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Biocompatible and bioactive coatings of Mn2+-doped β-tricalcium phosphate synthesized by pulsed laser deposition
Applied Surface Science 254 (2007) 1155–1159 www.elsevier.com/locate/apsusc
Biocompatible and bioactive coatings of Mn2+-doped b-tricalcium phosphate synthesized by pulsed laser deposition F. Sima a, G. Socol a, E. Axente a, I.N. Mihailescu a,*, L. Zdrentu b, S.M. Petrescu b, I. Mayer c a
National Institute for Lasers, Plasma and Radiation Physics, Box MG-54, RO-77125 Bucharest, Magurele, Romania b Institute of Biochemistry, Splaiul Independentei 296, Bucharest, Romania c Department of Inorganic and Analytical Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel Received 20 May 2007; received in revised form 1 August 2007; accepted 13 August 2007 Available online 21 August 2007
Abstract The extension of pulsed laser deposition to the synthesis on Ti substrates of b-tricalcium phosphate (b-TCP) coatings doped with manganese is reported. Targets sintered from two crystalline Mn-doped b-TCP powders (with the composition Ca2.9Mn0.1(PO4)2 and Ca2.8Mn0.2(PO4)2) were ablated with an UV KrF* (l = 248 nm, t 7 ns) laser source. X-ray diffraction and energy dispersive X-ray spectroscopy investigations showed that the films, while prevalently amorphous, had a Ca/P ratio of about 1.50–1.52. Scanning electron microscopy analyses revealed a rather homogeneous aspect of the coatings which were molded to the relief of the chemically etched Ti substrate. Fluorescence microscopy was applied to test the proliferation of mesenchymal stem cells grown on the obtained biostructures. Our investigations found that, even 14 days after cultivation, the synthesized films were not cytotoxic. On the contrary, they showed excellent bioactivity, as demonstrated by the neat spread of the cells over the entire surface of Mn-doped b-TCP. When tested in osteoprogenitor cell culture, the Ca2.8Mn0.2(PO4)2 samples revealed a higher potential for proliferation and better viability compared with Ca2.9Mn0.1(PO4)2. # 2007 Elsevier B.V. All rights reserved. Keywords: Mn:b-TCP thin films; Biocompatible and bioactive coatings; In vitro tests; PLD
1. Introduction Calcium phosphates (CaPs) are an important category of biomaterials and the predominant mineral component of bones [1]. This makes them extremely interesting for advanced implant applications in dentistry and orthopedics. Due to its versatility, low complication rate, and good long-term results, b-tricalcium phosphate (b-TCP) has recently become one of the most actively studied CaPs [2–5]. b-TCP has high biocompatibility with excellent osteoconductive qualities [6] and unique bioresorption properties [3,4]. It has been demonstrated that divalent manganese (Mn2+) ions substantially increase the affinity of integrins for binding to a bioactive surface, as transmembrane adhesion receptors connect the actin cytoskeleton to the extracellular matrix, and thus promote a
* Corresponding author. Tel.: +40 21 457 4491; fax: +40 21 457 4243. E-mail address: [email protected] (I.N. Mihailescu). 0169-4332/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2007.08.053
better functionalization of the prosthetic surface [7]. Yet CaPs are brittle in bulk and cannot be used in such applications unless they are deposited into thin films on a metallic support. Pulsed laser deposition (PLD) has proved to be a versatile technique for layer processing, particularly in the deposition of complex molecules of simple or doped calcium phosphates [8–10]. PLD has the unique ability to create a wide variety of coating morphologies ranging from amorphous to crystalline, and from smooth and dense to rough and porous [11]. The goals of this work were to obtain Mn:b-TCP thin films and prove the viability and bioactivity of pulsed laser deposited coatings on Ti substrates. A biological investigation by in vitro tests is mandatory in order to better understand the inner processes occurring at the bone tissue/material interface. In particular, to successfully replace human bones, it is necessary to test the way human bone marrow cells respond to biomaterials, since they are the type of cells that come into direct contact with the implants and give rise to bone-forming cells (osteoblasts).
