1

REVISED / EDITED

Electrodeposition of Silk Fibroin on Metal

Substrates

Devid Maniglio*, Walter Bonani, Gabrio Bortoluzzi, Eva Servoli, Antonella Motta,

Claudio Migliaresi

Department of Materials Engineering and Industrial Technologies and BIOtech Research Center, University of Trento,

via Mesiano 77, 38100, Trento, Italy.

devid.maniglio@ing.unitn.it Telephone: +39 0461 882751 Fax:+39 0461 883659

ABSTRACT

Silk fibroin is one of the most promising natural materials for tissue engineering,

having positive interactions with the biological environment, particularly in the

field of bone and cartilage regeneration. We developed a new approach to

creating hydrogels from water based fibroin solutions by applying an electric field

to effect protein migration and coagulation at the anode (Aluminium or Ti6Al4V

alloy) of an electrochemical cell. The process was easily controlled by the

voltage applied to the electrodes (3, 10, 30 V), solution concentration (1%, 2%,

2.6% w/v), time (up to 100 s) and electrode distance (1 - 6 mm). The hydrogel

thickness can be increased up to 60 µm and, depending on processing

conditions, porous coatings or compact films can be obtained. The ability of

electrodeposited fibroin hydrogels to coat metal objects with complex shape and

surface morphology, together with the acclaimed properties of fibroin, makes it a

promising technique to enhance the osteointegration of dental or orthopaedic

prostheses.

2

Keywords: electrodeposition, electrophoretic deposition, electrophoretic coating,

silk fibroin, electro-assisted assembly, natural polymers.

INTRODUCTION

The application of an electric field to induce ion migration in solution has been

widely used since its discovery. This procedure has been used to coat metallic

objects with a thin layer of another metal able to resist corrosion, wear or to

impart the object with a specific look. In the early 20th century the same principle

was applied to charged particles, to induce their migration under electric field in a

colloidal suspension towards one of the two electrodes (electrophoresis). The

main requirement is that the particles have to form a stable colloid suspension

and to carry a net charge. Thus, with a simple and cost-effective process, highly

homogeneous and conformal coatings of different materials (e.g. polymers,

pigments, dyes, ceramics and, of course, metals) can be applied to conductive

objects of any shape.1, 2

Natural polymers are polyelectrolites, and electrophoresis has been used for

analysis with respect to species, separation and recognition 3-5, the possibility to

employ an electric field to coat other materials has not been widely studied

except for polysaccharides 6-12. Many biopolymers are particularly attractive for

biomedical applications, where their intrinsic bioactive properties can be

favourably exploited fabricate biocompatible prostheses and tissue engineering

scaffolds. The conjugation of the mechanical properties of some metals (i.e.

titanium or titanium based alloys) with the acclaimed bioactive properties of

3

some biopolymer coatings could create surface engineered metal prostheses,

with optimal behaviour in terms of biocompatibility and cell interactions.

Recently, regenerated silk fibroin implants have been reported to induce a mild

inflammatory reaction in the body, exhibit antithrombogenic properties, and

promote cell adhesion and tissue repair 13-19. Fibroin can be processed in

different ways in order to prepare gels, powders, fibers or membranes 20-25 . As a

protein, silk fibroin is a polyampholyte with anionic and cationic side chains and

an isoelectric point around pH 4 26. Dissolved in water, silk fibroin has a net

negative charge, due to the negatively charged aminoacids (Aspartic Acid,

Glutamic acid) with respect to positively charged aminiacids (Lysine, Arginine). If

subjected to an electric field, the protein will experience a net force towards the

positive electrode and will accumulate on it, forming a gel-like adhering coating.

In this study we examined whether fibroin could be deposited onto the surface

of a positive electrode immersed in a fibroin solution in response to an applied

voltage. We examined the main variables controlling the process, compared the

results with the theoretical model proposed, and finally analyzed the deposited

fibroin.

