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Lasers in Dental Science

, Volume 2, Issue 2, pp 119–124 | Cite as

Biocompatibility of erbium chromium-doped yattrium-scandium-gallium-garnet (Er,Cr:YSGG 2780 nm) laser-treated titanium alloy used for dental applications (in vitro study)

  • Dalia A. Abd El daym
  • Mostafa E. Gheith
  • Nadia A. Abbas
  • Laila A. Rashed
  • Zeinab A. Abd El Aziz
Original Article
  • 232 Downloads

Abstract

The use of dental implants in the partial and complete edentulism has become the primary treatment regimen in the modern dentistry. Erbium chromium-doped yattrium-scandium-gallium-garnet (Er,Cr:YSGG) laser is most often used in dentistry in implant surgery and management of peri-implantitis. The aim of the present study was to assess the biocompatibility of Er,Cr:YSGG laser-treated titanium alloy (Ti-6Al-4V) and its surface characteristics to understand the impact of the laser on the titanium alloy surfaces.

Materials and methods

A total of 20 discs of titanium alloy (Ti-6Al-4V) were used. Ten discs were irradiated with Er,Cr:YSGG laser which was operating in a normal room atmosphere and temperature at power 2 W. Biocompatibility was investigated in vitro via MTT assay. Surface analysis of laser-treated and laser-untreated discs was examined with a scanning electron microscope.

Result

Laser-treated group showed superior cell viability compared to untreated group. No undesirable changes were observed by SEM.

Conclusion

We can conclude that Er,Cr:YSGG laser safely could improve the biocompatibility of dental implant.

Keywords

Biocompatibility Laser treatment Surface analysis Ti-6Al-4V 

Introduction

Researches on dental implants have been increased in the past few years and are expected to expand in the future due to the rising in the demand for cosmetic dentistry [1]. Pure titanium and titanium alloys are the metals that are most often used in the design of dental implants due to their high corrosion resistance [2], biocompatibility [3], and adequate mechanical properties [4].

Even if the grade four pure titanium possesses highest strength and hardness, its strength and hardness are lower than those of titanium alloys, in which alloying elements modify microstructures resulting in improvement of their mechanical and physical properties [5].

The success of dental implants depends on maintaining a stable attachment and efficient seal between the implant surface and the surrounding soft tissue and crestal bone [6]. The interaction of living cells with foreign material is a complicated matter but is a key for understanding the biocompatibility [7]. A material is considered biocompatible if it does not produce harmful or toxic reactions in the tissues that is in contact or adverse systemic reactions as a result of elements, ions, and/or compounds it release [8]. In vitro biocompatibility tests are performed outside a living organism to simulate biological reactions to materials when they are placed into the tissue of the body. They are repeatable, controllable, fast, and relatively simple, and there is no ethical problem [9].

Lasers have been expected to serve as an alternative or adjunctive treatment to conventional dental therapy [10, 11, 12, 13]. In oral implantology, lasers are useful in uncovering the implant, excision of mucosal hyperplasia, treatment of failed implant, and decontamination of implant surface [14].

Lasers are usually named for the “active medium” that is charged with energy inside the laser unit to create laser light. The YSGG laser receives its name from the elements that compose the crystal medium inside the laser system—yattrium, scandium, gallium, and garnet, doped with erbium and chromium (Er,Cr:YSGG). When the crystal is pumped with energy, a specific, monochromatic wavelength of light is emitted from the crystal and transferred to the target through a delivery system. In the case of the YSGG laser, the wavelength delivered from the laser through a fiber optic cable is 2780 nm [15, 16].

Erbium-based lasers are considered as cool lasers; therefore, they prevent burning, charring, and coagulation at the site of interaction and are safe to use directly on titanium surfaces. It is for this reason they are preferred for oral implantology procedures [17]. The aim of the present study was to assess the biocompatibility of Er,Cr:YSGG (2780 nm) laser surface-treated titanium alloy and its surface characteristics.

