In vitro investigation of protein adsorption and platelet adhesion oninorganic biomaterial surfaces

Huang Yana, Lu Xiaoyinga,*, Ma Jingwu a, Huang Nanb,**a State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, Chinab Institute of Biomaterials and Surface Engineering, Southwest Jiaotong University, Chengdu 610031, China

Applied Surface Science 255 (2008) 257–259

A R T I C L E I N F O

Article history:

Available online 5 July 2008

PACS:

87.85.jj

Keywords:

DLC and Ti

Surface property

Protein adsorption

Platelet adhesion

Blood compatibility

A B S T R A C T

The aim of this paper was to study the surface properties, protein adsorption and platelet adhesion

behaviors of diamond-like carbon (DLC) and titanium (Ti) films. The surface energy and microstructures

of these films were characterized by contact angle measurement and atomic force microscopy (AFM). A

modified Coomassie brilliant blue (CBB) protein assay was used to study the amount of adsorbed

proteins. Platelet adhesion was assessed by scanning electron microscopy (SEM). The AFM results show

that the DLC film is smoother than Ti. Protein adsorption results from CBB protein assay show that the

ratio of adsorbed albumin (Alb) to IgG (RA/I) on DLC is larger than Ti, which coincide with the sequence of

the ratio of interfacial tension between solid surface and Alb (gS,Alb) to interfacial tension between surface

and IgG (gS,IgG) (gS,Alb/gS,IgG). The DLC film has a preferential adsorption for Alb. The results suggest that

the ratio of gS,Alb/gS,IgG may indicate an Alb/IgG affinity ratio of materials. More platelets adhere on Ti film

than on DLC, which may correspond to the surface roughness of materials. The conclusion is the blood

compatibility of DLC seems to be better than Ti.

� 2008 Elsevier B.V. All rights reserved.

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1. Introduction

Diamond-like carbon (DLC) and titanium (Ti) are two kinds ofblood contacting biomaterials. DLC films have been used inartificial heart valves, stents, and rotary blood pumps for theirunique properties [1], and Ti has been popularly used to makeorthopedic joints and artificial heart valves. Thus the bloodcompatibility study for both DLC and Ti is very necessary.

Protein adsorption and platelet adhesion are two key issues inblood compatibility research. The interaction of proteins with abiomaterial surface occurs immediately after implantation, andthen platelets attach to the surfaces [2]. Because all these eventshappen on the materials surface, the surface chemical and physicalproperties of materials determine the protein adsorption andplatelet adhesion behavior.

In this article, the surface energy and microstructures of DLCand Ti films are first investigated by contact angle measurementand AFM. Then protein adsorption and platelet adhesion onmaterial surfaces are studied with a modified CBB protein assay

* Corresponding author. Tel.: +86 25 83793430; fax: +86 25 83792882.

** Corresponding author. Tel.: +86 28 87600625; fax: +86 28 87600625.

E-mail addresses: luxy@seu.edu.cn (X.Y. Lu), nhuang@263.com (N. Huang).

0169-4332/$ – see front matter � 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.apsusc.2008.06.192

and SEM. The relationship between surface properties and proteinadsorption and platelet adhesion behaviors are also discussed.

2. Materials and methods

2.1. Materials

DLC and Ti films were prepared on silicon wafers using a plasmaimmersion ion implantation and deposition (PIIID) system. Thecontent of sp3 bond in DLC is higher than 80%. The purity of Ti filmis as high as 99.9%. Both the samples and the parameters areprovided by the Institute of Biomaterials and Surface Engineering,Southwest Jiaotong University (China).

The proteins used were human albumin (Alb) and humanimmunoglobulin G (IgG) (Sigma, Dorset, UK). For each measure-ment, a fresh protein solution was prepared in 10 mM phosphatebuffered saline (PBS, pH 7.4). Deionized water was used to preparethe aqueous solutions. Dye reagent CBB G-250 was purchased fromSigma and prepared according to Bradford [3].

