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gelatin composite scaffold by collagen type I grafting for skin tissue engineering
Materials Science and Engineering C 34 (2014) 402–409
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Surface modification of nanofibrous polycaprolactone/gelatin composite scaffold by collagen type I grafting for skin tissue engineering Sneh Gautam a, Chia-Fu Chou b, Amit K. Dinda c, Pravin D. Potdar d, Narayan C. Mishra a,⁎ a
Department of Polymer & Process Engineering, Indian Institute of Technology Roorkee, India Institute of Physics, Academia Sinica, Taipei, Taiwan Department of Pathology, All India Institute of Medical Science, New Delhi, India d Department of Molecular Medicine & Biology, Jaslok Hospital & Research Centre, Mumbai, India b c
a r t i c l e
i n f o
Article history: Received 19 May 2013 Received in revised form 27 August 2013 Accepted 28 September 2013 Available online 5 October 2013 Keywords: Collagen type I Electrospinning Gelatin PCL Skin tissue engineering
a b s t r a c t In the present study, a tri-polymer polycaprolactone (PCL)/gelatin/collagen type I composite nanofibrous scaffold has been fabricated by electrospinning for skin tissue engineering and wound healing applications. Firstly, PCL/ gelatin nanofibrous scaffold was fabricated by electrospinning using a low cost solvent mixture [chloroform/ methanol for PCL and acetic acid (80% v/v) for gelatin], and then the nanofibrous PCL/gelatin scaffold was modified by collagen type I (0.2–1.5 wt.%) grafting. Morphology of the collagen type I-modified PCL/gelatin composite scaffold that was analyzed by field emission scanning electron microscopy (FE-SEM), showed that the fiber diameter was increased and pore size was decreased by increasing the concentration of collagen type I. Fourier transform infrared (FT-IR) spectroscopy and thermogravimetric (TG) analysis indicated the surface modification of PCL/gelatin scaffold by collagen type I immobilization on the surface of the scaffold. MTT assay demonstrated the viability and high proliferation rate of L929 mouse fibroblast cells on the collagen type I-modified composite scaffold. FE-SEM analysis of cell-scaffold construct illustrated the cell adhesion of L929 mouse fibroblasts on the surface of scaffold. Characteristic cell morphology of L929 was also observed on the nanofiber mesh of the collagen type I-modified scaffold. Above results suggest that the collagen type I-modified PCL/gelatin scaffold was successful in maintaining characteristic shape of fibroblasts, besides good cell proliferation. Therefore, the fibroblast seeded PCL/gelatin/collagen type I composite nanofibrous scaffold might be a potential candidate for wound healing and skin tissue engineering applications. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Skin is the outermost and the largest organ of our body which covers 8% of the total body mass [1]. Overall, it acts as a protecting barrier against microbial invasion, mechanical, chemical, osmotic and photo damage [1]. Skin defects and necrosis can result from burns, soft tissue trauma, ultraviolet radiation and chemical-contact [2]. The conventional therapies used to repair skin defects include autograft, allograft and xenograft [3,4]. Although autografts are used more frequently to repair skin defects, but often there are limitations of donor sites [5]. Alternatively, allografts can be obtained in abundance, though these are often related to high risk of immune rejection and disease transmission. To resolve these problems pertaining to conventional treatments, tissue engineered skin substitutes are used nowadays e.g. Integra [6–8], alloderm [9,10], apligraf [11,12] and epicel [13,14]. Major limitations
⁎ Corresponding author at: Department of Polymer & Process Engineering, IIT Roorkee, Saharanpur Campus, Saharanpur, U.P.-247 001, India. Tel.: +91 132 2714352; fax: +91 132 2714310. E-mail address: [email protected] (N.C. Mishra). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.09.043
of these tissue engineered skin substitutes are their high cost and low mechanical strength [15,16]. The use of Collagen type I is maximum among the various biomaterials applied for skin tissue regeneration [17,18]. Although collagen type I has been extensively used for skin tissue engineering including wound healing applications [18], but it is expensive, and the use of collagen type I as a major scaffold materials, increases the cost of tissue engineered skin substitutes. In this regard, the tissue engineered product will not be cost-effective [16]. Therefore, to address this problem, in our study, we aim to fabricate a tripolymer PCL/gelatin/collagen type I scaffold, by grafting collagen type I, in small quantity, on electrospun PCL/gelatin scaffold: thus, overall, the cost of scaffold will be reduced which will reduce the cost of skin ultimately. PCL/gelatin nanofibrous scaffold had already been investigated for skin tissue engineering in previous studies [1,19] where PCL was used for providing good mechanical strength to the scaffold [20–22], and gelatin [23] for good cell adhesion, proliferation and biodegradation [24,25]. In our body skin ECM is mainly composed of collagen type I (80– 85%) nanofibers [26] and collagen type I possesses different cell binding sequences such as Arg-Gly-Asp (RGD) and Gly-Phe-Hyp-Gly-Glu-Arg
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(GFOGER), which influence adhesion of fibroblasts to the scaffold [27]. Collagen type I based scaffold is well known for controlling many cellular functions of fibroblasts and keratinocytes, including cell shape, differentiation and migration due to the presence of RGD and GFOGER sequences [18]. It also helps in synthesizing a number of skin ECM proteins which enhances skin regeneration process. Therefore, collagen type I, if grafted on the PCL/gelatin nanofibers in small quantity, will make the scaffold to mimic the native skin ECM to some extent while reducing the overall cost of the scaffold. The tri-polymer PCL/gelatin/collagen type I nanofibrous scaffold, if fabricated, might provide better chemical cues to the fibroblasts for adhesion, proliferation and differentiation, which might result in regeneration of better quality skin substitute as compared to the skin regenerated from the PCL/gelatin scaffold. In this study, we fabricated a tri-polymer PCL/gelatin/collagen type I scaffold for skin tissue engineering applications. Firstly, PCL/gelatin nanofibrous scaffold was fabricated by electrospinning, using a low cost solvent mixture [chloroform/methanol for PCL and acetic acid (80% v/v) for gelatin] [22], and then the PCL/gelatin scaffold was modified by collagen type I (0.3% v/v acetic acid) grafting. The tri-polymer PCL/gelatin/collagen type I scaffold following the above economic technique, was fabricated for the first time by our group. To the best of our knowledge, there is no other study on fabricating such tri-polymer PCL/gelatin/collagen type I scaffold. Here, collagen type I has been used in very small quantity only for grafting process while fabricating nanofibrous tri-polymer PCL/gelatin/collagen-type-I composite scaffold: this might reduce the total cost of skin substitutes that is a need in the field of skin tissue engineering. 2. Materials and methods 2.1. Materials PCL pellets (MW = 80,000), gelatin (type B from bovine skin), and collagen (type I from calf skin) powder were from Sigma-Aldrich (St. Louis, MO). Glacial acetic acid was from Qualigens Fine Chemical, Mumbai, India. Chloroform and methanol were from Fisher Scientific, Mumbai, India. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) was from Hi-media laboratories Pvt. Limited, Mumbai, India. 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) (MTT), Dulbecco's modified eagle medium (DMEM) and phosphate buffer saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum was received from Hyclone, U.S.A., and L929 mouse fibroblast cell line was received from NCCS, Pune, India. Double distilled water was prepared in our lab and the same was used for preparing the polymer solution. 2.2. Preparation of PCL/gelatin and collagen type I solutions PCL and gelatin solutions were prepared as discussed previously [22]. After preparing the polymeric solutions, PCL and gelatin were mixed at 80:20 volume ratio and incubated for 48 h, then the gelatin solution was uniformly dispersed in PCL solution whereby an immiscible blend of PCL/gelatin was obtained and ready for electrospinning. For grafting, collagen type I has been dissolved in 0.3% (v/v) acetic acid by stirring (500 rpm, 20 min) at room temperature to prepare concentrations ranging from 0.2 to 1.5 wt.%. 2.3. Fabrication of electrospun PCL/gelatin composite scaffold Electrospinning was performed according to the protocol reported previously [22,28,29]. In brief, to start the electrospinning, polymer blend of PCL/gelatin was placed in a 3 mL plastic syringe fitted with a blunt tip needle of diameter 0.56 mm. The flow rate of polymer solution was controlled at 0.2 mL/h by a syringe pump (Model 11 Plus, Harvard Apparatus) and a distance of 10 cm between the needle and the collector was maintained throughout the electrospinning process. A high voltage of 22 kV was applied at the tip of the needle. The electrospun
