On the sulphonated PEEK for implant dentistry: Biological and physicochemical assessment

Materials Chemistry and Physics 223 (2019) 542–547

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Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys

On the sulphonated PEEK for implant dentistry: Biological and physicochemical assessment

T

Renata S. Bruma, Patricia R. Monichb, Fernanda Bertic, Márcio C. Fredelb, Luismar M. Portoc, Cesar A.M. Benfattia, Júlio C.M. Souzad,e,∗ a

Center for Research on Dental Implants (CEPID), School of Dentistry (ODT), Federal University of Santa Catarina (UFSC), Florianopolis, SC, 88040-900, Brazil Department of Mechanical Engineering (EMC), Federal University of Santa Catarina (UFSC), Florianopolis, SC, 88040-900, Brazil c Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900, Florianópolis, SC, Brazil d Center for MicroElectromechanical Systems (CMEMS-UMINHO), University of Minho, Guimarães, 4700-058, Portugal e Dept. of Dental Sciences, University Institute of Health Sciences (IUCS-CESPU), Gandra, 4585-116, Portugal b

H I GH L IG H T S

and physicochemical behavior of sPEEK was assessed in this study. • Biological modification of PEEK structure by sulphonation method decreased its wettability and cytocompatibility. • The • The release of sulfonic products from sPEEK can be toxic for human tissues. However, diferent sulphonation process should be studied to acess sPEEK toxicity.

A R T I C LE I N FO

A B S T R A C T

Keywords: PEEK sPEEK Cytotoxicity Biomaterials

Sulfonated PEEK (sPEEK) has been developed for drug delivery although there is still scarce evidence on its physicochemical and biological properties. The main aim of this study was to assess the cytotoxicity behavior of sPEEK processed for two different sulphonation times. PEEK was functionalized by sulphuric acid treatment for 1 or 1.5 h and then dissolved in dimethylsulfoxide (DMSO). The specimens were characterized by chemical, microstructural, thermophysical, hydrophobic, and roughness analysis. Cytotoxicity assays with fibroblasts followed ISO 10993-5. Thermophysical analyses by TGA and NMR revealed no statistical difference (p > 0,05) in the degree of sulphonation for both processing time points although chemical modifications were detected in the main polymer chain. PEEK surfaces were more hydrophilic than sPEEK surfaces and therefore biological assays revealed a higher cell proliferation on PEEK surfaces when compared to that recorded on sPEEK surfaces. Fibroblasts revealed a higher metabolic activity on PEEK than that on sPEEK specimens for 1 and 3 days of incubation. However, there was a significant increase in cell metabolic activity on sPEEK/DMSO of about 20% compared to that recorded on PEEK for 7 days of incubation. SPEEK specimens showed a limited cytocompatibility probably due to the release of sulfonic compounds to the surrounding medium. The sulphonation method should be adjusted considering different processing time and surface treatment to achieve a high biocompatibility performance of sPEEK for biomedical applications.

1. Introduction Polyetheretherketone (PEEK) has been extensively used as biomaterial since biological assays performed in the early 1980s [1]. Such polymeric group reveals attractive properties in the biomedical field such as high mechanical strength, resistance to chemical and radiation damage, thermal stability at high temperatures around 300 °C, radiolucency, and biocompatibility into bone [2–4]. In dentistry, PEEK has

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been used as healing and provisional abutment attached to titanium implants [5–7]. Some advantages of this material have been reported, such as reduced clinical work time, possibility of immediate esthetic and mechanical performance, adequate marginal healing, and low biofilm formation [8–11]. Peri-implant inflammatory reactions leading to bone loss is an issue in implant dentistry. The biofilm accumulation involving pathogenic bacterial species is a major etiologic factor in peri-implant

Corresponding author. Center for MicroElectromechanical Systems (CMEMS-UMINHO), University of Minho, Guimarães, 4700-058, Portugal. E-mail addresses: [email protected], [email protected] (J.C.M. Souza).

https://doi.org/10.1016/j.matchemphys.2018.11.027 Received 29 May 2018; Received in revised form 3 November 2018; Accepted 11 November 2018 Available online 12 November 2018 0254-0584/ © 2018 Elsevier B.V. All rights reserved.

