Glow discharge plasma pretreatment enhances osteoclast differentiation and survival on titanium plates

Hiroyuki Kawai^, , Yo Shibata and Takashi Miyazaki

Department of Oral Biomaterials and Technology, School of Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

Received 31 March 2003;  accepted 11 August 2003. ; Available online 6 November 2003.
Biomaterials
Volume 25, Issue 10 , May 2004, Pages 1805-1811

1. Abstract

Despite the fact that several reports have demonstrated osteoclast activity on various bioactive ceramics, osteoclast functions on surface-modified titanium have not come under focus. This study aimed to examine whether the increasing surface energy of glow discharge plasma (GDP) involved in protein adhesion containing the RGD (Arg-Gly-Asp) sequence affects osteoclast responses on titanium plates. We examined osteoclast differentiation and survival rates on titanium plates with and without GDP. The amounts of osteoclasts on titanium plates were not increased by GDP after 1 week. However, osteoclast differentiation was greatly activated by GDP pretreatment, as tartrate-resistant acid phosphatase synthesis significantly increased on the titanium plates with GDP. Additionally, since the presence of osteoclasts was detected only on the titanium plates with GDP, even after 4 h cultivation in a coculture test, the osteoclasts survival rate was increased by GDP pretreatment. As osteoclast responses were affected even on surface modified metallic materials, we concluded that novel approaches are needed not only for osteoclastic resorption on ceramic materials but also for osteoclast responses on surface-modified metallic materials.

Author Keywords: Titanium; Osteoclast; Metal surface treatment; Protein adsorption

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Specimen preparation

2.2. Cleaning of specimens

2.3. GDP

2.4. Osteoclast formation and differentiation on the specimens

2.5. Mouse bone marrow culture on the specimens

2.6. Actin ring formation

2.7. Reverse transcription-polymerase chain reaction

2.8. Synthesis of tartrate-resistant acid phosphatase on the specimens

2.9. Osteoclast survival on the specimens

2.10. Osteoclast preparation

2.11. Osteoclast survival on the specimens

2.12. Statistics

3. Results

3.1. Bone marrow culture on the specimens

3.1.1. Actin ring formation

3.1.2. RT-PCR

3.1.3. Trap synthesis on the specimens

3.2. Osteoclast survival on the specimens

4. Discussion

4.1. Osteoclast formation and differentiation on the specimens

4.2. Osteoclast survival

5. Conclusion

Acknowledgements

References

3. 1. Introduction

The stable bonding between mature bone and a material interface, known as osseointegration, is critical to the success of orthopedic and dental implants. Earlier studies of osteoconductivity of implant materials have focused on reactions of osteoblasts to the materials in vitro [1, 2 and 3]. However, since recent reports have indicated that continuous balanced osseous remodeling is essential for the successful maintenance of implant devices [4], osteoclastic resorption on various bioactive ceramics has been investigated [5, 6 and 7].

On the other hand, titanium and its alloys have been well established as primary metallic biomaterials to fabricate implant devices. We have already reported that the high adhesion and differentiation of osteoblasts on a titanium surface strongly depend on the binding of extra cellular matrix (ECM) proteins containing the RGD (Arg-Gly-Asp) sequence included in the culture medium through an integrin-mediated mechanism [8]. However, though several reports have indicated the importance of chemical properties, crystal structures, and grain sizes of bioactive ceramics in the activity of osteoclasts [9, 10, 11, 12 and 13], the responses of osteoclasts on modified-titanium surfaces have not been fully elucidated yet.

Differentiating osteoclasts are known to demonstrate morphological changes, polarization, and actin ring formation [7]. Nakamura et al. indicated that the presence of ECM proteins containing the RGD sequence was not sufficient for the actin ring formation of osteoclasts, because no rings developed in osteoclasts cultured on collagen gels or demineralized dentine [14]. Contrarily, Zreiqat et al. reported that in vitro expression of ECM proteins known to be associated with bone resorption were increased with high osteoblast differentiation onto the RGD modified titanium surfaces [4]. However, whether the binding of proteins containing the RGD sequence onto a titanium surface affects the actual response of osteoclast differentiation has not been examined.

Our previous studies demonstrated that glow discharge plasma (GDP) pretreatment of titanium plates enhanced the adhesion and differentiation of osteoblast-like cells [8]. Since GDP enhanced the surface energy of a titanium surface [15], increasing adhesion of cell-binding proteins containing the RGD sequence induced subsequent cell adhesion and differentiation. Therefore, we hypothesized that the GDP pretreatment might affect the responses of osteoclasts on titanium plates. In this study, we examined mouse bone marrow culture on titanium plates with and without GDP to investigate the formation and differentiation of osteoclasts on the titanium plates. The survival of differentiated osteoclasts prepared by the mouse coculture system on titanium plates with and without GDP was also investigated.

