Nanotexturing of titanium-based surfaces upregulates expression of bone sialoprotein and osteopontin by cultured osteogenic cells

Paulo Tambasco de Oliveira¹ and Antonio Nanci^,

Laboratory for the Study of Calcified Tissues and Biomaterials, Faculté de Médecine Dentaire, Université de Montréal, Pavillon Principal, Montréal, QC, Canada H3T 1J4

Received 13 May 2003;  accepted 21 May 2003. ; Available online 16 September 2003.
Biomaterials
Volume 25, Issue 3 , February 2004, Pages 403-413

1. Abstract

Bone formation around implants is influenced by surface geometry. Since cell/matrix/substrate interactions associated with cell signaling occur in the nanoscale dimension, we have evaluated the influence of nanotexturing of titanium-based surfaces on the expression of matrix proteins by cultured osteogenic cells at initial time points. Cells were obtained by enzymatic digestion of newborn rat calvaria and grown on titanium and titanium alloy discs with nanotextured or machined surfaces, and on glass coverslips for periods of 6 h, 1 day, and 3 days, under standard culture conditions. Cultures were processed for single or dual immunolabeling with monoclonal and/or polyclonal antibodies against bone sialoprotein (BSP), fibronectin (FN), osteopontin (OPN), type-I pro-collagen, or tubulin, followed by corresponding fluorophore-conjugated secondary antibodies. Some samples were processed for scanning electron microscope analysis of morphology and immunogold labeling. After 6 h, nanotextured surfaces exhibited up to a nine-fold increase in the proportion of cells with peripheral OPN labeling. At day 3, the proportion of OPN and BSP labeled cells was higher, and the intensity of immunoreactivity dramatically increased. No significant differences were observed in the expression pattern and the proportion of cells immunoreactive for FN or type-I pro-collagen. Our results demonstrate that nanotexturing of titanium-based surfaces upregulates the early expression of BSP and OPN in osteogenic cell cultures.

Author Keywords: Titanium; Nanotopography; Osteoblast; Immunolabeling; Bone sialoprotein; Osteopontin

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Titanium samples

2.2. Cell isolation and primary culture of osteogenic cells

2.3. Morphological analysis

2.4. Immunofluorescence

2.5. Colloidal gold immunocytochemistry

3. Results

3.1. Surface features

3.2. Cell morphology

3.3. Distribution of labeling

3.4. Quantitative analysis

3.5. Colloidal gold immunocytochemistry

4. Discussion

Acknowledgements

References

3. 1. Introduction

The ideal biomaterial should induce rapid, predictable and controlled healing of host tissues [1]. The surface of an implant plays a critical role in determining biocompatibility and, ultimately, integration because it is in direct contact with the tissues [2 and 3]. A long-standing hypothesis asserts that four material-related factors can influence events at bone-implant interfaces, that is, implant surface composition, surface energy, surface roughness, and surface topography [4]. While surface features can contribute to biomechanical retention, if they are to directly influence tissue repair, their characteristics must also take into consideration cellular and molecular ranges of activity [5 and 6].

Various surface textures have been created and used to successfully influence cell and tissue responses [7, 8, 9, 10, 11, 12, 13 and 14]. Among these, microtextured surfaces have been proposed to represent an important tool for establishing an advantageous, three-dimensional environment for osteogenic cells [14], and experimental data and early results from clinical trials show an effect on bone formation, at least during initial stages of osseointegration [15, 16, 17, 18 and 19].

It has been demonstrated that different cell types respond to nanotopography [20]. Cell/matrix/substrate interactions associated with cell signaling occur in the nanoscale level. Such signaling regulates cell attachment, spreading, migration, differentiation, and gene expression [21, 22, 23, 24 and 25]. Increased cellular responses have been reported in cell cultures grown on nanophase ceramics [26, 27, 28 and 29], polymer demixed nanotopography [30 and 31], and nanometer diameter carbon fibers [32]. Studies have also shown that nanoporous hydroxyapatite granules implanted in alveolar bone defects accumulate bone matrix proteins such as bone sialoprotein (BSP) and osteopontin (OPN) [33 and 34]. Indeed, focal deposition of such proteins is believed to represent an early step in bone formation onto hydroxyapatite and other biomaterials both in vivo and vitro [35].

