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Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material
Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Materials Letters journal homepage: www.elsevier.com/locate/matlet
Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material Sivakumar Singaravelu, Giriprasath Ramanathan, M.D. Raja, Sagar Barge, Uma Tirichurapalli Sivagnanam n Bioproducts Lab, CSIR-Central Leather Research Institute, Adyar, Chennai, India
art ic l e i nf o
a b s t r a c t
Article history: Received 8 January 2015 Accepted 19 March 2015
The preparation of a skin substitute with improved mechanical property and biocompatibility is needed for the advancement of tissue engineering. This is a first time report on the extraction of keratin (HK) from bovine horn and its use in the preparation of a composite biosheet by blending with chitosan (CH). The prepared biosheet exhibited better thermal and mechanical properties. Surface morphology revealed that the biosheet supported fibroblast cell attachment, and this result was corroborated with the MTT assay. Sustained release of the mupirocin (MP) from biosheet was observed during in vitro drug release studies. The enhancement of cell adhesion and proliferation of NIH 3T3 fibroblast has shown better biocompatibility with the HK-based biosheet. Thus HK-based biosheet have good potentials and could be used as wound dressing material. & 2015 Published by Elsevier B.V.
Keywords: Biomaterials Biomimetic Composite materials
1. Introduction Tissue engineering promotes the wound healing process as it mimics the function of the extracellular matrix, thus it improves and maintains the biological function of the material. It therefore plays a pivotal role in restoring the function of tissue or whole organs [1]. Throughout the world, the bovine horn, a by-product of the slaughter house and meat industry remains unused and lying as biowaste. Keratin (HK), a fibrous structural protein, is abundantly present in horns [2]. HK is a valuable biopolymer in the field of biomedicine as it exhibits properties such as biodegradability, biocompatibility [3] and good mechanical strength. Keratins are rich in cysteine residues which form covalent disulfide bonds and give rise to more durable structures [4]. The blending of HK with suitable polymer (chitosan) becomes an essential to achieve a biomaterial with ideal mechanical properties. The presence of cell binding motifs in keratin and the mucoadhesive nature of the chitosan make the blended material more suitable for tissue engineering application. Mupirocin (MP) was incorporated in HK-based material which absorbs exudates and simultaneously enhances the bioavailability of the drug at the wound site which in turn accelerates the healing rate. The HK-based material provides release of the drug continuously in sustained manner
n Corresponding author. Tel.: þ91 44 24420709, mobile: þ 91 99400 81005; fax: þ91 44 24911589. E-mail address: [email protected] (U.T. Sivagnanam).
which lessens the dressing frequency and reduces the trauma to the patients.
2. Materials and methods Bovine horn was collected from the slaughterhouse at Perambur, Chennai. Chitosan (CH) (low molecular weight) and all other chemicals were purchased from Sigma-Aldrich, Bangalore, India, unless specified otherwise. The mouse NIH-3T3 fibroblast was obtained from the National Centre for Cell Science (NCCS), Pune, Maharastra, India. Extraction of HK from bovine horn: The HK from bovine horn was extracted by the modified Shindai method (Briefly in Supporting information S1) [4–6]. Subsequently, the extracted solution was filtered and centrifuged. The supernatant was dialyzed extensively against deionized water using a dialysis bag (12,000 Da) for 3 days to get the HK dialysate. Fabrication of keratin–chitosan (HK–CH) biosheet: The extracted HK and 2% (w/v) CH solutions were mixed in different stoichiometric ratios (Table 1). 1.5 ml of ethylene glycol was added as plasticizer. Finally, the mixture was poured into a polyethylene tray (measuring 16 cm 18 cm) and air dried at room temperature to get the HK–CH biosheet. Similarly the drug loaded biosheets (HK–CH–MP) was prepared by incorporation of 50 mg of MP. Physicochemical characterization: The prepared biosheets were characterized using Fourier Transform Infrared Spectroscopy (FTIRABB 3000 spectrometer) The Amide І region (1700–1600 cm 1) was
http://dx.doi.org/10.1016/j.matlet.2015.03.088 0167-577X/& 2015 Published by Elsevier B.V.
