O R I G I N A L P A P E R
Osteogenic activities of polymeric soybean
oil-g-polystyrene membranes
R. Seda Tıg˘lı Aydın•Baki Hazer•Merve Acar•
Menems¸e Gu¨mu¨s¸dereliog˘lu
Received: 30 November 2012 / Revised: 18 February 2013 / Accepted: 30 April 2013 / Published online: 10 May 2013
Ó Springer-Verlag Berlin Heidelberg 2013
Abstract A novel biocompatible copolymer membrane was synthesized and characterized for use in guided bone regeneration using polymeric soybean oil-g-polystyrene (PSO-g-PS) graft copolymer which was successfully obtained by free radical polymerization of styrene initiated by PSO peroxide as a macroinitiator at 80°C. Osteoblastic cellular activities of MC3T3-E1 cells on PSO-g-PS membranes with different soybean oil composition (PSO-g-PS1, PSO-g-PS2, and PSO-g-PS3) were evaluated. Nuclear magnetic resonance (1H NMR) spectra showed that PSO inclusion (mol%) was found to be 27, 69, and 51 % for PSO-g-PS1, PSO-g-PS2, and PS3 membranes, respectively. Superior biocompatibility of the PSO-g-PS membranes was determined compared to polystyrene tissue culture plates (TCPS) as positive control. Cell proliferation was enhanced on PSO-g-PS2 and PSO-g-PS3 membranes compared to PSO-g-PS1 membranes (p \ 0.001), and a statistically significant higher ALP value of MC3T3-E1 cells on PSO-g-PS2 membranes (p \ 0.05) suggested that proliferation and differentiation of preos-teoblastic on PSO-g-PS membranes were enhanced with regard to soybean oil content within the membranes. Thus, the present study suggests that PSO-g-PS2
R. S. Tıg˘lı Aydın (&)
Department of Biomedical Engineering, Bu¨lent Ecevit University, Zonguldak 67100, Turkey e-mail: rseda.tigli@gmail.com
B. Hazer (&) M. Acar
Department of Chemistry, Bu¨lent Ecevit University, 67100 Zonguldak, Turkey e-mail: bhazer2@yahoo.com; bkhazer@karaelmas.edu.tr
M. Acar
e-mail: merwe802@hotmail.com M. Gu¨mu¨s¸dereliog˘lu
Department of Chemical Engineering, Hacettepe University, 06530 Beytepe/Ankara, Turkey e-mail: menemse@hacettepe.edu.tr
membranes, which showed a favorable biological environment for the preosteob-lastic cells, can be well suited for bone tissue engineering applications.
Keywords Soybean oil Styrene Cell–biomaterial interaction Bone tissue engineering
Introduction
The reconstruction of bone defects, which is a complex process influenced by age, bone structure, vascularization, defect morphology, and adjacent soft tissue [1,2] can be revealed by bone tissue engineering approach. Guided bone regeneration, which is a sub area of tissue engineering, has proven to be a well-established therapy to repair alveolar bone defects affected by periodontal diseases [2,3]. In this therapy, a barrier membrane is placed over bone defects with bone grafting materials to prevent in-growth of fibroblasts and to provide a space for osteogenesis within the underlying blood clot, which encourages bone growth [4,5].
Ideal properties for a barrier membrane should include biocompatibility, space-making effect, osteoinduction, time of absorption compatible with bone regener-ation, and clinical manageability [6–8]. Among diverse biomaterials, currently developed barrier membranes are classified as non-degradable (ePTFE, Gore-TexÒ) [6, 9] and degradable membranes (GuidorÒ, Bio-GideÒ) [6, 10]. Several studies reported that non-degradable membranes have better space-maintaining properties, but a second surgical procedure is required to remove the membranes after new bone generation [11], and another disadvantage is the early removal of membranes with regard to risk of infection [2, 12]. Although degradable membranes are reported as materials which can alter disadvantages of non-degradable membranes [13, 14], there are still challenges with respect to inflammatory reactions in the adjacent tissue, rapid degradation of membranes leading barrier of tissue invasion, and mechanical stability of membrane to sustain surgical treatment [5].
