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Author’s Accepted Manuscript Influence of glycosaminoglycans on the properties of thin films based on chitosan/collagen blends B. Kaczmarek, A. Sionkowska, J. SkopinskaWisniewska www.elsevier.com/locate/jmbbm
PII: DOI: Reference:
S1751-6161(18)30091-2 https://doi.org/10.1016/j.jmbbm.2018.02.006 JMBBM2683
To appear in: Journal of the Mechanical Behavior of Biomedical Materials Received date: 29 November 2017 Revised date: 1 February 2018 Accepted date: 5 February 2018 Cite this article as: B. Kaczmarek, A. Sionkowska and J. Skopinska-Wisniewska, Influence of glycosaminoglycans on the properties of thin films based on chitosan/collagen blends, Journal of the Mechanical Behavior of Biomedical Materials, https://doi.org/10.1016/j.jmbbm.2018.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Influence of glycosaminoglycans on the properties of thin films based on chitosan/collagen blends
B. Kaczmarek, A. Sionkowska, J. Skopinska-Wisniewska Department of Chemistry of Biomaterials and Cosmetics, Faculty of Chemistry, Nicolaus Copernicus University, Toruń, Poland Abstract Thin films based on chitosan, collagen, and glycosaminoglycans isolated from fish skin were obtained by solvent evaporation. The films were characterized by different analyses, e.g. surface free energy determination, swelling tests, roughness, mechanical and thermal measurements. Moreover, the degradation studies were carried out by the film treatment with collagenase. The results showed that the properties of the films based on chitosan and collagen can be modified by the glycosaminoglycans addition. It was noticed that the addition of glycosaminoglycans enhances the surface hydrophilicity and reduces surface free energy. Surfaces of films modified by glycosaminoglycans (GAGs) show more roughness which inhibits the risk of biofilm formation. The highest films swelling was obtained after 2h immersion in phosphate-buffered saline (PBS). After their immersion in PBS, the films were more elastic, which was assumed on the basis of the elongation at break values higher than in the case of films on a dry surface. The proposed films can create biocompatible coatings for biomedical applications.
Keywords: thin films, chitosan, collagen, glycosaminoglycans
Introduction Materials existing in the form of thin films can be obtained from natural or synthetic polymers [1-3]. To improve the compatibility of metallic implants with their surrounding environment after implantation into the human body, they can be covered by a thin layer of e.g. natural polymers [4]. It is beneficial to use them in order to obtain such a layer due to its biocompatibility. Collagen and chitosan can be used to obtain thin films for biomedical applications because they present significant biocompatibility. Moreover, they are miscible in different ratios [5]. Materials obtained from collagen and chitosan mixed in the 50/50 weight ratio exhibit the most appropriate properties for biomedical applications. Previous chitosan and
collagen blends research showed that the glycosaminoglycans (GAGs) addition enables the homogeneous mixture formation [6]. GAGs can be isolated from natural sources and constitute the components of the cells extracellular matrix. Their presence in the material improves the cells response in in vitro conditions [7]. The properties of such films, including their mechanical and thermal characteristics, surface free energy as well as their roughness, should be considered. Mechanical properties are meaningful because the material needs to be flexible to cover the implant surface [8]. Thermal properties must be measured due to the material behavior determination at~120 oC. Such temperature is required in the sterilization process and should not reduce the film stability [9]. However, the surface properties which determine the cells response after the material implantation are most important. A hydrophilic surface allows for the cells adhesion and, as a result, improves the film compatibility with surrounding tissues. Furthermore, a rough surface inhibits the biofilm formation, which is one of the main problems in implantation surgery [10]. The aim of the study was to obtain materials based on collagen and chitosan enriched with glycosaminoglycans isolated from Salmo salar fish skin. The biocompatibility studies were carried out previously with the human osteosarcoma cells [7]. The results showed the increase of cells viability on scaffolds with GAGs. To consider the film-forming properties of such a mixture, surface free energy, roughness, enzymatic degradation, swelling as well as mechanical and thermal properties were evaluated.
Materials and methods Collagen was isolated from rat tail tendons in our laboratory. Chitosan (Mv=5.4 x 105 g/mol; deacetylation degree: 77%) was purchased from Sigma-Aldrich Company (Poland). Polymers were dissolved in 0.1M acetic acid at 1% concentration each. The procedure of glycosaminoglycans (GAGs) isolation was according to the procedure reported previously. The skin from fish Salmo Salar (from Koral s. c., Tychy, Poland) was defatted in acetone for 48h. Obtained precipitate was dried at 60oC and dissolved in 100mM sodium acetate buffer containing 5mM EDTA and 5mM cysteine. Then papain was added (100mg per 1g of precipitate) and incubated at 60oC for 1h. Solution was boiling for 10 min and centrifuged at 10 000 x g for 15 min. Three volumes of saturated ethanol with sodium acetate was added and centrifuge again. Then it was died at 60oC. Obtained precipitate was dissolved in distiller water to obtain solution with 1% concentration. The isolated glycosaminoglycans mixture contains the hyaluronic acid and chondroitin sulfate [6].
