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Development of keratin–chitosan–gelatin composite scaffold for soft tissue engineering
Materials Science and Engineering C 45 (2014) 343–347
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Development of keratin–chitosan–gelatin composite scaffold for soft tissue engineering Prachi Kakkar a, Sudhanshu Verma b, I. Manjubala b, B. Madhan a,⁎ a b
Central Leather Research Institute (Council of Scientific and Industrial Research), Adyar, Chennai 600020, India Biomedical Engineering Division, School of Bio Sciences and Technology, VIT University, Vellore 632014, India
a r t i c l e
i n f o
Article history: Received 25 June 2014 Received in revised form 10 August 2014 Accepted 11 September 2014 Available online 16 September 2014 Keywords: Keratin Biomaterial Wound healing Gelatin Chitosan Tissue engineering
a b s t r a c t Keratin has gained much attention in the recent past as a biomaterial for wound healing owing to its biocompatibility, biodegradability, intrinsic biological activity and presence of cellular binding motifs. In this paper, a novel biomimetic scaffold containing keratin, chitosan and gelatin was prepared by freeze drying method. The prepared keratin composite scaffold had good structural integrity. Fourier Transform Infrared (FTIR) spectroscopy showed the retention of the native structure of individual biopolymers (keratin, chitosan, and gelatin) used in the scaffold. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) results revealed a high thermal denaturation temperature of the scaffold (200–250 °C). The keratin composite scaffold exhibited tensile strength (96 kPa), compression strength (8.5 kPa) and water uptake capacity (N1700%) comparable to that of a collagen scaffold, which was used as control. The morphology of the keratin composite scaffold observed using a Scanning Electron Microscope (SEM) exhibited good porosity and interconnectivity of pores. MTT assay using NIH 3T3 fibroblast cells demonstrated that the cell viability of the keratin composite scaffold was good. These observations suggest that the keratin–chitosan–gelatin composite scaffold is a promising alternative biomaterial for tissue engineering applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction There is an imminent need for the development of new and improved materials for soft tissue engineering applications. Over the last few decades, more importance has been given to materials, which are biologically active and that show better biocompatibility and biodegradability. These materials provide analogous environment to the extra cellular matrix (ECM) and provide an induced rate of synthesis or growth of new tissues. Several natural polymers viz collagen, chitosan, gelatin, and keratin possess the ability to induce the proliferation of cells and hence find their use as a biomaterial for a wide range of biomedical applications [1]. Amongst all biopolymers, collagen is a widely accepted material for tissue engineering applications. In view of its low antigenicity, excellent biocompatibility and biodegradability [2], collagen and collagen composites are widely used for soft tissue engineering applications such as wound healing [3,4], corneal implants [5], nerve regeneration [6] and drug delivery [7]. Chitosan is (1–4)-linked 2-amino-2-deoxy-b-glucan, a byproduct of N-deacetylation of chitin. It is a major constituent of crab and shrimp shells, and cuticles of insects [8,9]. Studies have shown the potential of
⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (B. Madhan).
http://dx.doi.org/10.1016/j.msec.2014.09.021 0928-4931/© 2014 Elsevier B.V. All rights reserved.
chitin and chitosan for being used as novel biomaterials. These carbohydrate polymers have high biocompatibility [10,11], wound healing capability [8,12] and anti microbial activity [13,14]. Gelatin is the denatured form of collagen. Gelatin has low antigenicity and it promotes cell adhesion, differentiation and proliferation [15]. Gelatin also possesses high cytocompatibility, which makes it a potential candidate as a biomaterial for various tissue engineering applications [16,17]. Keratin is a family of fibrous proteins, which is found abundantly in nature. It forms the main constituent of hair, wool, nail, horn and hooves of mammals, birds and reptiles [18]. Keratin contains cysteine amino acid residues (7–20%) [19]. The oxidation of these cysteine residues leads to inter- and intra-molecular covalent bonds which are responsible for the toughness of the keratin fibers. Keratin contains cell adhesion sequences RGD (Arg-Gly-Asp), and LVD (Leu-Asp-Val), which are also found in extra cellular matrix proteins like fibronectin [18,20]. Keratin also possesses cellular binding motifs like native ECM, which mimic the cellular attachment sites. Due to the presence of such properties, keratin can be used for the development of different tissue engineering constructs. Although keratin based scaffolds and films have been prepared without using any additive, the major drawback of these constructs is their brittleness [20]. Hence, no single polymer is considered best to meet all the requirements of tissue engineering constructs [21]. In the present work, we report the preparation of a new composite scaffold comprising keratin, gelatin and chitosan, which is comparable
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P. Kakkar et al. / Materials Science and Engineering C 45 (2014) 343–347
to collagen scaffold in terms of physico-chemical and biological properties.
2. Material and methods 2.1. Materials For extraction of keratin, we purchased sodium dodecyl sulfate (SDS) from Sisco Research Laboratories Pvt. Ltd., urea (mol. wt. 60.6), 2-mercaptoethanol (mol. wt. 78.13) and dialysis membrane (molecular weight cutoff — 14,000 Da) from HiMedia. For fabrication of scaffolds, chitosan with ≥75% degree of deacetylation, and gelatin were obtained from Sigma Aldrich, whereas acetic acid was purchased from Merk.
2.2. Methods 2.2.1. Extraction of keratin from bovine hooves The raw hooves obtained from animal slaughter house were washed thoroughly with distilled water, dried and pulverized. The extraction process reported by Yamauchi et al. [22], was adopted to extract keratin from the pulverized hooves. Defatting of the hooves was done with the help of soxhlet apparatus using a mixture of hexane and dichloromethane in the ratio of 1:1 v/v. About 10 g of defatted hooves was taken in a 500 ml conical flask and a mixture of 7 M urea, 6 g SDS and 15 ml βmercaptoethanol was added into it and incubated for 12 h in an orbital shaker at 60 °C. The resulting mixture was centrifuged at 6000 rpm for 15 min. Finally, the filtrate was collected and dialyzed against water for 7 days to obtain pure keratin with predominantly two molecular fractions at 45 and 60 Da.