4. 2. Materials and methods

2.1. Specimen preparation

The material used was JIS grade 2 titanium plates (30×30×1.0 mm KS-50, Kobe steel). The plates were sectioned to specimens with a dimension of 10×10×1.0 mm using a wire-type electric discharge-machining (W-EDM) device (
-0iA, FANUC). The surface of the specimens was gradually ground using waterproof polishing papers from #500 to #1200 under running water, and then polished with alumina particles of 0.3 
m average diameter.

2.2. Cleaning of specimens

The prepared specimens were ultrasonically cleaned in acetone, detergent solutions (7X, ICN) and pure distilled water for 15 min for each cleaning. Subsequently, the specimens were sterilized in an autoclave, then dried and stored in a desiccator for 24 h at 23°C and humidity 50%.

2.3. GDP

GDP was performed as in our previous study [8]. After setting the specimens in a holder in a chamber under argon gas replacement, GDP was processed under a vacuum of 8×10⁻³ Torr for 1 min.

2.4. Osteoclast formation and differentiation on the specimens

To investigate the formation and differentiation of osteoclasts, the specimens were subjected to mouse bone marrow cell culture [16] with and without GDP.

2.5. Mouse bone marrow culture on the specimens

Bone marrow cells were prepared from the tibia of 6-day-old ddY mice killed by cervical dislocation. Tibia were removed and dissected free of adhering tissues. The bone ends were cut off with scissors, and the marrow cavity flushed with
-minimal essential medium (
-MEM; Sigma, St. Louis, MO) by slowly injecting at one end of the bone using a sterile 25-G needle. The marrow cells from two animals were collected into a tube, centrifuged, and resuspended in 10 ml of
-MEM. Subsequently cells were seeded on the specimens with and without GDP in each well of 24-well culture dishes (Corning, Corning, NY) at a seeding density of 1.0×10⁶ cells/ml (erythrocytes not included). The culture medium used was
-MEM supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY), 1% antibiotics (200 U/ml of penicillin G and 100 
g/ml of streptomycin) in the presence of 10 

of freshly added 1
,25-dihydroxyvitamin D₃ (Wako Pure Chemical Industry Co., Osaka, Japan), a resorbing agent well known to stimulate the formation of osteoclasts. Cells were cultured in
-MEM containing 10% FBS with 50 ng/ml recombinant mouse macrophage colony-stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN) together with 100 ng/ml receptor activator of nuclear factor
B ligand (RANKL) (Pepro Tech, Pepro Tech EC, London) for 1 week in a humidified atmosphere of 95% air, 5% CO₂ at 37°C. The medium was replaced after 3 days in culture by fresh medium with newly added 1
,25-dihydroxyvitamin D₃.

2.6. Actin ring formation

The actin rings were visualized by staining F-actin with rhodamine-conjugated phalloigin [16]. Cells were fixed with 3.7% (v/v) formaldehyde in PBS(−) for 10 min and permeated by treatment with 0.1% Triton X-100 in PBS(−) for 1 min. Cells were incubated for 3 h with the rhodamine-conjugated phalloigin (Sigma) solution. The cells were washed with water, and the actin rings formed by osteoclasts were observed with fluorescence microscope (Olympus BX-50, Osaka, Japan).

2.7. Reverse transcription-polymerase chain reaction

For analyzing expression levels of calcitonin receptor (CTR), total cellular RNA was scraped from the specimens, with or without 10⁻⁸ 
1
,25(OH)₂D₃, for 48 h using Trizol solution (Life Technologies). First-strand complementary DNA was synthesized from total RNA with random primers and subjected to PCR amplification with EX Taq polymerase (Takara Biochemicals, Shiga, Japan) using specific PCR primers: mouse Emr1 (F4/80) (sense, 5′-AATCTTGGCCAAGAAGAGAC-3′ antisense, 5′-CAAGGCACGGACAATGTTGAGAAG-3′), mouse CTR (sense, 5′-TTTCAAGAACCTTAGCTGCCAGAG-3′, antisense, 5′-CAAGGCACGGACAATGTTGAGAAG-3′) and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sense, 5′-ACCACAGTCCATGCCATCAC-3′ and antisense, 5′-TCCACCACC-CTGT- TGCTGTA-3′). GAPDH was used as an internal control [17].

