DOI 10.1007/s11746-017-2954-6
ORIGINAL PAPER
Influence of Soybean Oil Blending with Polylactic Acid (PLA)
Films: In Vitro and In Vivo Evaluation
R. Seda Tıg˘lı Aydın1 · Elvan Akyol2 · Baki Hazer2
Received: 17 June 2016 / Revised: 6 January 2017 / Accepted: 6 January 2017 / Published online: 28 January 2017 © AOCS 2017
Introduction
In view of limited petroleum reserves in the future, poly-mers prepared from renewable resources have attracted great attention in recent years [1, 2]. The increasing atten-tion from the polymer community has encouraged scien-tists to work on vegetable oil-based polymers which play important roles in biomedical applications [3, 4]. Some critical properties of vegetable oil-based biomaterials (i.e. universal availability and low price, as well as superior compatibility properties) have increased their popular-ity as required biomaterials in biological applications [5,
6]. Soybean oil, which contains linoleic acid (51%), oleic acid (25%), palmitic acid (11%), linolenic acid (9%), and stearic acid (4%) residues [7], has been used with several biomaterials for a wide range of potential biomedical appli-cations [7–10]. Within these studies, several forms of soy-bean oil (i.e., conjugated soysoy-bean oil, epoxidized soysoy-bean oil, hydrogenated form polyols, auto-oxidized soybean oil) have been used for the production of polymeric oil-based biomaterials. Aydın et al. reported polymeric soybean oil-g-polystyrene (PSO-g-PS) as a potential candidate of guided bone regeneration films [7]. Miao et al. reported a new class of biocompatible polyurethanes prepared from soybean oil-based polyol, obtained from a mild, green, and simple synthetic route for suitable use in biomedical applications [8]. Hazer et al. demonstrated in vivo biocompatibility of the autoxidized and unoxidized unsaturated medium–long chain length (m–lcl) co-poly-3-hydroxyalkanoates (m-lcl-PHAs) derived from soybean oil acids [9]. Liu et al. dem-onstrated biocompatibility properties of elastomers with epoxidized soybean oil and epoxidized linseed oil [10]. Thus, synthesis of these materials has prompted scientists in the production of novel polymeric oil-based biomaterials for suitable biomedical applications.
Abstract Due to the great interest in oil-based polymers,
which are prepared from renewable resources, different forms and amounts of soybean oil-based PLA films were prepared and evaluated for their potential usage as a medi-cal biomaterial. Soybean oil, epoxidized soybean oil and auto-oxidized soybean oil were blended with PLA and PLA/oil films with appropriate oil amounts [2, 7, 14 and 20% (w/w)] were obtained by solvent casting. Thermal sta-bility and plasticization effect were determined by adjust-ing oil amounts and type. Epoxidized soybean oil blended films showed the smallest increase in elongation breaks (13–20%) and the highest decrease in thermal decompo-sition temperatures (364–327 °C) compared to other oil blended films. In vitro quantitative and qualitative cyto-toxicity results showed no reactivity (grade 0) for the L929 cells treated with 14% (w/w) oil blended PLA films. In vivo irritation and implantation tests concluded that 14% (w/w) oil blended PLA films were non-irritant. No erythema, no oedema reactions, no traumatic necrosis and foreign debris were observed. Thus, along with superior biocompatibility, PLA/oil films can replace petroleum-based products for several biomedical uses.
Keywords Soybean oil · Epoxidized soybean oil · PLA ·
Biocompatibility
* R. Seda Tığlı Aydın
rseda.tigli@gmail.com; seda.aydin@beun.edu.tr
1 Department of Biomedical Engineering, Bülent Ecevit
University, I˙ncivez, 67100 Zonguldak, Turkey
2 Department of Chemistry, Bülent Ecevit University, I˙ncivez,
Of all the potential products made from renewable resources, polylactic acid (PLA) is used worldwide as a biomaterial and has long been used for sutures and porous scaffolds [11]. However, the brittle nature of PLA limits its applications in some biomedical areas [11–13]. Thus, plasticization of PLA has been accomplished by some studies to obtain PLA with high elongation for suitable applications [14–19]. Robertson et al. studied in detail the compatibilization effect of conjugated soy-bean oil on PLA [15]. Chieng et al. [16], Emad et al. [17] and Fathilah et al. [18] reported the mechanical and thermal properties of epoxidized soybean oil-based PLA. Hazer studied the autoxidized soybean oil polymer and hydroxylated soybean oil polymer by means of mechani-cal properties, fracture surface analysis and antibacterial properties [19]. The efficiency of gold nanoparticles on the autoxidized soybean oil polymer has been investi-gated [20]. Nevertheless, a systematic approach to con-trol and evaluate the surface, bulk and biocompatibility properties of soybean oil-based PLA, with regard to dif-ferent amounts and forms of soybean oil blending, has not yet been reported. In this study, appropriate amounts of auto-oxidized soybean oil (polymerized soybean oil), epoxidized soybean oil and soybean oil were blended with PLA in order to maintain PLA/oil films by the sol-vent-casting method. PLA/oil films were characterized in terms of surface morphology, thermal and mechanical properties. In vitro and in vivo biocompatibility of the prepared films was evaluated by related cytotoxicity stud-ies in order to indicate a promising biomaterial candidate for suitable biomedical applications.