2.2.2. Fabrication of keratin composite scaffold To prepare keratin composite scaffold, about 120 mg of chitosan was dissolved in acidified water (using 50 mM acetic acid) to prepare chitosan solution. Gelatin solution was prepared by dissolving 240 mg of gelatin in hot water. Equimolar amount of chitosan and gelatin solutions were homogenized for 5 min. The extracted keratin solution (containing about 120 mg of keratin) was added drop by drop to the above mixture under continuous stirring to obtain a scaffold containing keratin, chitosan and gelatin in the ratio of 1:1:2 (w/w) respectively. The solution (about 48 ml) was homogenized for 30 min using Ultra Turrax IKA T25. The homogenized solution was frozen at − 40 °C overnight and lyophilized for 48 h to form a porous scaffold with dimensions of 10 × 10 × 0.9 cm3. The final concentration of keratin and chitosan in the homogenized solution was 2.5 mg/ml each and gelatin was 5 mg/ml.
2.4. Thermal analysis of composite scaffold Differential scanning calorimetric (DSC) analysis of prepared composite scaffold was performed using Universal V4.4A TA Instrument to observe the thermal degradation behavior. The sample was heated to a temperature range from 25 °C to 300 °C, at the rate of 5 °C/min. The calibration of the instrument was done by Indium standard and liquid nitrogen was flushed at the rate of 50 ml/min to the calorimetric cell. Thermo gravimetric analysis (TGA) of prepared composite scaffold was done using Universal V4.4A TA Instruments to check the thermal stability of the scaffold. The sample was heated at a temperature range from 20 °C to 800 °C at a rate of 20 °C/min in nitrogen atmosphere. 2.5. Porosity of the scaffolds The porosity (P) of the keratin composite and collagen scaffolds was measured by ethanol infiltration method [23]. Briefly, weight of the dry scaffolds (Wo) was recorded and then they were soaked in ethanol by exhausting the air bubbles. Then the scaffolds were taken out, surface ethanol wiped and were weighed immediately (We). The porosity of the scaffolds was defined as % porosity ¼ ½ðWe −Wo Þ=ρVs 100 where, ρ represents the density of ethanol at room temperature (0.789 mg/ml) and Vs is the volume of the scaffold which was calculated from the geometry of the scaffold. 2.6. Compressive strength and tensile strength analysis The compression and tensile strength of keratin composite and collagen scaffold were analyzed using Texture Analyzer Pro CT V1.4 Build 17 (Brookfield Engineering Labs, Inc). The respective samples were taken in triplicates. To analyze compression strength in dry state, circular punches were cut with thickness of 7–10 mm and diameter of 15 mm and compressed till the thickness of the sample reduced to 50% of the original thickness. For wet state analysis compression strength was determined for the sample immersed in phosphate buffer saline (pH-7.4) for 1 h and then analyzed in the same way as mentioned above. For tensile strength analysis, rectangular sections were cut with a cross-sectional area of approximately 7 × 4 mm2. For the analysis in dry state, the samples were clamped vertically, with a gauge length of 10 mm and tested with a trigger load of 7 g, at the test speed of 0.5 mm/s. All samples were stretched until failure. For the analysis in wet state, samples were immersed in phosphate buffer saline (pH-7.4) for about 1 h followed by the determination of tensile strength. 2.7. Water uptake studies of the composite scaffold
2.2.3. Fabrication of collagen scaffold To isolate type I collagen, the procedure given by Tanaka et al. [31] was followed. Isolated collagen was dialyzed against 0.05 M acetic acid and lyophilized to obtain a powder. To prepare the collagen scaffold, lyophilized collagen was dissolved in 0.05 M acetic acid to obtain 3 mg/ml concentration of collagen. To this solution, Triton X 100 was added and homogenized for about 20 min using Ultra Turrax IKA T25. This was then kept at −40 °C and lyophilized to get a porous scaffold.
The specimens of dimension 1 × 1 cm2 were cut in triplicate from each of the scaffolds (keratin composite scaffold and collagen as control) and dried under vacuum at 60 °C for 4 h. Subsequently, the weight of each sample (Wdry) was recorded and then immersed into PBS. After 24 h of incubation, the scaffolds were taken out, wiped on the surface and immediately weighed (Wwet). The water uptake capacity of the scaffold was calculated by using the following formula: Water uptake % ¼
h i Wwet –Wdry =Wdry 100:
2.3. Fourier Transform Infrared Spectra 2.8. Scanning Electron Microscopy Fourier Transform Infrared (FTIR) spectra of the composite scaffold, keratin, gelatin and chitosan were measured with ABB-FTIR, Model MB 3000 in the range of 500–4000 cm−1 with a resolution of 8 cm−1, at a scan rate of 32 scans/sample. Potassium bromide (KBr) was mixed with the composite scaffold, lyophilized keratin, chitosan and gelatin respectively to prepare the pellet for FTIR measurements.
The surface morphology, structural integrity and interconnectivity of the pores in the scaffold were observed using Scanning Electron Microscopy (SEM HITACHI-S3400N) at a magnification of 300×. The samples were prepared by sputter coating the scaffold surface with a thin layer of gold. The surface of the samples was scanned at 15 kV.