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While sarcoma and transformed osteoblasts are generally used in in vitro biocompatibility assays [12,13], it was osteoprogenitor cells, which are osteoblast precursors, that were used in these experiments. Interactions between two types of manganese-doped b-TCP (Mn:b-TCP) coatings and mesenchymal stem cells (MSCs) during in vitro differentiation of the latter were examined. MSCs were isolated from human bone marrow extracted from a 65-year-old male patient. They multiplied and were seeded at second passage on the surface of biomaterial samples and standard tissue culture material. To obtain human osteoblasts, the cells were grown in a differentiation medium. The effects of the biomaterials on cellular morphology, proliferation, and spread were examined 14 days later. 2. Experimental Samples of Ca3 xMnx(PO4)2 (x = 0, 0.1 and 0.2) were prepared through solid state reaction by crushing and mixing stoichiometric amounts of CaCO3, (NH4)2HPO4 and Mn(NO3)2 in an agate mortar. They were then heated in an alumina crucible to 300 8C for 3 h, recrushed, mixed, and reheated overnight to 1100 8C [14]. The Ca3(PO4)2 control sample was white, while the Mn-doped samples were pink. The XRD pattern of the samples revealed they had crystallized in the R3c rhombohedral structure characteristic of ß-TCP [15]. No reflections of any impurity phase were found. The lattice constants calculated for the Mn-doped samples were smaller than those known for Ca3(PO4)2 due to the substitution of Ca2+ by the smaller Mn2+ ions. The disk shaped targets were prepared by pressing the powders at 3 MPa into pellets, which were then sintered at 1000 8C for 6 h. The PLD experiments were conducted in a stainless steel reaction chamber using an UV KrF* excimer laser source (l = 248 nm, t 7.4 ns). Prior to any deposition, the chamber was evacuated down to a residual pressure of 10 4 Pa. The films were deposited in 13 Pa O2 on chemically etched Ti substrates heated to 400 8C. Fluence was set at 2 J cm 2 for each thin film deposition, which was in turn the combined result of 10,000 subsequent laser pulses. X-ray diffraction analyses were carried out with a PANalytical X’Pert PRO diffractometer equipped with a monochromator using Cu Ka radiation. The samples were scanned in the 2u range of 20–408. Morphological investigations of the synthesized products were performed using a scanning electron microscope Philips XL-20. The samples were coated with gold prior to examination. EDX analyses were also performed on all specimens. 2.1. Cell culture Second passage human MSC from the bone marrow of a 65year old male patient were cultivated on the synthesized materials at a density of 1 104 cells per 1 ml of medium in 24 well plates. The cells were negative for CD14, CD45, and CD34 and positive for CD13, CD29, and CD90, as revealed by Fluorescence-Associated Cell Sorting (FACS) analysis. Their
viability was confirmed by Tripan Blue dye exclusion assay (data not shown). The cells were cultured in direct contact with the surfaces for 14 days in an osteogenic medium (aMEM (Sigma) + 10% FCS, 82 mg/ml ascorbic acid magnesium phosphate salt, 100 nM dexametasone and 10 mM b-glycerophosphate) at 37 8C and 5% CO2. 2.2. Viability The cells were harvested and resuspended after counting at 106 cells/ml in FACS buffer. They were analyzed on a Beckton Dickinson FACSCalibur flow cytometer using an argon-ion laser emitting at 488 nm. The forward and side scatter characteristics of the cells were measured for each sample and displayed in a dot plot. Dead cells and debris were gated out and viability was statistically determined as a percentage of acquired cells (10,000 cells/sample) with the CellQuest Pro software. 