Materials and Methods:

Materials

Mechanical polished grade 5 Ti6Al4V Titanium alloy and Aluminium discs

(15mm diameter) kindly provided by Eurocoating S.p.a (Italy) were used as

4

substrates for the deposition.

Anhydrous Na2CO3 (minimum 99% from Sigma-Aldrich) and LiBr (98% from

Fluka Chemical) were used for sericin removal and fibroin dissolution. Slide-A-

Lyzer Cassettes from Pierce/Por, Biotech Cellulose Ester Films, 3500 MWCO

were used for fibroin solution dialysis.

Preparation of Fibroin Solutions

Bombyx mori cocoons were degummed in boiling water to remove sericin. The

silk was treated twice with 1.1 g/L and 0.4 g/mL Na2CO3 water solution at 98°C

for 1 h each (10 g silk in 1 liter of water), washed in de-ionised water and air-

dried 27. Once degummed, silk was dissolved in LiBr 9.3M (1g fibroin each 10 mL

solution) at 65°C overnight. The solution was then dialyzed in a Slide-A-Lyzer

Cassette against distilled water for three days, to remove the salt 28; the fibroin

concentration in the solution was measured with a UV/Vis spectrophotomer

(Nanodrop, ND1000). The concentration was adjusted by adding water to form

2.6%, 2.0% and 1.0% w/v solutions. Control fibroin gels were prepared according

to the procedure reported in literature 29. Briefly, gels were prepared by adding

drop by drop to the 2% fibroin water solution a 0.1M citric acid solution until pH

3.55 was reached.

Electrodeposition Apparatus

A power generator, an amperometer and a deposition chamber, containing the

5

protein-water solution and the positive and the negative electrodes, composed

the electrodeposition system. A scheme and the electrodeposition chamber are

shown in Figure 1 . The chamber was placed on a precision stage to control the

electrodes distance. Current was measured by means of an amperometer.

Film Deposition

Fibroin coatings were generated on the metal anodes by applying different

voltages (3V, 10V, 30V) for up to 2000 seconds to the electrodes immersed in

the fibroin-water solution (1.0%, 2.0%, 2.6% w/v). After the deposition, the anode

resulted coated by a gel-like fibroin material. To make the coating insoluble in

water, the fibroin gel was stabilized by immersion in a methanol-water solution

(80%) for 5 min and then rinsed with water. Following the methanol treatment,

the coated anode was freeze dried generating a gel layer with open porosity.

Characterization of the Hydrogel Coating

The FTIR spectra of electrodeposited fibroin and water fibroin solutions

were obtained on a FTIR-ATR Perkin Elmer Spectrum One in the

spectral region of 4000-600 cm-1 (average of 8 acquisitions, 2 cm-1

spectral resolution). The contribution of pure water was subtracted from

the spectra .

Cambridge Stereoscan 200 Scanning Electron Microscope (SEM) or

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Philips XL30 TMP Environmental Scanning Electron Microscope (ESEM)

were used to analyze the coating morphology.

The thickness of the deposited material was measured by means of optical

microscopy (Zeiss Axiotech at 100x magnification, resolution 2 µm) and by a

micrometer. Thickness was calculated by averaging the measurements obtained

on 5 different areas on freeze-dried samples. The values obtained with both

methods were compared each other and then with the theoretical model.

RESULTS AND DISCUSSION

It was demonstrated that fibroin could be deposited onto the anode surface of

an electrochemical cell. In these experiments a two parallel plate electrode

system was immersed in water fibroin solutions at constant electric potential

using different concentrations. After deposition, the electrodes were

disconnected from the power supply and removed from the solution. During the

process, especially at higher voltages, together with the deposition, bubbles

were generated because of the hydrolysis. Referring to the scheme of Figure 1a,

the first process on the metal electrode is the hydrolysis:

−+ ++→ eHOOH 442 22 (1)

thus, as the concentration of H+ was locally increased, the pH was lowered.