Samples preparation Chitosan and collagen solutions were mixed in the 50/50 weight ratio. During stirring, 1 and 5% addition of glycosaminoglycans solutions was added based on the chitosan and collagen amount. GAGs were also added to the chitosan solution as well as collagen. The mixtures were then placed in the incubator at 37oC for the solvent evaporation. Thin solid films of ~0.02 mm thickness were obtained. Films based on pure chitosan and collagen were also prepared and tested as the control samples.
Contact angle measurement Surface free energy (Ɣs), its polar (ƔsP) and dispersive (ƔsD) components can be calculated by the contact angle measurement in which non-covalent forces between the liquid and film surface are formed by Owens-Wendt method which enables the dispersive and polar components of surface free energy to be obtained [11]. The contact angles of two liquids: glycerol and diiodomethane were measured at a constant temperature using a goniometer equipped with a system of drop shape analysis (DSA 10 Control Unit, Krüss, Germany).
Atomic force microscope Topographic images were obtained using a multimode scanning probe microscope with a NanoScope IIIa controller (Digital Instruments, Santa Barbara, CA) operating in the tapping mode at room temperature. Surface images were acquired at fixed resolution (512 × 512 data points) using scan width 1 μm with a scan rate of 1.97 Hz. Silicon tips with spring constant 2–10 N/m were used. Roughness parameter such as the arithmetic average (Ra) and root mean square (Rq) was calculated from 5 μm × 5 μm scanned area using Nanoscope software.
Enzymatic degradation For the enzymatic degradation study, films of the determined weight were immersed in 1 ml of 0.1M Tris-HCl (pH=7.4) containing 50 mM CaCl2, and they were incubated at 37oC for 30 min. Then,1 ml of 0.1M Tris-HCl containing 50 units of collagenase from Clostridium histolyticum Type I (Sigma-Aldrich, Poland) was added to the solution with the immersed films. The samples were incubated at 37oC for 1h. The reaction was terminated by the addition of 0.2 ml 0.25M EDTA. The films were rinsed with distilled water and immersed in methanol for 3h. Then, they were rinsed by distilled water again, frozen, and lyophilized. The
percentage of weight loss was determined by calculating the difference between scaffolds weight before and after degradation [12].
Swelling Swelling behavior was measured by scaffolds immersion in phosphate-buffer saline (PBS) solution (pH=7.4) for 2, 24 and 48h. After each period of time immersed materials were gentle dried and weighted. Swelling ratios were then calculated using the equation [13]: (3) where ms(t) is the weight of the scaffolds after immersion in PBS for the period of time and ms(0) is the weight before immersion. Mechanical testing In order to perform mechanical testing, the samples were immersed in PBS solution for 2h (the immersion time was determined by previous swelling tests). Then, the samples were placed in the testing device to measure their mechanical parameters such as the Young modulus, tensile strength, and elongation at break.
Differential scanning calorimetry Differential scanning calorimetry measurements were made by DSC equipment (NETZSCH Phoenix DSC 204 F1). Heating rate was 10oC/min, from 20 to 250 oC in nitrogen atmosphere with flow 40 ml/min. The weight of samples was 1.0–1.5 mg and was measured using aluminum measuring pans.
Results and discussion Contact angle measurement The results of the contact angle measurement and surface free energy calculation are shown in Table 1. Such analysis is a technique to measure the integration possibilities with the surround tissue environment. The GAGs addition improves the contact angle for glycerin. It suggests that the hydrophilicity of the surface is enhanced because implemented GAGs chains contain hydrophilic groups. The dispersive component was in the range of 20-31 mJ/m2 for all the samples, and the polar component was much lower, in the range of 0.3-2.8 mJ/m2. The surface free energy is energy resulting from the “dangling bonds” exposed at material’s surface [14]. It controls the cell-biomaterial interactions and the energy value should be
minimize for better material integrations. It was noticed that the GAGs addition decreases the films surface free energy. The hydrophilic surface is characterized by the low surface free energy value what is beneficial for its application as a biomaterial film. Table 1. The angle for glycerin (θG), angle for diiodomethane (θD), surface free energy (Ɣs), its polar (ƔsP) and dispersive (ƔsD) components of films based on chitosan (CTS), collagen (Coll), and their mixture (CTS/Coll) with 1% or 5% glycosaminoglycans addition (1% GAG or 5% GAG respectively). Specimen
θG [o]
θD [o]
Ɣs [mJ/m2]
ƔsP [mJ/m2]
ƔsD [mJ/m2]
CTS
62.90 4.10
90.97 1.27
26.48 0.46
1.46 0.16
25.02 0.30
CTS+1%GAG
87.67 4.97
65.70 2.40
25.22 0.78
2.83 0.28
22.46 0.50
CTS+5%GAG
91.70 1.90
66.97 0.81
24.24 0.26
1.78 0.08
22.39 0.17
Coll
54.85 2.30
92.95 0.92
31.47 0.28
0.40 0.05
31.07 0.23
Coll+1%GAG
84.57 1.40
59.43 1.76
28.69 0.49
2.87 0.11
25.82 0.38
Coll+5%GAG
91.70 1.90
67.47 2.05
23.98 0.52
1.85 0.11
22.13 0.41
CTS/Coll
62.83 1.70
97.40 1.65
26.93 0.40
0.30 0.04
26.63 0.36
CTS/Coll+1%GAG
97.66 1.10
63.13 1.24
26.77 0.30
2.29 0.03
26.48 0.27
CTS/Coll+5%GAG
89.93 2.68
69.37 0.65
23.17 0.29
1.68 0.14
20.50 0.15
Atomic force microscope The atomic force microscopy analysis was used to evaluate the films surface morphology (Figure 1). The corresponding roughness values are listed in Table 2. It has been observed that the GAGs addition improves the surface roughness. Moreover, ternary blends of chitosan/collagen with GAGs present roughness higher than binary ones. The positively charged chitosan interacts with a negatively charged GAGs. As a result the complexes are formed and the organization of polymeric chains is changed. It has been reported that the chitosan deacetylation degree in the range of 70-90% facilitates the hydrogen bond formation, and consequently, the crystallinity formation is favored [15]. To consider the material in terms of its biomedical applicability it should be characterized by rough surface as a requirement. Such a property improves the cells adhesion to the film surface due to their flexible cell membrane. At the same time, the rough surface inhibits the biofilm formation due to the stiff bacteria cell wall where adhesion is possible only to flat surfaces [16].