2.3. ER-Tracker and actin staining On day 14 in culture, the osteoprogenitor cells were labeled in vivo by staining with a 1:1000 dilution of ER-Tracker BlueWhite DPX (Molecular Probes) dye in culture medium. The ER-Tracker specifically labels the cell endoplasmic reticulum making it possible to visualize the cells grown on opaque surfaces without a fixation step. After 30 min incubation, the cells were washed three times with fresh medium and visualized. Actin microfilament staining was performed in parallel on test samples and standard material. The cells were fixed in 4% p-formaldehyde at room temperature for 20 min. The samples were then washed with PBS and permeabilized with 0.2% Triton-X-100 diluted in PBS at room temperature for 3 min. The cells were washed once again in PBS, and F-actin was detected using Alexa Fluor 594-conjugated phalloidin (1:40 dilution; Molecular Probes) in PBS at 37 8C for 30 min. After three washes, the samples were mounted in Vectashield (Vector Laboratories) containing DAPI, a blue dye staining cell nuclei. Osteoprogenitor cells were visualized with a fluorescent microscope (Nikon Eclipse TS100). Images were taken using a Nikon Digital Light DS-SM camera. Basic image acquisition and analysis were performed using LuciaNet software. Advanced image analysis was performed using Adobe Photoshop v8.0. 3. Results and discussion The Mn:b-TCP coatings deposited by PLD on Ti substrates displayed X-ray diffraction patterns characteristic of the amorphous-poorly crystalline structures (Fig. 1a and b). All of the peaks that can be seen in Fig. 1 originated from the Ti substrate. Note that the human bone also consists of crystalline zones embedded in a prevalently amorphous medium. The SEM images (Fig. 2) show that both thin nanostructures exhibit the usual granular, rather compact morphology [9,10,16], and follow the relief of chemically etched Ti. The grains are in the micrometer range on average and display
F. Sima et al. / Applied Surface Science 254 (2007) 1155–1159
Fig. 1. XRD patterns of (a) Ca2.9Mn0.1(PO4)2 and (b) Ca2.8Mn0.2(PO4)2 thin films obtained by PLD.
droplets. As demonstrated, such features are specific of PLD and have a positive effect on osteointegration [8,17]. For all three samples, the results of EDX analyses indicate a Ca/P ratio in the range 1.50–1.52, very close to the Ca/P ratio of Ca3(PO4)2. Although the EDX method is less reliable than atomic absorption spectrometry, it confirms that PLD helps obtain thin films having the same composition as the initial powder.
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Irregular surfaces pose no problem in the biocompatible thin films deposited on metallic implants. On the contrary, it has been demonstrated that rough surfaces lead to a better osteointegration as compared with smooth implants [17]. To compare osteoprogenitor cell viability following contact with standard and tested materials, a cytometric analysis was performed to quantify the percentage of viable cells on each sample. Statistic data showed that the proportion of viable cells yielded by Ca2.8Mn0.2(PO4)2 thin films (36.15%) was higher than that on Ca2.9Mn0.1(PO4)2 thin films (27.09%) and almost as high as that produced by the standard material (Fig. 3). Two weeks on, the cells were tested using fluorescence microscopy. The cells were labeled with ER-Tracker, a fluorescent dye acting as a marker for the endoplasmic reticulum. The method has several major advantages: It makes it possible to microscopically analyze the cells, as the samples are opaque. Also, since the ER is a network-like intracellular organelle widely spread all over the cytoplasm, its labeling provides details on cell morphology and interaction with the surface. Besides, the cells can be analyzed live, as they do not need fixation pretreatment to be labeled. At the end of the 14-day interval, the cells almost entirely occupied the surfaces of both 0.2 Mn-doped b-TCP and the
Fig. 2. SEM images of (a) Ca2.9Mn0.1(PO4)2 and (b) Ca2.8Mn0.2(PO4)2 thin films obtained by PLD.
Fig. 3. FACS analysis of osteoprogenitor cells grown on (a) control material, and PLD-obtained thin films of (b) Ca2.9Mn0.1(PO4)2 and (c) Ca2.8Mn0.2(PO4)2.
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Fig. 4. ER-Tracker labeling of osteoprogenitor cells grown on (a) control material, and PLD-obtained thin films of (b) Ca2.9Mn0.1(PO4)2 and (c) Ca2.8Mn0.2(PO4)2.
Fig. 5. Osteoprogenitor cell actin filament staining on (a) standard cover slips, and on (b) Ca2.9Mn0.1(PO4)2 and (c) Ca2.8Mn0.2(PO4)2, both after 14 days in culture. The cells were fixed, permeabilized, and stained for actin using Alexa Fluor 594-conjugated phalloidin (red). The mounting media contained DAPI (blue), which stained cell nuclei.