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nanofibers were collected on a 1.5 cm × 1.5 cm flat aluminum plate. The electrospinning process was carried out at 25 ± 1 °C (room temperature) and 45% of humidity. 2.4. Modification of PCL/gelatin composite scaffold by collagen type I grafting The composite scaffolds of PCL/gelatin were surface modified by using collagen type I solutions at different concentrations in the range of 0.2–1.5 wt.%. For collagen type I grafting, firstly, PCL/gelatin composite scaffolds were immersed in 10 mL EDAC solution (10 mg/mL in PBS) and kept at 4°C for 4h to activate the carboxylic group of gelatin present in the composite scaffolds. After EDAC treatment, the scaffolds were reacted with different concentrations (0.2–1.5 wt.% in 0.3 v% acetic acid) of collagen type I at 4 °C for 24 h following the protocol of Ma and co-workers [30]. The collagen type I-modified (or modified henceforth) scaffolds were then air-dried overnight. The possible reactions during the process of surface modification have been shown in Fig. 1. 2.5. Characterization of electrospun composite scaffold The morphology of unmodified and collagen type I-modified PCL/ gelatin composite scaffolds were investigated by FE-SEM (Quanta 200F Model, FEI, the Netherlands) equipped with field-gun at an accelerating voltage of 15 kV. Before imaging, the scaffolds were coated with gold using a sputter coater (Biotech SC005, Switzerland). Fiber diameters of the scaffolds were calculated on the basis of FE-SEM images at 5 000× magnification. Five images were used for each sample and from each image, about 20 different fibers at 100 different locations were randomly selected and then average fiber diameter was determined by following the protocol of Yang and co-worker [31] using an image analysis software (Image J, NIH, USA). The surface pore sizes of unmodified and collagen type I-modified scaffolds were also calculated from the same FE-SEM images using Image J. To investigate the surface modification of PCL/gelatin composite scaffold by collagen type I grafting, TG, derivative TG (DTG) and FT-IR analysis were performed of unmodified and modified scaffold. The above analysis was also performed simultaneously by taking all components of the scaffold, i.e., PCL pellets, gelatin and collagen type I powder. TG analysis (EXSTAR, TG/DTA 6300) was performed to determine the thermal degradation pattern, where the samples were run from 22 to 700 °C at a scanning rate of 30 °C/min under a nitrogen atmosphere. FT-IR spectroscopy was recorded by Thermo Nicolet FT-IR (Nexus, USA) in transmittance mode with wavenumber range of 4000–500 cm− 1. The samples were prepared by processing compressed potassium bromide disk and the ratio of samples to KBr used for performing FT-IR analysis was 1 mg samples/900 mg KBr. 2.6. Cell behavior of L929 fibroblasts on collagen type I-modified PCL/gelatin composite scaffolds 2.6.1. Cell proliferation assay on composite scaffolds The cell proliferation on unmodified and collagen type I-modified PCL/gelatin composite scaffolds was determined by the colorimetric MTT assay using L929 mouse fibroblast cells at 1, 3 and 5days of cell culture. MTT assay is based on the ability of mitochondrial dehydrogenases of living cells to oxidize a tetrazolium salt (3-[4,5-dimethylthiazolyl-2y]-2,5-diphenyltetrazolium bromide) to an insoluble purple formazan product. The concentration of the purple formazan product is directly proportional to the number of metabolically active cells [32]. L929 mouse fibroblast cell line was maintained in DMEM with 10% FBS at 37 °C in a 5% CO2 incubator (BINDER, Germany). For positive control, cells were seeded into the well of tissue culture plate, without any scaffold. We cut all the scaffolds into pieces with dimensions of 5 mm × 5 mm × 1 mm, so that the well-surface of 96-well polystyrene tissue culture plate can entirely be covered with this scaffold. The
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Fig. 1. Schematic representation of the proposed reaction scheme for surface modification of PCL/gelatin composite scaffold by collagen type I grafting.
scaffolds were sterilized with UV light for 30 min both side each. After placing the sterilized scaffold into the well of 96-well polystyrene culture plate in triplicate, cell suspensions at a density of 5 × 103 cells/well, were pipetted directly over the scaffold, and left for 3–5 min in CO2 incubator, so that the cells get adhered only to the scaffold and had the least chance to dribble through the scaffold to the tissue-culture plate. DMEM media was then added into the well containing scaffolds and cells to have a final volume of 200μL, and then incubated for 1, 3 and 5days. After incubation period, the wells were washed with PBS and then freshly prepared culture media containing 0.5 mg/mL of MTT solution was added in each well. The plate was placed in 5% CO2 incubator at 37 °C for 4 h. After incubation period, the purple color formazan crystals were formed in the culture media due to the reduction of MTT salt by viable cells. The media was discarded and 200 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. After removing the scaffold from each well, absorbance was taken in a Biorad plate reader at 490 nm with the subtraction for plate absorbance at 650 nm.
change in fiber morphology. Fibers showed swollen morphology and dissolution of gelatin in PBS. All these studies indicate that the PCL/gelatin scaffolds are biodegradable in vitro and we know that collagen type I is a biodegradable and biocompatible polymer: we can conclude that our collagen type I-modified nanofibrous PCL/gelatin scaffold will also be biodegradable. 2.8. Statistical analysis The experimental results were represented as mean values with ± standard deviations (SD). The two-way analysis of variation (ANOVA) was also employed to determine the difference between the various scaffolds in cell proliferation assay (MTT assay). A p-value of less than 0.05 (p b 0.05) was considered to be of significant difference and a pvalue of greater than 0.05 (pN0.05) is considered for insignificant difference between the scaffolds. 3. Results and discussion
2.6.2. Cell attachment and morphological studies on composite scaffolds Morphological characteristics of L929 mouse fibroblast cells on unmodified and collagen type I-modified PCL/gelatin composite scaffolds were determined by FE-SEM analysis. The scaffolds (5 mm × 5 mm × 1 mm each) were placed in 96-well plate and soaked with 100 μL DMEM media overnight at 37 °C to make the scaffold surface more efficient for cell attachment before cell seeding. After PBS washing, scaffolds were incubated with L929 mouse fibroblast cells at a density of 5 × 103 cells/well for 1, 3 and 5 days. The media was changed every 24 h during cell culture. After incubation period, scaffolds were rinsed three times with PBS and fixed with 2.5% glutaraldehyde for 6h at 4°C, and then scaffolds were dehydrated with gradient concentration of ethanol (50%, 70%, 95%, and 100%) for 30min each at 4°C. Finally, the scaffolds were air-dried overnight and analyzed by FE-SEM to study the morphology of attached cell on unmodified and collagen type I-modified composite scaffolds. 2.7. Mechanical strength and degradability profile of the fabricated scaffolds Mechanical strength and degradability profile of the fabricated scaffolds can be determined using standard protocols [33–35] but didn't carry out these properties for the lack of experimental facility in our laboratory. The mechanical strength of PCL/gelatin scaffold had been determined in previous study, which shows high mechanical strength (tensile strength = 1.29 MPa; Young modulus = 30.8 MPa and Elongation at break = 1.38 mm/mm) [33]. Similar results have been reported by Gupta et al. [34] and Ghasemi-Mobarakeh et al. [23]. Here, we expect to have similar good mechanical strength for our tri-polymer PCL/gelatin/collagen type I scaffold. The biodegradation of PCL/gelatin scaffold has already been reported in previous studies. Ghasemi-Mobarakeh et al. [23] performed the biodegradation of nanofibrous PCL/gelatin scaffold and reported the swollen fiber morphology after 2 weeks of degradation study in the PCL/ gelatin scaffold (70:30). Kai et al. [35] studied the degradation behavior of PCL/gelatin (20:80) nanofibrous scaffold and reported a significant
3.1. Fiber morphology of composite scaffolds Fiber morphologies of unmodified and collagen type I-modified PCL/ gelatin composite scaffolds were analyzed by FE-SEM (Fig. 2). Fig. 2a and b shows randomly oriented nanofibers of unmodified PCL/gelatin composite scaffold with smooth surface. The average fiber diameter of the unmodified PCL/gelatin composite scaffold was determined as 440 ± 127 nm and the average pore size of 9.4 ± 1.7 μm. PCL/gelatin composite scaffolds modified by different concentrations of collagen type I (0.2–1.5 wt.%) also showed random orientation of nanofibers but with collapsed fiber morphology [Fig. 2(c-l)]. This may be due to the swelling of nanofiber of PCL/gelatin in the acidic aqueous solution of collagen type I during the surface modification (grafting) process. PCL/ gelatin composite scaffolds modified with 0.2–1.5 wt.% collagen type I show average fiber diameter in the range of 481–858 nm (Fig. 3a) with average pore size in the range of 7.9–1.6 μm, correspondingly (Fig. 3b). Figs. 2 and 3 indicate that by increasing the grafting concentration of collagen type I, it results in the increased average fiber diameter but decreased average pore size, respectively. The decrease in pore size may be due to the collapsed fiber morphology (Fig. 3b). 3.2. Fourier transform infrared (FT-IR) spectroscopy The FT-IR spectra of PCL, gelatin, collagen type I, unmodified and collagen type I-modified PCL/gelatin composite scaffold are shown in Fig. 4. PCL showed several characteristic bands at 2949cm−1 (asymmetric CH2 stretching), 2865 cm−1 (symmetric CH2 stretching), 1726 cm−1 (carbonyl stretching), 1293 cm−1 (C\O and C\C stretching), 1240 cm−1 (asymmetric C\O\C stretching) and 1170 cm−1 (symmetric C\O\C stretching) [36,37]. The gelatin is characterized by the bands at 3443 cm−1 due to N\H stretching of amide bond, C_O stretching at 1640 cm−1 (amide I), N\H bending at 1543 cm−1 (amide II), and 1245 cm−1 of amide III [37–39]. The IR spectrum of collagen type I shows the characteristic absorbance bands at 3312 cm− 1 (the stretching vibration of N\H group), 1652 cm−1 (amide I band from
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Fig. 2. FE-SEM micrograph of electrospun unmodified PCL/gelatin composite scaffold (a, b) and modified PCL/gelatin composite scaffold with different concentrations of collagen type I: 0.2 wt.% (c, d); 0.5 wt.% (e, f); 0.8 wt.% (g, h); 1.0 wt.% (i, j); 1.5 wt.% (k, l) at 10,000× and 20,000× magnifications.
the C_O stretching vibrations coupled to N\H bending vibrations), 1545 cm−1(amide II band due to the N\H bending vibrations coupled to C\N stretching vibrations) and 1240 cm−1 (amide III band from the combination peaks between N\H deformation and C\N stretching vibrations) [40]. In unmodified PCL/gelatin composite scaffold, all the characteristic bands of PCL and gelatin were observed but shifted
towards the lower wavenumbers. Further, in collagen type I-modified PCL/gelatin composite scaffold, all the characteristic bands of PCL, gelatin and collagen type I have been found but also get shifted towards the lower wavenumbers. The shifting of these absorbance bands towards the lower wavenumber and the presence of collagen type I absorbance bands in IR spectrum of modified PCL/gelatin composite scaffold
Fig. 3. Schematic showing the variation of (a) average fiber diameter and (b) average pore size on varying the concentrations of collagen type I; (Fiber diameter was increased and pore size was decreased by increasing the concentration of collagen type I).
