Materials Science and Engineering C 40 (2014) 242–247
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
PLA/chitosan/keratin composites for biomedical applications Constantin Edi Tanase a,⁎, Iuliana Spiridon b a b
Faculty of Medical Bioengineering, ‘Grigore T. Popa’ University of Medicine and Pharmacy, 9-13 Kogalniceanu Street, 700454 Iasi, Romania “Petru Poni” Institute of Macromolecular Chemistry, 41A Grigore Ghica Voda Alley, 700487 Iasi, Romania
a r t i c l e
i n f o
Article history: Received 21 October 2013 Received in revised form 3 March 2014 Accepted 21 March 2014 Available online 31 March 2014 Keywords: PLA Chitosan Keratin Biomaterials Mechanical properties In vitro studies
a b s t r a c t Novel composites based on PLA, chitosan and keratin was obtained via blend preparation. The goal of this contribution was to evaluate mechanical and in vitro behavior of the composites. The results point out composites with improved Young modulus and decreased tensile strength, significant increase in hardness (compared to PLA) and a good uptake of the surface properties. Biological assessments using human osteosarcoma cell line on these composites indicate a good viability/proliferation outcome. Hence preliminary results regarding mechanical behavior and in vitro osteoblast response suggest that these composites might have prospective application in medical field. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The interest for “green” materials with medical applications was increased due to both patients and medical world that search for solutions to their challenges such as the need for substitutes to replace or repair tissues or organs problems. The most biodegradable materials comprise synthetic polyesters such as poly(L-lactic acid) and poly(L-glycolic acid) and natural polymers such as chitosan, alginate, collagen, and fibrin [1]. Also, some inorganic materials e.g. hydroxyapatite or certain glasses have been used to obtain materials for hard tissue applications [2]. The interest for polyesters is due to their hydrolysable ester bonds and one of the most important constituent of this class is polylactic acid (PLA) which is derived fully from renewable resources. It has been used in biomedical applications but its application is somewhat limited by its inherently poor properties such as reduced impact strength and low thermal stability [3]. Poly(L-lactic acid) has been widely studied for use in biomedical applications such as sutures, scaffolds for tissue engineering, orthopedic devices, or drug delivery systems due to its biocompatibility and bioresorbability [4,5]. Chitosan is a polysaccharide obtained by the deacetylation of chitin and has many applications due to its price, excellent oxygen barrier properties, antimicrobial effects, biodegradability, biocompatibility,
⁎ Corresponding author. E-mail address: [email protected] (C.E. Tanase).
http://dx.doi.org/10.1016/j.msec.2014.03.054 0928-4931/© 2014 Elsevier B.V. All rights reserved.
antimicrobial activity and non-toxicity [6,7]. Due to easy processing method one may obtain films, fibers, gels and foams, as well as beads of different sizes and morphology with medical applications [8]. One important property is the fact that chitosan interacts with cells and cellular lysozyme degrades chitosan in vivo [9]. In the same time, various kinds of chitosan derivatives have medicine applications e.g. bone, cartilage, skin, nerve and blood vessel [7,10]. Literature data reported a good biocompatibility of PLA [9,11]. That is why, it is used in biomedical applications as internal body components, for implants and drug delivery systems [12]. Keratin is the major component of feathers. It is a structural protein characterized by high cystine content and a significant amount of hydroxyl amino acids, especially serine [13]. It's characterized by the presence of a range of noncovalent interactions (electrostatic forces, hydrogen bonds, hydrophobic forces) and covalent interactions (disulphide bonds), which are difficult to be damaged. The recent trends in biodegradable polymers indicate new development strategies and engineering to achieve polymeric materials with great interest both in the academic and industrial fields. It was found that the incorporation of functional fillers in the PLA matrix could improve the physical properties, as well as the surface characteristics of the matrix that are important for tissue engineering and artificial bone reconstruction. Motivated by our preliminary results [14], the purpose of this work is to investigate PLA–chitosan–keratin composites as biomaterials with potential applications in medicine, by means of mechanical and in vitro studies. Preliminary results regarding mechanical behavior and in vitro osteoblast response confirmed their potential for medical applications.
C.E. Tanase, I. Spiridon / Materials Science and Engineering C 40 (2014) 242–247
2. Materials and methods 2.1. Preparation of the composite film Before blend preparation, PLA (type 2002D, supplied by NatureWorks) pallets, chitosan (produced by Vanson Inc. with an average molecular weight of 1200 kDa and acetylation degree of 34%) and feather fibers were dried in a vacuum oven for 6 h at 50 °C. Compounding components were performed at 175 °C for 10 min at a rotor speed of 60 rpm using a fully automated laboratory Brabender station. Literature data [15] show that the addition of chitosan did not influence thermal properties and degree of crystallinity of PLA. Specimens for the mechanical characterization were prepared by compression molding using a Carver press. The compression molding was carried out at 175 °C with a pre-pressing step of 3 min at 50 atm and a pressing step of 2 min at 150 atm. A neat PLA sheet was prepared in the same conditions and acted as a reference. Composition and preparation of the samples are as follows: A111: 70% PLA and 30% chitosan; A121: 68% PLA, 30% chitosan and 2% keratin; A131: 66% PLA, 30% chitosan and 4% keratin. 2.2. DSC analysis Thermal characterization of composites has been performed with a TA Instruments Q20 Dynamic Scanning Calorimeter. All the samples were heated from 25 °C up to 200 °C with 10 °C/min, kept for 2 min and then cooled down to 25 °C with a cooling rate of 5 °C/min. An empty crucible was used as a reference material. All measurements were performed under N2 atmosphere. The degree of crystallinity of the PLA samples was obtained by dividing the melting enthalpy of the sample by 93.7 J/g [16], which is the estimated melting enthalpy of a pure PLA. The crystallinity of the composite materials was estimated as the function of PLA fraction in the composite and the melting enthalpy. 2.3. Characterization of composites Mechanical tests in terms of tensile and impact strength were performed. Thus, tensile strength measurements were carried out following ISO 527-2000 standard method, using an Instron 5 kN test machine operated at a crosshead speed of 30 mm/min. The unnotched Charpy impact strength was measured according to ISO 179-2010 using a Ceast apparatus provided with a hammer of 15 J. Seven specimens were tested for each material. The Vickers hardness tests were performed with a Shimadzu microhardness tester. A constant load of 4.903 N was applied for 12 s for all composite samples. Ten tests have been carried out for each sample and the average value is given.
