The role of surface wettability and surface charge of electrosprayed nanoapatites on the behaviour of osteoblasts

Acta Biomaterialia 6 (2010) 750–755

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Acta Biomaterialia journal homepage: www.elsevier.com/locate/actabiomat

The role of surface wettability and surface charge of electrosprayed nanoapatites on the behaviour of osteoblasts E.S. Thian a,b,*, Z. Ahmad c, J. Huang c, M.J. Edirisinghe c, S.N. Jayasinghe c, D.C. Ireland d, R.A. Brooks d, N. Rushton d, W. Bonfield b, S.M. Best b a

Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117 576, Singapore Department of Materials Science and Metallurgy, University of Cambridge, Pembroke Street, Cambridge CB2 3QZ, UK Department of Mechanical Engineering, University College London, Torrington Place, London WC1E 7JE, UK d Orthopaedic Research Unit, University of Cambridge, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK b c

a r t i c l e

i n f o

Article history: Received 9 February 2009 Received in revised form 18 June 2009 Accepted 7 August 2009 Available online 9 August 2009 Keywords: Carbonate Electrospraying Nanoapatites Osteoblasts Silicon

a b s t r a c t A new deposition method is presented, based on electrospraying, that can build bioceramic structures with desirable surface properties. This technology allows nanoapatite crystals, including hydroxyapatite (nHA), carbonate-substituted HA (nCHA) and silicon-substituted HA (nSiHA), to be electrosprayed on glass substrates. Human osteoblast cells cultured on nSiHA showed enhanced cell attachment, proliferation and protein expression, namely alkaline phosphatase, type 1 collagen and osteocalcin, as compared to nHA and nCHA. The modification of nanoapatite by the addition of silicon into the HA lattice structure renders the electrosprayed surface more hydrophilic and electronegatively charged. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The repair of a bone defect site remains a challenging issue for the orthopaedic, maxillofacial and dental surgeons. Currently, the most frequently used implant materials include titanium alloys, 316L stainless steel and cobalt–chromium alloys [1]. To improve the overall performance of skeletal prostheses, calcium phosphate coatings are often applied onto the implants, rendering the surfaces more bioactive and thereby encouraging bone fixation [2]. Hydroxyapatite (HA, Ca10(PO4)6(OH)2) is widely used in bone tissue engineering due to its biocompatibility, bioactivity and osteoconductivity [3]. The ability of the lattice structure of HA to accommodate substitutions of various cationic or anionic ions has been investigated extensively in recent years. Both carbonate-substituted HA (CHA) and silicon-substituted HA (SiHA) have shown to enhance bone apposition rate as compared to phase-pure HA [4–7]. Nanostructured HA has also been found to enhance osteoblast adhesion [8,9]. As such, the design of substituted HA with nanoscale features has the potential to lead to the develop-

ment of a novel coatings or coating compositions for orthopaedic, maxillofacial and dental applications. A number of techniques for the deposition of thin coatings have been investigated over recent years, and these include sputtering [10–12], electrospraying [13,14], pulsed laser deposition [15,16] and the sol–gel method [17,18]. Electrospraying appears to be a promising technique to deposit nanoapatites onto implant surfaces. As it is a room-temperature process using apatite suspension, it is now possible to retain nanoapatite crystals, in their original form, once the solvent has evaporated. The cell–biomaterial interaction is modulated by the surface characteristics of the material. Alterations in the surface chemistry, composition, roughness, wettability and charge can influence cell response, including adhesion, proliferation and differentiation [19–22]. For the current study, the behaviour of human osteoblast (HOB) cells cultured on these synthesized nanoapatites of HA, CHA and SiHA was evaluated in terms of proliferation as well as differentiation, with respect to surface wettability and surface charge. 2. Materials and methods

* Corresponding author. Address: Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117 576, Singapore. Tel.: +65 6516 5233; fax: +65 6779 1459. E-mail address: [email protected] (E.S. Thian).

