Accepted Manuscript

Modification of type I collagen on TiO2 surface using

electrochemical deposition

Nguyen Thanh Truc, Ho Hieu Minh, Ly Loan Khanh, Vo Minh

Thuy, Vo Van Toi, Tran Van Man, Huynh Cong Nhat Nam, Tran
Ngoc Quyen, Nguyen Thi Hiep

PII: S0257-8972(18)30286-X
DOI: doi:10.1016/j.surfcoat.2018.03.038
Reference: SCT 23218
To appear in: Surface & Coatings Technology
Received date: 22 September 2017
Revised date: 2 February 2018
Accepted date: 13 March 2018

Please cite this article as: Nguyen Thanh Truc, Ho Hieu Minh, Ly Loan Khanh, Vo Minh
Thuy, Vo Van Toi, Tran Van Man, Huynh Cong Nhat Nam, Tran Ngoc Quyen, Nguyen Thi
Hiep , Modification of type I collagen on TiO2 surface using electrochemical deposition.
The address for the corresponding author was captured as affiliation for all authors. Please
check if appropriate. Sct(2017), doi:10.1016/j.surfcoat.2018.03.038

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The goal of this research is to coat Col-1 onto the surface of Titanium (Ti) by employing
electrochemical method. The morphology, crystalline, and topography of Col-1 on Ti were
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evaluated using scanning electron microscope (SEM), X-ray diffraction (XRD), and atomic force
microscope (AFM) respectively. Subsequently, the scaffold’s in vitro performance was studied
using fibroblast cells.
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Modification Of Type I Collagen On TiO2 Surface Using Electrochemical Deposition

Nguyen Thanh Truc1#, Ho Hieu Minh1#, Ly Loan Khanh1#, Vo Minh Thuy1#, Vo Van Toi1, Tran

Van Man2, Huynh Cong Nhat Nam3, Tran Ngoc Quyen4,5 and Nguyen Thi Hiep1*

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Tissue Engineering and Regenerative Medicine Laboratory, Department of Biomedical

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Engineering, International University, Vietnam National University- Ho Chi Minh City (VNU-

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HCM), Ho Chi Minh City 700000, Vietnam.

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Department of Physical Chemistry, University of Science, VNU-HCM

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Laboratory of Oral-Maxillofacial Biology, Department of Dental Basic Sciences, Faculty of

Odonto-Stomatology, University of Medicine and Pharmacy Ho Chi Minh City, Viet Nam
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Institute of Applied Materials Science, Vietnam Academy of Science and Technology, 1A TL29,
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District 12, Hochiminh City 700000, Vietnam

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Graduate School of Science and Technology Viet Nam, Vietnam Academy of Science and

Technology, 1A TL29, District 12, Hochiminh City 700000, Vietnam

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(# Contribution of Authors is similar)

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Abstract
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Titanium (Ti) abutment is a supporting structure of dental implant which anchors and provides

retention for fixed or removable tooth restoration. However, bio-inert property of Ti leaves the soft

tissue to detach from the abutment, partly leading to implant failure. Meanwhile previous studies

have mainly focused on enhancing the osseointegration of Ti implant, the dislocation and imbalance

of dental implant still occur since the connective tissue defect at the abutment. This study aimed to

investigate the interaction between fibroblast and the modified Ti in which type 1 collagen (Col-1)
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was modified on Ti surface. Ti/Col-1 modification was performed by electrochemical deposition

(ECD) based on the polyelectrolyte property of Col-1 and conductivity of Ti under electric field.

Parameters optimized for ECD were investigated including Col-1 concentration, pH of electrolyte,

and deposition potential. Fibroblast’s cell adhesion/spreading, and cell viability/proliferation assays

were analyzed between the coated Ti/Col-1 with optimized ECD conditions and uncoated Ti.

Scanning Electron Microscope (SEM) observation indicated that Col-1 was modified successfully

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on Ti while fibroblast cell adhered and proliferated well in Ti/Col-1 compared with the uncoated Ti.

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This study suggested the influence of Col-1 modification on the attachment of fibroblast cells

around the Ti abutment.