Materials and Methods
Soybean oil (SBO) (MW: 3.2 Da) and epoxidized soy-bean oil (ESO) (MW: 3.0 Da) were locally purchased from Çotanak (Turkey) and the anti-oxidant adduct inside the commercial soybean oil was removed by leaching. Lactic acid monomer (d,l-Lactide) was obtained from
Sigma-Aldrich (Germany), and all the other chemicals (Sigma-Aldrich) were analytical grade and used without further purification.
Ring opening polymerization of PLA
Lactic acid monomer (d,l-Lactide) is polymerized via ring
opening polymerization (ROP) according to the procedure set out below. Briefly, 2 g of d,l-Lactide and toluene was
charged into a flame-dried Schlenk flask and the catalyst, 0.03 g of tin(II) octoate is added. Tin(II) octoate was dis-tilled under reduced pressure (0.5 mmHg) at 175 °C before
use. Then, argon gas was introduced through a needle into the tube for 3 min to expel the air. Finally, the flask was placed in an oil bath preheated at 110 °C for 24 h, and the reaction was finalized by adding cold chloroform, and the catalyst was filtered out by precipitating using chloroform. The crude polymer was dissolved in dichloromethane and poured into excess methanol to precipitate the polymer. Then, the precipitated polymer was dried under a vacuum at 50 °C for 24 h.
Auto‑oxidation of Polymerized Soybean Oil (PSO)
The polymerized soybean oil (PSO) was prepared due by auto-oxidation according to the previously reported pro-cedure in the literature [21]. 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. After 8 weeks, a gel polymer film associated with a waxy and viscous liq-uid was formed. Chloroform extraction of the crude poly-meric oil for 24 h at room temperature allowed the sepa-ration of the soluble part of the polymerized soybean oil from the gel. The scheme of the auto-oxidation process and the chemical structures of polymerized soybean oil (PSO), soy bean oil (SBO) and epoxidized soybean oil (ESO) are shown in Fig. 1.
Preparation of PLA/Oil (PLA/PSO, PLA/SBO, PLA/ESO) Films
PLA/oil polymeric blends were prepared by stirring appro-priate portions (w/w) of PLA and oil (PSO, SBO and ESO) overnight at room temperature. PLA/oil blends were prepared in chloroform in order to achieve 2, 7, 14 and 20% (w/w) oil in blends. Blends were assigned as PLA/ oil2, PLA/oil7, PLA/oil14 and PLA/oil20 which repre-sent the oil composition in the blends. Then, the solvent-casting technique was used in order to maintain PLA/ oil films with approximately 0.2 mm thickness. Briefly, blend solutions (10 mL) were poured into a Petri dish (Ø = 16 cm), then a transparent, smooth polymer film was obtained in 1–2 days of solvent evaporation. The film was subsequently dried under vacuum for a week at room temperature.
Characterization of PLA/Oil Films Gel Permeation Chromatography (GPC)
The molecular weights of prepared films were determined by gel permeation chromatography (GPC), Viscotek
GPCmax Auto sampler system (G2000H HR, G3000H HR and G4000H HR), and a Viscotek differential refractive index detector (THF flow rate of 1.0 mL/min). A single-pore GPC/SEC column (300 × 8 mm) (Product number CLM3004, Viscotek) was used. A calibration curve was generated with 8 PS standards having narrow molecular weight distribution (1.79 × 106, 9.25 × 105, 1.64 × 105,
1.2 × 105, 6.37 × 104, 2.91 × 104, 5.87 × 103, and 955 g/
mol of low polydispersity). Data were analyzed using Vis-cotek OmniSEC Omni–01 software.
FTIR‑ATR Analysis
Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra of PLA/oil films were recorded by using a Bruker IFS 66/s FTIR spectrophotometer (Ger-many) in the range of 400–4000 cm−1 (20 spectra/s, 4 cm−1
resolution).