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for each group. SPEEK-1 and sPEEK-2 were dissolved into dimetilsulphoxide (DMSO®, Synth, Brasil) at 10% wt/vol (sPEEK/solvent) by using sonication (Misonix - Sonicator 4000, USA) to obtain a homogeneous sPEEK solution. SPEEK/DMSO-1 and sPEEK/DMSO-2 solutions were poured separated in a silicon mold (Rhodorsil, RTV 4407 A/B, País), with wells of 6 mm in diameter and 10 mm in depth. Those solutions were dried at 70 °C for 72h. After curing for 72h, sPEEK/DMSO specimens were sterilized with ethylene oxide (Sterilab Company, Curitiba, Brazil). Each specimen was immersed in 30 μL phosphate buffered saline solution (PBS) into commercial tissue culture polystyrene (TCPS) wellplates for 168 h. PBS solution was changed after 24 h to avoid excessive decrease in pH. PEEK specimens (Invibio®, Batch: D0602, grau: NI1) produced by compression molding (at 300 °C for 30 min) were used as control group. An aluminum mold was labored, in order to produce specimens (5.5 × 10 mm) with compatible diameter to fit within TCPS wells for cell culture assays. Specimens were sterilized by ethylene oxide.

inflammatory reactions [8]. Several methods to prevent the biofilm accumulation comprises the use of therapeutic substances and procedures to decrease the content of pathogenic species [9–12]. The use of polymers as drug delivery systems have been proposed in literature although chemically methods of curing are required to avoid the damage of the therapeutic compounds. The sulphonation process of PEEK by chemical treatment using sulfuric acid treatment results in a sulphonated PEEK named sPEEK. The functionalized PEEK has been developed for application as proton exchange membranes in fuel cells [13–15]. The functionalization of PEEK can be useful to incorporate therapeutic compounds such as antimicrobials and growth factors for further drug delivery into the body [16–18]. Also, sPEEK membranes and coatings embedding particulate graft materials can enhance the bone healing [17–19]. Thus, PEEK sulfonation process can be useful to fabricate bioactive and absorbable composites incorporated with hydroxyapatite, beta-TCP, or xenogenic graft materials. Osteoinductive biomolecules and growth factors have also been incorporated into other biomaterials to enhance tissue healing [12]. Additionally, the use of antimicrobials into sPEEK can prevent continuous accumulation of biofilms and consequent infection in the early postoperative period [13]. SPEEK surface revealed antibacterial activity against S. aureus, E. coli, and S. mutans species [16,18,19]. The main aim of this study was to develop sPEEK membranes by sulphonation processing for application in implant dentistry. In this study, the functionalized PEEK was physicochemically and biological assessed by different techniques to assess properties proper as a drug delivery biomaterial.

2.2. Physical, chemical and topographical analysis Thermogravimetric analyses (TGA) of the PEEK-based specimens were carried under 20 mL/min nitrogen atmosphere from 20 up to 900 °C at a heating rate of 10 °C/min, using STA equipment (Netzsch, Germany) to determine the sulphonation degree of those samples. Measurements were performed in duplicate (n = 12) for each group. The degree of sulphonation (DS) was calculated after sPEEK curves analysis, assuming that the sPEEK first stage of thermal degradation was entirely caused by elimination of SO3H groups. For hydrogen nuclear magnetic resonance (1H NMR) analysis, sPEEK was dissolved in DMSO‑d6 (ca. 3% solution) for measurements (n = 6) using a NMR spectrometer (Varian AS-400, Oxford, UK). The DS was determined by integration of different aromatic signals. Fourier Transforming Infrared Spectroscopy (FTIR) analysis on PEEK-based specimens was performed with 4 cm−1 resolution using a FTL 2000 spectrometer (Bomem, Canada). FTIR analysis was performed at each stage of sample processing, which correspond to: i) PEEK; ii) PEEK after sulphonation for two different times (sPEEK-1 and sPEEK-2); iii) sPEEK-1 and -2 dissolved at DMSO (sPEEK/DMSO-1 and sPEEK/ DMSO-2); iv) sPEEK/DMSO-1 and sPEEK/DMSO-2 washed with PBS. The arithmetical roughness (Ra) of PEEK and sPEEK/DMSO-1 and -2 specimens were measured using an optical profilometer (DektakXT, Profilometer, Germany). Measurements were carried out three times for

2. Materials and methods 2.1. Synthesis of PEEK and sPEEK specimens Biomedical grade N1 PEEK (Invibio®, Batch: D0602, UK) was functionalized by sulphonation process using sulfuric acid treatment (Synth®, Brazil), as illustrated in Fig. 1. In a volumetric glass recipient, 1 g PEEK granules was solubilized in 25 mL of 98% sulfuric acid under continuous stirring for 18 h. The mixture was heated until 50 °C for 1 h (SPEEK-1) or 1.5 h (SPEEK-2). Then, the solution was gradually added to 500 mL of ice-cold distilled water resulting in the precipitation of a functionalized PEEK (sPEEK). The final product was filtered and washed with distilled water several times until the pH around 7. After that, the sPEEK was dried at 70 °C for 72h. This process was repeated 3 times