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Key words: Titanium abutment, Titanium modification, TiO2, collagen I, electrochemical deposition,
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fibroblast attachment.
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Corresponding author:
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Nguyen Thi Hiep (email: nthiep@hcmiu.edu.vn)

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Tel.: +84 2837 244 270; Fax: +84 2837 244 271.
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1. Introduction

The failures of dental implantation mainly related to the loss of integration, positioning failure,

biomechanical failures, and soft tissue defects [1]. Tissue defects are defined as the volume loss of

soft tissue or soft tissue detachment around the implant abutment [2-4]. The soft tissue containing

sulcular and junctional epithelia around the implants called peri-implant soft tissue, have similar

form and function as the periodontal tissue around the natural teeth [5]. This peri-implant tissue,

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however, is less vascular because of lacking the vessels from the periodontal ligament [6]. In fact,

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the peri-implant tissue seems similar to scar tissue which has less number of fibroblasts comparing

to the content of collagen. This minor number of fibroblast cells causes the poor cell response and

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tissue attachment to the implant material. In some cases, the infection at implanted site may occur in

the gap between abutment and detached soft tissue resulting in removal of such implants [7].
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Ti and its alloys have been used as a popularly metallic material for dental implantation since its
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biocompatibility and excellent resistance. This material’s favorable properties including

biocompatibility, corrosion resistance, and biochemical inertness commence from the chemical
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stability and structure of the titanium dioxide (TiO2) film, which spontaneously formed when Ti is
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exposed to oxygen [8-10]. Even though Ti’s bio-inert property is a positive factor to prevent the

rejection of implant from the body immunogenesis, it simultaneously hinders a weak cell-implant
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interaction between the gingival fibroblast and Ti abutment causing implant’s imbalance and
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dislocation [11, 12]. There are several efforts to modify the Ti surface with various types of

biopolymers and materials by different methods to restrict its bio-inert property [8], and avoid its

failure. However, most studies have been concentrated on building the bone-implant interface or

osseointegration of the implants whereas biomechanical failures and soft tissue defects received less

concern [13]. Thus, restricting Ti’s bio-inert property and stimulating the proliferation of gingival

fibroblasts were considered to be the optimization of soft tissue management in dental implant.
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Besides the methods used in previous studies such as plasma spraying, laser treatment, and acid-

etched which aimed to modify the implant surface’s topology and morphology for the

osseointegration [14, 15], organic molecule coating methods to immobilize proteins, enzymes and

peptides on biomaterials have currently become an interest. Ling et al. coated Col-I and Ca-P on Ti

using ECD method to obtain strong coating adhesion to Ti, the study reported the effective bone-

implant interface support by CaP/Col-1 [16]. Ao et al. dip coated Col-I onto Ti to increase bone

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formation [17, 18] while Nathalia et al. coated Col-I onto Ti using physical coating method and

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reported better fibroblast proliferation on the coated material [19]. In general, Col-1 stands out in

the role of biopolymer coating of Ti since Col-1 is the major structural protein in bone, a promising

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candidate for surface immobilization. This extracellular matrix (ECM) protein regulates various

aspects of cell behaviors including cell migration and attachment by interacting integrin located on
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cell membrane [20]. Col-1 has been used to enhance osteoblast adhesion, differentiation in vitro [21-
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24] and osseointegration in vivo [22-25] of Ti and Ti alloys by acting as mediator of osteoblastic cell

functions, including adhesion, differentiation, and ECM deposition [20, 21]. However, Col-1 also
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functions as a component of gingival fibroblast's ECM which assists to overcome the defects of soft
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tissue in Ti dental implantation [18, 26]. Effectively, because of the simple handling and easy

manipulation, electrochemical deposition method (ECD) was chosen to modification of Col-1 on Ti.
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ECD have been mentioned in previous researches for Ti surface modification in supporting
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osseointegration of implants. Specifically, ECD was applied in Strontium coating on Ti implant to

improve osseointegration in osteopenic models [27], modifying osteoconductive Calcium Phosphate

layer [28], and assisting mineralized collagen coating on Ti alloys to enhance osteoblast

proliferation [16]. However, there have been no previous study examining ECD method for coating

Col-1 on Ti to support fibroblast attachment.