Scanning Electron Microscopy (SEM) Imaging
The morphology and surface topography of PLA/oil films were visualized by using a SEM (FEI Quanta 200 FEG, USA) after coating with a thin gold–palladium alloy layer under vacuum.
Thermal Analysis
In order to evaluate the thermal characteristics of the PLA/ oil films, a differential scanning calorimeter (DSC; TA Q2000) was used. The melting temperatures (Tm) were evaluated between −60 °C and +170 °C at a heating rate of 10 °C/min. Thermal gravimetric analysis (TGA) of the films was assessed by a TA Q50 instrument to determine thermal degradation and decomposition temperatures (Td50(°C)) between 0 and 700 °C.
Mechanical Tests
The tensile strength and elongation breaks of PLA/oil films were measured for each of the five sheets with a gauge length of 20 mm using a universal testing machine (Zwick Roell, USA) at a crosshead speed of 100 mm/min.
Water Contact Angle Values
The wettability of the prepared PLA and PLA/oil films were assessed by water contact angle values. Water contact angles were measured by the sessile drop method at room temperature (Kruss DSA 100, Germany).
In Vitro Biocompatibility
In vitro biocompatibility assays of the prepared films were performed following the ISO10993-5 “Biological evalua-tion of medical devices: Part 5: Tests for in vitro cytotoxic-ity” standards. Prior to the tests, the films were prepared according to the procedure of sample preparation and ref-erence materials (ISO 10993-12: Biological evaluation of medical devices: Part 12: sample preparation and reference materials). Samples were cut into pieces (10 mm diameter) and sterilized by immersing them in 70% ethanol overnight. Samples were washed by sterilized buffer saline solution (pH 7.4) and then direct contact and extraction tests were performed in order to determine cytotoxicity. Qualitative and quantitative evaluation were performed according to the procedure given in ISO10993-5. In vitro biocompatibil-ity studies for both methods (direct contact and extraction) were carried out with a L-929 mouse fibroblastic cell line (S¸ap Enstitüsü, Ankara, Turkey). The cells were subcul-tured in flasks using DMEM (Sigma) supplemented with 10% (v/v) fetal bovine serum (FBS; Sigma) and 1% peni-cillin–streptomycin (Biological Industries, Israel) at 37 °C
Fig. 1 Schematic of polymer-ized soybean oil reaction and chemical structures of soybean oil and epoxidized soybean oil
in a humidified CO2 (5%) incubator. Cell culture studies were conducted in sterile 24-well tissue culture polysty-rene dishes, and the prepared films, 10 mm diameter and 100 µm thickness, were sterilized with 70% ethanol for 1 h, then equilibrated in sterile Dulbecco’s PBS (pH 7.4).
Extraction Method
As is defined in the ISO10993-5 standard procedure, PLA/ oil extracts (120 cm2 cross-sectional area with less than
0.5 mm height) were prepared by incubating in DMEM (20 mL) supplemented with 10% FBS at 37 °C for 24 h. Then, extracts were used for cell culture media. Briefly, a L929 cell suspension at a density of 2 × 104 cells/mL was
seeded in a 24-well plate. After 24 h of incubation, the cul-ture medium was aspirated, refreshed with extracts and incubated for 24 h. Then, the cells were quantitatively (cell metabolic activity) and qualitatively (microscopically; gen-eral morphology, vacuolization, detachment, cell lysis and film integrity) evaluated.
Direct Contact Method
According to the ISO10993-5 standard procedure, a L929 cell suspension at a density of 2 × 104 cells/mL was seeded
in a 24-well plate. After 24 h, the sterilized PLA/oil films were immersed in culture medium and incubated for 24 h. Then, the cells were quantitatively (cell metabolic activity) and qualitatively (microscopically; general morphology, vacuolization, detachment, cell lysis and film integrity) evaluated.
Determination of Cytotoxicity
For both methods, cytotoxicity was determined quanti-tatively and qualiquanti-tatively according to the ISO10993-5 standard procedure. The cells in the wells were quan-titatively assessed with 3-[4, 5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) formazan (Sigma, Germany) (MTT assay). In brief, the culture medium was aspirated and washed with 600 µL prewarmed PBS (pH 7.4). Then, 600 µL prewarmed culture medium sup-plemented with 60 µL MTT solution (2.5 mg/mL 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 µL of 0.04 M HCl in isopropanol solution was added to each well to dissolve the formazan crystals. The resulting solution was taken out and centrifuged at 15,871g for 2 min. The supernatant was used for measuring the optical density spectrophoto-metrically at 570 nm with reference to 690 nm using a microplate reader (Asys UVM 340; Austria). As positive control, wells were seeded with L929 cells at the same density in the absence of films/extracts, and empty wells (without cells) were used as negative controls. The data obtained from the negative controls were subtracted from the measured values. The number of viable cells was cor-related to the optical density values, and cell viability was then evaluated by normalizing the values to those from the positive control wells. As stated in ISO10993-5, a reduction of cell viability by more than 30% is considered a cytotoxic effect. Thus, 70% cell viability is considered as the limit for cytotoxicity.