Please cite this article as: Singaravelu S, et al. Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material. Mater Lett (2015), http://dx.doi.org/10.1016/j.matlet.2015.03.088i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99
S. Singaravelu et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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curve fitted into Gaussian line shapes for secondary structure analysis by Origin 6.0 software (Origin Lab, USA). Absorption band positions of individual components were used to identify α-helix, β-sheet, β-turn, and disordered structure of the protein. The percentages of different types of secondary structures were determined by adding the sum of absorptions for each and expressing their sums as a fraction of the total Amide I band area in Fig. 2(d). Scanning electron microscopy (SEM) was employed to study the surface morphology of the biosheets. Thermal properties were studied by Differential scanning calorimetry (DSC) and Thermo gravimetric analysis (DTA) (universal V4.4A TA instruments). Mechanical properties such as elongation at break and tensile strength were assessed using a Universal Testing machine (INSTRON model 1405). The swelling study and in vitro drug release studies were done according to the procedure followed from Naveen et al. [7–8]. Biocompatibility study: NIH 3T3 fibroblast cells were seeded over biosheets and were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS), supplemented with antibiotics at 37 1C in a humidified atmosphere of 5% CO2. At the end of 24 h, cell viability was assessed by
Table 1 Tensile properties of biosheet at various stoichiometric ratios of HK and CH. Sample no.
HK:CH ratio
Mean tensile strength (MPa)
Mean elongation at break (%)
Mean Young's modulus (MPa)
1. 2. 3.
1:1 1:2 1:3
7.40 7 0.437 16.3 7 0.872 21.147 0.864
16.03 7 0.548 9.377 0.482 6.197 0.548
0.4617 0.635 1.7327 0.723 3.1457 0.962
standard 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay [9]. Evaluation of antimicrobial activity: The HK–CH and HK–CH–MP (0.3 mg/cm2) biosheet were cut into 1 cm2 and evaluated against gram-positive Staphylococcus aureus (ATCC 11632) and gramnegative Escherichia coli (ATCC 10536) using agar well diffusion method [10].
3. Results and discussion Physiochemical characterization: The FTIR of CH, HK, and HK–CH biosheets are depicted in Fig. 1(a). The strong absorption band at 2600– 3300 cm 1 and 1142 cm 1 corresponds to the OH and polysaccharide structure of chitosan. The amide I and II bands were observed at 3300– 3400 cm 1 and 1550–1650 cm 1, corresponding to the peak of HK with peptide bonds (O¼C–N–H) were attributed by major peaks held in the above mentioned region with NH bending and CH stretching between the same, signifying the interaction between HK and CH biosheets [11]. For the secondary structure analysis, the Amide I band spectrum was curve fitted into the Gaussian line shapes (Fig. 2(b)) with absorption region with individual peak representing α-helix (1650–1657 cm 1), β-sheet (1612– 1645 cm 1), β-turn (1655–1675 cm 1), and disordered structure (1640– 1651 and (1670–1697 cm 1). Calculated peak area for the HK showed 29.04% α-helix, 37.91% β-sheet, 26.08% β-turn and 6.95% disordered structure. The increase in β-sheet content is also key factor for the mechanical property of HK [12]. TGA thermograms of HK–CH and HK–CH–MP biosheet are shown in Fig. 1(c). Initial weight losses were due to the evaporation of water. The second weight loss of 63% for HK–CH–MP and 45% for HK–CH biosheet was observed at 200–400 1C due to denaturation of
Fig. 1. (a) FTIR spectra, (b) secondary structure analysis of HK by FTIR, (c) DSC, (d) TGA of biosheets.
Please cite this article as: Singaravelu S, et al. Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material. Mater Lett (2015), http://dx.doi.org/10.1016/j.matlet.2015.03.088i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
S. Singaravelu et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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3
Fig. 2. SEM micrograph of HK, CH, HK–CH and HK–CH–MP biosheets and SEM morphology of NIH 3T3 fibroblasts at different magnification (a) 20 mm (b) 5 mm of HK–CH biosheet.
Fig. 3. (a) Swelling studies, (b) In vitro drug release study, (c) MTT assay and antibacterial activity of biosheet, (d) Escherichia coli, (e) Staphylococcus aureus.