243
were used as control surface. Cells were maintained under standard cell culture conditions (5% CO2, 95% humidity and 37 °C). The medium was changed every 2–3 days. Control cultures and seeded material samples were evaluated at days 1, 2 and 3 for cell viability/proliferation and at days 3 and 7 the samples were investigated via confocal laser scanning microscopy (CLSM; Leica SP2). 2.4.2. Cytotoxicity assay CellTiter 96®AQueous One Solution Cell Proliferation (Promega, Madison, WI) assay was used to study cell viability. The metabolic cell activity (an indirect measure of cytotoxicity) was measured by the conversion of MTS to formazan, which can be photometrically detected. MTS was mixed with fresh medium at the ratio of 1:10 and added to the cells for 1.5 h. The cells were placed in a CO2 incubator at 37 °C. After incubation time supernatants were transferred to a new microplate and optical density was measured photometrically at 492 nm in an ELISA 96 well-plate reader. All experiments were performed in triplicate and were treated and represented by their mean value and standard deviation parameters. The percentage cell viability was calculated according to the following equation: % cell viability ¼ 100 Abssample =Abscontrol ; where Abs sample is the absorbance of cells tested with various formulations and Abs control is the absorbance of reference cells (incubated on the culture media only). 2.4.3. Immunofluorescence analysis The cells were seeded 3.0 × 104 cells per cm2 in a 48-well plate. After preset time intervals, immunocytochemical staining was performed on whole samples. Briefly, the samples with cells were washed with PBS and fixed using a solution of 3.7% v/v paraformaldehyde. The fixed cells were permeabilized with buffered 0.5% v/v Triton X-100. Subsequently the cells were stained for nuclei with DAPI (0.4 μg mL−1) and cytoskeletal organization was revealed by actin labeling. F-actin filaments were stained with tetramethylrhodamine isothiocyanate (TRITC) conjugated phalloidin 0.2 μg mL−1 (Sigma, St. Louis, MO, USA). Labeled samples were examined by CLSM. 2.5. Statistics Statistical calculations and analyses were performed with the use of Prism 5 (GraphPad Software, Inc.) statistical software package. One-way analysis of variance (ANOVA) was employed to assess the statistical significance of results at a probability of error of 5% (*), 1% (**) and 0.1% (***). All experiments were repeated at least three times, and the results are presented as mean ± standard deviation (SD).
2.4. Biological tests 3. Results and discussions 2.4.1. Cell culture MG63 osteoblast-like cells (ATCC® no CRL-1427™, Rockville, MD-USA) were cultured in Dulbecco's modified Eagle's medium with 4500 mg L− 1 glucose (DMEM) from Gibco supplemented with 10% fetal bovine serum (FBS), 2 mM Glutamax I (Life Technologies) and 100 IU mL− 1 penicillin, 100 μg mL− 1 streptomycin. Cell cultures were sustained at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. The samples were sterilized using gamma irradiation according with ISO 11137—2.0 kGy at room temperature [17] and before using them in cell culture studies the samples were submerge in a diluted ethanol solution (70% v/v) for 15 min, followed by intensive washing in sterile phosphate buffer saline (PBS) solution. The cells were seeded over the sterile samples at a density of 3.0 × 104 cells per cm2 in a 48-well plate (Cellstar®, Greiner Bio-One) up to 7 days. Standard 48-well tissue culture plates (polystyrene)
3.1. DSC results The thermal behavior of the studied materials is presented in Fig. 1 and Table 1. Pure chitosan does not have melting properties and hence no endothermic peaks associated to melting process were detected, as other authors reported [18]. The thermogram of neat PLA revealed a glass transition temperature at 59.4 °C followed by a cold crystallization process peak at 127.8 °C and then melting peak at 152.97 °C, with an enthalpy of fusion of 13.62 J/mol. It was found that the addition of chitosan determined an increase of Tg to 60.2 °C, which can be explained by chitosan hindering movements of PLA chains. The presence of chitosan, which is a semicrystalline polymer, determines a loss of crystallinity in the PLA matrix, which drops from 20.61%
244
C.E. Tanase, I. Spiridon / Materials Science and Engineering C 40 (2014) 242–247
reflecting a solid-like behavior [14]. Keratin fibers serve as nucleation sites and probably polymer chains flexibility. The polymer volume immediately surrounding the keratin fibers has properties different from the bulk polymer [20]. During hardness test the stress transferability inside the biocomposite may be reduced due to the distance between the fibers decreases, i.e. work done will be gone to the deformation of matrix rather than fibers. 3.3. Water uptake The diffusion is a process by which small molecules are transported from one side of a system to another one as a result of random molecular motions. The water sorption is a complex mechanism which is influenced by swelling, molecular interactions, accessibility of sorption sites and material crystallinity and crosslinking, as well as of the presence of fillers. Using the above boundary conditions Fick's law has been used to give the time dependence of concentration, as shown below: Fig. 1. DSC thermograms (second run) of the studied materials.
to 18.05%. The presence of keratin in PLA chitosan system decreased Tg to 58.9 °C, while crystallinity increased to 19.17%. Apparently, the keratin decreases the Tg of PLA in PLA/chitosan system and facilitates crystallization of PLA. Probably crosslinking structures from keratin interfere with the crystallization process, many imperfect crystallites are formed and thus keratin serves as nucleating agents. 3.2. Mechanical properties Mechanical properties are important to the design biomaterials for medical applications. Chitosan incorporation into the PLA matrix improved Young modulus and decreased the tensile strength of PLA (Fig. 1). Polar interactions between ester functional groups of PLA and amine groups of chitosan were expected, but it is possible that processing conditions applied in our study may not have been sufficient for a reaction to occur between the chitosan and PLA, with significant effects on the mechanical properties. The addition of keratin resulted in an increase of impact strength and decrease of tensile properties compared to PLA/chitosan composite. The feather fibers appear to act as stress concentrators in the polymer matrix, thus reducing the crack initiation energy and consequently enhancing the impact strength of the composites. The structure of keratin, the primary constituent of chicken feathers, affects the chemical durability. Because of extensive crosslinking and strong covalent bonding within its structure, keratin provides high resistance to degradation [19]. The possible increase of the toughness may be due to microplastic deformation created around the feather particles. It is well-known that hardness and modulus of a polymeric material depend on its structure. As can be seen in Fig. 2, a significant rise in hardness has been obtained by adding of chitosan to PLA matrix. This suggests that the fiber surface becomes rougher. According with hardness values, the Young modulus of PLA chitosan composite is higher than that of PLA matrix. The incorporation of chitosan with or without keratin stabilized PLA that became consequently less brittle. The rheological studies have revealed that a decrease in the arrangement of PLA polymer chains as the content of keratin increases, A131 material Table 1 DSC parameters of PLA and composite materials. Sample
Tg (°C)
Tm (°C)
ΔHm (J/g)
X (%)
PLA A111 A123
59.4 60.2 58.9
152.97 153.45 150.67
19.32 24.17 26.98
20.61 18.05 19.17
Tm: melting temperature; ΔHm: melting enthalpy; X: crystallinity index; Tg: transition temperature.