Table 1 Characterization of PLA/oil membranes: GPC results, thermal, mechanical and wettability properties
a DSC analysis results (melting temperature)
b TGA analysis results (decomposition temperature at 50% weight loss)
Membranes Mn (Da) Mw (Da) MWD Tm (°C)a T
d50 (°C)b Tensile strength (MPa) Elongation break (%) Water contact angles (°)
PLA 78,307 138,066 1.8 156.7 364.2 34.69 13.61 83.8 ± 5.6 PLA-PSO2 43,383 133,657 2.9 150.7 363.4 27.67 27.29 86.1 ± 4.5 PLA-PSO7 40,932 105,684 2.6 148.4 359.2 18.54 38.97 88.1 ± 2.4 PLA-PSO14 51,885 115,657 2.6 146.7 359.5 13.15 40.57 88.7 ± 2.1 PLA-PSO20 45,141 111,612 2.5 146.3 357.7 12.19 43.54 94.4 ± 6.0 PLA-SOYA2 67,442 136,457 2.0 150.9 362.4 24.41 31.29 81.9 ± 3.0 PLA-SOYA7 61,429 131,865 2.2 148.0 359.4 17.68 31.99 83.3 ± 8.1 PLA-SOYA14 63,269 128,302 2.1 146.5 359.1 16.48 35.00 82.8 ± 1.9 PLA-SOYA20 66,382 132,302 2.1 144.0 357.6 12.56 39.22 81.3 ± 0.9 PLA-ESO2 54,697 119,094 2.2 151.6 356.9 18.59 13.60 79.7 ± 6.7 PLA-ESO7 67,075 133,165 2.0 151.0 335.0 15.30 13.62 78.0 ± 4.7 PLA-ESO14 65,070 129,394 2.0 150.5 334.9 12.86 15.60 78.4 ± 9.3 PLA-ESO20 67,881 142,230 2.3 145.7 327.7 12.09 20.90 72.8 ± 3.2
Qualitative evaluation was performed by staining cells by DAPI, and cell morphologies were observed under a fluorescence microscope (Olympus, Japan). Briefly, cells were rinsed twice with PBS, fixed in 2.5% (v/v) glutaral-dehyde in 0.1 M PBS (pH 7.4) for 10 min at 4 °C and per-meabilized in 0.1% Triton-X 100 for 5 min. Cell cytoskel-etal filamentous actin (F-actin) was visualized by treating the cells with Alexa Fluor 488 phalloidin, and cell nuclei were counterstained with DAPI for 30 min. Samples were visualized using a fluorescence microscope. Cytotoxicity was determined by evaluation of the change of cells from normal morphology, which were graded according to the ISO10993-5 standard, in terms of general morphology, vacuolization, detachment, cell lysis and film integrity. A numerical grade greater than 2 (mild reactivity), is consid-ered a cytotoxic effect (ISO10993-5).
In Vivo Biocompatibility
In vivo biocompatibility assays of the selected films (PLA, PLA-PSO14, PLA-SBO14, PLA-ESO14) were performed following the ISO10993-10 “Biological evaluation of med-ical devices: Part 10: tests for irritation and delayed-type hypersensitivity-Annex B” and ISO10993-6 “Biological evaluation of medical devices: Part 6: Tests for local effects after implantation” standards. Additionally, ISO 10993-2:2006 “Animal Welfare requirements” and ISO10993-12:2012 “Sample preparation and reference materials” standards were taken into account. The animal experiments were approved by the TÜBI˙TAK Animal Experiments Ethic Committee (Number: 16563500-111-85).
Irritation–Intracutaneous (intradermal) Reactivity Test
Healthy young 3 adult albino rabbits (female), weighing not less than 2 kg, were used. The method of extracts appli-cation proceeded with a polar solvent of PBS and a non-polar solvent of corn oil. After 72 h incubation at 37 °C, extracts (0.2 mL) were intradermally injected into the skins of fur-clipped animals at five different points of the test sites (left and right cranial and caudial end). After the intra-cutaneous injections, the appearance of each injected site was graded according to observations of the tissue reac-tion for erythema and edema after 24, 48 and 72 h instil-lation. Test scores were calculated according to the related standard.