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Table 1 List of characteristics peaks observed for composite scaffold in FTIR spectra. Characteristic functional groups
Observed peak values (cm−1)
Amide A (N–H) Amide I (C–O) Amide II (N–H & C–H) Amide III (C–N) –CH2 for pyranose ring –CH3 in amide Non-conjugated C`N
3359 1650 1542 1242 2935 1400 2364
bromide MTT assay (Sigma Aldrich), which is a colorimetric technique for determining the number of viable cells, that relies on the conversion of MTT to MTT formazan by the enzyme mitochondrial reductase of the viable cells [24]. The cell extracts from the scaffold were mixed with MTT solution and the absorbance at 550 nm was measured on a microplate reader (Biotek, USA). Fig. 1. Keratin composite scaffold prepared by freeze drying method.
2.9. Biocompatibility test with fibroblast cells NIH 3T3 fibroblast cell lines obtained from National Centre for Cell Science (NCCS), Pune, India, were used for the cell viability test. The cells were cultured and allowed to passage for 3 times. The passaged cells were seeded on the collagen and keratin composite scaffolds at a density of 1 × 105 cells/well. The scaffolds were incubated at 37 °C with 5% CO2 for 3 h. Dulbecco Modified Eagle Medium (Sigma Aldrich) containing 10% fetal bovine serum (Invitrogen), penicillin (50 unit/ml) and streptomycin (50 unit/ml) were added and incubated at 37 °C with 5% CO2. The medium was changed every 2 days and the scaffolds were observed under a microscope. Collagen scaffold was taken as control. After 3, 5 and 7 days of culture, cell viability was measured quantitatively using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Fig. 2. Fourier Transform Infrared (FTIR) spectroscopy of keratin composite scaffold and its individual components (keratin, chitosan and gelatin).
3. Results and discussion Biopolymers have always attracted researchers as a preferred choice for biomedical applications. Good mechanical stability and biocompatibility are the major properties which make these biopolymeric scaffolds suitable for soft tissue engineering applications such as wound healing. We have fabricated a porous 3-D keratin based composite scaffold consisting of keratin, chitosan and gelatin using freeze drying method (Fig. 1). Further characterization of the scaffold is presented in the following section. 3.1. Fourier Transform Infrared Spectra of composite scaffold The FTIR spectra of the keratin, chitosan, gelatin and the keratin composite scaffold are shown in Fig. 2. In keratin composite scaffold, the peaks observed near 3280 cm− 1 are characteristic of amide A, whereas amide I falls in the range of 1600–1700 cm−1, amide II near 1520 cm−1 and amide III in the range of 1220–1300 cm−1 [25]. The peak of amide A is due to stretching vibration of N–H bonds which is observed at 3359 cm−1. The peak of amide I falling at 1650 cm−1 is caused by stretching vibration of C–O bonds. N–H bending and C–H stretching vibration which are responsible for amide II band are observed at 1542 cm−1. At 1242 cm−1 the sharp peak of amide III is observed that is due to the phase combination of C–N stretching and N–H in plane bending and partial addition from C_C bending and C–C stretching vibration.
Fig. 3. Differential scanning calorimetric (DSC) analysis of keratin composite scaffold.
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P. Kakkar et al. / Materials Science and Engineering C 45 (2014) 343–347 Table 3 Water uptake and porosity of keratin composite scaffold and collagen scaffold. Scaffold
Water uptake % (Mean ± SD)
Porosity % (Mean ± SD)
Collagen Keratin composite
1985.06 ± 13.10 1796.52 ± 23.1
84.33 ± 2.11 86.86 ± 1.38
3.2. Thermal analysis of the composite scaffold The DSC thermogram of the keratin composite scaffold is shown in Fig. 3. The sharp endothermic peak observed at 91 °C is due to the water evaporation. The second endothermic peak observed in the range of 215 °C–230 °C is due to the denaturation of α-helix of keratin [29]. The peak observed in between 255 °C–280 °C is due to the denaturation of chitosan [30]. The thermogravimetric curve of the keratin composite scaffold sample is shown in Fig. 4A. The derivative of TGA curve (DTG) is shown in Fig. 4B. This analysis demonstrated that the weight loss in the temperature range of 79.69 °C–150 °C is due to the loss of moisture content. About 50% weight loss was observed at 361 °C. A weight loss of about 69% of the total weight that had taken place between 215 °C and 508.8 °C was due to the denaturation of keratin and chitosan. Hence, the thermal analysis of the keratin composite scaffold clearly demonstrates that the prepared scaffold can withstand high temperatures. 3.3. Compressive strength and tensile strength
Fig. 4. A) Thermogravimetric (TGA) analysis of keratin composite scaffold and B) derivative of TGA.
The peak at 2935 cm−1 in the spectrum of composite scaffolds corresponds to stretching vibration of CH2, which is attributed to the pyranose ring [26]. This spectrum confirms the presence of α helix with β turns with characteristic absorption band designating the peptide bonds (–CONH–) for amides I, II and III at 1650, 1542 and 1242 cm−1 respectively. Amide I represents α helix at 1650 cm−1 and β turns at 1542 cm−1. Literature also suggests that the absorption that took place at 1650 cm−1 represents α helix and the band between 1630–1515 cm−1 represents the presence of β turns [27,28]. The peaks at 1400 and 1041 cm−1 in the spectrum of composite scaffolds correspond to carboxyl –COOH and C–O stretching bands, respectively. The characteristic peaks of the keratin composite scaffold are listed in Table 1.