Although it has been proposed that a given cell type reacts in much the same way to the same topography made with different materials [20], little is known about the potential biologic effects of nanotextured metal implant surfaces. The aim of the present study was, therefore, to evaluate the early expression of collagenous and noncollagenous bone matrix proteins in osteogenic cells cultured on nanotextured titanium-based surfaces. A method for chemical deoxidation and controlled reoxidation using H₂SO₄/H₂O₂ that reproducibly results in the formation of a unique nanotopography [36] was applied to nanotexture commercially pure titanium (cpTi) and titanium alloy (Ti-6Al-4V). Expression of BSP, fibronectin (FN), OPN, and type-I collagen was investigated because these matrix constituents have been associated with initial events in bone formation [37, 38, 39, 40, 41 and 42]. The results obtained show that nanotexturing of titanium-based surfaces has a profound and rapid effect on the expression of OPN and BSP.

4. 2. Materials and methods

2.1. Titanium samples

cpTi and Ti-6Al-4V discs (14 mm in diameter), with a machined surface, were rinsed with toluene and then nanotextured by treating them with a solution consisting of equal volumes of concentrated H₂SO₄ and 30% aqueous H₂O₂ for 4 h (cpTi) and 1 h (Ti-6Al-4V) at room temperature (RT) using sterile conditions [36]. The cleaned, oxidized samples were rinsed with sterile dH₂O, air-dried and stored under ultraviolet light. Control, non-etched discs, were washed with 70% ethanol, rinsed in sterile dH₂O and also stored under ultraviolet light. The surfaces of some control and nanotextured discs were examined using a JEOL JSM-6700F field emission scanning electron microscope (SEM) operated at 1-2 kV.

2.2. Cell isolation and primary culture of osteogenic cells

Osteogenic cells were isolated by sequential trypsin/collagenase digestion of calvarial bone from newborn (2-4 days) Wistar rats (Charles River Canada, St-Constant, QC, Canada), as previously described [43 and 44]. All animal procedures were in accordance with guidelines of the Comité de déontologie de l'expérimentation sur les animaux of Université de Montréal. Cells were plated on machined surface and nanotextured discs and on Fisherbrand^® 12 mm-round glass coverslips (Fisher Scientific, Nepean, ON, Canada) in Falcon^® 24-well plates (Becton-Dickinson, Lincoln Park, NJ) at a cell density of 1.5×10⁴ cells/well. Cells were grown for periods of 6 h, 1 day, and 3 days in Gibco™ Minimum Essential Medium (MEM) with Earle's salts (Invitrogen, Burlington, ON, Canada) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Invitrogen) at 37°C in a humidified atmosphere with 5% CO₂.

2.3. Morphological analysis

For morphological analysis, cells were fixed for 1 h at RT in 5% glutaraldehyde buffered with 0.06 
sodium cacodylate, pH 7.2, and routinely processed for SEM [43 and 44]. Samples were examined without coating using a JEOL JSM-6700F or a JEOL JSM-5900LV SEM. Digital images were processed with Adobe Photoshop software.