Implantation Test
The assessment of the local effects after implantation of the prepared films was evaluated histologically by
implanted into dorsal subcutaneous tissue of 3 Sprague– Dawley rabbits (3–4 months, female). Silicon was used as negative control material. After 4 weeks of incubation, histopathological evaluation was graded according to the cell type and tissue responses. Under the conditions of this study, the test samples were considered a non-irritant (0.0–2.9), slight irritant (3.0 up to 8.9), moderate irritant (9.0–15.0) and severe irritant (>15) to the tissue as com-pared to the negative control sample. Clinical observations and gross pathological observations were also evaluated.
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 ANOVA in conjunction with Tukey’s post hoc test for multiple comparisons using the Graph-Pad Instant (Graph-Pad Software) statistics program. p < 0.05, p < 0.01, and p < 0.001 represent statistically significant, very significant,
and extremely significant values, respectively, whereas p > 0.05 represents no statistical significance.
Results and Discussion
Preparation and Characterization of PLA/Oil Films
PLA was blended with natural polymers obtained from renewable resources [auto-oxidized soybean oil, soybean oil (commercial) and epoxidized soybean oil (commer-cial)]. Polymeric soybean oil peroxide was obtained from auto-oxidation of soybean oil, which involves hydrogen abstraction from a methylene group between two double bonds in a polyunsaturated fatty acid chain,as has been given elsewhere [molecular weight (Mw) 9428 g/g-mole and polydispersity: 2.38] [21]. Schematic representation of auto-oxidation of soybean oil and chemical structures of soybean oil and epoxidized soybean oil are shown in Fig. 1. Presumably, there could be interactions between
the hydroperoxides of the soybean oil polymer and ester groups of PLA in view of the hydrogen bonding. In addi-tion, peroxide and epoxide groups of soybean oil polymer can enhance the solubility into the polyester, PLA. After blending appropriate amounts of oil (2, 7, 14 and 20%) with the PLA, PLA/oil films were maintained by the sol-vent casting method. Table 1 represents the GPC results of the prepared films. Etheric oxygen and epoxy groups, which are unique characteristic structures of PSO and ESO, were monitored via FTIR spectroscopy. Figure 2 shows
the FTIR spectra of the PLA/Oil films. The main regions of PLA and PLA/oil, –CH stretching at 3000–2850 cm−1,
C=O stretching at 1750–1745 cm−1, C–H bending at
1500–1400 cm−1 and –C–O stretching at 1100–1000 cm−1,
completely overlap with each other. The –C–O stretch-ing peak at 1100 cm−1 was known to be due to the etheric
oxygen and epoxy groups of PSO and ESO, respectively [7]. Thus, a small upward shift of –C–O stretching peak at 1080 cm−1 (neat PLA) was observed for the PLA/oil
films due to the etheric oxygen and epoxy groups of the oil.
Fig. 4 In vitro viability graphics of L929 cells cultured via a the extraction method, b the direct contact method. ***Statistically extremely sig-nificant difference (p < 0.001), **statistically very sigsig-nificant difference (p < 0.01), *statistically sigsig-nificant difference (p < 0.05)
This shift in the absorption peak indicates the miscibility and the interaction of PLA and ESO/PSO [16]. However, as seen from Fig. 2, PLA/SBO blends did not show any change at the –C–O stretching peak (1080 cm−1)
show-ing the lack of etheric oxygen or peroxy groups in soybean oil. Although the shifting indicates oil inclusion (wt%), the PLA/Oil2 blends did not show any shifting which may be due to the low amount of oil (2%) in the blends. However, a very small shifting was observed for the PLA/Oil20 blends regarding the low miscibility and interaction of PLA with the ESO/PSO groups. According to the physical observa-tions, the miscibility was not accomplished above 20% (w/w) of the oil content.
The film morphology of the PLA/oil films was visual-ized by SEM (Fig. 3). Homogenous distribution of the spherical oil droplets was observed on the PSO-blended PLA films. The amounts and the diameter of v oil drop-lets increased with increasing PSO content. Remarkable sizes of the PSO droplets on the PLA/PSO20 films were
observed (Fig. 3). SEM images demonstrated homoge-neous distribution of embedded SBO on the PLA/SBO films without any spherical oil droplets. Oil droplets were uncertain (embedded) and the structure of surfaces were observed as fragmented and sharp-cored (Fig. 3). For ESO blended films, homogenously distributed oil droplets were observed on the surfaces of the PLA/ESO2 and PLA/ PLAESO7 films. However, uncertain ESO droplets (as on PLA/SBO) were visualized on the PLA/ESO14 and PLA/ PLAESO20 films (Fig. 3).
