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Characterization of silk fibroin 3D composites modified by collagen
Journal of Molecular Liquids 215 (2016) 323–327
Contents lists available at ScienceDirect
Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Characterization of silk fibroin 3D composites modified by collagen A. Sionkowska a,⁎, K. Lewandowska a, M. Michalska a, M. Walczak b a b
Department of Chemistry of Biomaterials and Cosmetics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarin 7, 87-100 Toruń, Poland Department of Environmental Microbiology and Biotechnology, Faculty of Biology and Environment Protection Nicolas Copernicus University in Torun, Gagarin 9, 87-100 Torun, Poland
a r t i c l e
i n f o
Article history: Received 30 May 2015 Received in revised form 24 November 2015 Accepted 14 December 2015 Available online xxxx Keywords: Silk fibroin Collagen Biomaterials
a b s t r a c t Silk fibroin 3D composites with the addition of collagen were prepared through the lyophylisation process. The structure of composites was studied by ATR-FTIR technique and was observed by a scanning electron microscope. Mechanical properties were studied and compared with those of a silk fibroin 3D sponge. Moreover, miscibility studies of silk fibroin with collagen blends of different compositions were investigated using the viscometric method before the lyophylisation process. Viscometric studies indicate that silk fibroin/collagen blends are miscible at any composition at 25 °C. Scanning electron microscopy observations showed that in the lyophilisation process of silk fibroin/collagen blend a porous material can be obtained. The results showed that the addition of collagen to silk fibroin led to the decrease of tensile strength. However, Young modulus increased with an increasing amount of collagen. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Biomaterials play a very important role in tissue engineering [1]. 3D composites for biomedical application should have good biocompatibility, biodegradability and suitable mechanical properties. Good porosity and pore size are also important, they provide the space for cells to grow, transporting metabolites, nutrients and signal molecules. Porosity gives space and temporary mechanical support for tissue growth [1–3]. For obtaining scaffolds for medical application both natural and synthetic polymers were tested [4,5]. To improve and optimize both chemical and physical properties of biomaterials, polymers used for a scaffold can be mixed together [6–8]. For this purpose also two natural polymers can also be mixed. However, the appropriate solvent is required for the preparation of such blends. Miscible blending of two biopolymers with different physicochemical characteristics may lead to the development of a new biomaterial with unique properties that may present the advantages of each polymer and compensate for the disadvantages of each one [9]. Silk fibroin is a natural polymer produced by various species of silkworm and spiders [2]. Bombyx mori cocoons are usually used as a source of silk fibroin. Raw silk contains two proteins, silk fibroin and sericine. However, for the preparation of materials for biomedical application the degummed sericine can be removed [10]. Silk fibroin due to its good mechanical properties and biocompatibility has been used as a biomedical material for a long time [4,11]. The question is, whether silk fibroin can be mixed with another biopolymer, for example with collagen. Collagen is the most abundant protein in mammals. This natural polymer constitutes more than one-third of protein weight in tissue ⁎ Corresponding author. E-mail address: [email protected] (A. Sionkowska).
http://dx.doi.org/10.1016/j.molliq.2015.12.047 0167-7322/© 2015 Elsevier B.V. All rights reserved.
[12]. Collagen can be obtained from different sources such as cow skin or muscles, rat tail tendons, fish scales or skin and even from sea sponges [13]. Materials based on collagen are commonly used in tissue engineering [14,15]. Several materials based on two biopolymers, such as collagen and silk fibroin have already been prepared [16–21]. Composite fibres of collagen and dragline silk protein were also prepared by an electrospinning technique [16]. Some materials were prepared by dissolving collagen in a silk fibroin solution in 60 °C, however, it may cause adverse effects to collagen with a denaturation temperature below 60 °C [3,20,21]. The properties of collagen/silk fibroin scaffolds can be modified using methanol [17,20,21]. It is believed that those materials can be applied in biomedical fields. Although three-dimensional fibroin scaffolds have been prepared using a freeze–drying method previously, they still cannot meet the requirements of tissue engineering and further ideas and study are required. In particular, the results regarding the miscibility of these biopolymers were poorly published. The aim of this work was to study the interactions between silk fibroin and collagen by viscometry technique and to prepare 3D composites based on such blends. Our preliminary study showed that silk fibroin and collagen can be miscible depending on the pH of the solution and ionic strength. 2. Materials and methods 2.1. Preparation of silk fibroin and collagen mixtures Silk fibroin was obtained from B. mori cocoons (Jedwab Polski Sp. Z o.o. company) according to the method described by Kim et al. with slight modifications [22]. Worms were removed from cocoons, then empty cocoons were boiled for 1 h in an aqueous solution of 0.5%
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A. Sionkowska et al. / Journal of Molecular Liquids 215 (2016) 323–327
Na2CO3 twice. After the solution was removed, cocoons were boiled in 5% alkaline soap solution for 30 min and then for 20 min in distilled water. This procedure was repeated three times. After removing the sericine, silk fibroin was dried and dissolved in CaCl2:H2O:C2H5OH (molar ratio 1:8:2) at 80 °C for 4 h. The solution of silk fibroin was prepared with a concentration of 4% wt. Collagen was obtained from rat tail tendons. Tendons were washed in distilled water and dissolved in 0.1 M acetic acid for three days in 4 °C [23], the undissolved parts were removed by centrifugation for 10 min at 10,000 rpm. The completely frozen mixtures were lyophilized at −55 °C and 5 Pa for 48 h (ALPHA 1–2 LD plus, CHRIST, Germany). 1% wt solution was prepared by dissolving collagen in 0.1 M acetic acid. Solutions of these two polymers were mixed in weight ratios of silk fibroin to collagen: 90:10, 75:25 and 50:50. Pure silk fibroin was left as a control sample. Each sample was dialyzed in a cellulose tube (SERVAPOR) against distilled water for three days with deionized water changed every day. Finally, the dialysis mixtures were placed in a polystyrene container. The scaffolds were obtained during the lyophylization process for two days. The scaffolds obtained are shown in Fig. 1.