Fig. 1. Methods used for the manufacturing and analysis of sPEEK. 543

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2.4. Cell adhesion and morphology L929 cells were seeded on the top of PEEK and sPEEK/DMSO-1 and -2 specimens for 1, 3, and 7 days of incubation. After each experimental time point, the culture medium was removed and the specimens were washed three times with PBS. Then, 2.5% glutaraldehyde were added at 4 °C for 1 h to attach cells. PEEK and sPEEK/DMSO-1 and -2 specimens were dehydrated with alcohol gradual series (30%, 50%, 70%, 80%, and 100%), for 1 h each (except 80% alcohol dehydration that was overnight). Specimens were placed in a metallic support of Balzer's chamber and they were dried by critical point, considering at least three replacement of alcohol to CO2 liquid, using an EM CPD030 equipment (Leica, Germany). For microscopic analysis, surfaces were sputtercoated with a thin gold layer, and analyzed by secondary (SE) and backscattered (BSE) electrons mode at 10kV using a scanning electron microscope (SEM, JEOL JSM- 6390LV, Japan). 3. Results and discussion Fig. 2. Thermogravimetric curves for sPEEK-1 and -2.

3.1. Physicochemical characterization each specimen considering that each group was analyzed in triplicate (n = 9). The wettability of PEEK and sPEEK/DMSO-1 and -2 samples were evaluated by measuring the contact angle formed on the material surface by a droplet (1 uL) of distilled water by using a water contact angle goniometer (OCA 20, Data Physics, Germany). Measurements were carried out three times for each specimen considering that each group was analyzed in triplicate (n = 9).

TGA curves for sPEEK with two distinct weight loss events are shown in Fig. 2. The mass loss, at 100 °C occurred due to the removal of water molecules. The first stage of thermal degradation from 300 until 400 °C was attributed to the removal of sulfonic acid groups (SO3H), while the second stage of thermal degradation from 500 to 600 °C was related to the degradation of the PEEK chain. The mean degree of sulphonation (DS) for sPEEK-1 specimens were at 59%, while the DS of sPEEK-2 was around 56%. Nuclear Magnetic Resonance (H NMR) spectra are shown in Fig. 3. NMR spectra disclosed the presence of SO3H groups at sPEEK specimens once the introduction of sulfonic groups resulted in a distinct signal for protons at the E position, which appeared as a characteristic singlet at 7.5 ppm. The ratio between the peak area of HE (AHE) signal and the integrated peak area of the signals corresponding to all the other aromatic groups were used to calculate the sulphonation degree. The DS for sPEEK-1 ranged from 62 to 70%; while the DS for sPEEK-2 oscillated between 67 to 68%. There were no statistically significant differences

2.3. Cell viability and proliferation L929 cell line (immortalized murine cell line of fibroblasts) was used to assess the cytotoxicity of sPEEK specimens. Cells were maintained under a moist atmosphere at 37 °C with 5% CO2 and cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco®, USA) supplemented with 10% fetal bovine serum (Gibco®, USA) and 1% penicillin/ streptomicyin (Gibco®, USA). L929 cells were placed in 96-well places at a density of 2.5 × 104 cells/cm2. After 24h, cells were placed on the top of PEEK (control) and sPEEK/DMSO-1 and -2 and incubated for further 1, 3, and 7 days without renewing the culture medium to evaluate any toxic effect from the sPEEK/DMSO specimens. After each time point, cell metabolic activity was determined by MTS assay (Promega Corporation, USA) and dsDNA was quantified using PicoGreen dsDNA assay (Thermo Fischer, USA), following manufacturer's instructions. On the metabolic activity evaluation, PEEK and sPEEK/DMSO specimens and culture medium were removed and cells were washed three times with PBS. Then, 300 μL fresh medium and 60 μL of MTS reagent were added to each well; the well plates containing cells were incubated at 37 °C for 3 h. After incubation, 100 μL each well was transferred to a 96-well plate for optical density measurement by spectrophotometry using a MicroELISA at 490 nm (Thermo Plate TP Reader®, Brazil). All the tests were performed at four independent measurements (n=12). L929 proliferation was assessed after each time point removing samples and culture medium from well plates. Then, adhered L929 cells were washed three times with PBS solution and therefore 1 mL ultrapure water was added to each well at 37 °C, with 5% of CO2. Well plates contacting cells were stocked in a freezer (at −80 °C) until the analysis moment. Samples were defrozen at room temperature, requiring protection from light. PicoGreen reagent was added to each well to assess the amount of dsDNA following manufacturer's instruction. Finally, cell proliferation was assessed by fluorescence spectroscopy at 485/9 nm excitation and 528/20 nm emission by spectrophotometry (Microplate Reader M200 TECAN).

