Influence of MC3T3-E1 Preosteoblast Culture on the Corrosion and Biocompatibility of a T6 Treated AZ91 Alloy 1Emily K. Brooks, B.S., 2Menachem Tobias, M.S., and 1,2Mark Ehrensberger, Ph.D.
1Department of Biomedical Engineering, State University of New York at Buffalo, Buffalo, NY 2Department of Orthopaedic Surgery, State University of New York at Buffalo, Buffalo, NY
INTRODUCTION
METHODS
RESULTS CONTINUED
SIGNIFICANCE
REFERENCES
OBJECTIVES
RESULTS
CORROSION BIOCOMPATIBILITY/
BIOLOGY
1. To understand the corrosion of T6-AZ91 in a physiologically relevant media,
and to be aware of changes in electrochemical properties over time.
2. To investigate the biocompatibility of T6-AZ91.
3. To understand how the presence of cells may influence the corrosion
processes of T6-AZ91.
Material Artificially aged T6 treated Mg-9%Al-1%Zn alloy
Sample Preparation 3.8cm2 surface area coupons were wet sanded to 600
grit finish, sonicated, and placed under UV light for 30 minutes for sterilization.
Cell Culturing
• Used mouse preosteoblast MC3T3-E1 cells (ATCC #: CRL-2593)
• Cultured in osteogenic media (OM) – Alpha minimum essential medium
supplemented with 10% fetal bovine serum, and osteogenic reagents
• Seeded onto samples at 25,000 cells/cm2
• Media exchanges took place every 3rd day
Electrochemical Cell (Fig. 1)
• Allowed for concurrent cell culturing and electrochemical testing
• Connections were made to a Gamry ref 600 potentiostat
• Cells placed in humidified 37°C, 5%CO2 incubator (Fig. 2)
Electrochemical Impedance Spectroscopy (EIS)
• Performed at day 3 and day 21
• +/- 10mV (about OCP) from 100KHz-5mHz
• Results fit to a 2 layer circuit model (Fig. 3)
Inductively Coupled Plasma Mass Spectroscopy (ICPMS)
• Used to identify concentration of Mg ions released into OM
• 2mL samples were collected and underwent an acid digestion before
analysis using a Perkin Elmer Sciex model ELAN DRC-II
Scanning Electron Microscopy (SEM, Hitachi SU70)
• OM/AZ91 samples were allowed to air dry
• MC3T3/AZ91 samples were fixed with glutaraldehyde and dehydrated
• Elemental analysis of the corrosion product was completed using energy
dispersive X-ray spectroscopy (EDS)
Statistical Analysis
• 3 Samples were assessed at each condition
• Differences were determined using an ANOVA/Tukey’s post-hoc (p<0.05)
• Log transform data was used for Rs, Rin, Rout, Qin, Qout
Experimental Tests
Experimental Groups
• AZ91 samples were tested with and without cells cultured on the surface
• Analysis was performed after either 3 or 21 days of incubation
3 Day
No Cells
(OM/AZ91)
21 Day
No Cells
(OM/AZ91)
3 Day
With Cells
(MC3T3/AZ91)
21 Day
With Cells
(MC3T3/AZ91)
1E+0
1E+2
1E+4
1E+6
1E-3 1E-1 1E+1 1E+3 1E+5Frequency (Hz)
3 Day No Cells 3 Day Cells
21 Day No Cells 21 Day Cells
Imp
edan
ce (Ω
cm2) 3 Day OM/ AZ91
21 Day OM/AZ91
Fig. 4a
-80
-40
0
40
1E-3 1E-1 1E+1 1E+3 1E+5
Ph
ase
(deg
)
Frequency (Hz)
Fig. 4b
Electrochemical Impedance Spectroscopy Bode Plots
3 Day MC3T3/AZ91
21 Day MC3T3/AZ91
Bode plots resulting from EIS of OM/AZ91 and MC3T3/AZ91 after 3 and 21
days of incubation in OM. Figure 4a represents the impedance modulus curve, while figure 4b represents the phase angle curve..
Table 1: Atomic % as determined by EDS analysis
0 Days 3 Days 21 Days
Element Total Light Cracked Total Total
O 1.5 9.7 61.7 40.4 70.6
Mg 97.1 83.6 19.1 46.8 8.5
Al 1.1 5.8 8.1 6.9 7.8
P 0.8 8.1 4.4 8.4
Ca 0.2 2.6 1.3 4.3
Zn 0.1 0.2 0.4 0.2 0.2
Electrochemical Impedance Spectroscopy Circuit Analysis
0
500
1000
1500
Cells No Cells
Ω*c
m2
3D 21D 21D 3D MC3T3 OM
1E+0
1E+2
1E+4
1E+6
Cells No Cells
Ω*c
m2
3D 21D 3D 21D MC3T3 OM
DISCUSSION
SEM and EDS Results from OM/AZ91 Samples
0
50
100
Cells No Cells
3D 21D 3D 21D MC3T3 OM Fig. 11
0E+0
2E-5
4E-5
6E-5
Cells No Cells
S·sa /
cm2
3D 21D 3D 21D MC3T3 OM
0E+0
2E-5
4E-5
Cells No Cells
S·sa /
cm2
3D 21D 3D 21D
MC3T3 OM
0.0
0.5
1.0
Cells No Cells
3D 21D 3D 21D MC3T3 OM
0.0
0.5
1.0
Cells No Cells
3D 21D 3D 21D MC3T3 OM
Fig. 5: Rout Fig. 6: Rin
Fig. 5 and 6 report the values for the resistance of the outer layer and inner layer of
the circuit model for OM/AZ91 and MC3T3/AZ91 samples.