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Fig. 4. FT-IR patterns of (a) PCL, (b) gelatin, (c) collagen type I, (d) unmodified PCL/gelatin composite scaffold and (e) collagen type I-modified PCL/gelatin composite scaffold by 1.0 wt.% collagen I concentration.
suggest that the PCL/gelatin composite scaffold was successfully grafted by collagen type I.
3.3. Thermogravimetric (TG) analysis Fig. 5(a) represents the thermal degradation of PCL, gelatin, collagen type I, unmodified and collagen type I-modified PCL/gelatin composite scaffold. PCL showed single stage thermal degradation which starts from around 376 °C and almost completed at 480 °C [41]. The mass loss of gelatin [42] and collagen type I [43] occurred in two stages. The first stage in gelatin has been found between 50 and 200 °C and in collagen type I it was found between 28 and 200 °C, which is related to the loss of absorbed and bound water. The second stage of thermal degradation was found between 300 and 680 °C in gelatin while in collagen type I it was found in the range of 300–690 °C which is associated with the protein chain breakage and peptide bond rupture [42,43]. Both unmodified and collagen type I-modified PCL/gelatin composite scaffolds showed single-stage thermal degradation. In unmodified
PCL/gelatin composite scaffold, the main polymer degradation region started at 400 °C and almost completed at around 460 °C while collagen type I-modified PCL/gelatin composite scaffold started to degrade from 300 °C and finished entirely at 450 °C. Thermal behavior of all polymers and the scaffolds is depicted in Table 1, and the derivatives of weight loss for PCL, gelatin, collagen type I, unmodified and collagen type I-modified PCL/gelatin composite scaffold are shown in Fig. 5(b). Each peak in Fig. 5(b) represents the Tmax which corresponds to the maximum degradation rate at that particular temperature. Both gelatin and collagen type I are proteins and almost comprised of the same chemical structure. In derivative thermogravimetric (DTG) analysis, gelatin and collagen type I both exhibit two peaks: gelatin shows the maximum degradation rate at 194 °C and 331 °C while collagen type I at 121 °C and 361 °C. PCL, unmodified and collagen type I-modified PCL/gelatin composite scaffold reveal only single peak of fast thermal degradation at 436 °C in PCL and unmodified PCL/gelatin composite scaffold whereas this peak is shifted to 473 °C in collagen type I-modified PCL/gelatin composite scaffold. Thus, collagen type I-modified PCL/gelatin composite scaffold showed higher Tmax as compared to the unmodified PCL/gelatin scaffold. According to Lewandowska [44], if the measured lower Tmax of one component shifts towards the higher Tmax of another component then it shows some interactions (e.g., collagen type I binds PCL/gelatin through covalent bond as shown in Fig. 1) between these two components. In modified PCL/gelatin composite scaffold, lower Tmax of collagen type I has shifted towards the higher Tmax of unmodified PCL/gelatin composite scaffold that indicates chemical grafting of collagen type I on the surface of PCL/gelatin scaffold. Thus, the TGA and DTG results corroborated the surface modification of PCL/gelatin composite scaffold by collagen type I grafting which are also supported by FT-IR results.
3.4. Cell behavior of L929 fibroblast on the collagen type I-modified PCL/gelatin composite scaffolds 3.4.1. Cell proliferation assay on composite scaffolds Cell proliferation assay of L929 mouse fibroblast cells on the unmodified and collagen type I-modified composite scaffolds has been carried out for the period of 1, 3 and 5 days. After 5 days of cell culturing, the unmodified PCL/gelatin composite scaffold showed an increase in
Fig. 5. (a) TG analysis and (b) derivative curves of weight loss of (a) PCL, (b) gelatin, (c) collagen type I, (d) unmodified PCL/gelatin composite scaffold, (e) collagen type I-modified PCL/ gelatin composite scaffold by 1.0 wt.% collagen type I concentration.
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Table 1 The region and percentage of decomposition of unmodified and collagen type I-modified scaffolds along with PCL, Gelatin and collagen type I. Samples
Main region of decomposition (°C)
Percentage of mass decomposition
Tmax1(°C)
Tmax2 (°C)
Maximum degradation rate (mg/min)
Gelatin Collagen type I PCL Unmodified PCL/gelatin scaffold Collagen type I-modified (1.0 wt.%) PCL/gelatin scaffold
100–600 200–500 376–459 400–525 300–450
77 61 87.10 81.80 80.06
194 121 436 436 473
331 361 – –
0.93 2.17 3.26 2.89 2.10
cell proliferation as compared to the control but the collagen type I (0.2– 1.0 wt.%) modified PCL/gelatin composite scaffolds showed a significant higher proliferation rate as compared to control and unmodified scaffold (Fig. 6). This indicates that the surface of PCL/gelatin composite scaffold modified by collagen type I grafting enhances cell adhesion and proliferation of L929 mouse fibroblasts. Similar results were obtained in previous studies, which showed that different collagen substrates influenced the adhesion, proliferation and migration of HeLa cells and human skin fibroblast cells [45]. The cell proliferation rate over the composite scaffold modified by 1.5 wt.% collagen type I, was found highest after 1 day, but after 3 and 5 days of cell seeding, the cell proliferation rate goes on decreasing dramatically. The reason might be that, after 1 day of cell seeding, collagen type I (1.5 wt.%) microenvironment suited best to the cells, and therefore, the cells became very much confluent on the scaffold. However, the decrease in cell proliferation after 3 and 5days may be due to the following factors. Firstly, the confluency of cells (after one day) over the scaffold produces space stress on the cells and depletion in nutrients supply. Secondly, the highly collapsed morphology (Fig. 2k) of collagen type I (1.5 wt.%) modified scaffold produced very small pore size (1.6μm) which again leads to space stress as the cells cannot migrate inside the scaffold. We assume these two factors to be the reasons behind the decreased cell proliferation after 3 and 5 days of cell culturing. Among all the collagen type I-modified scaffolds (0.2– 1.5 wt.%), scaffold modified by 1.0 wt.% collagen type I showed the highest cell (L929 mouse fibroblasts) proliferation rate throughout the culture period i.e. up to 5 days. It is concluded that the cell growth and proliferation are significantly enhanced on the PCL/gelatin composite scaffold modified with collagen type I, particularly at 1.0 wt.%, which
suggests the possibility of using modified scaffold as a promising candidate for skin tissue engineering applications.
3.4.2. Cell attachment and morphological studies on composite scaffolds Cell morphology of L929 mouse fibroblasts on the unmodified and collagen type I-modified PCL/gelatin composite scaffolds was studied by FESEM. Despite cell adhered on both unmodified and modified composite scaffolds (Fig. 7), the cell expansion on all collagen type I-modified scaffolds is found to be greater than that on unmodified scaffold. After 3 days of cell seeding, cells started stretching and spreading over the unmodified PCL/gelatin composite scaffold, while after 5 days, all the cells became elongated and converted into spindle shape morphology, which indicates successful cell adhesion and proliferation of L929 mouse fibroblast cells. In all modified scaffolds by collagen type I (0.2– 1.5wt.%) grafting, cells gained the characteristic spindle-shape morphology within 3days, and started migrating inside the modified nanofibrous composite scaffolds (Fig. 8), while after 5 days of cell seeding, cells covered a large surface on the scaffold. PCL/gelatin composite scaffold modified by 1.0 wt.% collagen type I grafting demonstrated the highest expansion of L929 mouse fibroblast cells on the surface of the scaffold after 5days of cell seeding. From FE-SEM analysis of the cell-scaffold constructs, it is clear that cells comply well with the nanofiber-surface of the modified composite scaffold and are capable of migrating inside the scaffold. Overall, it indicates good adhesion, high proliferation rate and migration of L929 mouse fibroblasts cells on collagen type Imodified scaffolds. This result has corroborated MTT assay in confirming the potential of collagen type I-modified scaffold towards skin tissue engineering.