2.2. IR spectroscopy The interaction between functional groups of silk fibroin and functional groups of collagen and the structure of composites were evaluated by attenuated total reflection infrared spectroscopy using Nicolet iS10 equipped with an ATR device with diamond as a crystal. All spectra were recorded in absorption mode at 4 cm− 1 intervals and 64 scans.
2.3. Viscometric measurements Viscometric measurements of dilute polymer solution (c b 0.5%) were carried out in a controlled thermostatic bath at 25 ± 0.1 °C using the Ubbelohde capillary viscometer. The flow times were recorded with an accuracy ± 0.01 s. The flow time of each solution was determined as the average of several readings. Before measurements were taken the solutions were filtered through G1 sintered glass filters. The intrinsic viscosity and the interaction parameter values were determined according to the Heller procedure [24,25] from data obtained for solutions at 5 concentrations. The miscibility is estimated by comparison of the experimental and ideal values of bm and [η]m. The values of interaction parameters (bm) were obtained using the same methods as shown in previous papers [25,26].
2.5. Scanning electron microscopy The microstructure of scaffolds was studied using Scanning Electron Microscope (SEM) (LEO Electron Microscopy Ltd., England). Scaffolds were cut with a razor scalpel after being frozen in liquid nitrogen for 3 min. 3. Results and discussion 3.1. IR spectroscopy Infrared spectra were registered for sponge of silk fibroin and its blends with collagen. ATR-FTIR spectra of specimens studied are shown in Fig. 2. Silk fibroin and collagen are proteins, so both of them give similar spectra in FTIR spectroscopy. Although the miscibility of several polymers in solid state can be confirmed by FTIR spectra, in the blends of two natural polymers with a similar structure this technique is not a very powerful one. In Table 1 the position of typical bands in ATR-FTIR spectra for silk fibroin, collagen and their mixtures are presented. Silk fibroin displays bands at 1645, 1537 and 1241 cm−1, which are characteristic of the amide I, II and III bands of proteins. The amide I absorption arises predominantly from protein amide C_O stretching vibrations, the amide II absorption is made up of amide N–H bending vibrations and C–N stretching vibrations (60% and 40% contribution to the peak respectively); the amide III peak is complex, consisting of components from C–N stretching and N–H in plane bending from amide linkages, as well as absorptions arising from wagging vibrations from CH2 groups from the glycine backbone and proline side-chains. The amide groups have a characteristic absorption amide bands A and B in the region of 3400–3500 cm−1, however, these bands can be masked by the broad absorption band from the –OH group present in proteins. The amide A band for collagen was observed at 3381 cm−1, whereas for silk fibroin it was observed at 3283 cm−1. For silk fibroin/collagen blends with weight a ratio 50/50 it was observed at 3283 cm−1, the position of this band was similar to the position of amide A observed for silk fibroin. The position of amide I and amide II for silk fibroin and its blends with collagen were also similar. Small differences could only be observed for amide III. It seems that the FTIR technique cannot detect the inter-molecular interactions between silk fibroin and collagen. Usually the inter-molecular interaction between two different polymers through hydrogen bonding can be characterized by FTIR, because the specific interaction affects the local electron density and a corresponding frequency shift can be observed. For silk fibroin/collagen blends, a clear shift of amide bands was not observed. For this reason the viscometry studies of the interaction between silk fibroin and collagen in solution before the liophylisation process were conducted. On the basis of viscometric study one can assess the miscibility of two polymers in the blend in common solvent.
2.4. Mechanical properties 3.2. Viscometric measurements. Mechanical properties of SF/Col sponges were tested using a Zwick&Roell testing machine. 5 samples of each kind were placed between two discs and pressed. Young Modulus and tensile strength were then measured.