The addition of oils into PLA films has influenced the wettability of the obtained materials. Water contact angles indicated that the addition of ESO into the films has greatly reduced their hydrophobicity (Table 1). This may be due to the epoxide groups on the PLA/ESO films which led to the decreased water contact angles. Wettability was also dependent on the polarity of the polymer. High polarity results in increasing the wettability, i.e., decreasing the hydrophobicity of the surface layer of the polymer [22].
Fig. 5 Fluorescence images of L929 cells cultured via the extraction method (blue stained using DAPI, green stained using Alexa Fluor 488-phalloidin)
Previously, it was shown that epoxidized soybean oil-incor-porated PLA films reduced the contact angle value of the films [23]. As seen from Table 1, surprisingly, PSO addi-tion greatly increased the surface hydrophobicity which may be due to the PSO oil droplets on the PLA/PSO films, as observed in Fig. 3. Although, the peroxide groups on the PLA/PSO films can presumably be effective in the reduction of surface hydrophobicity, PSO droplets led to decreased wettability. Moreover, it was shown that the blending composition could be highly effective on surface wettability (Table 1). Although increasing the wettability of PLA has been suggested for the biocompatibility improve-ment of the strongly hydrophobic surface, it should be noted that the increase in the hydrophilicity will also favor the action of microorganisms on the materials [23] which lowers their antibacterial activity.
The thermal properties of the prepared PLA/Oil films were determined by DSC and TGA analysis. Melting
temperatures (Tm) and decomposition temperatures (Td) of copolymers are listed in Table 1, from which it can be seen that the increase in the oil content greatly decreases the melt-ing temperatures. These results not only indicate the exist-ence of oil in blends but also confirm that the Tm of PLA is greatly affected by the addition of oils, and also confirm the plasticizer effect of the oils which lowers the Tm. Moreover, no other different melting peak was observed from the ther-mograms (data not shown) which indicates no phase differ-ence in the films. Increasing temperature leads to the dissoci-ation of polymer chains starting from unstable groups, which is consistent with the value reported by other researchers [16,
18]. The TGA results indicate the decrease of decomposition temperatures at 50% weight loss (Td50) with increasing oil
content (Table 1). The results confirmed the existence of oil content in the films which also supports the DSC results. The significant decrease of Td50 in PLA/ESO films may be due to the low thermal stability of the ESO blended films.
Fig. 6 Fluorescence images of L929 cells cultured via the direct contact method (blue stained using DAPI, green stained using Alexa Fluor 488-phalloidin)
The results of stress–strain measurements are shown in Table 1 in order to evaluate the plasticization effect of oil in PLA/oil blends. As seen in Table 1, an increase in elongation breaks (%) and a decrease in tensile strength are exhibited due to the plasticization effect of the oils which is in good consistency with other reports [15–18]. The incre-ment of oil content greatly increases elongation breaks within the films, suggesting the films have better flexibility, thus facilitating their manipulation in surgical applications [7]. However, relatively small values of these increments were obtained for the PLA/ESO films compared to the other films which suggests a lower plasticizer effect.
In Vitro Biocompatibility
In vitro cytotoxicities of the prepared PLA/oil films were assessed by extraction and direct contact methods accord-ing to the ISO10993-5 standard protocol. General evalu-ations were carried out for both the methods in order to confirm the results. Cellular viabilities of L929 cells are demonstrated in Fig. 4. Since reduction of cell viability by more than 30% is considered a cytotoxic effect, biocom-patibility evaluations were performed regarding this crite-ria. Results obtained from the extraction method indicated that PLA/ESO films significantly decrease (p < 0.001) cell viability compared to the control group without showing any cytotoxic effect (Fig. 4a). Moreover, there are no sta-tistically significant differences between ESO films with different oil contents (p > 0.05) (Fig. 4a). However, accord-ing to the direct contact method values, there are no statis-tically significant values between the PLA/ESO films and the control group (p > 0.05) (Fig. 4b). Thus, the PLA/ESO films did not show any cytotoxic effect and the highest cel-lular viability value is obtained from PLA/ESO14 films (p < 0.05, SD) (Fig. 4b). Quantitative evaluations accord-ing to the extraction method showed a cytotoxic effect of PLA/PSO2 and PLA/PSO7 films (Fig. 4a). However, the results obtained from the direct contact method showed no toxicity for PLA/PSO2 and PLA/PSO7 films with consid-erable decreases of cellular viability (p < 0.001) (Fig. 4b). The highest cellular viability values are obtained from PLA/PSO14 films for both methods (p < 0.05, SD). Simi-lar results were obtained from PLA/SBO14 films (p < 0.05, SD) (Fig. 4). Additionally, the results obtained from the direct contact method showed no toxicity for PLA/SBO films. Consequently, according to both tests (extraction and direct contact), the PLA/Oil14 films showed considerably higher values of cellular viability (p < 0.05, SD) which suggests remarkable biocompatibility compared to other PLA/Oil (Oil2, Oil7 and Oil20) films. No significant values were determined between ESO14, PSO14 and SBO14 for the direct contact (p = 0.4140) and for extraction methods (p = 0.1090).