standard material (Fig. 4a and c), while those grown on 0.1 Mndoped b-TCP showed poorer proliferation and viability (Fig. 3b). The interaction of the cells with 0.1 Mn-doped bTCP (Fig. 4b) was not as intimate as in the case of using 0.2 Mn (Fig. 4c). The spread of osteoprogenitor cells was broader on 0.2 Mn-doped b-TCP, and the cell body was flatter, showing optimum interaction with the film. We next labeled the actin filaments with Alexa Fluor 594conjugated phalloidin to study the cell cytoskeleton pattern. The actin cytoskeleton favors extracellular matrix assembly, which is important for cell adhesion to a surface. Extracellular signals can alter the actin cytoskeleton and thereby the tension on the fibronectin fibrils, which will not expose their binding sites to the surface underneath. That is why the microscopic analysis of the actin filament pattern provides valuable qualitative data on how the tested materials influence cell adherence. When grown on standard material, the cells show a characteristic pattern of parallel stress fibers extending all over the cytoplasm and have a sharp contour. The 0.1 Mn-doped b-TCP is compatible with cell adhesion, but the actin filament pattern is indicative of an unstable interaction with the substrate. The filaments are not stretched and the cell membrane tends to get loose and fold (Fig. 5, arrow). By contrast, 0.2 Mn-doped b-TCP induces the formation of parallel actin filaments in the osteoprogenitor cells, which adhere, grow, and spread on the film surface (Fig. 5c). The developing osteoblasts have long dendrites and a sharp cell contour, indicating good interaction with the PLD-grown 0.2 Mn-doped b-TCP.
4. Conclusions Thin films of manganese-doped b-tricalcium phosphate were successfully pulsed laser deposited on chemically etched Ti substrates. Grazing incidence X-ray diffraction patterns showed that the films were in a mostly amorphous, poorly crystalline phase. SEM analyses showed rather homogeneous coatings that molded the rough relief of the chemically etched Ti substrate. A Ca/P ratio of about 1.50–1.52 was found in the samples by EDX. Our studies proved that none of the tested materials was cytotoxic, since both of them induced cell adherence and growth over a period of 14 days in culture. The samples of 0.2 Mn-doped b-TCP showed higher potential for proliferation and better viability when tested in osteoprogenitor cell culture than did those with a lower Mn content. In addition, the parallel pattern of actin filaments in 0.2 Mn-doped b-TCP showed that this biomaterial interacted better with the MSCs than did 0.1 Mn-doped b-TCP. Acknowledgements This work was performed in the framework of bilateral Agreement for Scientific Cooperation between the Israel Academy of Sciences and Humanities and Romanian Academy of Sciences under the theme ‘‘Thin films and structures for medical, chemical and biological applications’’. The authors thank DENTAURUM (Germany) and SAMO (Italy) for providing Ti substrates. The Romanian authors (F.S., G.S., E.A., I.N.M., L.Z., S.M.P) acknowledge partial support of this work under the Contract 42/2005 RO-NANOMED.
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References [1] M. Mathew, S. Takagi, J. Res. Natl. Inst. Stand. Technol. 1035 (2001) 106. [2] M. Hirata, H. Murata, H. Takeshita, T. Sakabe, Y. Tsuji, T. Kubo, Int. Orthop. 30 (6) (2006) 510–513. [3] R.D. Gaasbeek, H.G. Toonen, R.J. van Heerwaarden, P. Buma, Biomaterials 26 (33) (2005) 6713–6719. [4] N. Kondo, A. Ogose, K. Tokunaga, T. Ito, K. Arai, N. Kudo, H. Inoue, H. Irie, N. Endo, Biomaterials 26 (28) (2005) 5600–5608. [5] N. Kondo, A. Ogose, K. Tokunaga, H. Umezu, K. Arai, N. Kudo, M. Hoshino, H. Inoue, H. Irie, K. Kuroda, H. Mera, N. Endo, Biomaterials 27 (25) (2006) 4419–4427. [6] H.H. Horch, R. Sader, C. Pautke, A. Neff, H. Deppe, A. Kolk, Int. J. Oral Maxillofac. Surg. 35 (8) (2006) 708–713. [7] A. Armulik, G. Svineng, K. Wennerberg, R. Faessler, S. Johansson, Exp. Cell Res. 254 (2000) 55. [8] V. Nelea, I.N. Mihailescu, M. Jelinek, in: R. Eason (Ed.), Pulsed Laser Deposition of Thin Films: Applications-Led Growth of Functional Materials, John Wiley & Sons Inc, 2007,, p. 421 (chapter. 18).
