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Paper

Resorbable PCEC/gelatin-bismuth doped bioglass-graphene oxide bilayer membranes for guided bone regeneration

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Published 15 April 2019 © 2019 IOP Publishing Ltd
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1748-605X/14/3/035018

Abstract

Guided bone regeneration (GBR) is a therapeutic modality applied prior to dental implant placement to increase bone density at the defect site or during placement for directing bone growth around implant. In this study, an asymmetric, bilayer structure was prepared by covalently bonding a dense polycaprolactone-polyethylene glycol-polycaprolactone (PCEC) membrane layer with a hydrogel layer composed of bismuth doped bioactive glass (BG, 45S5) and graphene oxide (GO) particles incorporated in gelatin. Structural and mechanical properties (surface morphology and chemistry, thickness, degradation rate and tensile strength of GBR membranes) were studied. Membranes had a 3D structure having almost 1 mm thickness which is suitable for space filling. Highest tensile strength (TS) (1.71 ± 0.10 MPa, p < 0.001) was observed for membranes having the highest BG containing group (BG20) while lowest TS was observed (1.23 ± 0.11 MPa, p < 0.001) for BG8/GO2 samples. Similarly, hydrolytic degradation of BG20 involving bilayer structures was slower in phosphate buffered saline (PBS) (23% ± 5% in 4 weeks) than other GBR membranes while biodegraded at an equal rate in lipase (BG20 as 72% ± 3%, BG10 as 69% ± 1%, BG8/GO2 as 71% ± 7% and BG2/GO8 as 74% ± 8%). BG8/GO2, displayed lowest gelatin (GEL) release in PBS over 28 d period (175% ± 9% and 164% ± 10% mgGEL/gsample, p < 0.001). However, all bilayer membranes displayed a similar rate of degradation in lipase solution and also had similar mineral deposition ability in simulated body fluid. Significantly higher cell proliferation (p < 0.001) and osteogenic differentiation (p < 0.001) of human dental pulp stem cells were observed in BG20 and BG10 membrane groups than all other groups. On the other hand, GO presence decreased both mechanical and osteoinductive properties compared to pure BG counterparts. Collectively, amine introduced (aminolysis) synthetic dense PCEC layer was covalently bonded to composite hydrogel layer to obtain coherent bilayer membranes for GBR. They were successfully produced to have two layers designed to prevent fibrous tissue movement towards bone defect while enabling bone regeneration. BG20 membrane groups demonstrated higher calcium phosphate deposition TS, cellular growth and osteogenic differentiation.

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1. Introduction

Loss of bone quantity as a consequence of periodontal diseases, such as periodontitis, tooth loss and tumor resection, may require the use of biomaterials to revert the bone loss [1].

Consequently, dental implants are employed to encourage bone healing as well as replacing the missing function of tissues, such as tooth [2]. However, dental implants may also be in need of supporting structures to improve bone-implant adhesion while preventing micromovements and bacterial colonization of the implant which are important factors distrupting bone healing process [3]. For these reasons, biomaterials designed for Guided bone regeneration (GBR) became widely preffered approach for tissue engineering applications in dentistry [4].

GBR membranes are often produced as asymmetric bilayer structures in which a barrier layer prevents enterance of non-osteogenic cell populations to bone defect site while an osteogenic layer induces bone regeneration [5, 6]. Moreover, GBR bilayer membranes can also be produced from resorbable materials to improve healing process and eliminating the requirement of any further operation for their removal from the implanted site [7]. Furthermore, asymmetric nature of the bilayer membranes can be designed to release antibiotics, osteogenic factors and growth factors from the bone facing surface [810]. Henceforth, resorbable GBR bilayer membranes appeared as third generation biomaterials providing multiple functionality and effective bone healing [11].

In this study, a GBR bilayer membrane was produced thruogh covalently bonding poly-caprolactone-poly ethylene glycol-poly caprolactone (PCEC) triblock copolymer layer with a composite hydrogel layer of bismuth-doped BG and GO containing gelatin (GEL). PCEC is a bioresorbable polyester which is used in bone tissue engineering applications [12]. PCEC was selected for its lower glass-transition temperature (<0 °C) than body temperature for better elasticity, high flexibility and ability to undergo minimally invasive chemical surface treatment such as aminolysis [13]. In addition, GEL was selected for its ability to produce a porous hydrogel and its protein structure containing free amine groups that can undergo covalent bonding in the presence of a crosslinker such as glutaraldehyde [14]. Exploiting this possibility, PCEC thin membrane layer was aminolysed and subsequently interacted with GTA to obtain grafted aldehyde units to covalently attach and stabilize GEL-based hydrogel for stronger attachment of two layers. Thus, it was also aimed to develop a bilayer membrane with easier, robust and clinically managable handling.

In order to achieve high osteoinductivity, GEL hydrogel with bismuth-doped BG/GO composites was produced. BG has high mineral deposition capacity, biodegradability and osteoconductivity due to quick release of osteogenic ions such as silica, calcium and phosphate [15]. Additionally, BG inclusion increased mesenchymal stem cell growth [16], osteoblastic differentiation of stem cells [17], compressive strength in polymer-BG composite systems [1820]. As a result, BG was shown to improve bone adhesion and accelerate bone regeneration [21]. Moreover, bismuth is a heavy metal that was used in a large variety of medicinal compounds and known to possess hemostatic, antibacterial and anticancer properties [22]. When used as a dopant in BG structure, we have shown that bismuth enhanced the mechanical and biological properties of BG [23]. In addition, BG and GO were physically mixed for improving mechanical and osteogenic properties of the bilayer membrane. GO is also widely employed as nanomaterial due to its ability to improve overall hardness and mechanical properties of the nanocomposites [24]. Furthermore, GO was shown to positively affect cellular adhesion and osteogenic differentiation in our previous study [23]. BG-GO blends were also demonstrated to increase fracture toughness and cellular viability of MG63 osteoblastic cell line [25, 26]. To the best of our knowledge there is no study on a GBR bilayer having composition PCEC/GEL-BG-GO in literature. This novel composition with optimized aminolysis and subsequent covalent bonding will improve the applicability and success of GBR based treatment approaches. PCEC/GEL-BG-GO bilayer membranes were prepared by covalent binding of dense PCEC membrane with bare GEL, GEL-BG and GEL-BG-GO composite hydrogels. The structural, mechanical, mineral deposition capacity and biological properties of the membranes were analyzed.