the alpha helix in the HK and decomposition of CH. In DSC (Fig. 1 (d)), the endothermic peak was observed to shift from 175 1C of HK– CH to 145 1C of HK–CH–MP due to the presence of the drug [11]. The SEM micrograph shows the uniform, smooth morphology of all biosheets (Fig. 2) as HK, CH, HK–CH and HK–CH–MP. Increase in the tensile strength with increase in the amount of CH was observed. Based on Young's modulus of the HK–CH biosheet in
sample No. 3 (Table 1), a ratio was selected to incorporate MP into the biosheet [10]. Furthermore, the swelling ability of the biosheet will improve the swelling behavior of the wound dressing material only by enhancing the oxygen permeability, by absorption of wound exudates, and by keeping wounds dry. The profound change observed later was due to the swelling ability of the CH (Fig. 3
Please cite this article as: Singaravelu S, et al. Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material. Mater Lett (2015), http://dx.doi.org/10.1016/j.matlet.2015.03.088i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
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(a)). In this case, the degree of swelling was directly proportional to the composition of CH present in the sample. The HK–CH–MP biosheet showed good swelling behavior, and this can be attributed to the hydrophilic nature of the CH. This result strongly substantiates the contention that HK-based biosheet could be an alternative wound dressing material [10]. In vitro release studies indicated that the burst release of MP (32%) within the first hour was followed by sustained release of MP (64%) at the end of 92 h. Initial burst release may be due to the release of surface bound drug on the HK–CH–MP biosheet. (Fig. 3(b)) [8],[13]. Biocompatibility study: The developed HK–CH biosheet showed about 90% cell viability with better adhesion and proliferation of fibroblast cells (Fig. 3(c)). Moreover, it was observed that the HKfabricated biosheets had better compatibility than CH biosheets. HK has cell binding motifs (RGD (Arg-Gly-Asp), LDV (Leu-Asp-Val)), which might have aided easy adhesion and proliferation of fibroblast into the HK–CH biosheet (Fig. 2(a) and (d)) [11]. High cell viability indicates that the blended HK–CH biosheet has a good potential to be used as a wound dressing for tissue engineering applications [7]. Evaluation of Antimicrobial activity: Evaluation of the biosheets against two bacterial species is shown in Fig. 3(d) and (e). Clear zones were observed against both the tested organisms. The formation of clear zone implies gradual diffusion of MP from the biosheet [10]. 4. Conclusion The HK-based biosheet incorporated with MP would serve dual purposes the with high compatibility by not only supporting cell growth, but also by releasing an antibiotic in a sustained manner at the wound site by preventing infection, augmented bioavailability at the wound site and increased healing efficiency. Results of these emphasize that physical characteristics, such as biosheet
36 37 38 39 40 41 Acknowledgments 42 The author thanks UGC, New Delhi, India for financial support Q3 43 Q4 44 as part of the RGNF Fellowship. 45 46 47 Appendix A. Supporting information 48 49 Supplementary data associated with this article can be found in 50 the online version at http://dx.doi.org/10.1016/j.matlet.2015.03. 51 088. 52 53 References 54 55 [1] Reichl Stephan. Biomaterials 2009;30:6854–66. 56 [2] Rouse JG, Van Dyke ME. Materials 2010;3:999–1014. 57 [3] Boateng J, Matthews K. J Pharm Sci 2008;97:2892–923. [4] Nakamura, Arimoto M, Takeuchi K, Fujii T. Biol Pharm Bull 2002;25:569–72. Q5 58 [5] Fujii T, Ogiwara D, Arimoto M. Biol Pharm Bull 2004;27:89–93. 59 [6] Fujii T, Li D. J Biol Macromol 2008;8:48–55. 60 [7] Nagiah N, Madhavi L, Anitha R, Srinivasan NT, Sivagnanam UT. Polym Bull 61 2013;70:2337–58. [8] Nagiah N, Ramanathan G, Sobhana L, Sivagnanam UT, Srinivasan NT. Int J Polym 62 Mater 2014;63:583–5. [9] Mahmoudi Morteza, et al. ACS Nano 2011;5(9):7263–76. Q6 63 64 [10] Muthukumar T, Senthil R, Sastry TP. Colloids Surf B Biointerfaces 2013;102:694–9. 65 [11] Bhardwaj Nandana, Sow Wan Ting, Devi Dipali, Ng Kee Woei, Mandal Biman B, 66 Cho Nam-Joon. Integr Biol 2013:1–3. Q7 67 [12] Cardamone Jeanette M. J Mol Struct 2010;969:97–105. [13] Shanmugasundaram N, Sundaraseelan J, Uma S, Selvaraj D, Babu Mary. JBMR 68 Part B, Appl Biomater 2006;77 B(2)). Q8 69 morphology, thermal stability, mechanical property, and in vitro release profile, can be precisely directed to find application in tissue engineering and serve as efficient wound dressing material.