Table 2. The surface roughness of thin films based on chitosan (CTS), collagen (Coll) with and without glycosaminoglycans addition (GAGs). Specimen
Ra [nm]
Rq [nm]
CTS
4.24
5.33
CTS+1%GAG
4.27
5.34
CTS+5%GAG
5.92
7.10
Coll
3.64
4.59
Coll+1%GAG
5.04
6.12
Coll+5%GAG
9.80
11.90
CTS/Coll
11.20
15.90
CTS/Coll+1%GAG
13.80
17.20
CTS/Coll+5%GAG
18.00
23.00
Enzymatic degradation The enzymatic degradation can be the reason of the film weight loss after the material application to the human body. For the enzymatic degradation studies, collagenase was used (Figure 2). The results showed that the degradation by collagenase attained the highest degree for pure collagen-based films. The differences in the weight loss of those type of samples are in the range of standard deviation. Therefore, the GAGs addition shows no significant influence on the films degradation by collagenase; however, in the mixture with chitosan, it reduces the film weight changes. The GAGs added to the chitosan/collagen mixture stabilize the collagen by the electrostatic interactions of negatively charged GAGs chains and amphiphilic collagen. The decrease weight loss of films based on chitosan with GAGs addition is related with the stabilization process related with the complex formation.
Swelling The material swelling properties are important for the medical applications. It is necessary to study the swelling behavior in order to observe how the material will change after its placement into the aqueous environment. The analysis showed that the initial 2h immersion in PBS results in the highest films swelling (Figure 3). The films based on pure chitosan and collagen dissolved during immersion. The addition of glycosaminoglycans to pure chitosan and collagen, as well as to the chitosan/collagen mixture improved films stability. The
percentage swelling of chitosan-based blends was higher for films with 5% GAGs addition than with 1% in the initial 2h. The decrease of percentage swelling values in time was noticed for samples with 5% GAGs addition. It can be related with the initiated degradation process. The collagen-based films had the highest percentage swelling value after the initial 2h compared to 24 and 48h. Then the swelling decreased as a result of the sample degradation. The difference in the collagen-based film can be noticed with the higher amount of glycosaminoglycans added to the collagen. It improves the collagen film stability, where pure collagen has low stability in PBS solution. Compared the percentage swelling behavior for films based on chitosan/collagen mixture with 1 and 5% GAGs addition it can be noticed that the highest changes in the swelling behavior was observed after 24h for films with 1% GAGs. Nevertheless, it can be assumed that the addition of GAGs improves the films stability; however, the swelling was changed with the longer immersion time. The main goal of the GAGs addition was noticed as the stability improvement compared with the pure chitosan, collagen and chitosan/collagen blends.
Mechanical testing The mechanical properties of the films based on chitosan and collagen with GAGs addition were measured. The Young Modulus was determined for the films with dry surface (Figure 4) and for the samples in the swollen state (Figure 7). Moreover, the tensile strength (Figure 5 and 8) and elongation at break (Figure 6 and 9) were measured. The results showed that the films with the dry surface are characterized by the higher Young Modulus values. The swollen films have lower stiffness compared to the dry films. The Young Modulus values for the dry films was in the range ~1-1.75 GPa and for the swollen samples ~6-16 MPa. Such trend can be also noticed for the tensile strength values. The films were noticeably more elastic when their elongation at break values was compared. For the samples with dry surface it is in the range ~3-5% and for the swollen films ~15-40%. Such differences are related with the high swelling behavior of the films based on natural polymers. The proposed films have hydrophilic character and as a result of the water molecules absorption the elasticity is improved. The dry chitosan/collagen/GAGs films have higher mechanical parameters compared to those with wet surface. However, films in the swollen state are more elastic what can be observed as higher the percentage elongation at break values compared to the films with dry surface.