2. Materials and methods

2.1. Materials

Poly ethylene glycol (Mn = 4000 kDa), ε-caprolactone, gelatin (porcine skin, Type A, ~300 g bloom), L-glycine, lipase (porcine pancreas) were purchased from Sigma Aldrich (USA). Isopropanol (IPa), 1,6-hexanediamine (HDA), glutaraldehyde (GTA), 1,4-dioxane, 2,4-dinitrophenyl hydrazine, hydrochloric acid, phosphate buffered saline and carbonate buffer salts, tetraethyl orthosilicate, triethyl phosphate (TEP, Merck, Germany), calcium nitrate tetrahydrate, nitric acid, Bismuth (III) nitrate pentahydrate were obtained from Merck (Germany). Other solutions and media were reagent grade.

2.2. PCEC membrane preparation

PCEC triblock copolymer was synthesized through ring opening polymerization according to procedure presented in literature [27]. Obtained copolymer was precipitated in excessive cold ethanol, rinsed with deionized water and freeze-dried until use. PCEC was dissolved in dichloromethane (CH2Cl2, 10% w/v) and the obtained solution was further homogenized in a sonicator. The polymer solution (1.8 ml) was solvent casted in glass petri dish (phiv = 6 cm) and air-dried for 4 h. PCEC membranes were then freeze-dried and stored at room temperature (RT). Additionally, synthesized PCEC was analyzed with Proton Nuclear Magnetic Resonance (1H NMR) to determine its number average number of molecular weight (Mn) (supplementary table 1 is available online at stacks.iop.org/BMM/14/035018/mmedia).

2.3. Aminolysis of PCEC membranes

PCEC membrane surfaces were cleaned with ethanol prior to aminolysis. Briefly, an aminolysis solution was prepared by dissolving 10% w/v HDA in IPA) at RT. The membranes were then placed in aminolysis solution (20 ml/membrane) and magnetically stirred at RT with 150 rpm for 1 h. At the end of the reaction, membranes were thoroughly washed with phosphate-buffered saline (PBS, 0.1 M, pH 7.4).

In order to optimize the amount and timing of aminolysis procedure, PCEC membranes were treated with various concentrations of HDA (0–10% v/v yielding 0–4 mmols of –NH2 per cm2 respectively at λmax) for different time points (0–60 min with 15 min increments) prior to GTA immobilization. Shortly, after incubation in HDA solution, samples were rinsed twice with PBS, dipped in ninhydrin solution (1 M in ethanol) and then placed in atmospheric oven set at 60 °C and awaited until melting. Molten samples were dissolved in 1,4-dioxane and spectrum scan between 450 and 650 nm was performed to determine λmax (figure 2). Moreover, membranes were analyzed with 1H NMR to determine the effect of aminolysis on PCEC triblock structure.

2.4. Aldehyde pendant groups formation on PCEC membranes

PCEC membranes were directly placed in 2.5% (v/v) GTA solution after aminolysis and incubated at RT for 3 h (50 ml GTA solution per aminolysed membrane) and GTA was allowed to attach to amine groups produced on PCEC membranes in the previous step (figure 1). GTA immobilization was qualitatively observed by orange dinitrophenylhydrazone precipitation (S.figure 2). Concisely, 2, 4-dinitrophenyl hydrazine (DNP) was dissolved in methanol and acidified by dropwise addition of hydrochloric acid to obtain working solution (2% w/v). GTA immobilized PCEC membranes were placed in DNP solution and allowed to react for 1 h to form aldehyde groups on membranes. The membranes were then rinsed carefully with PBS and placed in carbonate buffer (CB, 0.1 M, pH 9.5) until use. CB was prepared by mixing sodium bicarbonate in dH2O (0.1 M NaHCO3) and sodium carbonate in dH2O (0.1 M Na2CO3) in 7:3 (v/v) ratio.

Figure 1.

Figure 1. Preparation of GBR bilayer membranes. Initial conditioning of dense PCEC membranes and qualitative analysis by ninhydrin and DNP (A), preparation of composite hydrogel (B) coating of PCEC with composite hydrogel (C). Note that homogenization process also includes GO for samples BG8/GO2 and BG2/GO8.

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Figure 2.

Figure 2. HDA treatment optimization by ninhydrin quantification (A), –NH2 quantification after HDA treatment (B) and SEM images of GBR bilayer membrane surfaces with cross-sectional images of samples (C).

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2.5. Fabrication of composite hydrogel/PCEC membrane

Composite hydrogels were prepared with GEL using BG and GO as nanoreinforcements. Briefly, BG and GO were grounded with ultrasonicator (25% amplitude for 5 min) in CB buffer to obtain very fine powder of BG and GO (Branson Sonifier SFX 550, USA). GEL was then dissolved in CB (5% w/v) at 37 °C containing BG and GO according to ratios given in table 2. Homogeneous solution of GEL and nanoreinforcements were poured in glass petri dish (4.8 ml) and PCEC membranes were placed directly on top of the casted solution. Samples were then incubated at 4 °C overnight to allow hydrogel formation. After that, composite hydrogel/PCEC membranes were incubated with 2.5% (w/v) GTA for 24 h, rinsed with PBS thoroughly to crosslink the hydrogel structure. Thereafter, they were introduced into 2.5% (w/v) glycine solution in deionized water and incubated for 4 h. Finally, composite hydrogel/PCEC membranes were freeze-dried after rinsing with PBS and stored in desiccator at 4 °C. All procedures are schematized in figure 1. All membranes (pure PCEC, aminolysed PCEC, GEL, GEL coated and composite GBR membranes) were analyzed with Attenuated Total Reflection/Fourier Transform Infrared Spectroscopy (ATR/FTIR) for structural analysis at mid-IR range (400–4000 cm−1) (Bruker Hyperion, Germany). Compositions of the samples are given in table 1.

Table 1.  Membrane groups prepared in the study.