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[9] A. Bigi, B. Bracci, F. Cuisinier, R. Elkaim, M. Fini, I. Mayer, I.N. Mihailescu, G. Socol, L. Sturba, P. Torricelli, Biomaterials 2381 (2005) 26. [10] M. Iliescu, V. Nelea, J. Werckmann, G. Socol, I.N. Mihailescu, I. Mayer, F. Cuisinier, Mater. Sci. Eng. C 105 (2007) 27. [11] H. Zeng, W.R. Lacefield, S. Mirov, J. Biomed. Mater. Res. 50 (2) (2000) 248–258. [12] T. Matsuura, R. Hosokawa, K. Okamoto, T. Kimoto, Y. Akagawa, Biomaterials 1121 (2000) 21. [13] P. Torricelli, M. Fini, G. Giavaresi, V. Borsari, A. Carpi, A. Nicolini, R. Giardino, Biomed. Pharmacother. 57 (2003) 57. [14] I. Mayer, F.J.G. Cuisinier, I. Popov, Y. Schleich, S. Gdalya, O. Burghaus, D. Reinen, Eur. J. Inorg. Chem. 1460 (2006) 7. [15] International Centre for Diffraction Data. Powder Diffraction File 2005, 09-0169. [16] E. Gyorgy, P. Toricelli, G. Socol, M. Iliescu, I. Mayer, I.N. Mihailescu, A. Bigi, J. Werckmann, J. Biomed. Mater. Res. A 353 (2004) 71. [17] D.M. Brunette, P. Tengvall, M. Textor, P. Thomsen (Eds.), Titanium in Medicine, Springer, Berlin, 2001.
Biocompatible and bioactive coatings of Mn2+-doped b-tricalcium phosphate synthesized by pulsed laser deposition F. Sima a, G. Socol a, E. Axente a, I.N. Mihailescu a,*, L. Zdrentu b, S.M. Petrescu b, I. Mayer c a
National Institute for Lasers, Plasma and Radiation Physics, Box MG-54, RO-77125 Bucharest, Magurele, Romania b Institute of Biochemistry, Splaiul Independentei 296, Bucharest, Romania c Department of Inorganic and Analytical Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel Received 20 May 2007; received in revised form 1 August 2007; accepted 13 August 2007 Available online 21 August 2007
Abstract The extension of pulsed laser deposition to the synthesis on Ti substrates of b-tricalcium phosphate (b-TCP) coatings doped with manganese is reported. Targets sintered from two crystalline Mn-doped b-TCP powders (with the composition Ca2.9Mn0.1(PO4)2 and Ca2.8Mn0.2(PO4)2) were ablated with an UV KrF* (l = 248 nm, t 7 ns) laser source. X-ray diffraction and energy dispersive X-ray spectroscopy investigations showed that the films, while prevalently amorphous, had a Ca/P ratio of about 1.50–1.52. Scanning electron microscopy analyses revealed a rather homogeneous aspect of the coatings which were molded to the relief of the chemically etched Ti substrate. Fluorescence microscopy was applied to test the proliferation of mesenchymal stem cells grown on the obtained biostructures. Our investigations found that, even 14 days after cultivation, the synthesized films were not cytotoxic. On the contrary, they showed excellent bioactivity, as demonstrated by the neat spread of the cells over the entire surface of Mn-doped b-TCP. When tested in osteoprogenitor cell culture, the Ca2.8Mn0.2(PO4)2 samples revealed a higher potential for proliferation and better viability compared with Ca2.9Mn0.1(PO4)2. # 2007 Elsevier B.V. All rights reserved. Keywords: Mn:b-TCP thin films; Biocompatible and bioactive coatings; In vitro tests; PLD
1. Introduction Calcium phosphates (CaPs) are an important category of biomaterials and the predominant mineral component of bones [1]. This makes them extremely interesting for advanced implant applications in dentistry and orthopedics. Due to its versatility, low complication rate, and good long-term results, b-tricalcium phosphate (b-TCP) has recently become one of the most actively studied CaPs [2–5]. b-TCP has high biocompatibility with excellent osteoconductive qualities [6] and unique bioresorption properties [3,4]. It has been demonstrated that divalent manganese (Mn2+) ions substantially increase the affinity of integrins for binding to a bioactive surface, as transmembrane adhesion receptors connect the actin cytoskeleton to the extracellular matrix, and thus promote a
* Corresponding author. Tel.: +40 21 457 4491; fax: +40 21 457 4243. E-mail address: [email protected] (I.N. Mihailescu). 0169-4332/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2007.08.053