All the plots of the reduced viscosity versus polymer concentration (curves not shown) show linear behaviour in the range of concentration studied, indicating that the intrinsic viscosity can be determined by
Fig. 1. Scaffolds obtained from: a) silk fibroin, b) SF/Coll (90:10), c) SF/Coll (75:25), d) SF/Coll (50:50).
A. Sionkowska et al. / Journal of Molecular Liquids 215 (2016) 323–327
Fig. 2. ATR-FTIR spectra of silk fibroin (SF), collagen (Col) and their mixtures with different SF/Col weight ratios.
linear extrapolation to zero concentration. The [η]exp m values were obtained according to Heller's procedure, which were acquired from the inverse of the Huggins and Kraemer equations [24]. This method gives some advantages such as high accuracy of determination of the intrinsic viscosity and slope constants because the slopes of the plots are smaller compared to the slopes of Huggins and Kraemer plots [24,25]. Using the criteria as proposed by Krigbaum et al. [27] and Garcia et al. [28] the parameters Δ[η]m and Δbm have been computed to establish the degree of miscibility in the silk fibroin/collagen blends. The plots of the miscibility parameter Δ[η]m and Δbm against the weight fraction of silk fibroin are shown in Figs. 3-5. It is obviously observed that the intrinsic viscosities of SF/Col blends do not follow the additive rule particularly well (Fig. 3). The experimental intrinsic viscosities are higher than the ideal values exhibiting positive deviation. According to Δ[η] criterion by Garcia [28] all silk fibroin/collagen blends are immiscible. This synergy could be explained by intermolecular interactions. The attractive force and/or electrostatic interactions between the polymeric components and solvent molecules in the blend solutions may cause the increase of intrinsic viscosity. Moreover, on the basis of these results, it is not possible to make a generalized conclusion about the miscibility of two polymers. This is due to the fact that the additivity rules, mentioned above, can be fulfilled in the solutions of polymers (polymers in solvent) for both, the immiscible and miscible polymer blends [25,29,30]. The plots of the viscosity interaction parameters versus the silk fibroin weight fraction in silk fibroin/collagen blends are illustrated in Fig. 5. As it can be seen from this figure the values of parameters are positive for all the investigated SF/Col blends. According to the two criteria, one can conclude that the SF/Col blends are miscible in all compositions. The miscibility is a result of the specific interaction between two polymeric components in solution.
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Fig. 3. Intrinsic viscosity ([η]) dependence on weight fraction of silk fibroin (wSF) in SF/Col blends. Dotted line indicates the ideal values of [η]id m which follow the Garcia criterion [28].
In the lyophilisation process of the silk fibroin/collagen blend the porous material was obtained. SEM observations showed that the pore size was from 20 to 150 μm for silk fibroin sponge. The porosity for silk fibroin sponges increased with the increase of addition of collagen in the mixture. However, the microstructure of silk fibroin sponge with the addition of collagen is less regular than without collagen and shows rather layered (sheet like) structures. It was also reported previously, that uniform pore distributions in a scaffold improved the mechanical properties, whereas the layered sheet structure of 3D sponges caused low tensile strength [19,20]. Biocompatibility of sponges needs to be confirmed by in vitro test. 3.4. Mechanical properties Mechanical properties of dry composites were tested at room temperature. Results of Young's modulus are shown in Fig. 7. Sponges made of pure silk fibroin (SF) have the lowest value of Young's modulus. Adding collagen (Col) to silk fibroin caused increasing elasticity of sponges. The highest value of Young's modulus was observed for sponges with the composition SF75:25Col. Our results are in sound agreement with the results published by Tiyaboonchai et al. [21] and Lu et al. [20] regarding silk fibroin/collagen scaffolds treated with methanol. As methanol treatment can cause changes in the secondary structure of proteins, we avoided methanol treatment in our studies. Results for tensile strength are shown in Fig. 8. The highest value of tensile strength was observed in sponges made of silk fibroin. However, several properties of scaffolds depend on the pH conditions of scaffold preparation [20]. Samples modified with collagen are characterized by lower tensile strength — even ten times lower than the samples made of pure silk fibroin (SF). The specimens (Fig. 1) were elastic and after
3.3. Scanning electron microscopy. Microstructure of sponges were tested using scanning electron microscopy (SEM). Images were made under 150 magnification (Fig. 6). Table 1 Characteristic bands in ATR-FTIR spectra and their surface for functional groups in silk fibroin sponge (SF) and mixtures (SF/Col) in different weight ratios. Observed vibrational frequencies wavenumber [cm− 1] Assignments
SF
SF:Col 90:10
SF:Col 75:25
SF:Col 50:50
H2O adsorbed, N—H (amide A) C—H (amide B) H2O adsorbed, CO (amide I) N—H (amide II), C—N C—N, N—H (amide III)
3283 3078 1645 1537 1241
3277 3067 1641 1516 1236
3294 3079 1650 1540 1242
3283 3078 1648 1536 1238
Fig. 4. Plot of criterion of Δ[η] versus weight fraction of silk fibroin (wSF) in SF/Col blends.