2.1. Electrospraying Nanohydroxyapatite (nHA), nanocarbonate-substituted HA (nCHA) containing 0.9 wt.% carbonate and nanosilicon-substituted

1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.08.012

E.S. Thian et al. / Acta Biomaterialia 6 (2010) 750–755

HA (nSiHA) containing 0.8 wt.% Si were first synthesized based on a room temperature wet precipitation reaction between calcium hydroxide and orthophosphoric acid solutions [23]. Carbon dioxide gas and tetraethoxysilane solution were used as the source for carbonate and Si, respectively. Electrospraying was then performed to produce a fine apatite spray using nanoapatite suspensions (diluted to 6.4 wt.% in ethanol) [24]. Briefly, the suspension was delivered through a stainless steel needle of inner diameter 500 lm via an automated syringe pump at a controlled flow rate of 7 ll min 1 in an electric field of 5.7 kV, where spray samples were then collected on glass microscope slides. All samples were later heat-treated at 400 °C for 4 h.

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secondary biotin-conjugated rabbit anti-mouse antibody (DAKO, UK) was added and incubated at 37 °C for 1 h. A further wash was conducted before incubating at 4 °C for 30 min in Texas redconjugated streptavidin (Vector Laboratories, Peterborough, UK). Fluorescein isothiocyanate-conjugated phalloidin (Sigma, Poole, UK) was later added at 37 °C for 1 h after the samples were washed, to stain the actin filaments. After washing for three times with 0.5% Tween 20/PBS solution for 5 min, TOTO-3 (Molecular Probes, Paisley, UK) was added at room temperature for 5 min to stain the cell nuclei. Samples were then given a final wash (5 min  3) before mounting under Vectashield antifade mountant and viewed under a Leica SP2 confocal laser scanning microscope (CLSM).

2.2. Characterization of nanoapatites 2.5. Cell proliferation A JEOL 6340 field emission scanning electron microscope (SEM), operating at an accelerating voltage of 5 kV, was used to image the nanoapatite morphology. A Philips PW1730 X-ray diffractometer (XRD), operating at 40 kW and 40 mA using Cu Ka radiation, was also used to characterize the phase purity of the synthesized nanoapatite. Data were collected over a 2h range of 24–36°, with a step size of 0.05° and a dwell time of 6 s. A KSV CAM 200 optical contact angle meter was used to measure the contact angle of the electrosprayed nanoapatites by the sessile drop method at room temperature. A 1.0 ll drop of deionized water was generated and deposited with a micrometric syringe on the sample’s surface. An image of the drop was then captured once stabilization was achieved. For each sample, a total of 10 drops were measured and averaged. A Zetasizer Nano ZS was used to determine the zeta potential of the nanoapatites at a laser excitation wavelength of 632 nm. For each sample, six replicate measurements were performed at room temperature and at pH 7.4 in an aqueous dispersion, using ethanol solution. A Nanoscope III atomic force microscope was used to measure the roughness of the electrosprayed nanoapatites, operating in tapping mode. For each sample, six replicate measurements were performed and averaged.

Alkaline phosphatase (ALP) activity was measured fluorimetrically at 355/460 nm, following the mixing of 50 ll of cell supernatant with 50 ll of ALP assay buffer containing 6,8-difluoro4-methylumbelliferyl phosphate and incubating at 37 °C for 15 min, thereby releasing the fluorescent compound 6,8-difluoro7-hydroxy-4-methylcoumarin.

2.3. Cell culture

2.7. Type 1 collagen

An in vitro cell culture model using HOB cells was used to evaluate how cells interact with the electrosprayed nanoapatites. These primary cells were obtained from single donor hip bone during joint-replacement surgery, following local ethical committee approval and informal consent. HOBs were cultured in McCoy’s 5A medium containing 10% human male AB serum, 1% glutamine and vitamin C (30 lg ml 1). nHA, nCHA and nSiHA samples were sterilized by soaking in ethanol for 2 days before being subjected to ultraviolet radiation for 30 min. Then 20,000 cells in 1 ml of culture medium were seeded on each sample, and incubated in a humidified atmosphere containing 5% carbon dioxide at 37 °C. Standard tissue culture plastic (TCP) samples were used as test controls. Three samples were used for each assay, and a total of three replications were performed for each sample.

