Tailoring the gelatin/chitosan electrospun scaffold for application in skin tissue engineering: an in vitro study
The nanofibrous structure containing protein and polysaccharide has good potential in tissue engineering. The present work aims to study the role of chitosan in gelatin/chitosan nanofibrous scaffolds fabricated through electrospinning process under optimized condition. The performance of chitosan in gelatin/chitosan nanofibrous scaffolds was evaluated by mechanical tests, scanning electron microscopy (SEM), Fourier transform infrared (FTIR) and in vitro cell culture on scaffolds with different gelatin/chitosan blend ratios. To assay the influence of chitosan ratio on biocompatibility of the electrospun gelatin/chitosan scaffolds for skin tissue engineering, the culturing of the human dermal fibroblast cells (HDF) on nanofibers in terms of attachment, morphology and proliferation was evaluated. Morphological observation showed that HDF cells were attached and spread well on highly porous gelatin/chitosan nanofibrous scaffolds displaying spindle-like shapes and stretching. The fibrous morphologies of electrospun gelatin/chitosan scaffolds in culture medium were maintained during 7 days. Cell proliferation on electrospun gelatin/chitosan scaffolds was quantified by MTS assay, which revealed the positive effect of chitosan content (around 30%) as well as the nanofibrous structure on the biocompatibility (cell proliferation and attachment) of substrates.
KeywordsGelatin/chitosan Blend ratio Nanofibers Skin HDF cells In vitro
In recent years, electrospinning as a reliable technique for production of biomimetic scaffolds containing large network of interconnected pores has gained great attention in the literature (Bhardwaj and Kundu 2010; Dabouian et al. 2018; Pezeshki-Modaress et al. 2014; Saeed et al. 2017).
The human body tissue is composed of cells and extracellular matrix (ECM) which provide proper structural components as well as controlling the body processes, performances and wound healings (Sell et al. 2010). The ECM contains highly hydrated macromolecular networks such as collagen and glycosaminoglycans (Wang et al. 2007). Tissue engineering provides constructs appropriate for tissue substitution. A crucial factor in tissue engineering is to design and fabricate a biocompatible and biodegradable scaffold for culturing or hosting cells and transplanting into the body to regenerate the neo-organs (Pietrucha and Marzec 2005).
The cells have to interact with the scaffolds’ structure in three dimensions. In natural ECM structure protein fibers’ diameters are smaller than the cells and could provide a direct contact with the cells in three-dimensional orientations. In summary, the tissue-engineered scaffold should provide the opportunity for to exchange the signals between cells and the microenvironment and also between the cells in regeneration process (Barnes et al. 2007). Therefore, electrospunnanofibrous substrates are good candidates for using as tissue-engineered scaffolds with nano-scale structure (Heydarkhan-Hagvall et al. 2008). Many research works have focused on proteins as biopolymers for fabrication of nanofibrous scaffolds. The components of natural tissues, collagen and GAGs are widely used for scaffold fabrication which serves as efficient substitutes for native ECM (Mottaghitalab et al. 2015; Zhong et al. 2005). Gelatin is a natural biopolymer which is notably similar to collagen and still less susceptible to degradation during electrospinning process and enjoy a great potential to conduct the migration, adhesion, growth and organization of cells during regeneration process (Heydarkhan-Hagvall et al. 2008; Mahboudi et al. 2015; Pant and Kim 2013; Pezeshki-Modaress et al. 2013; Sadeghi et al. 2018; Zandi et al. 2007). Chitosan including glucosamine and N-acetylglucosamine is a biocompatible and biodegradable polymer and in vivo assays have proven that chitosan-based biomaterials show non-inflammatory reaction after injection, implantation and ingestion in the human body (Barikani et al. 2014; Baxter et al. 2013; Jayakumar et al. 2011; Mao et al. 2003a). Scaffolds containing chitosan also benefit other useful properties such as wound healing property (because of structural similarity to glycosaminoglycans), reducing scars, hemostasis, antifungal and bacteriostasis character, which make it applicable as a dermal scaffold. Therefore, using the blend based on gelatin and chitosan to improve their individual properties could be applicable as scaffolding materials in tissue regeneration (Esfandiarpour-Boroujeni et al. 2016; Martínez-Camacho et al. 2011; Modaress et al. 2012; Pezeshki-Modaress et al. 2013; Rahman et al. 2013). It has been reported that a higher ratio of gelatin (> 50% w/w) in the gelatin/chitosan blended scaffolds resulted in better cell attachment and proliferation by considering the literature (Jafari et al. 2011; Modaress et al. 2012; Pezeshki-Modaress et al. 2013), but to the best of our knowledge there is no study on the influence of chitosan ratio on the nanofibrous scaffold properties in the literature. TFA/DCM (70/30) solvent system has been introduced as applicable solvent for electrospinning of gelatin/chitosan blends (Dhandayuthapani et al. 2010). Jafari et al. have fabricated gelatin/chitosan electrospun nanofibers using low molecular weight chitosan (Mw 1000 g mol−1) and exhibited the potential of produced nanofibers for skin tissue engineering (Jafari et al. 2011). Pezeshki et al. introduced the optimized conditions for electrospinning process of gelatin/chitosan at TFA/DCM (70/30) solvent system using response surface methodology (Pezeshki-Modaress et al. 2014). In this work, nanofibrous structures of gelatin/chitosan blends were fabricated and the effects of chitosan ratio (30, 40, 50 w/w) on the chemical, physical and biological property of the obtained nanofibers were studied.
