J Dent Res 82(6): 449-453, 2003
© 2003 International and American Associations for Dental Research
RESEARCH REPORT
Biomaterials & Bioengineering |
Osteoblast Precursor Cell Attachment on Heat-treated Calcium Phosphate Coatings
Y. Yang1,
J.D. Bumgardner2,
R. Cavin1,
D.L. Carnes3, and
J.L Ong1,4,*
1 University of Texas Health Science Center at San Antonio, Department of Restorative Dentistry, Division of Biomaterials, MSC 7890, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900;
2 Mississippi State University, Department of Agricultural and Biological Engineering, Box 9632, Mississippi State, MS 39762;
3 University of Texas Health Science Center at San Antonio, Department of Periodontics, MSC 7894, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900; and
4 University of Texas Health Science Center at San Antonio, Center for Clinical Bioengineering, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900;
*corresponding author, Ong{at}uthscsa.edu
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ABSTRACT
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The influence of properties of calcium phosphate (CaP) coatings on bone cell activity and bone-implant osseointegration is not well-established. This study investigated the effects of characterized CaP coatings of various heat treatments on osteoblast response. It was hypothesized that heat treatments of CaP coatings alter the initial osteoblast attachment. The 400°C heat-treated coatings were observed to exhibit poor crystallinity and significantly greater phosphate or apatite species compared with as-sputtered and 600°C heat-treated coatings. Similarly, human embryonic palatal mesenchyme (HEPM) cells, an osteoblast precursor cell line, seeded on 400°C heat-treated coatings, exhibited significantly greater cell attachment compared with Ti surfaces, as-sputtered coatings, and 600°C heat-treated coatings. The HEPM cells on Ti surfaces and heat-treated coatings were observed to attach through filopodia, and underwent cell division, whereas the cells on as-sputtered coatings displayed fewer filopodia extensions and cell damage. Analysis of the data suggested that heat treatment of CaP coatings affects cell attachment.
KEY WORDS: calcium phosphate coating • titanium • osteobalst • attachment • heat treatment
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INTRODUCTION
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Hydroxyapatite (HA) and calcium phosphate (CaP) coatings are successfully used in dental and orthopedic implant therapy. Commercial HA- or CaP-coated implants currently available are produced by plasma spray technology. Advantages of plasma spraying include a rapid deposition rate and sufficiently low cost (Herman, 1988). Nevertheless, there are problems associated with plasma-sprayed coatings, including poor adhesion and variation in bond strength between the coatings and metallic substrates (Filiaggi et al., 1991), as well as alterations in HA or CaP structure resulting from the coating process (Palka et al., 1998). The nature of the substrate plays an important role in the adhesion between plasma-sprayed coatings and metallic substrates (Yang and Ong, 2003). The bonding of plasma-sprayed HA or CaP coatings to the metallic substrate appears to be entirely mechanical, and reports indicate that a highly roughened substrate surface exhibits higher bond strength than a smooth substrate surface (Nimb et al., 1993).
Several experimental deposition processes were reported for producing HA and CaP coatings, including electrophoretic co-deposition (Dasarathy et al., 1993), sputter deposition (Ong et al., 1991;Wolke et al., 1994), and high-velocity oxy-fuel combustion spray deposition (Haman et al., 1995). These experimental processes have been developed in an attempt to improve the adhesive, compositional, and structural properties of the coatings. Cooley et al. (1992), Lacefield et al (1993), and de Groot et al. (1994) concluded that sputtering may be the method of choice for controlling the physical and chemical properties of HA or CaP coatings on dental and orthopedic implants. Studies have indicated significantly greater coating-metal interfacial strength for sputtered CaP coatings compared with commercially available plasma-sprayed HA coatings (40 MPa vs. 9 MPa) (Lucas et al., 1993). Additional characterizations indicated that sputtered CaP coatings were amorphous in the absence of heat treatment (Ong et al., 1992), but displayed an HA-type crystalline structure after post-deposition heat treatments (Ong and Lucas, 1994).