Type 1 collagen (COL1) production was determined by enzyme immunoassay (MetraTM CICP EIA kit, Quidel, Dorking, UK). The culture medium was first diluted with assay buffer at a ratio of 1:3. Next, 100 ll of rabbit anti-CICP was added and incubated at room temperature for 50 min. Wells were again washed three times, before 100 ll of reconstituted enzyme conjugate was added and incubated at 24 °C for 50 min. A final wash was performed, then 100 ll of working substrate solution was added and incubated at 24 °C for 30 min. Lastly, 50 ll of stop solution was added and fluorescence was read at 405 nm.

2.4. Cell attachment and spreading Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) with 1% sucrose for 15 min, washed with PBS and permeabilized at 4 °C for 5 min. Samples were then incubated with 1% bovine serum albumin (BSA)/PBS solution at 37 °C for 5 min to block the non-specific binding. Anti-vinculin monoclonal antibody (Sigma, Poole, UK) was added and incubated at 37 °C for 1 h. After washing with 0.5% Tween 20/PBS solution for 5 min, a

Cell number was determined using a CyQUANTÒ cell proliferation assay kit (Invitrogen, Paisley, UK). Briefly, culture medium was removed from the sample well and stored for later quantification of type 1 collagen and osteocalcin. Samples were washed three times with PBS solution, followed by a freeze–thaw cycle at 80 and 24 °C. Next, 300 ll of CyQUANTÒ buffer was added to each sample, sonicated for 5 min and centrifuged for 5 min at 1400 rpm to obtain the supernatant, before 100 ll of the cell supernatant was added to 100 ll of CyQUANTÒ GR dye, which exhibits strong fluorescence enhancement when bound to cellular nuclei acids. After 5 min incubation at room temperature, the microplate was read at 480/520 nm. The observed fluorescence intensity was then converted to cell number using a standard curve. 2.6. Alkaline phosphatase

2.8. Osteocalcin The osteocalcin (OC) level in the culture medium was measured by an enzyme immunoassay (MetraÒ Osteocalcin EIA kit, Quidel). Twenty-five microlitres of cell medium and 125 ll of anti-osteocalcin antibody were added to each well before incubating at room temperature for 2 h. Wells were then washed three times with 300 ll of wash buffer, following which 150 ll of reconstituted enzyme conjugate was added and incubated at room temperature for 1 h. Wells were again washed three times before 150 ll of working substrate solution was added and incubated at room temperature for 35 min. Finally, 50 ll of stop solution was added and fluorescence was read at 405 nm.

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2.9. Statistical analysis A two-tailed t-test was used to determine whether any significant differences existed between the mean values of the experimental groups. Differences between groups was considered to be significant at p < 0.05. 3. Results 3.1. Morphology of nanoapatites Micrometre-size relics were randomly deposited across the glass substrates by electrospraying. At high magnification, these structures comprised densely packed nanoapatite crystals, displaying a rod-like morphology of dimensions approximately 75 nm in length and 40 nm in width (Fig. 1). All nHA, nCHA and nSiHA exhibited similar morphology.

Fig. 2. XRD patterns of electrosprayed nanoapatites.

Table 1 Contact angle values for nHA, nCHA and nSiHA.

3.2. Phase composition of nanoapatites XRD analyses revealed the presence of several major HA peaks, thereby confirming that these nanoapatites were indeed crystalline and phase pure (Fig. 2). However, with the incorporation of carbonate and silicate ions in the HA lattice, the diffraction peaks between 2h = 31 and 33° became broader, implying that a smaller crystallite size was obtained in the nCHA and nSiHA. 3.3. Surface wettability of electrosprayed nanoapatites

a

Materials

Contact anglea (°)

nHA nCHA nSiHA

33.9 (3.1) 26.9 (3.1) 16.6 (3.0)

Errors are ± standard errors of the mean, with the values in parentheses.

Table 2 Zeta potential values for nHA, nCHA and nSiHA. Materials nHA nCHA nSiHA

Among the electrosprayed nanoapatites, nHA presented the highest contact angle value, followed by nCHA and then nSiHA (Table 1). a

Zeta potentiala (mV) 0.20 (0.09) 0.36 (0.11) 0.83 (0.14)

Errors are ± standard errors of the mean, with the values in parentheses.