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Fig. 5. Values of viscosity interaction parameters, Δbm, versus weight fraction of silk fibroin (wSF) in SF/Col blends. * — the value of bid m determined according to Krigbaum and Wall [25], ** —the value of bid m determined according to Garcia et al. [28].
compression by mechanical testing machine could turn back to the previous shape. The sponges based on the blend of silk fibroin and collagen have sufficient mechanical integrity to resist handling during implantation and in vivo loading. However, the mechanical properties depend on the water content in the specimen and even depend on the relative humidity of the environment in the laboratory, where the mechanical testing is done. For this reason the measurements of mechanical properties were conducted in the same relative humidity and in the same temperature. After treatment with methanol the specimens lost their elasticity and became more stiff and fragile.
Fig. 7. Young's Modulus of pure silk fibroin (SF) and silk fibroin modified with collagen (Col).
4. Conclusions In the lyophilisation process 3D porous material based on the blends of silk fibroin and collagen can be obtained. Viscometric method showed, that before the lyophilisation process the blends were miscible at any composition. Mechanical properties of the sponge were modified with the addition of collagen to silk fibroin. The addition of collagen to silk fibroin led to the decrease of tensile strength and to the increase of Young modulus. Sponges made of silk fibroin/collagen mixtures can be interesting materials for tissue engineering as scaffold to temporarily
Fig. 6. SEM images of (a) SF × 150, (b) SF90:10Col × 150, (c) SF75:25Col × 150, (d) SF50:50Col × 150.
A. Sionkowska et al. / Journal of Molecular Liquids 215 (2016) 323–327
Fig. 8. Tensile strength of pure silk fibroin (SF) and silk fibroin modified with collagen (Col).
support the formation of new tissue and organs. They can be also considered for wound dressing materials. Acknowledgments Financial support from the National Science Centre (NCN, Poland) (2015/18/M/ST5/00634) is gratefully acknowledged. References [1] A. Sionkowska, J. Kozłowska, Properties and modification of porous 3-D collagen/hydroxyapatite composites, Int. J. Biol. Macromol. 52 (2013) 250–259. [2] A. Sionkowska, A. Płanecka, Preparation and characterization of silk fibroin/chitosan composite sponges for tissue engineering, J. Mol. Liq. 178 (2013) 5–14. [3] Q. Lv, Q. Feng, K. Hu, F. Cui, Three-dimensional fibroin/collagen scaffolds derived from aqueous solution and the use for HepG2 culture, Polymer 46 (2005) 12662–12669. [4] J. Zhou, C. Cao, X. Ma, J. Lin, Electrospinning of silk fibroin and collagen for vascular tissue engineering, Int. J. Biol. Macromol. 47 (2010) 514–519. [5] G. Wang, X. Hu, W. Lin, C. Dong, H. Wu, Electrospun PLGA-silk fibroin-collagen nanafibrous scaffolds for nerve tissue engineering, In Vitro Cell. Dev. Biol. Anim. 47 (2011) 234–240. [6] M.A. de Moraes, M.F. Silva, R.F. Weska, M.M. Beppu, Silk fibroin and sodium alginate blend: miscibility and physical characteristics, Mater. Sci. Eng. C 40 (2014) 85–91. [7] J. Li, D. Barrow, H. Howell, S. Kalachandra, In vitro drug release study of methacrylate polymer blend system: effect of polymer blend composition, drug loading and solubilizing surfactants on drug release, J. Mater. Sci. Mater. Med. 21 (2010) 583–588. [8] P. Jetbumpenkul, P. Amornsudthiwat, S. Kanokpanont, S. Damrongsakkul, Balanced electrostatic blending approach – An alternative to chemical crosslinking of Thai silk fibroin/gelatin scaffold, Int. J. Biol. Macromol. 50 (2012) 7–13.
