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RESEARCH REPORT |
1 Dept. of Cellular and Molecular Biology and Pathology, "L. Califano",
2 Dept. of Dental and Maxillofacial Sciences,
3 Dept. of Biomorphological Sciences, and
4 CNR/IEOS "G. Salvatore", University of Naples "Federico II", via S. Pansini 5, 80131 Naples, Italy; and
5 Dept. of Periodontics, University of Pisa, Italy;
* corresponding author, lorposti{at}unina.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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2, 5, Vß3, and ß1 on TPS as compared with SB and S surfaces. An increase in alkaline phosphatase activity was detected only on SB and TPS surfaces. Analysis of cell apoptosis did not demonstrate any significant difference among the 3 different surfaces. The results indicate that titanium surface topography affects proliferation and differentiation of osteoblast-like SaOS-2 cells, suggesting that surface properties might be important for bone response around dental implants in vivo.
KEY WORDS: titanium surfaces • osteoblasts • cell growth • cell differentiation • cell apoptosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In particular, differentiation toward an osteoblastic phenotype is a multi-step process which is regulated by several factors, such as hormones and growth factors (Lian and Stein, 1993). We have recently studied the in vitro behavior of SaOS-2 cells, a well-characterized osteosarcoma human cell line, presenting clear osteoblast-like properties. We have shown that SaOS-2 cell differentiation is regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) (Postiglione et al., 2003).
Cell survival and proliferation on different titanium surfaces may also be regulated by apoptotic mechanisms. Apoptosis, or physiologic cell death, is based on an endogenous cell suicide mechanism, which can be selectively triggered by the cells in response to as-yet-largely-unknown stimuli (Cohen, 1993). Recently, it has been shown that titanium ions cause a preferential degradation of osteoclasts rather than osteoblasts, most likely by an apoptotic mechanism (Matsunaga et al., 2001). However, studies regarding the effects on apoptosis of surface topography of titanium dental implants are limited, and such effects, if any, are largely unknown.
To evaluate the biological role played in bone response by surface properties of titanium implants, in the present study we have investigated cell proliferation, differentiation, and apoptosis of SaOS-2 cells cultured on 3 different titanium surfaces (smooth, sandblasted, and titanium plasma-sprayed).
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Titanium Disks
Commercially pure titanium disks of 1 cm in diameter and 1 mm in thickness were supplied by Sweden & Martina S.p.A. (Due Carrare, Padova, Italy). Disks with 3 different surfaces of increasing roughness were used: a relatively smooth, machined surface (S); a sandblasted surface (SB); and a titanium plasma-sprayed surface (TPS). Titanium samples were subjected to a routine plasma cleaning to minimize surface contamination. Disks were then subjected to several steps of conventional solvent cleaning and placed in a cold-plasma reactor (Gambetti Kenologia, Milano, Italy). Furthermore, disks were treated by Ar plasma, with 100 W of power, a flow rate of 20 scc/m (standard cubic cm/minute) for 15 min, rinsed with distilled water, and autoclaved prior to cell culture experiments.
3H-thymidine Incorporation Assay
We estimated DNA synthesis by measuring 3H-thymidine incorporation. SaOS-2 cells were plated on titanium disks and cultured for up to 96 hrs. At given times (24, 48, 72, and 96 hrs), 100 µCi of 3H-thymidine was added to the cultures during the last 20 hrs of culture. Cells were harvested, counted in a Neubauer cytometer, and absorbed onto nitrocellulose paper; radioactivity was then counted in a ß-counter (Beckman LS1801, Milano, Italy). Results were expressed as mean incorporation at the experimental point (counts per min)/104 cells.
Cell Proliferation Assay
To determine the proliferation of SaOS-2 cells on titanium surfaces, we used the procedure described by Mosmann (1983). The assay is dependent on the cellular reduction of MTT [3-(4,5-dimethylthialzol-2-yl)-2,5-diphenyltetrazolium bromide] (Sigma), by the mitochondrial dehydrogenase of viable cells, to a blue formazan product which can be measured by a spectrophotometer. SaOS-2 cells were harvested by trypsin and plated on titanium disks, and the numbers of viable cells were determined at 24, 48, 72, and 96 hrs by the MTT assay. Titanium disks were transferred into 24-well plates and MTT (5 ng/mL) was added; disks were then incubated for 4 hrs at 37°C. The dye was eluted from the titanium disks with acidified isopropanol, and optical density was measured by a spectrophotometer (Beckman DU-40, Milano, Italy) at 570 nm. Titanium disks without cells and medium alone were used as negative controls.