Please cite this article as: Singaravelu S, et al. Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material. Mater Lett (2015), http://dx.doi.org/10.1016/j.matlet.2015.03.088i
1 2 3 4 5 6 7 8 9 10 11 12 13 Q2 14 15 16 Q1 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material Sivakumar Singaravelu, Giriprasath Ramanathan, M.D. Raja, Sagar Barge, Uma Tirichurapalli Sivagnanam n Bioproducts Lab, CSIR-Central Leather Research Institute, Adyar, Chennai, India
art ic l e i nf o
a b s t r a c t
Article history: Received 8 January 2015 Accepted 19 March 2015
The preparation of a skin substitute with improved mechanical property and biocompatibility is needed for the advancement of tissue engineering. This is a first time report on the extraction of keratin (HK) from bovine horn and its use in the preparation of a composite biosheet by blending with chitosan (CH). The prepared biosheet exhibited better thermal and mechanical properties. Surface morphology revealed that the biosheet supported fibroblast cell attachment, and this result was corroborated with the MTT assay. Sustained release of the mupirocin (MP) from biosheet was observed during in vitro drug release studies. The enhancement of cell adhesion and proliferation of NIH 3T3 fibroblast has shown better biocompatibility with the HK-based biosheet. Thus HK-based biosheet have good potentials and could be used as wound dressing material. & 2015 Published by Elsevier B.V.
Keywords: Biomaterials Biomimetic Composite materials
1. Introduction Tissue engineering promotes the wound healing process as it mimics the function of the extracellular matrix, thus it improves and maintains the biological function of the material. It therefore plays a pivotal role in restoring the function of tissue or whole organs [1]. Throughout the world, the bovine horn, a by-product of the slaughter house and meat industry remains unused and lying as biowaste. Keratin (HK), a fibrous structural protein, is abundantly present in horns [2]. HK is a valuable biopolymer in the field of biomedicine as it exhibits properties such as biodegradability, biocompatibility [3] and good mechanical strength. Keratins are rich in cysteine residues which form covalent disulfide bonds and give rise to more durable structures [4]. The blending of HK with suitable polymer (chitosan) becomes an essential to achieve a biomaterial with ideal mechanical properties. The presence of cell binding motifs in keratin and the mucoadhesive nature of the chitosan make the blended material more suitable for tissue engineering application. Mupirocin (MP) was incorporated in HK-based material which absorbs exudates and simultaneously enhances the bioavailability of the drug at the wound site which in turn accelerates the healing rate. The HK-based material provides release of the drug continuously in sustained manner
n Corresponding author. Tel.: þ91 44 24420709, mobile: þ 91 99400 81005; fax: þ91 44 24911589. E-mail address: [email protected] (U.T. Sivagnanam).
which lessens the dressing frequency and reduces the trauma to the patients.
2. Materials and methods Bovine horn was collected from the slaughterhouse at Perambur, Chennai. Chitosan (CH) (low molecular weight) and all other chemicals were purchased from Sigma-Aldrich, Bangalore, India, unless specified otherwise. The mouse NIH-3T3 fibroblast was obtained from the National Centre for Cell Science (NCCS), Pune, Maharastra, India. Extraction of HK from bovine horn: The HK from bovine horn was extracted by the modified Shindai method (Briefly in Supporting information S1) [4–6]. Subsequently, the extracted solution was filtered and centrifuged. The supernatant was dialyzed extensively against deionized water using a dialysis bag (12,000 Da) for 3 days to get the HK dialysate. Fabrication of keratin–chitosan (HK–CH) biosheet: The extracted HK and 2% (w/v) CH solutions were mixed in different stoichiometric ratios (Table 1). 1.5 ml of ethylene glycol was added as plasticizer. Finally, the mixture was poured into a polyethylene tray (measuring 16 cm 18 cm) and air dried at room temperature to get the HK–CH biosheet. Similarly the drug loaded biosheets (HK–CH–MP) was prepared by incorporation of 50 mg of MP. Physicochemical characterization: The prepared biosheets were characterized using Fourier Transform Infrared Spectroscopy (FTIRABB 3000 spectrometer) The Amide І region (1700–1600 cm 1) was
http://dx.doi.org/10.1016/j.matlet.2015.03.088 0167-577X/& 2015 Published by Elsevier B.V.