When the local pH of the solution was below the isoelectric point of fibroin, it

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reduced the net charge of fibroin and triggers protein gelation at the surface of

the anode. Assuming that the following pseudo first order reactions takes place,

coatingB

BAk

k

→

→2

1

(2)

the process can be described by the Miskovic-Stankovic model 13, 30 proposed for

a cathodic deposition of epoxy coatings, where A is water, B is hydronium, k1

and k2 are the rate constants of the first and second step of the deposition.

Setting the rate of the film growth as:

( )djjdt

d −= βδ (3)

where β is the electric field, jd, the dissolution current density, j, the current

density; Miskovic – Stankovic proposed the following equation:

[ ]

( )( )

tR

dtktk

eyjkk

AckkK

jjeeKj

0

12

0221

021

2

−

−−

=−

=

++−=

(4)

Where k1 is the electrolysis rate constant (A), k2, the deposition rate constant (B),

c, the concentration, j2, the current density on the deposited film, and δ0, the

starting thickness (equal to zero for a non coated surface). The current j, typically

bimodal behaviour, slightly increased from zero to a maximum value, then

progressively decreased with the reduction in the reaction rate due to the

shielding effect of the coating.

The combination of eq (3) and eq (4) and the subsequent integration gave the

8

following thickness-time relationship:

tjk

e

k

eK

tktk

212

0

12 11 ββδδ +

−−−+=−−

(5)

The SEM images of deposited fibroin after freeze-drying where; (a, b) refer to a

thick deposition (100 s) with open porosity and overlapping sheets. In thinner

depositions (c) (10 s) the structure has greater variability in the porosity,

revealing a more compact structure at lower deposition times. The porous

structure is maintained also in the bulk (d, e), emphasized in proximity of the

metal layer (particularly at lower concentrations) and including the formation of

beads together with overlapping pitted thin sheets. The contact between the

fibroin coating and the surface was characterized by the presence of small

anchoring fibrils (f). While the main porosity was due to the freeze-drying

process, the presence of large regions containing fibrils and beads or other

region where overlapping sheet were prevalent as reported by Tsukada et al 26.

These images were obtained from a 2% w/v solution with 3V applied voltage and

2mm electrodes distance, but are representative of all the other conditions

analysed.

The material obtained had an open porosity with randomly distributed pores.

The porosity was effected by the elimination of water from the gel and the gas

bubbling at the anode; the size of the pores ranged from a few microns to about

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100 µm. As the deposition proceeded, the coating formed an electrical insulate

on the anode and the deposition current progressively decreased. For short

deposition times, the coating was more compact and maintained a network-like

structure.

In some areas the structure showed an overlap of the large pitted sheets

(Figure 2 d, e) with many beads in the terminal portions. Shown in Figure 2 f is a

side-view of the structure with fibroin fibrils anchored to the metal surface. This

dependency of fibroin structure on pH was also described by Tsukada et al 26

who maintained that fibrous structures are expected at low pH, while at higher

pH overlapped sheet structures with larger pores are prevalent.

The IR spectroscopy of the electrodeposited coatings, before and after the

methanol stabilization, were compared with those of gels produced by solution

acidification via citric acid (Figure 3A). These gels were considered to be good

references as they were obtained at a pH lower than the iso-electric point, which

is proposed to occurs at the anode during the electrodeposition processes. For

the coating gel before methanol treatment, the strong absorption of amide I at

1644 cm-1 was attributed to a prevalent random coil conformation; however, after

being treated in methanol, the sample showed absorption bands at 1622 cm-1

(amide I) and 1265 cm-1 (amide III) that are representative of the β-sheet

conformation 31. It is reported in the literature, that with a shift to higher

crystallinity, the β-sheet structure enhances the stability of the coating in water

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32.

The IR spectrum of the electrodeposited gel after methanol treatment was similar

to the gel obtained by acidification, confirming the analogy of the molecular

assembly mechanism related to the increase of β-sheet content.