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[9] A. Sionkowska, Current research on the blends of natural and synthetic polymers: review”, Prog. Polym. Sci. 36 (2011) 1254–1276. [10] B.N. Prakash, S. Nivedita, R. Subrata, Use of sericin of Bombyx mori in the synthesis of silver nanoparticles, their characterization and application, Indian J. Fibre Text. Res. 36 (2011) 168–171. [11] X. Fei, M. Jiao, X. Du, Y. Yang, R. Zhang, Z. Shao, X. Zhao, X. Chen, Green synthesis of silk fibroin-silver nanoparticle composites with effective antibacterial and biofilmdisrupting properties, Biomacromolecules 14 (2013) 4483–4488. [12] A.M. Ferreira, P. Gentile, V. Chiono, G. Ciardelli, Collagen for bone tissue regeneration—review, Acta Biomed. 8 (2012) 3191–3200. [13] A. Sionkowska, B. Kaczmarek, K. Lewandowska, Modification of collagen and chitosan mixtures by the addition of tannic acid, J. Mol. Liq. 199 (2014) 318–323. [14] T. Muthukumar, P. Prabu, K. Ggosh, T.P. Sastry, Fish scale collagen sponge incorporated with Macrotyloma uniflorum plant extract as possible wound/burn dressing material, Colloids Surf. B: Biointerfaces 113 (2014) 207–212. [15] A. Sionkowska, Collagen based materials for biomedical applications: preparation and properties, Mater. Sci. Forum 706-709 (2012) 595–599. [16] B. Zhu, W. Li, R.V. Lewis, C.U. Segre, R. Wang, E-spun composite fibers of collagen and dragline silk protein: fiber mechanics, biocompatibility, and application in stem cell differentiation, Biomacromolecules 16 (2015) 202–213. [17] Y. Ni, Y. Jiang, J. Wen, H. Yu, W. Li, Construction of a functional silk-based biomaterial complex with immortalized chondrocytes in vivo, J. Biomed. Mater. Res. A 102 (2014) 1071–1078. [18] K. Sun, H. Li, R. Li, D. Li, C. Xu, Silk fibroin/collagen and silk fibroin/chitosan blended three-dimensional scaffolds for tissue engineering, Eur. J. Orthop. Surg. Traumatol. 25 (2014) 243–249. [19] S.Y. Kwon, J.W. Chung, H.J. Park, J.K. Park, Y.K. Seo, Silk and collagen scaffolds for tendon reconstruction, Proc. Inst. Mech. Eng. H J. Eng. Med. 228 (2014) 388–396. [20] Q. Lu, Q. Feng, K. Hu, F. Ciu, Preparation of three-dimensional fibroin/collagen scaffolds in various pH conditions, J. Mater. Sci. Mater. Med. 19 (2008) 629–634. [21] W. Tiyaboonchai, P. Chomchalao, S. Pongcharoen, M. Sutheerawattananond, P. Sobhon, Preparation and characterization of blended bombyxmori silk fibroin scaffolds, Fibers Polym. 12 (2011) 324–333. [22] U.J. Kim, J. Park, H.J. Kim, M. Wada, D.L. Kaplan, Three-dimensional aqueous-derived biomaterial scaffold from silk fibroin, Biomaterials 26 (2005) 2775–2785. [23] A. Sionkowska, J. Kozłowska, Characterization of collagen/hydroxyapatite composite sponges as a potential bone substitute, Int. J. Biol. Macromol. 47 (2010) 483–487. [24] W. Heller, Treatment of viscosity data on polymer solutions (an analysis of equations and procedures): I. Intrinsic viscosity and limiting slope constants, J. Colloid Sci. 9 (1954) 547–573. [25] K. Lewandowska, Viscometric studies in dilute solution mixtures of chitosan and microcrystalline chitosan with poly(vinyl alcohol), J. Solut. Chem. 42 (2013) 1654–1662. [26] A. Sionkowska, K. Lewandowska, A. Płanecka, Miscibility and physical properties of chitosan and silk fibroin mixtures, J. Mol. Liq. 198 (2014) 354–357. [27] W.R. Krigbaum, F.T. Wall, Viscosities of binary polymeric mixtures, J. Polym. Sci. 5 (1950) 505–514. [28] R. Garcia, O. Melad, C.M. Gómez, J.E. Figueruelo, A. Campos, Viscometric study on the compatibility of polymer-polymer mixtures in solution, Eur. Polym. J. 35 (1999) 47–55. [29] K. Lewandowska, The miscibility of poly(vinyl alcohol)/poly(N-vinylpyrrolidone) blends investigated in dilute solutions and solids, Eur. Polym. J. 41 (2005) 55–64. [30] K. Lewandowska, Miscibility and physical properties of chitosan and polyacrylamide blends, J. Mol. Liq. 209 (2015) 301–305.