2 2 Dð2nþ1Þ π t Mt 8 X 1 − l2 ¼ 1− 2 e 2 M∞ π ð2n þ 1Þ
ð1Þ
where: t is the time measured from when the concentration is changed, l is the plate thickness, Mo is the initial equilibrium mass and ΔM is the change in the mass from Mo to the new equilibrium mass Mt. The Eq. (1) describes the change in mass due to the movement of the diffusing species responding to the sudden change in pressure/humidity around the sample. The half thickness (l/2) is used as the diffusion length above since in a typical sorption experiment the entire plate is measured and the substance is assumed to diffuse uniformly to the central plane (Table 2). For Mt/M∞ b 0.5 rffiffiffiffiffiffiffiffi Mt 4 D·t ¼ · l π M∞
ð2Þ
and for Mt/M∞ N 0.5: Mt 8 −Dπ2 t ¼ 1− 2 ·e l2 : M∞ π
ð3Þ
Literature data reported that the PLA degradation occurs principally at the surface because of the absorption gradient of water [4], the evolution of this process being explained either via a bulk erosion mechanism, starting at the surface [21] or as a heterogeneous process [22]. It is well known that the presence of chitosan influences water uptake and diffusion coefficients [23]. The larger diffusion coefficient could be due to the presence of amorphous chitosan that create a new pathway for the water vapor to diffuse. Thus, in material comprising PLA and chitosan, both D2 and K2 (Mt/M∞ N 0.5) were less affected, while D1 and K1 were slowly affected. Addition of 4% keratin has improved the K1 value, as well as D1 (for for Mt/M∞ b 0.5), while for D2 and K2 (for Mt/M∞ N 0.5) slowly decreased. Our results suggest that water uptake process is more intense at the surface of material. We suppose that water molecule penetrates with difficulty the core of material due to the strong linkages between components of the studied materials, confirmed by rheological studies [14]. Literature data show that PLA has advantage to be processing in shapes for orthopedic applications and fabricated into scaffolds for replacement and regeneration of tissues, or devices for controlled delivery of biomolecules [24,25]. Also, it must be mentioned that, in vivo, the performance of the biomaterial-based structure is derived not only from the mechanical resistance of the biomaterial itself, but also from the complex mixture of biomaterial [26], surface
C.E. Tanase, I. Spiridon / Materials Science and Engineering C 40 (2014) 242–247
245
Fig. 2. Mechanical properties of composite materials. Data are presented as the mean ± SD; n = 7; ***p b 0.001 compared with PLA (PLA served as control).
properties being important to induce cellular attachment, differentiation and proliferation [27]. 3.4. Behavior of MG63 osteoblast-like cells The MTS tetrazolium compound is reduced by cells into a colored formazan product that is soluble in tissue culture medium. The quantity of the formazan product as measured at 492 nm is directly proportional to the number of living cells in a culture. The results of viability assay are represented in Fig. 3. As it can be seen the cell viability percent is comparable with the control (cell viability 100%) at 24 h and 48 h, respectively. After the second day when the cultures reached confluence and formed a dense cell layer, this layer was easily lost during routine medium change. This phenomenon explains the decrease in MTS reduction values observed at day 3. Even in this case the cell viability/proliferation reveals values appropriated to the control. From these results we can conclude that the cell viability/proliferation of the MG63 osteoblastlike cells seeded on the samples containing PLA–chitosan–keratin is similar to the control with a slightly variation which might be influenced by the material composition. Therefore, we can conclude that PLA–chitosan–keratin composites do not affect cell viability and indicate the potential use in bone tissue engineering. The ratio of components in composite materials used in this study was appropriate to
support the MG63 growth and offer proper space and support for cell proliferation. On the other hand, to evaluate the proliferation, distribution and cell adhesion of the cells, it was necessary to use CLSM. The CLSM images of the samples seeded with MG63 cells can be visualized in Fig. 4 at various time points, 72 h and 168 h respectively. The cytoskeletal organization is generally determined by actin staining with fluorescence labeledphalloidin and is used to evaluate the motility, spreading and shape of the cells [28]. Regarding the cell distribution and proliferation after 72 h on the samples, it can be observed that for samples A111 (Fig. 4A) and for samples A131 (Fig. 4C) the MG63 cells cover almost the entire sample surface. For the same time point (72 h) regarding sample A121 (Fig. 4B), it can be observed that MG63 cells present a good distribution but not similar with the other samples. At this time of culture, CLSM shows a suggestive proliferative cell population (Fig. 4-B—circled cells). These data are correlated with the results obtained in MTS assays. At 168 h after culture the cells formed multilayer's of flattened sheets,
Table 2 Diffusion coefficient values calculated form normalized mass changing vs. time.
A131 A121 A111 PLA a
K1a, b0.5
K2a, N0.5
l cm
D1, Mt/M∞ b 0.5, Eq. (2) cm2/s
D2, Mt/M∞ N 0.5, Eq. (3) cm2/s
1.29E−03 7.24E−04 9.84E−04 7.14E−04
−0.00075520 −0.00091237 −0.00081704 −0.00088931
0.098 0.101 0.093 0.089
2.4321E−06 1.4508E−06 1.6704E−06 1.1095E−06
7.3515E−07 7.4336E−07 7.1626E−07 7.1400E−07
K1 and K2 are the slopes of linearized Eqs. (2) and (3), respectively.
Fig. 3. Hardness as function of material composition.
246
C.E. Tanase, I. Spiridon / Materials Science and Engineering C 40 (2014) 242–247
materials reveal estimable biocompatibility features, demonstrating their potential use as substitute materials for bone tissue engineering. This preliminary investigation shows that the composition of PLA/chitosan/keratin materials is important to obtain materials with appropriate properties and careful tailoring of the keratin should modify mechanical properties of material and cellular adhesion. In the future follow-up study, more investigations will be done to further enhance the efficiency of cell bone formation in PLA/chitosan/keratin materials (Fig. 5).
4. Conclusions
Fig. 4. Mitochondrial activity measured via the MTS assay. Data are depicted as percentage of the untreated control. Triplicates were performed and the data represent means ± SD. *p b 0.05, **p b 0.01 and ***p b 0.001 compared to the untreated control.
covering completely the material surface for all samples (D—sample A111, E—sample A121 and F—sample A131). From Fig. 4 it can be seen that the cells have a wide connection with each other throughout cytoplasmic extensions, presenting an elongated shape, thereby emphasizing the distribution and proliferation of cells at different time points. The physical–chemical and topographical features of the biomaterial surface could influence the distribution of focal contact and cytoskeleton organization [29,27]. PLA–chitosan–keratin samples showed increased cell attachment and maintenance of cell numbers over the time periods (up to 168 h). As a final point the MTS viability/proliferation assays and CLSM analysis highlight a good cell viability, proliferation and distribution pointing out a good biocompatibility of the PLA–chitosan–keratin samples in contact with MG63 osteoblast-like cells. Hence these composite
In summary, new biomaterials based on PLA, chitosan and keratin composites were designed and evaluated. These composites with improved Young modulus and decreased tensile strength, significant increase in hardness (compared to PLA) and a good uptake of the surface properties were evaluated with regards of in vitro behavior. This study showed that PLA, chitosan and keratin composites support osteoblast attachment and proliferation during short-term culture indicating that these composites might be promising materials for medical application.