Extensive in vivo research indicates that HA-coated implants are biocompatible and may improve early performance when compared with non-coated Ti implants. This increase in performance is believed to result from more rapid osseointegration, the development of increased interfacial strength through early skeletal attachment, and increased bone-implant contact (Thomas and Cook, 1985;Cooley et al., 1992). Unfortunately, the performance-enhancing advantages of CaP coatings are mired in considerable controversy due to the lack of correlation between specific properties of the CaP surface and implant success. There has been a general lack of appreciation for the effects that variation in the chemical and physical characteristics of an HA or CaP coating may have on bone cell activity, and how this activity may influence the long-term success of implants (Steflik, 1994). This has led to many conflicting animal and clinical observations, and as a result, no consensus exists on the optimum coating properties required for an optimum rate of development of osseointegration. As such, the investigation of biological responses to these coatings needs to be critically evaluated. In this study, as-sputtered and heat-treated CaP coatings were characterized by x-ray diffraction (XRD) and x-ray photoelectron spectroscopy (XPS), and the effects of CaP coatings after heat treatment on initial bone cell attachment were evaluated.
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MATERIALS & METHODS
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Materials
Titanium (Ti) grade 2 (Metal Samples, Munford, AL, USA) disks, 15.30 mm in diameter and 2 mm thick, were used in this study. The Ti surfaces were prepared by wet-grinding with 240-, 400-, and 600-grit silicon carbide paper to a surface roughness of ra = 0.37 ± 0.01 µm, as measured by profilometry (Surtronic 3, Taylor-Hobson, UK). These surfaces were ultrasonically de-greased in benzene, acetone, and ethanol for 10 min each, and were rinsed with de-ionized water between applications of each solvent. Passivation was accomplished by exposure of the Ti samples to a 40% nitric acid solution at room temperature for 30 min (ASTM F86-76), after which the samples were rinsed with de-ionized water and air-dried. The cleaned Ti disks were divided into 4 groups. One group was left uncoated. Each of the 3 remaining groups was sputter-coated with a thin CaP layer.
Sputtering of CaP on cleaned Ti surfaces was performed by means of a CMS-18 radiofrequency magnetron sputtering system (Kurt J Lesker Company, Clairton, PA, USA). The target material used in the sputtering process was a four-inch copper disk coated with plasma-sprayed hydroxyapatite (APS Materials, Dayton, OH, USA). Sputter-deposition was accomplished by means of a process pressure of 1.0 
1.5 mbar and a sputtering power of 300 W for 2 hrs at a coating rate of 50 nm per hour. After being sputtered, the coated samples were left as-sputtered or subjected to post-deposition heat treatment at either 400°C or 600°C in a Thermolyne 48000 furnace (Barnstead International, Dubuque, IA, USA). All coated and uncoated samples were sterilized under UV light for a minimum of 24 hrs prior to experimentation.
X-ray diffraction (XRD)
We used a D8 Advanced x-ray diffractometor (Bruker, Madison, WI, USA) to characterize the structure of coatings. The x-ray diffractometer was equipped with a single Gobel mirror to yield a diffracted parallel beam while removing the Kß radiation. With a grazing incidence attachment, a 0.35° soller slit, and a LiF (100) flat crystal monochormator to improve resolution and peak-to-background ratios, triplicate coatings were analyzed by Cu K
radiation at 40 KV and 30 mA. The samples on Ti substrates were scanned from 25° (2
) to 35° (2) at a scan rate of 0.1° per min. Crystalline peak area in the range 25° to 35° was calculated, and the percent crystallinity of the coatings was quantified by correlation of the crystalline peak area to the known HA crystallinity standard curve, which we derived by mixing various ratios of 100% crystalline and amorphous commercial HA powder (Hitemco Medical Applications, Inc., Old Bethpage, NY, USA). Percent crystallinity of the coatings was analyzed by ANOVA, and difference was considered significant if P < 0.05.
X-ray Photoelectron Spectroscopy (XPS)
Elemental and chemical composition of the surface layers of the disks was determined by x-ray photoelectron spectroscopy (XPS) in a Physical Electronics Model 1600 surface analysis system (Eden Prairie, MN, USA). The system used an Mg electrode (K radiation at 1253.6 eV) at 15 kV and 300 W as the x-ray source. Duplicate samples were positioned at a 45° take-off angle with respect to the analyzer. Two spots, approximately 800 µm in diameter, were evaluated on each sample. Survey spectra were averaged from 10 scans with a pass energy of 46.95 eV. We used survey spectra to identify surface elements and to calculate their relative compositions in atomic percent. High-resolution spectra of elements were averaged from 15 scans with pass energy of 23.5 eV. The high-resolution spectra were used to identify the chemical states and to estimate percentages of chemical species present. Quantitative analyses were based on peak areas and atomic sensitivity factors with the use of Spectral Data Processor v2.3 software (XPS International, Mountain View, CA, USA). The Ca/P ratio of coatings from different treatments was analyzed by ANOVA, and difference was considered significant if P < 0.05.