Compression and tensile strength of both keratin composite and collagen (control) scaffolds were analyzed in dry and wet states. From the tensile strength curve, the ultimate tensile strength was calculated and compressive modulus was calculated from the load required for 50% compression of the scaffold. Ultimate tensile strength and compressive modulus of both the scaffolds in dry and wet conditions are presented in Table 2. We can observe that the ultimate tensile strength of the keratin composite scaffold in both dry and wet states is comparable to that of collagen scaffolds. The compressive modulus of keratin scaffolds especially in wet condition showed significantly higher values compared to collagen scaffolds prepared under similar conditions. Collagenous matrices have the innate ability to hold more water in comparison to keratin. Therefore collagen scaffold in wet condition could have resulted in greater swelling leading to lower resistance for compressive stress in comparison to keratin composite scaffold. 3.4. Water uptake capacity and porosity As the fabricated keratin composite scaffold was aimed for wound healing applications, the prepared scaffolds were tested for the water uptake capability. Porosity is related to water uptake capacity. The water uptake capacity and porosity of the keratin composite scaffold and collagen scaffold are given in Table 3. The keratin composite scaffold showed appreciable porosity (N 85%), which is comparable with that of collagen scaffold (84%), as estimated by ethanol infiltration method. Keratin composite scaffold also showed excellent water uptake capacity (N 1700%), placing it in a position almost comparable to collagen scaffold. These properties of keratin composite scaffolds are favorable for wound healing application.
Table 2 Tensile strength and compression of keratin composite scaffold and collagen scaffold. Analysis
Ultimate tensile strength (kPa) Compressive modulus (kPa)
Keratin composite scaffold
Collagen scaffold
Dry (Mean ± SD)
Wet (Mean ± SD)
Dry (Mean ± SD)
Wet (Mean ± SD)
95.69 ± 0.95 8.58 ± 0.50
10.06 ± 0.54 5.27 ± 0.55
107.33 ± 0.83 7.15 ± 0.86
12.46 ± 0.51 2.80 ± 0.44
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Fig. 5. Scanning Electron Microscopy of collagen (A) and keratin composite scaffold (B) at 300×.
3.5. Scanning Electron Microscopy The SEM images of the collagen and keratin composite scaffolds are shown in Fig. 5(A) and (B) respectively. Collagen scaffold (Fig. 5A) showed a highly porous structure, while keratin composite scaffold (Fig. 5B) also exhibited good porosity and well interconnected pores which would be very important for cell growth, proliferation and migration during the process of wound healing. 3.6. Biocompatibility–cell viability assay The NIH 3T3 cells cultured on the keratin composite scaffold as well as collagen (control) scaffold were monitored continuously for 7 days. The cell viability results of MTT assay (Fig. 6) performed after 3, 5 and 7 days showed no significant difference between the test sample and control, and after 7 days the cell viability of the composite scaffold was similar to that of collagen scaffold. This suggests that the prepared keratin composite scaffold has performance equivalent to collagen scaffolds with respect to the cell migration and proliferation rate on the scaffold. 4. Conclusions A novel keratin composite scaffold with chitosan and gelatin was prepared with a simple freeze drying process. The composite scaffold resulted in good thermal stability and all the functional groups of the individual constituent of the keratin composite remained intact as observed from FTIR results. The scaffold possessed good mechanical strength and water uptake capacity, apart from having micro-porous structure, which is likely to facilitate proliferation of cells. The composite scaffold was found to be biocompatible with fibroblast cells with
Fig. 6. Biocompatibility of keratin composite scaffold (KCG) and collagen scaffold.
high percentage of cell viability. Physico-chemical and biological characteristics of the keratin–chitosan–gelatin scaffold clearly demonstrated that it can be used as a potential alternative material for soft tissue engineering applications especially for wound healing. Acknowledgment The author is thankful to the Council of Scientific and Industrial Research (CSIR) (CSC 0134) for providing support in the form of Senior Research Fellowship (SRF). References [1] B. Srinivasan, R. Kumar, K. Shanmugam, U.T. Sivagnam, N.P. Reddy, P.K. Sehgal, J. Biomed. Mater. Res. B Appl. Biomater. 92B (2010) 5–12. [2] Y. Liu, L. Ma, C. Gao, Mater. Sci. Eng. C Mater. Biol. Appl. 32 (2012) 2361–2366. [3] V. Natarajan, N. Krithica, B. Madhan, P.K. Sehgal, J. Biomed. Mater. Res. B Appl. Biomater. 101B (2013) 560–567. [4] S. Perumal, S.k. Ramadass, B. Madhan, Eur. J. Pharm. Sci. 52 (2014) 26–33. [5] Y. Liu, L. Ren, Y. Wang, Mater. Sci. Eng. C Mater. Biol. Appl. 33 (2013) 196–201. [6] W. Xiao, X.Y. Hu, W. Zeng, J.H. Huang, Y.G. Zhang, Z.J. Luo, Injury 44 (2013) 941–946. [7] L. Sang, D. Luo, S. Xu, X. Wang, X. Li, Mater. Sci. Eng. C Mater. Biol. Appl. 31 (2011) 262–271. [8] H.-J. Tseng, T.-L. Tsou, H.-J. Wang, S.-h. Hsu, J. Tissue Eng. Regen. Med. 7 (2013) 20–31. [9] M. Kaya, T. Baran, A. Mentes, M. Asaroglu, G. Sezen, K. Tozak, Food Biophys. 9 (2014) 145–157. [10] R. Jayakumar, D. Menon, K. Manzoor, S.V. Nair, H. Tamura, Carbohydr. Polym. 