Plant oils are the suitable starting materials for polymers because of their abundance, the rich chemistry that their triglyceride structure provides, and their potential biodegradability [15–25]. Soybean oil, which is one of the polyunsaturated plant oils (MW approx. 874), contains linoleic acid (51 %), oleic acid (25 %), palmitic acid (11 %), linolenic acid (9 %), and stearic acid (4 %) residues [16]. Because of the poly unsaturated inclusion, soya oil is a drying oil and can easily be autoxidized to produce polymeric soya oil peroxides, which behave as a macroperoxy initiator for free radical polymerization of vinyl monomers [26]. Previously, several studies have been reported regarding the synthesis of graft copolymers based on polymeric soya oil peroxide [27–29]. C¸ akmakli et al. [27] reported the auto-oxidation of the unsaturated edible aliphatic oil (soya oil) to prepare polymeric oil peroxy initiators, and they successfully synthesized polymeric soybean oil-graft-methyl methacrylate (PSO-g-PMMA) by peroxidation, epoxida-tion and/or perepoxidaepoxida-tion of soybean oil, and subsequent graft copolymerizaepoxida-tion with MMA. Then, a biodegradability study was performed on PSO-g-PMMA copolymer, which has a non-biodegradable polymer grafted onto a biodegradable
polymer backbone, and results showed that the copolymer degraded by specific chain end scission [28]. Furthermore, Allı and Hazer [29] demonstrated the polymerization of auto-oxidized polymeric soybean oil with N-isopropyl acrylam-ide. Synthesis of these materials has prompted research into the production of soybean oil biomaterials for tissue regeneration since the effect of soybean oil on fibroblast and macrophage cell adhesion and spreading on polymeric membranes was shown [27]. Furthermore, Hazer et al. [30,31] reported in vivo biocompatibility of the autoxidized and unoxidized unsaturated medium–long chain length (m–lcl) co-poly-3-hydroxyalkanoates (m-lclPHAs) derived from soya oily acids by demonstrating no symptoms such as necrosis, abscess, or tumorigenesis in the vicinity of the implants. Moreover, Liu et al. [32] prepared elastomers with epoxidized soybean oil and epoxidized linseed oil and reported that the elastomers were all biocompatible and completely biodegradable after 3 months in vivo without any granulation or formation of scar tissues. Very recently, Wang et al. [33] reported a new series of soybean-based elastomers poly(epoxidized soybean oil-co-decamethylene diamine), and Miao et al. [34] reported a new class of biocompatible polyurethanes prepared from soybean-oil-based polyol for suitable use in biomedical applications.
In the present study, a novel soybean-oil-based biomaterial, polymeric soybean oil-g-polystyrene (PSO-g-PS) membrane was prepared, characterized, and the cellular activities on the membranes were evaluated with regard to a potential membrane candidate under the scope of guided bone regeneration.
Materials and methods Materials
Soybean oil was locally purchased (C¸ otanak, Turkey) and used as received. Styrene was obtained from Sigma-Aldrich (Germany) and it was purified by vacuum distillation over CaH2. All the other chemicals (Sigma-Aldrich, Germany) were
analytical grade and used without further purification.
Auto-oxidation of soybean oil and the peroxygen analysis
The polymeric soybean oil (PSO) was prepared due the auto-oxidation according to the procedure reported in the literature [27]. In brief, soybean oil (50 g) spread out in a Petri dish (Ø = 16 cm) was exposed to sunlight in the air at room temperature (8 weeks). Then, a gel polymer film associated with a waxy and viscous liquid was formed. Chloroform extraction of the crude polymeric oil for 24 h at room temperature allowed separation of the soluble part of the polymeric soybean oil from the gel. Peroxygen analysis of soluble part fractions was carried out by refluxing a mixture of 2-propanol (50 mL)/acetic acid (10 mL)/saturated aqueous solution of potassium iodide (1 mL) and 0.1 g of the polymeric sample for 10 min and titrating the released iodine against thiosulfate solution.
Graft copolymerization
Graft copolymers of PSO-g-PS were synthesized using PSO peroxides to initiate styrene monomer during free radical polymerization. In brief, for given amounts of PSO and styrene were charged separately into a Pyrex tube. Argon was introduced through a needle into the tube for about 3 min to expel the air. The tightly capped tube was then put into a water bath at 80°C for 1 day. Then, the contents of the tube were coagulated in methanol. The graft copolymer samples were dried overnight under vacuum at 30°C. Purification of copolymers was assessed using a typical fractional precipitation procedure [35]. Purified copolymers were dried under vacuum at room temperature. PSO-g-PS membranes were prepared by solvent-casting method. For this purpose, 0.5 g of PSO-g-PS was dissolved in 10 mL chloroform and the solution was poured into a glass Petri dish. The solvent was allowed to evaporate overnight at room temperature.
