Proliferation of Saos-2 and Ca9-22 cells on grooved and pillared titanium surfaces

Abstract

BACKGROUND:

Surface nanostructures in titanium (Ti) oral implants are critical for rapid osseointegration.

OBJECTIVE:

The purpose of this study was to evaluate the growth of osteoblast-like (Saos-2) and epithelial-like (Ca9-22) cells on nanopatterned Ti films.

METHODS:

Ti films with 500 nm grooves and pillars were fabricated by nanoimprinting, and seeded with Saos-2 and Ca9-22 cells. Cell viability and morphology were assessed by cell proliferation assay and scanning electron microscopy, respectively.

RESULTS:

As assessed after 1 hour, proliferation of Saos-2 cells was most robust on grooved films than on pillared and smooth films, in this order. These cells approximately doubled on grooved and pillared substrates in 24 hours and after 5 days, but not on smooth surfaces. In contrast, Ca9-22 cells favored smooth surfaces, followed by grooved and pillared films. Indeed, cells sparsely adhered to pillared films over 5 days of incubation (p < 0.05).

CONCLUSIONS:

The data show that Saos-2 and Ca9-22 cells respond differently to different nanostructures, and highlight the potential use of nanopatterns to promote bone regeneration or to prevent epithelial downgrowth at the implant-bone interface.

Keywords

Saos-2 Ca9-22 grooved implant pillared implant titanium nanopatterning cell proliferation

1. Introduction

Many methods have been developed to modify the surface of dental implants. For example, titanium implants have been roughened by plasma-spraying [1], grit-blasting [2], acid-etching [3,4], and anodization [5] to enhance osseointegration. Most of these surface-modified implants are now commercially available and proven to be >95% effective over 5 years [6]. However, the biological effects of surface topography are not precisely understood, although the topography of sandblasted and acid-etched titanium is known to influence the behavior of human osteoblast-like cells in vitro [7]. On the other hand, epithelial cell growth and focal adhesion are more robust on smooth surfaces rather than on rough surfaces [8]. Hence, the size and shape of surface nanostructures appear to be important determinants of the attachment and proliferation of osteoblast-like and epithelial-like cells.

Dental implants may also induce complications, of which the most serious is peri-implantitis [9,10]. Berglundh et al. [11,12] reported that peri-implantitis resembles periodontal disease. Indeed, soft tissue inflammatory lesions around dental implants were found to be more progressive in beagle dogs than that in teeth [13]. Therefore, preventing peri-implantitis is critical, especially since infection at the implant interface may also lead to implant failure by degrading newly formed ligaments and inducing fibrous encapsulation. However, specific treatments against peri-implantitis have not been developed, although non-surgical methods, plaque control, maintenance therapy, or localized drug therapy are generally effective to maintain oral health [14,15]. Surgery may be necessary in cases of deep pocket formation and peri-implant bone loss [16].

Nevins et al. [17] and Pecora et al. [18] demonstrated that implant collars with 8 μm or 12 μm microchannels affect the physical attachment of connective tissues that could inhibit epithelial downgrowth. Accordingly, connective tissue is established at a predetermined site to preserve coronal bone. Similarly, we have produced titanium-coated films with nano-grooved and nano-pillared surfaces [19–21], since micro- and nano-structures were found to also inhibit apical migration of epithelial cells and prevent loss of alveolar bone [22]. Furthermore, patterned implant collars may prevent peri-implantitis. The purpose of this study was to analyze the behaviors of human osteoblast-like and epithelial-like cells on nano-patterned surfaces, with a view to assess osteointegration and to evaluate the potential value of patterned dental implants or abutments.

2. Materials and methods

2.1. Fabrication of nano-patterned titanium-coated films

Nano-patterned titanium-coated films were fabricated according to Fig. 1. Briefly, a quartz master mold with grooves and holes was prepared (Kyodo International Inc., Kawasaki, Japan). Grooves had ridges of width 500 nm and height or depth 500 nm in a 5 × 5 mm² area, while holes were 500 nm wide and 500 nm deep. To obtain a grooved and pillared replica mold, a polycarbonate film (Sugawara Technology Corp., Tokyo, Japan) was pressed on to the master mold for 5 min at 0.2 MPa and 175 °C using an AH-1TC thermal nano-imprinting apparatus (Shimadzu Corp., Kyoto, Japan), and gradually cooled to 23 °C for 5 min as shown in Fig. 1a and b. Subsequently, the film was carefully peeled off and then sputter-coated with titanium on an HSR412 system (Shimadzu Corp.), as illustrated in Fig. 1c and d. Finally, the patterned Ti-film was gently transferred to a culture dish and sterilized under UV light for 30 min.

Fig. 1.

Fabrication of nano-patterned titanium-coated films. (a) A patterned master mold is set on a polycarbonate film, and (b) thermally pressed at 0.2 MPa and 175 °C. (c) The resulting replica mold is sputter-coated with titanium to obtain (d) a patterned Ti film.