ECD requires shorter time of preparation and operation than other methods like dipping and plasma-

polymerization [15, 29-32]. This method is an optimal choice for modification system, which
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supported Ti in respect to the fibroblast adhesion and soft tissue attachment. In the present study,

Col-1 was modified on Ti using ECD method to tackle the defects of soft tissue. In details, Col-1

suspension represented the electrolyte solution in this ECD system since being a polyelectrolyte

polymer whose net charge varies dependently on the pH [33]. Col-1 molecules bonded covalently on

Ti substrate as Cathode (-) by positive charge corresponding to a range of pH value [17]. Under

electric field, the high surface load of immobilized Col-1 molecules reduces the loss of protein [34]

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and forms a Col-1 coating layer on the substrate. Parameters in controlling ECD such as

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concentration of Col-1, pH of solution, and deposition potential were varied to optimize the Col-1

coating on Ti. After completing the ECD optimization, Ti substrates were modified by investigated

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ECD conditions. The biological properties of modified material in response to fibroblast including

cell adhesion, cell proliferation and cell viability were also analyzed.
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2. Materials and methods
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2.1. Preparation of Electrochemical deposition (ECD)

Cathode electrode: Ti alloy 6AL-4V foil (BAOJI Energy Titanium CO., China) was cut in strips
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with dimensions of 10 x 8 x 1.45 mm3 before ultrasonically washing in 70% ethanol and distilled
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water respectively for 10 minutes by Elma Ultrasonic Cleaning machine. Ti plates were then soaked

in 4 M NaOH solution for 2 hours at 90 0C to produce sodium titanate hydrogel layer [35, 36].
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Col-1 solution at different concentrations: Bovine Type I Collagen Ligament (Col-1) (Sigma
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Aldrich) was dissolved in 5 M acetic acid (Xilong Chemical Co., Ltd.) to achieve Col-1 solutions

with different concentrations (0.3 mg/ml, 0.5 mg/ml, and 0.7 mg/ml). These solutions were

fabricated to achieve pH 5.5, with 4 M NaOH (Xilong Scientific Co., Ltd.) and measured by

calibrated pH meter (ADWA, AD1000).

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Col-1 solutions at different pH: pH of 0.5 mg/ml Col-1 solutions were adjusted with 4 M NaOH

solution (Xilong Scientific Co., Ltd.) and measured by calibrated pH meter (ADWA, AD1000) to

achieve different pH: 4.5, 5.5, 6.5, and 7.5.

ECD model: Ti plate and Platinum (Pt) net acted as Cathode (-) and Anode (+) respectively. These

two electrodes were placed opposite at a distance of 15 mm in a 100 ml beaker contained Col-1

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solution. Time for ECD was set at 20 minutes, then the coated Ti plates were then rinsed with

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distilled water and dried at room temperature.

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2.2 Investigation the effects of varied ECD parameters:

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Col-1 concentration: Col-1 solutions at 0.3, 0.5, and 0.7 mg/ml with pH 5.5 were used as electrolyte

to coat three separate Ti plates, followed ECD model.

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Solution pH: 0.5 mg/ml Col-1 solutions at different pH (4.5, 5.5, 6.5, and 7.5) were used as

electrolyte to coat four separate Ti plates, followed ECD model.

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Deposition potential: 0.5 mg/ml Col-1 solutions at pH 5.5 was used as electrolyte to coat six
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separate Ti plates, at different voltage (0.5, 1, 1.5, 2, 2.5, and 3 V), followed ECD model.
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All the samples were investigated respectively to varied parameters as being mentioned in Table I.

2.3 Characterization of the coating Ti/Col-1

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After coating process, four replicate specimens of each Ti, TiO2, and Ti/Col-1 having dimensions of
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10 x 8 x 1.45 mm3 were dried at room temperature. Then they were examined with a scanning

electron microscope (SEM, JEOL JSM-IT100) at 5 and 16 kV to observe the morphology of

collagen on Ti surface of different parameters at magnifications 50X and 5,000X. To quantitatively

measure and analyse the surface roughness of Ti surfaces on the nanometer scale, an atomic force

microscope (AFM, Bruker) was used to record the images of one sample of TiO2 and Ti/Col-1.

Sample size for AFM analysis was similar as mention in SEM. The images were taken in tapping

mode using a scanning probe with the resonant frequency around 400 kHz and a typical spring
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constant of 40 N/m, the scan areas were 10µm x 10µm. Roughness surface profiles were statistically

determined by Gwyddion software (version 2.49). In addition, to confirm the presence of Col-1 on

coated Ti, X-ray diffraction (XRD) profiles of control sample (untreated Ti), coated Ti/Col-1 and

pure Col-1 were acquired by XRD machine (Thermal Nicolet 6700, Cu Kα, 0.02 deg per step).