2.2. Methods

2.2.1. Surface energy and interfacial energy measurements

The surface energy of DLC and Ti films were determined bycontact angle measurement using a CAM200 optical contact angle

Table 2Concentration of adsorbed Alb and IgG on material surfaces

Protein Material

DLC Ti

Alb (mg/cm2) 2.45 2.89

IgG (mg/cm2) 1.78 3.12

RA/I 1.38 0.93

Y. Huang et al. / Applied Surface Science 255 (2008) 257–259258

system (KSV Inc., Finland). Ultrapure water and glycerol dropletswere deposited on each sample surface separately and the contactangles were measured subsequently. Three different regions ofthe surface were used for each kind of liquid. The measured resultswere averaged. The surface energy of DLC and Ti films werecalculated according to the equation in reference [4]. The interfacialenergy between different surfaces and different proteins (Alb, IgG)were worked out from the surface energy measured here referringto the formula in reference [4–6].

2.2.2. AFM characterization

The surface topographies of DLC and Ti film were observed witha commercial AFM (Nanoscope IIIa, Digital Instrument Corp., USA).The operating mode was tapping mode, the scanning rate was 1 Hzand the scanning area was 2 mm � 2 mm.

2.2.3. Protein adsorption

Quantitative evaluation of protein adsorption was studiedusing CBB protein assay [7,8]. At first, the absorbencies of proteinstandards (1, 2, 3, 4 and 5 mg/ml) were measured using a UV-4100UV/vis spectrophotometer (Hitachi Company, Japan) at twowavelengths, and standard curves were drawn respectively forAlb and IgG. For each protein, 3 samples were used (n = 3). After thesizes of samples were measured (about 0.8 cm � 0.8 cm), thesamples were placed into tubes containing 2 ml protein solution(3 mg/ml). After incubating at 37 8C for 1 h, 1 ml solution waspipetted out from each tube and mixed with 3 ml CBB G-250solution and then the absorbencies were measured. At last, theconcentration of proteins adsorbed on the material surfaces andthe value of adsorbed Alb to IgG (RA/I) on DLC and Ti film werecalculated.

2.2.4. Platelet adhesion

The samples (0.8 cm � 0.8 cm) were soaked in 2 ml platelet richplasma (PRP) for 1 h at 37 8C. Thereafter, the samples were rinsedwith a 0.9% NaCl solution carefully to remove weakly adherentplatelets. Adherent platelets were then fixed in 2.5% glutaralde-hyde solution for 12 h, then dehydrated using a graded ethanolseries (50, 60, 70, 80, 90 and 100 vol.% of ethanol) for 10 min each.The dried samples were then coated with gold and examined bymeans of SEM (E-3000N type, Toshiba Company, Japan).

3. Results and discussion

3.1. Surface energy and interfacial energy results

The value of contact angle and calculated surface energy (gS) areshown in Table 1. gp

S and gdS represent the polar component and

dispersive component of the surface energy, respectively. Thewater contact angle on Ti surface is larger than DLC, whichindicates that Ti surface is more hydrophobic than DLC. The DLCfilm has a higher polar and dispersive component of the surfaceenergy than Ti, and also has a higher total surface energy. Using gp

S

and gdS values, the interfacial tensions gS,Alb and gS,IgG between

material surfaces and proteins were calculated and also shown inTable 1. It can be seen that the ratio of gS,Alb/gS,IgG of DLC is larger

able 1urface energy measurement results

Contact angle (8) Surface energy (dyn/cm) Interfacial energy

Water Glycerol gdS gp

S gS gS,Alb (dyn/cm) gS,IgG (dyn/cm) gS,Alb/gS,IgG

LC 61.37 � 1.47 53.64 � 0.86 17.28 23.25 40.53 3.04 2.91 1.04

i 73.92 � 2.96 68.23 � 3.35 13.55 16.83 30.38 6.57 6.50 1.01

TS

D

T

than Ti, which indicates that in the Alb and IgG binary system, theDLC film has a higher Alb/IgG affinity ratio than Ti.

3.2. AFM results

Fig. 1 shows surface morphologies for 2 mm � 2 mm size scan ofthree kinds of material surfaces. The surface morphology of the Tifilm exhibits a dense insular appearance (Fig. 1b), which isdifferent from the DLC film (Fig. 1a). The root mean squared (RMS)roughness of Ti (2.49 nm) is much lager than DLC films (0.11 nm).

