Clinica Chimica Acta 294, 1–26. Witte, F., Fischer, J., Nellesen, J., Crostack, H.A., Kaese, V., Pisch, A., Beckmann, F., Windhagen, H., 2006. In vitro and in vivo corrosion measurements of magnesium alloys. Biomaterials 27, 1013–1018. Witte, F., Kaese, V., Haferkamp, H., Switzer, E., Meyer-Lindenberg, A., Wirth, C.J., Windhagen, H., 2005. In vivo corrosion of four magnesium alloys and the associated bone response. Biomaterials 26, 3557–3563. Wan, Y., Xiong, G., Luo, H., He, F., Huang, Y., Zhou, X., 2008. Preparation and characterization of a new biomedical magnesium–calcium alloy. Materials and Design 29, 2034–2037. Yang, G.F., 2011.Controlling the Mechanical and Biodegradation Characteristics of Mg–Ca–Zn Alloy for Biomedical Application, Master’s Thesis. University of Science & Technology Thesis.
Available online at www.sciencedirect.com
www.elsevier.com/locate/jmbbm
Research Paper
The modification of microstructure to improve the biodegradation and mechanical properties of a biodegradable Mg alloy Hyung-Seop Hana, Yin Minghuia, Hyun-Kwang Seoka, Ji-Young Byunb, Pil-Ryung Chac, Seok-Jo Yangd, Yu Chan Kima,n a
Center for Biomaterials, Korea Institute of Science & Technology, Seoul 136-650, South Korea Materials/Devices Division, Korea Institute of Science & Technology, Seoul 136-650, South Korea c School of Advanced Materials Engineering, Kookmin University, Seoul 136-702, South Korea d Department of Mechatronics Engineering, Chungnam National University, Deajeon 305-764, South Korea b
ar t ic l e in f o
abs tra ct
Article history:
The effect of microstructural modification on the degradation behavior and mechanical
Received 15 October 2012
properties of Mg–5 wt%Ca alloy was investigated to tailor the load bearing orthopedic
Received in revised form
biodegradable implant material. The eutectic Mg/Mg2Ca phase precipitated in the as-cast
5 December 2012
Mg–5 wt%Ca alloy generated a well-connected network of Mg2Ca, which caused drastic
Accepted 17 December 2012
corrosion due to a micro galvanic cell formed by its low corrosion potential. Breaking the
Available online 28 December 2012
network structure using an extrusion process remarkably retarded the degradation rate of
Keywords:
the extruded Mg–5 wt%Ca alloy, which demonstrates that the biocompatibility and
Magnesium
mechanical properties of Mg alloys can be enhanced through modification of their
Extrusion
microstructure. The results from the in vitro and in vivo study suggest that the tailored
Microstructure
microstructure by extrusion impede the deterioration in strength that arises due to the
Biodegradation
dynamic degradation behavior in body solution.
Mechanical properties
1.
Introduction
Over the past two decades, bioabsorbable polymeric devices have been utilized in many aspects of orthopaedic surgery. Since bioabsorbable implants have the advantage of dissolving in a biological environment after a certain period of functional use, these represent an appropriate solution by means of their cost, convenience, and aesthetic reasons, all of which are favorable to patients. However, these biodegradable polymers have relatively low strength and an n
Corresponding author. Tel.: þ82 29585457; fax: þ82 29585449. E-mail address: [email protected] (Y.C. Kim).