2.4 In vitro studies

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2.4.1 Cell maintenance

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Mouse fibroblast L929 cell line (ATCC) was used to evaluate the biological properties of

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experimental materials. The cells were maintained in culture flask containing DMEM media,

supplemented with 10% (v/v) FBS (Fetal Bovine Serum) and 1% PS (penicillin/streptomycin) at

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37 °C, 95% humidity, and 5% CO2 (Incubator, Binder). Cells were dissociated using trypsin-EDTA
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(GIBCO), were then centrifuged and resuspended in medium. The culture medium was changed

every two days. After sterilization, 2 samples of each group (Ti; TiO2; Ti/Col-1) were placed in 24-
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well plate and seeded with L929 fibroblast cells (5 x 104 cells per well), then incubated at 37oC for 1
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hour and 1 day correspondingly.

2.4.2 Sterilization
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Ti, TiO2, and Ti/Col-1 were sterilized by UV for 20 minutes following 70% ethanol washing for 20
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minutes, finally rinsed thoroughly with PBS. Before testing, the sterilized samples were keep in

clean bench to avoid the contamination.

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2.4.3 SEM observation

After indicated incubation time (1 hour or 1 day), samples were washed with PBS, then fixed in 4 %

glutaraldehyde (GA) for 20 minutes; and rinsed thoroughly with PBS before being dehydrated with

series of ethanol (50%, 60%, 70%, 80%, 90%, 95% and 100%) at room temperature. Once being

dried, the samples were sputter-coated to examine under SEM (JEOL JSM-IT100) at 5 and 16 kV,

with magnifications of 100X and 8000X.

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2.4.4 MTT assay

Three types of Ti surfaces (untreated Ti, TiO2 and Ti/Col-1) after being sterilized were separately

placed in a 24-well plate. Mouse fibroblasts L929 were seeded into each well at 5 x 104 cells/ml and

1 ml per well. Cell viability was measured at day 1, 3, and 5 by adding 200 µl of 3-(4, 5-

dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide solution (MTT, Sigma) to each well and

incubating for 4 hours. Samples were removed carefully without disturbance of the formazan layer

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on top the solution once dark purple insoluble formazan was formed. Ti samples were then placed

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on a new 24-well plate. Next, 1 ml of dimethyl sulfoxide (DMSO, Sigma) was added into each well

to dissolve the insoluble formazan on Ti surfaces. Then the transparently colorful solution was taken

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out to be read by UV-visible spectroscopy at 630 nm wavelength with DMSO as control solution.

The absorbance of each solution was measured 3 times automatically and average value was
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calculated.
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2.4.5 Statistical analysis

All experiments by MTT assay were performed in triplicate. The means and standard deviations (SD)
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for each set of data were calculated. One-way ANOVA with Tukey’s post-hoc test were used to
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compare between groups using SPSS v.23 (IBM) with the level of significance being 0.05.
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3. Results

In this study, Ti was modified with Col-1 using ECD method, and then the morphology of treated
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TiO2 was observed by SEM to investigate the optimized coating parameters. The presence of Col-1

was also confirmed by XRD. Lately, optimized Ti/Col-1 substrates were tested in vitro for fibroblast

cell adhesion/spreading and cell viability/proliferation by MTT assay.

3.1 Optimization of ECD

Ti surface was massively covered with TiO2 layer (porous topography) after NaOH treatment (Fig.

2b) in comparison to non-treated Ti (Fig. 2a).

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3.1.1 Effects of Col-1’s concentration

SEM images showed a layer of Col-1 deposition on all samples of which Col-1 concentration varied

from 0.3-0.7 mg/ml (Fig. 3a-c). The morphology of Col-1 varied respectively with increasing Col-1

concentration. Particularly, when Col-1 concentration achieved 0.3 mg/ml, formed Col-1 layer on Ti

surface was thinly dispersed and scattered and did not cover the porous surface fully (Fig. 3a). As

the concentration increased to 0.5 mg/ml, Ti/Col-1 samples achieved an even distribution and thin

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layer of Col-1 (Fig. 3b), with completely Col-1 fulfilled porous TiO2 surface. Similarly, as Col-1

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concentration increased to 0.7 mg/ml (Fig. 3c), the morphology of Ti/Col-1 sample was denser than

the sample at 0.5 mg/ml (Fig. 3c). However, bristling precipitation occurred at Col-1 0.7 mg/ml,

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which created an uncontrollable Col-1 coating layer in some areas covered by aggregated Col-1 and

empty areas with porous TiO2. It was rather a low effective ECD while Ti/Col-1 at 0.7 mg/ml of
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Col-1 yielded an aggregated coating layer that prevented cellular bioactivity. It was necessary to
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produce a coherent thin Col-1 coating and support the cell attachment in which Col-1 concentration

at 0.5 mg/ml was satisfied for an optimized Ti/Col-1 modification.