82 (2010) 227–232. [11] E. Khor, L.Y. Lim, Biomaterials 24 (2003) 2339–2349. [12] M.P. Ribeiro, P.I. Morgado, S.P. Miguel, P. Coutinho, I.J. Correia, Mater. Sci. Eng. C 33 (2013) 2958–2966. [13] G.J. Tsai, W.H. Su, J. Food Prot. 62 (1999) 239–243. [14] G.J. Tsai, Z.Y. Wu, W.H. Su, J. Food Prot. 63 (2000) 747–752. [15] H.-J. Wang, L. Di, Q.-S. Ren, J.-Y. Wang, Materials 2 (2009) 613–635. [16] M.A. Vandelli, F. Rivasi, P. Guerra, F. Forni, R. Arletti, Int. J. Pharm. 215 (2001) 175–184. [17] S. Young, M. Wong, Y. Tabata, A.G. Mikos, J. Control. Release 109 (2005) 256–274. [18] M. Feughelmann, Keratin, in: K.JI (Ed.), Encyclopedia of Polymer Science and Engineering, Wiley, New York, 1985. [19] L.M. Dowling, W.G. Crewther, D.A. Parry, Biochem. J. 236 (1986) 705–712. [20] R.C. Marshall, D.F.G. Orwin, J.M. Gillespie, Electron Microsc. Rev. 4 (1991) 47–83. [21] J. Manuel Fernandez, M. Silvina Molinuevo, M. Susana Cortizo, A.M. Cortizo, J. Tissue Eng. Regen. Med. 5 (2011) E126–E135. [22] A. Yamauchi, T. Kusunoki, A. Kohda, Y. Konishi, J. Biomed. Mater. Res. 31 (1996) 439–444. [23] C. Xu, W. Lu, S. Bian, J. Liang, Y. Fan, X. Zhang, Sci. World J. 2012 (2012) (695137695137). [24] G. Zund, Q. Ye, S.P. Hoerstrup, A. Schoeberlein, A.C. Schmid, J. Grunenfelder, P. Vogt, M. Turina, Eur. J. Cardiothorac. Surg. 15 (1999) 519–524. [25] M.F. Zeng, Z.P. Fang, C.W. Xu, J. Membr. Sci. 230 (2004) 175–181. [26] A. Aluigi, M. Zoccola, C. Vineis, C. Tonin, F. Ferrero, M. Canetti, Int. J. Biol. Macromol. 41 (2007) 266–273. [27] A. Pawlak, M. Mucha, Thermochim. Acta 409 (2004) 95–97. [28] E. Wojciechowska, A. Wlochowicz, A. Weselucha-Birczynska, J. Mol. Struct. 511 (1999) 307–318. [29] S. Patel, N. Gheewala, A. Suthar, A. Shah, Int. J. Pharm. Pharm. Sci. 1 (2009) 38–46. [30] M. Spei, R. Holzem, Colloid Polym. Sci. 265 (1987) 965–970. [31] S. Tanaka, G. Avigad, E.F. Eikenberry, B. Brodsky, J. Biol. Chem. 263 (1988) 17650–17657.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Development of keratin–chitosan–gelatin composite scaffold for soft tissue engineering Prachi Kakkar a, Sudhanshu Verma b, I. Manjubala b, B. Madhan a,⁎ a b
Central Leather Research Institute (Council of Scientific and Industrial Research), Adyar, Chennai 600020, India Biomedical Engineering Division, School of Bio Sciences and Technology, VIT University, Vellore 632014, India
a r t i c l e
i n f o
Article history: Received 25 June 2014 Received in revised form 10 August 2014 Accepted 11 September 2014 Available online 16 September 2014 Keywords: Keratin Biomaterial Wound healing Gelatin Chitosan Tissue engineering
a b s t r a c t Keratin has gained much attention in the recent past as a biomaterial for wound healing owing to its biocompatibility, biodegradability, intrinsic biological activity and presence of cellular binding motifs. In this paper, a novel biomimetic scaffold containing keratin, chitosan and gelatin was prepared by freeze drying method. The prepared keratin composite scaffold had good structural integrity. Fourier Transform Infrared (FTIR) spectroscopy showed the retention of the native structure of individual biopolymers (keratin, chitosan, and gelatin) used in the scaffold. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) results revealed a high thermal denaturation temperature of the scaffold (200–250 °C). The keratin composite scaffold exhibited tensile strength (96 kPa), compression strength (8.5 kPa) and water uptake capacity (N1700%) comparable to that of a collagen scaffold, which was used as control. The morphology of the keratin composite scaffold observed using a Scanning Electron Microscope (SEM) exhibited good porosity and interconnectivity of pores. MTT assay using NIH 3T3 fibroblast cells demonstrated that the cell viability of the keratin composite scaffold was good. These observations suggest that the keratin–chitosan–gelatin composite scaffold is a promising alternative biomaterial for tissue engineering applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction There is an imminent need for the development of new and improved materials for soft tissue engineering applications. Over the last few decades, more importance has been given to materials, which are biologically active and that show better biocompatibility and biodegradability. These materials provide analogous environment to the extra cellular matrix (ECM) and provide an induced rate of synthesis or growth of new tissues. Several natural polymers viz collagen, chitosan, gelatin, and keratin possess the ability to induce the proliferation of cells and hence find their use as a biomaterial for a wide range of biomedical applications [1]. Amongst all biopolymers, collagen is a widely accepted material for tissue engineering applications. In view of its low antigenicity, excellent biocompatibility and biodegradability [2], collagen and collagen composites are widely used for soft tissue engineering applications such as wound healing [3,4], corneal implants [5], nerve regeneration [6] and drug delivery [7]. Chitosan is (1–4)-linked 2-amino-2-deoxy-b-glucan, a byproduct of N-deacetylation of chitin. It is a major constituent of crab and shrimp shells, and cuticles of insects [8,9]. Studies have shown the potential of
⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (B. Madhan).
http://dx.doi.org/10.1016/j.msec.2014.09.021 0928-4931/© 2014 Elsevier B.V. All rights reserved.
