Calcium Phosphate Coating of Nickel

The 11th Iranian Chemical Engineering Congress (ICHEC11) November 28-30, 2006, Tehran, Iran

PUMC Zinc/Calcium Phosphate Coating of Nickel–tungsten Amorphous Nanocomposites. Coating Procedure and Release of Nickel in Human Cell Cultures. H. Khoshvaght1, M.R. Arshadi2, A. Khoshvaght3, M. Pouralamdari4, and M.G. Hosseini5

1

Electrochemistry Research Laboratory, Department o f chemistry, Sharif University of Technology, Tehran, Iran Corresponding Author E-mail: [email protected]

Abstract Nickel–tungsten amorphous nanocomposites (NiW-ANC) were coated with zinc/calcium phosphate by dipping in oversaturated zinc/calcium phosphate solution. The layer thickness (typically 1–15 mm) can be varied by choice of the immersion time. The porous nature of the layer of microcrystals makes it mechanically stable enough to withstand strong bending of the material. This layer may improve the biocompatibility of NiW-ANC, particularly for osteosynthetic devices by creating a more physiological surface and by restricting a potential nickel release. The adherence of human leukocytes (peripheral blood mononuclear cells and polymorphonuclear neutrophil granulocytes) and platelets to the zinc/calcium phosphate layer was analyzed in vitro. In comparison to non-coated NiW-ANC, leukocytes and platelets showed a significantly increased adhesion to the coated NiW-ANC. Keywords: NiW-ANC, Metallic implants, Zinc/Calcium phosphates, Coating

1

Master of Science from Sharif University of Technology, Tehran, Iran professor of chemistry of Sharif University of Technology, Tehran, Iran 3 Master of Science Student from University of Mashhad, Mashhad, Iran 4 Master of Science from Sharif University of Technology, Tehran, Iran 5 Assistant of professor of Tabriz University, Tabriz, Iran 2


1. Introduction The interest in electrodeposited alloys of tungsten and molybdenum with iron group metals is due to their specific magnetic, electrical, mechanical, thermal and corrosion properties[1-2]. Nanocomposites metallic materials are generally limited in practical applications because of their severe brittleness. It has not yet been well understood whether their brittle behavior is due to an intrinsic feature of nanocomposites materials, or whether this is due to processing difficulties for the fine grain sizes such as an imperfect consolidation of the Nanocomposites powders [3]. On the other hand, electrodeposition is a superior technique for producing Nanocomposites materials having grain sizes anywhere from the essentially amorphous to nanoscaled materials for the grain sizes of about 5-50 nm in bulk form or as coatings with no post-processing requirements. Amorphous nanocomposites (ANC) on the basis of nickel–tungsten (chemical formula NiW; Nitinol®) possess interesting properties that have stimulated great interest in clinical medicine. In clinical use are stents for various applications [5–7], orthodontic wires [8] and staples for foot surgery. Other applications like osteosynthetic devices [9] and bone substitution materials [10] have also been suggested [4]. These excellent mechanical properties are certainly of great potential use in clinical medicine, but the high nickel content of the material has triggered studies on the biocompatibility of this material. Of course, the problems of acute nickel toxicity [11] and of allergic reaction [12] have to be addressed for all the above devices. However, some open questions remain about possible release of nickel from nickel–tungsten alloys. For instance, cytokine synthesis was induced in vitro in monocytes and microvascular endothelium cells [13] and in peripheral blood mononuclear cells (PBMC) [14], corrosion products of NiW gave adverse effects on smooth muscle cells [6], a local accumulation of nickel was observed [15], fluoride was found to enhance the corrosion rate of NiW [16], and the surface .Finish appears to play a significant role for nickel ion release [17]. In general, from reviewing the literature it can be concluded that nickel–tungsten amorphous nanocomposites are useful for many medical devices but, as they contain so much nickel, there remain reservations about the long-term performance of implants. To extend the potential for osteosynthetic devices made of nickel–tungsten amorphous nanocomposites (NiW-ANC), we have coated the material with calcium phosphate (a) to further improve the biocompatibility in bone contact, following the strategies developed for femoral and dental endoprostheses, and (b) to further suppress the release of nickel that occurs initially within the first days and possibly after mechanical load application at the implantation site.