References [1] R. Langer, D.A. Tirrell, Designing materials for biology and medicine, Nature 428 (6982) (2004) 487–492. [2] A.R. Boccaccini, J.J. Blaker, Bioactive composite materials for tissue engineering scaffolds, Expert Rev. Med. Devices 2 (3) (2005) 303–317. [3] R. Bhardwaj, A.K. Mohanty, Advances in the properties of polylactides based materials: a review, J. Biobased Mater. Bioenergy 1 (2) (2007) 191–209. [4] C. Saiz-Arroyo, Y. Wang, M.A. Rodriguez-Perez, N.M. Alves, J.F. Mano, In vitro monitoring of surface mechanical properties of poly( L -lactic acid) using microhardness, J. Appl. Polym. Sci. 105 (2007) 3858–3864. [5] Qizhi Chen, Chenghao Zhu, George A. Thouas, Progress and challenges in biomaterials used for bone tissue engineering: bioactive glasses and elastomeric composites, Prog. Biomater. 1 (2) (2012) 1–22.
Fig. 5. CLSM images (DAPI and TRITC) at 72 h (A—samples A111, B—samples A121 and C—samples A131) and 168 h (D—samples A111, E—samples A121 and F—samples A131) after MG63 cells cultured on PLA–chitosan–keratin samples.
C.E. Tanase, I. Spiridon / Materials Science and Engineering C 40 (2014) 242–247 [6] R. Jayakumar, M. Prabaharan, P.T. Sudheesh Kumar, S.V. Nair, H. Tamura, Biomaterials based on chitin and chitosan in wound dressing applications, Biotechnol. Adv. 29 (2011) 322–337. [7] In-Yong Kim, Seog-Jin Seo, Hyun-Seuk Moon, Mi-Kyong Yoo, In-Young Park, Bom-Chol Kim, Chong-Su Cho, Chitosan and its derivatives for tissue engineering applications, Biotechnol. Adv. 26 (2008) 1–21. [8] Scott P. Noel, Harry Courtney, Joel D. Bumgardner, Warren O. Haggard, Chitosan films. A potential local drug delivery system for antibiotics, Clin. Orthop. Relat. Res. 466 (2008) 1377–1382. [9] K. Tomihata, Y. Ikada, In vitro and in vivo degradation of films of chitin and its deacetylated derivatives, Biomaterials 18 (1997) 567–575. [10] C.E. Tanase, M.I. Popa, L. Verestiuc, Biomimetic chitosan–calcium phosphate composites with potential applications as bone substitutes: preparation and characterization, J. Biomed. Mater. Res. B Appl. Biomater. 100 (3) (2012) 700–708. [11] P.U. Rokkanen, O. Bostman, E. Hirvensalo, E.A. Makela, E.K. Partio, H. Patiala, S.I. Vainionpaa, K. Vihtonen, P. Tormala, Bioabsorbable fixation in orthopaedic surgery and traumatology, Biomaterials 21 (2000) 2607–2613. [12] C.A. Mills, M. Navarro, E. Engel, E. Martinez, M.P. Ginebra, J. Planell, A. Errachid, J. Samitier, Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications, J. Biomed. Mater. Res. A 76 (2006) 781–787. [13] K.M. Arai, R. Takahashi, Y. Yokote, K. Akahane, Amino acid sequence of feather keratin from fowl, J. Biochem. 132 (1983) 501–507. [14] Iuliana Spiridon, Oana Maria Paduraru, Mirela Fernanda Zaltariov, Raluca Nicoleta Darie, Influence of keratin on polylactic acid/chitosan composite properties. Behavior upon accelerated weathering, Ind. Eng. Chem. Res. 52 (29) (2013) 9822–9833. [15] J. Bonilla, E. Fortunati, M. Vargas, A. Chiralt, J.M. Kenny, Effects of chitosan on the physicochemical and antimicrobial properties of PLA films, J. Food Eng. 119 (2013) 236–243. [16] R.G. Liao, B. Yang, W. Yu, C.X. Zhou, Isothermal cold crystallization kinetics of polylactide/nucleating agents, J. Appl. Polym. Sci. 104 (1) (2007) 310–317.
247
[17] M. Bernkopf, Sterilisation of bioresorbable polymer implants, Med. Device Technol. 18 (3) (May-June 2007). [18] C. Swapna Joseph, K.V. Harish Prashanth, N.K. Rastogi, A.R. Indiramma, S. Yella Reddy, K.S.M.S. Raghavarao, Optimum blend of chitosan and poly-(ε-caprolactone) for fabrication of films for food packaging applications, Food Bioprocess Technol. 4 (2011) 1179–1185. [19] S.C. Mishra, N.B. Nayak, A. Satapathy, Investigation on biowaste reinforced epoxy composites, J. Reinf. Plast. Compos. 29 (19) (2010) 3016. [20] J.R. Barone, Polyethylene/keratin fiber composites with varying polyethylene crystallinity, Compos. Part A 36 (2005) 1518–1524. [21] H. Tsuji, A. Mizuno, Y. Ikada, J. Appl. Polym. Sci. 77 (2000) 1452. [22] S.M. Li, H. Garreau, M. Vert, J. Mater. Sci. Mater. Med. 1 (1990) 131. [23] Vitor M. Correlo, Elisabete D. Pinho, Iva Pashkuleva, Mrinal Bhattacharya, Nuno M. Neves, Rui L. Reis, Water absorption and degradation characteristics of chitosan-based polyesters and hydroxyapatite composites, Macromol. Biosci. 7 (2007) 354–363. [24] J.M. Anderson, M.S. Shive, Biodegradation and biocompatibility of PLA and PLGA microspheres, Adv. Drug Deliv. Rev. 28 (1997) 5–24. [25] M. Denti, P. Randelli, D. Lo Vetere, M. Moioli, M. Tagliabue, Bioabsorbable interference screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction: clinical and computerized tomography results of four different models. A prospective study, J. Orthop. Traumatol. 5 (2004) 151–155. [26] Y. Luo, G. Engelmayr, D.T. Auguste, L.S. Ferreira, J.M. Karp, R. Saigal, R. Langer, Threedimensional scaffolds, in: R. Lanza, R. Langer, J.P. Vacanti (Eds.), Principles of Tissue Engineering, 3rd ed., Elsevier, Amsterdam, 2007, pp. 359–373. [27] K. Anselme, Osteoblast adhesion on biomaterials, Biomaterials 21 (7) (2000) 667–681. [28] D.E. Ingber, I.I. Tensegrity, How structural networks influence cellular information processing networks, J. Cell Sci. 116 (2003) 1397–1408. [29] K. Anselme, M. Bigerelle, B. Noel, E. Dufresne, D. Judas, A. Iost, P. Hardouin, Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses, J. Biomed. Mater. Res. 49 (2000) 155–166.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
PLA/chitosan/keratin composites for biomedical applications Constantin Edi Tanase a,⁎, Iuliana Spiridon b a b
Faculty of Medical Bioengineering, ‘Grigore T. Popa’ University of Medicine and Pharmacy, 9-13 Kogalniceanu Street, 700454 Iasi, Romania “Petru Poni” Institute of Macromolecular Chemistry, 41A Grigore Ghica Voda Alley, 700487 Iasi, Romania
a r t i c l e
i n f o
Article history: Received 21 October 2013 Received in revised form 3 March 2014 Accepted 21 March 2014 Available online 31 March 2014 Keywords: PLA Chitosan Keratin Biomaterials Mechanical properties In vitro studies