chitin and chitosan for being used as novel biomaterials. These carbohydrate polymers have high biocompatibility [10,11], wound healing capability [8,12] and anti microbial activity [13,14]. Gelatin is the denatured form of collagen. Gelatin has low antigenicity and it promotes cell adhesion, differentiation and proliferation [15]. Gelatin also possesses high cytocompatibility, which makes it a potential candidate as a biomaterial for various tissue engineering applications [16,17]. Keratin is a family of fibrous proteins, which is found abundantly in nature. It forms the main constituent of hair, wool, nail, horn and hooves of mammals, birds and reptiles [18]. Keratin contains cysteine amino acid residues (7–20%) [19]. The oxidation of these cysteine residues leads to inter- and intra-molecular covalent bonds which are responsible for the toughness of the keratin fibers. Keratin contains cell adhesion sequences RGD (Arg-Gly-Asp), and LVD (Leu-Asp-Val), which are also found in extra cellular matrix proteins like fibronectin [18,20]. Keratin also possesses cellular binding motifs like native ECM, which mimic the cellular attachment sites. Due to the presence of such properties, keratin can be used for the development of different tissue engineering constructs. Although keratin based scaffolds and films have been prepared without using any additive, the major drawback of these constructs is their brittleness [20]. Hence, no single polymer is considered best to meet all the requirements of tissue engineering constructs [21]. In the present work, we report the preparation of a new composite scaffold comprising keratin, gelatin and chitosan, which is comparable
344
P. Kakkar et al. / Materials Science and Engineering C 45 (2014) 343–347
to collagen scaffold in terms of physico-chemical and biological properties.
2. Material and methods 2.1. Materials For extraction of keratin, we purchased sodium dodecyl sulfate (SDS) from Sisco Research Laboratories Pvt. Ltd., urea (mol. wt. 60.6), 2-mercaptoethanol (mol. wt. 78.13) and dialysis membrane (molecular weight cutoff — 14,000 Da) from HiMedia. For fabrication of scaffolds, chitosan with ≥75% degree of deacetylation, and gelatin were obtained from Sigma Aldrich, whereas acetic acid was purchased from Merk.
2.2. Methods 2.2.1. Extraction of keratin from bovine hooves The raw hooves obtained from animal slaughter house were washed thoroughly with distilled water, dried and pulverized. The extraction process reported by Yamauchi et al. [22], was adopted to extract keratin from the pulverized hooves. Defatting of the hooves was done with the help of soxhlet apparatus using a mixture of hexane and dichloromethane in the ratio of 1:1 v/v. About 10 g of defatted hooves was taken in a 500 ml conical flask and a mixture of 7 M urea, 6 g SDS and 15 ml βmercaptoethanol was added into it and incubated for 12 h in an orbital shaker at 60 °C. The resulting mixture was centrifuged at 6000 rpm for 15 min. Finally, the filtrate was collected and dialyzed against water for 7 days to obtain pure keratin with predominantly two molecular fractions at 45 and 60 Da.
2.2.2. Fabrication of keratin composite scaffold To prepare keratin composite scaffold, about 120 mg of chitosan was dissolved in acidified water (using 50 mM acetic acid) to prepare chitosan solution. Gelatin solution was prepared by dissolving 240 mg of gelatin in hot water. Equimolar amount of chitosan and gelatin solutions were homogenized for 5 min. The extracted keratin solution (containing about 120 mg of keratin) was added drop by drop to the above mixture under continuous stirring to obtain a scaffold containing keratin, chitosan and gelatin in the ratio of 1:1:2 (w/w) respectively. The solution (about 48 ml) was homogenized for 30 min using Ultra Turrax IKA T25. The homogenized solution was frozen at − 40 °C overnight and lyophilized for 48 h to form a porous scaffold with dimensions of 10 × 10 × 0.9 cm3. The final concentration of keratin and chitosan in the homogenized solution was 2.5 mg/ml each and gelatin was 5 mg/ml.
2.4. Thermal analysis of composite scaffold Differential scanning calorimetric (DSC) analysis of prepared composite scaffold was performed using Universal V4.4A TA Instrument to observe the thermal degradation behavior. The sample was heated to a temperature range from 25 °C to 300 °C, at the rate of 5 °C/min. The calibration of the instrument was done by Indium standard and liquid nitrogen was flushed at the rate of 50 ml/min to the calorimetric cell. Thermo gravimetric analysis (TGA) of prepared composite scaffold was done using Universal V4.4A TA Instruments to check the thermal stability of the scaffold. The sample was heated at a temperature range from 20 °C to 800 °C at a rate of 20 °C/min in nitrogen atmosphere. 2.5. Porosity of the scaffolds The porosity (P) of the keratin composite and collagen scaffolds was measured by ethanol infiltration method [23]. Briefly, weight of the dry scaffolds (Wo) was recorded and then they were soaked in ethanol by exhausting the air bubbles. Then the scaffolds were taken out, surface ethanol wiped and were weighed immediately (We). The porosity of the scaffolds was defined as % porosity ¼ ½ðWe −Wo Þ=ρVs 100 where, ρ represents the density of ethanol at room temperature (0.789 mg/ml) and Vs is the volume of the scaffold which was calculated from the geometry of the scaffold. 2.6. Compressive strength and tensile strength analysis The compression and tensile strength of keratin composite and collagen scaffold were analyzed using Texture Analyzer Pro CT V1.4 Build 17 (Brookfield Engineering Labs, Inc). The respective samples were taken in triplicates. To analyze compression strength in dry state, circular punches were cut with thickness of 7–10 mm and diameter of 15 mm and compressed till the thickness of the sample reduced to 50% of the original thickness. For wet state analysis compression strength was determined for the sample immersed in phosphate buffer saline (pH-7.4) for 1 h and then analyzed in the same way as mentioned above. For tensile strength analysis, rectangular sections were cut with a cross-sectional area of approximately 7 × 4 mm2. For the analysis in dry state, the samples were clamped vertically, with a gauge length of 10 mm and tested with a trigger load of 7 g, at the test speed of 0.5 mm/s. All samples were stretched until failure. For the analysis in wet state, samples were immersed in phosphate buffer saline (pH-7.4) for about 1 h followed by the determination of tensile strength. 2.7. Water uptake studies of the composite scaffold
2.2.3. Fabrication of collagen scaffold To isolate type I collagen, the procedure given by Tanaka et al. [31] was followed. Isolated collagen was dialyzed against 0.05 M acetic acid and lyophilized to obtain a powder. To prepare the collagen scaffold, lyophilized collagen was dissolved in 0.05 M acetic acid to obtain 3 mg/ml concentration of collagen. To this solution, Triton X 100 was added and homogenized for about 20 min using Ultra Turrax IKA T25. This was then kept at −40 °C and lyophilized to get a porous scaffold.