Materials and methods

Cylindrical rod of conventional biomedical titanium alloy Ti-6Al-4V of 6-mm diameter was used. The composition of Ti alloy used in the study is given in Table 1.
Table 1

Elemental chemical composition of Ti-6Al-4V (weight %)

Element

C

Si

Fe

O

V

Al

Ti

Wt%

0.14

0.01

0.16

0.17

3.97

6.36

Bal

The rod was cut into 20 discs with a thickness of 2 mm using a silicon carbide cutoff wheel at 3800 rpm under continuous flowing coolant. Discs were mounted on Teflon molds to facilitate finishing, polishing, and laser treatment. The exposed surface of the samples was finished with different grades of silicon carbide grit papers up to 2400 grit in a single direction to obtain similar morphology for all samples. Final polishing was carried out with alumina paste. All discs were washed with distilled water, dried, and then sterilized in alcohol before the experiment.

Laser treatment

Laser surface treatment of ten discs was carried out using Er,Cr:YSGG laser (Waterlase MD, Biolase Technology, USA) that was operated in a normal room atmosphere and temperature. Table 2 summarizes the laser parameters used in the study. A pulse frequency was fixed at 20 pulses per second. The delivery system consists of a fiber optic tube terminating in a zirconia tip (600 μm in diameter). Water cooling system 40% water and 60% air was used. Titanium discs were irradiated and hand guided at constant distance of 0.5 to 1 mm with the laser system (Fig. 1). Each disc was irradiated in parallel movements moving the laser beam continuously and not staying too long in one spot. The disc was scanned once to standardize the treatment time. The angle created by the laser beam and the disc surface was approximately 90°. All treatments were performed by the same experienced operator.
Table 2

Laser parameters used in the study

Laser type

Wave length (nm)

Power (W)

Energy (mJ)

Spot size (mm)

Fluence (J/cm2)

Frequency (Hz)

Power density (W/cm2)

Total time (s)

Pulse duration (μs)

Dose (J/cm2)

Er,Cr:YSGG

2780

2

100

0.6

35.4

20

707

20

50

14,140

Fig. 1

Titanium disc irradiated and hand guided at constant distance of 0.5 to 1 mm with the laser system

Surface analysis

The discs were examined under a scanning electron microscope to assess the surface characteristics of the machined and laser-treated titanium discs before cytotoxicity test. All samples were introduced into the vacuum chamber SEM (Model Quanta 250 FEG) and observed at ×2400 magnification.

Cytotoxicity

Direct cell proliferation assay was performed with human fibroblast (HFB4) cells that were obtained from the Holding company for Production of Vaccines, Sera and Drugs (VACSERA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. MTT stock solution (5 mg mL−1) was made by dissolving 50 mg of MTT salt in 10 mL of sterile phosphate-buffered saline. The solution was filtered through a 0.22-μm polyethersulfone (PES) membrane filter, kept in the dark, and refrigerated at < 4 °C. HFB4 cells were harvested using 0.25% trypsin-EDTA solution in phosphate-buffered saline. The cells were incubated at 37 °C with 5% CO2 in a humidified atmosphere. Ti-6Al-4V discs were removed from the Teflon mold and autoclaved. Twenty sterile laser-treated and untreated discs were placed in a 24-well plate, ten discs for each group. HFB4 cells were seeded on each disc and incubated at 37 °C for 3 h considering cells exposed to untreated samples (+ve control). Afterwards, 1 mL of culture medium was added to each well to cover the discs’ surfaces. Tissue culture was placed in a 12-well plate which was considered as cells not exposed to any material (−ve control). Two days after incubation, the discs were removed with sterile tweezers and 500 μL of the MTT solution was added to each well. Incubation was performed at 37 °C for 4 h in an incubator containing CO2. During incubation, MTT is reduced by the action of succinate dehydrogenase present in cell mitochondria that results in the production of insoluble purple formazan crystals in the cytoplasm of the cells. Then, 1 mL of isopropyl alcohol was added to each well. It dissolves the purple crystals and gives a purple color to the medium. The absorbance (optical density) of the solution was read at 570-nm wavelength using an ELISA microplate reader (Statfax 2100, USA) and calculated using the equation below (1):
$$ \mathrm{Cell}\ \mathrm{viability}\ \left(\%\right)=\frac{\mathrm{Absorbance}\ 570\ \mathrm{nm}\ \mathrm{of}\ \mathrm{treated}\ \mathrm{cells}\ }{\mathrm{Absorbance}\ 570\ \mathrm{nm}\ \mathrm{of}\ \mathrm{control}\ \mathrm{untreated}\ \mathrm{cells}}\times 100 $$
(1)