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3.1.2 Effects of pH
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Fig. 3 demonstrates SEM images of Ti/Col-1 morphology after coating with separated solutions at
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various electrolytes’ pH value (4.5; 5.5; 6.5; 7.5). All samples expressed the formation of Col-1

layer on their surface; however, morphology of this layer was significantly different among the
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samples. Fig. 3a shows the aggregation of Col-1 on Ti (pH 4.5) whereas the sample treated with

higher pH (5.5) (Fig. 4b) showed a better coating layer, which evenly and widely fulfilled the porous

Ti structure. It was proved that the Col-1 molecules charged more positively when reaching nearly

to the isoelectric point (iP) at which pH varied from 5.5-7 depending on the source of Col-1 [33].

Therefore, as the pH solution were increased to 6.5 (Fig. 4c) and 7.5 (Fig. 4d), Col-1 were not

formed smoothly on the surface of Ti since the charges were alternated due to increasing pH. In
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some areas on Ti surface of Fig. 4d, there were disorganization of Col-1 deposition and a lot of

empty spaces comparing to Fig. 2b. It was concluded that pH of the electrolyte affected directly the

net charge of Col-1. Therefore, the optimized pH solution was selected to be 5.5 for better

controlling of Col-1’s net charge and directing the Col-1 molecules to the cathode Ti.

3.1.3 Effects of deposition potential

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As the voltage increased from 0.5 to 3.0 V (Fig. 5a-f), there was a difference in Col-1 morphology

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among the coated Ti/Col-1. Theoretically, the higher the voltage at a distinct deposition time of 20

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minutes, the more positively charged collagen molecules tended to deposit on the Ti surface.

However, the coated Ti/Col-1 at 2.0 V (Fig. 5d) had significantly better distribution of collagen

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coating because it showed the thickest collagen layer scattered thoroughly on Ti surface compared
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with other Ti/Col-1 coated at 0, 0.5, 1, 1.5, 2.5, and 3 V (Fig. 5a, b, c, e, f). Except coating at 2.0 V,

the contrast between the TiO2 porous surface and the cover of Col-1 layer on the other samples that
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reflected the empty spaces left after coating were observed easily. In brief, the optimized
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electrochemical potential was optimized at 2.0 V since producing satisfied collagen coating.

3.2 Morphology and Topology of TiO2 and Ti/Col-1

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The results of an AFM surface analysis on treated TiO2 and coated Ti/Col-1 were shown in Table 2
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and Fig. 6. Five parameters for characterizing Ti surface include: average roughness (Ra), root mean

square roughness (RMS) roughness (Rq), maximum height (Rmax), Skewness (Rskw) and Kurtosis
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(Rkur) [37-39]. The sample’s surface topography was analyzed by Ra, Rq and Rmax, while the surface

morphology was evaluated by Rskw and Rkur. The surface topography of untreated TiO2 and Ti/Col-1

was significantly different; the roughness parameters of TiO2 were lower than Ti/Col-1, i.e, Ra of

22.7 nm and 44.5 nm and Rq of 29.4 nm and 59.6 nm on the 10µm x 10µm of scanning areas of

TiO2 and Ti/Col-1, respectively. Also, the Rmax data recorded from scanning area of Ti/Col-1

(Rmax=587 nm) was higher than TiO2 (Rmax=293.6 nm) remarking the totally different between the
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two surface topography. Obviously, the roughness of Ti/Col-1 exhibited was nearly 2 times greater

than TiO2. For characterizing the surface morphology, the degree of symmetry and the sharpness of

the rough surface profile are measured by Skewness and Kurtosis [37-39]. TiO2 had Rskew close to 0

and Rkur far from 3 (Rkur=0.95) indicating a symmetrical distribution of platykurtic peaks and valleys,

while Ti/Col-1 had Rskew of 0.5 and Rkur far from 3 ( Rkur=1.87) showing less symmetrical

distribution of platykuritc peaks and valleys than TiO2. Specifically, the peaks formed on Ti/Col-1

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were sharper and the valleys were broader than TiO2. Modification of Ti with Col-1 resulted in a

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greater roughness surface in comparison with TiO2.