1751-6161/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jmbbm.2012.12.007
& 2012 Elsevier Ltd. All rights reserved.
unpredictable degradation rate, resulting in sudden collapse during their degradation in a living body (Hofmann, 1995; Claes and Ignatius, 2002). Moreover, biodegradable polymers decrease the pH value of body fluid upon dissolution, which is not desirable for the promotion of bone growth (Bo¨stman and Pihlajama¨ki, 2000). Recently, the use of magnesium as a biodegradable metal has been considered as a promising biomedical material due to its unique properties, namely: a larger fracture toughness than that of ceramics and an elastic modulus closer to that of
journal of the mechanical behavior of biomedical materials 20 (2013) 54 –60
natural bone compared with other commonly used metallic implants (Staiger et al., 2006). Therefore, Mg alloys can minimize or bypass the ‘‘stress-shield’’ phenomenon which exists in the current metallic implants made of stainless steel or titanium alloys (Staiger et al., 2006).Moreover, as the fourth most abundant cation, magnesium is essential to human metabolism, and is an essential nutrient in the human body. In particular, it has important influences on nerve, muscle, bone and heart function (Saris et al., 2000). Magnesium is a degradable (or bioabsorbable) metal in the environment of the human body because its highly negative standard electrode potential induces much faster corrosion than is the case for other metallic materials, thus rendering additional surgical operations for its removal unnecessary. However, the intense chemical activity of magnesium would lead to an upsurge of corrosion in corrosive media containing Cl (Inoue et al., 2002; Lee et al., 2009), which can cause the plethoric production of hydrogen gas during in vivo degradation (Staiger et al., 2006; Levesque et al., 2003). Recently, magnesium alloys, such as AZ31, AZ91, WE43, and LAE442 have been investigated for the purpose of improving the corrosion resistance of magnesium for bone implant applications (Witte et al., 2006, 2005; Duygulu et al., 2007). However, the above magnesium–aluminum or magnesiumrare-earth alloys are potentially toxic because aluminum has been reported to be related to brain disease and Alzheimer’s disease (Saris et al., 2000), and rare-earth elements, such as yttrium, may cause liver toxicity. Therefore, it is necessary to develop a new magnesium alloy for biomedical application which has good biocompatibility, good corrosion resistance and good mechanical properties. In particular, it is desirable that such beneficial properties are achieved with the addition of the fewest trace elements possible. Calcium may be one of the best alloying elements to develop a Mg based biodegradable alloy for orthopaedic applications (Kim et al., 2008; Wan et al., 2008; Li et al., 2008) because it is a major component of human bone and essential in chemical signaling between cells. Nie and Muddle (1997) suggested that the Mg–Ca system has good potential in the sense that it has a useful combination of strength, creep resistance, castability, corrosion resistance, and cost because the equilibrium inter metallic phase (Mg2Ca) is structurally analogous with the magnesium matrix. Recently, Li et al. (2008) reported that only Mg alloy with Ca content less than 3 wt% was available for bioabsorbable implants because Mg alloys with larger Ca content were very corrosive and brittle, in spite of their relatively high strength. Previously, most research has involved changing the alloying elements or adjusting the amount of alloying elements to improve the mechanical and corrosion properties of Mg alloys. In this study, we propose a new route to improve both the mechanical properties and biodegradability of a Mg–Ca alloy; i.e., tailoring the microstructure. We achieved good corrosion resistance in a Mg–Ca alloy by tailoring the microstructure, even for a Ca content of up to 5 wt%. We revealed the relationship between the rapid corrosion of Mg–Ca alloys in a simulated body fluid (SBF) and its microstructure, and modified the microstructure to improve the corrosion, biocompatibility, and mechanical properties based on the identified relationship.
2.
Materials and methods
2.1.
Material preparation
55
A binary Mg–5 wt%Ca alloy was prepared by melting and casting under an Ar atmosphere in a vacuum/inert gas atmosphere furnace. Before the Ar gas was introduced to the furnace, the oxygen and moisture in the furnace were removed by applying a vacuum of 103 torr. A STS430 crucible filled with high purity Mg (99.98%) and Ca (99.95%) was kept at 850 1C for 120 min in order to melt the alloy completely. The molten alloy was cast into a STS430 steel mold after stirring for 5 min. The as-cast ingots were preheated for some hours before extrusion, and then extruded into a rod at about 350 1C with an extrusion ratio of 25:1, and cooled in air.