3.4. Surface charge of electrosprayed nanoapatites The zeta potential values of nHA, nCHA and nSiHA are shown in Table 2. The highest and lowest values were obtained for nHA and nSiHA, respectively, with nCHA presenting an intermediate value.

Table 3 Roughness values for nHA, nCHA and nSiHA.

3.5. Surface roughness of electrosprayed nanoapatites From Table 3, it was found that nSiHA has the highest roughness value, followed by nCHA and then nHA. However, the roughness values of these nanoapatites were statistically insignificant.

a

Materials

Roughnessa (nm)

nHA nCHA nSiHA

38 (12) 45 (15) 48 (12)

Errors are ± standard errors of the mean, with the values in parentheses.

3.6. Cell attachment and spreading Immunofluorescence staining at day 1 revealed that cells were spreading and stretching well since actin bundles were apparent on all electrosprayed nanoapatites (Fig. 3). In addition, more vinculin adhesion plaques were observed for cells that had attached on nSiHA as compared to nHA and nCHA, indicating that cell adherence was more enhanced on nSiHA. 3.7. Cell proliferation

Fig. 1. SEM morphology of electrosprayed nHA.

The CyQUANTÒ assay revealed that the number of HOB cells on both nHA and nSiHA increased from day 2 to day 8, but those on nCHA appeared to decrease (Fig. 4). Generally, cells multiplied significantly on nHA and nSiHA as compared to nCHA at each time point. The number of cells growing on nHA and nSiHA decreased after day 8 whilst those on nCHA seemed to increase slightly. Normally, a decrease in cell proliferation signals that cells are advancing into the differentiation stage since a reciprocal relationship between proliferation and differentiation has been described in the development of osteoblast phenotype [25].

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Fig. 3. CLSM images of nuclei (stained blue), actin bundles (stained green) and adhesion plaques (stained red) in HOB cells at day 1 on (a) nHA, (b) nCHA and (c) nSiHA.

70000 nCHA

nHA

nSiHA

14

60000

nCHA

nHA

c,d,e

nSiHA

12

c,d

50000

d d

d,e

40000

b,d

10

c,d,e

8

b,d

-2

Cell Density

TCP

b,d

ALP (x 10 nmol/cell)

TCP

30000

d a

20000

6 4 d

a

2

10000

0

0 Day 2

Day 8

Day 14

d a

ND Day 2

Day 8

Day 14

Culture Period

Culture Period Fig. 4. Proliferation of HOB cells on TCP (acting as a control), nCHA, nHA and nSiHA samples. (a) p < 0.05 for nCHA between groups; (b) p < 0.05 for nHA between groups; (c) p < 0.05 for nSiHA between groups; (d) p < 0.05 when comparing nHA and nSiHA to nCHA within groups; and (e) p < 0.05 when comparing nSiHA to nHA within groups.

Fig. 5. Quantitative measurement of ALP activity on TCP (acting as a control), nCHA, nHA and nSiHA samples. (a) p < 0.05 for nCHA between groups; (b) p < 0.05 for nHA between groups; (c) p < 0.05 for nSiHA between groups; (d) p < 0.05 when comparing nHA and nSiHA to nCHA within groups; and (e) p < 0.05 when comparing nSiHA to nHA within groups. ND, not detected for nCHA at day 2.

3.8. Alkaline phosphatase

3.9. Type 1 collagen

The levels of ALP activity of HOBs cultured on various electrosprayed nanoapatites are shown in Fig. 5. Cells growing on nSiHA produced significantly higher amounts of ALP activity at days 8 and 14 when compared to nHA and nCHA. As for nCHA, ALP activity was only detectable from day 8 onwards.

A significant quantity of COL1 protein was expressed on nSiHA at days 8 and 14 than on nHA (Fig. 6). In contrast, HOBs cultured on nCHA started to secrete COL1 only from day 8 onwards. The level of COL1 expression seemed to level out or slow down on nSiHA at day 14.