Please cite this article as: Singaravelu S, et al. Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material. Mater Lett (2015), http://dx.doi.org/10.1016/j.matlet.2015.03.088i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99
S. Singaravelu et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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curve fitted into Gaussian line shapes for secondary structure analysis by Origin 6.0 software (Origin Lab, USA). Absorption band positions of individual components were used to identify α-helix, β-sheet, β-turn, and disordered structure of the protein. The percentages of different types of secondary structures were determined by adding the sum of absorptions for each and expressing their sums as a fraction of the total Amide I band area in Fig. 2(d). Scanning electron microscopy (SEM) was employed to study the surface morphology of the biosheets. Thermal properties were studied by Differential scanning calorimetry (DSC) and Thermo gravimetric analysis (DTA) (universal V4.4A TA instruments). Mechanical properties such as elongation at break and tensile strength were assessed using a Universal Testing machine (INSTRON model 1405). The swelling study and in vitro drug release studies were done according to the procedure followed from Naveen et al. [7–8]. Biocompatibility study: NIH 3T3 fibroblast cells were seeded over biosheets and were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS), supplemented with antibiotics at 37 1C in a humidified atmosphere of 5% CO2. At the end of 24 h, cell viability was assessed by
Table 1 Tensile properties of biosheet at various stoichiometric ratios of HK and CH. Sample no.
HK:CH ratio
Mean tensile strength (MPa)
Mean elongation at break (%)
Mean Young's modulus (MPa)
1. 2. 3.
1:1 1:2 1:3
7.40 7 0.437 16.3 7 0.872 21.147 0.864
16.03 7 0.548 9.377 0.482 6.197 0.548
0.4617 0.635 1.7327 0.723 3.1457 0.962
standard 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay [9]. Evaluation of antimicrobial activity: The HK–CH and HK–CH–MP (0.3 mg/cm2) biosheet were cut into 1 cm2 and evaluated against gram-positive Staphylococcus aureus (ATCC 11632) and gramnegative Escherichia coli (ATCC 10536) using agar well diffusion method [10].
3. Results and discussion Physiochemical characterization: The FTIR of CH, HK, and HK–CH biosheets are depicted in Fig. 1(a). The strong absorption band at 2600– 3300 cm 1 and 1142 cm 1 corresponds to the OH and polysaccharide structure of chitosan. The amide I and II bands were observed at 3300– 3400 cm 1 and 1550–1650 cm 1, corresponding to the peak of HK with peptide bonds (O¼C–N–H) were attributed by major peaks held in the above mentioned region with NH bending and CH stretching between the same, signifying the interaction between HK and CH biosheets [11]. For the secondary structure analysis, the Amide I band spectrum was curve fitted into the Gaussian line shapes (Fig. 2(b)) with absorption region with individual peak representing α-helix (1650–1657 cm 1), β-sheet (1612– 1645 cm 1), β-turn (1655–1675 cm 1), and disordered structure (1640– 1651 and (1670–1697 cm 1). Calculated peak area for the HK showed 29.04% α-helix, 37.91% β-sheet, 26.08% β-turn and 6.95% disordered structure. The increase in β-sheet content is also key factor for the mechanical property of HK [12]. TGA thermograms of HK–CH and HK–CH–MP biosheet are shown in Fig. 1(c). Initial weight losses were due to the evaporation of water. The second weight loss of 63% for HK–CH–MP and 45% for HK–CH biosheet was observed at 200–400 1C due to denaturation of
Fig. 1. (a) FTIR spectra, (b) secondary structure analysis of HK by FTIR, (c) DSC, (d) TGA of biosheets.
Please cite this article as: Singaravelu S, et al. Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material. Mater Lett (2015), http://dx.doi.org/10.1016/j.matlet.2015.03.088i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
S. Singaravelu et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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3
Fig. 2. SEM micrograph of HK, CH, HK–CH and HK–CH–MP biosheets and SEM morphology of NIH 3T3 fibroblasts at different magnification (a) 20 mm (b) 5 mm of HK–CH biosheet.
Fig. 3. (a) Swelling studies, (b) In vitro drug release study, (c) MTT assay and antibacterial activity of biosheet, (d) Escherichia coli, (e) Staphylococcus aureus.
