In vitro corrosion behavior and skin sensitizing potential of Mg-Zn alloy
Weilin Yua, Daoyun Chena*, Yaohua He a**, Hairong Tao a, Yan Zhang a, Yao Jiang a, Xiaonong Zhang b and Shaoxiang Zhang b
a,Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, P.R. China
b,State Key Laboratory of Metal Matrix Composites, School of Materials Science and Engineering, Shanghai Jiaotong University, Shanghai 200240, P.R. China
*Corresponding author
Professor Yaohua He is Corresponding Author. His other information is as following:
Address: Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, P.R. China
Tel: +86 21 64369181
Fax: +86 21 64369181
Email:
**Corresponding author
Professor Daoyun Chen is Corresponding Author. His other information is as following:
Address: Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Shanghai 200233, P.R. China
Tel: +86 21 64369181
Fax: +86 21 64369181
Email:
These authors contributed equally to this work.
Abstract
Magnesium(Mg)-based alloys have attracted great attention in respect of biodegradable biomaterials due to their intriguing characteristic of biodegradability and biocompatibility.In thepresent study, Magnesium-Zinc (Mg-Zn) alloy was developed as a novel magnesium alloy in attempt to enhance corrosion resistance of Mg-based materials. The in vitro corrosion behavior was determined by electrochemical corrosion test and static immersion test, its skin sensitizing potential was evaluated by local skin toxicity, allergenic rate and skin reaction scores..Results showed that Mg-Zn alloys possess more highly corrosion potential and pitting potential than pure Mg samples. The corrosion rate of Mg-Zn alloys in simulated body fluid (SBF) was great lower than that of pure Magnesium materials. Hydroxyapatite (HA) was the main corrosion product of Mg-Zn alloys. The local skin toxicity test and skin sensitizing potential test confirmed that Mg-Zn alloys do not have obvious irritant effects on skin. In conclusion, the addition of Zinc element can significantly enhance the corrosion resistance of Mg-based alloys and the Mg-Zn alloy can be recommended as a promising candidate for biodegradable materials.
Keywords:
Magnesium-Zinc alloys; Corrosion resistance; Immersion test; Skin sensitizing potential test
Introduction
Magnesium(Mg)-based alloy was regard as a promising candidate for biodegradable bone implantsbecause of their outstanding biological performance and their biodegradability in bioenvironment[1-3].In addition, Mg alloys have close mechanical properties to natural bone. Therefore, it can minimize the “stress shielding” phenomena caused by current metallic implants made of stainless steel or titanium alloy. However, the rapid corrosion rate of Mg alloys in human body fluid and the release of hydrogen gas upon degradation have limited their clinical applications[4, 5]. Besides, skin sensitizing reactions to metal implants were reported more often after the introduction of aseptic surgery.Moreover, there is a strong belief that the corrosion of metallic implant materials causes the development of contact dermatitis[6, 7].
It has been considered that the corrosion resistance of Mg can be improved by various elements alloying technique. Witte et al. suggested that the corrosion resistance of Mg can be enhanced by alloying with some rare earth elements[8, 9]. However, the potentially toxic effects of rare earth elements on cells are reported. For example, it is believed that use of such elements aspraseodymium (Pr), cerium (Ce), or yttrium (Y), could lead to hepatotoxicity [10]. Al is harmful to neurons and osteoblasts, and it is associated with dementia and Alzheimer’s disease [11, 12]. In the design of degradable biomedical materials, elements with potential toxic effects should be avoided[13, 14]. Therefore, the potential toxic elements should be excluded from the future generation of biomedical grade Mg alloys.
Recently, Mg alloy components consisting of nutrient elements have attracted great attention in the research of biomedical materials,because these elements are required by human body and it possibly possess good biocompatibility.Research on binary Magnesium Calcium (Mg-Ca) alloys indicated that a Mg-Ca binary alloy with 0.6-1.0 wt.% Ca showed good mechanical properties and corrosion resistance [15], whereas a further increase in Ca content led to deterioration in these properties[16]. Manganese (Mn) and Zinc (Zn) were also chosen as alloying elements due to their good biocompatibility [17, 18]. It has been shown that the corrosion resistance of a Mg-1.0 Mn-1.0 Zn alloy in simulated body fluid (SBF) is better than that of WE43 alloy [19].