a b s t r a c t Novel composites based on PLA, chitosan and keratin was obtained via blend preparation. The goal of this contribution was to evaluate mechanical and in vitro behavior of the composites. The results point out composites with improved Young modulus and decreased tensile strength, significant increase in hardness (compared to PLA) and a good uptake of the surface properties. Biological assessments using human osteosarcoma cell line on these composites indicate a good viability/proliferation outcome. Hence preliminary results regarding mechanical behavior and in vitro osteoblast response suggest that these composites might have prospective application in medical field. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The interest for “green” materials with medical applications was increased due to both patients and medical world that search for solutions to their challenges such as the need for substitutes to replace or repair tissues or organs problems. The most biodegradable materials comprise synthetic polyesters such as poly(L-lactic acid) and poly(L-glycolic acid) and natural polymers such as chitosan, alginate, collagen, and fibrin [1]. Also, some inorganic materials e.g. hydroxyapatite or certain glasses have been used to obtain materials for hard tissue applications [2]. The interest for polyesters is due to their hydrolysable ester bonds and one of the most important constituent of this class is polylactic acid (PLA) which is derived fully from renewable resources. It has been used in biomedical applications but its application is somewhat limited by its inherently poor properties such as reduced impact strength and low thermal stability [3]. Poly(L-lactic acid) has been widely studied for use in biomedical applications such as sutures, scaffolds for tissue engineering, orthopedic devices, or drug delivery systems due to its biocompatibility and bioresorbability [4,5]. Chitosan is a polysaccharide obtained by the deacetylation of chitin and has many applications due to its price, excellent oxygen barrier properties, antimicrobial effects, biodegradability, biocompatibility,
⁎ Corresponding author. E-mail address: [email protected] (C.E. Tanase).
http://dx.doi.org/10.1016/j.msec.2014.03.054 0928-4931/© 2014 Elsevier B.V. All rights reserved.
antimicrobial activity and non-toxicity [6,7]. Due to easy processing method one may obtain films, fibers, gels and foams, as well as beads of different sizes and morphology with medical applications [8]. One important property is the fact that chitosan interacts with cells and cellular lysozyme degrades chitosan in vivo [9]. In the same time, various kinds of chitosan derivatives have medicine applications e.g. bone, cartilage, skin, nerve and blood vessel [7,10]. Literature data reported a good biocompatibility of PLA [9,11]. That is why, it is used in biomedical applications as internal body components, for implants and drug delivery systems [12]. Keratin is the major component of feathers. It is a structural protein characterized by high cystine content and a significant amount of hydroxyl amino acids, especially serine [13]. It's characterized by the presence of a range of noncovalent interactions (electrostatic forces, hydrogen bonds, hydrophobic forces) and covalent interactions (disulphide bonds), which are difficult to be damaged. The recent trends in biodegradable polymers indicate new development strategies and engineering to achieve polymeric materials with great interest both in the academic and industrial fields. It was found that the incorporation of functional fillers in the PLA matrix could improve the physical properties, as well as the surface characteristics of the matrix that are important for tissue engineering and artificial bone reconstruction. Motivated by our preliminary results [14], the purpose of this work is to investigate PLA–chitosan–keratin composites as biomaterials with potential applications in medicine, by means of mechanical and in vitro studies. Preliminary results regarding mechanical behavior and in vitro osteoblast response confirmed their potential for medical applications.
C.E. Tanase, I. Spiridon / Materials Science and Engineering C 40 (2014) 242–247
2. Materials and methods 2.1. Preparation of the composite film Before blend preparation, PLA (type 2002D, supplied by NatureWorks) pallets, chitosan (produced by Vanson Inc. with an average molecular weight of 1200 kDa and acetylation degree of 34%) and feather fibers were dried in a vacuum oven for 6 h at 50 °C. Compounding components were performed at 175 °C for 10 min at a rotor speed of 60 rpm using a fully automated laboratory Brabender station. Literature data [15] show that the addition of chitosan did not influence thermal properties and degree of crystallinity of PLA. Specimens for the mechanical characterization were prepared by compression molding using a Carver press. The compression molding was carried out at 175 °C with a pre-pressing step of 3 min at 50 atm and a pressing step of 2 min at 150 atm. A neat PLA sheet was prepared in the same conditions and acted as a reference. Composition and preparation of the samples are as follows: A111: 70% PLA and 30% chitosan; A121: 68% PLA, 30% chitosan and 2% keratin; A131: 66% PLA, 30% chitosan and 4% keratin. 2.2. DSC analysis Thermal characterization of composites has been performed with a TA Instruments Q20 Dynamic Scanning Calorimeter. All the samples were heated from 25 °C up to 200 °C with 10 °C/min, kept for 2 min and then cooled down to 25 °C with a cooling rate of 5 °C/min. An empty crucible was used as a reference material. All measurements were performed under N2 atmosphere. The degree of crystallinity of the PLA samples was obtained by dividing the melting enthalpy of the sample by 93.7 J/g [16], which is the estimated melting enthalpy of a pure PLA. The crystallinity of the composite materials was estimated as the function of PLA fraction in the composite and the melting enthalpy. 2.3. Characterization of composites Mechanical tests in terms of tensile and impact strength were performed. Thus, tensile strength measurements were carried out following ISO 527-2000 standard method, using an Instron 5 kN test machine operated at a crosshead speed of 30 mm/min. The unnotched Charpy impact strength was measured according to ISO 179-2010 using a Ceast apparatus provided with a hammer of 15 J. Seven specimens were tested for each material. The Vickers hardness tests were performed with a Shimadzu microhardness tester. A constant load of 4.903 N was applied for 12 s for all composite samples. Ten tests have been carried out for each sample and the average value is given.