the alpha helix in the HK and decomposition of CH. In DSC (Fig. 1 (d)), the endothermic peak was observed to shift from 175 1C of HK– CH to 145 1C of HK–CH–MP due to the presence of the drug [11]. The SEM micrograph shows the uniform, smooth morphology of all biosheets (Fig. 2) as HK, CH, HK–CH and HK–CH–MP. Increase in the tensile strength with increase in the amount of CH was observed. Based on Young's modulus of the HK–CH biosheet in
sample No. 3 (Table 1), a ratio was selected to incorporate MP into the biosheet [10]. Furthermore, the swelling ability of the biosheet will improve the swelling behavior of the wound dressing material only by enhancing the oxygen permeability, by absorption of wound exudates, and by keeping wounds dry. The profound change observed later was due to the swelling ability of the CH (Fig. 3
Please cite this article as: Singaravelu S, et al. Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material. Mater Lett (2015), http://dx.doi.org/10.1016/j.matlet.2015.03.088i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
S. Singaravelu et al. / Materials Letters ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
(a)). In this case, the degree of swelling was directly proportional to the composition of CH present in the sample. The HK–CH–MP biosheet showed good swelling behavior, and this can be attributed to the hydrophilic nature of the CH. This result strongly substantiates the contention that HK-based biosheet could be an alternative wound dressing material [10]. In vitro release studies indicated that the burst release of MP (32%) within the first hour was followed by sustained release of MP (64%) at the end of 92 h. Initial burst release may be due to the release of surface bound drug on the HK–CH–MP biosheet. (Fig. 3(b)) [8],[13]. Biocompatibility study: The developed HK–CH biosheet showed about 90% cell viability with better adhesion and proliferation of fibroblast cells (Fig. 3(c)). Moreover, it was observed that the HKfabricated biosheets had better compatibility than CH biosheets. HK has cell binding motifs (RGD (Arg-Gly-Asp), LDV (Leu-Asp-Val)), which might have aided easy adhesion and proliferation of fibroblast into the HK–CH biosheet (Fig. 2(a) and (d)) [11]. High cell viability indicates that the blended HK–CH biosheet has a good potential to be used as a wound dressing for tissue engineering applications [7]. Evaluation of Antimicrobial activity: Evaluation of the biosheets against two bacterial species is shown in Fig. 3(d) and (e). Clear zones were observed against both the tested organisms. The formation of clear zone implies gradual diffusion of MP from the biosheet [10]. 4. Conclusion The HK-based biosheet incorporated with MP would serve dual purposes the with high compatibility by not only supporting cell growth, but also by releasing an antibiotic in a sustained manner at the wound site by preventing infection, augmented bioavailability at the wound site and increased healing efficiency. Results of these emphasize that physical characteristics, such as biosheet
36 37 38 39 40 41 Acknowledgments 42 The author thanks UGC, New Delhi, India for financial support Q3 43 Q4 44 as part of the RGNF Fellowship. 45 46 47 Appendix A. Supporting information 48 49 Supplementary data associated with this article can be found in 50 the online version at http://dx.doi.org/10.1016/j.matlet.2015.03. 51 088. 52 53 References 54 55 [1] Reichl Stephan. Biomaterials 2009;30:6854–66. 56 [2] Rouse JG, Van Dyke ME. Materials 2010;3:999–1014. 57 [3] Boateng J, Matthews K. J Pharm Sci 2008;97:2892–923. [4] Nakamura, Arimoto M, Takeuchi K, Fujii T. Biol Pharm Bull 2002;25:569–72. Q5 58 [5] Fujii T, Ogiwara D, Arimoto M. Biol Pharm Bull 2004;27:89–93. 59 [6] Fujii T, Li D. J Biol Macromol 2008;8:48–55. 60 [7] Nagiah N, Madhavi L, Anitha R, Srinivasan NT, Sivagnanam UT. Polym Bull 61 2013;70:2337–58. [8] Nagiah N, Ramanathan G, Sobhana L, Sivagnanam UT, Srinivasan NT. Int J Polym 62 Mater 2014;63:583–5. [9] Mahmoudi Morteza, et al. ACS Nano 2011;5(9):7263–76. Q6 63 64 [10] Muthukumar T, Senthil R, Sastry TP. Colloids Surf B Biointerfaces 2013;102:694–9. 65 [11] Bhardwaj Nandana, Sow Wan Ting, Devi Dipali, Ng Kee Woei, Mandal Biman B, 66 Cho Nam-Joon. Integr Biol 2013:1–3. Q7 67 [12] Cardamone Jeanette M. J Mol Struct 2010;969:97–105. [13] Shanmugasundaram N, Sundaraseelan J, Uma S, Selvaraj D, Babu Mary. JBMR 68 Part B, Appl Biomater 2006;77 B(2)). Q8 69 morphology, thermal stability, mechanical property, and in vitro release profile, can be precisely directed to find application in tissue engineering and serve as efficient wound dressing material.
Please cite this article as: Singaravelu S, et al. Preparation and characterization of keratin-based biosheet from bovine horn waste as wound dressing material. Mater Lett (2015), http://dx.doi.org/10.1016/j.matlet.2015.03.088i