2.4. Immunofluorescence

Cells were fixed for 15 min at RT using 4% paraformaldehyde in 0.1 
sodium phosphate buffer (PB), pH 7.2. After washing in PB, they were processed for immunofluorescence labeling. Briefly, they were permeabilized with 0.5% Triton X-100 in PB for 15 min followed by blocking with 1% ovalbumin in PB for 30 min. Primary monoclonal and/or polyclonal antibodies to BSP, FN, OPN, type-I pro-collagen, and tubulin (for detecting cell outlines) were used (Table 1), followed by corresponding Alexa Fluor 488 (green fluorescence)—or 594 (red fluorescence)—conjugated goat secondary antibodies (Molecular Probes, Eugene, OR) at a working dilution of 1:200. Dual labeling was done with a 1:1 mixture of two primary antibodies followed by a 1:1 mixture of corresponding secondary antibodies. Mixtures were prepared to yield the same working dilutions as the ones used in single labeling (Table 1). Replacement of the primary antibody with PB was used as control. For type-I pro-collagen labeling, incubations with pre-immune IgG were also carried out. All antibody incubations were performed in a humidified environment for 60 min at RT. Between each incubation step, the samples were washed in PB (3×5 min). Before mounting for microscope observation, samples were briefly washed with dH₂O. Metal discs were mounted facing up on glass slides, while a glass coverslip was mounted with Airvol 205 (Airproducts, Allentown, PA) on the surface containing cells. Control glass coverslips were mounted face down with Airvol 205 (Airproducts) on glass slides. The samples were then examined by epiluminescence under a conventional fluorescence microscope (Axiophot; Carl Zeiss, Oberkochen, Germany), using Plan-Neofluar objectives (×40, numerical aperture 0.75; and ×100, numerical aperture 1.30). For quantitative analysis, at least 20 microscopic fields at ×100, comprising between 300 and 400 cells, were randomly selected. The proportions of immunoreactive cells were determined in 6-h samples, as a major aim was to evaluate the influence of surface texture on the expression of matrix proteins at a very early time interval. In the case of FN, the proportion of immunoreactive cells at day 1 was also quantified since assembly of fibrillar FN takes up to 8 h to initiate [47]. Proportions of immunoreactive cells were expressed as the mean percentage per microscopic field±standard deviation and comparisons were carried out using the non-parametric Kruskal-Wallis test for independent samples. If the result of the Kruskal-Wallis test was "significant", i.e. occurrence of at least one significant difference, the Fisher's least significant difference multiple comparisons procedure, computed on ranks rather than data, was carried out [48]. Photographic recordings were made on Kodak Elite Chrome 400 reversal film (Eastman Kodak, Rochester, NY). Double photographic exposure was done for the simultaneous visualization of dual labeling. Slides were then digitally recorded at 1200 dpi using an Epson Expression 1600 scanner and processed with Adobe Photoshop software. The results described below are representative of three sets of primary cultures.

Table 1. Primary antibodies used and their working dilutions

2.5. Colloidal gold immunocytochemistry

Cell fixation, permeabilization, and blocking were carried out as described above. Cells were then incubated with a rabbit anti-OPN antibody (LF123, Table 1) for 60 min, followed by blocking with 1% ovalbumin for 15 min and incubation with a protein A-gold complex for 30 min, all in a humidified environment and at RT. The complex was prepared as previously described [49] using colloidal gold particles of 8-10 nm. After washing in PB, cells were then fixed in 5% glutaraldehyde for 15 min, post-fixed in 1% osmium tetroxide for 60 min, and routinely processed for observation, without coating, in a JEOL 6700F field emission SEM operated at 1-2 kV.

5. 3. Results

3.1. Surface features

At low magnification, the surface of both alloy (Fig. 1B and D) and cpTi (Fig. 1F and H) exhibited shallow grooves resulting from machining and polishing. These were less frequent on cpTi and were not significantly affected by the nanotexturing treatment. At high magnification, the machined metal surfaces did not reveal any topographical features ( Fig. 1A and E). However, chemical treatment of their surfaces reproducibly resulted in the formation of a unique texture characterized by nanopitting (diameter in the 10 nm range) with a honeycomb-like appearance (Fig. 1C and G).

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Fig. 1. Scanning electron micrographs of cells grown on diverse titanium surfaces. Control samples (A, Ti alloy, and E, cpTi) exhibit a smooth surface, while etched ones (C, Ti alloy, and G, cpTi) are characterized by a unique nanotopography. At 6 h, no significant differences were detected in cell shape between control (B and F) and nanotextured (D and H) surfaces. The predominant cell shape was polygonal, with cells showing either thin cytoplasmic extensions or large veil-like ones.

3.2. Cell morphology

Cell morphology on titanium surfaces cannot be assessed with transmitted light. For this reason, epifluorescence labeling of tubulin was used to approximate cell outlines. Indeed, cells grown on glass exhibited tubulin-defined contours that matched those observed by phase contrast. In addition, cell morphology was also studied by SEM (see below).