Membranes GEL (w/v% in CB) Bi-BG (% of GEL) GO (% of GEL)
Pure PCECa 0 0 0
PCEC-NH2b 0 0 0
PCEC-GELc 5 0 0
BG20 5 20 0
BG10 5 10 0
BG8/GO2 5 8 2
BG2/GO8 5 2 8

aand bshow only PCEC membranes, plain and surface treated, respectively. cPCEC-GEL bilayer membrane of two only polymers (PCEC and GEL).

2.6. Characterization of bilayer membranes

2.6.1. Structural and mechanical characterization

Surface morphology, PCEC membrane thickness, composite hydrogel thickness (dry) and porosity of the samples were analyzed with Field Emission Scanning Electron Microscopy after coated with Au-Pd conductive layer (FESEM, FEI QUANTA 400 F Field Emission SEM, USA). In addition, GBR membranes were tested for water uptake over time (n = 4, until no change in weight was observed) for various time points (1, 24 and 48 h). The equation given below was used to determine water uptake capacity (equation (1)):

Equation (1)

Composite hydrogel coating thickness was also determined in wet form after 48 h in PBS using digital micrometer (n = 3, Mitutoyo, Japan) and in dry form with SEM. For mechanical analysis, samples were cut into dog-bone shape having same macroscale dimensions as referenced by ASTM D1708 (S.figure 3). The samples were then wetted in PBS at 37 °C for 24 h and then analyzed for tensile properties such as ultimate tensile strength (UTS) and elastic modulus (E) (n = 4) until break.

Figure 3.

Figure 3. Water uptake values of GBR membranes (n = 4) used in the study (A) and contact angle measurements of the samples in dry form (B). Statistical significance was displayed as (***) p < 0.001, error bars: Standard deviation.

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2.6.2. Degradation study and chemical characterization

GEL degradation occurs by hydrolysis of GTA crosslinks as reported elsewhere [28]. Degradation of the samples was determined by incubating them separately in PBS and lipase solution. For degradation in PBS, samples were punched to obtain cylindrical membranes (phiv = 10 mm), weighed (n = 4) and recorded as initial (t0) weights. The membranes were then inserted in PBS solution (10 mg membrane ml−1) and placed in 37 °C for 4 weeks. For degradation study in lipase solution, they were incubated in 110 U l−1 lipase solution in 37 °C for 2 weeks. Then, the collected samples were freeze-dried and weighed at pre-determined time intervals to calculate weight loss (%) using equation (2):

Equation (2)

Moreover, prior to PBS refreshment, an aliquot of 250 μl was collected from each incubation solution for further analysis such as GEL release as a degradation product. Amount of GEL released was determined with bicinchoninic acid assay and normalized with total weight of the samples at the given time point [5].

2.6.3. Contact angle measurement

Static contact angle (θ) measurements (sessile drop) were done to ascertain the overall surface hydrophilicity of the membranes as well as the effect of BG and GO inclusion (n = 3). Shortly, membranes were taped on glass slides and a drop of 3 ul deionized water was dropped on each sample using a motor-driven syringe at 25 °C (Attension, Biolin Scientific, Sweden).

2.6.4. Mineral nucleation study

Cylindrical membranes were sterilized in 70% (v/v) ethanol in deionized water for 30 min and UV irradiation for 1 h. Air-dried samples were then placed in simulated body fluid (SBF) prepared as 10 mg membrane ml−1 according to the method given by Kokubo and Takairi [29]. At the end of pre-determined time intervals (at 1, 7 and 14 d), membranes were removed, rinsed with 70% ethanol in deionized water, freeze-dried and stored in desiccator at 4 °C. Finally, membrane surfaces were analyzed with FESEM to detect calcium phosphate (CaP) precipitates and the precipitates were additionally analyzed with Energy Dispersive x-ray Spectroscopy attached to FESEM device for determination of elemental composition and Ca/P ratio.

2.6.5. Cell culture study

Membranes used for cell culture studies were prepared and sterilized as explained in section 2.6.4. For cell proliferation study, the membranes were seeded with Dental Pulp Stem Cells (DPSC, passage #4) at a density of 10 000 cells cm−2. After 4 h, membranes were placed in another plate containing growth media (Dulbecco's MEM (DMEM low glucose) containing 10% v/v Fetal Bovine Serum (FBS) and 1% v/v penicillin-streptomycin cocktail) and incubated in growth media for 1, 4 and 7 d. At the end of each period, membranes (n = 5) were thoroughly rinsed with PBS and placed in 10% v/v Alamar Blue containing growth media for 4 h (ThermoFisher, USA). Absorbance of the reduced dye at each well was measured at 570 and 600 nm and %reduction of the dye was calculated according to supplier's protocol. Increase in %reduction in time was interpreted as increase in cell number (proliferation) on the GBR membranes. In addition, 4 h post-seeding cellular viability assay can be interpreted as a cellular attachment survey on the GBR membranes. Samples (n = 3) from each group were selected randomly, rinsed twice with PBS and fixed by 4% paraformaldehyde for 30 min at the end of 7th day of incubation. A sample from each group (n = 1) was further dehydrated through graded ethanol series (30% v/v to absolute ethanol in PBS) and hexamethyl disilazane to observe morphology of the cells on the GBR membranes with SEM examination. Furthermore, fixed DPSCs on GBR membranes (n = 2) were then stained by actin dye (FITC-phalloidin (green), Thermo Fisher, USA) and nuclei dye (DRAQ5 (red), Cell Signaling Technologies, USA) in order to determine cellular morphology, attachment and colonization on the GBR membranes through Confocal Laser Scanning Microscopy (Zeiss, Germany).