Polymer characterization
Nuclear magnetic resonance (1H NMR) spectrum of PSO and PSO-g-PS was recorded in CDCl3 at 17 °C with a tetramethylsilane internal standard using a
400 MHz/54 mm Ultra Shield Plus NMR (Burker, Ultra long hold time). FTIR analysis of PSO and PSO-g-PS polymers were performed using ATR-FTIR spectrometer (Perkin Elmer Pyris 1) in the range of 400–4,000 cm-1. The molecular weight of the polymeric samples was determined by gel permeation chromatog-raphy (GPC) with a Waters model 6000A solvent delivery system with a model 401 refractive index detector and a mode 730 data module and with two Ultrastyragel linear columns in series. Chloroform was used in the elution at a flow rate of 1.0 mL min-1. A calibration curve was generated with polystyrene standards. In order to evaluate thermal characteristics of PSO-g-PS copolymers, a differential scanning calorimeter (TA Q2000, DSC) was used. The glass transition temperatures (Tg1and Tg3) were evaluated between -100°C and ?140 °C at a heating rate of
10°C/min (first heating) under nitrogen atmosphere, and samples were held at the final temperature for 1 min to eliminate the thermal history applied to the samples. After being cooled to -100°C, they were then reheated to 140 °C at a rate of 10°C/min (second heating). Thermal gravimetric analysis (TGA) of the copolymers was assessed by (TA Q50, TGA) instrument to determine thermal degradation between 0 and 700°C. Biodegradation tests of copolymers were carried out in lipase solution (0.1 mg/mL) in PBS (pH 7.4) containing 0.02 % sodium azide (NaN3). Incubations were performed in a humidified incubator at 37 °C for 14 days.
Dry weights of dehydrated samples were measured at first and second weeks of incubation. Degradation was determined as the percentage of weight loss (WL)
according to Eq. (1).
WLð Þ ¼% ðW0 WÞ=W0
100 ð1Þ
where, W0is initial weight, and W is weight of matrix after degradation.
The wettability of the copolymer samples was assessed by water contact angles measured by sessile drop method at room temperature (Kru¨ss DSA 100, Germany).
The tensile strength, strain at failure, and elastic modulus of PSO-g-PS membranes were measured for each of the five sheets with a gauge length of 20 mm using a universal testing machine (Zwick Roell, USA) at crosshead speed of 100 mm/min. Cell culture studies
Cell culture studies were carried out with MC3T3-E1, mouse osteogenic cell line (Riken Cell Bank, Ibaraki, Japan). The cells were subcultured in flasks using minimum essential medium (aMEM) (Sigma Co.) supplemented with 10 % (v/v) fetal bovine serum (FBS, Sigma Co.) and 1 % penicillin–streptomycin (Biological Industries, Ashrat, Israel). The cells, maintained at 37°C in a humidified CO2(5 %)
atmosphere (Heraus Instruments, Heraus, Germany), were dissociated with 0.25 % trypsin–ethylenediaminetetraacetic acid (trypsin–EDTA) (Sigma), centrifuged, and resuspended in medium prior to cell seeding. All the cell culture studies were carried out in Laminar Flow Cabinet (Bioair, Type II Laminar Flow Cabinet, Italy). Sterilization and cell seeding
Cell culture studies were conducted in sterile 24-well tissue culture polystyrene (TCPS) dishes in stationary conditions. Prior to cell culture experiments, 24-well TCPS were precoated with 70 % ethanol soaked Parafilm for wrapping the plates, and placed under UV light during 30 min for sterilization. Prepared membranes, which have 15 mm diameter and 100 lm thickness, were sterilized with 70 % ethanol for 1 h, equilibrated in sterile Dulbecco’s PBS (pH 7.4) and then, were immersed in conditioning medium (DMEM-F12 supplemented with 10 % (v/v) FBS) for 2 h prior to cell seeding. Then, 1 mL of cell suspension in osteogenic medium (aMEM ? 10 % FBS containing 50 lg/mL ascorbic acid and 10 mM b-glycerol phosphate) was added to each well to maintain 2 9 104cells mL-1 inoculation density for each membrane. The culture medium was replenished every 2 days.