2. Materials and Methods 2.1. NiW-ANC Electroplating Process A 500 ml bath was set-up, and the applied DC current density. Before each experiment, samples were cut and a connecting rod of the same material was attached at the back of each sample to allow for an external electrical connection. Each specimen was fixed in an epoxy resin (Dexter Hysol, resin EE4183, hardener HD3561). Each sample was then wet-polished with silicon carbide paper from 180 to 2400 grit finish, polished again with an aqueous suspension of Al2O3 of 0.3 nm and then with 0.05 nm grain size. Finally, the electrodes were washed again with high-purity water bath; the nickel–tungsten alloy electrodeposition was conducted using current pulse plating method (Fig1B and C). Current pulse plating method

was

used

to

control

surface

topography

and

composition

of

the

deposits.

A

BUEHLER/METASERV mounting press was used for preparation of specimens. The result reported is an average of three experiments performed under identical conditions. 2.2. PUMC Zinc/Calcium Phosphate Coating Procedure NiW-ANCs were cut into rectangular pieces (typical dimensions: 10-10-0.3 mm3) and cleaned with acetone (10 min), ethanol (10 min) and distilled water (rinsing).The plates were then boiled for 60 min in 30% aqueous H2O2 and rinsed again with distilled water etching was performed with 4 m aqueous KOH for 30 min at 120°C, followed by multiple rinsing with distilled water. It is also possible to etch the material with saturated aqueous Ca(OH)2 solution instead of KOH (170°C, 20 h, closed vessel). After this etching step, the plates were immersed at 54°C in supersaturated calcification solution (SCS) containing the following ion concentrations (Table1) : Table1. Supersaturated calcification solution

Slurry of zinc oxide was first prepared with water. Nitric acid and phosphoric acid were added successively with constant stirring. Finally calcium carbonate was added to the mixture in small installments under stirring till it dissolved completely. Phosphate coatings such as these not only improve


the corrosion inhibition of the metal surfaces, they also increase the adhesion of lacquers subsequently applied to the surface. In addition, they are able in certain cases to contribute towards improving the properties of metal sheets for cold forming and for seep drawing (Fig1A and B). Cell seeding experiments were performed for nickel–tungsten amorphous nanocomposites (NiWANC) and PUMC zinc/calcium phosphate coating of nickel–tungsten amorphous nanocomposited materials.

Fig1. A) EDS analyze of PUMC zinc/calcium phosphate, B) SEM image of PUMC zinc/calcium phosphate and NiW-ANC and C) EDS analyze of NiW-ANC

2.3. Instruments Surface morphology of the deposit was tested with scanning electron microscope (model XL30 made in Philips CO.) in 20 kV. The composition of the deposits on top surface was measured by energy dispersive spectroscopy (EDS) equipped in the SEM instrument. The morphology of the deposits were determined using the atomic force microscope (AFM) and scanning tunneling microscope (STM). The AFM and STM were performed using the Digital ÑM TERMOSCOPES auto probe. The surfaces were scanned using contact mode of scanning. The Ni+2 content was determined using inductively coupled plasma spectroscopy (ICP) having VARIAN/VISTA-PRO CCP simultaneous ICP-OES system. The X-ray diffraction (XRD) experiments were carried out on the X-ray rotating powder diffractometer, model of Xpert (PHILIPS, Netherlands) using Cu- Ka operating at potential 40 kV, tube current 30 mA and scan rate 6°/ min. The composition of the deposits was determined with a PHILIPS PW2404 X-ray fluorescence (XRF) analyzer calibrated with bulk samples of nickel and tungsten. The morphology of the deposits also was determined using the scanning tunneling microscope (STM). 2.4. Isolation of leucocyte fractions Polymorphonuclear neutrophil leukocytes (PMN) and PBMC were isolated by a single-step procedure. Briefly, ethylene diaminetetraacetic acid (EDTA)-anticoagulated peripheral blood (9 ml) was diluted with an equal volume of 0.9% aqueous NaCl and carefully overlain on a double-gradient formed by layering 10 ml of aqueous polysucrose/sodium diatrizoate adjusted on 10 ml Histopaque 1119 in 50 ml tubes .The tubes were subsequently centrifuged at 700g for 30 min at room temperature. After