Cell Attachment Study
The cell attachment study was conducted with ATCC CRL 1486 human embryonic palatal mesenchyme (HEPM) cells, an osteoblast precursor cell line, and the VybrantTM cell adhesion assay (Molecular Probes, Eugene, OR, USA). HEPM cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 7% FBS, penicillin (5000 units/mL-1), streptomycin (5000 µg/mL-1), and fungizone (250 µg/mL) in an atmosphere of 5% CO2 95% air at 100% humidity and 37°C. Medium was changed twice a week. HEPM cells from pre-confluent cultures were harvested with 0.25% trypsin-1 mM EDTA (GibcoBRL, Life Technologies, Grand Island, NY), centrifuged, and re-suspended in serum-free DMEM. The cells were seeded onto triplicate control or coated Ti disks in six-well culture plates at a density of 10,000 cells per disk and incubated as just described for 180 min. Non-adherent cells were removed, and the samples were washed twice in serum-free DMEM. The medium removed was saved, and the volume was recorded. Calcein AM was added to an aliquot of the initial cell-seeding solution, as well as to the solution containing the non-adherent cells, to a final concentration of 5 µM. A 100-µL aliquot of each solution containing calcein AM was mixed with 100 µL of phosphate-buffered saline (PBS) in a 96-well plate and incubated at 37°C for 120 min. Fluorescence was determined by means of a SPECTRAmax GEMINI XS microplate reader (Molecular Devices Corp, Sunnyvale, CA, USA) at an absorbance maximum of 494 nm and an emission maximum of 517 nm, and was proportional to cell number. We determined the percent cell attachment by subtracting the non-adherent cell number from the number of cells seeded and then dividing by the number of cells seeded. Mean cell attachment for the different surfaces was analyzed by ANOVA, and differences were considered significant if P < 0.05. For consistency, the cell attachment study was performed on two different occasions.
Scanning Electron Microscopy (SEM)
After 3 hrs of incubation, the cells were washed 3 times with PBS, then were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for a minimum of 1 hr at room temperature. The specimens were then washed 3 times with 0.1 M cacodylate buffer, followed by post-fixation with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hr at room temperature. After fixation, the specimens were dehydrated through an ethanol series, an ethanol-freonTF series, and subsequently were critical-point-dried in a Bomar 1500 critical-point dryer (Ladd Research, Williston, VT, USA). After being dried, the specimens were mounted on aluminum cylinders and sputter-coated with a thin layer of 60% gold 40% palladium by means of a Technics Hummer V sputter coater (Technics, San Jose, CA, USA). The specimens were then evaluated in a JEOL JSM-840A (JEOL USA, Inc., Peabody, MA, USA) scanning electron microscope with an acceleration voltage of 15 kV.
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RESULTS
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X-ray Diffraction
X-ray diffraction patterns of as-sputtered CaP coatings, 400°C heat-treated CaP coatings, and 600°C heat-treated CaP coatings are shown in Fig. 1A
. It was observed that as-sputtered CaP coatings were amorphous, and 600°C heat-treated CaP coatings were highly crystalline. The 400°C heat-treated CaP coatings exhibited very poor crystallinity, with the presence of only one small crystalline peak at 32.9°. Percent crystallinity of as-sputtered CaP coatings, 400°C heat-treated CaP coatings, and 600°C heat-treated CaP coatings was 0%, 1.9 ± 0.4%, and 66.4 ± 2.8%, respectively (Table. It was observed that as-sputtered CaP coatings were amorphous, and 600°C heat-treated CaP coatings were highly crystalline. The 400°C heat-treated CaP coatings exhibited very poor crystallinity, with the presence of only one small crystalline peak at 32.9°. Percent crystallinity of as-sputtered CaP coatings, 400°C heat-treated CaP coatings, and 600°C heat-treated CaP coatings was 0%, 1.9 ± 0.4%, and 66.4 ± 2.8%, respectively (Table).
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Figure 1. Structural and compositional analysis of CaP coatings of different treatments. (A) Representative x-ray diffraction analyses of as-sputtered Ca coatings, 400°C heat-treated CaP coatings, and 600°C heat-treated CaP coatings. (B) Representative high-resolution O 1s spectra of CaP-coated surfaces by x-ray photoelectron spectroscopy. The O 1s were curve-fitted showing two peaks at 532 eV and 531 eV.