243
were used as control surface. Cells were maintained under standard cell culture conditions (5% CO2, 95% humidity and 37 °C). The medium was changed every 2–3 days. Control cultures and seeded material samples were evaluated at days 1, 2 and 3 for cell viability/proliferation and at days 3 and 7 the samples were investigated via confocal laser scanning microscopy (CLSM; Leica SP2). 2.4.2. Cytotoxicity assay CellTiter 96®AQueous One Solution Cell Proliferation (Promega, Madison, WI) assay was used to study cell viability. The metabolic cell activity (an indirect measure of cytotoxicity) was measured by the conversion of MTS to formazan, which can be photometrically detected. MTS was mixed with fresh medium at the ratio of 1:10 and added to the cells for 1.5 h. The cells were placed in a CO2 incubator at 37 °C. After incubation time supernatants were transferred to a new microplate and optical density was measured photometrically at 492 nm in an ELISA 96 well-plate reader. All experiments were performed in triplicate and were treated and represented by their mean value and standard deviation parameters. The percentage cell viability was calculated according to the following equation: % cell viability ¼ 100 Abssample =Abscontrol ; where Abs sample is the absorbance of cells tested with various formulations and Abs control is the absorbance of reference cells (incubated on the culture media only). 2.4.3. Immunofluorescence analysis The cells were seeded 3.0 × 104 cells per cm2 in a 48-well plate. After preset time intervals, immunocytochemical staining was performed on whole samples. Briefly, the samples with cells were washed with PBS and fixed using a solution of 3.7% v/v paraformaldehyde. The fixed cells were permeabilized with buffered 0.5% v/v Triton X-100. Subsequently the cells were stained for nuclei with DAPI (0.4 μg mL−1) and cytoskeletal organization was revealed by actin labeling. F-actin filaments were stained with tetramethylrhodamine isothiocyanate (TRITC) conjugated phalloidin 0.2 μg mL−1 (Sigma, St. Louis, MO, USA). Labeled samples were examined by CLSM. 2.5. Statistics Statistical calculations and analyses were performed with the use of Prism 5 (GraphPad Software, Inc.) statistical software package. One-way analysis of variance (ANOVA) was employed to assess the statistical significance of results at a probability of error of 5% (*), 1% (**) and 0.1% (***). All experiments were repeated at least three times, and the results are presented as mean ± standard deviation (SD).
2.4. Biological tests 3. Results and discussions 2.4.1. Cell culture MG63 osteoblast-like cells (ATCC® no CRL-1427™, Rockville, MD-USA) were cultured in Dulbecco's modified Eagle's medium with 4500 mg L− 1 glucose (DMEM) from Gibco supplemented with 10% fetal bovine serum (FBS), 2 mM Glutamax I (Life Technologies) and 100 IU mL− 1 penicillin, 100 μg mL− 1 streptomycin. Cell cultures were sustained at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. The samples were sterilized using gamma irradiation according with ISO 11137—2.0 kGy at room temperature [17] and before using them in cell culture studies the samples were submerge in a diluted ethanol solution (70% v/v) for 15 min, followed by intensive washing in sterile phosphate buffer saline (PBS) solution. The cells were seeded over the sterile samples at a density of 3.0 × 104 cells per cm2 in a 48-well plate (Cellstar®, Greiner Bio-One) up to 7 days. Standard 48-well tissue culture plates (polystyrene)
3.1. DSC results The thermal behavior of the studied materials is presented in Fig. 1 and Table 1. Pure chitosan does not have melting properties and hence no endothermic peaks associated to melting process were detected, as other authors reported [18]. The thermogram of neat PLA revealed a glass transition temperature at 59.4 °C followed by a cold crystallization process peak at 127.8 °C and then melting peak at 152.97 °C, with an enthalpy of fusion of 13.62 J/mol. It was found that the addition of chitosan determined an increase of Tg to 60.2 °C, which can be explained by chitosan hindering movements of PLA chains. The presence of chitosan, which is a semicrystalline polymer, determines a loss of crystallinity in the PLA matrix, which drops from 20.61%
244
C.E. Tanase, I. Spiridon / Materials Science and Engineering C 40 (2014) 242–247
reflecting a solid-like behavior [14]. Keratin fibers serve as nucleation sites and probably polymer chains flexibility. The polymer volume immediately surrounding the keratin fibers has properties different from the bulk polymer [20]. During hardness test the stress transferability inside the biocomposite may be reduced due to the distance between the fibers decreases, i.e. work done will be gone to the deformation of matrix rather than fibers. 3.3. Water uptake The diffusion is a process by which small molecules are transported from one side of a system to another one as a result of random molecular motions. The water sorption is a complex mechanism which is influenced by swelling, molecular interactions, accessibility of sorption sites and material crystallinity and crosslinking, as well as of the presence of fillers. Using the above boundary conditions Fick's law has been used to give the time dependence of concentration, as shown below: Fig. 1. DSC thermograms (second run) of the studied materials.
to 18.05%. The presence of keratin in PLA chitosan system decreased Tg to 58.9 °C, while crystallinity increased to 19.17%. Apparently, the keratin decreases the Tg of PLA in PLA/chitosan system and facilitates crystallization of PLA. Probably crosslinking structures from keratin interfere with the crystallization process, many imperfect crystallites are formed and thus keratin serves as nucleating agents. 3.2. Mechanical properties Mechanical properties are important to the design biomaterials for medical applications. Chitosan incorporation into the PLA matrix improved Young modulus and decreased the tensile strength of PLA (Fig. 1). Polar interactions between ester functional groups of PLA and amine groups of chitosan were expected, but it is possible that processing conditions applied in our study may not have been sufficient for a reaction to occur between the chitosan and PLA, with significant effects on the mechanical properties. The addition of keratin resulted in an increase of impact strength and decrease of tensile properties compared to PLA/chitosan composite. The feather fibers appear to act as stress concentrators in the polymer matrix, thus reducing the crack initiation energy and consequently enhancing the impact strength of the composites. The structure of keratin, the primary constituent of chicken feathers, affects the chemical durability. Because of extensive crosslinking and strong covalent bonding within its structure, keratin provides high resistance to degradation [19]. The possible increase of the toughness may be due to microplastic deformation created around the feather particles. It is well-known that hardness and modulus of a polymeric material depend on its structure. As can be seen in Fig. 2, a significant rise in hardness has been obtained by adding of chitosan to PLA matrix. This suggests that the fiber surface becomes rougher. According with hardness values, the Young modulus of PLA chitosan composite is higher than that of PLA matrix. The incorporation of chitosan with or without keratin stabilized PLA that became consequently less brittle. The rheological studies have revealed that a decrease in the arrangement of PLA polymer chains as the content of keratin increases, A131 material Table 1 DSC parameters of PLA and composite materials. Sample
Tg (°C)
Tm (°C)
ΔHm (J/g)
X (%)
PLA A111 A123
59.4 60.2 58.9
152.97 153.45 150.67
19.32 24.17 26.98
20.61 18.05 19.17
Tm: melting temperature; ΔHm: melting enthalpy; X: crystallinity index; Tg: transition temperature.