Differential scanning calorimetry
Table 3. The maximum temperature of the process (T) and enthalpy of the processes (ΔH) measured during the samples heating by differential scanning calorimetry. Specimen
T [oC]
ΔH [mW/mg]
CTS
66.9
0.5920
CTS+1%GAG
67.7
0.6407
CTS+5%GAG
75.0
0.8530
Coll
61.5
0.7227
Coll+1%GAG
59.4
0.2992
Coll+5%GAG
61.8
0.4961
CTS/Coll
56.0
0.9344
CTS/Coll+1%GAG
62.5
0.9860
CTS/Coll+5%GAG
74.5
2.9350
Differential scanning calorimetry was used to determine the maximum temperatures of the process (T) and enthalpy of the processes (ΔH) which occurred during the samples heating. All the measurement results carried out for films with and without glycosaminoglycans are shown in Table 3. The occurring peak on the DSC curve is associated with the presence of water in the material and its release during heating. From the process enthalpy values, the amount of water present in the structure can be determined. The highest content of water was found for the chitosan and collagen mixture with 5% GAGs addition. Their addition to the mixtures containing chitosan improves the films thermal stability which is observed by increasing the maximum temperature of the process. The glycosaminoglycans presence in the collagen films results in the decrease of water content; however, the significant changes in maximum temperature values were not observed.
Conclusions The use of glycosaminoglycans isolated from fish skin is eco-friendly due to the biodegradable waste management standards since the skin can be applied for biomedical purposes. Thin films based on chitosan and collagen with the GAGs addition can be obtained by solvent evaporation. The GAGs presence modifies the films properties. Their presence reduces the film surface free energy and improves its roughness, which can inhibit the biofilm formation on the films after implantation. Such materials were also more stable in a PBS solution and resistant to degradation by collagenase. It can be assumed that the films
properties were improved in comparison to the properties exhibited by samples without GAGs. The proposed materials can be applied as biocompatible thin covers; however, it is expected that more detailed and advanced studies of cells as well as bacteria adhesion on their surface will be carried out in the near future.
Acknowledgement Financial support from the National Science Centre (NCN, Poland) Grant No UMO2015/19/N/ST8/02176 is gratefully acknowledged.
References [1] C.J. Buchko, L.C. Chen, Y. Shen, D.C. Martin, Processing and microstructural characterization of porous biocompatible protein polymer thin films, Polym. 40 (1999) 73977407. [2] R.N. Tharanathan, Biodegradable films and composite coatings: past, present and future, Trends Food Sci. Technol. 14 (2003) 71-78. [3] A. Dufresne, M. R. Vignon, Improvement of starch film performances using cellulose microfibrils, Macromolecules 31 (1998) 2693-2696. [4] B. O’Brien, W. Carroll, The evolution of cardiovascular stent materials and surfaces in response to clinical drivers: A review, Acta Biomater. 5 (2009) 945-958. [5] K. Lewandowska, A. Sionkowska, S. Grabska, B. Kaczmarek, M. Michalska, The miscibility of collagen/hyaluronic acid/chitosan blends investigated in dilute solutions and solids, J. Mol. Liq. 220 (2016) 726-730. [6] B. Kaczmarek, A. Sionkowska, K. Łukowicz, A.M. Osyczka, The cells viability study on the composites of chitosan and collagen with glycosaminoglycans isolated from fish skin, Mat. Lett. 206 (2017) 166-168. [7] B. Kaczmarek, A. Sionkowska, A.M. Osyczka, Collagen-based scaffolds enriched with glycosaminoglycans isolated from skin of Salmo salar fish, Polym. Test. 62 (2017) 132-136. [8] P.X. Ma, Biomimetic materials for tissue engineering, Adv. Drug Del. Rev. 60 (2008) 184-198. [9] R. Landers, U. Hubner, R. Schmelzeisen, R. Mulhaupt, Rapid prototyping of scaffolds derived from thermoreversible hydrogels and tailored for applications in tissue engineering, Biomaterials 23 (2002) 4437-4447. [10] M.M. Gentleman, E. Gentleman, The role of surface free energy in osteoblast– biomaterial interactions, Int. Mater. Rev. 59 (2014) 417-429.
[11] J. Skopinska-Wisniewska, A. Sionkowska, A. Kaminska, A. Kaznica, R. Jachimiak, T. Drewa, Surface characterization of collagen/elastin based biomaterials for tissue regeneration, Appl. Surf. Sci.255 (2009) 8286-8292. [12] J. Kozlowska, A. Sionkowska, Effects of different crosslinking methods on the properties of collagen–calcium phosphate composite materials, Int. J. Biol. Macromol. 74 (2015) 394403. [13] A. Sionkowska, B. Kaczmarek, Modification of 3D materials based on chitosan and collagen blends by sodium alginate, Mol. Cryst. Liq. Cryst. 640 (2016) 39-45. [14] M.M. Gentleman, E. Gentleman, The role of surface free energy in osteoblast– biomaterial interactions, Int. Mater. Rev. 59 (2014) 417-429. [15] B.W.S. Souza, M.A. Cerqueira, A. Casariego, A.M.P. Lima, J.A. Teixeira, A.A. Vicente, Effect of moderate electric fileds in the permeation properties of chitosan coatings, Food Hydrocoll. 23 (2009) 2110-2115. [16] A.C. Ionescu, S. Hahnel, G. Cazzaniga, M. Ottobelli, R.R. Braga, M.C. Rodrigues, E. Brambilla, Streptococcus mutans adherence and biofilm formation on experimental composites containing dicalcium phosphate dihydrate nanoparticles, J. Mater. Sci. Mater. Med. 28 (2017) doi: https://doi.org/10.1007/s10856-017-5914-7.