centrifugation, two distinct leukocyte cell layers (PBMC and PMN including eosinophil granulocytes) were obtained above the bottom sediment of erythrocytes. The cell layers were carefully aspirated and both PMN and PBMC were transferred to separate 50ml tubes which were subsequently filled with phosphate °

buffered saline. Centrifugation followed at 200g for 15 min at 4 C. After this first washing contaminating erythrocytes within the PMN fraction were removed by hypotonic lysis using 0.3% NaCl for 2 min at room temperature. After reconstitution of physiological osmotic strength in the PMN fraction both cell populations were washed again with PBS. This method led to more than 95% pure and viable PMN or PBMC. Isolated cells were adjusted to 1×106 cells ml-1 in cell culture medium. 2.5. Preparation of platelets Platelets were isolated from peripheral EDTA-anticoagulated blood via the preparation of plateletrich plasma (PRP). PRP was obtained by centrifugation of blood which was previously diluted with an °

equal volume of PBS containing 1.5% EDTA (PBS/EDTA) at 200g for 30 min at 24 C. Subsequently, the °

supernantant (PRP) was aspirated and centrifuged at 1285g for 20min at 24 C. The supernatant was discarded and the pellet was washed to remove residual EDTA. 2.6. Cell seeding experiments Coated and non-coated NiW-ANC samples were autoclaved (120 °C steam for 3 h) and were each placed in one tissue culture plate. Subsequently, 1ml of the respective cell suspension was added to the coated and non-coated samples. Cell culture followed for 24 h (37°C, 5% CO2, humanized atmosphere). Cell adherence to the sample surface was determined using fluorescence microscopy. For fluorescence analysis, the samples were carefully removed from the culture plates and washed three times with RPMI1640. Subsequently, adherent cells were stained. After stained cells were photographed using a fluorescence microscope and a digital camera.

3. Results and Discussion The surface of a coated NiW-ANC plates are shown in Fig.1, 2 and 3 .By EDX spectroscopy, AFM, SEM and X-ray powder diffraction, we found that the layer consists mainly of Sholzite crystals (CaZn2(PO4)2.2H 2O) with some content of Parasholzite crystals (CaZn 2(PO4)2.2H2O). A side view reveals

the porous nature of the film (Fig.1B). Structure and morphology of the PUMC zinc/calcium phosphate layer on NiW-ANC was identical with that on NiW. The layer is able to withstand the strong bending that is characteristic (Fig.1B). This is probably due to the high porosity of the interdigitated crystal layer that is able to accommodate extension and compression. Also adhesive strength was observed.


Fig. 2. XRD patterns of Layer of phosphate on NiW-ANC a) after 60 second b) after 20 min

Fig3. (A) Surface of NiW-ANC (top view). (B) Surface of NiW-ANC (side view). (C) PUMC-Zinc/calcium phosphate layer on NiW-ANC (top view), (D layer of zinc/calcium phosphate on NiW-ANC after immersion (top view).

Structural investigations by electron microscopy and EDX spectroscopy have revealed that the zinc/calcium phosphate coating in fact consists of two layers, as known for such zinc/calcium phosphate coatings. The first has a thickness of 1 µm or less and is directly bound to the metal surface. The second layer grows on top of the first one. In Fig. 2A, XRD of the first layer and in Fig. 2B, of the second layer can be seen. Analyses experiments and subsequent analysis by SEM and EDX have shown that the first layer always remains on the metal substrate, unless scratching is performed with a very hard object, like a steel needle. As EDX shows that the first layer also consists of zinc phosphate, it will still act as a barrier towards nickel ion release by incorporating the nickel ions into the apatite structure (ICP analyses of human cell cultures). A sufficient blocking of the Ni release from NiW-ANC is obviously a prerequisite for