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TCP

nCHA

nHA

nSiHA

c,d,e

5

d,e

b,d

-3

-1

COL1 (x 10 ng mL /cell)

6

4

d

d

d a

3 2 1 ND

0

Day 2

Day 8

Day 14

Culture Period Fig. 6. Quantitative measurement of COL1 production on TCP (acting as a control), nCHA, nHA and nSiHA samples. (a) p < 0.05 for nCHA between groups; (b) p < 0.05 for nHA between groups; (c) p < 0.05 for nSiHA between groups; (d) p < 0.05 when comparing nHA and nSiHA to nCHA within groups; and (e) p < 0.05 when comparing nSiHA to nHA within groups. ND, not detected for nCHA at day 2.

3.10. Osteocalcin OC was detected as early as day 2 for cells attaching on nSiHA, and increased with culturing time (Fig. 7). In contrast, for nHA and nCHA, the onset of OC expression was seen only from day 8 and 14, respectively. A higher level of OC release was observed on cells seeded on nSiHA as compared with nHA and nCHA at all time points. 4. Discussion Electrospraying provides the ability to deposit nHA, nCHA and nSiHA crystals onto glass substrates. For the current work, the effects of surface wettability and surface charge of these nanoapatites on cell adhesion, proliferation and differentiation were evaluated. The static contact angle measurements revealed that, among the nanoapatites studied, nSiHA has the lowest contact angle, suggesting that it is more hydrophilic than nHA and nCHA. Conversely, nHA has the highest contact angle, rendering it hydrophobic in nature. At a physiological pH of 7.4 these nanoapatites exhibited a net negative charge, as obtained from the zeta potential measurements. More importantly, nSiHA was more electronegatively charged as compared to nHA and nCHA. There was no significant difference in the roughness values of the various nanoapatites, as determined by the atomic force microscopy measurements. As

3.5

TCP

nCHA

nHA

c,d,e

nSiHA

2.5

c,d,e

b,d

-5

-1

OC (x 10 ng mL /cell)

3

2 1.5

b,d

d,e

a

such, one is able to assume that these electrosprayed nanoapatites exhibited identical topography. In this regard, the cell response is primarily governed by the surface chemistry (wettability and charge) of the nanoapatites. These findings demonstrated that the underlying material surface characteristics, namely wettability and charge, could be used to control cell behaviour. The current data indicated that cell attachment was greatly enhanced on more wettable substrata, which was the case for nSiHA, since more distinct and thick vinculin adhesion plaques were observed within cells. Similar results were obtained by other studies, indicating the positive influence of hydrophilicity towards cell adhesion [26,27]. This effect led to a significant increase in cell viability and proliferation on nSiHA. It was also postulated that certain proteins (mainly positive charge groups) adsorbed more effectively to nSiHA due to its higher negative surface charge. These adsorbed proteins are likely to trigger specific mRNA for gene expression, thereby carry the coding information to the ribosome sites of protein synthesis in the cell. There was clear evidence in this study that nSiHA encouraged the appropriate cell signalling to stimulate osteoblast outgrowth. The protein expressions of ALP, COL1 and OC were statistically higher on nSiHA throughout culturing, implying that nSiHA induced osteoblast differentiation and maturation at early time points. However, the exact mechanism by which how Si affects gene expression remains unknown and needs further investigation. When cell confluence is reached, osteoblasts will start to differentiate, and this phenomenon is observed on nSiHA at early time points. ALP activity is an early marker of osteoblast differentiation in vitro which is known to increase during cell differentiation, marking the mature phenotype of bone-forming cells. This pattern was observed in the study when HOB cells were cultured on nSiHA. A similar profile was observed on nCHA and nHA, but at later time points. The increased of ALP activity is then followed by increased synthesis of bone matrix protein COL1 and the finally deposition of non-collagenous extracellular matrix protein OC. OC is a late differentiation marker for osteoblast and its existence denotes the onset of matrix mineralization. The presence of COL1 and OC proteins was significantly increased on nSiHA, thereby suggesting that surface wettability (hydrophilic surface is preferred) and surface charge (electronegatively charged surface is preferred) affect bone formation by influencing osteoblast proliferation and differentiation. Interestingly, cell viability was lower on nCHA as compared to nHA, despite its better wettability and higher negative surface charge. This phenomenon was due to the high solubility rate of nCHA among the studied nanoapatites, owing to its relatively low crystallinity, rendering the surface unfavourable for cell attachment [24]. This effect induced lower cell attachment and thereby delayed osteoblastic proliferation and protein expression. In principle, it is difficult to evaluate whether surface topography or surface chemistry is more influential towards cell response since both parameters are indispensable. Generally, it may not seem to be so crucial how the cell–material interaction is influenced so long as the surface leads to improved material characteristics, favouring cell activity.