Cytocompatibility
The cytotoxicity evaluation was performed by direct contact assay. In brief, 1 mL of MC3T3-E1 cell suspension at a density of 2 9 104cells mL-1 was seeded in 24-well plate. Then, sterilized polymer membranes were immersed in culture medium and exposed to MC3T3-E1 cells for 1, 2 and 4 days. As positive control, wells were seeded with MC3T3-E1 cells at the same density in the absence of polymer membranes, and empty wells (without cells) were used as negative controls. Cells on wells were quantitatively assessed with 3-[4, 5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) formazan (Sigma, Germany) at different culture periods (MTT assay) at selected time intervals (1st, 2nd, and 4th day). In brief, culture medium was aspirated and washed with 600 lL prewarmed PBS (pH 7.4). Six hundred microlitre prewarmed culture medium supplemented with 60 lL MTT solution (2.5 mg mL-1 MTT dissolved in PBS) was added to each sample, which were incubated at 37°C for 3 h. Then, the medium was removed from each
well and 400 lL of 0.04 M HCl in isopropanol solution was added to each well to dissolve formazan crystals. The resulting solution was taken out and centrifuged at 13,000 rpm for 2 min. The supernatant was used for measuring optical density spectrophotometrically at 570 nm with reference to 690 nm using a microplate reader (Asys UVM 340, Austria). The data obtained from negative controls were subtracted from measured values. The number of viable cells was correlated to optical density values, and cell viability was then evaluated by normalizing the values to those from positive control wells.
Cell attachment and proliferation
MC3T3-E1 cells were seeded onto polymer membranes and cultured as described above for 2 and 4 h to determine the cell attachment on polymer membranes by crystal violet assay. In brief, the medium was aspirated and cells were washed twice in Dulbecco’s PBS (DPBS). Then, the cells on membranes were fixed with 250 lL of glutaraldehyde (2.5 % v/v) at 4°C for 20 min and stained with crystal violet (0.5 w/v in 20 % methanol) for 30 min. After washing gently with water, the crystal violet was solubilized with 250 lL of 0.5 % sodium dodecyl sulfate (SDS), and the absorbance was measured at 570 nm using a Labomed Double Beam UV–visible spectrophotometer. As a positive control, wells were seeded with MC3T3-E1 cells at the same density in the absence of polymer membranes, and empty polymer membranes (without cells) were used as negative controls. The data obtained from negative controls were subtracted from measured values. The number of attached cells was correlated to optical density values, and cell attachment was then evaluated by determining the ratio of number of cells attached to membranes to the initial cell seeding numbers. MC3T3-E1 cell proliferation on polymer membranes was assessed by MTT assay as described above. Cells seeded on 24-well TCPS were used as control.
Microscopic imaging
After crystal violet staining, cells on polymer membranes were visualized by optical microscope (Olympus, Japan). Cytoskeleton organization on polymer membranes was assessed at the end of 4 h, 1, 2, and 4 days of incubation period. Cells were rinsed twice with PBS, fixed in 2.5 % (v/v) glutaraldehyde in 0.1 M PBS (pH 7.4) for 10 min at 4°C and permeabilized in 0.1 % Triton-X 100 for 5 min. Cell cytoskeletal filamentous actin (F-actin) was visualized by treating the cells with Alexa Fluor 488 phalloidin (Invitrogen, USA) for 20 min, and cell nuclei were counterstained with propidium iodide for 5 min. Samples were visualized using fluorescence microscope (Olympus, Japan).
Alkaline phosphatase (ALP) assay
To evaluate the osteogenic differentiation capacity of the MC3T3-E1 cells, alkaline phosphatase activity (ALP) measurements [36,37] were done at the first week of incubation period. In brief, the membranes were washed thrice with PBS (pH 7.4)
and freeze-dried at -80°C (Christ, Germany). Then, membranes were cut with scissors and homogenized by sonicating in the presence of 1 % Triton-X 100. The sample lysate was centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was assayed for ALP activity, using p-nitrophenyl phosphate (pNPP) as substrate at 37°C for 30 min. After stopping reaction with 0.02 N NaOH, hydrolysis of substrate to p-nitro phenol was measured spectrophotometrically at 405 nm using microplate reader (Asys UVM 340, Australia).
