Electrospinning of polycaprolactone (PCL) with collagen coating from environmentally benign solvent: Preliminary physico-chemical studies for skin substitute
Kajal Ghosal1 *, Sabu Thomas1, Nandakumar Kalarikkal1, Arumugam Gnanamani2 1 2
Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kerala
Microbiology Division, Central Leather Research Institute (CSIR), Adyar, Chennai
*Correspond to Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Priyadarsini Hills, Kottayam, Kerala, India-686560 E-mail:
[email protected] 1
Abstract: Fabrication of nanofibers with some biomaterials based on natural materials (collagen) through electrospinning is an important area for research. The effect of collagen coating on polycaprolactone (PCL) nanofibers surface was studied here. In this work, PCL nanofibers with titanium dioxide (TiO2) nanopowder were used for the development of active wound dressings. We used here glacial acetic acid as a solvent, an environmentally benign solvent. The prepared nanofibers were coated by collagen by soaking scaffold in 10 mg/mL and 20 mg/ml collagen solution overnight. The samples produced were subjected to contact angle measurements, SEM, FTIR, XRD and for mechanical strength determination. Nanofibers in the range of 200–800 nm were produced. The other study confirmed the physical interaction between collagen and PCL. Hydrophilicity of PCL nanofibers was increased. That was confirmed by observing contact angle values. Hydrophilic surface of scaffold is necessary for biomedical applications. FTIR have proved the presence of amide group on PCL structure which will facilitate cell adhesion and proliferation. SEM images have clearly proved the formation of nanofibers as well as attachment of collagen with PCL nanofibers. XRD has shown crystalline nature of PCL polymer. PCL can impart more mechanical strength and incorporation of collagen has decreased the tensile strength at some extent.
Key Words: Electrospinning; PCL; Collagen; TiO2; Wound dressing.
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Introduction Open injuries will lead to serious bacterial wound infections, including gas gangrene and tetanus, and these in turn may delay in return to normal activities, and the possibility of a life-threatening illness. Wound infection can become a serious concern if injured patients get late definitive care or if the number of injured persons exceeds available trauma care capacity. Thus wound management is very essential in order to minimize the above said consequences and treatment costs. From the ancient time, a myriad of dressings [1] have been applied to cover up the wound. Till date there is a continuous effort to develop more suitable wound dressing material. A series of biodegradable polymer scaffolds are used for wound dressing, tissue engineering, and drug delivery. Polyesters, the main family of synthetic biodegradable polymers, have got vast applicability in biomedical field. Most of the biomedical application includes polyglycolide, polylactide, polycaprolactone[2], and their copolymers. Besides these aliphatic polyesters, different types of synthetic polymers are also being explored [3] by biomedical researchers with a goal to find out a strong yet flexible polymer scaffold with long durability. Our approach in this context is to develop a more advanced wound dressing material of core sheath nanofiber type. Specifically, we intend to obtain the material through electrospinning technique [4] with PCL and antimicrobial TiO2 as the core whereas collagen acts as the sheath. PCL [5] will act as structural components which attribute good mechanical property and collagen give a moist environment to the wound bed and effectively manage the wound exudates. Collagen, a natural extracellular matrix (ECM), is a component found in many tissues, such as bone, skin, tendon, ligament, and other connective tissues. Due to excellent assembled structure, abundance availability in nature, and degradability in biological environments, it has gained wide applications in tissue engineering [6]. The major limitation of collagen is its poor mechanical properties which may be overcome by blending it with PCL. Incorporation of metal oxide nanoparticles like TiO2 is to improve the antimicrobial properties of the designed material. Inorganic materials have shown excellent bacterial resistance and thermal stability over organic antibacterial materials. Due to their nano-scale size, inorganic nanoparticles exhibit improved physical, chemical and biological properties. TiO2 nanostructures have been extensively studied as antimicrobial agents. Nowadays there is a growing interest for use of TiO2 nanoparticles in polymeric scaffold. Barzegary et al. [7] have shown antibacterial effects of TiO2. 3
Materials Reconstituted Type I Collagen of bovine skin was obtained as a gift from Dr. A. Gnanamani, Central Leather Research Institute, Adyar, Chennai. PCL (Mw 80,000) was obtained from Sigma Aldrich, USA. Glacial Acetic acid, Analytical Grade was obtained from SRL, Mumbai. TiO2 nanopowder of 20 nm size range was purchased from Sigma-Aldrich.