Membranes randomly selected for osteogenic differentiation analysis (n = 5) were placed initially in growth media for 3 d to allow cellular colonization on them. After that, membranes were incubated in osteogenic medium (0.01 M β-glycerophosphate, 50 μg ml−1 ascorbic acid and 10−7 M dexamethasone in growth medium) for 1, 2 and 3 weeks. At the end of each period, membranes were rinsed with PBS, then PBS was replaced with sterile deionized water. Freeze-thaw cycles were applied three times using alternating at −80 °C and 37 °C. Then, lysates were collected and Alkaline Phosphatase Activity (ALP Activity), intracellular calcium concentration (ICa), intracellular orthophosphate concentration (IPa) and total DNA concentration in the cell lysates were determined. Briefly, lysates were reacted with para-nitrophenyl phosphate for 1 h at 37 °C to acquire 4-nitrophenol as a product of ALP. Hence, ALP activity was obtained by colorimetric quantification (molarity) of 4-nitrophenol at OD405 [30]. In addition, lysates were treated with o-cresophthalein complexone to determine ICa as described elsewhere [5]. At the end of the treatment, ICa for each membrane was determined spectroscopically at OD560. Furthermore, intracellular orthophosphate concentration was quantified by molybdenum blue method. Simply, 25 μl lysate aliquots were combined with ammonium molybdate (50 μl), ascorbic acid (25 μl) and 100 μl dH2O to give phosphomolybdic acid as the final product which was spectroscopically measured at OD650. ALP activity, ICa and IPa concentrations were normalized with total DNA concentration of respective membrane determined through Hoechst 33258 DNA quantification protocol provided by supplier (ThermoFisher, USA).

2.7. Statistical analysis

Data presented in the study were analyzed for their normality prior to one way analysis of variance (SPSS v.24, IBM, USA). All data were found normally distributed (Shapiro-Wilk Test of Normality). Statistical differences were analyzed with Tukey's post hoc comparisons at p < 0.05 (*) and p < 0.001 (***) (SPSS v.24, IBM, USA). Linear fit model to approximate the linear region for mechanical analysis was also used and regression values for each sample at linear region were determined. All data were presented as mean ± standard deviation.

3. Results

Preparation of bismuth-doped BG (1.32 ± 0.45 μm, 0.975 M 45S5 Bioglass and 0.025 M Bismuth Oxide) and GO (nano-sized, 1.84 g cm−3, obtained by graphite oxidation [31]) was done as described in our previous study [23]. In order to observe the functional groups of BG, GO and groups formed during aminolysis of PCEC, ATR/FTIR analysis was conducted (S.figure 2(A)). Primary amine groups (occurring after aminolysis of PCEC chains) had characteristic N–H vibration bands in spectra at 3300 cm−1. Additionally, presence of GEL in the bilayer structures led to C=O stretching of amide I bonding at 1637 cm−1, amide II N–H vibration at around 1551 cm−1 and C–H vibration at 3073 cm−1 (S.figure 2(A)). PCEC displayed specific –C–O–C– vibration bands in –OCH2CH2– repeated units at 1660 cm−1 and ester C=O vibrations at 1241 cm−1. Furthermore, bands appeared at 2939 and 2868 cm−1 were attributed to –CH2– vibrations in PCL homosequences present in triblock PCEC copolymer. In addition, the bilayer GBR membrane preparation involved crosslinking reaction in between aminolysed GBR membrane and GEL. Therefore, it is imperative to determine the change in functional groups of both PCEC and GEL at different pH levels to calibrate the environmental conditions for achieving efficient PCEC-GEL bonding. Therefore, PCEC and GEL were reacted at pH 7.4 and pH 9.5 and then analyzed with ATR/FTIR (S.figure 2(A)). Amine functional groups appeared at the same absorption bands in spectrum for both samples and GEL. However, PCEC-GEL crosslinked at pH 9.5 had larger ATR/FTIR band frequency and area in FTIR spectra compared to pH 7.4 counterpart at GEL-specific bands (S.figure 2(A)). Groups were analyzed using ATR/FTIR to define functional groups present in the GBR membranes (S.figure 2(B)). All groups displayed similar peaks in their spectra for PCEC, GEL and BG (S.figure 2(B)). –Si–O–Si– bending mode at 451 cm−1, amorphous –P2O5– vibration at 581 cm−1 and –Si–O– vibrations at 955 cm−1 were ascribed to BG. On the other hand, BG8/GO2 and BG2/GO8 samples showed additional absorption bands representing GO at 1598 cm−1 for C=O vibration and –OH stretching vibration at 3184 cm−1 (S.figure 2(B)).

PCEC membranes were qualitatively analyzed with DNP and ninhydrin adsorption both prior to aminolysis and after aminolysis (S.figure 2(C)). Non-treated PCEC membrane was in white/transparent form while aminolyzed samples appeared black after ninhydrin adsorption. Amine groups had a direct correlation with ninhydrin amount adsorbed [32] (figure 2(A)). Exploiting this relationship, total amine groups on the PCEC membranes after aminolysis were determined as 1.16 ± 0.05 mmol cm−2 after 1 h of incubation in HDA (figure 2(B)). Mn of PCEC and change in Mn after aminolysis is summarized in S.table 1. It was observed that aminolysis led in a small decrease in MnPCEC (from 30 to 28 kDa). Additionally, increment in –H intensity belonging to methyl groups of PEG intrablock –(OCH2)– was observed (S.figure 1). In addition, covalent bond was formed between PCEC-NH2 and GEL through GTA groups introduced on PCEC membranes (figure 1). The active aldehyde groups were detected with DNP analysis and orange precipitates of dinitrophenylhydrazone were formed [33] (S.figure 2(C)). Since dinitrophenylhydrazone particles are non-soluble, aldehyde groups were not quantified colorimetrically.

Dense PCEC membranes were fabricated by solvent casting. Their surface morphology is shown in figure 2(C). At the end of aminolysis, a rough surface without pore openings in the bulk was observed. Electrographs of GBR bilayer membranes showed high porosity on the composite GEL layer in which BG and BG/GO particles were trapped in GEL forming pore walls (figure 2(C)). Furthermore, cross-sectional electrographs exhibited PCEC membrane, composite GEL layer and pores formed throughout the GEL layer (figure 2(C)). In order to determine sample thicknesses, SEM images of GBR membranes (n = 3) were analyzed using ImageJ (NIH, USA). BG8/GO2 samples were found the thinnest while BG10 samples were the thickest in dry form (table 2). In addition, BG20 samples were found to be thickest and BG10 the thinnest in wet form however, no statistical difference was observed for the samples tested.

Table 2.  Thicknesses of GBR membranes in dry and wet conditions (n = 3).