The IR spectra of freeze dried materials were also analyzed. The spectrum of

the fibroin solution was compared with those on the coating (with and without

methanol treatment) after freeze-drying (Figure 3B). The strong absorption

bands at 1642-1644 cm-1 (amide I) and 1235 cm-1 (amide III) is indicative of a

random coil conformation and/or β turns and bends structures, indicating that the

electrodeposition did not induce significant changes in the secondary structure of

fibroin. Conversely, the gel after methanol treatment presented absorption bands

at 1624 cm-1 (amide I) and a characteristic shoulder band at 1265 cm-1, which

were attributed to the β-sheet protein conformation.

The effect of applied voltage, distance between the electrodes and

concentration of the solution were analysed with respect to the deposition

thickness and the current density. The variation in the current density with

solution concentration at different applied voltages is shown in Figure 4 (these

curves are highly reproducible). As described by Miskovic-Stankovic, in the case

of cathodic deposition, the process was compatible with the presence of two

pseudolinear reactions: a monotonic decreasing function (30V Figure 4C) or a

function with a rapid increment followed by a slow decreasing stage at 3 V. In the

first stage of the process, the current-time graph, a single short spike (10V

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curves), generally attributed to water discharge phenomena at the electrodes,

was observed. The deposition could be described by focusing on the second part

of the curves, in which the deposition contribution is dominant, to ensure the full

control of the process.

Another important parameter in the process is the distance between the two

electrodes. When the specific mass of the deposition, after drying, was plotted

against the electrode distance an inverse proportionality of the two variables was

observed. The deposition kinetics decreased because of the reduction of the

electric field due to the shielding effect of the deposited fibroin (Figure 5a). The

concentration of fibrin was critical because it is proportional to the amount of

polyelectrolytes available in the solution; thus, a higher current was developed by

increasing the charged species in solution. In Figure 5b, the deposition mass

(upon drying) deposited at different concentrations was plotted versus time

showed the dependency of the process, starting from the concentration after

dialysis (2.6 %) and diluting it to 2% and 1%.

To compare the process evolution using equation (5) proposed by Miskovic-

Stankovic, the thickness of the deposition after freeze drying was measured by

means of optical microscopy and a micrometer. No tests on gel-like coatings

could be done because of the difficulties encountered evaluating their thickness.

Although, the micrometer measurements seemed to give lower values than

those obtained by optical microscopy (probably due to a contact compression,

not present in the optical measurement), the data were in good agreement with

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the theoretical model (Figure 6). These results confirmed the suitability of the

model for anodic deposition of cationic polyelectrolytes, at least in the case of

freeze-dried fibroin. Based on the agreement of the data with the model, the

material growth on the electrodes can be controlled by adjusting the conditions to

obtain both thin layers and thick depositions.

CONCLUSIONS

In an aqueous solution after the regeneration, fibroin is negatively charged and

thus undergoes electrophoresis under an applied electric field. Its structure-

sensitivity to acid pH changes permitted coagulation in the proximity of the

anode, which led to the deposition of a gel-like material on the positive electrode

in layers of variable thickness, while no deposition took place at the negative

electrode. The process was very fast (in the order of seconds or minutes,

depending on the desired deposited mass) and highly reproducible. This method

is a novel approach to fibroin processing, as it permits solution gelation without

chemically changing the pH of the bulk solution, and thus, the possibility to

process it in a wide variety of devices. This technique could be employed in the

self assembly of fibroin and to coat metallic objects of any shape with a fibroin

layer or to obtain three-dimensional self supporting scaffolds. Fibroin

electrodeposition could be used to coat orthopaedic or dental prostheses to

enhance their bioactivity by stimulating the osteointegration process.