Results

The descriptive statistics of the cell viability assessment for the control cells not exposed to any material (−ve control), the cells exposed to untreated samples (+ve control), and the cells exposed to laser-treated samples are summarized in Table 3 and shown in Fig. 2.
Table 3

Descriptive statistics of the cell viability assessment (%) for the different test groups

Column

Mean

SD

SE

CI

Range

Min

Max

Control

98.767

0.306

0.176

0.759

0.6

98.5

99.1

Untreated sample

71.9

8.296

3.71

10.301

22.5

58.9

81.4

Laser treated

84.843

7.77

2.937

7.186

23.4

69.2

92.6

Fig. 2

A bar chart showing mean cell viability values for the different test groups

Results of one way ANOVA revealed that there were very high significant differences among the three groups (F value = 13.022, P value ≤ 0.001). The results of Tukey’s pairwise multiple comparison procedure between the different groups are summarized in Table 4.
Table 4

Tukey’s pairwise multiple comparison procedure for significant ANOVA between the different groups

Comparison

Diff of means

q

P

Control vs. untreated sample

26.867

7.137

< 0.001*

Control vs. laser treated

13.924

3.914

0.042*

Laser treated vs. untreated sample

12.943

4.288

0.026*

*Significant difference at P ≤ 0.05

Surface morphology

Figures 3 and 4 are showing two morphologically different SEM images for laser-treated and untreated discs. The laser-treated image showed sealing of the scratches due to machining and polishing. No voids, inclusions, pits, or microfractures were observed in laser-treated discs. The SEM image of untreated discs showed scratches caused by machining and polishing.
Fig. 3

Electron microscopic image of untreated disc

Fig. 4

Electron microscopic image of laser-treated disc

Discussion

Er,Cr:YSGG (2780 nm) is a hard tissue laser used currently in dentistry and it is a free-running pulsed laser that allows precise bone sectioning and ablation with minimal thermal effects upon the adjacent tissues [18]. Er,Cr:YSGG laser exhibits almost no absorption of laser irradiation in titanium and thus prevent excessive energy transformation in the form of heat development. It can be used safely on implant surfaces with adequate water spray without increase in temperature [19]. During irradiation, parameters such as output power, energy, dose, and duration should be considered. The power setting chosen was 2 W, 20 Hz over a period of 20 s, and the energy dose 100 mJ/pulse to allow adequate dose to be delivered to the titanium alloy surface without undesirable results [20]. The total energy represents the total energy utilized during the procedure. The focal distance of 0.5–1 mm was maintained to avoid bringing the laser tip too close to titanium surface as the closest space between the fiber tip and the spot represents a higher power density (www.lasertrainingcenter.net). The laser irradiation was in parallel movements moving the laser continuously not staying too long in one spot to ensure positive laser effect [21].