Alkaline Phosphatase Measurements
Alkaline phosphatase (ALP) was determined with p-nitrophenylphosphate as a substrate. SaOS-2 cells plated on titanium disks were analyzed on the 2nd, 7th, and 14th days of culture. Cells were scraped into 500-µL ice-cold harvest buffer (10 mM Tris HCl, pH 7,4, 0,2% NP-40, and 2 mM phenylmethylsulfonyl fluoride, PMSF). Enzymatic activity was measured by an automatic analyzer (Hitachi 747, Boehringer Mannheim, Indianapolis, IN, USA). The results were expressed as UI/(enzyme activity)/104 cells.
Enzyme-linked Immunoassay in situ
SaOS-2 cells were plated on titanium surfaces and cultured for up to 96 hrs. At given times (6, 24, 48, 72, 96 hrs), cells were fixed on titanium surfaces by 50% (vol/vol) methanol-acetone for 10 min at room temperature and air-dried. Disks were incubated with calcium-and magnesium-free phosphate-buffered saline (PBS)/0.5% bovine serum albumin (BSA), for 2 hrs at 4°C, then filled with 50 µL of one of the following rabbit anti-sera: anti-collagen I (CoI), -fibronectin (FN), -vitronectin (VN), and -tenascin (TN) (Chemicon, Temecula, CA, USA), in PBS/0.5% BSA and 0.2% Tween 20 and allowed to react for 1 hr at room temperature. Plates were then washed with PBS, filled with 50 µL horseradish peroxidase-conjugated anti-rabbit IgG in PBS, 0.2% Tween 20, allowed to react for 1 hr, washed again with PBS, and filled with 150 µL of 1 mg/mL o-phenylenediamine, 0.006% hydrogen peroxide, 0.1 M citrate buffer, pH 5.0. After 30 min of incubation, the absorbance at 450 nm was measured by a spectrophotometer. Titanium disks without cells and disks coated with purified extracellular matrix (ECM) were used as controls. To evaluate the effects of serum on ECM deposition by SaOS-2 cells, we used serum-starved cells, cultured in DMEM 0.5% BSA, as controls.
Flow Cytometric Analysis
SaOS-2 cells plated on titanium surfaces and cultured for up to 96 hrs were harvested by treatment with trypsin/PBS and incubated with specific monoclonal antibodies against integrin subunits, such as anti-2 (kindly donated by Dr. A.E.G.Kr. von dem Borne, Amsterdam, the Netherlands), anti-5 (Telios, San Diego, CA, USA), anti-Vß3 (Telios), and anti-ß1 (kindly donated by Dr. M.E. Hemler, Boston, MA, USA), for 1 hr at 4°C, in 0.5% BSA in PBS. Cells were then washed in the same buffer and incubated with the second fluorescein-conjugated antibody for 30 min at 4°C. Finally, cells were suspended in BSA/PBS and analyzed by flow cytometry (FACScan, Becton Dickinson, Mountain View, CA, USA). Non-specific IgGs of the same isotype were used as a negative control. Results were expressed as fold of expression, as compared with the negative control.
For apoptosis analysis, propidium iodide staining was used. SaOS-2 cells plated on titanium surfaces and cultured for up to 96 hrs were harvested by trypsin, pelleted, washed twice with PBS, fixed and permeabilized with cold 70% ethanol, and then stored at 4°C. A 1-mL quantity of propidium iodide staining solution (50 µg/mL in PBS, pH 7.4) containing 0.5 mg/mL DNase-free RNase was added to 2 x 106 cells (30 min, room temperature), and the DNA content of the cells was analyzed by flow cytometry. Apoptosis analysis was performed with the use of CELL LYSIS software.
Statistical Analysis
Results were calculated as mean ± SD. Statistical analysis was performed by the Student t test, and the differences were considered significant with "p" values less than 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). The numbers of cells on the 3 different titanium surfaces were measured by the MTT assay. The number of cells increased slightly up to 96 hrs of culture on SB and TPS surfaces, while cell proliferation was significantly higher on S surfaces after 72 and 96 hrs (p < 0.03 vs. SB; p < 0.04 vs. TPS) (Fig. 1B).