2.2.
Mechanical test
Mechanical properties were evaluated by compression testing. Casting samples were machined according to a f 3 6 mm cylinder shape compression system with a strain rate of 0.6 103 s1. In order to deteriorate strength as arises through dynamic degradation behavior, compression tests were performed on the sample after adequate immersion testing. At least four samples were tested for each condition.
2.3.
Immersion test
For the immersion tests, the specimens were put in a beaker containing SBF. Before the test, the pH was adjusted to 7.4. The degradation rate of the samples was estimated by measuring the amount of generated hydrogen with a funnel. After the immersion tests, the corrosion morphology was analyzed with a Scanning Electron Microscopy (SEM, HITACHI S4200) and X-ray diffractometry (XRD, Bruker D8).
2.4.
In vitro biocompatibility test
Biocompatibility for cytotoxicity was evaluated by in vitro cell experiments. An MEM serum free solution was used as the extraction medium and the extracts were prepared with a surface area to extraction medium ratio of 0.7 cm2/ml under the conditions of 37 1C and 5 vol% CO2 in humidified air for 1–7 days, respectively. The extracts were collected, centrifuged, and filtered before storing at 4 1C prior to the cytotoxicity test. Mouse fibrosarcoma L929 cells and Human osteoblast-like HOS cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin at 37 1C in a humidified atmosphere of 5% CO2. In vitro cytotoxicity tests were carried out by indirect contact as suggested by the ISO 10993-5 standards. A MEM medium, and MEM medium containing 0.64% phenol, acted as negative and positive controls, respectively. Cells were incubated in 96-well flatbottomed cell culture plates with 5 103 cells/100 ml medium in each well, and were incubated for 12 h to allow attachment. All wells were aspirated and 100 ml of extracts and controls (positive and negative) were added; thus 3 replicates for each sample. All wells were then incubated for 24 h. After
journal of the mechanical behavior of biomedical materials 20 (2013) 54 –60
incubation, XTT assay was carried out by using CCK-8 kit (Dojindo/Japan) according to the manufacturer’s directions.
2.5.
In vivo biocompatibility test
This study was approved by the Animal Care and Use Committee of Chungnam National University and eight adult male Sprague Dawley rats weighing approximately400 g were used (Orient Bio, Gyunggi, South Korea). Rats were anesthetized with 0.8 ml ketamine with 0.01 ml lumpun and the implantation was performed in the dorsal abdominal region to evaluate hydrogen evolution in vivo. Bar shaped specimens (20 mm (length) 5 mm (width) 1 mm (height)) of as-cast Mg–5 wt%Ca alloy and extruded Mg–5 wt%Ca were implanted in the rats and analyzed after 12 weeks.
3.
Results and discussion
3.1.
Mechanical properties
Extrusion was adopted in order to control microstructural parameters without changing the composition. Fig. 1(a) and (b) show the typical microstructures of as-cast and extruded Mg–5 wt%Ca. As-cast Mg–5 wt%Ca is composed of the primary a-Mg and eutectic Mg/Mg2Ca phases. High contrast represents the Mg2Ca phase in the eutectic phase which constructs a well-connected network in the whole specimen. On the other hand, the secondary phase with fine lamellar contrasts was observed in the extruded Mg–5 wt%Ca alloy and refinement of the microstructure and dispersion of the lamellar phase were observed after extrusion. Fig. 2 shows the XRD patterns obtained from as-cast and extruded Mg–5 wt%Ca alloys. The X-ray diffraction spectrums of both the as-cast and extruded Mg–5 wt%Ca alloy include the diffraction peaks corresponding to the Mg2Ca phase as well as strong a-Mg peaks, which implies that the phase of the extruded sample is the same as that of the as-cast alloy. During the extrusion process, the phase compositions of the alloy did not change. The effect of different microstructures on mechanical properties and biodegradable behavior before and after extrusion was investigated with compression tests and hydrogen evolution tests, respectively. Fig. 3 shows the yield strength (YS) and ultimate compression strength (UCS) of as-cast and
extruded Mg–5 wt%Ca alloys. For comparison, pure Mg data have also been included. The YS and UCS increased from 1570.75 MPa and 3771.85 MPa to 15077.50 MPa and 202710.0 MPa with the addition of 5 wt% Ca, respectively. Also, the extrusion was enhanced by up to 18079.00 MPa and 393718.25 MPa, respectively. From the viewpoint of mechanical strength, the extruded Mg–5 wt%Ca alloy may be considered as a load bearing implant, but the biodegradation rate in body solution should be also considered for its use as a biomaterial.