Von Kossa analysis
The mineralization of cells on membranes was determined by the von Kossa analysis as described previously [36,38]. In brief, membranes at the second week of culture were removed, washed once with PBS, and then, soaked in ice cold absolute ethanol for 20 min. After washing with deionized water, 5 % AgNO3(0.5 g/mL)
was added onto cells and incubated for 30 min in dark environment. Then, membranes were washed with deionized water and exposed to UV for 2 min. Statistical analysis
All data are expressed as means ± standard deviations. Three similar experiments were done which were carried out in triplicate. Statistical analysis was performed by one-way analysis of variance (ANOVA) in conjunction with Tukey’s post hoc test for multiple comparisons using Graph-Pad Instant (GraphPad Software) statistics program with significance at p \ 0.05. p \ 0.05, p \ 0.01, and p \ 0.001 represent statistically significant, very significant, and extremely significant values, respec-tively, whereas p [ 0.05 represents statistically no significant values.
Results and discussion Auto-oxidation of soybean oil
Polymeric soybean oil containing peroxide/hydroperoxide groups (1.3 wt%) was obtained in the air at room temperature, since polymeric soybean oil peroxide was obtained from auto-oxidized soybean oil, which involves hydrogen abstraction from a methylene group between two double bonds in a polyunsaturated fatty acid chain. Soya oil polymerization reactions via auto-oxidation have been given elsewhere [27]. Molecular weight (Mw) and polydispersity of auto-oxidized PSO were determined from GPC as 9,428 g/g-mole and 2.38, respectively, which are also reported before [27]. Figure1a, b represents FTIR spectra of soybean oil and PSO after auto-oxidation, respectively. As seen from the figure, the characteristic peaks observed at 805 cm-1 (epoxide groups) and 3,300 cm-1 (hydroperoxide groups) clearly indicate auto-oxidation of soybean oil. Figure1c demonstrates 1H NMR spectrum of PSO. The characteristic peaks of the auto-oxidized polymeric soybean oil were marked on the spectrum which confirms the PSO segments in the copolymer structure. Characteristic peaks as indicated in Fig.1c (d, ppm): –CH– of
soybean oil at 2.8, 2.4, 1.9, 1.4, and 0.9 ppm and the peaks at 4.1–4.4 ppm originate from the protons in the methylene groups of the triglyceride. The vinylic protons are detected at 5.3 ppm.
Graft copolymerization and characterization
The peroxide groups in auto-oxidized soybean oil let PSO to be used as an initiator of styrene during copolymerization. Then, cross-linked and soluble graft copolymer fractions were isolated by means of chloroform extraction. Soluble fractions of the graft copolymers were fractionally precipitated to determine the yield of the graft copolymers. Table1 represents copolymerization conditions and copolymer Fig. 1 FTIR spectra of a soybean oil, b auto-oxidized polymeric soybean oil (PSO). c1H NMR spectrum
of auto-oxidized polymeric soybean oil (PSO). (The characteristic peaks of the auto-oxidized polymeric soybean oil have been marked on the1H NMR spectrum.)
analysis results as well as molecular weights and polydispersity of the copolymers. Although higher initial amounts of PSO in monomer solution yields higher amount of graft copolymer, optimum polymerization yield was achieved for PSO-g-PS2 copolymer. Figure2a represents FTIR spectra of g-PS1, g-PS2, and PSO-g-PS3 copolymers. The peaks at 1,743 and 1,600 cm-1showed the presence of PSO and polystyrene, respectively. By comparison of the characteristic bands of PSO at 1,743 cm-1 and styrene at 1,600 cm-1 (Fig.2a), PSO amounts (wt%) in the copolymers were calculated using a calibration curve due to the methodology reported before [39], and the results are reported in Table1.1H NMR spectra of copolymer samples are demonstrated in Fig.2b (PSO-g-PS1), Fig.2c (PSO-g-PS2), and Fig.2d (PSO-g-PS3). All samples of PSO-g-PS copolymers contained characteristic peaks as indicated in Fig.2b–d (d, ppm): the aromatic protons of styrene at 6.0–7.6 ppm and the broad signals from 1 to 3 ppm are assignable to CH and CH2protons of the main chain. The characteristic peaks for all copolymers at
2.8, 2.4, 1.9, 1.4, and 0.9 ppm indicate –CH2– of soybean oil in which PSO
inclusion (mol%) was found to be 27, 69 and 51 % (Table1) by taking the ratio of signals at 6.5 ppm (styrene) and 0.9 ppm (PSO) for PSO-g-PS1 (Fig.2b), PSO-g-PS2 (Fig.2c), and PSO-g-PS3 (Fig.2d), respectively.