4. Conclusions Tri-polymer PCL/gelatin/collagen type I nanofibrous composite scaffold was successfully fabricated by grafting collagen type I, in small quantity, on electrospun PCL/gelatin scaffold. Among all collagen type I-modified (0.2–1.5 wt.%) scaffolds, grafting with 1.0 wt.% collagen type I, illustrated the highest proliferation for L929 mouse fibroblast cells as confirmed by MTT assay. Good cell adhesion and characteristic morphology of fibroblast was observed through FESEM of cell-scaffold construct. The fibroblast seeded PCL/gelatin/collagen type I composite nanofibrous scaffold might be a potential candidate for wound healing and skin tissue engineering applications.
Acknowledgments
Fig. 6. The proliferation of L929 mouse fibroblast cells on PCL/gelatin scaffolds: unmodified and modified with various concentrations of collagen type I (0.2–1.5 wt.%). Significant difference between different scaffolds was denoted as *(p b 0.05). (∗) unmarked bars show insignificant difference between the scaffolds (p N 0.05).
Chia-Fu Chou and Narayan C. Mishra acknowledge the support from the Academia Sinica Nano Program and the National Science Council, Taiwan, R.O.C. (99-2112-M-001-027-MY3) to carry out this research work. We are thankful to the technical support from Jaslok Hospital and Research Centre, Mumbai, and particularly to Mr. Sachin Ramdas Chaugule for his assistance to carry out biocompatibility study. We are also thankful to Sweta K. Gupta and acknowledge her suggestions.
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Fig. 7. FE-SEM images of L929 mouse fibroblast cells on (a, b and c) unmodified PCL/gelatin scaffold after 1, 3 and 5 days; (d, e and f) collagen type I (0.2 wt.%) modified scaffold after 1, 3 and 5 days; (g, h and i) collagen type I (0.5 wt.%) modified scaffold after 1, 3 and 5 days; (j, k and l) collagen type I (0.8 wt.%) modified after 1, 3 and 5 days; (m, n and o) collagen type I (1.0 wt.%) modified scaffold after 1, 3 and 5 days; (p, q and r) collagen type I (1.5 wt.%) modified scaffold after 1, 3 and 5 days at 1000× magnification.
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Fig. 8. Cell migration of L929 mouse fibroblasts inside the nanofibers of collagen type I-modified (1.0 wt.%) PCL/gelatin composite scaffold (a) after 3 days and (b) after 5 days of cell seeding.
References [1] E.J. Chong, T.T. Phan, I.J. Lim, Y.Z. Zhang, B.H. Bay, C.T. Lim, S. Ramakrishna, Acta Biomater. 3 (2007) 321–330. [2] S. Bo¨ttcher-Haberzeth, T. Biedermann, E. Reichmann, Burn 36 (2010) 450–460. [3] R. Murugan, Z.M. Huang, F. Yang, S. Ramakrishna, J. Nanosci. Nanotechnol. 7 (2007) 4595–4603. [4] S.G. Kumbar, S.P. Nukavarapu, R. James, L.S. Nair, C.T. Laurencin, Biomaterials 29 (2008) 4100–4107. [5] S.K. Gupta, A.K. Dinda, P.D. Potdar, N.C. Mishra, Mater. Sci. Eng. C 33 (2013) 4032–4038. [6] Y. IV, J. Burke, J. Biomed. Mater. Res. 14 (1980) 65–81. [7] R. Sheridan, M. Hegarty, R.G. Tomkins, J.F. Burke, Eur. J. Plast. Surg. 17 (1994) 91–93. [8] D. Heimbach, A. Luterman, J. Burke, Ann. Surg. 208 (1988) 313–320. [9] D. Wainwright, M. Madden, A. Luterman, J. Hunt, W. Monafo, D. Heimbach, R. Kagan, K. Sittig, A. Dimick, D. Herndon, J. Burn Care Rehabil. 17 (1996) 124–136. [10] R.R. Lorenz, R.L. Dean, D.B. Hurley, J. Chuang, M.J. Citardi, Laryngoscope 113 (2003) 496–501. [11] A.F. Falabella, I.C. Valencia, W.H. Eaglatein, L.A. Schachner, Arch. Dermatol. 136 (2000) 1225–1230. [12] D.P. Fivenson, L. Scherschun, L.V. Cohen, Plast. Reconstr. Surg. 112 (2003) 584–588. [13] C.C. Compton, Eur. J. Pediatr. Surg. 2 (1922) 216–222. [14] R. Gobet, M. Raghunath, S. Altermatt, Surgery 121 (1997) 654–661. [15] D.M. Supp, S.T. Boyce, Clin. Dermatol. 23 (2005) 403–412. [16] R.F. Pereira, C.C. Barrias, P.L. Granja, P.J. Bartolo, Nanomedicine 8 (2013) 603–621. [17] B.-S. Kim, I.-K. Park, T. Hoshiba, H.-L. Jiang, Y.-J. Choi, T. Akaike, C.-S. Cho, Prog. Polym. Sci. 36 (2011) 238–268. [18] D. Brett, Wounds 20 (2008) 347–353. [19] R.S. Tığlı, N. Kazaroğlu, B. Mav˙ı¸s, M. Gümü¸sderel˙ıo˘glu, J. Biomater. Sci. Polym. Ed. (2011) 207–223. [20] N. Bölgen, Y.Z. Mencelog˘ lu, K. Acatay, I. Vargel, E. Piskin, J. Biomater. Sci. Polym. Ed. 16 (2005) 1537–1555. [21] L.A. Bosworth, S. Downes, Polym. Degrad. Stab. 95 (2010) 2269–2276. [22] S. Gautam, A.K. Dinda, N.C. Mishra, Mater. Sci. Eng. C 33 (2013) 1228–1235. [23] L. Ghasemi-Mobarakeh, M.P. Prabhakaran, M. Morshed, M.H. Nasr-Esfahani, S. Ramakrishna, Biomaterials 29 (2008) 4532–4539.
[24] J. Ratanavaraporn, S. Damrongsakkul, N. Sanchavanakit, T. Banaprasert, Sorada Kanokpanont, J. Met. Mater. Miner. 16 (2006) 31–36. [25] K. Rupali, B. Amrita, D. Girish, J. Adv. Sci. Res. 2 (2011) 14–23. [26] G.S. Schultz, G. Ladwig, A. Wysocki, World Wide Wounds, 2005. Available on line at: www.worldwidewounds.com/2005/august/Schultz/Extrace-Matric-Acute-ChronicWounds.html (accessed September, 2012). [27] S.T. Khew, Y.W. Tong, Biomacromolecules 8 (2007) 3153–3161. [28] A. Greiner, J.H. Wendorff, Angew. Chem. Int. Ed. 46 (2007) 5670–5703. [29] V. Chaurey, P.C. Chiang, C. Polanco, Y.H. Su, C.F. Chou, N. Swami, Langmuir 26 (2010) 19022–19026. [30] Z. Ma, C. Gao, Y. Gong, J. Shen, Biomaterials 26 (2005) 1253–1259. [31] Y. Yang, X. Zhu, W. Cui, X. Li, Y. Jin, Macromol. Mater. Eng. 249 (2009) 611–619. [32] M.A. Alvarez-Perez, V. Guarino, V. Cirillo, L. Ambrosio, Biomacromolecules 11 (2010) 2238–2246. [33] Y. Zhang, H. Ouyang, C.T. Lim, S. Ramakrishna, Z.M. Hung, J. Biomed. Mater. Res. B Appl. Biomater. 72B (2004) 156–165. [34] D. Gupta, J. Venugopal, M.P. Prabhakaran, V.R. Dev, S. Low, A.T. Choon, S. Ramakrishna, Acta Biomater. 5 (2009) 2560–2569. [35] D. Kai, M.P. Prabhakaran, B. Stahl, M. Eblenkanp, E. Wintermantel, S. Ramakrishna, Nanotechnology 23 (2012) 1–10. [36] Y. Wan, X. Lu, S. Dalai, J. Zhang, Thermochim. Acta 487 (2009) 33–38. [37] Y.C. Lim, J. Johnson, Z. Fei, Y. Wu, D.F. Farson, J.J. Lannutti, H.W. Choi, L.J. Lee, Biotechnol. Bioeng. 108 (2011) 116–126. [38] G.S. Al-Saidi, A. Al-Alawi, M.S. Rahman, N. Guizani, Int. Food Res. J. 19 (2012) 1167–1173. [39] M. Meskinfam, M.S. Sadjadi, H. Jazdarreh, World Acad. Sci. Eng. Technol. 52 (2011) 395–398. [40] C. Tangsadthakun, S. Kanokpanont, N. Sanchavanakit, T. Banaprasert, S. Damrongsakkul, J. Met. Mater. Miner. 16 (2006) 37–44. [41] A. Mohamed, V.L. Finkenstadt, S.H. Gordon, G. Biresaw, D.E. Palmquist, P. Rayas-Duarte, J. Appl. Polym. Sci. 110 (2008) 3256–3266. [42] C. Peña, K. Caba, A. Eceiz, R. Ruseckait, I. Mondragon, Bioresour. Technol. 101 (2010) 6836–6842. [43] V. Mano, R.E. Silva, Mater. Res. 10 (2007) 165–170. [44] K. Lewandowska, Thermochim. Acta 493 (2009) 42–48. [45] S.L. Schor, J. Cell Sci. 41 (1980) I59–I75.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Surface modification of nanofibrous polycaprolactone/gelatin composite scaffold by collagen type I grafting for skin tissue engineering Sneh Gautam a, Chia-Fu Chou b, Amit K. Dinda c, Pravin D. Potdar d, Narayan C. Mishra a,⁎ a
Department of Polymer & Process Engineering, Indian Institute of Technology Roorkee, India Institute of Physics, Academia Sinica, Taipei, Taiwan Department of Pathology, All India Institute of Medical Science, New Delhi, India d Department of Molecular Medicine & Biology, Jaslok Hospital & Research Centre, Mumbai, India b c
a r t i c l e
i n f o
Article history: Received 19 May 2013 Received in revised form 27 August 2013 Accepted 28 September 2013 Available online 5 October 2013 Keywords: Collagen type I Electrospinning Gelatin PCL Skin tissue engineering
a b s t r a c t In the present study, a tri-polymer polycaprolactone (PCL)/gelatin/collagen type I composite nanofibrous scaffold has been fabricated by electrospinning for skin tissue engineering and wound healing applications. Firstly, PCL/ gelatin nanofibrous scaffold was fabricated by electrospinning using a low cost solvent mixture [chloroform/ methanol for PCL and acetic acid (80% v/v) for gelatin], and then the nanofibrous PCL/gelatin scaffold was modified by collagen type I (0.2–1.5 wt.%) grafting. Morphology of the collagen type I-modified PCL/gelatin composite scaffold that was analyzed by field emission scanning electron microscopy (FE-SEM), showed that the fiber diameter was increased and pore size was decreased by increasing the concentration of collagen type I. Fourier transform infrared (FT-IR) spectroscopy and thermogravimetric (TG) analysis indicated the surface modification of PCL/gelatin scaffold by collagen type I immobilization on the surface of the scaffold. MTT assay demonstrated the viability and high proliferation rate of L929 mouse fibroblast cells on the collagen type I-modified composite scaffold. FE-SEM analysis of cell-scaffold construct illustrated the cell adhesion of L929 mouse fibroblasts on the surface of scaffold. Characteristic cell morphology of L929 was also observed on the nanofiber mesh of the collagen type I-modified scaffold. Above results suggest that the collagen type I-modified PCL/gelatin scaffold was successful in maintaining characteristic shape of fibroblasts, besides good cell proliferation. Therefore, the fibroblast seeded PCL/gelatin/collagen type I composite nanofibrous scaffold might be a potential candidate for wound healing and skin tissue engineering applications. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Skin is the outermost and the largest organ of our body which covers 8% of the total body mass [1]. Overall, it acts as a protecting barrier against microbial invasion, mechanical, chemical, osmotic and photo damage [1]. Skin defects and necrosis can result from burns, soft tissue trauma, ultraviolet radiation and chemical-contact [2]. The conventional therapies used to repair skin defects include autograft, allograft and xenograft [3,4]. Although autografts are used more frequently to repair skin defects, but often there are limitations of donor sites [5]. Alternatively, allografts can be obtained in abundance, though these are often related to high risk of immune rejection and disease transmission. To resolve these problems pertaining to conventional treatments, tissue engineered skin substitutes are used nowadays e.g. Integra [6–8], alloderm [9,10], apligraf [11,12] and epicel [13,14]. Major limitations