3.2.
Biodegradation behavior
Fig. 4 shows the results of the hydrogen evolution test for the as-cast and extruded Mg–5 wt%Ca alloys. As the dissolution of one Mg atom produces one hydrogen molecule, the measured hydrogen volume can be converted into the amount of dissolved Mg. As shown in Fig. 4, the hydrogen evolution rate of the as-cast sample was much higher than that of the extruded sample in both longitudinal and radial directions, i.e., the degradation rate of the as-cast sample was higher than that of the extruded sample. The degradation rate of as-cast Mg–5 wt%Ca alloy was much higher than that of the as-cast and extruded pure magnesium. However, A
As-cast Mg-5wt%Ca
A: Mg B: Mg2Ca
A B A
Relative Intensity (a.u.)
56
A BB
A
B
A
A B
A
As-extruded Mg-5wt%Ca A A
B
10
20
A BB
30
B A
40
A
50 2-Theta
B A
BB
60
70
A
80
90
Fig. 2 – XRD pattern obtained from as-cast and extruded Mg–5 wt%Ca alloy.
Fig. 1 – SEM image of Mg–5 wt%Ca alloys: (a) as-cast and (b), (c) extruded sample (longitudinal and radial direction, respectively).
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extruded Mg–5 wt%Ca alloy showed similar degradation rate to the extruded pure magnesium. The detailed microstructures of the as-cast and extruded Mg–5 wt%Ca samples after immersion test were investigated to clarify why extrusion caused such a big change in the degradation rate. Fig. 5(a) shows the SEM image of the as-cast Mg–5 wt%Ca sample after immersion testing. The interconnected Mg2Ca dissolved drastically due to the formation of a micro galvanic cell between Mg and Mg2Ca. Kim et al. (2008) reported that the corrosion potential of Mg2Ca (1.54 VSCE) is higher than that of Mg (2.37 VSHE), which can cause a micro-
450
Mg (casting)
400
Mg (extrusion)
200 150 100 50 0 YS
UCS
Fig. 3 – Yield strength (YS) and ultimate compression strength (UCS) of as-cast and extruded pure Mg and Mg–5 wt%Ca alloys.
H2 Evolution [ml/cm2]
2.0
As cast Pure Mg Extruded Pure Mg As cast Mg- 5wt % Ca Extruded Mg- 5wt % Ca - radial direction Extruded Mg- 5wt % Ca - longitudinal direction
1.5
1.0
0.5
3 Compression stress H2 evolution
200
2
150
1.5 100
1
50
0.5 0
0 0
0
20
40 60 Immersion time [hr]
Fig. 4 – Hydrogen evolution rates of specimens.