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Table. Percent Crystallinity from X-ray Diffraction Patterns and Percent of Oxygen Species from High-resolution X-ray Photoelectron Spectroscopy Spectra of Calcium Phosphate Coatings (mean ± standard deviation; n = 3)
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X-ray Photoelectron Spectroscopy (XPS)
XPS analysis indicated a calcium/phosphorus (Ca/P) ratio of 1.6 ± 0.2 for as-sputtered CaP coatings. This ratio was significantly lower than the Ca/P ratio for 400°C heat-treated (2.6 ± 0.3) and 600°C heat-treated CaP coatings (2.5 ± 0.2). A single peak fit was observed for the high-resolution Ca 2p3/2 peak for all samples and treatments. The binding energy of the Ca 2p3/2 peak was approximately 347 eV, which corresponded closely to Ca-oxide-, -hydroxide-, -phosphate-, and -carbonate-type species. Two peaks were found to fit the high-resolution 2p3 peak. The peak at approximately 132.7 eV corresponded to (PO4)-3-type species, and the peak at approximately 133.7 corresponded to H(PO4)-2 type species. No statistical difference between CaP of different heat treatments was observed between the two phosphate species peak areas (p = 0.7). In addition, two peaks were also found to fit the O 1s peak (Fig. 1B
). Oxygen bonded in phosphate and apatite was associated with the peak at approximately 532 eV, while oxygen in oxide, hydroxide, and carbonate was associated with the peak at approximately 531 eV. Statistical analyses indicated significant differences in the areas of the two oxygen peaks for coatings heat-treated at 400°C and 600°C (p = 0.004). The CaP coatings heat-treated at 400°C exhibited the highest relative amount of oxygen in phosphate and apatite species as compared with the non-heat-treated and 600°C heat-treated CaP samples (p 
0.05) (Table).
Cell Attachment
Fig. 2 displays HEPM cell attachment expressed as a percent of total cells plated for the Ti surface and each of the CaP-coated groups. HEPM cells seeded onto CaP coatings after 400°C heat treatment (64.9 ± 4.2%) exhibited significantly greater (P < 0.001) cell attachment than HEPM cells seeded onto the Ti surfaces (36.1 ± 3.7%), as-sputtered CaP coatings (25.5 ± 6.0%), and CaP coatings after 600°C heat-treatment (34.1 ± 2.0%). No significant difference in cell attachment was observed between the as-sputtered CaP coatings, and CaP coatings after 600°C heat-treatment, or between these surfaces and the Ti surface.
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Figure 2. HEPM cell attachment on heat-treated CaP coatings. Data were expressed as the mean ± SD (n = 6). Asterisk (*) indicated significant difference (p < 0.001).
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SEM
Changes in cell morphology were observed when HEPM cells were seeded onto CaP coatings of different heat treatments. Fig. 3 displays representative morphology of HEPM cells attached to the Ti surface and to CaP-coated surfaces after 3 hrs. HEPM cells were observed to attach to Ti and CaP surfaces through strand-like and sheet-like filopodia (Figs. 3A to 3D), although the cells on as-sputtered CaP coatings (Fig. 3B) had fewer filopodia than the cells on the heat-treated CaP surfaces (Figs. 3C, 3D). In addition, HEPM cells apparently undergoing cell division were observed on the Ti surface, as well as on the heat-treated CaP coatings (Figs. 3E, 3G, 3H). By contrast, many cells appeared damaged on the as-sputtered CaP coating (Fig. 3F).
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Figure 3. Representative SEM micrographs of attached cell morphology on: (A) control Ti surfaces (X4000), (B) as-sputtered CaP coatings (X4000), (C) 400C heat-treated CaP coatings (X2500), and (D) 600°C heat-treated CaP coatings (X4000). Representative SEM micrographs displaying dividing cells on: (E) control Ti surfaces (X2500), (F) as-depostied CaP coatings (X900), (G) 400°C heat-treated CaP coatings (X2500), and (H) 600°C heat-treated CaP coatings (X2500). Damaged cells were observed on as-sputtered CaP coatings. Bar = 5 µm.