GBR membrane Dry thickness (mm) Wet thickness (mm)
BG20 0.38 ± 0.01 0.97 ± 0.06
BG10 0.41 ± 0.02 0.86 ± 0.16
BG8/GO2 0.38 ± 0.02 0.94 ± 0.05
BG2/GO8 0.39 ± 0.03 0.91 ± 0.03

The water uptake analysis of GBR membranes revealed an equilibrium of swelling at the end of 48 h for each sample (figure 3(A)). After incubation in PBS at 37 °C for 48 h, GEL samples (bare) showed statistically the highest (p < 0.001) water uptake at app. 714% ± 46% PCEC membrane (bare) displayed the lowest at app. 33%. In addition, PCEC-NH2 membranes (bare) had statistically higher water uptake (app. 79% ± 17%) than the PCEC membranes. Moreover, GBR membranes had similar water uptake values such as 644% ± 57% for BG20, 627% ± 36% for BG10, 646% ± 83% for BG8/GO2 and 668% ± 50% for BG2/GO8 (figure 3(A)). Additionally, static surface contact angles of GBR membranes revealed similar θ degrees at around 80° as shown in figure 3(B) despite the fact that BG2/GO8 samples displayed a slightly higher θ as 87° ± 0.3°.

GEL samples degraded at a significantly higher rate (p < 0.001) than other samples in PBS (figure 4(A)). At day 14, GEL had completely degraded while other groups had lower weight loss; PCEC-GEL app. 48.5%, BG20 22.7%, BG10 22.7%, BG8/GO2 27.3% and BG2/GO8 28.4%. In addition, PCEC-GEL underwent statistically higher (p < 0.001) hydrolytic degradation at the end of 28 d whereas PCEC and PCEC-NH2 had statistically the lowest (p < 0.001) weight loss (figure 4(A)). The lipase solution was prepared to mimic a bone defect environment having a local maximum lipase concentration as 110 U l−1 as determined by Martins et al [34]. GBR membranes showed lower rate of enzymatic degradation (p < 0.05) than PCEC-GEL and GEL (figure 4(B)). GEL samples, unlike to hydrolytic degradation in PBS, had completely degraded at the end of incubation in lipase solution at the end of 1 week. Concurrently to PBS degradation, aliquots were collected from degradation media to determine total GEL release during degradation (figure 4(C)). It was observed that GEL release from the samples were comparable until 14th day. Moreover, PCEC-GEL resulted in statistically the highest GEL release at 14th, 21st and 28th days (figure 4(C)). In addition, BG20 and BG8/GO2 exhibited statistically the lowest (p < 0.001) GEL release (174.80 ± 8.60 and 164.14 ± 9.70 mgGEL/gsample, respectively). As the composite layer of GBR membranes degraded, GEL release was observed.

Figure 4.

Figure 4. Degradation in PBS (A), enzymatic degradation in lipase (B), GEL release during degradation in PBS (C). Statistical significance was displayed as significant differences at p < 0.05 (*) and p < 0.001 (***), n = 4 for all studies, error bars: Standard deviation.

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Mechanical tests revealed that application of tensile force did not lead to detachment of two layers (S.figure 3). All membranes followed linear deformation until break. The failure stresses and E values are given in table 3. According to the results obtained, PCEC and PCEC-NH2 samples provided statistically the lowest tensile properties (p < 0.05). In addition, GEL layer led to a decrease in UTS of PCEC-NH2 as well as E (table 3). On the contrary, inclusion of nanoreinforcements such as BG and GO, improved UTS and E substantially (S.figure 3). Among the GBR membranes, only BG20 samples showed significantly higher UTS than PCEC-GEL (p < 0.001) and also BG20 and BG10 appeared to have similar UTS and E values. It was also noted that decrease in BG amount in samples led to a decrease in UTS and thus E values although no statistical difference was observed (p < 0.001). However, increment in GO amount as BG amount declines might compensate the loss of UTS as shown in table 3. Increasing the GO presence in the GBR membranes from 2% (w/w) to 8% (w/w) reinforced the tensile properties more than BG in same amount (table 3).

Table 3.  Mechanical properties of the samples (n = 4, mean ± standard deviation).

GBR membranes Ultimate tensile strength (MPa) Tensile modulus (E, MPa)
PCEC (bare) 1.00 ± 0.27 8.74 ± 1.40
PCEC-NH2 (bare) 0.91 ± 0.31 8.55 ± 1.32
PCEC-GEL 0.64 ± 0.21 7.99 ± 1.28
BG20 1.71 ± 0.10 16.48 ± 1.60
BG10 1.59 ± 0.11 12.67 ± 1.05
BG8/GO2 1.23 ± 0.11 10.48 ± 1.55
BG2/GO8 1.34 ± 0.16 12.08 ± 0.71

SBF incubation of membranes for various times resulted in CaP precipitation. GBR membranes showed highly bioactive surfaces without exception (figure 5). CaP precipitates formed on the pore walls where BG or BG/GO were embedded (white circles and closer views in figure 5). Ca/P ratios obtained at the end of the analysis differed greatly (inserts on figure 5). BG20 samples had precipitates with a Ca/P ratio of 1.66, while BG10, BG8/GO2 and BG2/GO8 had ratios; 1.77, 1.74 and 1.61, respectively.

Figure 5.

Figure 5. SEM images of samples after mineral nucleation study for various time points. White dashed circle represents the magnified area as shown in the smaller image. Ca/P ratios were obtained with EDX analysis. (BG2/GO8 had no Day1 EDX value due to too much background noise and small amount of CaP precipitation).

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In cellular viability study, DPSC viability on pure PCEC membrane was taken as positive control. Although DPSC percent viability was found to be higher on PCEC-NH2 membranes compared to PCEC, there was no statistical difference. GBR membranes, GEL and PCEC-GEL showed significantly higher (p < 0.001) cell attachment than pure PCEC and PCEC-NH2 but they did not differ from each other (figure 6(A)). Additionally, PCEC-NH2 showed a significantly elevated (p < 0.001) cellular viability than PCEC at 7th day of incubation (figure 6(B)). Moreover, all other samples resulted in statistically higher (p < 0.001) cellular viability starting as early as 1st day of incubation until the end of study. Furthermore, GBR membranes containing BG, GO and BG/GO exhibited considerably high dye %reduction such as 23.9% for BG20, 24.4% for BG10, 24.3% for BG8/GO2 and 23.6% for BG2/GO8 at the end of 7th day of incubation (figure 6(B)).

Figure 6.