Epifluorescence tubulin labeling revealed that cells grown both on metal and glass had variable morphology and dimensions (Fig. 3D), and became progressively larger and more spread over the 3-day culture period. At 6 h, cells were attached and partially spread on all surfaces, showing predominantly a polygonal morphology with relatively short cytoplasmic extensions. Some rounded cells were also observed. At days 1 and 3, cell shape and dimensions were variable, and polygonal, stellate and fusiform cells were present (Fig. 3H-L). Some of them emitted long cytoplasmic extensions, which frequently contacted others. Typical migrating cell morphology, with leading and trailing edges, was observed mainly in areas of low cell densitiy. At day 3, areas of confluency appeared to be more prominent on the nanotextured metal surfaces.

At 6 h, SEM revealed no significant differences in cell shapes between control and nanotextured metal surfaces (compare in Fig. 1, B with D and F with H). The predominant shape was polygonal, with some cells showing thin and large finger-like projections and others a uniform, pericellular cytoplasmic spreading. At higher magnification, two structural features were particularly noteworthy. Portions of some lamellar projections appeared reticulated ( Fig. 2A-C). Such a structural modification was generally found at one pole of the cells, thus suggesting an association with directional motility. The other feature was the presence of numerous filipodia along the periphery of the cells (Fig. 2D-F). Although these were found on control surfaces, they were more abundant on nanotextured ones ( Fig. 2D). These filipodia appeared to span some of the larger surface topographical features. Their surface was not smooth and some of its irregularities attached to the ridges of the nanocavitations created by the etching treatment ( Fig. 2E and F). There was no evident extracellular accumulation of matrix at this very early time interval.

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Fig. 2. Scanning electron micrographs of cells grown on normal (A) and nanotextured surfaces (B-F). (A-C) Portions of the cytoplasm of some cells (asterisk) exhibited a reticulated appearance. (C) Higher magnification in the vicinity of the dotted area in (B). Cells on all metal surfaces extended filipodia; however, these were more abundant on nanotextured Ti alloy (D, arrowheads) and on nanotextured cpTi (B, arrowheads, E and F) surfaces. Their outlines appeared to follow the walls of the nanocavitations (F, arrows) created by the etching treatment. (F) High magnification of the boxed area in (E). N, nucleus.

3.3. Distribution of labeling

For all surfaces, the various primary antibodies labeled different proportions of cells and yielded different fluorescence intensities. However, remarkable differences were observed in the pattern of OPN and BSP expression in cells grown on nanotextured surfaces.

Generally, OPN was localized in a perinuclear tubular network, vesicular structures, and punctuate deposits throughout the cytoplasm (Fig. 3). Some cells showed labeling of a large, juxtanuclear region reminiscent of the Golgi area in active osteoblasts. Strikingly, after 6 h on etched surfaces, an important proportion of OPN-positive cells exhibited an overexpression of peripheral cytoplasmic labeling, which resembled the reported perimembranous intracellular OPN [50, 51 and 52]. This labeling appeared as focal subplasmalemmal accumulations of immunofluorescence with a dense, finely granular texture, and was often observed together with juxtanuclear labeling ( Fig. 3C and E-G). The peripheral OPN labeling pattern was only rarely observed on control surfaces and, when present, the intensity of immunofluorescence was much lower (compare in Fig. 3, G with H). Noteworthy, labeling with monoclonal and polyclonal OPN antibodies did not necessarily colocalize over the peripheral focal deposits. Dual labeling and double exposure with polyclonal anti-tubulin ( Fig. 3E and F), -BSP, or -type-I pro-collagen antibodies, which allowed visualization of the cell outline, suggested that sometimes there were also extracellular accumulations of OPN, in addition to the peripheral cytoplasmic labeling. The frequency and intensity of intra- and extracellular peripheral labeling increased over the 3-day culture period ( Fig. 3J and K). The extracellular OPN labeling resembled the tracks of FN and other matrix molecules left behind by migrating fibroblasts in cell culture [53]. No extracellular accumulation of OPN was evident on both metal and glass control surfaces at any time points.