The specimens of dimension 1 × 1 cm2 were cut in triplicate from each of the scaffolds (keratin composite scaffold and collagen as control) and dried under vacuum at 60 °C for 4 h. Subsequently, the weight of each sample (Wdry) was recorded and then immersed into PBS. After 24 h of incubation, the scaffolds were taken out, wiped on the surface and immediately weighed (Wwet). The water uptake capacity of the scaffold was calculated by using the following formula: Water uptake % ¼
h i Wwet –Wdry =Wdry 100:
2.3. Fourier Transform Infrared Spectra 2.8. Scanning Electron Microscopy Fourier Transform Infrared (FTIR) spectra of the composite scaffold, keratin, gelatin and chitosan were measured with ABB-FTIR, Model MB 3000 in the range of 500–4000 cm−1 with a resolution of 8 cm−1, at a scan rate of 32 scans/sample. Potassium bromide (KBr) was mixed with the composite scaffold, lyophilized keratin, chitosan and gelatin respectively to prepare the pellet for FTIR measurements.
The surface morphology, structural integrity and interconnectivity of the pores in the scaffold were observed using Scanning Electron Microscopy (SEM HITACHI-S3400N) at a magnification of 300×. The samples were prepared by sputter coating the scaffold surface with a thin layer of gold. The surface of the samples was scanned at 15 kV.
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Table 1 List of characteristics peaks observed for composite scaffold in FTIR spectra. Characteristic functional groups
Observed peak values (cm−1)
Amide A (N–H) Amide I (C–O) Amide II (N–H & C–H) Amide III (C–N) –CH2 for pyranose ring –CH3 in amide Non-conjugated C`N
3359 1650 1542 1242 2935 1400 2364
bromide MTT assay (Sigma Aldrich), which is a colorimetric technique for determining the number of viable cells, that relies on the conversion of MTT to MTT formazan by the enzyme mitochondrial reductase of the viable cells [24]. The cell extracts from the scaffold were mixed with MTT solution and the absorbance at 550 nm was measured on a microplate reader (Biotek, USA). Fig. 1. Keratin composite scaffold prepared by freeze drying method.
2.9. Biocompatibility test with fibroblast cells NIH 3T3 fibroblast cell lines obtained from National Centre for Cell Science (NCCS), Pune, India, were used for the cell viability test. The cells were cultured and allowed to passage for 3 times. The passaged cells were seeded on the collagen and keratin composite scaffolds at a density of 1 × 105 cells/well. The scaffolds were incubated at 37 °C with 5% CO2 for 3 h. Dulbecco Modified Eagle Medium (Sigma Aldrich) containing 10% fetal bovine serum (Invitrogen), penicillin (50 unit/ml) and streptomycin (50 unit/ml) were added and incubated at 37 °C with 5% CO2. The medium was changed every 2 days and the scaffolds were observed under a microscope. Collagen scaffold was taken as control. After 3, 5 and 7 days of culture, cell viability was measured quantitatively using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Fig. 2. Fourier Transform Infrared (FTIR) spectroscopy of keratin composite scaffold and its individual components (keratin, chitosan and gelatin).
3. Results and discussion Biopolymers have always attracted researchers as a preferred choice for biomedical applications. Good mechanical stability and biocompatibility are the major properties which make these biopolymeric scaffolds suitable for soft tissue engineering applications such as wound healing. We have fabricated a porous 3-D keratin based composite scaffold consisting of keratin, chitosan and gelatin using freeze drying method (Fig. 1). Further characterization of the scaffold is presented in the following section. 3.1. Fourier Transform Infrared Spectra of composite scaffold The FTIR spectra of the keratin, chitosan, gelatin and the keratin composite scaffold are shown in Fig. 2. In keratin composite scaffold, the peaks observed near 3280 cm− 1 are characteristic of amide A, whereas amide I falls in the range of 1600–1700 cm−1, amide II near 1520 cm−1 and amide III in the range of 1220–1300 cm−1 [25]. The peak of amide A is due to stretching vibration of N–H bonds which is observed at 3359 cm−1. The peak of amide I falling at 1650 cm−1 is caused by stretching vibration of C–O bonds. N–H bending and C–H stretching vibration which are responsible for amide II band are observed at 1542 cm−1. At 1242 cm−1 the sharp peak of amide III is observed that is due to the phase combination of C–N stretching and N–H in plane bending and partial addition from C_C bending and C–C stretching vibration.
Fig. 3. Differential scanning calorimetric (DSC) analysis of keratin composite scaffold.