Fig. 7: Qout Fig. 8: Qin
Fig. 7 and 8 report the values for the magnitude of the constant phase element for
both the outer layer and inner layer of the circuit model. Fig. 9: αout
Fig. 10: αin
Fig. 9 and 10 report the values for the exponent of the constant phase element for
both the outer layer and inner layer of the circuit model.
ICPMS Results for Mg Ion Concentration
Fig. 11 shows the Mg ions released to
solution between 0-3 days and 18-21
days of incubation for OM/AZ91 and
MC3T3/AZ91 samples. Grey bars
represent Mg ions present in the base
OM.
Figs. 12a-c are representative SEMs of OM/AZ91 samples after 0 (a), 3 days (b),
or 21 days (c) of incubation, cracked surface indicates corrosion product formation.
Table 1 displays the EDS surface analysis results for OM/AZ91 samples.
Fig. 13a-d shows
representative scanning
electron micrographs of
MC3T3/AZ91 samples after 3
(13ab) and 21 days of cell
culture (13cd). Good cell
adhesion and viability is
observed at both time points.
Intermittent openings in the
cellular layer were found at
21 days as indicated by the
yellow circle in Fig. 13c.
SEM and EDS Results
from MC3T3/AZ91
Samples
Ag/AgCl reference
electrode
Agar salt bridge
connecting
reference and
working chambers
Graphite rod
counter electrode
AZ91 sample
working electrode
Potentiostats
Chambers
Fig. 2 Fig. 1
RS
Rout Rin
CPEout CPEin Rs – Solution Resistance
Rout – Outer Layer Resistance
Rin – Inner Layer Resistance
CPE – Constant Phase Element
Q – Magnitude of the CPE
α – Exponent of the CPE
Fig. 3
Fig. 12a Fig. 12b Fig. 12c
Fig. 13a Fig. 13b
Fig. 13c Fig. 13d
Magnesium (Mg) and its alloys are a class of biodegradable metals which have
gained increasing interest as orthopaedic implant materials.1,2 Clinical use has
been prevented because of fast corrosion in the physiological environment.
Attempts have been made to control the corrosion processes, but there has
been limited success relating in vitro test results to in vivo corrosion rates.3,4
Cellular attachment is necessary for the success of an orthopaedic device, but
it is not well understood how the presence of cells may be affecting Mg
corrosion. This study explores Mg corrosion with a monolayer of cells cultured
on the surface. All tests have been completed using an artificially aged T6
treated Mg-9%Al-1%Zn (AZ91) alloy.
Corrosion of OM/AZ91 Samples
• The reported increase in Rin and Rout and decrease in Mg ion release from 3
to 21 days indicates a reduction in the corrosion rate for OM/AZ91.
• SEM and EDS results are evidence of formation of an insoluble corrosion
product consisting of Mg and Ca phosphates.
• As the corrosion product covers the surface, it acts as a barrier to corrosion
reactions, resulting in lower corrosion rates at 21 days compared to 3 days.
Corrosion of MC3T3 Samples
• At 3 days samples with cells have higher Rin and Rout, as well as lower Qin
and Qout when compared to OM/AZ91 samples. These changes are
consistent with a thicker more protective layer forming on MC3T3/AZ91.
• Increased resistance may be due to the physical barrier of cells, or
increased precipitation of proteins.
• Between 3 and 21 days, Rout and Rin decrease, Qout and Qin increase, and
there is an increase in Mg ions released to solution. These changes suggest
an increase in the corrosion rate for MC3T3/AZ91 samples over time.
• The decrease in corrosion resistance is likely a result of H2 bubbles
disrupting the protective qualities of the cell layer. The overall reaction for Mg corrosion is: 𝑀𝑔 + 2𝐻2𝑂 → 𝑀𝑔2+ + 2𝑂𝐻− +𝐻2. We believe H2 bubbles
burst through the cell layer, leaving areas of the sample surface exposed,
ultimately accelerating corrosion.
• After 21 days the corrosion rates of OM/AZ91 and MC3T3/AZ91 are similar.
Biocompatibility
• AZ91 samples showed good biocompatibility at
both 3 and 21 day time points. Multiple cell layers
covered the surface by 21 days.
• The cell to cell and cell to substrate adhesions
were maintained (Fig. 14) even as the underlying
metal corroded.
Fig. 14
• Samples with cells on the surface have a higher initial corrosion resistance,
but the corrosion taking place produced changes in the morphology of the
cell layer over time, reducing its protective qualities.
• Corrosion and biocompatibility of
Mg and its alloys can be
considered part of a feedback
loop; where the corrosion
resistance is dependent on
biological activity, just as the
adhesion of cells is dependent on
the rate of corrosion.
• Corrosion testing with cells seeded on the surface of a Mg material may
provide more realistic conditions for predicting in vivo corrosion of Mg, and
contribute to finding a more accurate correlation between in vitro and in vivo
corrosion rates.
1. Staiger, M. P., et al. (2006). "Magnesium and its alloys as orthopedic biomaterials: a
review." Biomaterials 27(9): 1728-1734.
2. Barfield, W. R., et al. (2012). "The potential of magnesium alloy use in orthopaedic
surgery." Current Orthopaedic Practice 23(2): 146-150.
3. Walker, J., et al. (2012). "Magnesium alloys: predicting in vivo corrosion with in vitro
immersion testing." J Biomed Mater Res B Appl Biomater 100(4): 1134-1141.
4. Kirkland, N. T., et al. (2012). "Assessing the corrosion of biodegradable magnesium
implants: a critical review of current methodologies and their limitations." Acta
Biomater 8(3): 925-936.