Contents lists available at ScienceDirect
Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq
Characterization of silk fibroin 3D composites modified by collagen A. Sionkowska a,⁎, K. Lewandowska a, M. Michalska a, M. Walczak b a b
Department of Chemistry of Biomaterials and Cosmetics, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Gagarin 7, 87-100 Toruń, Poland Department of Environmental Microbiology and Biotechnology, Faculty of Biology and Environment Protection Nicolas Copernicus University in Torun, Gagarin 9, 87-100 Torun, Poland
a r t i c l e
i n f o
Article history: Received 30 May 2015 Received in revised form 24 November 2015 Accepted 14 December 2015 Available online xxxx Keywords: Silk fibroin Collagen Biomaterials
a b s t r a c t Silk fibroin 3D composites with the addition of collagen were prepared through the lyophylisation process. The structure of composites was studied by ATR-FTIR technique and was observed by a scanning electron microscope. Mechanical properties were studied and compared with those of a silk fibroin 3D sponge. Moreover, miscibility studies of silk fibroin with collagen blends of different compositions were investigated using the viscometric method before the lyophylisation process. Viscometric studies indicate that silk fibroin/collagen blends are miscible at any composition at 25 °C. Scanning electron microscopy observations showed that in the lyophilisation process of silk fibroin/collagen blend a porous material can be obtained. The results showed that the addition of collagen to silk fibroin led to the decrease of tensile strength. However, Young modulus increased with an increasing amount of collagen. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Biomaterials play a very important role in tissue engineering [1]. 3D composites for biomedical application should have good biocompatibility, biodegradability and suitable mechanical properties. Good porosity and pore size are also important, they provide the space for cells to grow, transporting metabolites, nutrients and signal molecules. Porosity gives space and temporary mechanical support for tissue growth [1–3]. For obtaining scaffolds for medical application both natural and synthetic polymers were tested [4,5]. To improve and optimize both chemical and physical properties of biomaterials, polymers used for a scaffold can be mixed together [6–8]. For this purpose also two natural polymers can also be mixed. However, the appropriate solvent is required for the preparation of such blends. Miscible blending of two biopolymers with different physicochemical characteristics may lead to the development of a new biomaterial with unique properties that may present the advantages of each polymer and compensate for the disadvantages of each one [9]. Silk fibroin is a natural polymer produced by various species of silkworm and spiders [2]. Bombyx mori cocoons are usually used as a source of silk fibroin. Raw silk contains two proteins, silk fibroin and sericine. However, for the preparation of materials for biomedical application the degummed sericine can be removed [10]. Silk fibroin due to its good mechanical properties and biocompatibility has been used as a biomedical material for a long time [4,11]. The question is, whether silk fibroin can be mixed with another biopolymer, for example with collagen. Collagen is the most abundant protein in mammals. This natural polymer constitutes more than one-third of protein weight in tissue ⁎ Corresponding author. E-mail address: [email protected] (A. Sionkowska).
http://dx.doi.org/10.1016/j.molliq.2015.12.047 0167-7322/© 2015 Elsevier B.V. All rights reserved.
[12]. Collagen can be obtained from different sources such as cow skin or muscles, rat tail tendons, fish scales or skin and even from sea sponges [13]. Materials based on collagen are commonly used in tissue engineering [14,15]. Several materials based on two biopolymers, such as collagen and silk fibroin have already been prepared [16–21]. Composite fibres of collagen and dragline silk protein were also prepared by an electrospinning technique [16]. Some materials were prepared by dissolving collagen in a silk fibroin solution in 60 °C, however, it may cause adverse effects to collagen with a denaturation temperature below 60 °C [3,20,21]. The properties of collagen/silk fibroin scaffolds can be modified using methanol [17,20,21]. It is believed that those materials can be applied in biomedical fields. Although three-dimensional fibroin scaffolds have been prepared using a freeze–drying method previously, they still cannot meet the requirements of tissue engineering and further ideas and study are required. In particular, the results regarding the miscibility of these biopolymers were poorly published. The aim of this work was to study the interactions between silk fibroin and collagen by viscometry technique and to prepare 3D composites based on such blends. Our preliminary study showed that silk fibroin and collagen can be miscible depending on the pH of the solution and ionic strength. 2. Materials and methods 2.1. Preparation of silk fibroin and collagen mixtures Silk fibroin was obtained from B. mori cocoons (Jedwab Polski Sp. Z o.o. company) according to the method described by Kim et al. with slight modifications [22]. Worms were removed from cocoons, then empty cocoons were boiled for 1 h in an aqueous solution of 0.5%
324
A. Sionkowska et al. / Journal of Molecular Liquids 215 (2016) 323–327
Na2CO3 twice. After the solution was removed, cocoons were boiled in 5% alkaline soap solution for 30 min and then for 20 min in distilled water. This procedure was repeated three times. After removing the sericine, silk fibroin was dried and dissolved in CaCl2:H2O:C2H5OH (molar ratio 1:8:2) at 80 °C for 4 h. The solution of silk fibroin was prepared with a concentration of 4% wt. Collagen was obtained from rat tail tendons. Tendons were washed in distilled water and dissolved in 0.1 M acetic acid for three days in 4 °C [23], the undissolved parts were removed by centrifugation for 10 min at 10,000 rpm. The completely frozen mixtures were lyophilized at −55 °C and 5 Pa for 48 h (ALPHA 1–2 LD plus, CHRIST, Germany). 1% wt solution was prepared by dissolving collagen in 0.1 M acetic acid. Solutions of these two polymers were mixed in weight ratios of silk fibroin to collagen: 90:10, 75:25 and 50:50. Pure silk fibroin was left as a control sample. Each sample was dialyzed in a cellulose tube (SERVAPOR) against distilled water for three days with deionized water changed every day. Finally, the dialysis mixtures were placed in a polystyrene container. The scaffolds were obtained during the lyophylization process for two days. The scaffolds obtained are shown in Fig. 1.
2.2. IR spectroscopy The interaction between functional groups of silk fibroin and functional groups of collagen and the structure of composites were evaluated by attenuated total reflection infrared spectroscopy using Nicolet iS10 equipped with an ATR device with diamond as a crystal. All spectra were recorded in absorption mode at 4 cm− 1 intervals and 64 scans.