better functionalization of the prosthetic surface [7]. Yet CaPs are brittle in bulk and cannot be used in such applications unless they are deposited into thin films on a metallic support. Pulsed laser deposition (PLD) has proved to be a versatile technique for layer processing, particularly in the deposition of complex molecules of simple or doped calcium phosphates [8–10]. PLD has the unique ability to create a wide variety of coating morphologies ranging from amorphous to crystalline, and from smooth and dense to rough and porous [11]. The goals of this work were to obtain Mn:b-TCP thin films and prove the viability and bioactivity of pulsed laser deposited coatings on Ti substrates. A biological investigation by in vitro tests is mandatory in order to better understand the inner processes occurring at the bone tissue/material interface. In particular, to successfully replace human bones, it is necessary to test the way human bone marrow cells respond to biomaterials, since they are the type of cells that come into direct contact with the implants and give rise to bone-forming cells (osteoblasts).
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While sarcoma and transformed osteoblasts are generally used in in vitro biocompatibility assays [12,13], it was osteoprogenitor cells, which are osteoblast precursors, that were used in these experiments. Interactions between two types of manganese-doped b-TCP (Mn:b-TCP) coatings and mesenchymal stem cells (MSCs) during in vitro differentiation of the latter were examined. MSCs were isolated from human bone marrow extracted from a 65-year-old male patient. They multiplied and were seeded at second passage on the surface of biomaterial samples and standard tissue culture material. To obtain human osteoblasts, the cells were grown in a differentiation medium. The effects of the biomaterials on cellular morphology, proliferation, and spread were examined 14 days later. 2. Experimental Samples of Ca3 xMnx(PO4)2 (x = 0, 0.1 and 0.2) were prepared through solid state reaction by crushing and mixing stoichiometric amounts of CaCO3, (NH4)2HPO4 and Mn(NO3)2 in an agate mortar. They were then heated in an alumina crucible to 300 8C for 3 h, recrushed, mixed, and reheated overnight to 1100 8C [14]. The Ca3(PO4)2 control sample was white, while the Mn-doped samples were pink. The XRD pattern of the samples revealed they had crystallized in the R3c rhombohedral structure characteristic of ß-TCP [15]. No reflections of any impurity phase were found. The lattice constants calculated for the Mn-doped samples were smaller than those known for Ca3(PO4)2 due to the substitution of Ca2+ by the smaller Mn2+ ions. The disk shaped targets were prepared by pressing the powders at 3 MPa into pellets, which were then sintered at 1000 8C for 6 h. The PLD experiments were conducted in a stainless steel reaction chamber using an UV KrF* excimer laser source (l = 248 nm, t 7.4 ns). Prior to any deposition, the chamber was evacuated down to a residual pressure of 10 4 Pa. The films were deposited in 13 Pa O2 on chemically etched Ti substrates heated to 400 8C. Fluence was set at 2 J cm 2 for each thin film deposition, which was in turn the combined result of 10,000 subsequent laser pulses. X-ray diffraction analyses were carried out with a PANalytical X’Pert PRO diffractometer equipped with a monochromator using Cu Ka radiation. The samples were scanned in the 2u range of 20–408. Morphological investigations of the synthesized products were performed using a scanning electron microscope Philips XL-20. The samples were coated with gold prior to examination. EDX analyses were also performed on all specimens. 2.1. Cell culture Second passage human MSC from the bone marrow of a 65year old male patient were cultivated on the synthesized materials at a density of 1 104 cells per 1 ml of medium in 24 well plates. The cells were negative for CD14, CD45, and CD34 and positive for CD13, CD29, and CD90, as revealed by Fluorescence-Associated Cell Sorting (FACS) analysis. Their