Fig. 3. H NRM of sulfonated PEEK samples of sPEEK-1 and sPEEK-2. 544

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Fig. 4. FTIR spectrum of (A) pure PEEK (black curve); (A) sPEEK-1 (red curve); (A) sPEEK-2 (blue curve); (B) sPEEK/DMSO-1 (red curve); (B) sPEEK/DMSO-2 (blue curve); (B) sPEEK/DMSO-1 with PBS rinsing (red curve); (B) sPEEK/DMSO-2 with PBS rinsing (blue curve).

Montero et al. (2016) [19] performed thermal and chemical analyses of PEEK sulphonated for 1, 1.5, 2, 2.5, and 3 h. In that previous study, DS of PEEK was around 62 and 65% for 1 h and 2 h considering results of TG curves, NMR, and ion-exchange capacity. Those values are higher than the ones found in the present study (58.7% for 1 h and 55.8% for 1.5 h of sulphonation). FTIR spectra was able to validate the sulphonation process as found in previous studies [16–18]. Additionally, chemical modifications resultant from DMSO and PBS immersion were assessed by FTIR. Considering sPEEK can be modified in different organic solvents such as DMSO, the incorporation of therapeutic and bioactive compounds can be a potential strategy for biomedical applications [20]. The chemical modification of sPEEK can occur in PBS depending on the degree of sulphonation. The chemical modification of sPEEK is related to the ion exchange phenomenon by deprotonation of H+ from sPEEK due to the presence of SO3H groups. On the results shown in the present study, sPEEK/DMSO-2 was more susceptible to the structural changes occurred through such process, which could also explain the differences noticed in biological performance between sPEEK/DMSO-1 and sPEEK/DMSO-2. Our preliminary results indicated that sPEEK/DMSO membranes produced with sPEEK sulfonated for 2 or 3 h were not suitable for cytotoxicity assays and, therefore, additional studies were not performed within this group.

between the two groups (p > 0,05). Although DS between sPEEK-1 and sPEEK-2 has not statistically differed, FT-IR spectra between samples were different. That was the reason why sPEEK-1 and sPEEK-2 were selected to be dissolved in DMSO and have their biological behavior assessed in this study. FTIR spectra (Fig. 4) revealed bands attributed to the PEEK main chain (1650, 1490, 1158 and 925 cm−1) at almost all conditions as shown in Fig. 4. Also, bands associated with SO3H groups (1255, 1080, 1024, and 709 cm−1) were present in the groups in which the sulphonation has been accomplished (B-F). The main modification in sPEEK immersed in DMSO was confirmed by the band at 795 cm−1. PBS rinsing does not seem to cause important structure changes on sPEEK/ DMSO-1 although PBS has abruptly affected FTIR spectra for group G (sPEEK/DMSO-2). The mean values of water contact angle and roughness recorded on PEEK-based specimens are shown in Fig. 5. There were no statistical differences between sPEEK groups (p < 0.05). However, the water contact angle values for PEEK surfaces were lower when compared to sPEEK. Roughness means values have also exhibited statistical significant difference (p = 0 < 0.05) between PEEK and sPEEK groups. Regarding the results obtained for thermal and chemical analysis, the sulphonation degree of sPEEK test specimens is partially in agreement with previous studies that used similar procedures for both processing and physicochemical characterization [13,19]. For instance,

3.2. Biological behavior The cytotoxicity of sPEEK/DMSO-1 and -2 specimens were assessed according to ISO 10993-5. The metabolic activity of L929 cells in contact with the PEEK-based specimens are shown in Fig. 6. Results were normalized, considering PEEK (control group) as 100% for each experimental time. At the first day, the metabolic activity of cells grown on sPEEK/DMSO-1 was around 9% lesser than that recorded on PEEK while sPEEK/DMSO-2 was 9.3% higher when compared to PEEK. Both groups have shown statistical different values from the control. On the third day of incubation, the metabolic activity of cells grown on the test groups did not show statistical differences from the control group, considering that cells grown on PEEK revealed only 5% higher metabolic activity when compared to that for test groups. On the seventh day of incubation, the cell metabolic activity on sPEEK/DMSO-1 remained stable while there was a significant increase in cell metabolic activity on sPEEK/DMSO-2 of about 20% compared to that recorded on PEEK. Regarding cell proliferation, the dsDNA mean values of cells grown on sPEEK/DMSO-1 and SPEEK/DMSO-2 was around 71% of that recorded on PEEK for the first day of incubation (Fig. 7). At the third day

Fig. 5. (A) Ra roughness and (B) water contact angle mean values for PEEK and sPEEK. 545

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Fig. 6. Metabolic activity of L929 cells culture on direct contact with PEEK; SPEEK/DMSO-1 SPEEK/DMSO-2. * indicates significant difference compared to control PEEK.