⁎ Corresponding author at: Department of Polymer & Process Engineering, IIT Roorkee, Saharanpur Campus, Saharanpur, U.P.-247 001, India. Tel.: +91 132 2714352; fax: +91 132 2714310. E-mail address: [email protected] (N.C. Mishra). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.09.043
of these tissue engineered skin substitutes are their high cost and low mechanical strength [15,16]. The use of Collagen type I is maximum among the various biomaterials applied for skin tissue regeneration [17,18]. Although collagen type I has been extensively used for skin tissue engineering including wound healing applications [18], but it is expensive, and the use of collagen type I as a major scaffold materials, increases the cost of tissue engineered skin substitutes. In this regard, the tissue engineered product will not be cost-effective [16]. Therefore, to address this problem, in our study, we aim to fabricate a tripolymer PCL/gelatin/collagen type I scaffold, by grafting collagen type I, in small quantity, on electrospun PCL/gelatin scaffold: thus, overall, the cost of scaffold will be reduced which will reduce the cost of skin ultimately. PCL/gelatin nanofibrous scaffold had already been investigated for skin tissue engineering in previous studies [1,19] where PCL was used for providing good mechanical strength to the scaffold [20–22], and gelatin [23] for good cell adhesion, proliferation and biodegradation [24,25]. In our body skin ECM is mainly composed of collagen type I (80– 85%) nanofibers [26] and collagen type I possesses different cell binding sequences such as Arg-Gly-Asp (RGD) and Gly-Phe-Hyp-Gly-Glu-Arg
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(GFOGER), which influence adhesion of fibroblasts to the scaffold [27]. Collagen type I based scaffold is well known for controlling many cellular functions of fibroblasts and keratinocytes, including cell shape, differentiation and migration due to the presence of RGD and GFOGER sequences [18]. It also helps in synthesizing a number of skin ECM proteins which enhances skin regeneration process. Therefore, collagen type I, if grafted on the PCL/gelatin nanofibers in small quantity, will make the scaffold to mimic the native skin ECM to some extent while reducing the overall cost of the scaffold. The tri-polymer PCL/gelatin/collagen type I nanofibrous scaffold, if fabricated, might provide better chemical cues to the fibroblasts for adhesion, proliferation and differentiation, which might result in regeneration of better quality skin substitute as compared to the skin regenerated from the PCL/gelatin scaffold. In this study, we fabricated a tri-polymer PCL/gelatin/collagen type I scaffold for skin tissue engineering applications. Firstly, PCL/gelatin nanofibrous scaffold was fabricated by electrospinning, using a low cost solvent mixture [chloroform/methanol for PCL and acetic acid (80% v/v) for gelatin] [22], and then the PCL/gelatin scaffold was modified by collagen type I (0.3% v/v acetic acid) grafting. The tri-polymer PCL/gelatin/collagen type I scaffold following the above economic technique, was fabricated for the first time by our group. To the best of our knowledge, there is no other study on fabricating such tri-polymer PCL/gelatin/collagen type I scaffold. Here, collagen type I has been used in very small quantity only for grafting process while fabricating nanofibrous tri-polymer PCL/gelatin/collagen-type-I composite scaffold: this might reduce the total cost of skin substitutes that is a need in the field of skin tissue engineering. 2. Materials and methods 2.1. Materials PCL pellets (MW = 80,000), gelatin (type B from bovine skin), and collagen (type I from calf skin) powder were from Sigma-Aldrich (St. Louis, MO). Glacial acetic acid was from Qualigens Fine Chemical, Mumbai, India. Chloroform and methanol were from Fisher Scientific, Mumbai, India. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) was from Hi-media laboratories Pvt. Limited, Mumbai, India. 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) (MTT), Dulbecco's modified eagle medium (DMEM) and phosphate buffer saline (PBS) were obtained from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum was received from Hyclone, U.S.A., and L929 mouse fibroblast cell line was received from NCCS, Pune, India. Double distilled water was prepared in our lab and the same was used for preparing the polymer solution. 2.2. Preparation of PCL/gelatin and collagen type I solutions PCL and gelatin solutions were prepared as discussed previously [22]. After preparing the polymeric solutions, PCL and gelatin were mixed at 80:20 volume ratio and incubated for 48 h, then the gelatin solution was uniformly dispersed in PCL solution whereby an immiscible blend of PCL/gelatin was obtained and ready for electrospinning. For grafting, collagen type I has been dissolved in 0.3% (v/v) acetic acid by stirring (500 rpm, 20 min) at room temperature to prepare concentrations ranging from 0.2 to 1.5 wt.%. 2.3. Fabrication of electrospun PCL/gelatin composite scaffold Electrospinning was performed according to the protocol reported previously [22,28,29]. In brief, to start the electrospinning, polymer blend of PCL/gelatin was placed in a 3 mL plastic syringe fitted with a blunt tip needle of diameter 0.56 mm. The flow rate of polymer solution was controlled at 0.2 mL/h by a syringe pump (Model 11 Plus, Harvard Apparatus) and a distance of 10 cm between the needle and the collector was maintained throughout the electrospinning process. A high voltage of 22 kV was applied at the tip of the needle. The electrospun
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nanofibers were collected on a 1.5 cm × 1.5 cm flat aluminum plate. The electrospinning process was carried out at 25 ± 1 °C (room temperature) and 45% of humidity. 2.4. Modification of PCL/gelatin composite scaffold by collagen type I grafting The composite scaffolds of PCL/gelatin were surface modified by using collagen type I solutions at different concentrations in the range of 0.2–1.5 wt.%. For collagen type I grafting, firstly, PCL/gelatin composite scaffolds were immersed in 10 mL EDAC solution (10 mg/mL in PBS) and kept at 4°C for 4h to activate the carboxylic group of gelatin present in the composite scaffolds. After EDAC treatment, the scaffolds were reacted with different concentrations (0.2–1.5 wt.% in 0.3 v% acetic acid) of collagen type I at 4 °C for 24 h following the protocol of Ma and co-workers [30]. The collagen type I-modified (or modified henceforth) scaffolds were then air-dried overnight. The possible reactions during the process of surface modification have been shown in Fig. 1. 2.5. Characterization of electrospun composite scaffold The morphology of unmodified and collagen type I-modified PCL/ gelatin composite scaffolds were investigated by FE-SEM (Quanta 200F Model, FEI, the Netherlands) equipped with field-gun at an accelerating voltage of 15 kV. Before imaging, the scaffolds were coated with gold using a sputter coater (Biotech SC005, Switzerland). Fiber diameters of the scaffolds were calculated on the basis of FE-SEM images at 5 000× magnification. Five images were used for each sample and from each image, about 20 different fibers at 100 different locations were randomly selected and then average fiber diameter was determined by following the protocol of Yang and co-worker [31] using an image analysis software (Image J, NIH, USA). The surface pore sizes of unmodified and collagen type I-modified scaffolds were also calculated from the same FE-SEM images using Image J. To investigate the surface modification of PCL/gelatin composite scaffold by collagen type I grafting, TG, derivative TG (DTG) and FT-IR analysis were performed of unmodified and modified scaffold. The above analysis was also performed simultaneously by taking all components of the scaffold, i.e., PCL pellets, gelatin and collagen type I powder. TG analysis (EXSTAR, TG/DTA 6300) was performed to determine the thermal degradation pattern, where the samples were run from 22 to 700 °C at a scanning rate of 30 °C/min under a nitrogen atmosphere. FT-IR spectroscopy was recorded by Thermo Nicolet FT-IR (Nexus, USA) in transmittance mode with wavenumber range of 4000–500 cm− 1. The samples were prepared by processing compressed potassium bromide disk and the ratio of samples to KBr used for performing FT-IR analysis was 1 mg samples/900 mg KBr. 2.6. Cell behavior of L929 fibroblasts on collagen type I-modified PCL/gelatin composite scaffolds 2.6.1. Cell proliferation assay on composite scaffolds The cell proliferation on unmodified and collagen type I-modified PCL/gelatin composite scaffolds was determined by the colorimetric MTT assay using L929 mouse fibroblast cells at 1, 3 and 5days of cell culture. MTT assay is based on the ability of mitochondrial dehydrogenases of living cells to oxidize a tetrazolium salt (3-[4,5-dimethylthiazolyl-2y]-2,5-diphenyltetrazolium bromide) to an insoluble purple formazan product. The concentration of the purple formazan product is directly proportional to the number of metabolically active cells [32]. L929 mouse fibroblast cell line was maintained in DMEM with 10% FBS at 37 °C in a 5% CO2 incubator (BINDER, Germany). For positive control, cells were seeded into the well of tissue culture plate, without any scaffold. We cut all the scaffolds into pieces with dimensions of 5 mm × 5 mm × 1 mm, so that the well-surface of 96-well polystyrene tissue culture plate can entirely be covered with this scaffold. The