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REFERENCES

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16. Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R. L.; Chen, J.; Lu, H.; Richmond, J.; Kaplan, D. L., (2003). Silk-based biomaterials, Biomaterials 24, (3): 401-16. 17. Ramesh Dandu, Hamidreza Ghandehari, and Joseph Cappello Characterization of Structurally Related Adenovirus-laden Silk-elastinlike Hydrogels J. Bioact. Compat. Polym.,2008 23: 5-19. 18. Horan, R. L.; Antle, K.; Collette, A. L.; Wang, Y.; Huang, J.; Moreau, J. E.; Volloch, V.; Kaplan, D. L., (2005). In vitro degradation of silk fibroin, Biomaterials 26, (17): 3385-3393. 19. Unger, R. E.; Wolf, M.; Peters, K.; Motta, A.; Migliaresi, C.; Kirkpatrick, C. J., (2004). Growth of human cells on a non-woven silk fibroin net: a potential for use in tissue engineering, Biomaterials 25, (6): 1069-1075. 20. Chen, G.; Zhou, P.; Mei, N.; Chen, X.; Shao, Z.; Pan, L.; Wu, C., (2004). Silk fibroin modified porous poly (e-caprolactone) scaffold for human fibroblast culture in vitro, Journal of Materials Science: Materials in Medicine 15, (6): 671-677. 21. Li, M.; Lu, S.; Wu, Z.; Tan, K.; Minoura, N.; Kuga, S., (2002). Structure and properties of silk fibroin-poly(vinyl alcohol) gel, International Journal of Biological Macromolecules 30, (2): 89. 22. Min, B. M.; Lee, G.; Kim, S. H.; Nam, Y. S.; Lee, T. S., (2004). Electrospinning of silk fibroin nanofibers and its effect on the adhesion and spreading of normal human keratinocytes and fibroblasts in vitro, Biomaterials 25, (7): 1289-1297. 23. Motta, A.; Fambri, L.; Migliaresi, C., (2002). Regenerated silk fibroin films: Thermal and dynamic mechanical analysis, Macromolecular Chemistry and Physics 203, (10-11): 1658-1665. 24. dal Pra, I.; Petrini, P.; Charini, A.; Bozzini, S.; Fare, S.; Armato, U., (2003). Silk Fibroin-Coated Three-Dimensional Polyurethane Scaffolds for Tissue Engineering: Interactions with Normal Human Fibroblasts, Tissue Engineering 9, (6): 1113-1121. 25. Meinel, L.; Hofmann, S.; Karageorgiou, V.; Kirker-Head, C.; McCool, J.; Gronowicz, G.; Zichner, L.; Langer, R.; Vunjak-Novakovic, G.; Kaplan, D. L., (2005). The inflammatory responses to silk films in vitro and in vivo, Biomaterials 26, (2): 147-155. 26. Masuhiro Tsukada, G. F. N. M. G. A., (1994). Preparation and application of porous silk fibroin materials, Journal of Applied Polymer Science 54, (4): 507-514. 27. Valluzzi, R.; He, S. J.; Gido, S. P.; Kaplan, D., (1999). Bombyx mori silk fibroin liquid crystallinity and crystallization at aqueous fibroin–organic solvent interfaces, International Journal of Biological Macromolecules 24, (2): 227-236. 28. Magoshi, J.; Magoshi, Y.; Nakamura, S., (1985). Crystallization, liquid crystal, and fiber formation of silk fibroin, J. Appl. Polymer Sci 41: 187-204. 29. Fini, M.; Motta, A.; Torricelli, P.; Giavaresi, G.; Nicoli Aldini, N.; Tschon, M.; Giardino, R.; Migliaresi, C., (2005). The healing of confined critical size

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cancellous defects in the presence of silk fibroin hydrogel, Biomaterials 26, (17): 3527-3536. 30. Miškovic-Stankovic, V. B., (2002). The mechanism of cathodic electrodeposition of epoxy coatings and the corrosion behaviour of the electrodeposited coatings, Journal of the Serbian Chemical Society 67, (5): 305-324. 31. Wilson, D.; Valluzzi, R.; Kaplan, D., (2000). Conformational Transitions in Model Silk Peptides, Biophys. J. 78, (5): 2690-2701. 32. Tsukada, M., Gotoh, Y., Nagura, M., Minoura, N., Kasai, N., Freddi, G., structural-changes of silk fibroin membranes induced by immersion in methanol aqueous-solutions, Journal Of Polymer Science: Part B-Polymer Physics, 32, (5): 961-968.