3.3. Protein adsorption results

The amount of adsorbed proteins on material surfaces obtainedfrom CBB protein assay are listed in Table 2. The DLC film adsorbsless protein than Ti for both Alb and IgG. Some researches showedthat more proteins adsorb on the surface with the higher surfaceenergy [4]. However, in this study, Ti film with a lower surfaceenergy adsorbs more protein than DLC. The roughness of materialsmay have a greater influence on protein adsorption than surfaceenergy for these two materials. The Ti film surface is rougher thanDLC, and more proteins are adsorbed on the Ti surface. The order ofcalculated ratio of RA/I is DLC > Ti, which coincides with the orderof gS,Alb/gS,IgG in Table 1. The results suggest that the ratio of gS,Alb/gS,IgG may indicate the protein adsorption property of materials. Aprevious study has reported that a protein adsorption ratio equal to1 means no selective adsorption occupied on material surface [4].In contrast to Ti film, which shows no selective adsorption of Alb(the RA/I is 0.93), the DLC film exhibits a preferential adsorption forAlb (the RA/I is 1.38). This property of preferential adsorption of Albis regarded as beneficial for blood compatibility of biomaterials [4].

3.4. Platelet adhesion

The morphology of the adherent platelets on DLC and Tisurfaces is displayed in Fig. 2, which is the result of randomlyselected SEM fields. On DLC surface, only a few platelets adherentand are isolated (Fig. 2a). The platelets are in round shape and withonly slight pseudopodia can be observed (Fig. 2b). In contrast,much more platelet adhesion and aggregation can be observed onthe Ti film surface compared to the DLC (Fig. 2c), and plateletspreading is observed (Fig. 2d). According to Goodman et al. [9], theactivated patterns of platelets can be classified to five classes: (a)discoid or round; (b) dendritic; (c) early pseudopodial, spreaddendritic; (d) intermediate pseudopodial, spreading; (e) fullyspread. Thus the adherent platelets on DLC surface are in loweractivated state, while the platelets on Ti are significantly activated.

Fig. 1. AFM images of material surface: (a) DLC and (b)Ti.

Fig. 2. SEM morphology of adherent platelets on material surfaces: (a) DLC, 1000� magnification, (b) DLC, 10,000� magnification, (c) Ti, 1000� magnification and (d) Ti,

10,000� magnification.

Y. Huang et al. / Applied Surface Science 255 (2008) 257–259 259

This may attribute to the surface morphology of the materials. Tisurface is rougher than the DLC film, and the insular structure canentrap platelets, causing adhesion and deformation.

Researches have shown that the adsorbed Alb can resist plateletadhesion to material surfaces [10], while adsorbed IgG will induceplatelet coagulation, and then leads to thrombosis [2]. The Alb/IgGratio can be regarded as an index when assessing the adhesion ofplatelets to material surfaces. The higher the ratio is, the lower thenumber of adherent platelets. In this study, the Alb/IgG ratio of DLCis higher than Ti, and more platelet are adhered on Ti surface. Theresult of platelet adhesion is in agreement with that of proteinadsorption.

4. Conclusions

The quantitative evaluation results from CBB protein assayshow that the ratio of Alb to IgG (RA/I) adsorbed on DLC surfaces islarger than Ti, which is in coincidence with the order of gS,Alb/gS,IgG.The ratio of gS,Alb/gS,IgG may indicate an Alb/IgG affinity ratio ofmaterials. DLC film has a preferential adsorption for Alb. Theamount of adsorbed platelets on Ti film surface is much more thanthat on DLC, which may contribute to its large roughness. Theblood compatibility of DLC seems to be better than Ti. This may

attribute to different interfacial energy between materials andproteins and surface roughness of DLC and Ti film.

Acknowledgements

This paper is supported by NSFC Project 30470478, 30770583and 60121101, Open Foundation of State Key Laboratory ofBioelectronics of Southeast University, and the Scientific ResearchFoundation of Graduate School of Southeast University, China.

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