Gelatin type B and chitosan of medium weight were purchased from Sigma-Aldrich (USA). N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), trifluoroacetic acid (TFA), dichloromethane (DCM) and ethanol were purchased from Merck (Germany). DMEM/F12 medium, FBS, trypsin/EDTA, l-glutamine, and penicillin/streptomycin were purchased from Gibco, Canada.
Preparation of polymer solution
Chitosan solution of 5% (w/v) and gelatin solution of 15% (w/v) were prepared by dissolving them in a co-solvent system of TFA and DCM (70:30) as previously reported (Pezeshki-Modaress et al. 2014). The two solutions were agitated overnight at room temperature to achieve homogeneous solutions with different ratios of 50/50, 60/40 and 70/30 (gelatin/chitosan). The solution was poured into a 5 mL syringe and was subjected to the electrospinning process using a horizontal system with a cylindrical collector covered by aluminum foil (Co881007 NYI, ANSTCO, Iran) at 30 °C. Electrospinning was performed at 27 kV applied voltage and 0.5 mL/h flow rate. The distance between the needle tip and collector was 100 mm. The electrospun nanofibers were kept at 4 °C and dry condition until further characterization.
Morphologies of the fibers
The morphology and diameters of electrospun nanofibers of gelatin/chitosan were investigated using scanning electron microscopy (SEM, VEGA, TESCAN, Czech) after gold sputter coating. The diameter of electrospun nanofibers was measured using image analysis software (Image J 1.42q, National Institute of Health, USA). At least 200 different fibers were used to determine the MFD and SDF.
Measurement of porosity
The values are expressed as the means ± standard error (n = 3).
The tensile mechanical properties were tested with a mechanical tester (SANTAM, STM-20, Iran). The samples were rectangular disks (30 × 10 mm2) with a thickness of around 40 µm tested at a constant tensile deformation rate of 5 mm/min in the dry state at room temperature. The stress and elongation-at-break were determined. The values are expressed as the means ± standard error (n = 3) (Mao et al. 2003b; Modaress et al. 2012).
Fourier transform infrared (FTIR) analysis
The chemical structure of the gelatin/chitosan nanofibers was analyzed by Fourier transform infrared spectroscopy (FTIR) using a BRUKER FTIR spectrophotometer (EQUINOX 55, Germany). The infrared spectra of the samples were measured over a wavelength range of 4000–400 cm−1.
Crosslinking and sterilization
The nanofibrous scaffolds were chemically crosslinked using 0.02 g N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) (Merck, Germany) in 10 mL pure ethanol for 24 h, and then sterilized with 70% ethanol for 4 h. They were rinsed several times in phosphate buffer solution (PBS) to remove traces of ethanol. The cross-linked electrospun scaffolds were kept at 4 °C under dry condition for further assessment.