Methods Preparation of PCL-TiO2 nanofibers with and without coating All formulations details (F1-F5) are listed in Table 1. Scaffolds were fabricated using the electrospinning technique. Nanofibers of PCL with and without TiO2 nanopowders were prepared using glacial acetic acid as a solvent. TiO2 (0.5% w/v and 1% w/v) was dissolved in glacial acetic acid at room temperature and was stirred for 1 hr. The suspension was sonicated for 15 min. For F1, PCL at a concentration of 8% (w/v) was dissolved in glacial acetic acid. For F2, PCL at a concentration of 10% (w/v) was added to 0.5% (w/v) TiO2 suspension and for F5, PCL at a concentration of 10% (w/v) was dissolved in 1.0% (w/v) TiO2 suspension. All solutions were again stirred for 24 hr. These solutions were sonicated for 15 min before electrospinning. PCL/TiO2 and control PCL solution were taken in a 10 mL syringe fitted with (23G) flat tip metal needle. The flow rate was set under 2.0 mL per hr through a syringe pump. A high voltage of around 20.0 kV from a high voltage supply (Modle AYRA N801, Goldstar, New Delhi, India) was applied between the metal needle tip and a grounded collector at a temperature of 25°C, and 65 ± 5% relative humidity. The gap between needle and collector was kept at 15 cm. F1, F2 and F5 samples were prepared using the above procedure. To obtain F3 and F4, Collagen coating over the PCL-TiO2 nanofibers scaffold was done by immersing F2 overnight into a different concentration of collagen solutions. The amounts of collagen were 10 mg/mL and 20 mg/ml in glacial acetic acid to prepare F3 and F4 respectively. Afterward, the constructs were washed three times with polybutylene succinate (PBS) and kept air-dried [8].
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XRD The X-ray measurements of nanofibers (F2 and F5) were carried out using Bruker D8 Advance X-ray diffractometer [9] with the scanning rate of 2° per min using Cu Ka radiation. The XRD data were collected in the 2θ range of 5°–60°.
Fourier transform infrared (FTIR) spectra An FTIR spectroscopic analysis of electrospun material was made using Spectrum One (PerkinElmer, USA model). FTIR spectra were recorded in the wave number range of 4000–400 cm−1 at room conditions.
Contact angle measurement Biomaterials will come into contact with water, blood, and other body fluids during their use. So it is desirable to check them for their wettability while intending to produce materials for biomedical applications such as scaffolds for wound healing or skin regeneration, cellular proliferation or tissue engineering. This can be done by checking the contact angle which is made by a liquid on the surface of the electrospun matrix. Here, surface contact angle measurements for all formulations in the presence of acetic acid were made according to the methods summarized [10] using contact angle meter (Holmarc Optomechatronics).
Tensile test For measurement of mechanical strength of the electrospun nanofibers (F1-F5), tensile testing was carried out using universal tensile machine (INSTRON 1408) in accordance [11] with American Standards for Testing Methods (ASTM) D882-97with a 500 N load cell at a speed of 1 mm/min onto the specimen. The nanofibers matrices were prepared with width 5 mm and gauge length of 20 mm. The thickness of the samples was around 0.2 mm.
SEM Surface morphology investigation of the electro spun fiber was done under a scanning electron microscope (SEM) (S3400NSEM, HITACHI). Prior to scanning under the SEM, the samples 5
were sputter coated with gold using a fine coater (E1010, HITACHI). On the basis of the SEM photographs, the diameters of fibers were analyzed using analysis software Digimizer VR . The materials were sputter-coated with a gold layer to avoid charging [12].
Results and Discussions Contact angle measurement Contact angle measurements have given a clear view about the hydrophilic and hydrophobic surface of scaffolds. When the study was done using less concentration of PCL as for F1, the contact angle was 86° and for F2 formulation, the contact angle increased to 90°. F2 formulation was coated with different concentration of collagen to get formulations F3 and F4. When contact angle study was done, it has been seen that there was reduction of contact angle upto 45° for F3 and 42° for F4. For F5, contact angle was 90° again. Collagen coating has increased the hydrophilicity of scaffold. As a result, it traps the water molecules and contact angle reduced. So collagen coating may act as a suitable biomaterial scaffold for biomedical applications [13]. All contact angle values are given in Table 2.
FTIR Analysis Typical bands such as N–H stretching at 3273 cm−1 for amide A, C–H stretching at 2919 cm−1 for amide B, C=O stretching at 1600–1700 cm−1 for amide I, N–H deformation at 1500–1550 cm−1 for amide II and N–H deformation at 1200–1300 cm−1 for amide III in collagen and PCL/collagen nanofiber scaffolds were found (Figures 1). The amide I, II and III band regions of the spectrum are directly related to polypeptide conformation. The amide I band, is a sensitive marker of polypeptide secondary structure. There are C–N stretching vibrations and N–H bending vibrations as minor vibration modes of the amide I band. The vibrational frequency of each C–O bond depends on the strength of the carbonyl oxygen and interactions between amide units, both of which are influenced by local peptide conformation leading to modification of secondary structure [8]. Amide groups were observed in collagen and PCL/collagen nanofiber scaffolds. These amide groups and carboxyl groups supported fibroblast attachment and proliferation in skin tissue engineering.