3.3 XRD prolife of Ti-Col I

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To confirm the Col-1 coating on the porous TiO2 surface, the pure Col-1, TiO2 (Ti/NaOH) and
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Ti/Col-1 (Ti/TiO2/Col-1) were compared by their XRD pattern. Fig. 7 shows the XRD profiles of

untreated Ti (Ti/TiO2), TiO2 (Ti/NaOH) and Ti/Col-1 (Ti/TiO2/Col-1). The peaks of Ti/NaOH
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crystalline structure were indexed according to Lindahl, C., et al [40]. It was found that diffraction
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from TiO2 after treating with NaOH (Ti/NaOH) lacked the peak at 220 and had one more peak at 330

comparing to untreated Ti (Ti/TiO2). Otherwise, the XRD pattern of Ti/Col-1 was nearly the same as
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untreated Ti (Ti/TiO2) except having one more peak at 180, which indicated the deposition of Col-1
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on its surface

3.5 In vitro studies

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In vitro studies among Ti; TiO2 and Ti/Col-1 were performed in order to observe their interaction

with fibroblast cell, and confirm whether NaOH treatment or Col-1 modification affected the

fibroblast cell’s attachment and proliferation. The fibroblast behaviors on each sample were

evaluated based on in vitro characteristics including cell adhesion, cell proliferation and cell

viability test.

3.5.1 Cell adhesion

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Followed previous study on investigating cell adhesion on coated Ti surface [41], the SEM images

of untreated Ti (Fig. 8a), TiO2 (Fig. 8b) and Ti/Col-1 (Fig. 8c) substrates were recorded after

reseeding with L929 fibroblast for 1 hour. The number of cell was different among three samples

(Fig. 8a-c) reflecting the compatibility of the substrate’ surface to the cell. The Ti/Col-1 sample (Fig.

8c) showed the largest number of cell adhered whereas Ti and TiO2 showed similar cell number (Fig.

8a, b). Regarding the cell morphology, Ti/Col-1 (Fig. 8c) indicated that the cell-sample interface

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was significantly supported through the growth of lamellipodia and filopodia. Cell adhesion on

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untreated Ti was also confirmed since having the filopodia (Fig. 8a), it was showed that the

filopodia from the fibroblast linked to the substrates to the cell surface by formation of focal

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adhesion. Simultaneously, there was no evidence for cell focal adhesions on TiO2. Neither filopodia

nor lamellipodia were identified from cell morphology (Fig. 8b). It suggested lower capacity of TiO2
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in biological functions than Ti/Col-1 and Ti. In conclusion, Ti/Col-1 was evaluated to be more
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bioactive for cell adhesion.

3.5.2 Cell spreading

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Cell spreading among Ti, TiO2 and Ti/Col-1 was evaluated after 1 day of incubation. SEM images
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(Fig. 8 and 9) shows that the number of cell in three samples were increased fully on the substrate,

however, cell morphology was clearly different. The edge of cell on Ti substrate (Fig. 9a) appeared
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the lamellipodia that traced a sign of strong adhesion and a ready-state of migration. This was also
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identified in Ti/Col-1 after 1 hour of incubation (Fig. 8c). On the other hand, there was a change in

cell morphology on TiO2 substrate (Fig. 9b). The cell spread and stretched but at a restraint state,

lamellipodia or filopodia stilled hindered reflecting the weak cell-TiO2 interaction. Fig. 9c shows the

most significant in cell morphology since that cell spread and stretched largely, and began to have

cell-cell interaction. It can be concluded that the speed and capacity of cell spreading on Ti/Col-1

was higher than the others. These samples were collected after 1 day of cell incubation, with

corresponding Ti/Col, Ti, and TiO2 in the order of reducing adhesion strength.
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3.5.3 Cell viability and proliferation

MTT was used to evaluate cell viability and proliferation after 1, 3 and 5 days of Ti, TiO 2, and

Ti/Col-1. The results of MTT assay are shown in Fig. 10. It is shown that the number of viable cells

on three types of surfaces kept increasing after each incubation period indicating that all these three

surfaces are suitable for fibroblast cell proliferation. However, Ti/Col-1 always had the highest cell

number for all 1, 3, and 5 days, whereas the Fig.s for TiO2 were always lowest. At day 3 and 5, there

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was no significant difference between Ti and Ti/Col-1.

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4. Discussion

Ti and its alloys have been popularly applied in dental and orthopedics implantation by its TiO 2 film

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that prevents the corrosion, and the bio-inert property [12]. However, to enhance its bioactivity such
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as stimulating the cell-implant interaction, Ti surface needs the modification. The increase in cell-

implant interaction results in the reduction in risk of implant’s dislocation or implant’s rejection.
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Therefore, Col-1, functions as an ECM of gingival fibroblast [18], was introduced to Ti implant
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using ECD method to augment the bioactive behavior of the scaffold, attracting cell migration to

implant site, and forming cell-implant interaction.