2.8. Synthesis of tartrate-resistant acid phosphatase on the specimens

Cytochemical staining for TRAP is widely used for identifying osteoclast activity in vivo and in vitro [16]. Naphthol AS-MX phosphate (5 mg, Sigma, St. Louis, MO) was resolved in 0.5 ml of N,N-dimethylformamide (Wako). Thirty milligrams of fast red violet LB salt (Sigma) and 50 ml of 0.1 
sodium acetate buffer (pH 5.0) containing 50 m
sodium tartrate were added to the mixture (the TRAP-staining solution). Adherent cells on the specimens were fixed with 3.7% (v/v) formaldehyde in Ca²⁺- and Mg²⁺-free phosphate buffered saline (PBS(−)) for 10 min, fixed again with ethanol-acetone (50:50, v:v) for 1 min, and incubated with the TRAP-staining solution for 1 min at room temperature. TRAP-positive osteoclasts appeared as red cells. The TRAP-stained areas on specimens with and without GDP were calculated by a NIH image program (developed at the US National Institutes of Health and available from the Internet by anonymous FTP from zippy. nimh.nih.gov from the National Technical Information Service, Springfield, VA, part no. PB95-500195GEI).

2.9. Osteoclast survival on the specimens

To evaluate survival of the differentiated osteoclasts on the specimens, we prepared functionally differentiated osteoclasts using mouse coculture in collagen gel [16]. Cells were cultured on specimens with and without GDP.

2.10. Osteoclast preparation

To obtain functionally differentiated osteoclasts, a collagen gel culture was developed. Type I collagen solution was obtained from Nitta Gelatin Co., Osaka, Japan. The collagen solution, 5× concentrated
-MEM and 200 m
HEPES buffer (pH 7.4) containing 2.2% NaHCO₃ (7:2:1, v:v:v) were quickly mixed at 4°C. A 10-cm culture dish (Corning) was coated with 4 ml of the collagen mixture at 4°C. The dish was put in a CO₂ incubator for 30 min to make the aqueous type I collagen gelatinous at 37°C. Primary osteoblastic cells (2.0×10⁶ cells/ml) and bone marrow cells (2.0×10⁷ cells/ml) were cocultured on a collagen gel-coated dish in 15 ml of
-MEM containing 10% (v/v) FCS and 10⁻⁸ 
1
,25-dihydroxyvitamin D₃. The medium was changed every 2 days. After culturing for 1 week, the dish was treated with 4 ml of 0.2% bacterial collagenase (Wako) for 20 min at 37°C in a shaking waterbath (60 cycle/min). The cells released from the dish were collected by centrifugation at 250g for 5 min and suspended in 10 ml of
-MEM containing 10% FCS.

2.11. Osteoclast survival on the specimens

The specimens were placed in 24 well culture plates with 500 
l cell suspension and incubated at 5% CO₂ atmosphere 37°C for 1 and 4 h. Adherent cells on the specimens were visualized by staining F-actin with rhodamine-conjugated phalloigin as described above. We also examined RT-PCR to detecting osteoclasts on the specimens after 1 and 4 h cultivation. The details of the RT-PCR method have been described elsewhere in this article.

2.12. Statistics

All data were confirmed at least six times in repeated investigations.

Results of the tests were expressed as mean±SD of six specimens (n=6). TRAP stained areas on the specimens were analyzed statistically by Student's t-test. Significant differences were considered to exist when p<0.01.

5. 3. Results

3.1. Bone marrow culture on the specimens

3.1.1. Actin ring formation

After cultivation, numerous giant cells that had developed actin ring formation were observed on the specimens both with and without GDP (Fig. 1). Since mature osteoclasts demonstrate intracellular polarization and actin ring formation, the adherent giant cells seemed to be osteoclasts. No differences were observed between specimens with or without GDP.

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Fig. 1. Actin ring formation of cells in the bone marrow culture test on the specimens. Arrows indicates osteoclasts formation having actin rings. Bars: 100 
m. Left panel: actin ring formation of cells on the specimen without GDP. Right panel: actin ring formation of cells on the specimen with GDP.

3.1.2. RT-PCR

The expression of a CTR indicates the presence of osteoclasts on the specimens. CTR expression was detected on specimens with or without GDP, thus confirming that osteoclasts were formed on the specimens. However, since the expression levels of CTR were not different between specimens with or without GDP (Fig. 2), indicating that the amounts of osteoclasts on the specimens were equivalent.

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Fig. 2. RT-PCR of the bone marrow culture test for the expression levels of CTR on the specimens.

3.1.3. Trap synthesis on the specimens

Despite the fact that, a difference of CTR expression was not detected on specimens with or without GDP, the analysis of NIH images indicated that the TRAP-stained areas on specimens with GDP were significantly higher (45.5±9.5%) (p<0.01) than those on specimens without GDP (13.9±5.5%) (Fig. 3). Since TRAP activity on the specimens with GDP was higher compared with those without GDP, osteoclast differentiation on specimens with GDP was much greater than those of specimens without GDP.

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Fig. 3. Analysis of TRAP areas on the specimens in the bone marrow culture test. The TRAP stained areas on the specimens were analyzed statistically by Student's t-test. Error bars indicate mean±standard deviation of six specimens (n=6). Significant differences were considered to exist when ^*p<0.01.

