Research Article

Evaluation of Cytotoxicity of Polyetheretherketone (PEEK) as a Dental Implant Material

Roseline Meshramkar*, Rhea Mahajan, Lekha KP and Ramesh Nadiger
Department of Prosthodontics, SDM College of Dental Sciences and Hospital, India


*Corresponding author: Roseline Meshramkar, Department of Prosthodontics, SDM College of Dental Sciences and Hospital, Dharwad-580009, Karnataka, India


Published: 29 Mar, 2018
Cite this article as: Meshramkar R, Mahajan R, Lekha KP, Nadiger R. Evaluation of Cytotoxicity of Polyetheretherketone (PEEK) as a Dental Implant Material. J Dent Oral Biol. 2018; 3(2): 1127.

Abstract

Purpose: PEEK (polyetheretherketone) is a synthetic polymer being used increasingly as a dental implant material due to its iso-elastic nature and enhanced mechanical properties. This study analysed the cytotoxicity and biocompatibility of PEEK which helps to improve its bioactivity and ensure widened clinical prospects in future.
Methods: Samples of PEEK dental implant material were added to murine T3T fibroblasts which were cultured in Dulbecco’s modified eagle medium at 37ºc. After 24 hours incubation at 37ºc and 5% carbon dioxide, the medium was replaced with 200 micro litre of medium which contained extracts of PEEK implant material. Cell morphology was analysed using Motic Inverted Microscope.
Results: The result for biocompatibility of PEEK as a dental implant material when evaluated using Colony-forming unit fibroblast assay, was positive showing no signs of cytotoxicity.
Conclusion: With analogous physical and mechanical properties to bone, PEEK has proved to be a potent biocompatible dental implant material.
Keywords: PEEK (Polyetheretherketone); Dental implants; Cytotoxicity; Fibroblasts; Colony formation assa1


Introduction

PEEK (Polyetheretherketone) is a synthetic thermoplastic, tooth colored polymer. Due to its prime property of iso-elasticity with bone and its radiolucent nature, PEEK has benefited the field of orthopaedics in the form of spine and hip implants and it shows comparable promise as a dental implant material mainly because of its superior physical properties such as stress shielding.
In dentistry, PEEK is presently being used in a variety of applications ranging from fabrication of fixed crowns and bridges, components of removable partial dentures, implant abutments and dental implants. Among these, there is maximal degree of interplay with bio-mechanical requirements in case of replacing a tooth in to or multiple teeth using dental implants [1].
Some case reports have suggested prevalence of allergy positive reactions against titanium reagents along with similar reports on materials such as chromium, mercury, palladium and nickel [2]. Ceramic based materials are seen to be also seen to exhibit inconsistent biologic behavior as dental restorations [3]. Although such reports are scanty, they give us all the more reason to evaluate the cytotoxicity of PEEK as an exceptional, bio inert substitute to these conventional implant materials such as titanium and zirconia. Recognition of an implant material as biocompatible nowadays depends on a large number of factors, such as: Absence of cytotoxicity, mutagenicity and carcinogenicity. The exclusion of its allergenic properties, physical and chemical stability and biological ‘inertia’ in a biological environment is essential [4].
Although the mechanical performance of such materials can be assessed readily, in vivo performance and biocompatibility must be scrutinized before prosthesis can be safely implanted [5]. Therefore, it becomes essential for us to assess the cytotoxic and biocompatible properties of a relatively new biomaterial such as PEEK to ensure its long term safety as a dental implant material.