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Fig. 3. Immunolabeled preparations of osteogenic cells grown on glass and on metal surfaces at 6 h (A-G) and at day 3 (H-L). (A) Osteopontin (OPN)-positive cells show a similar labeling pattern on glass (A) and Ti alloy (B); the Golgi area (G) and some cytoplasmic granules are immunofluorescent. (C and D) Dual labeling for OPN (C) and tubulin (D) reveals that not all cells express OPN. Based on cell outlines defined by tubulin labeling, cells indicated by arrows have an abundant peripheral OPN labeling. (E and F) Dual labeling OPN (red) and tubulin (green) of cells grown on nanotextured Ti alloy surfaces. Double exposure reveals that some have only red fluorescence in their periphery. Regions where the two proteins co-distribute appear in yellow. The red fluorescent area in F, marked with an asterisk, represents extracellular accumulation of OPN. (G) Osteopontin labeling of a cell grown on nanotextured cpTi, illustrating the typical pattern of peripheral cytoplasmic accumulations (arrowheads). (H) These accumulations are much less prominent in cells grown on control surfaces, even after 3 days. Upregulation of OPN is clearly observed in cells at day 3 on nanotextured surfaces (cf. I and J). (K and L) Dual labeling OPN (green) and BSP (red) and dual exposure of immunoreactive cells on nanotextured cpTi (K) and nanotextured Ti alloy (L). In (K), OPN tracks (asterisk) are observed adjacent to some osteoblastic cells. (L) shows extracellular accumulation of BSP (asterisk) in a subconfluent area. N, nucleus, BARS=20 
m.

BSP labeling was distinctively and mainly detected in the juxtanuclear Golgi area and as punctuate deposits throughout the cytoplasm. Noteworthy, etched surfaces exhibited some immunoreactive cells with peripheral labeling in cytoplasmic extensions at 6 h. By 3 days, nanotexturing resulted in a readily apparent increase in the proportion of BSP immunoreactive cells. In addition, both the intracellular (Fig. 3K and L) and extracellular ( Fig. 3L) immunolabeling signals were more intense. Focal, extracellular accumulation of BSP-reactive matrix was also observed on control titanium discs, but in a lesser quantity and with a weaker staining intensity. Double labeling for BSP and OPN revealed that the majority of BSP-positive cells also expressed OPN. Although there was a colocalization of labeling for these two proteins over the Golgi area and some granules, extracellular colocalization was rarely observed.

At 6 h on all surfaces, FN immunoreactive cells exhibited variable amounts of a dot and/or a fibrillar immunolabeling, which varied in intensity. Although these labeling patterns are suggestive of initial extracellular assembly, the possibility that some of the dot fluorescence represents intracellular labeling cannot be ruled out. Distinctively, on nanotextured surfaces, some cells were associated with very intense dot fluorescence. FN tracks, with a faint granular pattern, were frequently detected on the substrate surface adjacent to cells with a strong perinuclear labeling. Regions of high cell density exhibited an extracellular, fibrillar FN network, which extended from one cell to another and appeared to be more prominent on glass than on metal surfaces.

On all surfaces, cells immunoreactive for type-I pro-collagen exhibited two distinct labeling patterns. Cells exhibited either (1) well-defined juxtanuclear labeling and punctuate deposits throughout the cytoplasm (at 6 h only) or (2) diffuse labeling around the nucleus (at 6 h and in all immunoreactive cells at days 1 and 3). These distinctive labeling patterns for type-I pro-collagen was not observed when the primary antibody was replaced with pre-immune IgG.

Tubulin labeling was detected throughout the whole cytoplasm, with a more intense fluorescence around the nucleus (Fig. 3D-F).

3.4. Quantitative analysis

Different proportions of osteogenic cells were immunoreactive for BSP, FN, OPN, type-I pro-collagen, and tubulin at 6 h (Table 2). Statistical significant differences were detected in the proportion of cells exhibiting a peripheral distribution of OPN labeling, with a three- to four-fold increase on nanotextured cpTi and Ti-6Al-4V compared to control metal surfaces (
=0.1%) and up to a nine-fold increase compared to glass (
=0.1%). Approximately, 90% of cells with peripheral OPN also showed labeling in the Golgi area. On all surfaces, almost half the cells were labeled for OPN and type-I pro-collagen, whereas only about 1/6 of cells were immunoreactive for BSP and 1/5 for cytoplasmic FN. Extracellular FN was associated with almost all cells and tubulin was ubiquitously expressed.