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P. Kakkar et al. / Materials Science and Engineering C 45 (2014) 343–347 Table 3 Water uptake and porosity of keratin composite scaffold and collagen scaffold. Scaffold
Water uptake % (Mean ± SD)
Porosity % (Mean ± SD)
Collagen Keratin composite
1985.06 ± 13.10 1796.52 ± 23.1
84.33 ± 2.11 86.86 ± 1.38
3.2. Thermal analysis of the composite scaffold The DSC thermogram of the keratin composite scaffold is shown in Fig. 3. The sharp endothermic peak observed at 91 °C is due to the water evaporation. The second endothermic peak observed in the range of 215 °C–230 °C is due to the denaturation of α-helix of keratin [29]. The peak observed in between 255 °C–280 °C is due to the denaturation of chitosan [30]. The thermogravimetric curve of the keratin composite scaffold sample is shown in Fig. 4A. The derivative of TGA curve (DTG) is shown in Fig. 4B. This analysis demonstrated that the weight loss in the temperature range of 79.69 °C–150 °C is due to the loss of moisture content. About 50% weight loss was observed at 361 °C. A weight loss of about 69% of the total weight that had taken place between 215 °C and 508.8 °C was due to the denaturation of keratin and chitosan. Hence, the thermal analysis of the keratin composite scaffold clearly demonstrates that the prepared scaffold can withstand high temperatures. 3.3. Compressive strength and tensile strength
Fig. 4. A) Thermogravimetric (TGA) analysis of keratin composite scaffold and B) derivative of TGA.
The peak at 2935 cm−1 in the spectrum of composite scaffolds corresponds to stretching vibration of CH2, which is attributed to the pyranose ring [26]. This spectrum confirms the presence of α helix with β turns with characteristic absorption band designating the peptide bonds (–CONH–) for amides I, II and III at 1650, 1542 and 1242 cm−1 respectively. Amide I represents α helix at 1650 cm−1 and β turns at 1542 cm−1. Literature also suggests that the absorption that took place at 1650 cm−1 represents α helix and the band between 1630–1515 cm−1 represents the presence of β turns [27,28]. The peaks at 1400 and 1041 cm−1 in the spectrum of composite scaffolds correspond to carboxyl –COOH and C–O stretching bands, respectively. The characteristic peaks of the keratin composite scaffold are listed in Table 1.
Compression and tensile strength of both keratin composite and collagen (control) scaffolds were analyzed in dry and wet states. From the tensile strength curve, the ultimate tensile strength was calculated and compressive modulus was calculated from the load required for 50% compression of the scaffold. Ultimate tensile strength and compressive modulus of both the scaffolds in dry and wet conditions are presented in Table 2. We can observe that the ultimate tensile strength of the keratin composite scaffold in both dry and wet states is comparable to that of collagen scaffolds. The compressive modulus of keratin scaffolds especially in wet condition showed significantly higher values compared to collagen scaffolds prepared under similar conditions. Collagenous matrices have the innate ability to hold more water in comparison to keratin. Therefore collagen scaffold in wet condition could have resulted in greater swelling leading to lower resistance for compressive stress in comparison to keratin composite scaffold. 3.4. Water uptake capacity and porosity As the fabricated keratin composite scaffold was aimed for wound healing applications, the prepared scaffolds were tested for the water uptake capability. Porosity is related to water uptake capacity. The water uptake capacity and porosity of the keratin composite scaffold and collagen scaffold are given in Table 3. The keratin composite scaffold showed appreciable porosity (N 85%), which is comparable with that of collagen scaffold (84%), as estimated by ethanol infiltration method. Keratin composite scaffold also showed excellent water uptake capacity (N 1700%), placing it in a position almost comparable to collagen scaffold. These properties of keratin composite scaffolds are favorable for wound healing application.
Table 2 Tensile strength and compression of keratin composite scaffold and collagen scaffold. Analysis
Ultimate tensile strength (kPa) Compressive modulus (kPa)
Keratin composite scaffold
Collagen scaffold
Dry (Mean ± SD)
Wet (Mean ± SD)
Dry (Mean ± SD)
Wet (Mean ± SD)
95.69 ± 0.95 8.58 ± 0.50
10.06 ± 0.54 5.27 ± 0.55
107.33 ± 0.83 7.15 ± 0.86
12.46 ± 0.51 2.80 ± 0.44
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Fig. 5. Scanning Electron Microscopy of collagen (A) and keratin composite scaffold (B) at 300×.
3.5. Scanning Electron Microscopy The SEM images of the collagen and keratin composite scaffolds are shown in Fig. 5(A) and (B) respectively. Collagen scaffold (Fig. 5A) showed a highly porous structure, while keratin composite scaffold (Fig. 5B) also exhibited good porosity and well interconnected pores which would be very important for cell growth, proliferation and migration during the process of wound healing. 3.6. Biocompatibility–cell viability assay The NIH 3T3 cells cultured on the keratin composite scaffold as well as collagen (control) scaffold were monitored continuously for 7 days. The cell viability results of MTT assay (Fig. 6) performed after 3, 5 and 7 days showed no significant difference between the test sample and control, and after 7 days the cell viability of the composite scaffold was similar to that of collagen scaffold. This suggests that the prepared keratin composite scaffold has performance equivalent to collagen scaffolds with respect to the cell migration and proliferation rate on the scaffold. 4. Conclusions A novel keratin composite scaffold with chitosan and gelatin was prepared with a simple freeze drying process. The composite scaffold resulted in good thermal stability and all the functional groups of the individual constituent of the keratin composite remained intact as observed from FTIR results. The scaffold possessed good mechanical strength and water uptake capacity, apart from having micro-porous structure, which is likely to facilitate proliferation of cells. The composite scaffold was found to be biocompatible with fibroblast cells with
Fig. 6. Biocompatibility of keratin composite scaffold (KCG) and collagen scaffold.
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