a
b
c
d
Figure 1. The AFM images of a) Coll+5%GAG b) CTS+5%GAG c) CTS/Coll+1%GAG d) CTS/Coll+5%GAG with 1 μm resolution.
weight loss [%]
25 20 15 10 5 0
Figure 2. The weight loss [%] of films based on chitosan (CTS), collagen (Coll) with 1 or 5% glycosaminoglycans (GAGs) after degradation by collagenase.
700
swelling [%]
600 500 400 300 200 100
2h 24h 48h
0
Figure 3. The film swelling behavior [%] based on chitosan (CTS), collagen (Coll) with glycosaminoglycans (GAG) after 2, 24 and 48h immersion time in PBS.
Emod [GPa]
2 1.5 1 0.5 0
Figure 4. The Young’s Modulus values measured of films with dry surface. 80 70
σmax [MPa]
60 50 40 30 20 10 0
Figure 5. The tensile strength values measured of films with dry surface.
6 5
dl [%]
4 3 2 1 0
Figure 6.The elongation at break value of films with dry surface.
Figure 7. The Young’s Modulus values measured for swollen films.
5
σmax [MPa]
4 3 2 1 0
Figure 8. The tensile strength values measured for swollen films.
45 40 35 dl [%]
30 25 20 15 10 5
0
Figure 9. The elongation at break values measured for swollen films.
PII: DOI: Reference:
S1751-6161(18)30091-2 https://doi.org/10.1016/j.jmbbm.2018.02.006 JMBBM2683
To appear in: Journal of the Mechanical Behavior of Biomedical Materials Received date: 29 November 2017 Revised date: 1 February 2018 Accepted date: 5 February 2018 Cite this article as: B. Kaczmarek, A. Sionkowska and J. Skopinska-Wisniewska, Influence of glycosaminoglycans on the properties of thin films based on chitosan/collagen blends, Journal of the Mechanical Behavior of Biomedical Materials, https://doi.org/10.1016/j.jmbbm.2018.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Influence of glycosaminoglycans on the properties of thin films based on chitosan/collagen blends
B. Kaczmarek, A. Sionkowska, J. Skopinska-Wisniewska Department of Chemistry of Biomaterials and Cosmetics, Faculty of Chemistry, Nicolaus Copernicus University, Toruń, Poland Abstract Thin films based on chitosan, collagen, and glycosaminoglycans isolated from fish skin were obtained by solvent evaporation. The films were characterized by different analyses, e.g. surface free energy determination, swelling tests, roughness, mechanical and thermal measurements. Moreover, the degradation studies were carried out by the film treatment with collagenase. The results showed that the properties of the films based on chitosan and collagen can be modified by the glycosaminoglycans addition. It was noticed that the addition of glycosaminoglycans enhances the surface hydrophilicity and reduces surface free energy. Surfaces of films modified by glycosaminoglycans (GAGs) show more roughness which inhibits the risk of biofilm formation. The highest films swelling was obtained after 2h immersion in phosphate-buffered saline (PBS). After their immersion in PBS, the films were more elastic, which was assumed on the basis of the elongation at break values higher than in the case of films on a dry surface. The proposed films can create biocompatible coatings for biomedical applications.
Keywords: thin films, chitosan, collagen, glycosaminoglycans
Introduction Materials existing in the form of thin films can be obtained from natural or synthetic polymers [1-3]. To improve the compatibility of metallic implants with their surrounding environment after implantation into the human body, they can be covered by a thin layer of e.g. natural polymers [4]. It is beneficial to use them in order to obtain such a layer due to its biocompatibility. Collagen and chitosan can be used to obtain thin films for biomedical applications because they present significant biocompatibility. Moreover, they are miscible in different ratios [5]. Materials obtained from collagen and chitosan mixed in the 50/50 weight ratio exhibit the most appropriate properties for biomedical applications. Previous chitosan and
collagen blends research showed that the glycosaminoglycans (GAGs) addition enables the homogeneous mixture formation [6]. GAGs can be isolated from natural sources and constitute the components of the cells extracellular matrix. Their presence in the material improves the cells response in in vitro conditions [7]. The properties of such films, including their mechanical and thermal characteristics, surface free energy as well as their roughness, should be considered. Mechanical properties are meaningful because the material needs to be flexible to cover the implant surface [8]. Thermal properties must be measured due to the material behavior determination at~120 oC. Such temperature is required in the sterilization process and should not reduce the film stability [9]. However, the surface properties which determine the cells response after the material implantation are most important. A hydrophilic surface allows for the cells adhesion and, as a result, improves the film compatibility with surrounding tissues. Furthermore, a rough surface inhibits the biofilm formation, which is one of the main problems in implantation surgery [10]. The aim of the study was to obtain materials based on collagen and chitosan enriched with glycosaminoglycans isolated from Salmo salar fish skin. The biocompatibility studies were carried out previously with the human osteosarcoma cells [7]. The results showed the increase of cells viability on scaffolds with GAGs. To consider the film-forming properties of such a mixture, surface free energy, roughness, enzymatic degradation, swelling as well as mechanical and thermal properties were evaluated.