Table 2. Proportionsa of immunoreactive cells at 6 h on control and etched (E) surfaces

3.5. Colloidal gold immunocytochemistry

At 6 h on nanotextured titanium, there were some areas contiguous with cells that were decorated with numerous gold particles (Fig. 4). Although the particles indicated the presence of OPN at these sites, these protein accumulations did not result in any morphological texture discernable by scanning microscopy. These areas often gave the appearance of tracks associated with cell projections. Few, randomly distributed, particles were present on the metal beyond these regions.

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Fig. 4. Immunogold preparation of a 6-h cell culture grown on nanotextured titanium and labeled for osteopontin. Note the presence of an intensely labeled area, here outlined by arrows, adjacent to a cell (asterisks) and the paucity of gold particles on the titanium surface beyond.

6. 4. Discussion

The results of this study, comparing nanotextured and machined titanium-based surfaces and glass, show that nanotexturing of titanium-based surfaces has a profound and rapid effect on the expression of noncollagenous bone matrix proteins by calvaria-derived osteogenic cells. To our knowledge, this is the first study to demonstrate that nanotextured metals can influence the very early stages of in vitro osteogenesis. There was an overexpression of OPN and BSP both intra- and extracellularly. In addition, nanotextured surfaces exhibited a higher proportion of cells with a peripheral cytoplasmic distribution of OPN as early as 6 h. No major differences were observed in the expression pattern and the proportion of cells immunoreactive for FN or type-I pro-collagen. However, there occasionally were some FN immunoreactive cells that exhibited an intense reactivity on the nanotextured surfaces.

Nanotextured surfaces were created using an H₂SO₄/H₂O₂ mixture for deoxidation and controlled reoxidation of metals [36]. This chemical treatment eliminates surface contaminants and results in a consistent and reproducible titanium oxide surface layer, which is characterized by unique nanopitted topography. In addition, increased number of hydroxyl groups is expected to take place on H₂O₂-treated titanium surfaces [54], an increase that has been correlated with enhancement of apatite nucleation and protein adsorption [54, 55 and 56]. The formation of an apatite layer and adsorption of selected proteins enhances osteoblast adhesion and matrix production on biomaterial surfaces [27, 28 and 57]. Since apatite formation does not take place over a 3-day, short-term culture period under the conditions we have used, the observed overexpression of OPN and BSP on nanotextured surfaces must, therefore, result from (1) the direct response of calvarial cells to topographic features [20], (2) an indirect effect resulting from an enhanced adsorption of serum- and/or cell-derived bioactive molecules, or (3) their synergistic action [58]. Irrespective of the nature of the inductive event, i.e. topography, protein or both, these ultimately all derive from the nanotexturing of titanium-based surfaces. The resulting cell/matrix/substrate interactions clearly influence signal transduction pathways and alter BSP and OPN gene expression, protein secretion and distribution. Further studies using serum-free conditions should permit to evaluate the potential contribution of proteins adsorbed from serum. On the other hand, evaluation of paracrine/autocrine activity of proteins produced by cultured cells during the short culture time will require patterning of the disc surface with selected proteins.