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Fig. 1. Schematic representation of the proposed reaction scheme for surface modification of PCL/gelatin composite scaffold by collagen type I grafting.
scaffolds were sterilized with UV light for 30 min both side each. After placing the sterilized scaffold into the well of 96-well polystyrene culture plate in triplicate, cell suspensions at a density of 5 × 103 cells/well, were pipetted directly over the scaffold, and left for 3–5 min in CO2 incubator, so that the cells get adhered only to the scaffold and had the least chance to dribble through the scaffold to the tissue-culture plate. DMEM media was then added into the well containing scaffolds and cells to have a final volume of 200μL, and then incubated for 1, 3 and 5days. After incubation period, the wells were washed with PBS and then freshly prepared culture media containing 0.5 mg/mL of MTT solution was added in each well. The plate was placed in 5% CO2 incubator at 37 °C for 4 h. After incubation period, the purple color formazan crystals were formed in the culture media due to the reduction of MTT salt by viable cells. The media was discarded and 200 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. After removing the scaffold from each well, absorbance was taken in a Biorad plate reader at 490 nm with the subtraction for plate absorbance at 650 nm.
change in fiber morphology. Fibers showed swollen morphology and dissolution of gelatin in PBS. All these studies indicate that the PCL/gelatin scaffolds are biodegradable in vitro and we know that collagen type I is a biodegradable and biocompatible polymer: we can conclude that our collagen type I-modified nanofibrous PCL/gelatin scaffold will also be biodegradable. 2.8. Statistical analysis The experimental results were represented as mean values with ± standard deviations (SD). The two-way analysis of variation (ANOVA) was also employed to determine the difference between the various scaffolds in cell proliferation assay (MTT assay). A p-value of less than 0.05 (p b 0.05) was considered to be of significant difference and a pvalue of greater than 0.05 (pN0.05) is considered for insignificant difference between the scaffolds. 3. Results and discussion
2.6.2. Cell attachment and morphological studies on composite scaffolds Morphological characteristics of L929 mouse fibroblast cells on unmodified and collagen type I-modified PCL/gelatin composite scaffolds were determined by FE-SEM analysis. The scaffolds (5 mm × 5 mm × 1 mm each) were placed in 96-well plate and soaked with 100 μL DMEM media overnight at 37 °C to make the scaffold surface more efficient for cell attachment before cell seeding. After PBS washing, scaffolds were incubated with L929 mouse fibroblast cells at a density of 5 × 103 cells/well for 1, 3 and 5 days. The media was changed every 24 h during cell culture. After incubation period, scaffolds were rinsed three times with PBS and fixed with 2.5% glutaraldehyde for 6h at 4°C, and then scaffolds were dehydrated with gradient concentration of ethanol (50%, 70%, 95%, and 100%) for 30min each at 4°C. Finally, the scaffolds were air-dried overnight and analyzed by FE-SEM to study the morphology of attached cell on unmodified and collagen type I-modified composite scaffolds. 2.7. Mechanical strength and degradability profile of the fabricated scaffolds Mechanical strength and degradability profile of the fabricated scaffolds can be determined using standard protocols [33–35] but didn't carry out these properties for the lack of experimental facility in our laboratory. The mechanical strength of PCL/gelatin scaffold had been determined in previous study, which shows high mechanical strength (tensile strength = 1.29 MPa; Young modulus = 30.8 MPa and Elongation at break = 1.38 mm/mm) [33]. Similar results have been reported by Gupta et al. [34] and Ghasemi-Mobarakeh et al. [23]. Here, we expect to have similar good mechanical strength for our tri-polymer PCL/gelatin/collagen type I scaffold. The biodegradation of PCL/gelatin scaffold has already been reported in previous studies. Ghasemi-Mobarakeh et al. [23] performed the biodegradation of nanofibrous PCL/gelatin scaffold and reported the swollen fiber morphology after 2 weeks of degradation study in the PCL/ gelatin scaffold (70:30). Kai et al. [35] studied the degradation behavior of PCL/gelatin (20:80) nanofibrous scaffold and reported a significant
3.1. Fiber morphology of composite scaffolds Fiber morphologies of unmodified and collagen type I-modified PCL/ gelatin composite scaffolds were analyzed by FE-SEM (Fig. 2). Fig. 2a and b shows randomly oriented nanofibers of unmodified PCL/gelatin composite scaffold with smooth surface. The average fiber diameter of the unmodified PCL/gelatin composite scaffold was determined as 440 ± 127 nm and the average pore size of 9.4 ± 1.7 μm. PCL/gelatin composite scaffolds modified by different concentrations of collagen type I (0.2–1.5 wt.%) also showed random orientation of nanofibers but with collapsed fiber morphology [Fig. 2(c-l)]. This may be due to the swelling of nanofiber of PCL/gelatin in the acidic aqueous solution of collagen type I during the surface modification (grafting) process. PCL/ gelatin composite scaffolds modified with 0.2–1.5 wt.% collagen type I show average fiber diameter in the range of 481–858 nm (Fig. 3a) with average pore size in the range of 7.9–1.6 μm, correspondingly (Fig. 3b). Figs. 2 and 3 indicate that by increasing the grafting concentration of collagen type I, it results in the increased average fiber diameter but decreased average pore size, respectively. The decrease in pore size may be due to the collapsed fiber morphology (Fig. 3b). 3.2. Fourier transform infrared (FT-IR) spectroscopy The FT-IR spectra of PCL, gelatin, collagen type I, unmodified and collagen type I-modified PCL/gelatin composite scaffold are shown in Fig. 4. PCL showed several characteristic bands at 2949cm−1 (asymmetric CH2 stretching), 2865 cm−1 (symmetric CH2 stretching), 1726 cm−1 (carbonyl stretching), 1293 cm−1 (C\O and C\C stretching), 1240 cm−1 (asymmetric C\O\C stretching) and 1170 cm−1 (symmetric C\O\C stretching) [36,37]. The gelatin is characterized by the bands at 3443 cm−1 due to N\H stretching of amide bond, C_O stretching at 1640 cm−1 (amide I), N\H bending at 1543 cm−1 (amide II), and 1245 cm−1 of amide III [37–39]. The IR spectrum of collagen type I shows the characteristic absorbance bands at 3312 cm− 1 (the stretching vibration of N\H group), 1652 cm−1 (amide I band from
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Fig. 2. FE-SEM micrograph of electrospun unmodified PCL/gelatin composite scaffold (a, b) and modified PCL/gelatin composite scaffold with different concentrations of collagen type I: 0.2 wt.% (c, d); 0.5 wt.% (e, f); 0.8 wt.% (g, h); 1.0 wt.% (i, j); 1.5 wt.% (k, l) at 10,000× and 20,000× magnifications.
the C_O stretching vibrations coupled to N\H bending vibrations), 1545 cm−1(amide II band due to the N\H bending vibrations coupled to C\N stretching vibrations) and 1240 cm−1 (amide III band from the combination peaks between N\H deformation and C\N stretching vibrations) [40]. In unmodified PCL/gelatin composite scaffold, all the characteristic bands of PCL and gelatin were observed but shifted
towards the lower wavenumbers. Further, in collagen type I-modified PCL/gelatin composite scaffold, all the characteristic bands of PCL, gelatin and collagen type I have been found but also get shifted towards the lower wavenumbers. The shifting of these absorbance bands towards the lower wavenumber and the presence of collagen type I absorbance bands in IR spectrum of modified PCL/gelatin composite scaffold
Fig. 3. Schematic showing the variation of (a) average fiber diameter and (b) average pore size on varying the concentrations of collagen type I; (Fiber diameter was increased and pore size was decreased by increasing the concentration of collagen type I).