2.2. Scanning electron and laser microscopy of surfaces

Patterned Ti films were sputter-coated with Pt-Pd on an E-1030 system (Hitachi High-Tech Fielding Corp., Tokyo, Japan), and imaged on an S-4000 scanning electron microscope (Hitachi High-Tech Fielding Corp., Tokyo, Japan). Surface topography and line profiles were also obtained on a VK-X200 3D laser microscope (Keyence, Osaka, Japan).

2.3. Cell culture

Saos-2 (RBRC-RCB3688, RIKEN Cell Bank, Tsukuba, Japan) and Ca9-22 cells (RCB1976, RIKEN Cell Bank, Tsukuba, Japan) were cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air, in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum (CELLect Gold, MP Biomedicals Inc., Solon, OH, USA) and 1% penicillin-streptomycin-amphotericin B (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Cultures were checked daily, and media were changed twice a week. At 70% confluence, cells were detached using Accumax^TM (Funakoshi Co., Ltd., Tokyo, Japan), counted on a hemocytometer, and used in subsequent analyses.

2.4. Cell adhesion and proliferation

Patterned Ti-films were immersed in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% penicillin-streptomycin-amphotericin B, seeded with Saos-2 or Ca9-22 cells at 5,000 cells/cm², and incubated for 1 hour, 24 hours, and 5 days at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. Cells were then fixed with 2.5% glutaraldehyde, stained with Giemsa, and imaged on an ECLIPSE E200 optical microscope (Nikon, Tokyo, Japan). Adherent cells in 5 × 5 mm² fields were counted in ImageJ (NIH, Bethesda, MD, USA). Error bars indicate standard deviation from n = 6.

2.5. Scanning electron microscopy of adherent cells

Patterned Ti films seeded with cells for 24 hours were rinsed with phosphate-buffered saline to remove non-adherent cells, fixed with 2.5% glutaraldehyde, and dehydrated sequentially in 50%, 60%, 70%, 80%, 90%, 95%, and 100% ethanol for 5 min each, followed by critical point drying at 37 °C. Finally, specimens were sputter-coated with Pt-Pd and imaged on a scanning electron microscope.

2.6. Statistical analysis

Data were analyzed in GraphPad Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA), and are reported as mean ± standard deviation. Groups were compared by one-way analysis of variance with Tukey’s multiple comparison post-hoc test, with p < 0.05 considered statistically significant.

3. Results

3.1. Fabrication of nano-patterned titanium-coated films

Scanning electron micrographs of the patterned Ti films (Fig. 2a and b) were acquired. Laser microscopy (Fig. 2c and d) confirmed the generation of uniform ridges and grooves with width 500 nm and height 500 nm, as well as uniform pillars with width 500 nm and height 500 nm.

Fig. 2.

(a, b) Scanning electron micrographs of Ti surfaces with (a) 500 nm grooves and (b) 500 nm pillars. (c, d) Laser micrographs of (c) a Ti film with grooves 500 nm high and 500 nm wide and of (d) a Ti film with pillars 500 nm high and 500 nm wide.

3.2. Cell proliferation

The number of Saos-2 cells attached to the Ti films after 1 hour, 24 hours, and 5 days is shown in Fig. 3. There were more attached cells on grooved and pillared films than on smooth films, even after only 1 hour (p < 0.05), with no significant difference (p > 0.05) in attachment between smooth film and grooved and pillared films. After 24 hours and 5 days, the number of cells adhered to the grooved surfaces was higher than those adhered to pillared or smooth films (p < 0.05).

Fig. 3.

Proliferation of Saos-2 cells on patterned Ti films over 5 days. Cells generally accumulated on all surfaces with time. *, significantly higher (p < 0.05) than all other groups; **, significantly lower (p < 0.05) than all other groups, as tested by Tukey’s multiple comparison test.

Significantly fewer Ca9-22 cells adhered to the pillared Ti films as compared to the smooth and grooved surfaces (Fig. 4). Attachment to pillared films was consistently low up to 5 days (p < 0.05). The cell adhesion was comparable between smooth and grooved Ti films at 1 hour (p > 0.05), but was higher in the former at 24 hours and 5 days (p < 0.05).

Fig. 4.

Proliferation of Ca9-22 cells on patterned Ti-films over 5 days. Cells accumulated on smooth and grooved films with time, but not on pillared films. *, significantly higher (p < 0.05) than all other groups; **, significantly lower than all other groups (p < 0.05), as tested by Tukey’s multiple comparison test. (p < 0.05).

3.3. Scanning electron microscopy of filopodia in attached cells

Filopodia were observed in scanning electron micrographs of Saos-2 cells attached to grooved and pillared films after 24 hours (Fig. 5). Most cells had extended filopodia to the top of the ridges on the film (Fig. 5b) or around pillars (Fig. 5c). Ca9-22 cells growing on smooth films extended filopodia in all directions (Fig. 6). Ca9-22 cells growing on grooved films extended filopodia parallel to the ridges on the film, while those growing on pillared films extended few filopodia.

Fig. 5.