Thermal properties of the synthesized PSO-g-PS copolymers were determined by DSC and TGA analysis. Glass transition temperatures (Tg) and decomposition
temperatures (Td) of copolymers are listed in Table2. As seen from this table, Tg
values of copolymers were decreased due to the increase of soybean oil content in the copolymer, which may be due to the plasticization effect of PSO in the copolymers that has been observed by lowering the glass transition to 68–84°C compared with 100°C for homo-PS [25]. Thermal decomposition temperatures were shifted to higher values with lower PSO-g-PS decomposition percentages with regard to increase in PSO content within the copolymer (Table2).
Biodegradability of the PSO-g-PS membranes were quantitatively tested, and results indicated that PSO-g-PS2 and PSO-g-PS3 membranes showed gravimetri-cally 6 and 4 % weight loss, respectively; while PSO-g-PS1 and homo-PS membranes had no remarkable weight loss at the end of 14 days of incubation period (Table3). The degree of degradation of the copolymers was related with the Table 1 Results and conditions for the polymerization of styrene initiated by PSO
Copolymer PSO-g-PS PSO (g) Styrene (g) Polymer total (g) Yield soluble wt%
Molecular weight PSO in copolymer Mw 9 104 MWD wta% molb%
PSO-g-PS1 0.2 5.0 3.4 65.4 63.91 3.73 23 27
PSO-g-PS2 2.0 10.0 11.0 91.6 43.08 3.83 68 69
PSO-g-PS3 7.0 30.0 32.0 86.5 32.69 3.82 62 51
a
Calculated from their FTIR spectra (by comparison with peaks at 1,743 cm-1(PSO) and 1,600 cm-1 (styrene), Fig.2)
b Calculated from their NMR spectra (by comparison with peaks at 6.5 ppm (styrene) and 0.9 ppm
Fig. 2 aFTIR spectra of PSO-g-PS1, PSO-g-PS2, and PSO-g-PS3 copolymers,1H NMR spectra of bPSO-g-PS1, c PSO-g-PS2, and d PSO-g-PS3 copolymers
Table 2 Thermal properties of the PSO-g-PS graft copolymers
Copolymer DSC TGA Tg1(°C) Tg3(°C) Td(°C) PSO-g-PS (wt%) a PSO-g-PS1 -35 84 280 5.9 PSO-g-PS2 -32 68 296 4.9 PSO-g-PS3 -32 75 290 5.4 a Decomposed PSO-g-PS (wt%) at Td
Table 3 Degradation, wettability, and mechanical properties of the graft copolymers Copolymers Degradation (%) Water contact
angle (degree) Tensile strength (MPa) Strain at failure (%) Elastic modulus (MPa) 7th day 14th day PSO-g-PS1 0 0 37.1 ± 5.2 30.7 ± 1.1 1.29 ± 0.08 2033.3 ± 124.7 PSO-g-PS2 2.9 5.9 93.0 ± 1.4 17.9 ± 0.8 1.70 ± 0.17 370.5 ± 82.1 PSO-g-PS3 1.3 4.0 81.6 ± 2.0 20.7 ± 0.7 1.65 ± 0.20 812.5 ± 88.4
PSO amount in the copolymer, and results clearly concluded that the copolymer PSO-g-PS can undergo enzymatic degradation due to its degradable PSO fragments since PS is not biodegradable. Previously, it has been reported that PSO-g-PMMA copolymer degraded enzymatically through PSO fragments by specific chain end scission, [28] in which our results were in a good correlation. Table3shows water contact angle values of copolymers to evaluate wettability of membranes. According to the results, contact angles of PSO-g-PS membranes were dramatically increased due to the increment of PSO content within the copolymer, which clearly explored that PSO significantly increases hydrophobicity of membranes. The mechanical properties of PSO-g-PS membranes including their tensile strength, elastic modulus, and strain at failure are summarized in Table3. The results showed that, when PSO content was increased within the copolymer, the tensile strength and elastic modulus of the membranes decreased. However, an increase in strain failure due to the increment of PSO content within membranes suggested the membranes with better flexibility with PSO content increment, thus facilitating their manip-ulation in surgical applications.