Qualitative examinations were performed and evalu-ations were carried out according to the ISO10993-5 standard. Figures 5 and 6 show fluorescence microscope images of L929 cells taken after treatment of the extrac-tion and direct contact methods, respectively. Table 2 set out in vitro qualitative morphological grading and reactiv-ity for the extraction and direct contact tests according to ISO10993-5. According to the standard, grading above 2 is considered as a cytotoxic effect. As seen from Figs. 5 and
6, no discrete intracytoplasmatic granules, no cell lysis and no reduction of cell growth were observed for cells treated with PLA films and cytotoxicity graded as 0 (Table 2). Cells treated with PLA/ESO films showed changes in mor-phology (approximately 10% of the cells are round), and some malformed or degenerated cells were observed under the specimens (Figs. 5, 6). Thus, slight reactivity (grade 1) was determined for PLA/ESO films. However, accord-ing to the direct contact test, PLA/ESO14 was scored as 0, indicating no reactivity around or under the films. Accord-ing to Figs. 5 and 6, not more than 50% growth inhibition was observable with loosely attached lysed cells and the
Table 2 In vitro biocompatibility of films
Qualitative morphological grading and reactivity for extraction and direct contact tests according to ISO10993-5
a None (0): discrete intracytoplasmatic granules, no cell lysis, no
reduction of cell growth. Slight (1): not more than 20% of the cells are round, loosely attached and without intracytoplasmatic granules, or show changes in morphology; occasional lysed cells are present; only slight growth inhibition observable. Mild (2): not more than 50% of the cells are round, devoid of intracytoplasmatic granules, no extensive cell lysis; not more than 50% growth inhibition observable
b None (0): no detectable zone around or under specimen. Slight (1):
some malformed or degenerated cells under specimen. Mild (2): zone limited to area under specimen
Membranes Extraction Direct contact Gradea Reactivityb Gradeb Reactivityb
Control 0 None 0 None
PLA 0 None 0 None
PLA-PSO2 2 Mild 2 Mild
PLA-PSO7 2 Mild 2 Mild
PLA-PSO14 1 Slight 0 None
PLA-PSO20 1 Slight 1 Slight
PLA-SBO2 1 Slight 1 Slight
PLA-SBO7 1 Slight 1 Slight
PLA-SBO14 0 None 1 Slight
PLA-SBO20 1 Slight 2 Mild
PLA-ESO2 1 Slight 1 Slight
PLA-ESO7 1 Slight 1 Slight
PLA-ESO14 1 Slight 0 None
zone limited to the area under the PLA/PSO2 and PLA/ PSO7 films; thus, mild reactivity (grade 2) was determined. According to the extraction test, PSO14 and PSO20 films showed slight reactivity (grade 1) with no more than 5% of cells being round and loosely attached. However, simi-lar to PLA/ESO14, PLA/PSO14 was scored as 0, indicat-ing n” reactivity around or under the films. The results obtained from the extraction test indicate PLA/SBO films were slightly reactive against cells, 5–10% loosely attached lysed cells (grade 1), except for PLA/SBO14 films graded “0”. Moreover, according to direct contact test, some mal-formed or degenerated cells under PLA/SBO films except for PLA/SBO20, where mild reactivity was observed (grade 2). Consequently, according to in vitro qualitative examinations, all PLA/oil films are evaluated as biocom-patible following the ISO10993-5 standard. Thus, PLA/oil films can be regarded as novel biomaterials obtained from renewable resources. Moreover, both quantitative and qual-itative results showed no reactivity for the cells treated with PLA/oil14 films. Thus, in vivo examinations were assessed with PLA/oil14 films according to related standards.