In vitro human dermal fibroblast cell culture
HDF cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% l-glutamine in T-75 flask tissue culture. Four and six passage cells were used in all the experiments. The cells were cultured at 37 °C and 5% CO2. The culture medium was refreshed every 72 h. At confluence, fibroblast cells were harvested and subcultivated in the same medium. The cells were separated with 0.05% trypsin/EDTA, centrifuged, and re-suspended in medium. The sterile nanofibrous scaffolds were incubated in culture medium overnight in order to check contamination, increase protein adsorption and cell attachment onto the nanofibers. The density of 1 × 104 cells/cm2 in culture medium (DMEM/F12, Gibco, Canada), containing 10% fetal bovine serum, was seeded on the scaffold in a 24-well culture plates. The medium was replaced regularly every 48 h; the culture process was carried out in an incubator at 37 °C with 5% CO2. All experiments were run in triplicate. Cell proliferation on film served as reference and control substrates. The samples were analyzed by DAPI staining, MTS and scanning electron microscopy (SEM).
To evaluate the cell proliferation and metabolic activity on scaffolds, the MTS (Promega, G5421) assay was performed according to the manufacturer’s instructions. Briefly, HDF cells were seeded at a density of 1 × 10 4 cells/cm2 on scaffolds. The medium was changed every 2 days. At days 1, 3, 7 and 14, the scaffolds were transferred into new wells and the MTS solution was added into each well, after which the plates were incubated in the dark at 37 °C for 3 h. The absorbance of the solution was measured at 490 nm. The experiments were run in triplicate.
Scanning electron microscopy and DAPI staining
As far as the study of the morphological characteristics of cells cultured onto the nanofibrous matrices and also maintaining the fibrous structure of gelatin/chitosan substrate at cell culture medium were concerned, SEM observations were carried out. The morphological characteristics of the cells cultured onto the nanofibrous matrices were studied through scanning electron microscopy. After growing for 1 and 7 days, the cellular constructs of the HDF cells were harvested, washed with PBS to remove non-adherent cells and then fixed with 2.5% glutaraldehyde overnight at 4 °C, dehydrated through a series of graded alcohol solutions (50, 70, 80, 90, 95 and 100%) and then vacuum-dried overnight. Dry cellular constructs were sputter coated with gold and observed by SEM at an accelerating voltage of 15 kV. For DAPI staining process, samples were fixed for 2 h in a 10% PBS/neutral-buffered formalin solution (pH 7.4) at 25 °C. Subsequently, samples were washed in d.d.H2O and dehydrated in a graded alcohol series. Then, the samples were stained with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, D8417), after which they were visualized utilizing an Olympus fluorescent microscope (BX51 with Olympus DP72 digital camera).
Results and discussion
Morphology of the scaffolds
The MFD and SDF of electrospun gelatin/chitosan scaffolds
Physical and mechanical properties of gelatin/chitosan scaffolds
The electrospun gelatin (Chi 0) spectrum illustrated several characteristic absorption bands at 1670 cm−1 for amid 1 (C=O) stretching vibration, 1550 cm−1 for amid 2 (N–H) bending vibration, 1465 cm−1 for CH2 bending, 1262 cm−1 for amid 3 (C–N) stretching vibration and 1160 cm−1 for –C–O stretching (Al-Saidi et al. 2012; Bin Ahmad et al. 2011; Lai et al. 2012; Nguyen and Lee 2010). The electrospun chitosan (Chi 100) spectrum exhibited strong peak at 1680 cm−1 with a shoulder around 1645 cm−1 and a peak at 1566 cm−1 for C=O stretching vibration, vibration of amine group and ammonium ions, respectively (Haider et al. 2010; Qian et al. 2011). The peak at 1803 represents CO–F group. The Chi 100 spectrum displayed also some peaks at 1442, 1415 and 1351 cm−1 for C–H bending vibration, 1093 and 1157 cm−1 for C–O stretching vibration (Bin Ahmad et al. 2011; Dhandayuthapani et al. 2010; Yin et al. 2003).
The FTIR spectra of the nanofibrous gelatin/chitosan blends exhibited characteristic absorption bands at 1680 and 1554 cm−1 representing the carboxyl and amine groups.
The shifting and broadening of both 1680 and 1554 cm−1 peaks for gelatin/chitosan blend spectrum in comparison of pure gelatin and chitosan spectrum revealed the formation of hydrogen bonding between chitosan and gelatin. The hydroxyl, carboxyl and amine groups of gelatin could form hydrogen bond with hydroxyl and amine groups of chitosan. These interactions could lead to polyanionic–polycationic complex (Bin Ahmad et al. 2011; Dhandayuthapani et al. 2010; Qian et al. 2011). The gelatin/chitosan blends spectrum show absorption bands at 1093 and 1415 cm−1 representing the C–O stretching (represent the saccharide structure of chitosan) and C–H bending vibration, respectively, which is absent in pure gelatin spectrum. There is another important peak at 1803 cm−1 in Chi 100 and gelatin/chitosan blends spectrum for CO–F groups which is absent in pure gelatin spectrum.