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A B

Figure 1 Deposition system scheme (A) and picture (B). The apparatus has a

voltage generator, an amperometer and a deposition chamber.

Amperometer

Voltage generator

start/stop button

Deposition chamber

17

a b

c d

e f

Figure 2 The final SEM structure of fibroin coatings deposited after different deposition times

30 µm 30 µm

30 µm 100 µm

10 µm 30 µm

18

200 0 1800 1600 1 400 1200 10 00 800

A

P eaks a ttribution A m ide I1697 cm -1 ß shee ts1644 cm -1 R andom co il1622 cm -1 ß shee ts

A m ide III1265 cm -1 ß shee ts1239 cm -1 R andom co il1228 cm -1 ß shee ts

105 0 -10801228

126 5

N on treated e lectrodeposited ge l G el obta ined by citric acid treatm ent E lectrodeposited ge l m ethanol treated

1644

W av enum b ers [cm -1]

Tra

smitt

ance

IR spectra o f th e depo sited g els

1622

16971 239

2000 1600 1200 800

1697

Peaks attribution

Amide I1697 cm-1 β sheets1642 cm-1 β turns and bends1624 cm-1 β sheets

Amide III1265 cm-1 β sheets1232 cm-1 Random coil

Comparison between freeze-dried fibroin solution and gels

Wavenumbers [cm-1]

Tra

smitt

ance

fibroin solution non treated gel methanol treatted gel

1642

1624

1265

1232

1050-1080

B

Figure 3 ATR-FTIR spectra. A: comparison between fibroin solution, the electrodeposited gels (before and after methanol treatment) and the gel obtained by fibroin solution acidification. B: comparison between fibroin solution after freeze drying and the electrodeposited gels (with or without methanol treatment) after freeze drying.

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0 10 20 30 40 50 60 70 80 90 100 1100,000

0,002

0,004

0,006

0,008

0,010

0,012

0,014

0,016 Conc 1.0% Conc 2.0% Conc 2.6%

J [m

A/m

m2 ]

time [s]

Fibroin electrodeposition, 3 V applied voltage A

0 20 40 60 80 1000,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14 Conc 1.0% Conc 2.0% Conc 2.6%

J

[mA

/mm

2 ]

time [s]

Fibroin electrodeposition, 10V applied voltage B

20

0 10 20 30 40 50 60 700,00

0,05

0,10

0,15

0,20

0,25

0,30 Conc 1.0% Conc 2.0% Conc 2.6%

J [m

A/m

m2 ]

time [s]

Fibroin electrodeposition, 30V applied voltage C

Figure 4 Current-time plot of fibroin deposition on aluminum, 2mm electrodes distance at different solution concentration (1.0%, 2.0%, 2.6% w/v). Voltage difference applied at the electrodes are; A: 3V; B: 10V; C: 30V. Instrumental

error is ±(1% value + 2 µA).

21

1 2 3 4 5 60

2

4

6

8

10

12

de

posi

tion

[ µg/

mm

2 ]

electrodes distance [mm]

Distance dependency

(a)

0 30 60 90 120 150 180 210 240 270 300 330 360 390 4200

2

4

6

8

10

12

14

16

18

20

22

Concentration dependency (3V)

time [s]

Dep

ositi

on [

µg/m

m2 ]

1.0 % 2.0 % 2.6 %

(b)

Figure 5 Correlation between deposition mass and electrodes distances after 30 min (a) and deposition kinetics at different fibroin concentrations (b).

22

0 10 20 30 40 50 60-10

0

10

20

30

40

50

60

70

Deposition Thickness

tj

k

e

k

eK

tktk

212

0

12 11 ββδδ +

−−−+=−−

δ [m

m]

deposition time [s]

Micrometer Optical microscopy Theoretical prediction

Figure 6 Comparison of the deposition thicknesses, measured by means of optical microscopy and micrometer on freeze-dried sample (1%, 3V) with the theoretical model. The parameters δ0, β, k1 , k2 of the theoretic model are calculated by simple fitting current vs time plot with Eq(4).

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