development of long-lasting implants when it coated by PUMC zinc/calcium phosphate. Self-healing of a scratched surface after immersion into SCS for a second time absorbed (5b).Note the reconstruction of the original zinc/calcium phosphate layer. Fig.1. Mechanical stability of the zinc/calcium phosphate layer towards bending (left: uncoated NiW; right: coated NiW). Minor amounts of Ni ions (such as 0.5 ppm) were reported to induce allergic pathomechanisms in highly sensitized subjects [18] fistrongly adverse reactions of osteoblasts to nickel ions were observed in vitro [19]. In addition, there is a self-healing capacity of the material. As most extracellular body liquids (like blood serum or saliva) are oversaturated with respect to zinc/calcium phosphate precipitation, a surface that is capable of nucleating the crystallization will induce zinc/calcium phosphate precipitation. This can be demonstrated in vitro if a scratched surface is immersed into a supersaturated calcium phosphate solution (SCS) for a second time.The new layer has coated the defect in the topmost layer by reprecipitation of crystals (Fig.4 ).This can also be unequivocally demonstrated by EDX and SEM. The attachment of different cells to the implant surface is a prerequisite for osteointegration of an implant. Fig.4. representative microphotography of PBMC attached to a coated (A) and (B) non-coated NiW sample. After calcein-staining PBMC appear as green fluorescents cells. That representative microphotography of PMN attached to a coated (C) and a non-coated (D) NiW sample. That representative microphotography of platelets attached to a coated (E) and a non-coated (F) NiW sample. Here, we compare the capacity of peripheral blood leukocytes and platelets to adhere to zinc/calcium phosphate-coated or non-coated NiW samples since these cells will immediately get into close contact with implant surfaces. Additionally, they provide growth factors necessary to initiate and accelerate tissue repair and regeneration.

Fig4. (A abd B) Representative microphotography of PBMC attached to a coated and a non-coated NiW sample. After calcein-staining PBM appear as green fluorescent cells. (C and D) Representative microphotography of PMN attached to a coated and a non-coated NiW sample. After calcein-staining PMN appear as green fluorescent cells. (E and F) Representative microphotography of platelets attached to a coated and a noncoated NiW sample. After calcein-stainingplatelets appear as green fluorescent cells.


As shown in Figs.4, an increased adherence of leukocytes and platelets to the zinc/calcium phosphate coated samples compared to the non-coated samples was observed throughout. This increased cellular attachment was most pronounced for PMN and platelets (Figs.4B and D). Since the PBMC fraction contained considerable amounts of platelets, the increase in adherent platelets to the coated surfaces dominated the respective increase in PBMC (Fig.4B). However, also PBMC adherent to non-coated NiW surfaces showed small rosettes of platelets around the cells. Fig.5 shows the adherences of PBMCs using SEM. zinc/calcium phosphate coatings are regularly applied to metallic biomedical implants to improve osteointegration. The increased adherence of leukocytes and platelets to the phosphate-coated samples can be ascribed to an increase in surface area and a relatively rougher surface morphology compared to the non-coated metal surface. This attachment and accumulation of leukocytes (especially neutrophil granulocytes and platelets) may also induce activation of these cells and may lead to the release of cytokines, growth factors and lipid mediators into the microenvironment. On the one hand, mediators such as plateletderived factors, transforming growth factors, and insulin-like growth factors as well as leukocyte-derived cytokines. Control natural wound healing and bone formation; on the other hand, especially PMN accumulation has also been considered as a major component in the pathogenesis of in fiammatory tissue injuries. PMN may react with zinc/calcium phosphate surface structures which they cannot phagocytose and thereby release large quantities of reactive oxygen species, lytic enzymes or proinfiammatory cytokines.

Fig5. a) Self-healing and reconstruction of the original zinc/calcium layer in Human Cell Cultures and b) representative SEM of PBMC attached to coated NiW-ANC plate

However, an appropriate host response will obviously depend on the nature and the quantities of released mediators. Thus, an increased adhesion of leukocytes is desired for implant contact and tissue integration but does not assure improved biocompatibility in bone contact.

4. Conclusions A layer of zinc/calcium phosphate was precipitated on NiW-ANCs.The layer shows a sufficient mechanical stability. Even if the topmost layer is removed, there remains a thin surface layer that induces


zinc phosphate crystallization in contact with oversaturated zinc/calcium phosphate solutions (similar to blood or saliva).The properties of the NiW-ANC are improved. The gentle method of zinc/calcium phosphate deposition offers ways to control the layer thickness, the crystallographic phase (by variation of the coating solution and its pH) and potentially to incorporate biologically active compounds into the layer (like antibiotics or bone-growth factors).

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