Materials and methods Collagen was isolated from rat tail tendons in our laboratory. Chitosan (Mv=5.4 x 105 g/mol; deacetylation degree: 77%) was purchased from Sigma-Aldrich Company (Poland). Polymers were dissolved in 0.1M acetic acid at 1% concentration each. The procedure of glycosaminoglycans (GAGs) isolation was according to the procedure reported previously. The skin from fish Salmo Salar (from Koral s. c., Tychy, Poland) was defatted in acetone for 48h. Obtained precipitate was dried at 60oC and dissolved in 100mM sodium acetate buffer containing 5mM EDTA and 5mM cysteine. Then papain was added (100mg per 1g of precipitate) and incubated at 60oC for 1h. Solution was boiling for 10 min and centrifuged at 10 000 x g for 15 min. Three volumes of saturated ethanol with sodium acetate was added and centrifuge again. Then it was died at 60oC. Obtained precipitate was dissolved in distiller water to obtain solution with 1% concentration. The isolated glycosaminoglycans mixture contains the hyaluronic acid and chondroitin sulfate [6].
Samples preparation Chitosan and collagen solutions were mixed in the 50/50 weight ratio. During stirring, 1 and 5% addition of glycosaminoglycans solutions was added based on the chitosan and collagen amount. GAGs were also added to the chitosan solution as well as collagen. The mixtures were then placed in the incubator at 37oC for the solvent evaporation. Thin solid films of ~0.02 mm thickness were obtained. Films based on pure chitosan and collagen were also prepared and tested as the control samples.
Contact angle measurement Surface free energy (Ɣs), its polar (ƔsP) and dispersive (ƔsD) components can be calculated by the contact angle measurement in which non-covalent forces between the liquid and film surface are formed by Owens-Wendt method which enables the dispersive and polar components of surface free energy to be obtained [11]. The contact angles of two liquids: glycerol and diiodomethane were measured at a constant temperature using a goniometer equipped with a system of drop shape analysis (DSA 10 Control Unit, Krüss, Germany).
Atomic force microscope Topographic images were obtained using a multimode scanning probe microscope with a NanoScope IIIa controller (Digital Instruments, Santa Barbara, CA) operating in the tapping mode at room temperature. Surface images were acquired at fixed resolution (512 × 512 data points) using scan width 1 μm with a scan rate of 1.97 Hz. Silicon tips with spring constant 2–10 N/m were used. Roughness parameter such as the arithmetic average (Ra) and root mean square (Rq) was calculated from 5 μm × 5 μm scanned area using Nanoscope software.
Enzymatic degradation For the enzymatic degradation study, films of the determined weight were immersed in 1 ml of 0.1M Tris-HCl (pH=7.4) containing 50 mM CaCl2, and they were incubated at 37oC for 30 min. Then,1 ml of 0.1M Tris-HCl containing 50 units of collagenase from Clostridium histolyticum Type I (Sigma-Aldrich, Poland) was added to the solution with the immersed films. The samples were incubated at 37oC for 1h. The reaction was terminated by the addition of 0.2 ml 0.25M EDTA. The films were rinsed with distilled water and immersed in methanol for 3h. Then, they were rinsed by distilled water again, frozen, and lyophilized. The
percentage of weight loss was determined by calculating the difference between scaffolds weight before and after degradation [12].
Swelling Swelling behavior was measured by scaffolds immersion in phosphate-buffer saline (PBS) solution (pH=7.4) for 2, 24 and 48h. After each period of time immersed materials were gentle dried and weighted. Swelling ratios were then calculated using the equation [13]: (3) where ms(t) is the weight of the scaffolds after immersion in PBS for the period of time and ms(0) is the weight before immersion. Mechanical testing In order to perform mechanical testing, the samples were immersed in PBS solution for 2h (the immersion time was determined by previous swelling tests). Then, the samples were placed in the testing device to measure their mechanical parameters such as the Young modulus, tensile strength, and elongation at break.
Differential scanning calorimetry Differential scanning calorimetry measurements were made by DSC equipment (NETZSCH Phoenix DSC 204 F1). Heating rate was 10oC/min, from 20 to 250 oC in nitrogen atmosphere with flow 40 ml/min. The weight of samples was 1.0–1.5 mg and was measured using aluminum measuring pans.