80
0.5
1
1.5 Time (hr)
2
2.5
3
450
3 Compression stress
400
H2 evolution
350 300
2.5 2
250
1.5
200 150
1
100
0.5
50 0 0
0.0
2.5
H2 evolution (ml/cm2)
250
250
20
40 60 Time (hr)
80
H2 evolution (ml/cm2)
Mg5wt%Ca (extrusion)
300
Compression strength (MPa)
Mg5wt%Ca (casting)
350
Comrpession strength (MPa)
Strength (MPa)
500
galvanic circuit between the Mg2Ca phase (cathode) and Mg matrix (anode). In our current work (Yang, 2011), however, the open circuit potential (OCP) of Mg2Ca in Hanks’ solution with a pH of 7.4 and at a temperature of 37 1C is lower by 360 mV than that of the Mg matrix, which explains the reason for the drastic corrosion along well-connected Mg2Ca in SBF as shown in Fig. 5(a). In the extruded sample, however, the well-connected network structure of Mg2Ca was broken during deformation as shown in Fig. 1(a) and (b), which explains why the degradation rate was remarkably retarded as shown in Fig. 4(b). The anisotropic degradable behavior was also investigated. Corrosion in the longitudinal direction was faster than that in the radial direction because the inter metallic compound Mg2Ca was connected along the
0 100
Fig. 6 – Variation of compression strength by immersion time for the (a) as-cast and (b) extruded Mg–5wt%Ca specimens.
Fig. 5 – Microstructure after immersion tests: (a) as-cast and (b) extruded Mg–5 wt%Ca alloy.
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journal of the mechanical behavior of biomedical materials 20 (2013) 54 –60
longitudinal direction while the connection along the radial direction was broken, as shown in Fig. 1(a) and (b). Another important point obtained from the results of this study is that the anisotropic microstructure with broken lamellar distribution of Mg2Ca formed by extrusion can also reduce the deterioration in strength caused by the dynamic degradation in human body. To assess the degree of deterioration in strength by immersion in SBF, compression tests were performed for the samples with various immersion times. Fig. 6 shows the variation of compression strength with immersion time for the as-cast and extruded Mg–5 wt%Ca specimens. The deterioration in strength with immersion time remarkably decreased for the extruded specimens as compared with the as-cast specimens. For example, the compression test results obtained at the same amount of hydrogen evolution (1 ml/cm2) for the as-cast and extruded Mg–5 wt%Ca specimens, which corresponds to 1.5 h for the as-cast sample and 20 h for the as-extruded sample, respectively, show that the UCS decreased from 202710.0 MPa to 12977.75 MPa for the as-cast sample and from 393718.25 MPa to 360712.5 MPa for the as-extruded sample, respectively. The ratio of UTS before and after immersion testing is about 60% for the as-cast sample and
about 90% for the as-extruded sample, respectively. As the above comparison is based on the same amount of corrosion, the reduced deterioration in strength may originate from the anisotropic and broken lamellar microstructure of Mg2Ca in the extruded sample rather than retarded degradation rate. It can be explained that the changes in microstructure by extrusion transmutes the degradation behavior into a uniform mode. Fig. 7 shows the scanning electron microscopy (SEM) micrographs recorded from the side surfaces of the fractured samples before and after the immersion tests for the as-cast and extruded Mg–5 wt%Ca specimens. All specimens (both as-cast and extruded) failed in shear mode, which means that the microstructure change and corrosion do not affect the deformation mode. Therefore, the results displayed in Figs. 6 and 7 demonstrate that the anisotropic and broken lamellar microstructure formed by extrusion can effectively prevent the deterioration in strength caused by the dynamic degradation in a human body.
3.3.
Biocompatibility
Since excessive ion leaching from biodegradable implants may produce undesirable effects, biocompatibility is another
Fig. 7 – Scanning electron microscopy (SEM) micrographs recorded from the side surfaces of the fractured samples before and after immersion testing of the as-cast and extruded Mg–5 wt%Ca specimens: (a) as-cast Mg–5 wt%Ca alloy, (b) as-cast Mg–5 wt%Ca alloy after immersion tested for 1.5 h, (c) as-extruded Mg–5 wt%Ca alloy and (d) as-extruded Mg–5 wt%Ca alloy after immersion tested for 20 h.