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SEM study Figure 2 displays the SEM micrographs of electrospun matrices obtained from the PCL and collagen/PCL blends. Fibers are clearly formed with globule structures. The diameters of the fibers were calculated using image analysis software, Digimizer VR . The average diameter was found to be in between 200-400 nm for the nanofibers of formulation F1. Other formulations diameter was in between 300-800 nm. SEM image of Figure 2(a) showed clearly the formation of fibers but with beads. It was believed that the concentration of the PCL polymer is still too small. Although the concentrations were high enough to obtain a suitable viscosity, it was too small to initiate a high degree of entanglement during solvent evaporation in the jet [14]. This entanglement was needed for fiber formation and locally the charge density increased seriously because of solvent evaporation. Along with the concentration of polymer, solvent may have some effect over the nanofiber formation. Di-electric constant of the solvent was main parameter which will effect nanofiber formation. Sometimes presence of metallic particle can change the solution conductivity and fibers formation may get effected. Here as a metallic nanoparticle, we have used TiO2 nanopowders which may change the solution conductivity and fibers formation may get effected. SEM images of Figure 2(b) showed the formations of nanofibers without beads. Collagen coating has shown some cross linking with the PCL nanofibers and it was cleary observed in Figure 2(c) and 2(d).
XRD The diffractograms in Figure 3 show that PCL/TiO2 combinations (F2 and F5) exhibits sharp peaks at 22° which can be detected on the diffractogram of PCL typical of a crystalline material. No characteristic peaks of TiO2 were detected indicating a good mix of TiO2 with the polymer matrix.
Tensile Strength Incorporation of PCL has increased tensile strength whereas presence of collagen has decreased the mechanical strength of this scaffold at some extent. Values are given in Table 2.
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Table 1. Formulations details on PCL, Collagen and TiO2 S No Collagen 1
PCl
TiO2
(mg/ml)
(mg/ml)
(%)
F1
0
80
0
F2
0
100
0.5
F3
10
100
0.5
F4
20
100
0.5
F5
0
100
1.0
Table 2. Contact angles and Tensile Tests values for all formulations
S. No
Contact
Tensile
Angles (°)
strength
n=3
(MPa) n=3
F1
86 ± 3.02
1.76 ± 0.30
F2
90 ± 2.84
1.93 ± 0.43
F3
45 ± 2.00
1.81 ± 0.29
F4
42 ± 2.04
1.75 ± 0.45
F5
90 ± 3.33
2.03 ± 0.22
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Figure 1. FTIR images of different formulations; (a) Collagen (b) PCL (c) F3 (d) F4
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Figure 2. SEM images of different formulations; (a)F1 (b) F2 (c) F3 (d) F4
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Figure 3. XRD image of F2 and F5 formulations Conclusion Electrospinning of PCL with TiO2 and without TiO2 was carried out using acetic acid and it produced nanofibers with diameter in the range of 200-800 nm. Biomaterials obtained from natural sources such as collagen demonstrated the feasibility and efficacy of coating over nanofibers of synthetic biomaterials. A better hydrophilicity of the collagen-coated nanofibers has been seen than the uncoated ones. Presence of collagen over the nanofibers showed the presence of amide group over PCL fibers which will help in cell adhesion and proliferation. TiO2 was well dispersed in PCL scaffold. So no characteristics peaks have been observed in XRD. Tensile strength of the scaffold decreased at some extent in the presence of collagen. Still effects of processing parameters such as needle diameter, polymer concentration, solvent, metallic particle and applied voltage need to be investigated fully on the electrospinning of PCL polymer.
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Acknowledgements The research has been sponsored by the University Grants Commission, India under the UGC D. S. Kothari postdoctoral fellowship scheme.
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Electrospun PCL nanofibers: It’s Modification towards Skin Tissue Engineering Kajal Ghosal, Sabu Thomas Centre for Nanoscience and Nanotechnology, Mahatma Gandhi University, Kerala
Synthetic polymer based nanofiber fabricated by electrospinning process were continuously used in tissue engineering for repairmen or restoration of all malfunctioned or lost tissue/organs. Nowadays the concepts of hybrid nanofibers are gaining importance towards improved biocompatibility. In our study, Titanium di-oxide (TiO2) incorporated PCL nanofibers were produced via electrospinning process using suitable solvent. Then electrospun fibers were coated with natural polymer collagen in an attempt to make it more biocompatible for skin tissue engineering. The produced samples were investigated for different analysis such as contact angle measurements, SEM, FTIR, XRD, tensile strength etc. Produced nanofibers were also tested for biological responses in terms of MTT assay and cell adhesion assay using human dermal fibroblasts (HDF). SEM study confirmed production of nanofibers. FTIR study showed the related functional groups of chemicals whereas XRD revealed nanoparticle distribution within nanofibers. Tensile strength tests presented different mechanical strength for nanofibers. MTT assay and cell adhesion showed that nanofibers were also biocompatible. Keywords: Synthetic polymer, Natural Polymer, Nanoparticle, Bio-compatibility, Tissue Engineering
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