Zn is one of the most abundant nutritionally essential elements in the human body and has basically safety for biomedical applications[20]. In Zn deficiency, almost all the physiological functions are strongly perturbed[21]. Further, Zn is known as an essential element in bone formation, which is involved in the mineralization of bone matrix, increased ALP activity of osteoblast cells[22-25].Zinc-doped hydroxyapatite (HA) improved osteoblast cell adhesion compared with the undoped hydroxyapatite[26]. In addition, it has confirmed that Zn can effectively strengthen Mg through a solid solution harden mechanism[27].Therefore, Zn probablyis a promising alloying element to develop biodegradable Mg-based alloys for the clinical applications. In this study, a patented Mg-Zn alloy[28]was developed as a novel magnesium alloy, the in vitrocorrosion behavior and the skin sensitizing potential were evaluated in this paper.
Materials and methods
Materials and animals
Mg-Zn alloy and high purity Mg were produced and provided by Shanghai Aoruiji Medical Technology Limited Company. The chemical composition of Mg-Zn alloy and high purity Mg is shown in Table 1. Poly-L-lactic acid (PLLA) samples were provided by Shanghai Jingtong Medical Technology Limited Company. Stainless steel materials were provided by Shanghai Pudong Medical Instrument Limited Company. Disk samples withacross-section area of 1cm2 and a height of 2mm were used for electrochemical corrosion and static immersion test.Disk samples with a diameter of 4 mm and a height of 2 mm were used for skin toxicity and skin sensitizing potential test. All samples were ultrasonically cleaned in distilled water, followed by sterilization with 29kGy of 60Co radiation.
Forty-five New Zealand rabbits (weighing 2.5 ± 3.2 kg) were provided by the Laboratory Animal Centre of the Sixth People’s Hospital of Shanghai Jiaotong University. All procedures were approved by the Animal Care and Use Committeeof theuniversity.
Electrochemical corrosion analysis
The electrochemical corrosion tests were carried out at 37±0.5 ºC in simulated body fluid (SBF, containing 6.800 gl-1 NaCl,0.200 gl-1 CaCl, 0.400 gl-1 KCl, 0.100 gl-1 MgSO4, 2.200 gl-1 NaHCO3, 0.126 gl-1Na2HPO2 and 0.026 gl-1 NaH2PO4) using a three electrode corrosion measurement system (PARSTAT2273).A saturated calomel electrode (SCE) was used as the reference and the counter platinum electrode. The corrosion rate of the samples was calculated by extrapolating the polarization curve according to ASTM-G 102-89[29]. At the same time, electrochemical impedance spectroscopy (EIS) analysis was carried outat open circuit potential with a perturbing signal of 5 mV. The frequency varied from 100 to 1 MHz. The EIS results were analyzedusing ZsimpWin 3.10 Echem software.
Static immersion test
The immersion tests were performed in SBF following the procedure described by Kokubo T[30].The solution temperaturewas kept at 37±0.5ºC using a water bath. The pH value of the solutionwasmonitored during the immersion test(PHS-3C pH meter, Lei-ci, Shanghai).After 3 and 30 days of immersion, samples were removed out of the solution, rinsed with distilled water and dried in air. The surface morphology after immersion was tested using scanning electron microscopy (SEM) equipped with an energy dispersive spectrometer (EDX) attachment and X-ray diffractometry (XRD, Rigaku DMAX 2400). The corrosion rate was calculated according to ASTM-G31-72[31]..
Local skin toxicity and skin sensitizing potential test
15 adults New Zealand rabbits were randomly divided into 3 groups for local skin toxicity test. Group 1: Mg-Zn alloy samples; Group 2: PLLA samples; Group 3: stainless steel samples. The back hair (120cm2) of the rabbits was removed by barium sulfate. 3 samples from the same group were attached to 1 side of the back skin(The back of each rabbit attached 6 samples from 2 different group) and covered with medical Mepore dressing (AoMei YiXin, Beijing, China). After 4 hours, 48 hours, 72 hours and 7 days, the animals’ behavior, feeding, superficial knowledge, the eye, the mucous membrane, breath, four limb activity and body weight were observed.
The skin sensitizing potential test was performed in a similar method that is described previously[32]. Briefly,30 adult rabbits were randomly divided into 3 groups (10 animals of each group). 24 hours before experiments, the back hair (15×8cm) was removed by barium sulfate. In experimental group, the gauzes (2.5cm×2.5cm) immersed with extraction medium of Mg-Zn alloys were attached to the backside of the rabbits(Fig.1A) and covered with medical Mepore dressing(Fig.1B). Gauzes immersed with 1% 2,4-Dinitrochlorobenzene were taken as positive control and the dry gauzes (without immersed with any solution ) were taken as negative control. The gauzes were removed after 6 hours. The skin reaction was observed immediately and after 24 hours, 48 hours and 72 hours after gauzes removal. For the grading of the skin reaction, the erythema classification according to Magnusson-Kligman test was used (0: no reaction; 1: mild redness, no swelling; 2: moderate and diffuse redness, no swelling; 3: intensive redness and swelling; 4: necrosis). A skin reaction graded greater than zero was defined as erythema.