Results and discussion Contact angle measurement The results of the contact angle measurement and surface free energy calculation are shown in Table 1. Such analysis is a technique to measure the integration possibilities with the surround tissue environment. The GAGs addition improves the contact angle for glycerin. It suggests that the hydrophilicity of the surface is enhanced because implemented GAGs chains contain hydrophilic groups. The dispersive component was in the range of 20-31 mJ/m2 for all the samples, and the polar component was much lower, in the range of 0.3-2.8 mJ/m2. The surface free energy is energy resulting from the “dangling bonds” exposed at material’s surface [14]. It controls the cell-biomaterial interactions and the energy value should be
minimize for better material integrations. It was noticed that the GAGs addition decreases the films surface free energy. The hydrophilic surface is characterized by the low surface free energy value what is beneficial for its application as a biomaterial film. Table 1. The angle for glycerin (θG), angle for diiodomethane (θD), surface free energy (Ɣs), its polar (ƔsP) and dispersive (ƔsD) components of films based on chitosan (CTS), collagen (Coll), and their mixture (CTS/Coll) with 1% or 5% glycosaminoglycans addition (1% GAG or 5% GAG respectively). Specimen
θG [o]
θD [o]
Ɣs [mJ/m2]
ƔsP [mJ/m2]
ƔsD [mJ/m2]
CTS
62.90 4.10
90.97 1.27
26.48 0.46
1.46 0.16
25.02 0.30
CTS+1%GAG
87.67 4.97
65.70 2.40
25.22 0.78
2.83 0.28
22.46 0.50
CTS+5%GAG
91.70 1.90
66.97 0.81
24.24 0.26
1.78 0.08
22.39 0.17
Coll
54.85 2.30
92.95 0.92
31.47 0.28
0.40 0.05
31.07 0.23
Coll+1%GAG
84.57 1.40
59.43 1.76
28.69 0.49
2.87 0.11
25.82 0.38
Coll+5%GAG
91.70 1.90
67.47 2.05
23.98 0.52
1.85 0.11
22.13 0.41
CTS/Coll
62.83 1.70
97.40 1.65
26.93 0.40
0.30 0.04
26.63 0.36
CTS/Coll+1%GAG
97.66 1.10
63.13 1.24
26.77 0.30
2.29 0.03
26.48 0.27
CTS/Coll+5%GAG
89.93 2.68
69.37 0.65
23.17 0.29
1.68 0.14
20.50 0.15
Atomic force microscope The atomic force microscopy analysis was used to evaluate the films surface morphology (Figure 1). The corresponding roughness values are listed in Table 2. It has been observed that the GAGs addition improves the surface roughness. Moreover, ternary blends of chitosan/collagen with GAGs present roughness higher than binary ones. The positively charged chitosan interacts with a negatively charged GAGs. As a result the complexes are formed and the organization of polymeric chains is changed. It has been reported that the chitosan deacetylation degree in the range of 70-90% facilitates the hydrogen bond formation, and consequently, the crystallinity formation is favored [15]. To consider the material in terms of its biomedical applicability it should be characterized by rough surface as a requirement. Such a property improves the cells adhesion to the film surface due to their flexible cell membrane. At the same time, the rough surface inhibits the biofilm formation due to the stiff bacteria cell wall where adhesion is possible only to flat surfaces [16].
Table 2. The surface roughness of thin films based on chitosan (CTS), collagen (Coll) with and without glycosaminoglycans addition (GAGs). Specimen
Ra [nm]
Rq [nm]
CTS
4.24
5.33
CTS+1%GAG
4.27
5.34
CTS+5%GAG
5.92
7.10
Coll
3.64
4.59
Coll+1%GAG
5.04
6.12
Coll+5%GAG
9.80
11.90
CTS/Coll
11.20
15.90
CTS/Coll+1%GAG
13.80
17.20
CTS/Coll+5%GAG
18.00
23.00
Enzymatic degradation The enzymatic degradation can be the reason of the film weight loss after the material application to the human body. For the enzymatic degradation studies, collagenase was used (Figure 2). The results showed that the degradation by collagenase attained the highest degree for pure collagen-based films. The differences in the weight loss of those type of samples are in the range of standard deviation. Therefore, the GAGs addition shows no significant influence on the films degradation by collagenase; however, in the mixture with chitosan, it reduces the film weight changes. The GAGs added to the chitosan/collagen mixture stabilize the collagen by the electrostatic interactions of negatively charged GAGs chains and amphiphilic collagen. The decrease weight loss of films based on chitosan with GAGs addition is related with the stabilization process related with the complex formation.
Swelling The material swelling properties are important for the medical applications. It is necessary to study the swelling behavior in order to observe how the material will change after its placement into the aqueous environment. The analysis showed that the initial 2h immersion in PBS results in the highest films swelling (Figure 3). The films based on pure chitosan and collagen dissolved during immersion. The addition of glycosaminoglycans to pure chitosan and collagen, as well as to the chitosan/collagen mixture improved films stability. The
percentage swelling of chitosan-based blends was higher for films with 5% GAGs addition than with 1% in the initial 2h. The decrease of percentage swelling values in time was noticed for samples with 5% GAGs addition. It can be related with the initiated degradation process. The collagen-based films had the highest percentage swelling value after the initial 2h compared to 24 and 48h. Then the swelling decreased as a result of the sample degradation. The difference in the collagen-based film can be noticed with the higher amount of glycosaminoglycans added to the collagen. It improves the collagen film stability, where pure collagen has low stability in PBS solution. Compared the percentage swelling behavior for films based on chitosan/collagen mixture with 1 and 5% GAGs addition it can be noticed that the highest changes in the swelling behavior was observed after 24h for films with 1% GAGs. Nevertheless, it can be assumed that the addition of GAGs improves the films stability; however, the swelling was changed with the longer immersion time. The main goal of the GAGs addition was noticed as the stability improvement compared with the pure chitosan, collagen and chitosan/collagen blends.