In clinical situation, the damaged part of the oral mucosa has a lower metabolic rate and proliferation status than healthy oral mucosa due to low number of viable cells present in damaged tissue. There are a number of different assays that can be used to measure the viability and proliferation status of cells exposed to test materials in vitro to assess the material’s relative toxicity [22]. The MTT assay is the most common test to evaluate the cytotoxicity of dental materials because it is a rapid and inexpensive method [23]. It determines the ability of viable cells to convert the soluble tetrazolium salt (MTT) into purple formazan precipitate which can be quantified spectrophotometrically and is directly proportional to the number of viable cells [24]. Adequate contact between cells and test material is very important in biological evaluation of materials. The contact between cells and material can be achieved in three ways: direct contact, indirect contact, and contact through extract. This study is based on the direct contact of the material with the culture medium by growing the cells directly on the titanium alloy samples. Surface characteristics of the material are important because if the cells adhere to the surface of the material, consequently, they will grow well [9]. The surface quality of the implant affects the response of osteoblasts to the titanium surface which plays an important role in the initial biocompatibility of the implant [25].

In the current study, both laser-treated and untreated samples revealed a significant decrease in cell viability compared to control cells; however, the cytotoxicity behavior in laser-treated samples was superior to that in untreated samples. This could be attributed to the change in the surface topography of the laser-treated samples proved by SEM. This change could increase cell spreading and migration and decrease focal contacts among cells which promoted the rate of mitosis and cell proliferation and enhanced cell adhesion to the samples [26]. Several studies showed that laser-treated surfaces reduce β phase formation in titanium alloy which increase corrosion resistance and enhance the chemical inertness of the laser-treated surface compared to the untreated surface [27]. It could be an influential factor in cell response to laser-treated surface that improve its biocompatibility.

Applications of non-contact laser systems have gained popularity in implant dentistry providing less postoperative pain, less bleeding, and faster healing [28, 29]. They have also proven a worthwhile method for the treatment of peri-implantitis [30, 31]. In peri-implantitis treatment, the implant surface is directly exposed to the laser. However, depending on laser parameters and surface properties of dental implants, monochromatic laser light can cause profound alterations in the morphology of an implant surface. A study on the effects of laser irradiation on machined and anodized titanium discs reported that machined and anodized Ti disc surfaces were affected by irradiation with Er,Cr:YSGG laser at powers over 3 W [21]. A previous study showed that Er,Cr:YSGG did not cause significant changes on a Ti plasma spray-coated implants even at 6 W [31].

In the present study, SEM revealed no undesirable changes on the surface of machined titanium alloy after irradiation with an Er,Cr:YSGG laser at 2 W. No voids, inclusions, pits, or microfractures were observed in laser-treated discs. It showed sealing of scratches due to machining and polishing.

Conclusion

Er,Cr:YSGG laser could safely improve biocompatibility of dental implant. The result of the present study shows that laser treatment had significant effect on biocompatibility of titanium alloy. However, more testing researches that mimic the clinical situations are necessary to describe suitable laser settings for different surface modifications of dental implants to give clinicians safer guidelines for laser-assisted therapy in implant dentistry.