Results
Electrochemical corrosion behavior of Mg-Zn alloy
Fig.2 illustrates the electrochemical polarization curve of Mg-Zn alloy and pure Mg. It is believed that the pitting potential (Ept) is an indication of the effectiveness of the corrosion to resistance. The increased Ept value of Mg-Zn alloys indicating that Mg-Zn alloys have more corrosion resistance that pure Mg samples. The corrosion potential (Ecorr/V)and electrochemical corrosion rate (mm·yr-1) obtained from the polarization curve were showed in the Table 2. Results indicated that the corrosion potential of Mg-Zn alloys were significantly higher than that of pure Mg samples, whereas the corrosion rate test from the Mg-Zn alloys were great lower than that tested from the Pure Mg materials.
Static immersion measure
Fig.3 shows the degradation rate of the Mg-Zn alloy and pure Mg after 3 days and 30 days immersion in SBF. On 3 days time point, the degradation rate of Mg-Zn alloy was great lower than that of pure Mg, which is consistent with the results found in the electrochemical corrosion experiments. At the beginning of immersion, there was a mass of gas bubble formed in both the Mg-Zn alloy and pure Mg group, but it significantly decreased after 24h and almost disappeared after 3 days immersion (data did not show).
The surface morphology of the samples after immersion in SBF was observed by SEM (Fig.4). There were a number of cracks and corrosion pit can be found on the surface of pure Mg(Fig 4A and 4C), while Mg-Zn alloy showed uniform corrosion surface and no obvious corrosion pit occurred (Fig 4B and 4D). EDS analysis was conducted on corrosion layer, the results indicate that the corrosion products mainly composed of O,Mg, P, andCa (Fig.5). Fig.6 shows the XRD patterns of Mg-Zn alloy after immersion in SBF for 3 days and 30 days. The data suggested that, at days of 3, Mg was the mass of corrosion products and at days of 30, magnesium hydroxide [Mg(OH)2] and hydroxyapatite (HA) were the main corrosion products deposited on surface of Mg-Zn alloy.
The pH variation of the SBF solution in which the Mg-Zn alloy and pure Mg samples were immersed is shown in Fig.7. The pH value was increased rapidly from 7.44 to 8.8 within the first 20h, then it increased slowly and maintained at the value of 9.22 on the following immersion periods.
Local skin toxicity and skin sensitizing potential test
All animals were survived in the local skin toxicity test.During the observation period of 7 days, their behavior, feed, superficial knowledge,the eye, the mucous membrane, breath, the four limb activity and body weight all had no toxic response.It is indicated Mg-Zn allays do not have acute skin toxicity for clinical applications. The allergenic rate and the skin reaction scores of various samples were show in Table 3. During the period of 7 days, 80% animals in 2,4-Dinitrochlorobenzene group showed erythema and smelling to different degree (Fig.8A), whereas the erythema and swelling have not appeared in Mg-Zn alloys group (Fig.8B). The results confirmed that Mg-Zn alloys do not have obvious irritant effects on skin.
Discussion
Mg is anessential element in human body, its advantages of biodegradation and biocompatible are recognized as the potential candidates for the applications of biodegradable biomaterials as the bone implants and cardiovascular stents. However, the rapid corrosion characteristic of pure Mg is a critical drawback for its clinical applications. One of the effective measurements to reduce corrosion rate of Mg is elements alloying, especially those containing nutrimental elements. Zn is also considered as a highly essential element for human and has a solution harden effects on magnesium alloys[21, 33].In the investigation of Mg-Mn-Zn alloys indicated that Zn can be absorbed by the surrounding tissue and body fluid[3]. In thisstudy, we confirmed that alloying Zn element could great enhance in vitro corrosion behavior of Mg-based alloys. Further, no significant skin toxicity and skin sensitizing potential were detected in Mg-Zn alloys.