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= S, 2B = SB, 2C = TPS).
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). Serum-starved cells, cultured in DMEM/0.5% BSA, still produced ECM components, albeit in lower amounts, thus showing that matrix production was not due to the deposition of components present in the serum. Also in the absence of serum, the total amount of ECM deposited on TPS was significantly higher compared with that on SB and S surfaces (p < 0.05) (data not shown). To evaluate the receptors involved in the adhesion of SaOS-2 cells to titanium surfaces, we studied the expression of 2, 5, Vß3, and ß1 integrin subunits by flow cytometric analysis in SaOS-2 cells cultured for up to 96 hrs on titanium disks. All integrin subunits increased between 24 and 96 hrs of culture on the 3 different surfaces. However, there was a higher expression on TPS surfaces as compared with SB and S surfaces; the difference was statistically significant after 96 hrs of culture (p < 0.05) (Fig. 3B).
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) demonstrated a significant increase in ALP activity, in SaOS-2 cells cultured on rough surfaces, after 7 days of culture. ALP production remained significantly higher up to 14 days of culture (p < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Cell proliferation was higher on S than on SB and TPS surfaces, in agreement with the observations of Martin et al. (1995). Cell survival on the different titanium surfaces may also be regulated through apoptotic mechanisms. The analysis of cellular apoptosis did not show any significant difference (p > 0.05) in cell death rates among cells cultured on the 3 titanium surfaces. Analysis of these data, therefore, confirms that the lower number of cells observed on rough surfaces was indeed due to a lower proliferation of SaOS-2 cells on this type of surface rather than to an increased cell death rate.
Several studies suggest that differentiation toward an osteoblastic phenotype correlates with a decrease in cell proliferation and an increase in alkaline phosphatase (ALP) activity, as well as in extracellular matrix (ECM) production (Lian and Stein, 1993). In particular, in SaOS-2 cells, ALP activity can be considered as an osteoblastic phenotypic marker and, thus, an index of osteoblastic differentiation (Farley et al., 1991; Postiglione et al., 2003). Indeed, in the SaOS-2 cells, when proliferation slows, the expression of osteoblastic functions, including ALP and collagen production, increases (Rodan et al., 1987). Analysis of our data shows that the increase in ALP activity was related to surface roughness, being the highest when the cells were cultured on TPS surfaces.
Collagen I (CoI), fibronectin (FN), vitronectin (VN), and tenascin (TN) are among the components of the ECM produced by osteoblasts during differentiation, and CoI is the major component of bone connective tissue (Owen et al., 1990). The analysis of ECM production in SaOS-2 cells demonstrated that the synthesis of CoI, FN, VN, and TN varied depending on the type of surface. Particularly, the matrix was deposited on TPS primarily in the first hours of culture (6–24 hrs), whereas matrix deposition on SB and S surfaces took place progressively between 6 and 96 hrs. Among the 3 surfaces, TPS displayed the higher induction of ECM synthesis and organization, proving to be the best surface for ECM production by osteoblast-like cells. Cells also synthesized CoI, FN, and TN on S and SB surfaces, while VN was not produced on S and SB disks, therefore being the ECM component most sensitive to surface characteristics.
To investigate whether the increase in matrix deposition had a functional correlation, we analyzed the expression of the integrin receptors for ECM components. In particular, 2ß1, 5ß1, and Vß3 represent the main RGD-dependent receptors for CoI, FB, and VN, respectively (Springer, 1990). In agreement with data on ECM production, we found a higher expression of these receptors on rough compared with smooth surfaces.
In summary, these results show that there is an inverse correlation between cell proliferation and differentiation on the 3 titanium surfaces investigated. In fact, SaOS-2 cells proliferate better on smooth surfaces, while rough surfaces promote their differentiation toward an osteoblastic phenotype. Therefore, analysis of our data suggests that rough surfaces may favor a better biological outcome of dental implants in vivo, since they seem to induce differentiation toward an osteoblastic phenotype, enhancing bone healing and the long-term maintenance of osseointegration. Further studies are needed to support this hypothesis.
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ACKNOWLEDGMENTS |
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Received January 31, 2002; Last revision May 28, 2003; Accepted June 24, 2003
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REFERENCES |
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