Materials and Methods

Test sample preparation
T3T mouse connective tissue fibroblastic cell line was used to study the cytotoxicity of PEEK dental implant material in vitro. PEEK was acquired in the form of granules (2 press Bio HPP clear-granules SP Dental, Pune, India). We took three test samples each containing four sterilized (autoclaved) PEEK granules with equal mass by volume ratio. The comparison regarding fibroblastic cell viability was done by analysing test samples against untreated murine (T3T) fibroblasts which were used as a control group (Figure 1).
Cell culture
The PEEK granules were immersed in 7 ml of culture medium for 24 hours at 37ºC to extract any cytotoxic substances. Murine normal fibroblast cells (NCCS, Pune) were cultured at 37ºC under a humidified atmosphere of 5% CO2 and 95% air and were grown in DMEM (Dulbecco’s modified eagle medium) High Glucose medium (HIMEDIA Laboratories, Mumbai) supplemented with 10% fetal bovine serum (HIMEDIA Laboratories, Mumbai), 1% Antibiotic Antimycotic solution (HIMEDIA Laboratories, Mumbai) (Figure 2).
Colony formation assay
A qualitative assessment of the cell cultures was performed using the Colony Formation Assay. The fibroblastic cells were grown up to 80% confluence and were trypsinized and seeded in 6-well plates in triplicate at a density of 500 cells/well for 2 days at 37°C (Figure 3).
Analysis of cell morphology
After 48 hours of incubation, the changes in the cell morphology were captured under objective of Motic Inverted Microscope with 10MP resolution camera with the help of Motic Image PLUS 2.0 (Figure 4 and 5).


Figure 1

Another alt text

Figure 1
Autoclaved peek granules.

Figure 2

Another alt text

Figure 2
Murine t3t fibroblasts cultured with peek extract.

Figure 3

Another alt text

Figure 3
Colony formation assay carried out in 6 -well plates.

Figure 4

Another alt text

Figure 4
Viable t3t fibroblasts with control group.

Figure 5

Another alt text

Figure 5
Viable t3t fibroblasts with peek test sample.

Results

Table 1
Comparison between optical density of fibroblastic cells seen in control group and test samples (PEEK group). The murine cells exposed to the test sample exhibited no morphological alterations or a significant reduction in cell number and cell death. The qualitative evaluation showed no statistical difference in the cell number between the control and the test specimen.


Discussion

The physical and biologic properties of the implant materials adjudge the long term success of implants. Considering that oral implants thrive on close contact between implant surface and oral epithelium, it is crucial for us to evaluate the cytotoxic effect of any novel bio material [6]. The conventionally used implant materials such as Titania (TiO2) and Zirconia (ZrO2) are still not up to scratch in terms of an ideal material which replaces hard biological tissue [7], whereas PEEK is emerging as a feasible candidate for the same.
Usually, for in vitro toxicity tests, some cells are plated in a well of a cell-culture dish where they attach, forming the so-called test system. The material to be tested is then placed in this test system. If the material is not cytotoxic, the viable cells will remain attached to the well with time [8].
Colony Formation Assay (CFA) is one of the most popular and standard recognized qualitative test for determination of cytotoxic effects of a given material, among many other tests such as MTT assay, cell proliferation assay, cell transformation assay.
Even though CFA poses a tiring and time consuming attempt at counting the number of colonies manually or evaluating them under the microscope, it is still regarded as a gold standard test [9]. To exclude the possibility of any inconsistency with the results, we made sure that the granules used for the samples were of similar size and comparable mass by volume ratio.
Using the colony formation assay, also known as Colony Formation Unit-Fibroblast (CFU-F), we analysed the results for cytotoxicity of PEEK. It was observed that the fibroblasts showed no remarkable morphologic alterations. The cell viability observed in the test sample was neither increased because of the presence of PEEK granules nor decreased as in comparison to the control group. The in vitro interaction of mice fibroblasts with untreated PEEK showed no overt cytotoxic or mutagenic effects. Various studies affirm the biocompatibility of PEEK using tests for mutagenesis like Ames test [4]. This makes untreated PEEK not only a biocompatible, but also a bio-inert material. Morrison et al conducted a similar study comparing biocompatibility of PEEK and epoxy resin, which confirmed that it showed no significant cytotoxicity when it was assessed quantitatively in terms of cell protein content, leakage of cytosolic lactate dehydrogenase (LDH) activity through damaged cell membranes, intracellular reduced glutathione (GSH) content and MTT assay [5].
In 2002, another study used SV40 rat osteoblasts and 3T3 mouse fibroblasts in direct contact with PEEK material and revealed that there were no effects on the morphology of the osteoblasts nor was there any evidence of a negative influence on the 3T3 proliferation rate or cytotoxic effects on the osteoblasts in the MTT assay. On the contrary, there was even evidence of stimulation of the osteoblast protein content which has resulted in discussion that PEEK might have a favorable effect on bone growth (osteointegration) [4].
Despite the stable chemical nature of PEEK which makes it an attractive endo-prosthetic material, chemical surface inertness does not account for a sound interfacial biocompatibility and PEEK requires a surface modification prior to its application in vivo [10]. Studies have proved that silane-coupled PEEK-HA had in general improved biomechanical properties than untreated PEEK and did not show cytotoxicity in vitro [11].
Another study showed improved biocompatibility of PEEK modified specifically through the methods of plasma technology [10]. Therefore, to evaluate future scope of PEEK as a dental implant material, further research is required to analyse satisfactory results for more desirable physical properties along with minimal cytotoxic effects.