3.2. Osteoclast survival on the specimens

After 1 h cultivation, actin ring formations of the cells were observed both on specimens with and those without GDP (Fig. 4A). Additionally, since the expression of CTR was detected on both specimens (Fig. 5), osteoclasts still adhered to both forms of specimen.

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Fig. 4. Actin ring formation of cells in an osteoclast survival test on the specimens. Arrows indicate osteoclast formations having actin rings. Bars: 100 
m. (A) Actin ring formation on the specimens after 1 h incubation. Left panel: actin ring formation of the cells on the specimen without GDP. Right panel: Actin ring formation of the cells on the specimen with GDP. (B) Actin ring formation on the specimens after 4 h incubation. Left panel: actin ring formation of the cells on the specimen without GDP. Right panel: actin ring formation of the cells on the specimen with GDP.

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Fig. 5. RT-PCR of osteoclast survival test for the expression levels of CTR on the specimens.

However, after 4 h cultivation, because actin ring formations of the cells were not observed on specimens without GDP (Fig. 4B), and the expression of CTR was detected only on specimens with GDP ( Fig. 5), osteoclasts remained only on the specimens with GDP, even after 4 h of cultivation.

6. 4. Discussion

The objective of this study was to examine whether the increasing surface energy of GDP, involved in protein adhesion containing the RGD sequence included in the culture medium, affects osteoclast response on titanium plates.

4.1. Osteoclast formation and differentiation on the specimens

We previously demonstrated that GDP pretreatment of titanium plates enhanced adhesion and differentiation of osteoblast-like cells. Since GDP enhanced the surface energy of a titanium surface, increasing adhesion of cell-binding proteins containing the RGD sequence induced subsequent cell adhesion and differentiation [8 and 15].

Zreiqat et al. reported that expression protein levels for osteocalcin, type I collagen, and bone sialoprotein involved in osteoblast differentiation were up-regulated on a RGD-modified titanium surface [4]. The expression of these proteins is tightly linked in osteoblastic differentiation [18, 19, 20 and 21]. In addition, since osteocalcin is essential during bone calcification and resorption [4], it could be assumed that binding the RGD peptides regulated osteoclast differentiation on a titanium surface. The adherent osteoclasts on the specimens were derived from the hematopoietic stem cells contained in mouse bone marrow. Thus, we assumed that the increasing osteoblast differentiation of GDP stimulated osteoclast differentiation on the titanium plate.

4.2. Osteoclast survival

Our latest study demonstrated that large amounts of adsorbed proteins containing the RGD-sequence on titanium plates with GDP mediated strong bonding between substrate and integrin receptors of osteoblastic cells. The strong bonding of the cells activated high stress fiber formation and cytoskeletal changes of osteoblastic cells [8]. Additionally, the initial adhesion of osteoclasts is also mediated by specific membrane receptors, mainly
_v
3-integrin, which is the vitoronectin or fibronectin receptor that recognizes the binding domains of the RGD sequence. Integrin also interacts with cytoskeletal proteins at the site of adhesion. Subsequently, on a primary attachment site, osteoclast cells tightly adhere between integrin and cytoskeletal elements [22].

Since the osteoclast-RGD sequence regulates osteoclast adhesion, we could assume that osteoclasts strongly adhered to the titanium plates with GDP compared to those without GDP, as well as increasing the osteoblast-titanium binding of GDP. Therefore, osteoclasts remained even after 4 h of cultivation on titanium plates because of the increasing cell-titanium binding strength of GDP.

Since osteoclastic differentiation and the survival rate increased on the titanium plates pretreated with GDP, it was obvious that the increasing surface energy of GDP involved in adsroption of proteins containing the RGD sequence affected osteoclastic responses on titanium plates.

7. 5. Conclusion

In conclusion, in addition to chemical properties, crystal structures, and the grain sizes of the bioactive ceramics previously demonstrated, osteoclast responses were also affected, even in the surface conditions of metallic materials. Normal bone remodeling depends on the coordinated activity of osteoblasts and osteoclasts, and on the complex regulatory interactions between these cells and the extra cellular matrix. Therefore, novel approaches are needed not only for osteoclastic resorption on ceramic materials but also for osteoclastic responses on surface-modified metallic materials.

8. Acknowledgements

This study was conducted by the Department of Oral Biomaterials and Technology, School of Dentistry, Showa University. The authors are grateful to Dr. Naoyuki Takahashi and Dr. Kanami Itoh for their technical assistance (Department of Biochemistry, School of Dentistry, Showa University). The authors also gratefully acknowledge the financial support received from a Grant-in-Aid for Scientific Research (B) of The Ministry of Education, Culture, Sports, Science and Technology.

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