viability was confirmed by Tripan Blue dye exclusion assay (data not shown). The cells were cultured in direct contact with the surfaces for 14 days in an osteogenic medium (aMEM (Sigma) + 10% FCS, 82 mg/ml ascorbic acid magnesium phosphate salt, 100 nM dexametasone and 10 mM b-glycerophosphate) at 37 8C and 5% CO2. 2.2. Viability The cells were harvested and resuspended after counting at 106 cells/ml in FACS buffer. They were analyzed on a Beckton Dickinson FACSCalibur flow cytometer using an argon-ion laser emitting at 488 nm. The forward and side scatter characteristics of the cells were measured for each sample and displayed in a dot plot. Dead cells and debris were gated out and viability was statistically determined as a percentage of acquired cells (10,000 cells/sample) with the CellQuest Pro software. 2.3. ER-Tracker and actin staining On day 14 in culture, the osteoprogenitor cells were labeled in vivo by staining with a 1:1000 dilution of ER-Tracker BlueWhite DPX (Molecular Probes) dye in culture medium. The ER-Tracker specifically labels the cell endoplasmic reticulum making it possible to visualize the cells grown on opaque surfaces without a fixation step. After 30 min incubation, the cells were washed three times with fresh medium and visualized. Actin microfilament staining was performed in parallel on test samples and standard material. The cells were fixed in 4% p-formaldehyde at room temperature for 20 min. The samples were then washed with PBS and permeabilized with 0.2% Triton-X-100 diluted in PBS at room temperature for 3 min. The cells were washed once again in PBS, and F-actin was detected using Alexa Fluor 594-conjugated phalloidin (1:40 dilution; Molecular Probes) in PBS at 37 8C for 30 min. After three washes, the samples were mounted in Vectashield (Vector Laboratories) containing DAPI, a blue dye staining cell nuclei. Osteoprogenitor cells were visualized with a fluorescent microscope (Nikon Eclipse TS100). Images were taken using a Nikon Digital Light DS-SM camera. Basic image acquisition and analysis were performed using LuciaNet software. Advanced image analysis was performed using Adobe Photoshop v8.0. 3. Results and discussion The Mn:b-TCP coatings deposited by PLD on Ti substrates displayed X-ray diffraction patterns characteristic of the amorphous-poorly crystalline structures (Fig. 1a and b). All of the peaks that can be seen in Fig. 1 originated from the Ti substrate. Note that the human bone also consists of crystalline zones embedded in a prevalently amorphous medium. The SEM images (Fig. 2) show that both thin nanostructures exhibit the usual granular, rather compact morphology [9,10,16], and follow the relief of chemically etched Ti. The grains are in the micrometer range on average and display
F. Sima et al. / Applied Surface Science 254 (2007) 1155–1159
Fig. 1. XRD patterns of (a) Ca2.9Mn0.1(PO4)2 and (b) Ca2.8Mn0.2(PO4)2 thin films obtained by PLD.
droplets. As demonstrated, such features are specific of PLD and have a positive effect on osteointegration [8,17]. For all three samples, the results of EDX analyses indicate a Ca/P ratio in the range 1.50–1.52, very close to the Ca/P ratio of Ca3(PO4)2. Although the EDX method is less reliable than atomic absorption spectrometry, it confirms that PLD helps obtain thin films having the same composition as the initial powder.
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Irregular surfaces pose no problem in the biocompatible thin films deposited on metallic implants. On the contrary, it has been demonstrated that rough surfaces lead to a better osteointegration as compared with smooth implants [17]. To compare osteoprogenitor cell viability following contact with standard and tested materials, a cytometric analysis was performed to quantify the percentage of viable cells on each sample. Statistic data showed that the proportion of viable cells yielded by Ca2.8Mn0.2(PO4)2 thin films (36.15%) was higher than that on Ca2.9Mn0.1(PO4)2 thin films (27.09%) and almost as high as that produced by the standard material (Fig. 3). Two weeks on, the cells were tested using fluorescence microscopy. The cells were labeled with ER-Tracker, a fluorescent dye acting as a marker for the endoplasmic reticulum. The method has several major advantages: It makes it possible to microscopically analyze the cells, as the samples are opaque. Also, since the ER is a network-like intracellular organelle widely spread all over the cytoplasm, its labeling provides details on cell morphology and interaction with the surface. Besides, the cells can be analyzed live, as they do not need fixation pretreatment to be labeled. At the end of the 14-day interval, the cells almost entirely occupied the surfaces of both 0.2 Mn-doped b-TCP and the
Fig. 2. SEM images of (a) Ca2.9Mn0.1(PO4)2 and (b) Ca2.8Mn0.2(PO4)2 thin films obtained by PLD.