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In ECD, more number of Col-1 molecules charging positively resulted in more Col-1 deposited on
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cathode Ti (-). The influence of deposition parameters such as: concentration of Col-1, pH of

electrolyte and deposition potential which varies the total amount of electric charge was investigated.
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The increase in concentration and depostition potential resulted in more positively charged Col-1

molecules participating in ECD process, thus more Col-1 deposited on Ti surface after coating.

Values of Col-1 concentration and deposition potential refered from previous researches of ECD

modification on Ti implants and Col-1 coating method [16, 17]. Additionally, Col-1 has isoelectric

point (iP) varying correspondingly to pH of solution, therefore positive charge of Col-1 solution

relies on its pH. This iP is around 5-7 depending on the source of Col-1 and the used buffer [42, 43],
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and Col-1 will charge positively if pH of solution is lower than iP and vice versa. pH of Col-1

electrolyte solution was studied around its iP values to optimize the postive charge. Another reason

for pH investigation was to avoid the denaturation of Col-1. At low pH condition, Col-1 is denatured

since the triple helix structure can be loosened and changed to gelatin that easily decomposes in

aqueous environment [42]. Therefore, optimization of Col-1 coating on Ti by ECD method included

these three parameters.

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Adding to ECD variables optimizing, Ti was also treated with NaOH 4M before deposition process

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to produce the porous topography TiO2. The natural TiO2 layer is formed spontaneously on Ti as

exposing to the air [44], however, NaOH treatment helps to produce a fine nanoscale network

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structure on the surface of Ti, which enhances the adhesion strength and bioactivity of the implants

[16, 45]. The optimized ECD parameters were at Col-1 concentration, pH, and deposition potential
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equaled 0.5 mg/ml, 5.5, and 2.0 V respectively, which provided successfully coherent thin Col-1
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coating on TiO2 surface. AFM images (Fig.6) indicated the nanoscale surface of TiO2 and Ti/Col-1

and proved that the coating of Col-1 on TiO2 substrate resulting in a higher roughness morphology
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compared with the uncoated substrate.

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To confirm the Col-1 coating on the porous TiO2 surface, the pure Col-1, TiO2 (Ti/NaOH) and

Ti/Col-1 (Ti/TiO2/Col-1) were compared by their XRD pattern since XRD has been studied on
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collagen and remarked Col-1 differently from almost other protein [46]. XRD detects the
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composition of the sample on an atomic level, the presence of Col-1 on the Ti surface was determied

since having one more peak at 180 comparing with the uncoated Ti and TiO2 in XRD profile (Fig. 7).

The peaks in TiO2 (Ti/NaOH) were not identified in crystalline structure detected in Ti/Col-1 profile

although Ti had gone through NaOH treatment before coating with Col-1. Otherwise, the XRD

pattern of Ti/Col-1 was nearly the same as untreated Ti (Ti/TiO2) except one more peak at 180

demonstrating the deposition of Col-1. The Ti/Col-1 fabricated was then seeded with fibroblasts to

test for cell adhesion/spreading, proliferation, and viability. The results (Fig. 8 and 9) showed that
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Ti/Col-1 was the most favorable matrix for cell adhesion evidenced by the growth of lamellipodia, a

cytoskeletal protein actin projection found at the edge of mobile cell [47] and filopodia that

functions as a sensor of cell when contacting new environment [48]. Both lamellipodia and filopodia

have been believed to be motor for migration process of cell [48, 49], hence, their appearance

proved Col-1 layer on Ti providing not only an appropriate matrix for fibroblast adhesion but also

migration. Cell viability (Fig. 10) results from MTT assay yielded that Ti/Col-1 had great potential

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for fibroblast cell proliferation compared with untreated Ti and TiO2. As a result, ECD was proved

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to be an effective method to modify Ti with Col-1 acting as scaffold for fibroblast attachment in this

study and offering a new direction for future research on dental implantation.