2.3. Viscometric measurements Viscometric measurements of dilute polymer solution (c b 0.5%) were carried out in a controlled thermostatic bath at 25 ± 0.1 °C using the Ubbelohde capillary viscometer. The flow times were recorded with an accuracy ± 0.01 s. The flow time of each solution was determined as the average of several readings. Before measurements were taken the solutions were filtered through G1 sintered glass filters. The intrinsic viscosity and the interaction parameter values were determined according to the Heller procedure [24,25] from data obtained for solutions at 5 concentrations. The miscibility is estimated by comparison of the experimental and ideal values of bm and [η]m. The values of interaction parameters (bm) were obtained using the same methods as shown in previous papers [25,26].
2.5. Scanning electron microscopy The microstructure of scaffolds was studied using Scanning Electron Microscope (SEM) (LEO Electron Microscopy Ltd., England). Scaffolds were cut with a razor scalpel after being frozen in liquid nitrogen for 3 min. 3. Results and discussion 3.1. IR spectroscopy Infrared spectra were registered for sponge of silk fibroin and its blends with collagen. ATR-FTIR spectra of specimens studied are shown in Fig. 2. Silk fibroin and collagen are proteins, so both of them give similar spectra in FTIR spectroscopy. Although the miscibility of several polymers in solid state can be confirmed by FTIR spectra, in the blends of two natural polymers with a similar structure this technique is not a very powerful one. In Table 1 the position of typical bands in ATR-FTIR spectra for silk fibroin, collagen and their mixtures are presented. Silk fibroin displays bands at 1645, 1537 and 1241 cm−1, which are characteristic of the amide I, II and III bands of proteins. The amide I absorption arises predominantly from protein amide C_O stretching vibrations, the amide II absorption is made up of amide N–H bending vibrations and C–N stretching vibrations (60% and 40% contribution to the peak respectively); the amide III peak is complex, consisting of components from C–N stretching and N–H in plane bending from amide linkages, as well as absorptions arising from wagging vibrations from CH2 groups from the glycine backbone and proline side-chains. The amide groups have a characteristic absorption amide bands A and B in the region of 3400–3500 cm−1, however, these bands can be masked by the broad absorption band from the –OH group present in proteins. The amide A band for collagen was observed at 3381 cm−1, whereas for silk fibroin it was observed at 3283 cm−1. For silk fibroin/collagen blends with weight a ratio 50/50 it was observed at 3283 cm−1, the position of this band was similar to the position of amide A observed for silk fibroin. The position of amide I and amide II for silk fibroin and its blends with collagen were also similar. Small differences could only be observed for amide III. It seems that the FTIR technique cannot detect the inter-molecular interactions between silk fibroin and collagen. Usually the inter-molecular interaction between two different polymers through hydrogen bonding can be characterized by FTIR, because the specific interaction affects the local electron density and a corresponding frequency shift can be observed. For silk fibroin/collagen blends, a clear shift of amide bands was not observed. For this reason the viscometry studies of the interaction between silk fibroin and collagen in solution before the liophylisation process were conducted. On the basis of viscometric study one can assess the miscibility of two polymers in the blend in common solvent.
2.4. Mechanical properties 3.2. Viscometric measurements. Mechanical properties of SF/Col sponges were tested using a Zwick&Roell testing machine. 5 samples of each kind were placed between two discs and pressed. Young Modulus and tensile strength were then measured.
All the plots of the reduced viscosity versus polymer concentration (curves not shown) show linear behaviour in the range of concentration studied, indicating that the intrinsic viscosity can be determined by
Fig. 1. Scaffolds obtained from: a) silk fibroin, b) SF/Coll (90:10), c) SF/Coll (75:25), d) SF/Coll (50:50).
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Fig. 2. ATR-FTIR spectra of silk fibroin (SF), collagen (Col) and their mixtures with different SF/Col weight ratios.
linear extrapolation to zero concentration. The [η]exp m values were obtained according to Heller's procedure, which were acquired from the inverse of the Huggins and Kraemer equations [24]. This method gives some advantages such as high accuracy of determination of the intrinsic viscosity and slope constants because the slopes of the plots are smaller compared to the slopes of Huggins and Kraemer plots [24,25]. Using the criteria as proposed by Krigbaum et al. [27] and Garcia et al. [28] the parameters Δ[η]m and Δbm have been computed to establish the degree of miscibility in the silk fibroin/collagen blends. The plots of the miscibility parameter Δ[η]m and Δbm against the weight fraction of silk fibroin are shown in Figs. 3-5. It is obviously observed that the intrinsic viscosities of SF/Col blends do not follow the additive rule particularly well (Fig. 3). The experimental intrinsic viscosities are higher than the ideal values exhibiting positive deviation. According to Δ[η] criterion by Garcia [28] all silk fibroin/collagen blends are immiscible. This synergy could be explained by intermolecular interactions. The attractive force and/or electrostatic interactions between the polymeric components and solvent molecules in the blend solutions may cause the increase of intrinsic viscosity. Moreover, on the basis of these results, it is not possible to make a generalized conclusion about the miscibility of two polymers. This is due to the fact that the additivity rules, mentioned above, can be fulfilled in the solutions of polymers (polymers in solvent) for both, the immiscible and miscible polymer blends [25,29,30]. The plots of the viscosity interaction parameters versus the silk fibroin weight fraction in silk fibroin/collagen blends are illustrated in Fig. 5. As it can be seen from this figure the values of parameters are positive for all the investigated SF/Col blends. According to the two criteria, one can conclude that the SF/Col blends are miscible in all compositions. The miscibility is a result of the specific interaction between two polymeric components in solution.