Figure 6. Cellular attachment in terms of dye % reduction results 4 h post seeding (A) and cell density as % reduction results for 1, 4 and 7 d (B). Statistical difference between groups at significance levels: p < 0.001 (***), n = 5 for all studes, error bars: Standard deviation.

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The osteoinductive capacity of GBR membranes was tested with ALP activity, ICa and IPa of DPSCs seeded on membranes (figure 7). DPSCs on BG20 and BG10 had peaked ALP activity (2.93 ± 0.65 mol/gDNA/min and 2.45 ± 0.43 mol/gDNA/min, respectively), ICa (0.17 ± 0.03 gCa2+/mgDNA and 0.14 ± 0.02 gCa2+/mgDNA, respectively) and IPa (1.48 ± 0.42 gPO43−/mgDNA and 0.99 ± 0.39 gPO43−/mgDNA) at the end of 2nd week with a significant difference from the first week (p < 0.001) (figure 7). Highest ALP activity of cells on BG8/GO2, BG2/GO8 and PCEC-GEL was observed at the end of 3rd week (figures 6(C)–(E)). BG8/GO2 and BG2/GO8 resulted in 1.93 ± 0.22 mol/gDNA/min and 2.11 ± 0.26 mol/gDNA/min ALP activity, respectively (figure 7(A)). These values were numerically lower than that observed for DPSCs on BG20 and BG10. Likewise, ICa results of DPSCs seeded on BG8/GO2 and BG2/GO8 were 0.05 ± 0.01 gCa2+/mgDNA and 0.09 ± 0.02 gCa2+/mgDNA, respectively (figure 7(B)). IPa values for these groups were 0.40 ± 0.09 gPO43−/mgDNA and 0.61 ± 0.09 gPO43−/mgDNA, respectively (figure 7(C)). It was noteworthy that initial (1st week) ICa results were high for BG8/GO2 and BG2/GO8, then these values decreased in 2nd week and finally peaked in 3rd week.

Figure 7.

Figure 7. ALP activity (A), ICa (B) and IPa (C) for 3 weeks. Statistical difference between groups at significance levels: p < 0.05 (*) and p < 0.001 (***), n = 5 for all studies, error bars: Standard deviation.

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Owing to immense autofluorescence, confocal analysis could only be conducted on BG20 samples (S.figure 4). DPSCs were fluorescently stained employing FITC-phalloidin (f-actins, green) and DRAQ5 (nuclei, red). In the given image, the DPSCs were located on the surface of BG20 (S.figure 4(A)) and the associated cell groups were observed at the z-stack of the surface up to 60 μm depth (S.figure 4(B)). Then, very high number of DPSCs were found at the bottom of the layer (approximately 600 μm), situated in an area between composite GEL layer and dense PCEC layer. Pore walls (green arrows) could be observed due to autofluorescence (S.figure 4(C)) further displaying highly porous GEL structure enabling cellular colonization.

SEM images of GBR membranes with and without cells are given in figure 8. According to electrographs, surfaces of the bilayer samples that were not seeded with cell appeared porous (figures 8(A), (D), (G) and (J) for BG20, BG10, BG8/GO2 and BG2/8, respectively). Additionally, a thick layer of cell sheet was visible on the surfaces of cell seeded groups (figure 8, green arrow). When the samples were further analyzed at higher magnifications (figures 8(C), (F), (I) and (L) for BG20, BG10, BG8/GO2 and BG2/8, respectively) it was clearly observed that DPSCs attached on membranes, proliferated and formed a confluent layer on the GBR membranes (red arrow: individual detailed DPSCs).

Figure 8.

Figure 8. SEM images of BG20 without DPSC (A), DPSC seeded BG20 (B) and DPSC in pores (C) were shown. Similar order of images was presented for BG10 (D)–(F), BG8/GO2 (G)–(I) and BG2/GO8 (J)–(L), respectively (Green arrow heads show dense cell layers, red arrow heads show filopodia of cells).

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4. Discussion

Maintaining an adequate bone volume plays a critical role in dental implant applications. Defected bone around the implant is expected to support mechanical loading during operation, insertion of implant and implant stability [35]. Therefore, various regenerative modalities were employed in literature to increase implant success rate by improving reconstruction at deficient zone prior to implantation and/or at peri-implant area during implantation [36]. In recent studies, periosteal distraction (PD), distraction osteogenesis, block grafting (BO) combined with onlay grafting modality were studied as pre-prosthetic implantation procedures [37, 38]. However, these techniques obligate secondary modalities to successfully induce bone regeneration despite the fact that natural and biological processes are brought about [3941]. Additionally, nanoreinforcements such as autologous grafts, natural or synthetic demineralized bone-like powders (of bone allografts, CaPs, BGs and various doped minerals) and GBR membranes were studied by many research groups and applied in clinic [42, 43]. Although success rate is high with grafting techniques and GBR, donor site morbidity of autologous BO or powders, tendency to migrate and handling problems associated due to working with powders appeared as the main disadvantages [44].

In this study composite GBR membranes which possess flexibility, defined dimensions, porosity and osteogenic capacity were produced. Therefore, it is highly expected that the prepared GBR membranes will be handled surgically with ease. Moreover, they may promote stability by inhibiting ingrowth of the soft tissue and primary wound closure with the help of PCEC layer while inducing bone growth through space filling ability and osteogenic ion release of the composite layer.

The PCEC layer was obtained as a dense structure and after aminolysis FTIR bands (S.figure 2(A)) belonging to amide groups were observed that infer aminolysis was accomplished. Furthermore, DNP and ninhydrin treatment displayed visual proof that GTA was bonded to surface amine and exhibited free aldehyde functional groups (S.figures 2(A) and (C)). DNP precipitated as hydrazone (orange precipitate) on the GTA treated PCEC layers following aminolysis proving free aldehyde groups were present and functional on the surface. Additionally, PCEC-NH2 membranes turned purple more validating the presence of amine groups as ninhydrin was reduced to hydrindantin (purple) after reacting with α-amino groups formed on PCEC surface. Moreover, ninhydrin quantification was done to determine the rate of maximum aminolysis and total amine formation (figures 2(A) and (B)) suggesting that maximum period of HDA exposure should be 60 min since further exposure brought about structural disruption (figure 2(B)). The amount of amine groups found on the PCEC-NH2 was obtained as 1.16 ± 0.05 mmol cm−2 NH2 which was significantly higher than given in the literature (6 × 10−4 mmol cm−2 with poly (lactic acid)) [45]. Therefore, compared to PCL aminolysis as intensively studied in literature, we have achieved higher aminolysis rate probably owing to labile ester bonding between PCL and PEG units thus enabling robust amide formation when HDA and PCEC interacted as schematized in figure 9 [46]. In addition, high aminolysis rate induced a stronger bonding between osteogenic composite hydrogel layer and dense PCEC membrane (further discussed later).