1 0.5 0

ND ND Day 2

5. Conclusions

ND Day 8

Day 14

Culture Period Fig. 7. Quantitative measurement of OC expression on TCP (acting as a control), nCHA, nHA and nSiHA samples. (a) p < 0.05 for nCHA between groups; (b) p < 0.05 for nHA between groups; (c) p < 0.05 for nSiHA between groups; (d) p < 0.05 when comparing nHA and nSiHA to nCHA within groups; and (e) p < 0.05 when comparing nSiHA to nHA within groups. ND, not detected for nCHA at days 2 and 8, and nHA at day 2.

On the basis of the current findings, electrosprayed nanoapatites were able to stimulate initial attachment, proliferation and differentiation of osteoblast cells in vitro. Enhanced protein expression, namely of ALP, COL1 and OC, at an early time point was observed with the substitution of silicon into the HA crystal lattice. This effect was due to the chemical modification of the nanoapatite, which significantly influenced the surface wettability

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and increased the surface charge. Electrospraying has proven to be an excellent technique for depositing bioactive nanobiomaterials. Acknowledgement The authors acknowledge support from the Engineering and Physical Sciences Research Council, United Kingdom under Grant Numbers GR/S97873 and GR/S97880, and Ministry of Education Academic Research, Singapore under Grand Number R-265-000311-133. Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figure 3, is difficult to interpret in black and white. The full colour images can be found in the on-line version, at doi: 10.1016/j.actbio.2009.08.012. References [1] Hench LL. Biomaterials. Science 1980;208:826–31. [2] Lacefield WR. Materials characteristics of uncoated/ceramic-coated implant materials. Adv Dent Res 1999;13:21–6. [3] LeGeros RZ, LeGeros JP. Dense hydroxyapatite. In: Hench LL, Wilson J, editors. An introduction to bioceramics. Singapore: World Scientific Publishing; 1999. p. 139–80. [4] Doi Y, Shibutani T, Moriwake Y, Kajimoto T, Iwayama Y. Sintered carbonate apatites as bioresorbable bone substitutes. J Biomed Mater Res 1997;39: 603–10. [5] Porter AE, Patel N, Brooks RA, Best SM, Rushton N, Bonfield W. Effect of carbonate-substitution on the ultrastructural characteristics of hydroxyapatite implants. J Mater Sci Mater Med 2005;16:1–9. [6] Patel N, Best SM, Bonfield W, Gibson IR, Hing KA, Damien E, et al. A comparative study on the in vivo behavior of hydroxyapatite and silicon substituted hydroxyapatite granules. J Mater Sci Mater Med 2002;13: 1199–206. [7] Porter AE, Patel N, Skepper JN, Best SM, Bonfield W. Effect of sintered silicatesubstituted hydroxyapatite on remodelling processes at the bone–implant interface. Biomaterials 2004;25:3303–14. [8] Evis Z, Sato M, Webster TJ. Increased osteoblast adhesion on nanograined hydroxyapatite and partially stabilised zirconia composites. J Biomed Mater Res 2006;78A:500–7. [9] Sato M, Sambito MA, Aslani A, Kalkhoran NM, Slamovich EB, Webster TJ. Increased osteoblast functions on undoped and yttrium-doped nanocrystalline hydroxyapatite coatings on titanium. Biomaterials 2006;27:2358–69. [10] Jansen JA, Wolke JGC, Swann S, van der Waerden JPCM, de Groot K. Application of magnetron sputtering for the producing ceramic coatings on implant materials. Clin Oral Implant Res 1993;4:28–34.

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