Table 1

Another alt text

Table 1
Comparison between optical density of fibroblastic cells seen in control group and test samples (PEEK group).

Conclusion

By the course of this experimental study we concluded that PEEK can be utilized as a suitable biomechanical and chemically stable dental implant material. When studied under colony formation assay with living fibroblasts, it showed negligible alterations to the cell morphology or number clearly indicating that it is not cytotoxic in nature.


References

  1. Ramamoorthi M, Verma V, Sheikh Z. Dental biomaterials and a novel composite of Zirconia and Poly Ether Ether Ketone [PEEK] for dental implants. IDJSR. 2015;2(4):16-22.
  2. Hosoki M, Nishigawa K, Miyamoto Y, Ohe G, Matsuka Y. Allergic contact dermatitis caused by titanium screws and dental implants. J Prosthodont Res. 2016;60(3):213-9.
  3. Kerem Kilic, Bulent Kesim, Zeynep Sumer, ZubeydePolat, ServetKesim. In vitro cytotoxicity of all-ceramic substructural materials after aging. J Dent Sci. 2013;8(3):231-8.
  4. Katzer A, Marquardt H, Westendorf J, Wening JV, von Foerster G. Polyetheretherketone cytotoxicity and mutagenicity in vitro. Biomaterials. 2002;23(8):1749-59.
  5. Morrison C, Macnair R, MacDonald C, Wykman A, Goldie I, Grant MH. In vitro biocompatibility testing of polymers for orthopaedic implants using cultured fibroblasts and osteoblasts. Biomaterials. 1995;16(13):987-92.
  6. Miura S, Takebe J. Biological behavior of fibroblast-like cells cultured on anodized-hydrothermally treated titanium with a nanotopographic surface structure. J Prosthodont Res. 2012;56(3):178-86.
  7. Marchi J, Ussui V, Delfino CS, Bressiani AH, Marques MM. Analysis in vitro of the cytotoxicity of potential implant materials. I: Zirconia-titania sintered ceramics. J Biomed Mater Res B Appl Biomater. 2010;94(2):305-11.
  8. Gociu M, Pătroi D, Prejmerean C, Păstrăv O, Boboia S, Prodan D, Moldovan M. Biology and cytotoxicity of dental materials: an in vitro study. Rom J Morphol Embryol. 2013;54(2):261-5.
  9. Katz D, Ito E, Lau KS, Mocanu JD, Bastianutto C, Schimmer AD, et al. Increased efficiency for performing colony formation assays in 96-well plates: novel applications to combination therapies and high-throughput screening. Biotechniques. 2008;44(2):ix-xiv.
  10. 10. Briem D, Strametz S, Schroder K, Meenen NM, Lehmann W, Linhart W, et al. Response of primary fibroblasts and osteoblasts to plasma treated polyetheretherketone (PEEK) surfaces. J Mater Sci: Mater Med. 2005;16(7):671-7.
  11. Rashidi AR, Wahit MU, Abdullah MR, Abdul Kadir MR. The Effect of Silane on the Biomechanical Properties of PEEK/HA Composite. Adv Mat Res. 2015;1125:426-31.