Figure 9.

Figure 9. Reaction mechanism employed in bilayered GBR production.

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SEM analysis of the surface revealed that the BG and BG/GO particles were observed as agglomerates in GEL layer although particles and gelatin were homogenously blended prior to preparation of composite GEL layers. A similar finding was presented by Masoudi Rad et al (2017) that β-tricalcium phosphate particles were dispersed in the fibers of poly glycerol sebacate/PCL fibers [47]. GBR membranes, regardless of constitution of composite layer, displayed a wet thickness at around 1 mm as presented in table 2 leading to a dimensional change almost 2.5 folds compared to dry samples. In addition, the water uptake capacity of the GBR membranes was around 650% of the dry versions implying that dimensional and volumetric change in GBR membranes may allow press-fitting at the defected zone providing a highly porous and 3D structure. Since space fitting ability can be pronounced more with a hydrogel layer, the membranes may then encourage cellular invasion as well as adsorption of blood inducing rapid wound closure and stability through clotting. Such structure thus will create a bone-similar zone effectively enhancing nutrient exchange for colonized cells and discouraging movement of soft tissue [48]. Therefore, highly flexible GBR membranes produced in this study can be recommended for post-prosthetic peri-implant therapy and/or in combination with pre-prosthetic modalities.

Despite the fact that highly bioactive BG and BG/GO were present in the GEL structure, contact angle analysis resulted in close values for GBR samples and PCEC membranes. Xing et al (2014) showed that absence of divalent metal ions such as Ca2+ could lead to increased number of free functional groups in GEL that pure GEL might display better initial hydrophilicity [49]. Moreover, Sikalo et al (2005) pointed out that even though initial contact with water droplets may play a role in defining hydrophilicity of a material, the movement of water on surface and into the structure appeared more significant [50]. Therefore, water uptake capacities of GBR membranes can be taken into account in determination of overall hydrophilicity of the structure in addition to sessile drop contact angle measurement. BG or BG/GO inclusion increased only 'initial' water contact angle values compared to PCEC-GEL owing to absence of free functional groups. However, as the water moves into the structure in time during water uptake analysis, GBR membranes showed greater expansion and water uptake capacity.

During aminolysis procedure, a faster rate of degradation could be observed as a consequence of HDA acting on ester bonds [45]. Therefore, the hydrolytic degradation analysis in PBS revealed that aminolysis treatment further enhanced biodegradation rate of PCEC although a significant difference could not be observed at the end of 4th week (figure 4(A)). Additionally, GBR membranes having either GEL or its composite with BG and BG/GO layer displayed remarkably lower rate of degradation (p < 0.001) implying that the bilayer structure improved overall chemical stability of GEL after membrane production. On the other hand, PCEC-GEL samples had faster weight loss (p < 0.001) in PBS environment than the composite counterparts (figure 4(A)). Coincidentally with the case of contact angle values, presence of nanoreinforcements might have triggered a slower rate of water exchange initially and allowed extended entanglement which in return decreased the rate of degradation as well as GEL release [51]. A similar trend was observed in lipase environment. All GBR membranes had similar enzymatic degradation rate at the end of 28th day (figure 4(B)) while PCEC-GEL displayed a significantly higher rate of degradation than that of GBR membranes (p < 0.05). Furthermore, lipase environment brought about rapid degradation of PCEC-NH2 entailing the effect of aminolysis on the membrane. Overall, GBR samples maintained structural integrity throughout the degradation studies. In addition, release of GEL during the hydrolytic degradation in PBS was also investigated and BG20 and BG8/GO2 provided lower release (p < 0.001) than BG10 and BG2/GO8 counterparts (figure 4(C)).

GBR membranes are often in direct contact with blood and saliva therefore UTS in wet state is the major parameter to achieve a functional and easily applicable GBR membrane [52, 53]. The wet tensile analysis revealed that the GBR membranes (mechanically strongest BG20 is presented in S.figure 3) failed at 10% strain under tensile stress (S.figure 3). Macroscopically, the wet GEL layer did not slide as bulk on the PCEC membrane implying a strong bonding between layers. Furthermore, it was determined that PCEC-NH2 led to a slight decrease in UTS compared to PCEC (table 3) proving that aminolysis brought about scissions at ester bonds in the backbone to form amide as described in figure 9. These results were also further confirmed by the SEM images in which a drastic change in surface morphology was observed(figure 2). Furthermore, FTIR bands appeared at amide specific wavelengths after aminolysis and increase in intensity of these bands following the aminolysis in CB to form PCEC-GEL proved the successful aminolysis and bilayer formation (S.figures 2(A), (B)). PCEC-GEL structure led to decrease in UTS and E values that infer bilayer membrane comprised of PCEC and GEL was weaker and flexible (table 3). On the other hand, introduction of the nanoreinforcements such as BG and BG/GO immensely improved the UTS and E values (table 3). Mechanical analysis affirmed that BG20 had the highest yield strength while appearing the most stiff material. However, GO included samples exhibited significantly lower UTS and E than that of BG20 and lower UTS than that of BG10 (p < 0.001). Kamguyan et al (2017) argued that despite surface chemistry and surface roughness analyses suggested extensive similarity in the various combinations of poly (dimethylsiloxane)/hydroxyapatite composites, bulk properties differ significantly [54]. Mangal et al (2015) analyzed nanoparticle/polymer composites of silica (SiO2) and poly ethylene/poly methyl methacrylate in terms of chain entanglement and plasticization effect through phase stability [55]. Parallel to these findings, we can speculate that BG20 could be in a threshold concentration in initial blending process and BG particles could hinder chain movement and embellish chain entanglement in the composite structures more efficiently than BG/GO combination. As a result, lower degradation rate, GEL release and better tensile properties were achieved with GBR samples having BG only than the ones with BG/GO. In addition, GBR samples incubated in SBF for 1 and 5 d demonstrated similar outcomes in terms of mineral nucleation at the beginning since initial nucleation of SBF depends dominantly on surface chemistry [56]. Then, as the complete wetting occurred, reinforcements were started to be released which was further accelerated by GEL release and supersaturation was achieved in accordance with the released concentrations of calcium and phosphate species in parallel with extensive discussions in literature [57, 58]. Therefore, BG20 sample exhibited largest area covered by CaP precipitates as shown in figure 5 while BG2/GO8 showed least precipitation on the surface. Figure 5 shows Ca/P ratios of all samples and these ratios were close to 1.60, which is dominant in hydroxyapatite phase as indicated by [59].