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Fig. 4. FT-IR patterns of (a) PCL, (b) gelatin, (c) collagen type I, (d) unmodified PCL/gelatin composite scaffold and (e) collagen type I-modified PCL/gelatin composite scaffold by 1.0 wt.% collagen I concentration.
suggest that the PCL/gelatin composite scaffold was successfully grafted by collagen type I.
3.3. Thermogravimetric (TG) analysis Fig. 5(a) represents the thermal degradation of PCL, gelatin, collagen type I, unmodified and collagen type I-modified PCL/gelatin composite scaffold. PCL showed single stage thermal degradation which starts from around 376 °C and almost completed at 480 °C [41]. The mass loss of gelatin [42] and collagen type I [43] occurred in two stages. The first stage in gelatin has been found between 50 and 200 °C and in collagen type I it was found between 28 and 200 °C, which is related to the loss of absorbed and bound water. The second stage of thermal degradation was found between 300 and 680 °C in gelatin while in collagen type I it was found in the range of 300–690 °C which is associated with the protein chain breakage and peptide bond rupture [42,43]. Both unmodified and collagen type I-modified PCL/gelatin composite scaffolds showed single-stage thermal degradation. In unmodified
PCL/gelatin composite scaffold, the main polymer degradation region started at 400 °C and almost completed at around 460 °C while collagen type I-modified PCL/gelatin composite scaffold started to degrade from 300 °C and finished entirely at 450 °C. Thermal behavior of all polymers and the scaffolds is depicted in Table 1, and the derivatives of weight loss for PCL, gelatin, collagen type I, unmodified and collagen type I-modified PCL/gelatin composite scaffold are shown in Fig. 5(b). Each peak in Fig. 5(b) represents the Tmax which corresponds to the maximum degradation rate at that particular temperature. Both gelatin and collagen type I are proteins and almost comprised of the same chemical structure. In derivative thermogravimetric (DTG) analysis, gelatin and collagen type I both exhibit two peaks: gelatin shows the maximum degradation rate at 194 °C and 331 °C while collagen type I at 121 °C and 361 °C. PCL, unmodified and collagen type I-modified PCL/gelatin composite scaffold reveal only single peak of fast thermal degradation at 436 °C in PCL and unmodified PCL/gelatin composite scaffold whereas this peak is shifted to 473 °C in collagen type I-modified PCL/gelatin composite scaffold. Thus, collagen type I-modified PCL/gelatin composite scaffold showed higher Tmax as compared to the unmodified PCL/gelatin scaffold. According to Lewandowska [44], if the measured lower Tmax of one component shifts towards the higher Tmax of another component then it shows some interactions (e.g., collagen type I binds PCL/gelatin through covalent bond as shown in Fig. 1) between these two components. In modified PCL/gelatin composite scaffold, lower Tmax of collagen type I has shifted towards the higher Tmax of unmodified PCL/gelatin composite scaffold that indicates chemical grafting of collagen type I on the surface of PCL/gelatin scaffold. Thus, the TGA and DTG results corroborated the surface modification of PCL/gelatin composite scaffold by collagen type I grafting which are also supported by FT-IR results.
3.4. Cell behavior of L929 fibroblast on the collagen type I-modified PCL/gelatin composite scaffolds 3.4.1. Cell proliferation assay on composite scaffolds Cell proliferation assay of L929 mouse fibroblast cells on the unmodified and collagen type I-modified composite scaffolds has been carried out for the period of 1, 3 and 5 days. After 5 days of cell culturing, the unmodified PCL/gelatin composite scaffold showed an increase in
Fig. 5. (a) TG analysis and (b) derivative curves of weight loss of (a) PCL, (b) gelatin, (c) collagen type I, (d) unmodified PCL/gelatin composite scaffold, (e) collagen type I-modified PCL/ gelatin composite scaffold by 1.0 wt.% collagen type I concentration.
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Table 1 The region and percentage of decomposition of unmodified and collagen type I-modified scaffolds along with PCL, Gelatin and collagen type I. Samples
Main region of decomposition (°C)
Percentage of mass decomposition
Tmax1(°C)
Tmax2 (°C)
Maximum degradation rate (mg/min)
Gelatin Collagen type I PCL Unmodified PCL/gelatin scaffold Collagen type I-modified (1.0 wt.%) PCL/gelatin scaffold
100–600 200–500 376–459 400–525 300–450
77 61 87.10 81.80 80.06
194 121 436 436 473
331 361 – –
0.93 2.17 3.26 2.89 2.10
cell proliferation as compared to the control but the collagen type I (0.2– 1.0 wt.%) modified PCL/gelatin composite scaffolds showed a significant higher proliferation rate as compared to control and unmodified scaffold (Fig. 6). This indicates that the surface of PCL/gelatin composite scaffold modified by collagen type I grafting enhances cell adhesion and proliferation of L929 mouse fibroblasts. Similar results were obtained in previous studies, which showed that different collagen substrates influenced the adhesion, proliferation and migration of HeLa cells and human skin fibroblast cells [45]. The cell proliferation rate over the composite scaffold modified by 1.5 wt.% collagen type I, was found highest after 1 day, but after 3 and 5 days of cell seeding, the cell proliferation rate goes on decreasing dramatically. The reason might be that, after 1 day of cell seeding, collagen type I (1.5 wt.%) microenvironment suited best to the cells, and therefore, the cells became very much confluent on the scaffold. However, the decrease in cell proliferation after 3 and 5days may be due to the following factors. Firstly, the confluency of cells (after one day) over the scaffold produces space stress on the cells and depletion in nutrients supply. Secondly, the highly collapsed morphology (Fig. 2k) of collagen type I (1.5 wt.%) modified scaffold produced very small pore size (1.6μm) which again leads to space stress as the cells cannot migrate inside the scaffold. We assume these two factors to be the reasons behind the decreased cell proliferation after 3 and 5 days of cell culturing. Among all the collagen type I-modified scaffolds (0.2– 1.5 wt.%), scaffold modified by 1.0 wt.% collagen type I showed the highest cell (L929 mouse fibroblasts) proliferation rate throughout the culture period i.e. up to 5 days. It is concluded that the cell growth and proliferation are significantly enhanced on the PCL/gelatin composite scaffold modified with collagen type I, particularly at 1.0 wt.%, which
suggests the possibility of using modified scaffold as a promising candidate for skin tissue engineering applications.
3.4.2. Cell attachment and morphological studies on composite scaffolds Cell morphology of L929 mouse fibroblasts on the unmodified and collagen type I-modified PCL/gelatin composite scaffolds was studied by FESEM. Despite cell adhered on both unmodified and modified composite scaffolds (Fig. 7), the cell expansion on all collagen type I-modified scaffolds is found to be greater than that on unmodified scaffold. After 3 days of cell seeding, cells started stretching and spreading over the unmodified PCL/gelatin composite scaffold, while after 5 days, all the cells became elongated and converted into spindle shape morphology, which indicates successful cell adhesion and proliferation of L929 mouse fibroblast cells. In all modified scaffolds by collagen type I (0.2– 1.5wt.%) grafting, cells gained the characteristic spindle-shape morphology within 3days, and started migrating inside the modified nanofibrous composite scaffolds (Fig. 8), while after 5 days of cell seeding, cells covered a large surface on the scaffold. PCL/gelatin composite scaffold modified by 1.0 wt.% collagen type I grafting demonstrated the highest expansion of L929 mouse fibroblast cells on the surface of the scaffold after 5days of cell seeding. From FE-SEM analysis of the cell-scaffold constructs, it is clear that cells comply well with the nanofiber-surface of the modified composite scaffold and are capable of migrating inside the scaffold. Overall, it indicates good adhesion, high proliferation rate and migration of L929 mouse fibroblasts cells on collagen type Imodified scaffolds. This result has corroborated MTT assay in confirming the potential of collagen type I-modified scaffold towards skin tissue engineering.