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Fig. 8 – Cell viability expressed as a percentage of the viability of cells in the control after up to 7 days of culture in as-cast and extruded Mg–5 wt%Ca alloys: (a) Mouse fibrosarcoma L929 cells and (b) Human osteoblastlike HOS.
59
crucial parameter for the use of medical devices and their component materials. In order to identify the level of biocompatibility, in vitro cytotoxicity tests were carried out by indirect contact as suggested by the ISO 10993-5 standards. Fig. 8 shows the viability of mouse fibrosarcoma L-929(Fig. 8(a)), and human osteoblast-like HOS (Fig. 8(b)) expressed as a percentage of the viability of cells cultured after being cultured in as-cast and extruded Mg–5 wt%Ca alloy extraction medium solutions for up to 7 days. It can be seen that for both types of cells, the extract of as-cast Mg5 wt%Ca leads to significantly reduced cell viability compared to the pure magnesium. This is due to the faster corrosion rate of the as-cast Mg–5 wt%Ca alloy. However, the extract of extruded Mg–5 wt%Ca alloy shows no toxicity in the 7-day culture. This result indicates that biocompatibility can be also enhanced through retardation of the degradation rate by changing the microstructure. Fig. 9 shows the X -ray images of biodegraded as-casted and extruded Mg–Ca implants four weeks post-implantation. The size of implanted samples used in this preliminary test was relatively large to show the clear difference in corrosion rate and hydrogen evolution. The trend of in vivo corrosion was similar to that of in vitro result and the images clearly showed the difference in amount of corroded samples and accumulated hydrogen gas within the tissue. Although it was smaller for the extruded Mg–Ca alloys, rats implanted with both cast and extruded Mg–Ca showed clinically visible gas bubbles. The smaller gas bubbles observed for extruded Mg–Ca started to disappear after two weeks and rats implanted with extruded Mg–Ca alloy did not show visible gas bubbles clinically after 12 weeks. The gas bubbles formed within the first week after surgery and persisted for the entire 12 weeks period for as-cast Mg–Ca alloy. All animals survived until the expected sacrifice date and there were no adverse effects caused by the gas bubbles. Typical methods of modifying the mechanical and corrosion properties of alloys have been via alloy design, but the toxicity of adding elements, which imposes much difficulty on alloy design, should be essentially considered for their use as biomaterials. In this
Fig. 9 – Biodegraded samples implanted on dorsal abdominal region of Sprague-Dawley rats for 4 weeks: (a) as-cast Mg–5 wt%Ca alloy and (b) extruded Mg–5 wt%Ca alloy.
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sense, tailoring of the microstructure by a process of deformation is one of the most effective methods of improving the biodegradation and biomechanical properties while keeping the mechanical properties, without further considering elemental toxicity. Based on this concept, we invented a new method to improve the corrosion and mechanical properties of Mg alloys; that is, tailoring of the microstructure.
4.
Conclusions
Phases and their spatial distribution, which are the constituents of microstructure, are demonstrated to be the crucial factor in the biodegradation behavior of magnesium alloys. A well-connected network of Mg2Ca in the as-cast Mg–5 wt%Ca binary alloy drastically increased the degradation rate due to micro-galvanic cells formed by its low corrosion potential. The extrusion and deformation processes refine primary Mg grains and break the network structure of Mg2Ca, which resulted in a significant retardation of the degradation rate. The microstructure modification by extrusion improved the mechanical properties in both an ambient environment and body solution. Grain refinement and breaking the network structure of the relatively brittle and corrosive Mg2Ca phase increased mechanical strength in an ambient environment and retarded abrupt deterioration in strength during degradation in a body solution. All the above results demonstrate that tailoring of the microstructure by applying a proper deformation process can be one of the most effective methods of improving the biodegradation properties without elemental toxicity.
Acknowledgments This research was financial support by ‘‘KIST project (2E23090, 2E23720)’’ and ‘‘Seoul R&BD program, Seoul Development Institute, Republic of Korea (SS100008)’’.
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