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Surface parameters modification by multilayer coatings deposition for biomedical applications

Studies are presented of the surface parameters of various multilayer coatings, namely, TiN, CrN, (Ti, Cr)N, TiN/TiC10N90, TiN/TiC20N80 deposited by means of Arc-PVD on stainless steel (1H18N9), as well as of the same coatings with an additional Al2O3 film deposited by reactive magnetron sputtering (RMS). The surface thickness, roughness and topography are estimated. Other parameters, such as the surface free energy (SFE) and fractional polarity are determined by means of the Wu and the Owens-Wendt-Rabel-Kaelble methods. Experiments are carried out on the in vitro cell/material interaction (in a fibroblasts culture) in order to determine the materials biomedical response. The results show some correlation between the surface properties and cell adhesion. The best biological response parameters (cell number, proliferation function, morphology) are obtained in the case of coatings with the highest values of the polar part component of the SFE and the fractional polarity, such as TiN, TiN/TiC10N90 and oxide coatings.


Introduction
Nearly all materials used for dental or orthopedic prostheses and coronary stents are pure metals or metal alloys. One way of solving the problem of improving the characteristics of metal prostheses is the deposition of multifunctional coatings on their working parts. Oxide and nitride multilayer coatings have been used for orthopedic and dental implants due to their high hardness and wear and corrosion resistance. The biocompatibility of these biomaterials is tested before surgical implantation. One criterion for the suitability of a given material for bio-applications involving close tissue contact is the physicochemical reactivity of the surface. In vitro studies of cell adhesion on various material and coating surfaces are the basic tools to determine the material surface/tissue response on a cellular level [1,2]. The effects of the materials composition, surface chemistry and surface topography on the cell adhesion and proliferation have been largely studied. The substrate roughness affects significantly the cell attachment, adhesion, proliferation and differentiation [3]. The surface energy is also a fundamental property that can have influence on the cell behavior [4]. The aim of the present study was to perform a comparative analysis of the surface parameters of various types of multilayer coatings with respect to the direct cell adhesion, spreading and proliferation on the tested surface in in vitro tests.

Materials and methods
The substrates used for coatings deposition were stainless steel 1H18N9 samples. The substrates were cleaned in an ultrasonic bath by means of a standard technology. The TiN, CrN, (Ti,Cr)N, TiN/TiC 10 N 90 , TiN/TiC 20 N 80 coatings were deposited by Arc-PVD. The process was described in detail in our previous study [5]; its main parameters were as follows: reactive atmosphere N 2 , deposition pressure 8 10 -2 Pa, arc current 100 A, substrate bias 150 V, deposition temperature 593 K, thickness of deposited coatings 2 -5 μm. The Al 2 O 3 coating deposition was performed by reactive magnetron sputtering (RMS) in a high-vacuum pumping system at a pressure of about 10 -5 mBar. The main process parameters were: magnetron discharge power 1-8 kW, oxygen source power 1 kW, deposition rate 8 μm/hour [6]. The surface topography and adhered cell morphology was observed by scanning electron microscopy (SEM, S-450, Hitachi, Japan) and atomic force microscopy (AFM Q-Scope-250, Quesant Instrument Corporation, USA). An analysis of was made of the surface parameters, namely, surface roughness, surface free energy, fractional polarity. The surface roughness was measured by means of a Hommel T-2000 profilometer. The roughness and thickness values of the coatings investigated are presented in table 1. The contact angles were measured by means of a tensiometric method. Prior to these measurements, the samples were ultrasonically cleaned in acetone and deionized water and dried. The advancing water contact angle was measured by Wilhelm's method (Kruss K12) at a temperature 20 o C [6]. Standard liquids were used with well-known values of the surface tension, and the dispersion and polar interaction components, such as water, formamide, diiodo methane, ethylene glycol, α-bromo naphthalene and glycerol. The surface free energy (SFE) and its polar and dispersion components were determined by means of the Wu [7] and Owens-Wendt-Rabel-Kaelble [8] methods.
The experiments on cytotoxicity and cytocompatibility were carried out in vitro, in a culture of fibroblasts. In process of cell cultivation with coated samples, the cell cytology, morphology and proliferation activity were determined after 24 h and 3 and 5 days of cultivation. Rat hypodermic cellular tissue was extracted to obtain the initial fibroblast culture. The suspension of extracted cells was centrifuged at 750 rev/min during 15 min. The sowing cell area was 3 10 5 cell/ml density of cultural medium. The fibroblast cultivation with 3 ml of Dulbecco Modified Eagle's Medium

Surface free energy (SFE) estimation
The calculation of the surface free energy of solids from contact angle measurements is based on the surface energy balance condition: The values of the surface free energy and its polar and dispersion components calculated by Wu method for two liquids and the Owens-Wendt-Rabel-Kaeble's method for the liquid system α-bromonaphthalene-formamide-ethylene glycol-diidomethane-glycerol-water were determined from contact angle measurements at 20 o C. The advancing water contact angles of the samples were in the range 60-70 degrees at standard condition and the surface energies values changed from 40-50 mN/m depending on the material and its surface properties (table 2).The polar part of SFE and the fractional polarity vary from 3.9 -4.7 (0.086-0.107) for (TiCr)N and TiN/TiC 20 N 80 to 7.0-8.2 (0.152-0.17) for TiN, TiN/TiC 10 N 90 and 9.9(0.24) for oxide coatings. Figure 1 shows the adhered cell morphology for TiN, TiN/TiC 10 N 90 , TiN/TiC 20 N 80 coatings after 5 days cultivation. The cell counts by hemocytometry demonstrated the characteristic features of cell spreading and proliferation on the control and sample surface after 1-, 3-and 5-day stay in the culture. The fibroblast cells were well spread both on the control and on the coated surfaces. The cell morphology was typical for cells on a coated surface. The fibroblast cells had polygonal forms -spindle and oblong with cytoplasm micro-filaments. The nucleus was large with chromatin. There existed a large number of cells at the mythos stage. Moreover, a large amount of naked nuclear cell clumps was observed for the TiN/TiC 20 N 80 coatings. Differences were detected in the cell

Results and discussion
Previous studies have examined the surface energy effect on cell functions, such as adhesion, proliferation and differentiation. In some cases, the cell functions are enhanced on hydrophilic surfaces, in other cases, on hydrophobic. The average cell area after 24 h has a maximum at a contact water angle of 60 o [9].

Conclusion
The initial cell behavior on the biomaterial interface will influence the cell differentiation, proliferation and extra cellular matrix formation. The surface topography, roughness, energy and chemistry are the main factors affecting the cell growth and function. We performed a comparative analysis of the cell adhesion on the surface of the samples with multilayer coatings and a study of the surface parameters influence on the cell/material interaction in in vitro tests. The results show some correlation between the surface properties and cell adhesion. The best biological response parameters (cell number, proliferation function, morphology) were obtained in the case of coatings with the highest parameters of the polar part component of SFE and the fractional polarity, such as TiN, TiN/TiC 10 N 90 and oxide coatings. Higher surface energy and fractional polarity result in a greater number of attached fibroblast cells, and higher cell activity and proliferation ratio.