Cell culture studies
Cell viability, cellular adhesion, and proliferation
Mouse MC3T3-E1 cells were used to evaluate the potentials of PSO-g-PS membranes as a potential candidate of biomaterials for guided bone regeneration. Cell responses to PSO-g-PS membranes were determined with respect to presence of soybean oil within the membranes. No cytotoxicity was shown when MC3T3 cells were cultured in the presence of PSO-g-PS membranes for 1, 2, and 4 days (Fig.3). As seen in Fig.3, the fractions of viable cells, normalized to the positive control (TCPS), were almost 100 % for all copolymers with no statistically significant differences (p [ 0.05) during 4 days of incubation period. Results
indicated that no leachable toxic molecules were released from the copolymers, leading the good cytocompatibility of membranes.
MC3T3-E1 cell adhesion on copolymer membranes were evaluated and monitored after 2 and 4 h of incubation period (Fig.4). As shown in Fig.4e, j, the presence of soybean oil first decreased the cellular adhesion (Fig.4e) which may be due to the hydrophobic character of soybean oil (Table3). It has been reported that hydrophobic factors can reduce cell adhesion [40], thus the same effect was initially observed. Then, cellular adhesion was sharply increased by the content of soybean oil (Fig.4f). At 4 h, statistically very significant amounts of cellular attachment on PSO-g-PS2 and PSO-g-PS3 membranes was obtained (p \ 0.01) compared to PSO-g-PS1 membranes, indicating supportive effect of soybean oil on cell attachment (Fig.4j). Moreover, the value of cellular attachment on PSO-g-PS2 membranes was almost reached to the value for the positive control (TCPS) with a
Fig. 4 Cellular attachment on PSO-g-PS copolymers. Microscopic images of crystal violet stained MC3T3-E1 cells on a PSO-g-PS1, b PSO-g-PS2, c PSO-g-PS3, and d TCPS after 2 h incubation (magnification 940). Fluorescence microscope images [red stained using propidium iodide, green stained using Alexa Fluor 488-phalloidin) of MC3T3-E1 cells on f PSO-g-PS1, g PSO-g-PS2, h PSO-g-PS3, and iTCPS after 4 h incubation (magnification 940)], the blurred images are caused by membrane opacity. Normalized MC3T3 cell attachment e at 2 h, j at 4 h on PSO-g-PS1, PSO-g-PS2, and PSO-g-PS3 membranes compared with cell-seeded TCPS as positive control. Statistical differences between groups (n = 3, TCPS is control group, ***p \ 0.001, *p \ 0.05; PSO-g-PS1 is control group,xxxp\ 0.001)
small statistical difference (p \ 0.05). Similar supportive results with regard to soybean oil on cell viability and cell attachment were reported which confirms our results [32]. Figure 4a–d, f–i demonstrates the morphological character of attached MC3T3-E1 cells on copolymeric membranes and TCPS. Unlike at 2 h (Fig.4a–d), after 4 h incubation, cells on all types of membranes started to get in touch with each other with a similar cell morphology compared to TCPS (Fig.4f–i).
Proliferation of MC3T3-E1 cells on PSO-g-PS copolymers were evaluated and assessed by MTT assay (Fig.5b). As demonstrated in Fig.5b, for the first 2 days, metabolic activities of MC3T3-E1 cells on PSO-g-PS1, PSO-g-PS2, and PSO-g-PS3 membranes were lower compared to TCPS (p \ 0.001). Moreover, for the first 2 days, there is no statistically significant difference among the metabolic activities of MC3T3-E1 cells on PSO-g-PS1, PSO-g-PS2, and PSO-g-PS3 membranes (p [ 0.05) and PSO-g-PS2 and PSO-g-PS3 membranes (p [ 0.05). TCPS is generally evaluated as a control and should be interpreted as demonstrating the maximum proliferation ability. However, at the end of 4 days, a sharp increase of optical density (OD) values was achieved especially for PSO-g-PS2 and PSO-g-PS3 membranes, while OD values of TCPS was decreased (Fig.5b) which is a real effect as cells become crowded and make a confluent layer through TCPS (after 4 days). As seen from Fig.5b, cell proliferation was enhanced on PSO-g-PS2 and PSO-g-PS3 membranes compared to PSO-g-PS1 membranes (p \ 0.001) and suggesting the superior effect of soybean oil on osteoblastic cell proliferation. Lukyanova et al. [41] reported the effect of soybean-oil-based scaffold on Chinese hamster ovary (CHO) fibroblast cell growth, and results suggested that soybean oil induces an environment which favors cell migration.