2 2 Dð2nþ1Þ π t Mt 8 X 1 − l2 ¼ 1− 2 e 2 M∞ π ð2n þ 1Þ
ð1Þ
where: t is the time measured from when the concentration is changed, l is the plate thickness, Mo is the initial equilibrium mass and ΔM is the change in the mass from Mo to the new equilibrium mass Mt. The Eq. (1) describes the change in mass due to the movement of the diffusing species responding to the sudden change in pressure/humidity around the sample. The half thickness (l/2) is used as the diffusion length above since in a typical sorption experiment the entire plate is measured and the substance is assumed to diffuse uniformly to the central plane (Table 2). For Mt/M∞ b 0.5 rffiffiffiffiffiffiffiffi Mt 4 D·t ¼ · l π M∞
ð2Þ
and for Mt/M∞ N 0.5: Mt 8 −Dπ2 t ¼ 1− 2 ·e l2 : M∞ π
ð3Þ
Literature data reported that the PLA degradation occurs principally at the surface because of the absorption gradient of water [4], the evolution of this process being explained either via a bulk erosion mechanism, starting at the surface [21] or as a heterogeneous process [22]. It is well known that the presence of chitosan influences water uptake and diffusion coefficients [23]. The larger diffusion coefficient could be due to the presence of amorphous chitosan that create a new pathway for the water vapor to diffuse. Thus, in material comprising PLA and chitosan, both D2 and K2 (Mt/M∞ N 0.5) were less affected, while D1 and K1 were slowly affected. Addition of 4% keratin has improved the K1 value, as well as D1 (for for Mt/M∞ b 0.5), while for D2 and K2 (for Mt/M∞ N 0.5) slowly decreased. Our results suggest that water uptake process is more intense at the surface of material. We suppose that water molecule penetrates with difficulty the core of material due to the strong linkages between components of the studied materials, confirmed by rheological studies [14]. Literature data show that PLA has advantage to be processing in shapes for orthopedic applications and fabricated into scaffolds for replacement and regeneration of tissues, or devices for controlled delivery of biomolecules [24,25]. Also, it must be mentioned that, in vivo, the performance of the biomaterial-based structure is derived not only from the mechanical resistance of the biomaterial itself, but also from the complex mixture of biomaterial [26], surface
C.E. Tanase, I. Spiridon / Materials Science and Engineering C 40 (2014) 242–247
245
Fig. 2. Mechanical properties of composite materials. Data are presented as the mean ± SD; n = 7; ***p b 0.001 compared with PLA (PLA served as control).
properties being important to induce cellular attachment, differentiation and proliferation [27]. 3.4. Behavior of MG63 osteoblast-like cells The MTS tetrazolium compound is reduced by cells into a colored formazan product that is soluble in tissue culture medium. The quantity of the formazan product as measured at 492 nm is directly proportional to the number of living cells in a culture. The results of viability assay are represented in Fig. 3. As it can be seen the cell viability percent is comparable with the control (cell viability 100%) at 24 h and 48 h, respectively. After the second day when the cultures reached confluence and formed a dense cell layer, this layer was easily lost during routine medium change. This phenomenon explains the decrease in MTS reduction values observed at day 3. Even in this case the cell viability/proliferation reveals values appropriated to the control. From these results we can conclude that the cell viability/proliferation of the MG63 osteoblastlike cells seeded on the samples containing PLA–chitosan–keratin is similar to the control with a slightly variation which might be influenced by the material composition. Therefore, we can conclude that PLA–chitosan–keratin composites do not affect cell viability and indicate the potential use in bone tissue engineering. The ratio of components in composite materials used in this study was appropriate to
support the MG63 growth and offer proper space and support for cell proliferation. On the other hand, to evaluate the proliferation, distribution and cell adhesion of the cells, it was necessary to use CLSM. The CLSM images of the samples seeded with MG63 cells can be visualized in Fig. 4 at various time points, 72 h and 168 h respectively. The cytoskeletal organization is generally determined by actin staining with fluorescence labeledphalloidin and is used to evaluate the motility, spreading and shape of the cells [28]. Regarding the cell distribution and proliferation after 72 h on the samples, it can be observed that for samples A111 (Fig. 4A) and for samples A131 (Fig. 4C) the MG63 cells cover almost the entire sample surface. For the same time point (72 h) regarding sample A121 (Fig. 4B), it can be observed that MG63 cells present a good distribution but not similar with the other samples. At this time of culture, CLSM shows a suggestive proliferative cell population (Fig. 4-B—circled cells). These data are correlated with the results obtained in MTS assays. At 168 h after culture the cells formed multilayer's of flattened sheets,
Table 2 Diffusion coefficient values calculated form normalized mass changing vs. time.
A131 A121 A111 PLA a
K1a, b0.5
K2a, N0.5
l cm
D1, Mt/M∞ b 0.5, Eq. (2) cm2/s
D2, Mt/M∞ N 0.5, Eq. (3) cm2/s
1.29E−03 7.24E−04 9.84E−04 7.14E−04
−0.00075520 −0.00091237 −0.00081704 −0.00088931
0.098 0.101 0.093 0.089
2.4321E−06 1.4508E−06 1.6704E−06 1.1095E−06
7.3515E−07 7.4336E−07 7.1626E−07 7.1400E−07
K1 and K2 are the slopes of linearized Eqs. (2) and (3), respectively.
Fig. 3. Hardness as function of material composition.
246
C.E. Tanase, I. Spiridon / Materials Science and Engineering C 40 (2014) 242–247
materials reveal estimable biocompatibility features, demonstrating their potential use as substitute materials for bone tissue engineering. This preliminary investigation shows that the composition of PLA/chitosan/keratin materials is important to obtain materials with appropriate properties and careful tailoring of the keratin should modify mechanical properties of material and cellular adhesion. In the future follow-up study, more investigations will be done to further enhance the efficiency of cell bone formation in PLA/chitosan/keratin materials (Fig. 5).
4. Conclusions
Fig. 4. Mitochondrial activity measured via the MTS assay. Data are depicted as percentage of the untreated control. Triplicates were performed and the data represent means ± SD. *p b 0.05, **p b 0.01 and ***p b 0.001 compared to the untreated control.
covering completely the material surface for all samples (D—sample A111, E—sample A121 and F—sample A131). From Fig. 4 it can be seen that the cells have a wide connection with each other throughout cytoplasmic extensions, presenting an elongated shape, thereby emphasizing the distribution and proliferation of cells at different time points. The physical–chemical and topographical features of the biomaterial surface could influence the distribution of focal contact and cytoskeleton organization [29,27]. PLA–chitosan–keratin samples showed increased cell attachment and maintenance of cell numbers over the time periods (up to 168 h). As a final point the MTS viability/proliferation assays and CLSM analysis highlight a good cell viability, proliferation and distribution pointing out a good biocompatibility of the PLA–chitosan–keratin samples in contact with MG63 osteoblast-like cells. Hence these composite
In summary, new biomaterials based on PLA, chitosan and keratin composites were designed and evaluated. These composites with improved Young modulus and decreased tensile strength, significant increase in hardness (compared to PLA) and a good uptake of the surface properties were evaluated with regards of in vitro behavior. This study showed that PLA, chitosan and keratin composites support osteoblast attachment and proliferation during short-term culture indicating that these composites might be promising materials for medical application.