Fig. 3. FACS analysis of osteoprogenitor cells grown on (a) control material, and PLD-obtained thin films of (b) Ca2.9Mn0.1(PO4)2 and (c) Ca2.8Mn0.2(PO4)2.
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Fig. 4. ER-Tracker labeling of osteoprogenitor cells grown on (a) control material, and PLD-obtained thin films of (b) Ca2.9Mn0.1(PO4)2 and (c) Ca2.8Mn0.2(PO4)2.
Fig. 5. Osteoprogenitor cell actin filament staining on (a) standard cover slips, and on (b) Ca2.9Mn0.1(PO4)2 and (c) Ca2.8Mn0.2(PO4)2, both after 14 days in culture. The cells were fixed, permeabilized, and stained for actin using Alexa Fluor 594-conjugated phalloidin (red). The mounting media contained DAPI (blue), which stained cell nuclei.
standard material (Fig. 4a and c), while those grown on 0.1 Mndoped b-TCP showed poorer proliferation and viability (Fig. 3b). The interaction of the cells with 0.1 Mn-doped bTCP (Fig. 4b) was not as intimate as in the case of using 0.2 Mn (Fig. 4c). The spread of osteoprogenitor cells was broader on 0.2 Mn-doped b-TCP, and the cell body was flatter, showing optimum interaction with the film. We next labeled the actin filaments with Alexa Fluor 594conjugated phalloidin to study the cell cytoskeleton pattern. The actin cytoskeleton favors extracellular matrix assembly, which is important for cell adhesion to a surface. Extracellular signals can alter the actin cytoskeleton and thereby the tension on the fibronectin fibrils, which will not expose their binding sites to the surface underneath. That is why the microscopic analysis of the actin filament pattern provides valuable qualitative data on how the tested materials influence cell adherence. When grown on standard material, the cells show a characteristic pattern of parallel stress fibers extending all over the cytoplasm and have a sharp contour. The 0.1 Mn-doped b-TCP is compatible with cell adhesion, but the actin filament pattern is indicative of an unstable interaction with the substrate. The filaments are not stretched and the cell membrane tends to get loose and fold (Fig. 5, arrow). By contrast, 0.2 Mn-doped b-TCP induces the formation of parallel actin filaments in the osteoprogenitor cells, which adhere, grow, and spread on the film surface (Fig. 5c). The developing osteoblasts have long dendrites and a sharp cell contour, indicating good interaction with the PLD-grown 0.2 Mn-doped b-TCP.
4. Conclusions Thin films of manganese-doped b-tricalcium phosphate were successfully pulsed laser deposited on chemically etched Ti substrates. Grazing incidence X-ray diffraction patterns showed that the films were in a mostly amorphous, poorly crystalline phase. SEM analyses showed rather homogeneous coatings that molded the rough relief of the chemically etched Ti substrate. A Ca/P ratio of about 1.50–1.52 was found in the samples by EDX. Our studies proved that none of the tested materials was cytotoxic, since both of them induced cell adherence and growth over a period of 14 days in culture. The samples of 0.2 Mn-doped b-TCP showed higher potential for proliferation and better viability when tested in osteoprogenitor cell culture than did those with a lower Mn content. In addition, the parallel pattern of actin filaments in 0.2 Mn-doped b-TCP showed that this biomaterial interacted better with the MSCs than did 0.1 Mn-doped b-TCP. Acknowledgements This work was performed in the framework of bilateral Agreement for Scientific Cooperation between the Israel Academy of Sciences and Humanities and Romanian Academy of Sciences under the theme ‘‘Thin films and structures for medical, chemical and biological applications’’. The authors thank DENTAURUM (Germany) and SAMO (Italy) for providing Ti substrates. The Romanian authors (F.S., G.S., E.A., I.N.M., L.Z., S.M.P) acknowledge partial support of this work under the Contract 42/2005 RO-NANOMED.
F. Sima et al. / Applied Surface Science 254 (2007) 1155–1159
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