In vitro cell adhesion and proliferation
Based on our results, gelatin/chitosan nanofibrous scaffolds were fabricated using electrospinning at optimized condition with average fiber diameter in the range 180–196 nm. The fibrous morphologies of electrospun gelatin/chitosan scaffolds in culture medium were remained intact during 7 days. The electrospun gelatin/chitosan scaffolds have porosity around 92% and tensile strength of 1.1 MPa. The FTIR spectroscopy analysis demonstrated the presence of chitosan in nanofibrous structure. To assay the bioactivity of scaffolds, the attachment, morphology and proliferation of HDF cells on electrospun gelatin/chitosan scaffolds were analyzed. The morphological observation showed that HDF cells attached and spread well on gelatin/chitosan nanofibrous scaffolds exhibiting spindle-like shape. The SEM micrograph also revealed that cross-linked electrospun gelatin/chitosan blends were able to maintain their nanofibrous morphology in culture medium and provide steady physical and chemical support for cell growth. The MTS results demonstrated the significant beneficially influence of chitosan and also the nanofibrous morphological on the HDF cell proliferation. Considering the MTS assay, porosity and also the mechanical property of gelatin/chitosan nanofibers with different ratios, it could be stated that Chi 30 (70/30 gelatin/chitosan composition) has a good potential for using in skin tissue engineering application.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Al-Saidi G, Al-Alawi A, Rahman M, Guizani N (2012) Fourier transform infrared (FTIR) spectroscopic study of extracted gelatin from shaari (Lithrinus microdon) skin: effects of extraction conditions. Int Food Res J 19:1167–1173Google Scholar
- Dhandayuthapani B, Krishnan UM, Sethuraman S (2010) Fabrication and characterization of chitosan-gelatin blend nanofibers for skin tissue engineering. J Biomed Mater Res Part B Appl Biomater 94:264–272Google Scholar
- Esfandiarpour-Boroujeni S, Bagheri-Khoulenjani S, Mirzadeh H (2016) Modeling and optimization of degree of folate grafted on chitosan and carboxymethyl–chitosan progress. Biomaterials 5:1–8Google Scholar
- Jafari J, Emami SH, Samadikuchaksaraei A, Bahar MA, Gorjipour F (2011) Electrospun chitosan–gelatin nanofiberous scaffold: Fabrication and in vitro evaluation. Bio-Med Mater Eng 21:99–112Google Scholar
- Nguyen T-H, Lee B-T (2010) Fabrication and characterization of cross-linked gelatin electro-spun nano-fibers. Commun Netw 2Google Scholar
- Pant HR, Kim CS (2013) Electrospun gelatin/nylon-6 composite nanofibers for biomedical applications. Polym Int 62:1008–1013Google Scholar
- Pezeshki-Modaress M, Rajabi-Zeleti S, Zandi M, Mirzadeh H, Sodeifi N, Nekookar A, Aghdami N (2013) Cell loaded gelatin/chitosan scaffolds fabricated by salt-leaching/lyophilization (SLL) for skin tissue engineering: in vitro and in vivo study. J Biomed Mater Res Part AGoogle Scholar
- Pezeshki-Modaress M, Zandi M, Mirzadeh H (2014) Fabrication of gelatin/chitosan nanofibrous scaffold: process optimization and empirical modeling. Polym IntGoogle Scholar
- Sadeghi A, Pezeshki-Modaress M, Zandi M (2018) Electrospun polyvinyl alcohol/gelatin/chondroitin sulfate nanofibrous scaffold: fabrication and in vitro evaluation. Int J Biol MacromolGoogle Scholar
- Saeed SM, Mirzadeh H, Zandi M, Barzin J (2017) Designing and fabrication of curcumin loaded PCL/PVA multi-layer nanofibrous electrospun structures as active wound dressing. Prog Biomater 1–10Google Scholar
- Zandi M, Mirzadeh H, Mayer C (2007) Effects of concentration, temperature, and pH on chain mobility of gelatin during early stages of gelation. Iran Polym J 16:861Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.