Figure5a represents MC3T3-E1 cells grown on PS1, PS2, PSO-g-PS3 membranes, and TCPS after 1, 2, and 4 days of culture period. As shown in Fig.5a, cellular morphologies of MC3T3-E1 cells on PSO-g-PS membranes were dramatically changed compared to TCPS, especially on days 1 and 2. Cells on PSO-g-PS membranes tend to get contact with other cells in a spindle-like morphology compared to cells grown on TCPS (Fig.5a). Moreover, there were visible thin actin stress fibers with prominent focal contacts which may lead to important consequences for cell processes such as migration and proliferation [42]. Cytoskeleton organization in the fully spread MC3T3-E1 cells on PSO-g-PS membranes showed enhanced cell migration and cell proliferation at the end of 4 days of incubation.
ALP activity and mineral deposition
Investigation and identification of cellular activities on a biomedical material provide useful data to develop new therapeutic applications [43, 44]. For bone regeneration, the activities of ALP and deposit minerals in vitro are dependent to the synthesis of ECM [36,37]. MC3T3-E1 cells are osteogenic cell line and have the capacity to differentiate into osteoblasts and deposit minerals in vitro [45]. ALP activity, which is the marker associated with ECM development, has been routinely used as an early marker of osteoblasts differentiation [42]. ALP activity of MC3T3-E1 cells and mineral deposition on PSO-g-PS membranes was characterized to
evaluate osteoblastic differentiation (Fig.6). As demonstrated in Fig.6a, ALP activities of MC3T3-E1 cells on PSO-g-PS2 membranes showed the greatest statistically different value (p \ 0.05) compared to all. Moreover, a statistically significant higher ALP value of MC3T3-E1 cells on PSO-g-PS2 membranes compared to PSO-g-PS3 membranes (p \ 0.05) were obtained, suggesting the effect of soybean oil on ALP activity (Fig.6a).
Fig. 5 aFluorescent microscope images (red stained using propidium iodide, blue stained using Alexa Fluor 488-phalloidin) of MC3T3-E1 cells on PSO-g-PS1, PSO-g-PS2, PSO-g-PS3 membranes, and TCPS at 1st, 2nd, and 4th days of incubation (magnification 940), the blurred images are caused by membrane opacity. b MTT assay of PSO-g-PS1, PSO-g-PS2, PSO-g-PS3 membranes, and TCPS. Statistical differences between groups (n = 3, ***p \ 0.001; TCPS is control group,xxxp\ 0.001; PSO-g-PS1 is
Von Kossa staining was used to visualize mineralized nodule formation on cell-seeded membranes. Different levels of staining (black-brown areas) were observed in each group (Fig.6b). As shown in Fig.6b, mineral deposition (black-brown areas) was maximized at PSO-g-PS2 membranes. Results concluded that soybean-oil-based membranes not only increased cell proliferation, but also enhanced differentiation of osteoblasts. Taking all these data into account, PSO-g-PS2 membranes showed a favorable biological environment which induces functions of the preosteoblastic cells. All these information may provide a scientific basis for the use of PSO-g-PS membranes as a barrier membranes used in bone and periodontal tissue engineering applications.
Conclusion
In this study, a novel barrier membrane based on soybean oil was investigated in terms of improving our understanding on the roles of different material character-istics in guided tissue regeneration. PSO-g-PS membranes were successfully prepared using auto-oxidized soybean oil as a macroperoxy initiator for free radical polymerization of styrene. Cellular behavior of MC3T3-E1 cells on membranes Fig. 6 aALP activity of MC3T3-E1 cells cultured on PSO-g-PS1, PSO-g-PS2, PSO-g-PS3 membranes, and TCPS after 1 week of incubation. Statistical differences between groups (n = 3, *p \ 0.05; TCPS is control group). b von Kossa stained PSO-g-PS1, PSO-g-PS2, and PSO-g-PS3 membranes after 2 weeks of incubation. Black-brown areas indicate mineral deposition on membranes
were evaluated in terms of cell attachment, proliferation, and differentiation. Results concluded that PSO-g-PS2 (the highest molar PSO inclusion in copolymers; 69 %) membranes were a good candidate of a supportive membrane due to their cytocompatibility and in vitro degradability for use in biomedical applications. However, further in vivo studies should be performed to highlight in vivo absorbable characteristics of the membranes which is a very challenging area concerning the development of guided bone regeneration membranes.
Acknowledgments The Authors thank to TU¨ BI˙TAK (Grant No. 211T016) and Bu¨lent Ecevit University (Grant# 2011-10-3-02) for financial support.
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