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DISCUSSION
This study investigated the effect of heat treatment of sputtered CaP coatings on HEPM cell attachment. Other studies suggest that the bioactivity of CaP enhances bone formation rates and bone-implant bonding through the dissolution of the ceramic (de Groot et al., 1994;Ong et al., 2002;ter Brugge et al., 2002) that produces solution-mediated events affecting bone cell activity (Okamoto et al., 1998;Chang et al., 1999), organic matrix deposition (Ferraz et al., 1999), and mineral precipitation and resorption (Wolke et al., 1994;Gross et al., 1997). The dissolution characteristics of CaP ceramics are determined by the crystallinity of the material (Wolke et al., 1994;Gross et al., 1997). Because the crystallinity of CaP coatings is affected by heat treatment (Ong and Lucas, 1994; Ong et al., 1998), this study examined CaP coatings of various crystallinities. It was observed from x-ray diffraction analysis that as-sputtered CaP coatings were amorphous and 600°C heat-treated CaP coatings were crystalline. The 400°C heat-treated CaP coatings were observed to be poorly crystallinized, indicating the influence of post-deposition heat treatment temperature on crystallinity. The Ca/P ratio for as-sputtered CaP coatings was observed to be significantly lower than the Ca/P ratio for heat-treated CaP surfaces, suggesting a more calcium-enriched surface after heat treatments. In addition, XPS analysis also indicated significantly greater phosphate and apatite species on CaP coatings after 400°C heat-treatment (40 ± 3%) as compared with as-sputtered CaP coatings (30 ± 5%, p = 0.004) and CaP coatings after 600°C heat treatment (31 ± 5%, p < 0.05).
HEPM cell attachment was similar on the titanium surfaces and the as-sputtered and 600°C heat-treated CaP coatings. By contrast, HEPM cell attachment on 400°C heat-treated CaP coatings was significantly greater than cell attachment on uncoated titanium surfaces as well as on as-sputtered and 600°C heat-treated CaP coatings. This increase in cell attachment was likely related to the increase of phosphate and apatite species. The phosphate and apatite groups on 400°C heat-treated CaP surfaces were significantly greater than the other surfaces evaluated in this study, suggesting that the presence of phosphate and apatite groups was critical for cell attachment. In addition, the crystallinity of 400°C heat-treated CaP surfaces (1.9 ± 0.4%) was greater than that of as-sputtered CaP surfaces (0%), but significantly less than that of 600°C heat-treated CaP surfaces (66.4 ± 2.8%), suggesting that the degree of surface crystallinity may be an important factor for initial cell attachment.
Analysis of the SEM data indicated that HEPM cells attached to the Ti surfaces and heat-treated CaP coatings through strand-like and sheet-like filopodia, suggesting excellent biocompatibility. By contrast, HEPM cells on as-sputtered CaP coatings displayed fewer filopodia, often appeared damaged, and failed to exhibit apparent cell division. These observations are consistent with those from other studies reporting that amorphous CaP coatings prohibit osteoblast proliferation and differentiation (ter Brugge and Jansen, 2002), and that certain concentrations of calcium and phosphate ion pairs induce osteoblast apoptosis (Adams et al., 2001). CaP coatings show extensive dissolution during the initial 300 min of exposure to aqueous environments through the release of calcium and phosphate ions from active dissolution sites (Nancollas and Tucker, 1994) or polynucleation centers (Christoffersen, 1980), with a rate virtually independent of fluid dynamics. It is worth noting that the amorphous phase dissolves more rapidly than the apatite-like phases (Nancollas and Tucker, 1994). This probably explains why HEPM cell attachment was lower on the amorphous, as-sputtered CaP coatings than on the partially crystalline 400°C heat-treated CaP coatings. The fact that cell attachment on the 400°C heat-treated surfaces was greater than on the more crystalline 600°C heat-treated surfaces suggests that perhaps there is an optimum crystalline content and ligand of a CaP coating that promote cell attachment, and possibly other cell activities.
Although the HEPM cell attachment observed in this study is consistent with that in other studies reporting greater cell attachment on HA and CaP coatings than on Ti surfaces (Okamoto et al., 1998; Chang et al., 1999;Takebe et al., 2000), analysis of the data suggests that an optimum HA surface exists. This study, as well as previous studies (Ong et al., 1991, 1997;Ong and Lucas, 1994; HREF="#WOLKE-ETAL-1994">Wolke et al., 1994), suggests that there exists an optimal amorphous-crystalline content for CaP coatings, and demonstrates the need for additional studies relating the physical and chemical characteristics of CaP surfaces to effects on cellular activity.
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ACKNOWLEDGMENTS
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This study was funded by grants from the National Institute of Health (Grant Nos. 1RO1AR46581 and 1S10RR016879).
Received July 11, 2002;
Last revision February 11, 2003;
Accepted March 3, 2003
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Y. Yang, K.-H. Kim, C.M. Agrawal, and J.L. Ong
Influence of Post-deposition Heating Time and the Presence of Water Vapor on Sputter-coated Calcium Phosphate Crystallinity
J. Dent. Res.,
October 1, 2003;
82(10):
833 - 837.
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ABSTRACT
INTRODUCTION