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Fig. 3. Intrinsic viscosity ([η]) dependence on weight fraction of silk fibroin (wSF) in SF/Col blends. Dotted line indicates the ideal values of [η]id m which follow the Garcia criterion [28].
In the lyophilisation process of the silk fibroin/collagen blend the porous material was obtained. SEM observations showed that the pore size was from 20 to 150 μm for silk fibroin sponge. The porosity for silk fibroin sponges increased with the increase of addition of collagen in the mixture. However, the microstructure of silk fibroin sponge with the addition of collagen is less regular than without collagen and shows rather layered (sheet like) structures. It was also reported previously, that uniform pore distributions in a scaffold improved the mechanical properties, whereas the layered sheet structure of 3D sponges caused low tensile strength [19,20]. Biocompatibility of sponges needs to be confirmed by in vitro test. 3.4. Mechanical properties Mechanical properties of dry composites were tested at room temperature. Results of Young's modulus are shown in Fig. 7. Sponges made of pure silk fibroin (SF) have the lowest value of Young's modulus. Adding collagen (Col) to silk fibroin caused increasing elasticity of sponges. The highest value of Young's modulus was observed for sponges with the composition SF75:25Col. Our results are in sound agreement with the results published by Tiyaboonchai et al. [21] and Lu et al. [20] regarding silk fibroin/collagen scaffolds treated with methanol. As methanol treatment can cause changes in the secondary structure of proteins, we avoided methanol treatment in our studies. Results for tensile strength are shown in Fig. 8. The highest value of tensile strength was observed in sponges made of silk fibroin. However, several properties of scaffolds depend on the pH conditions of scaffold preparation [20]. Samples modified with collagen are characterized by lower tensile strength — even ten times lower than the samples made of pure silk fibroin (SF). The specimens (Fig. 1) were elastic and after
3.3. Scanning electron microscopy. Microstructure of sponges were tested using scanning electron microscopy (SEM). Images were made under 150 magnification (Fig. 6). Table 1 Characteristic bands in ATR-FTIR spectra and their surface for functional groups in silk fibroin sponge (SF) and mixtures (SF/Col) in different weight ratios. Observed vibrational frequencies wavenumber [cm− 1] Assignments
SF
SF:Col 90:10
SF:Col 75:25
SF:Col 50:50
H2O adsorbed, N—H (amide A) C—H (amide B) H2O adsorbed, CO (amide I) N—H (amide II), C—N C—N, N—H (amide III)
3283 3078 1645 1537 1241
3277 3067 1641 1516 1236
3294 3079 1650 1540 1242
3283 3078 1648 1536 1238
Fig. 4. Plot of criterion of Δ[η] versus weight fraction of silk fibroin (wSF) in SF/Col blends.
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Fig. 5. Values of viscosity interaction parameters, Δbm, versus weight fraction of silk fibroin (wSF) in SF/Col blends. * — the value of bid m determined according to Krigbaum and Wall [25], ** —the value of bid m determined according to Garcia et al. [28].
compression by mechanical testing machine could turn back to the previous shape. The sponges based on the blend of silk fibroin and collagen have sufficient mechanical integrity to resist handling during implantation and in vivo loading. However, the mechanical properties depend on the water content in the specimen and even depend on the relative humidity of the environment in the laboratory, where the mechanical testing is done. For this reason the measurements of mechanical properties were conducted in the same relative humidity and in the same temperature. After treatment with methanol the specimens lost their elasticity and became more stiff and fragile.
Fig. 7. Young's Modulus of pure silk fibroin (SF) and silk fibroin modified with collagen (Col).
4. Conclusions In the lyophilisation process 3D porous material based on the blends of silk fibroin and collagen can be obtained. Viscometric method showed, that before the lyophilisation process the blends were miscible at any composition. Mechanical properties of the sponge were modified with the addition of collagen to silk fibroin. The addition of collagen to silk fibroin led to the decrease of tensile strength and to the increase of Young modulus. Sponges made of silk fibroin/collagen mixtures can be interesting materials for tissue engineering as scaffold to temporarily
Fig. 6. SEM images of (a) SF × 150, (b) SF90:10Col × 150, (c) SF75:25Col × 150, (d) SF50:50Col × 150.
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Fig. 8. Tensile strength of pure silk fibroin (SF) and silk fibroin modified with collagen (Col).
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