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Nevertheless, in case of working on molecular level, a limitation of ECD method is to apply only for

modification of extremely thin coating layer on base metal, namely Ti. When coating Col-1 layer
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on Ti substrate, Col-1 molecules bonded covalently on Ti substrate as Cathode (-) by positive
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charges. The increasing Col-1 molecules adhered on Ti surface hide the contact areas of Cathode (-)

with later ones, therefore it is impossible to control uniformity of thick coating. In fact, ECD process
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without parameter optimization will cause a lumpy and uneven coating that is not appreciated for
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cellular activity.
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5. Conclusion
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The optimization of ECD condition for modification of Col-1 on TiO2 was at concentration of 0.5

mg/ml, pH 5.5, and deposition potential of 2 V. Ti, TiO2, and Ti/Col-1 samples expressed the

biocompatibility by cell adhesion and cell spreading in vitro. However, Ti/Col-1 showed rapid cell

adhesion and proliferation. For further research, in vivo characteristics of coated Ti/Col-1 are

intended to study more other biological properties.

Acknowledgment
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This research was supported by L’Oreal UNESCO for Women in Science – National Fellowship

2016.

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Tables

Table I The labels of the samples whose differently ECD parameters were evaluated

Table II AFM surface roughness analysis of TiO2 and Ti/Col-1 (Unit: nm)

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Figure Captions

Fig. 1 Electrochemical deposition (ECD) model for modification of Col-1 on Ti

Fig. 2 Ti surface (a) before and (b) after NaOH treatment at 10,000X (Scale bar: 1 µm)

Fig. 3 SEM morphology of Ti/Col-1 surfaces at different Col-1 concentration: (a). 0.3 mg/ml, (b).

0.5 mg/ml, and (c). 0.7 mg/ml (E = 2.5V; t = 20 mins; d = 1.5cm; pH = 5.5) at 5,000X (Scale bar: 5

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Fig. 4 SEM morphology of Ti/Col-1 surfaces at different pH (a). 4.5, (b). 5.5, (c). 6.5, and (d). 7.5

([Col-1] = 0.5 mg/ml, E = 2.5 V; t = 20 mins; d = 1.5 cm) at 5,000X (Scale bar: 5 µm)

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Fig. 5 SEM morphology of Ti/Col-1 surfaces at different voltage values: (a). 0.5, (b). 1.0, (c). 1.5,
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(d). 2.0, (e). 2.5, and (f). 3.0 V ([Col] = 0.5 mg/ml; pH = 5.5; t = 20 mins; d = 1.5cm) at 5,000X

(Scale bar: 5 µm)

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Fig. 6 Three dimensional and two dimensional AFM topography images and cross-sectional surface
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profiles of (a) TiO2 and (b) Ti/Col-1

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Fig. 7 XRD pattern of collagen coating on the porous TiO2

Fig. 8 SEM micrographs of L929 Fibroblast cell reseeding on (a) untreated Ti, (b) TiO2 porous
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surface and (c) coated Ti/Col-1 after 1 hour at magnification of 100x, 1000x, and 8000x (Scale bar:
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100 µm, 10 µm, and 2 µm respectively)

Fig. 9 SEM micrographs of L929 Fibroblast cell reseeding on (a) untreated Ti, (b) TiO2 porous

surface and (c) coated Ti with Collagen-1 after 1 day at magnification of 100x, 1000x, and 8000x

(Scale bar: 100 µm, 10 µm, and 2 µm respectively)

Fig. 10 MTT assay results of Ti, TiO2, and Ti/Col-1. Note: Asterisk (*) indicates p < 0.05 between

groups using one-way ANOVA with Tukey’s post-hoc test

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Table 1

Col-1 concentration Deposition

Parameter Electrolytes’ pH
[mg/ml] potential [V]

0.3 5.5 2.5

Col-1 concentration
0.7 5.5 2.5
[mg/ml]
0.9 5.5 2.5

0.5 4.5 2.5

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0.5 5.5 2.5
Electrolytes’ pH

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0.5 6.5 2.5

0.5 7.5 2.5

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0.5 5.5 0.5

0.5 5.5 1.0

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Deposition 0.5 5.5 1.5
potential [V] 0.5 5.5 2
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0.5 5.5 2.5

0.5 5.5 3.0

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Table II AFM surface roughness analysis of TiO2 and Ti/Col-1 (Unit: nm)

Ra Rq Rmax Rskew Rkur

TiO2 22.7 29.4 293.6 0.09 0.95
Ti/Col-1 44.5 59.6 587 0.5 1.87

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Highlights

 The optimization of electrochemical deposition method to modify Titanium surface with

collagen type 1 is proposed.
 The mechanism relies on surface immobilization of collagen type 1 under electric field.
 The biological properties of modified material in response to fibroblast cells were analyzed.
 Titanium coated with collagen type 1 showed a promising in vitro result that initiates in vivo
experiment presently.

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