Notes

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Seth S, Kalra P (2013) Effect of dental implant parameters on stress distribution at bone-implant interfaces. Inter J Sci Res 2:121–124Google Scholar
  2. 2.
    Abdel-Hady Gepreel M, Niinomi M (2013) Biocompatibility of Ti-alloys for long-term implantation. J Mech Behav Biomed Mate 20:407–415CrossRefGoogle Scholar
  3. 3.
    Yamazoe J, Nakagawa M, Matono Y, Takeuchi A, Ishikawa K (2007) The development of Ti alloys for dental implant with high corrosion resistance and mechanical strength. Dent Mater J 26:260–267CrossRefPubMedGoogle Scholar
  4. 4.
    Piotrowski B, Baptista AA, Patoor E, Bravetti P, Eberhardt A, Laheurte P (2014) Interaction of bone-dental implant with new ultra low modulus alloy using a numerical approach. Mater Sci Engin C 38:151–160CrossRefGoogle Scholar
  5. 5.
    Koike M, Hummel SK, Ball JD, Okabe T (2012) Fabrication of titanium removable dental prosthesis frameworks with a 2-step investment coating method. J Prosthet Dent 107:393–399CrossRefPubMedGoogle Scholar
  6. 6.
    Baltriukiene D, Sabaliuskas V, Balciusas E, Melninkaitis A, Liufkelskene E, Bukelskiene V (2014) The effect of laser-treated titanium surface on human gingival fibroblast behavior. J Biomed Mater Res A 102(3):713–720CrossRefPubMedGoogle Scholar
  7. 7.
    Kearns VR, Williams RL, Mirvakily F, Doberty PJ, Martin N (2013) Guided gingival fibroblast attachment to titanium surface: an in vitro study. J Clin Periodontol 40(1):99–108CrossRefPubMedGoogle Scholar
  8. 8.
    O’Brien WJ (2009) Dental materials and their selection, 4th edn. Quintessence Publishing, Chicago, p 23Google Scholar
  9. 9.
    Frankova J, Pivodova V, Ruzicka F, Tomankova K, Vrlkova J (2013) Comparing biocompatibility of gingival fibroblast and bacterial strains on a different modified titanium discs. J Biomed Mater Res A 101(10):2915–2924CrossRefPubMedGoogle Scholar
  10. 10.
    Arisan V, Karabuda CZ, Ozdemir T (2010) Implant surgery using bone- and mucosa-supported stereolithographic guides in totally edentulous jaws: surgical and post-operative outcomes of computer-aided vs.standard techniques. Clin Oral Implants Res 21(9):980–988PubMedGoogle Scholar
  11. 11.
    Nickenig H-J, Wichmann M, Schlegel KA, Nkenke E, Eitner S (2010) Radiographic evaluation of marginal bone levels during healing period, adjacent to parallel-screw cylinder implants inserted in the posterior zone of the jaws, placed with flapless surgery. Clin Oral Implants Res 21(12):1386–1393CrossRefPubMedGoogle Scholar
  12. 12.
    Pourzarandian A, Watanabe H, Aoki A, Ichinose S, Sasaki KM, Nitta H, Ishikawa I (2004) Histological and TEM examination of early stages of bone healing after Er:YAG laser irradiation. Photomed Laser Surg 22(4):342–350CrossRefPubMedGoogle Scholar
  13. 13.
    Yoshino T, Aoki A, Oda S, Takasaki AA, Mizutani K, Sasaki KM, Kinoshita A, Watanabe H, Ishikawa I, Izumi Y (2009) Long-term histologic analysis of bone tissue alteration and healing following Er:YAG laser irradiation compared to electrosurgery. J Periodontol 80(1):82–92CrossRefPubMedGoogle Scholar
  14. 14.
    Garg AK (2007) Lasers in dental implantology: innovation improves patient care. Dent Implantol 18:57–61Google Scholar
  15. 15.
    Featherstone JD (2000) Caries detection and prevention with laser energy. In: Convissar FA (ed) Dent Clin North Am, vol 44. Saunders, Philadelphia, pp 955–966Google Scholar
  16. 16.
    Gimbel CB (2000) Hard tissue procedures. In: Convissar FA (ed) Dent Clin North Am, vol 44. Saunders, Philadelphia, pp 931–948Google Scholar
  17. 17.
    Kim S-W, Kwon Y-H, Chung J-H, Shin S-I, Herr Y (2010) The effect of Er:YAG laser irradiation on the surface microstructure and roughness of hydroxyapatite-coated implant. J Periodontal Implant Sci 40:276–282CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Matsumoto K, Hossain MM, Kawano H, Kimura Y (2002) Clinical assessment of Er,Cr: YSGG laser application for cavity preparation. J Clin Laser Med Surg 20:17–21CrossRefPubMedGoogle Scholar