In Vivo Biocompatibility
In vivo cytotoxicities of the prepared PLA/oil14 films were assessed by an irritation-intracutaneous
reactivity test and implantation using the ISO10993-10 and ISO10993-6 standards, respectively. General evalua-tions were done for both methods (irritation and implan-tation) in order to confirm the results. According to the irritation reactivity test results, no erythema and no edema reactions were observed after PLA/oil14 film treatment due to the applied tests (ISO10993-10). Thus, irrita-tion response was scored as 0 which indicates the negli-gible response category of irritation. Table 3 represents the results obtained from the implantation test which are evaluated according to histological examinations. As seen from Table 3, after 4 weeks of implantation tests, cell types and responses were scored in order to determine local biological effects after implantation. Rare [1–5/ per high-powered (×400) field] lymphocytes and narrow band fibrosis were observed for all samples after PLA/ oil14 implantations (ISO10993-10). Additionally, neovas-cularization with minimal capillary proliferation (focal, 1–3 buds) was observed for 1 sample after PLA/SBO14 implantation. Thus, overall average scores were deter-mined as 2.33 for PLA/SBO14 and 2.0 for PLA, PLA/ PSO14 and PLA/ESO14 films, respectively. These results and both clinical and gross pathological observations con-cluded that PLA/oil14 films were “non-irritant” (Table 3) and can be evaluated as a biocompatible biomaterial for biomedical uses.
Table 3 In vivo
biocompatibility of membranes
Evaluation of implantation test: histological evaluation (cell type/response). Determined according to ISO10993-6
a Used to determine irritant ranking shown below as the conclusion. A negative difference is recorded as
zero. Non-irritant (0.0–2.9), slight irritant (3.0 –8.9), moderate irritant (9.0–15.0), severe irritant (>15)
Membrane PLA PLA-PSO14 PLA-SOYA14 PLA-ESO14
Animal number 1 2 3 1 2 3 1 2 3 1 2 3
Cell type/response Grading Grading Grading Grading
Inflammation 0 0 0 0 0 0 0 0 0 0 0 0 Polymorphonuclear cells 0 0 0 0 0 0 0 0 0 0 0 0 Lymphocytes 1 1 1 1 1 1 1 1 1 1 1 1 Plasma cells 0 0 0 0 0 0 0 0 0 0 0 0 Macrophages 0 0 0 0 0 0 0 0 0 0 0 0 Giant cells 0 0 0 0 0 0 0 0 0 0 0 0 Necrosis 0 0 0 0 0 0 0 0 0 0 0 0 Sub-total (X2) 1 1 1 1 1 1 1 1 1 1 1 1 Neovascularisation 0 0 0 0 0 0 0 0 1 0 0 0 Fibrosis 1 1 1 1 1 1 1 1 1 1 1 1 Fatty infiltrate 0 0 0 0 0 0 0 0 0 0 0 0 Sub-total 1 1 1 1 1 1 1 1 2 1 1 1 Total 2 2 2 2 2 2 2 2 3 2 2 2 Group total-averagea 2 2 2.33 2 Test (-) control 1 1 1 1 Traumatic necrosis 0 0 0 0 0 0 0 0 0 0 0 0 Foreign debris 0 0 0 0 0 0 0 0 0 0 0 0
Conclusion
Soybean oil was auto-oxidized, and polymerized soy-bean oil, soysoy-bean oil and epoxidized soysoy-bean oil were blended with PLA. After blending 2, 7, 14 and 20% (w/w) oils (PSO, SBO and ESO) with PLA, polymeric blends were solvent-casted and PLA/oil films were successfully obtained. The existence of oil and oil miscibility was char-acterized by FTIR spectra of the prepared films. Surface morphology was examined by SEM images, which dem-onstrated homogenous distribution of spherical PSO drop-lets on the PLA/PSO films, whereas no droplet structure of SBO was observed and the ESO was homogenously distributed within the films. Thermal analysis and mechani-cal tests not only confirmed the existence of oil within the films but also concluded that PLA/ESO films greatly lower the thermal stability and the plasticizing effect. In vitro quantitative and qualitative cytotoxicity results showed no reactivity (grade 0) for the cells treated with PLA/oil14 films. Results obtained from implantation and irritation tests showed no erythema and no edema reactions and, according to implantation test scores, the PLAoil14 films were evaluated as non-irritant without any observation of traumatic necrosis and foreign debris. Consequently, PLA/ oil films could serve as promising environmentally friendly biomaterials which can re-emphasize the idea of using renewable resources for several biomedical uses.
Acknowledgements This study was financially supported by the Turkish Scientific Research Council (Grant Number: 213M375) and Bülent Ecevit University Research Fund (Grant Number: 2014-39971044-02). Authors thank TÜBI˙TAK-MAM for performing ani-mal experiments (Implantation and Irritation tests).
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