Magnesium with low corrosion potential, easily corroded in SBF according to following equation[34].
anodic reaction:Mg→Mg+2e
cathodic reaction:2H2O +2e→H2+2OH-
Mg2++2H2O→Mg(OH)2
From above equation, it can be deduced that hydrogen gas would be generated when Mg alloys immersed in SBF. The gas bubble generation is confirmed in present study, and most of the gas was formed during the first 24h, then gradually disappeared after 3 days immersion. In addition, according to above equation, Mg(OH)2 is a corrosion products in the Mg immersion test. Mg(OH)2 is hardly soluble in water , but easily deposited on the surface of the corrosion material and formed a protective membrane. EDS analysis confirmed the existence of Mg(OH)2 on the surface of corroded Mg alloys in this immersion test. However, this protective membrane consist of Mg(OH)2 is apt to be corroded bychloride ions (Cl-) in SBF, which transforms Mg(OH)2 into soluble MgCl2[15], results in excess OH- ions and the ascend of pH value in the solution. In high pH value environment, if the solution contains ions such as PO43-, Ca2+, etc. HA [Ca10(PO4)6(OH)2] and Mg(OH)2 are likely formed on the surface of materials[35]. In current study, XRD analysis demonstrated a great deal of HA [Ca10(PO4)6(OH)2] and Mg(OH)2adhered on the surface of sample. Previous studies [4,16] have reported thatthe corrosion layer containing such magnesium-substituted calciumphosphate compounds on surface of Mg are beneficial to osteoinductivityand osteoconductivity, predicting the good biocompatibility ofmagnesium[8, 36].Kuwahara et al. have reported that Mg immersed in Hank’s solution, the corrosion products on their surface might be someamorphous (Ca0.86Mg0.14)10(PO4)6(OH)2, a rather complicated compound[37]. In view of the similar ion concentrations in the SBF usedin this study to those in Hank’s solution, there might be someamorphous phosphates containing magnesium/calcium formed, as Kuwaharaindicated.
Current immersion test confirmed that, in first 3 days, the corrosion rate of Mg-Zn alloys in SBF was great lower than those of pure Mg. These results are consistent with the previous findings of electrochemical rate obtained from the polarization curve. Further, this investigation showed that the addition of Zn increased the Ept value, indicating that the corrosion layers on the Mg-Zn alloys sample were more protective than those on the pure Mg sample. Taken together, the data from this paper suggests that the addition of Zn can regulate the corrosion rate and enhance the corrosion resistance of Mg alloys.
Both Mg and Zn are the essential elements of human body and take part in almost all physiological functions. Therefore, theoretically, both ions are basically safety for biomedical applications. The degradation of Mg alloys in vivo is a corrosion process. The corrosion particles have been considered responsible for the development of contact dermatitis and aseptic loosening of implants[38, 39]. These corrosion products appeared in the form of debris, inorganic metal salts, metal oxides or organometallic complex, which could lead to contact dermatitis in different way.Further, it is also confirmed that released metal ions from corroding implants results in allergies more frequently than uncorroded implants[32, 38, 40]. In current study, no obvious local skin toxicity or erythema have been observed in Mg-Zn alloys group. It indicated that Mg-Zn alloys do not have acute irritant effects to skin and possess basic safety for biomedical application.
Conclusions
In this paper, Mg-Zn alloys were investigated as a degradable biomaterials. Thein vitro corrosion behavior was determined by electrochemical corrosion and static immersion test and their skin sensitizing potential was analyzed by local skin toxicity, allergenic rate and skin reaction scores.The findings can be concluded that :(1) Mg-Zn alloys showed high corrosion potential and pitting potential than pure Mg; (2) Mg-Zn alloys revealed significantly lower corrosion rate in SBF than pure Mg materials; (3) HA and Mg(OH)2are the main corrosion products of Mg-Zn alloys in SBF; (4) the addition of Zn element can enhance the corrosion resistance of Mg-based alloys; (5) Mg-Zn alloys do not have obvious irritant effects on skin.
Acknowledgements
This work was supported by the Natural Science Foundation of China (No. 81271998andNo. 81271961).
Figure legends
Fig.1
The skin sensitizing potential test: gauzes of immersed with extraction medium of Mg-Zn alloys were attached to the backside of the rabbits(A) and covered with medical Mepore dressing(B)
Fig.2
The electrochemical polarization curve of Mg-Zn and pure Mg in SBF(Ept1:Pitting potential of Mg-Zn; Ept2,Pitting potential of pure Mg).
Fig.3
Static degradation rates of pure Mg and Mg-Zn alloy immersion in SBF for 3days and 30days.
Fig.4
The corrosion morphology of pure Mg and Mg-Zn alloy following immersion in SBF at 3 days and 30 days(A-B: immersion in SBF for 3 days; C-D: immersion in SBF for 30 days).
Fig.5
The surface corrosion products of pure Mg and Mg-Zn alloys following immersion in SBF(A-B: immersion in SBF for 3 days; C-D: immersion in SBF for 30 days).
Fig.6
The XRD pattern of Mg-Zn alloy immersion in SBF. At days of 3, Mg was the mass of corrosion products, at days of 30, the main corrosion products were Mg(OH)2 and HA.
Fig.7
The pH value of SBF during 72 h static immersion.