4. Conclusions Tri-polymer PCL/gelatin/collagen type I nanofibrous composite scaffold was successfully fabricated by grafting collagen type I, in small quantity, on electrospun PCL/gelatin scaffold. Among all collagen type I-modified (0.2–1.5 wt.%) scaffolds, grafting with 1.0 wt.% collagen type I, illustrated the highest proliferation for L929 mouse fibroblast cells as confirmed by MTT assay. Good cell adhesion and characteristic morphology of fibroblast was observed through FESEM of cell-scaffold construct. The fibroblast seeded PCL/gelatin/collagen type I composite nanofibrous scaffold might be a potential candidate for wound healing and skin tissue engineering applications.
Acknowledgments
Fig. 6. The proliferation of L929 mouse fibroblast cells on PCL/gelatin scaffolds: unmodified and modified with various concentrations of collagen type I (0.2–1.5 wt.%). Significant difference between different scaffolds was denoted as *(p b 0.05). (∗) unmarked bars show insignificant difference between the scaffolds (p N 0.05).
Chia-Fu Chou and Narayan C. Mishra acknowledge the support from the Academia Sinica Nano Program and the National Science Council, Taiwan, R.O.C. (99-2112-M-001-027-MY3) to carry out this research work. We are thankful to the technical support from Jaslok Hospital and Research Centre, Mumbai, and particularly to Mr. Sachin Ramdas Chaugule for his assistance to carry out biocompatibility study. We are also thankful to Sweta K. Gupta and acknowledge her suggestions.
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Fig. 7. FE-SEM images of L929 mouse fibroblast cells on (a, b and c) unmodified PCL/gelatin scaffold after 1, 3 and 5 days; (d, e and f) collagen type I (0.2 wt.%) modified scaffold after 1, 3 and 5 days; (g, h and i) collagen type I (0.5 wt.%) modified scaffold after 1, 3 and 5 days; (j, k and l) collagen type I (0.8 wt.%) modified after 1, 3 and 5 days; (m, n and o) collagen type I (1.0 wt.%) modified scaffold after 1, 3 and 5 days; (p, q and r) collagen type I (1.5 wt.%) modified scaffold after 1, 3 and 5 days at 1000× magnification.
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Fig. 8. Cell migration of L929 mouse fibroblasts inside the nanofibers of collagen type I-modified (1.0 wt.%) PCL/gelatin composite scaffold (a) after 3 days and (b) after 5 days of cell seeding.
References [1] E.J. Chong, T.T. Phan, I.J. Lim, Y.Z. Zhang, B.H. Bay, C.T. Lim, S. Ramakrishna, Acta Biomater. 3 (2007) 321–330. [2] S. Bo¨ttcher-Haberzeth, T. Biedermann, E. Reichmann, Burn 36 (2010) 450–460. [3] R. Murugan, Z.M. Huang, F. Yang, S. Ramakrishna, J. Nanosci. Nanotechnol. 7 (2007) 4595–4603. [4] S.G. Kumbar, S.P. Nukavarapu, R. James, L.S. Nair, C.T. Laurencin, Biomaterials 29 (2008) 4100–4107. [5] S.K. Gupta, A.K. Dinda, P.D. Potdar, N.C. Mishra, Mater. Sci. Eng. C 33 (2013) 4032–4038. [6] Y. IV, J. Burke, J. Biomed. Mater. Res. 14 (1980) 65–81. [7] R. Sheridan, M. Hegarty, R.G. Tomkins, J.F. Burke, Eur. J. Plast. Surg. 17 (1994) 91–93. [8] D. Heimbach, A. Luterman, J. Burke, Ann. Surg. 208 (1988) 313–320. [9] D. Wainwright, M. Madden, A. Luterman, J. Hunt, W. Monafo, D. Heimbach, R. Kagan, K. Sittig, A. Dimick, D. Herndon, J. Burn Care Rehabil. 17 (1996) 124–136. [10] R.R. Lorenz, R.L. Dean, D.B. Hurley, J. Chuang, M.J. Citardi, Laryngoscope 113 (2003) 496–501. [11] A.F. Falabella, I.C. Valencia, W.H. Eaglatein, L.A. Schachner, Arch. Dermatol. 136 (2000) 1225–1230. [12] D.P. Fivenson, L. Scherschun, L.V. Cohen, Plast. Reconstr. Surg. 112 (2003) 584–588. [13] C.C. Compton, Eur. J. Pediatr. Surg. 2 (1922) 216–222. [14] R. Gobet, M. Raghunath, S. Altermatt, Surgery 121 (1997) 654–661. [15] D.M. Supp, S.T. Boyce, Clin. Dermatol. 23 (2005) 403–412. [16] R.F. Pereira, C.C. Barrias, P.L. Granja, P.J. Bartolo, Nanomedicine 8 (2013) 603–621. [17] B.-S. Kim, I.-K. Park, T. Hoshiba, H.-L. Jiang, Y.-J. Choi, T. Akaike, C.-S. Cho, Prog. Polym. Sci. 36 (2011) 238–268. [18] D. Brett, Wounds 20 (2008) 347–353. [19] R.S. Tığlı, N. Kazaroğlu, B. Mav˙ı¸s, M. Gümü¸sderel˙ıo˘glu, J. Biomater. Sci. Polym. Ed. (2011) 207–223. [20] N. Bölgen, Y.Z. Mencelog˘ lu, K. Acatay, I. Vargel, E. Piskin, J. Biomater. Sci. Polym. Ed. 16 (2005) 1537–1555. [21] L.A. Bosworth, S. Downes, Polym. Degrad. Stab. 95 (2010) 2269–2276. [22] S. Gautam, A.K. Dinda, N.C. Mishra, Mater. Sci. Eng. C 33 (2013) 1228–1235. [23] L. Ghasemi-Mobarakeh, M.P. Prabhakaran, M. Morshed, M.H. Nasr-Esfahani, S. Ramakrishna, Biomaterials 29 (2008) 4532–4539.
[24] J. Ratanavaraporn, S. Damrongsakkul, N. Sanchavanakit, T. Banaprasert, Sorada Kanokpanont, J. Met. Mater. Miner. 16 (2006) 31–36. [25] K. Rupali, B. Amrita, D. Girish, J. Adv. Sci. Res. 2 (2011) 14–23. [26] G.S. Schultz, G. Ladwig, A. Wysocki, World Wide Wounds, 2005. Available on line at: www.worldwidewounds.com/2005/august/Schultz/Extrace-Matric-Acute-ChronicWounds.html (accessed September, 2012). [27] S.T. Khew, Y.W. Tong, Biomacromolecules 8 (2007) 3153–3161. [28] A. Greiner, J.H. Wendorff, Angew. Chem. Int. Ed. 46 (2007) 5670–5703. [29] V. Chaurey, P.C. Chiang, C. Polanco, Y.H. Su, C.F. Chou, N. Swami, Langmuir 26 (2010) 19022–19026. [30] Z. Ma, C. Gao, Y. Gong, J. Shen, Biomaterials 26 (2005) 1253–1259. [31] Y. Yang, X. Zhu, W. Cui, X. Li, Y. Jin, Macromol. Mater. Eng. 249 (2009) 611–619. [32] M.A. Alvarez-Perez, V. Guarino, V. Cirillo, L. Ambrosio, Biomacromolecules 11 (2010) 2238–2246. [33] Y. Zhang, H. Ouyang, C.T. Lim, S. Ramakrishna, Z.M. Hung, J. Biomed. Mater. Res. B Appl. Biomater. 72B (2004) 156–165. [34] D. Gupta, J. Venugopal, M.P. Prabhakaran, V.R. Dev, S. Low, A.T. Choon, S. Ramakrishna, Acta Biomater. 5 (2009) 2560–2569. [35] D. Kai, M.P. Prabhakaran, B. Stahl, M. Eblenkanp, E. Wintermantel, S. Ramakrishna, Nanotechnology 23 (2012) 1–10. [36] Y. Wan, X. Lu, S. Dalai, J. Zhang, Thermochim. Acta 487 (2009) 33–38. [37] Y.C. Lim, J. Johnson, Z. Fei, Y. Wu, D.F. Farson, J.J. Lannutti, H.W. Choi, L.J. Lee, Biotechnol. Bioeng. 108 (2011) 116–126. [38] G.S. Al-Saidi, A. Al-Alawi, M.S. Rahman, N. Guizani, Int. Food Res. J. 19 (2012) 1167–1173. [39] M. Meskinfam, M.S. Sadjadi, H. Jazdarreh, World Acad. Sci. Eng. Technol. 52 (2011) 395–398. [40] C. Tangsadthakun, S. Kanokpanont, N. Sanchavanakit, T. Banaprasert, S. Damrongsakkul, J. Met. Mater. Miner. 16 (2006) 37–44. [41] A. Mohamed, V.L. Finkenstadt, S.H. Gordon, G. Biresaw, D.E. Palmquist, P. Rayas-Duarte, J. Appl. Polym. Sci. 110 (2008) 3256–3266. [42] C. Peña, K. Caba, A. Eceiz, R. Ruseckait, I. Mondragon, Bioresour. Technol. 101 (2010) 6836–6842. [43] V. Mano, R.E. Silva, Mater. Res. 10 (2007) 165–170. [44] K. Lewandowska, Thermochim. Acta 493 (2009) 42–48. [45] S.L. Schor, J. Cell Sci. 41 (1980) I59–I75.