Mechanical testing The mechanical properties of the films based on chitosan and collagen with GAGs addition were measured. The Young Modulus was determined for the films with dry surface (Figure 4) and for the samples in the swollen state (Figure 7). Moreover, the tensile strength (Figure 5 and 8) and elongation at break (Figure 6 and 9) were measured. The results showed that the films with the dry surface are characterized by the higher Young Modulus values. The swollen films have lower stiffness compared to the dry films. The Young Modulus values for the dry films was in the range ~1-1.75 GPa and for the swollen samples ~6-16 MPa. Such trend can be also noticed for the tensile strength values. The films were noticeably more elastic when their elongation at break values was compared. For the samples with dry surface it is in the range ~3-5% and for the swollen films ~15-40%. Such differences are related with the high swelling behavior of the films based on natural polymers. The proposed films have hydrophilic character and as a result of the water molecules absorption the elasticity is improved. The dry chitosan/collagen/GAGs films have higher mechanical parameters compared to those with wet surface. However, films in the swollen state are more elastic what can be observed as higher the percentage elongation at break values compared to the films with dry surface.
Differential scanning calorimetry
Table 3. The maximum temperature of the process (T) and enthalpy of the processes (ΔH) measured during the samples heating by differential scanning calorimetry. Specimen
T [oC]
ΔH [mW/mg]
CTS
66.9
0.5920
CTS+1%GAG
67.7
0.6407
CTS+5%GAG
75.0
0.8530
Coll
61.5
0.7227
Coll+1%GAG
59.4
0.2992
Coll+5%GAG
61.8
0.4961
CTS/Coll
56.0
0.9344
CTS/Coll+1%GAG
62.5
0.9860
CTS/Coll+5%GAG
74.5
2.9350
Differential scanning calorimetry was used to determine the maximum temperatures of the process (T) and enthalpy of the processes (ΔH) which occurred during the samples heating. All the measurement results carried out for films with and without glycosaminoglycans are shown in Table 3. The occurring peak on the DSC curve is associated with the presence of water in the material and its release during heating. From the process enthalpy values, the amount of water present in the structure can be determined. The highest content of water was found for the chitosan and collagen mixture with 5% GAGs addition. Their addition to the mixtures containing chitosan improves the films thermal stability which is observed by increasing the maximum temperature of the process. The glycosaminoglycans presence in the collagen films results in the decrease of water content; however, the significant changes in maximum temperature values were not observed.
Conclusions The use of glycosaminoglycans isolated from fish skin is eco-friendly due to the biodegradable waste management standards since the skin can be applied for biomedical purposes. Thin films based on chitosan and collagen with the GAGs addition can be obtained by solvent evaporation. The GAGs presence modifies the films properties. Their presence reduces the film surface free energy and improves its roughness, which can inhibit the biofilm formation on the films after implantation. Such materials were also more stable in a PBS solution and resistant to degradation by collagenase. It can be assumed that the films
properties were improved in comparison to the properties exhibited by samples without GAGs. The proposed materials can be applied as biocompatible thin covers; however, it is expected that more detailed and advanced studies of cells as well as bacteria adhesion on their surface will be carried out in the near future.
Acknowledgement Financial support from the National Science Centre (NCN, Poland) Grant No UMO2015/19/N/ST8/02176 is gratefully acknowledged.
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a
b
c
d
Figure 1. The AFM images of a) Coll+5%GAG b) CTS+5%GAG c) CTS/Coll+1%GAG d) CTS/Coll+5%GAG with 1 μm resolution.
weight loss [%]
25 20 15 10 5 0
Figure 2. The weight loss [%] of films based on chitosan (CTS), collagen (Coll) with 1 or 5% glycosaminoglycans (GAGs) after degradation by collagenase.
700
swelling [%]
600 500 400 300 200 100
2h 24h 48h
0
Figure 3. The film swelling behavior [%] based on chitosan (CTS), collagen (Coll) with glycosaminoglycans (GAG) after 2, 24 and 48h immersion time in PBS.
Emod [GPa]
2 1.5 1 0.5 0
Figure 4. The Young’s Modulus values measured of films with dry surface. 80 70
σmax [MPa]
60 50 40 30 20 10 0
Figure 5. The tensile strength values measured of films with dry surface.
6 5
dl [%]
4 3 2 1 0
Figure 6.The elongation at break value of films with dry surface.
Figure 7. The Young’s Modulus values measured for swollen films.
5
σmax [MPa]
4 3 2 1 0
Figure 8. The tensile strength values measured for swollen films.
45 40 35 dl [%]
30 25 20 15 10 5
0
Figure 9. The elongation at break values measured for swollen films.