Surface treated membrane (PCEC-NH2) appeared more favorable in terms of cellular attachment than PCEC. In addition, GBR membranes having prominent GEL layer displayed almost 2.5 folds increase in cellular attachment compared to monolayer membranes (figure 6(A)). The surface of GBR membranes, the bare and composite GEL layers, were highly suitable for cellular attachment (p < 0.001). Moreover, the GBR membranes again showed a significantly greater cellular growth than membranes (p < 0.001, figure 6(B)). Chen et al (2015) displayed that GEL presence in a GBR membrane resulted in alleviation of inflammatory response during degradation, resulting in permitting good cellular growth [60]. It was also shown that GEL exposed the anchoring amino acid sequences for integrin mediated cellular attachment [61]. Furthermore, GEL-based hydrogels were found to have suitable surface chemistry for platelet aggregation at the defect site and hemostatic ability [62, 63]. Another important point is that PCEC-NH2 surface was also considerably favorable than PCEC and allowed better cellular proliferation rate (p < 0.001). Better surface hydrophilicity of PCEC-NH2 layer played a critical role at both cellular attachment and significantly enhanced cellular growth as previously shown in literature [64].

The GBR membranes showed extensive autofluorescence due to crosslinking agent used. GTA was shown to cause autofluorescence when employed to create covalent bonding in between amine groups [65]. Consequently, we were unable to collect confocal images except for the BG20 (S.figure 4). DPSCs seeded on BG20 appeared more on the bottom of the composite GEL layer than the surface. The confocal analysis resulted showed that BG20 was biocompatible and enabled colonization by the DPSCs. In addition, SEM images showed that all the GBR bilayer membranes could sustain cellular attachment on the surface irrespectively of BG or BG/GO presence (figure 8). Therefore, GEL layer might have played a more critical role in maintaining cellular attachment due to biochemical composition of GEL [66]. Obtained electrographs showed the distribution of DPSCs on composite hydrogel part of GBR membranes. DPSCs displayed a confluent layer on the membranes and formed a thin layer on the surface features (grooves and pores) of membranes. A similar change in morphology of DPSCs was observed by Mangano et al (2010) on titanium surfaces that were textured after acidic/alkaline treatments [67]. An et al (2015) demonstrated that DPSCs agglomerates could be seen as flattened regions [68]. They also displayed extensive CaP formation at the sites of confluent DPSC regions. Alike to DPSCs, other stem cells also display fibroblastic phenotype such as mesenchymal stem cells. Song et al (2015) observed that bone marrow derived mesenchymal stem cells could cover the surface of tricalcium phosphate scaffolds and cluster on each other leading to similar electrographs as seen in figure 8 [69].

Effect of GBR membranes on osteogenic differentiation of DPSCs was also investigated through ALP activity, intracellular calcium and intracellular orthophosphate concentration (figures 7(A)–(C)). Collectively, the significantly highest two osteogenic response of DPSCs seeded on GBR membranes was observed on BG20 and BG10. DPSCs on these samples also displayed peaked osteogenic differentiation as early as 2nd week while other GBR samples including PCEC-GEL peaked at 3rd week (p < 0.001). The overall effect of BG on greater osteogenic differentiation capacity can be speculated as the presence of Bi, CaO and P2O5 in the structure which allowed increased concentration of calcium and phosphate species in the cell culture environment [23, 70]. On the other hand, silica presence might further encourage cellular differentiation towards osteogenic phenotype [71]. When BG10, BG8/GO2 and BG2/GO8 samples were compared, it was observed that although GO showed good biocompatibility, GO presence did not improve the osteogenic response. Consequently, we proposed that biochemical spatiotemporal dynamics in both cells as well as in extracellular environment were pronounced more than the mechanical properties and surface chemical characteristics in the present study as previously discussed by [72, 73]. Therefore, we acknowledged BG20 and BG10 as the most robust and reliable GBR membranes which may perform efficiently as a primary or secondary modality in dental implant therapy.

5. Conclusion

Most importantly, GBR membranes produced in this study were designed to meet the strongly anticipated requirements of a successful dental support membrane, namely space filling ability, preventing soft tissue invasion and maintaining barrier function which should last for several months owing to presence of PCEC membrane, showing osteoinductive and osteoconductive properties, being highly resorbable and flexible and easy to apply by a single operation by the surgeon. Asymmetrical bilayer GBR membranes composed of a dense PCEC thin membrane and a highly porous composite GEL layer with BG or BG/GO were produced successfully. Through aminolysis, a robust bonding was achieved between synthetic PCEC membrane and osteogenic composite hydrogel layer. According to results obtained, BG containing GBR membranes performed mechanically, biochemically and biologically significantly better than other membranes. BG/GO blend containing GBR membranes appeared inferior to BG only membranes in terms of mineral deposition ability, DPSC osteogenic differentiation and degradation in PBS. Meanwhile, BG20 and BG10 were shown to improve significantly intracellular calcium and orthophosphate presence and ALP activity. Furthermore, these membranes displayed extensive CaP precipitation thus better mineral deposition property than BG/GO counterparts. Consequently, BG20 and BG10 hold potential for use as a GBR modality in dental implant therapy. Further studies in vivo are required to validate in vitro results.

Acknowledgments

Authors would like thank Center of Excellence in Biomaterials and Tissue Engineering as well as in Middle East Technical University Central Laboratory for their support. DPSCs were kindly provided by Dr Reza moonesi Rad, METU.

Conflict of Interest

Authors state that there are no conflicts to declare.

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