Fig. 8. SEM micrographs revealed L929 cells adherence on PEEK at day 1(A) and 7(B); on sPEEK/DMSO-1 at day 1(C) and 7 (D); on sPEEK/DMSO-2 at day 1(E) and 7(F).

Considering cytotoxicity assays, PEEK specimens were assessed as control group, once PEEK is well-known as a non-toxic biomaterial [21,22]. Metabolic activity assays revealed that sPEEK did not kill L929 cells, although it interfered on the cell behavior which were confirmed mainly for sPEEK/DMSO-2. SPEEK have already been assessed in contact with other cells lines, such as MG-63 osteoblast like cells and mouse MC3T3-E1 pre-osteoblasts, and therefore cell behavior was not affected by sPEEK specimens [16,17]. Thus, Zhao et al. (2013) [16] evaluated sPEEK cytotoxicity in contact with MC3T3-E1 pre-osteoblasts, comparing to PEEK (control). In that previous study, sPEEK was rinsed with water (sPEEK-W) or acetone (sPEEK-WA) although no dissolution in DMSO was tested. The cell metabolic activity by MTT (570 nm) was higher on sPEEK-WA than that on sPEEK-W or PEEK for 1,3,7, and 14 days of incubation. It is important to highlight that fibroblasts commonly spread proper on smooth surfaces than on rough ones [23]. It could be due the fact that fibroblasts need to establish a stronger network on smooth surfaces to stabilize their growth on the surface [24]. Even though PEEK exhibited a rougher surface compared to that for sPEEK/DMSO-1 and sPEEK/DMSO-2, fibroblast spreading was higher on PEEK surface than on sPEEK/DMSO surfaces. Also, wettability can influence the initial cell adherence on biomaterial surfaces, as much fibroblast attachment seems to be higher also on hydrophilic surfaces [25]. Once PEEK specimens were more hydrophilic than sPEEK/DMSO-1 and sPEEK/DMSO2, it could explain the significant reduction of cell proliferation by DNA concentration analysis.

Fig. 7. Proliferation of L929 cells cultured on direct contact with PEEK; sPEEK/ DMSO-1 and sPEEK/DMSO-2. * indicates significant difference compared to control PEEK.

of evaluation, the dsDNA mean values of cells grown on sPEEK/DMSO1 decreased to around 66% while the dsDNA mean values of cells grown on sPEEK/DMSO-2 increased to 82% in comparison to the mean values recorded on the control group. When considering the seventh day of assessment, the dsDNA mean values of cells grown on sPEEK/DMSO-1 increased to around 73% while the dsDNA mean values of cells grown on sPEEK/DMSO-2 remained at about 76% in comparison to the mean values recorded on the control group. At the three experimental time points of evaluation, the test groups (sPEEK/DMSO-1 and sPEEK/ DMSO-2) have shown statistical significant decrease in cell proliferation, compared to the control (p < 0.05). SEM images (Fig. 8) showed that the control group (PEEK) exhibited morphological aspects of a rougher surface when compared to that for sPEEK/DMSO-1 and sPEEK/DMSO-2, as shown in Fig. 8. L929 cells with rounded shape were detected on PEEK, sPEEK/DMSO-1, and sPEEK/DMSO-2 surfaces after the first day of cell culture. At the end of seven days of cell culture, L929 cells were morphologically adhered and spread on PEEK (Fig. 8C) and sPEEK/DMSO-1 (Fig. 8D). Unlike, L929 cells maintained a rounded morphology on sPEEK/DMSO-2 surfaces after seven days of culture (Fig. 8F).

4. Conclusions PEEK was functionalized by sulphonation method for two different processing times. The resultant functionalized sPEEK can be modified by using DMSO to incorporate therapeutic compounds for biomedical applications. However, the sulphonation process for 1.5 h resulted in sPEEK with limited cytocompatibility when in contact with fibroblasts. 546

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The degree of sulphonation and additional post-processing immersion in isotonic solutions could stabilize sPEEK surfaces to stimulate the migration of cells. Further biological assays should clarify the cytocompatibility of sPEEK related to the degree of sulphonation and modification of surfaces by immersion in isotonic solutions.

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