Scanning electron micrographs of attached Saos-2 cells after 24 hours on (a) a smooth Ti film, (b) a Ti film with 500 nm grooves, and (c) a Ti film with 500 nm pillars. Cells extended filopodia parallel to grooves and around pillars.

Fig. 6.

Scanning electron micrographs of Ca9-22 cells after 24 hours on (a) a smooth Ti film, (b) a Ti film with 500 nm grooves, and (c) a Ti film with 500 nm pillars. Cells extended filopodia in all four directions on smooth films, and parallel to grooves in grooved films. Few filopodia were extended on pillared films.

3.4. Growth and colonization

To visualize colonization, Ti films were seeded with Saos-2 and Ca9-22 cells for 5 days and then fixed with 2.5% glutaraldehyde and stained with Giemsa stain (Fig. 7). The substrates were densely colonized by Saos-2 cells, and cells were most confluent on grooved films. Conversely, fewer Ca9-22 cells colonized pillared Ti-films compared to other surfaces.

Fig. 7.

Colonization of patterned films after 5 days. Saos-2 and Ca9-22 cells were fixed in 2.5% glutaraldehyde and stained with Giemsa. Substrates were found to be densely colonized by Saos-2 cells, although cells were most confluent on grooved films. On the contrary, fewer Ca9-22 cells were observed on pillared Ti-films than on other surfaces.

4. Discussion

Dental implants are an established treatment for complete and partial edentulism. However, the success of this treatment largely depends on stable osseointegration into the alveolar bone [23]. Multidisciplinary research has enhanced surface modifications of dental implants to enhance direct bonding to the bone tissue. However, the molecular and cellular mechanisms regulating osseointegration are not fully understood yet. In the present study, we investigated the behavior of osteoblast-like and epithelial-like cells cultured on nano-patterned surfaces in vitro to clarify the mechanisms by which these cells attach to implant surfaces.

The patterned films were produced by imprinting against a mold, which is more convenient than laser interference lithography, UV nanoimprinting, milling by computer-aided design/computer-aided manufacturing, and other commonly-used processes [24–27]. Shrinkage and expansion were reduced compared to that in other techniques. Replica mold manufacturing is one of the most important patterning modalities.

After 24 hours and 5 days, the number of Saos-2 cells attached to the grooved surfaces was highest, followed by the number attached to the pillared and smooth films (p < 0.05). This observation is consistent with previous evidence supporting the effects of surface topography on cell adhesion, alignment, morphology, proliferation, and differentiation [28,29]. Osteoblast-like cells growing on a 500 nm-wide groove, deposit well-aligned calcium phosphate at the interface between cells and substrate [30], a significant result considering that osteocytes in lamellar bone are aligned to osteocyte lacunae [31]. Additionally, canaliculi crossing cement lines were observed connecting with osteocytes in the interstitial bone, or in other osteons. This implies that 500 nm nano-patterned structures that are similar to lamellar features promote osteoblast cell adhesion and proliferation, and thus may influence early osteointegration or modeling and remodeling of bone tissue. Remarkably, significantly fewer Ca9-22 cells adhered to pillared Ti films than to smooth and grooved surfaces. Attachment to pillared films was sparse up to 5 days (p < 0.05). On the other hand, attachment was comparable between smooth and grooved Ti films after 1 hour (p > 0.05), but higher in the former at 24 hours and 5 days (p < 0.05), which corroborates previous findings [32,33]. Epithelial cells on zirconium and titanium were observed selectively proliferating on smooth or machined surfaces compared to rough surfaces [34]. In contrast, rough substrates such as pitted devices restricted epithelial cell proliferation. Importantly, epithelial cell proliferation is considered a risk factor for peri-implantitis [35], and is thus critical for controlling epithelial cell attachment and proliferation.

Consistent with our findings, Igarashi et al. reported that osteoblasts favor rough surfaces, although their adhesion and proliferation are enhanced via unknown mechanisms [36], while epithelial cells adhere to and proliferate more efficiently on smooth surfaces than on rough surfaces [37].

Ca9-22 cells may have suppressed epithelial cell proliferation. The varying behavior of osteoblast-like and epithelial-like cells on smooth, grooved, or pillared structures implies that proper use of nano-patterns may prevent epithelial downgrowth and peri-implantitis. Therefore, we anticipate that patterned surfaces could serve as a practical tool in dental implants or abutments for minimizing complications related to dental implants.

5. Conclusion

We prepared grooved and pillared Ti sheets by thermal nano-imprinting. These sheets elicit different behaviors in osteoblast-like and epithelial-like cells, and thus should improve osseointegration but prevent epithelial downgrowth. We anticipate that this study will expand the choice of surface for dental implants and abutments and help prevent peri-implantitis.

Footnotes

Acknowledgements

This work was supported by JSPS KAKENHI grant numbers 26670829, 25463047, 18H06285 and 19K21374. Laser microscopy was performed at OPEN FACILITY, Hokkaido University Sousei Hall.

Conflict of interest

The authors declare that there are no conflicts of interest to report.

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