References [1] R. Langer, D.A. Tirrell, Designing materials for biology and medicine, Nature 428 (6982) (2004) 487–492. [2] A.R. Boccaccini, J.J. Blaker, Bioactive composite materials for tissue engineering scaffolds, Expert Rev. Med. Devices 2 (3) (2005) 303–317. [3] R. Bhardwaj, A.K. Mohanty, Advances in the properties of polylactides based materials: a review, J. Biobased Mater. Bioenergy 1 (2) (2007) 191–209. [4] C. Saiz-Arroyo, Y. Wang, M.A. Rodriguez-Perez, N.M. Alves, J.F. Mano, In vitro monitoring of surface mechanical properties of poly( L -lactic acid) using microhardness, J. Appl. Polym. Sci. 105 (2007) 3858–3864. [5] Qizhi Chen, Chenghao Zhu, George A. Thouas, Progress and challenges in biomaterials used for bone tissue engineering: bioactive glasses and elastomeric composites, Prog. Biomater. 1 (2) (2012) 1–22.
Fig. 5. CLSM images (DAPI and TRITC) at 72 h (A—samples A111, B—samples A121 and C—samples A131) and 168 h (D—samples A111, E—samples A121 and F—samples A131) after MG63 cells cultured on PLA–chitosan–keratin samples.
C.E. Tanase, I. Spiridon / Materials Science and Engineering C 40 (2014) 242–247 [6] R. Jayakumar, M. Prabaharan, P.T. Sudheesh Kumar, S.V. Nair, H. Tamura, Biomaterials based on chitin and chitosan in wound dressing applications, Biotechnol. Adv. 29 (2011) 322–337. [7] In-Yong Kim, Seog-Jin Seo, Hyun-Seuk Moon, Mi-Kyong Yoo, In-Young Park, Bom-Chol Kim, Chong-Su Cho, Chitosan and its derivatives for tissue engineering applications, Biotechnol. Adv. 26 (2008) 1–21. [8] Scott P. Noel, Harry Courtney, Joel D. Bumgardner, Warren O. Haggard, Chitosan films. A potential local drug delivery system for antibiotics, Clin. Orthop. Relat. Res. 466 (2008) 1377–1382. [9] K. Tomihata, Y. Ikada, In vitro and in vivo degradation of films of chitin and its deacetylated derivatives, Biomaterials 18 (1997) 567–575. [10] C.E. Tanase, M.I. Popa, L. Verestiuc, Biomimetic chitosan–calcium phosphate composites with potential applications as bone substitutes: preparation and characterization, J. Biomed. Mater. Res. B Appl. Biomater. 100 (3) (2012) 700–708. [11] P.U. Rokkanen, O. Bostman, E. Hirvensalo, E.A. Makela, E.K. Partio, H. Patiala, S.I. Vainionpaa, K. Vihtonen, P. Tormala, Bioabsorbable fixation in orthopaedic surgery and traumatology, Biomaterials 21 (2000) 2607–2613. [12] C.A. Mills, M. Navarro, E. Engel, E. Martinez, M.P. Ginebra, J. Planell, A. Errachid, J. Samitier, Transparent micro- and nanopatterned poly(lactic acid) for biomedical applications, J. Biomed. Mater. Res. A 76 (2006) 781–787. [13] K.M. Arai, R. Takahashi, Y. Yokote, K. Akahane, Amino acid sequence of feather keratin from fowl, J. Biochem. 132 (1983) 501–507. [14] Iuliana Spiridon, Oana Maria Paduraru, Mirela Fernanda Zaltariov, Raluca Nicoleta Darie, Influence of keratin on polylactic acid/chitosan composite properties. Behavior upon accelerated weathering, Ind. Eng. Chem. Res. 52 (29) (2013) 9822–9833. [15] J. Bonilla, E. Fortunati, M. Vargas, A. Chiralt, J.M. Kenny, Effects of chitosan on the physicochemical and antimicrobial properties of PLA films, J. Food Eng. 119 (2013) 236–243. [16] R.G. Liao, B. Yang, W. Yu, C.X. Zhou, Isothermal cold crystallization kinetics of polylactide/nucleating agents, J. Appl. Polym. Sci. 104 (1) (2007) 310–317.
247
[17] M. Bernkopf, Sterilisation of bioresorbable polymer implants, Med. Device Technol. 18 (3) (May-June 2007). [18] C. Swapna Joseph, K.V. Harish Prashanth, N.K. Rastogi, A.R. Indiramma, S. Yella Reddy, K.S.M.S. Raghavarao, Optimum blend of chitosan and poly-(ε-caprolactone) for fabrication of films for food packaging applications, Food Bioprocess Technol. 4 (2011) 1179–1185. [19] S.C. Mishra, N.B. Nayak, A. Satapathy, Investigation on biowaste reinforced epoxy composites, J. Reinf. Plast. Compos. 29 (19) (2010) 3016. [20] J.R. Barone, Polyethylene/keratin fiber composites with varying polyethylene crystallinity, Compos. Part A 36 (2005) 1518–1524. [21] H. Tsuji, A. Mizuno, Y. Ikada, J. Appl. Polym. Sci. 77 (2000) 1452. [22] S.M. Li, H. Garreau, M. Vert, J. Mater. Sci. Mater. Med. 1 (1990) 131. [23] Vitor M. Correlo, Elisabete D. Pinho, Iva Pashkuleva, Mrinal Bhattacharya, Nuno M. Neves, Rui L. Reis, Water absorption and degradation characteristics of chitosan-based polyesters and hydroxyapatite composites, Macromol. Biosci. 7 (2007) 354–363. [24] J.M. Anderson, M.S. Shive, Biodegradation and biocompatibility of PLA and PLGA microspheres, Adv. Drug Deliv. Rev. 28 (1997) 5–24. [25] M. Denti, P. Randelli, D. Lo Vetere, M. Moioli, M. Tagliabue, Bioabsorbable interference screws for bone-patellar tendon-bone anterior cruciate ligament reconstruction: clinical and computerized tomography results of four different models. A prospective study, J. Orthop. Traumatol. 5 (2004) 151–155. [26] Y. Luo, G. Engelmayr, D.T. Auguste, L.S. Ferreira, J.M. Karp, R. Saigal, R. Langer, Threedimensional scaffolds, in: R. Lanza, R. Langer, J.P. Vacanti (Eds.), Principles of Tissue Engineering, 3rd ed., Elsevier, Amsterdam, 2007, pp. 359–373. [27] K. Anselme, Osteoblast adhesion on biomaterials, Biomaterials 21 (7) (2000) 667–681. [28] D.E. Ingber, I.I. Tensegrity, How structural networks influence cellular information processing networks, J. Cell Sci. 116 (2003) 1397–1408. [29] K. Anselme, M. Bigerelle, B. Noel, E. Dufresne, D. Judas, A. Iost, P. Hardouin, Qualitative and quantitative study of human osteoblast adhesion on materials with various surface roughnesses, J. Biomed. Mater. Res. 49 (2000) 155–166.