  19. 19.
    Gomez-Santos L, Arnabat-Dominguez J, Sierra-Rebolledo A, Gay-Escoda C (2010) Thermal increment due to Er,Cr:YSGG and CO2 laser irradiation of different implant surfaces. A pilot study. Med Oral Patol Oral Cir Bucal 15:782–787CrossRefGoogle Scholar
  20. 20.
    Lee JH, Heo SJ, Koak JY, Kim SK, Lee SJ, Lee SH (2008) Cellular responses on anodized titanium discs after laser irradiation. Lasers Surg Med 40:738–742CrossRefPubMedGoogle Scholar
  21. 21.
    Park JH, Heo SJ, Koak JY, Kim SK, Han CH, Lee JH (2012) Effects of laser irradiation on machined and anodized titanium disks. Int J Oral Maxillofac Implants 27:265–272PubMedGoogle Scholar
  22. 22.
    Wataha JC, Lockwood PE, Bouillaguet S, Noda M (2003) In vitro biological response to core and flowable dental restorative materials. Dent Mater 19:25–31CrossRefPubMedGoogle Scholar
  23. 23.
    Issa Y, Watts DC, Brunton PA, Waters CM, Duxbury AJ (2004) Resin composite monomers alter MTT and LDH activity of human gingival fibroblasts in vitro. Dent Mater 20:12–20CrossRefPubMedGoogle Scholar
  24. 24.
    Bonakdar S et al (2010) Preparation and characterization of polyvinyl alcohol hydrogels crosslinked by biodegradable polyurethane for tissue engineering of cartilage. Mater Sci Eng C 30(4):636–643CrossRefGoogle Scholar
  25. 25.
    Kim HJ, Kim SH, Kim MS, Lee EJ, Oh HG, Oh WM, Park SW, Kim WJ, Lee GJ, Choi NG, Koh JT, Dinh DB, Hardin RR, Johnson K, Sylvia VL, Schmitz JP, Dean DD (2005) Varying Ti-6Al-4V surface roughness induces different early morphologic and molecular responses in MG63osteoblast-like cells. J Biomed Mater Res A 74(3):366–373CrossRefPubMedGoogle Scholar
  26. 26.
    Nothdurft FP, Fontana D, Ruppenthal S, May A, Aktas C, Mehraein Y (2014) Differential behavior of fibroblasts and epithelial cells on structured implant abutment materials: a comparison of materials and surface topographies. Clin Implant Dent Relat Res 16:640–648Google Scholar
  27. 27.
    Atapour M, Pilchak A, Frankel GS, Williams JC, Fathi MH, Shamanian M (2010) Corrosion behavior of Ti-6Al-4V with different thermomechanical treatments and microstructures. Corrosion 66(6):1–9CrossRefGoogle Scholar
  28. 28.
    Martin E (2004) Lasers in dental implantology. Dent Clin North Am 48:999–1015CrossRefPubMedGoogle Scholar
  29. 29.
    Kreisler M, Gotz H, Duschner H (2002) Effect of Nd:YAG, Ho: YAG, Er:YAG, CO2, and GaAIAs laser irradiation on surface properties of endosseous dental implants. Int J Oral Maxillofac Implants 17:202–211PubMedGoogle Scholar
  30. 30.
    Kotsovilis S, Karoussis IK, Trianti M, Fourmousis I (2008) Therapy of peri-implantitis: a systematic review. J Clin Periodontol 35:621–629CrossRefPubMedGoogle Scholar
  31. 31.
    Miller RJ (2004) Treatment of the contaminated implant surface using Er,Cr:YSGG laser. Implant Dent 13:165–170CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Dalia A. Abd El daym
    • 1
  • Mostafa E. Gheith
    • 2
  • Nadia A. Abbas
    • 3
  • Laila A. Rashed
    • 4
  • Zeinab A. Abd El Aziz
    • 5
  1. 1.Dental Research Center, Ministry of Health and PopulationCairoEgypt
  2. 2.Department of Laser Applications in Dental Surgeries, National Institute of Laser Enhanced ScienceCairo UniversityCairoEgypt
  3. 3.Department of Prosthodontic, Faculty of Oral and Dental MedicineCairo UniversityCairoEgypt
  4. 4.Department of Medical Biochemistry and Molecular Biology Faculty of MedicineCairo UniversityCairoEgypt
  5. 5.Corrosion Control and Surface Protection DepartmentCentral Metallurgical Research and Development Institute (CMRDI)CairoEgypt

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