OPN is generally regarded as a multifunctional extracellular matrix protein involved in cell adhesion and migration and in the regulation of mineral deposition [42, 59, 60, 61, 62 and 63]. It has recently been proposed that an intracellular form of OPN, so-called perimembranous intracellular OPN, is an integral component of a hyaluronan-CD44-ERM (ezrin, radixin, moesin) attachment complex [50, 51 and 52]. This complex is believed to be involved in the migration of fetal calvarial cells and embryonic fibroblasts in vitro. Nanotextured titanium surfaces remarkably sustained not only the growth and differentiation of a higher proportion of cells exhibiting peripheral cytoplasmic labeling for OPN, but also overexpression of this matrix protein. The dual labeling patterns suggest that the cells in which OPN is upregulated could represent pre-osteoblasts, differentiated osteoblasts, and fibroblasts. The presence of adjacent OPN tracks strongly suggests that peripheral OPN immunolabeling is associated with migrating cells. These OPN tracks resemble the ones of FN and other matrix molecules, left behind by migrating fibroblasts in culture [53]. However, peripheral OPN immunoreactive cells did not always (particularly at 6 h) exhibit the typical morphology of migrating cells, that is, broad lamellar leading and long filamentous trailing edges. In the present study, OPN tracks were generally associated with an arcuate region of the cell body apparent in the immunofluorescence preparations or short, stumpy processes visible in the SEM. In these areas, the region of the cell bordering the tracks appeared disrupted. Such an alteration has been shown to occur at the trailing edge of migrating cells where the membrane is less well supported by the cytoskeleton [64].

Expression of BSP is generally believed to initiate in newly differentiated osteoblasts [41]. However, it has recently been reported that early progenitors transiently express BSP [65]. In newborn rat calvarial cell culture, about 20% of cells enzymatically released from the second and third digests express BSP before plating; although the presence of early progenitors cannot be excluded, a large proportion of these cells are most likely differentiated osteoblasts and/or osteocytes [66]. Since progenitors represent less than 1% of cells isolated from rat calvaria under standard culture conditions [67], the intra- and extracellular overexpression of BSP observed at day 3 on nanotextured surfaces must reflect an upregulation of the synthetic activity and secretion in differentiated osteoblasts. Although cell crowding prevented quantification at day 3, straightforward observation readily revealed the presence of more BSP-positive cells on nanotextured titanium, suggesting that the surface obtained following H₂SO₄/H₂O₂ treatment may, in addition to stimulating osteoblast activity, also influence the osteoblast differentiation sequence.

In summary, the chemical treatment used in the present study was originally developed for covalently linking selected molecules with known biological activity to oxidized titanium surfaces in order to guide and promote tissue healing [36]. However, it is clear that the nanotopography produced, by itself, has significant biological effects on cultured osteogenic cells, a finding consistent with results from in vitro studies with nanostructured ceramics, polymers, and carbon fibers [26, 27, 28, 29, 30, 31 and 32]. BSP and OPN contain Arg-Gly-Asp (RGD) sites, a common recognition sequence for integrins expressed by osteoblasts [24, 62, 63, 68 and 69]. Thus, the enhanced, early deposition of these two multifunctional matrix proteins resulting from nanotexturing can influence osteoblastic cell adhesion and migration. It may also influence the kinectics of progenitor or differentiating cells and, ultimately, the rate of bone formation [70]. Therefore, it will be interesting to follow-up the effects of nanotexturing of titanium surfaces with studies aimed at evaluating if such surfaces influence later stages of in vitro osteogenesis (i.e. nodule formation) and bone repair in vivo. Collectively, the results described herein open the door towards a new generation of dental and orthopedic implants, in which nanotexture can directly signal cells and act as an "intelligent" surface.

7. Acknowledgements

This work was supported by the Canadian Institutes of Health Research (CIHR). Paulo Tambasco de Oliveira was the recipient of a post-doctoral fellowship (00/11604-4) from FAPESP (Brazil), and was also supported by the University of São Paulo (Brazil). We would like to express particular thanks to Sylvia Francis Zalzal for performing cell isolations, and for her judicious advice on cell culture experiments. The authors also thank Dr. Larry W. Fisher (NIH) for providing the polyclonal antibodies against BSP (LF-100) and OPN (LF-123), and Drs. Malcolm Collins and Paul Bornstein (University of Washington at Seattle, WA) for an antibody to recombinant N-propeptide of the
1 (I) human collagen chain. The mouse monoclonal anti-rat OPN (MPIIIB10-1) and anti-rat BSP (WVID1-9C5) antibodies, developed by Michael Solursh and Ahnders Franzen, were obtained from the Developmental Studies Hybridoma Bank formed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences (Iowa City, IA 52242).

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Corresponding author. Tel.: [phone]; fax: [phone]

¹ Present address: Faculty of Dentistry, University of São Paulo, Ribeirão Preto, Brazil.

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