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.