Edited by: Ilkwon Oh, Korea Advanced Institute of Science & Technology (KAIST), South Korea
Reviewed by: Senentxu Lanceros-Mendez, University of Minho, Portugal; Cesare Stefanini, Sant'Anna School of Advanced Studies, Italy
This article was submitted to Bionics and Biomimetics, a section of the journal Frontiers in Bioengineering and Biotechnology
†These authors have contributed equally to this work
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The aim of this study is the analysis and characterization of a hydrolyzed keratin-based biomaterial and its processing using electrospinning technology to develop
In order to reproduce a functional engineered
Several synthetic and natural biomaterials have been tested and modified in order to mimic extra-cellular matrix (ECM) characteristics. Traditionally, synthetic polymers have been used because they can be processed more easily with different bioprinting technologies in different complex structure in order to mimic at meso- and micro- level the properties of targeted tissues (De Maria et al.,
Keratin is an interesting natural biomaterial that can be used for scaffold fabrication. It is a fibrous protein with a structure similar to collagen, and it is the main component of the epidermal corneal layer, nails and appendages such as hairs, horns, feathers, and wool. Keratin contains many molecules of the aminoacid cysteine that enables disulfide bridges formations. Disulfide bridges boost the hydrogen bonds action in maintaining a close cohesion of keratin chains, wrapping in a structure similar to a helix. These bonds, provide stiffness and strength to hair, nails and horns (Zhang et al.,
During the extraction process, keratin is degraded, chemically or enzymatically, in units of smaller dimensions with the breaking of disulphide bridges (hydrolyzed keratin) thereby losing its characteristics of rigidity and insolubility in water. Only through enzymatic hydrolysis, it is possible to control and obtain subunits of desired molecular weight (Tsuda and Nomura,
Keratin and its hydrolizates have tripeptides sequences Arg-Gly-Asp (RGD) and Leu-Asp-Val (LDV) that could bind with cell surface ligands acting as the ECM that facilitates cell-cell and cell-matrix interactions promoting cell adhesion and supporting cell proliferation (Wang et al.,
The weak point of keratin-based materials is their poor mechanical properties (Yamauchi et al.,
Homogeneous films with smooth surfaces were obtained, from mixtures of keratin and gelatin, which showed a high miscibility between the two components (Rouse and Van Dyke,
In this work, we produced and characterized films made of hydrolyzed-keratin-based biomaterials in terms of their chemical and physical features (swelling, contact angle, mechanical properties, and surface charge density). These materials were processed using the electrospinning technique to develop a suitable substrates for
Hydrolyzed keratin was gently provided by Consortium SGS (Santacroce sull'Arno, Italy), a company that processes animal byproducts. In this specific case, keratin was obtained from chicken feathers processed by alkaline hydrolysis (MW > 10 kDa) containing about 1% of free amino acids. The final concentration was 35° Brix.
A 10% w/v aqueous solution of gelatin from porcine skin, type A (Sigma-Aldrich, Italy) was prepared dissolving gelatin powder by continuous stirring at 50°C for 1 h. (3-Glycidyloxypropyl)-trimethoxysilane (GPTMS) (Sigma-Aldrich, Italy) was used as cross-linking agent.
Gelatin (G) and keratin hydrolizates (K) solutions were mixed at different volume ratios: K:G 1:1, 1:2, 2:1. GPTMS was added in a ratio of 6% w/v respect to the final volume, and the resulting solutions were stirred for 40 min at 50°C. Films from the three different solutions were prepared by casting in a plastic petri dish of 3 cm diameter and dried for 24 h at 37°C.
Solutions for electrospinning were prepared starting from a 10% w/v gelatin type A solution in 50% w/v (in deionized water) 2,2,2-Trifluoroethanol (TFE) (Sigma-Aldrich, Italy) (Ki et al.,
Three samples for each type of film were dipped in deionized water and at specific time points (every 30 min for the first 4 h, then every 1 h until 6 h and finally at 24 h, 30 h, and 4 days) their weight (using a Metler Toledo AE240 balance) and area (using ImageJ software after image acquisition with Canon A620 digital camera) were measured. The swelling percentage was calculated according to Equation (1):
where pi is the weight (or area) measured at the
where pf is the final weight of dried samples.
Static contact angle was measured using the sessile drop method, with a 5 μl double distilled water droplet at room temperature. Images were acquired with a horizontal optical microscope equipped with a digital camera and the CAM 200 software. The test was performed on dried and hydrated samples; for each angle reported, at least five measurements on different surface locations were averaged.
Surface potential or charge can have influence on cell adhesion (Chang et al.,
The surface charge density was calculated by modeling the system as a capacitor, with three different configuration reported in
In this work, electrospun structures were fabricated from the three different solutions using a Linari electrospinning system (Linari Engineering Ltd., Pisa, Italy), by varying the applied potential, the flow rate, the distance between the needle and the collector and the electrospinning process duration (time), in order to find the optimal working parameters (
Investigated electrospinning parameters.
Applied potential (kV) | 20 | 30 | 40 |
Distance from collector (cm) | 10 | 15 | 20 |
Time (h) | 1/2 | 1 | 2 |
K:G 1:1 sample (2 h deposition). Scale bar = 1 cm.
Mechanical characterization of both casted films and electrospun structures was carried out performing uniaxial tensile tests using a universal machine Zwick-Roell Z005 ProLine equipped with a 100N load cell. Three rectangular shaped specimens for each type of structure were tested. Samples were pulled with a strain rate of 10%/min of the initial length until failure. Load F (N) and elongation Δx (m) were recorded and from the stress–strain graphs the elastic modulus E (Pa), the failure stress σmax, (Pa), the corresponding failure strain εr, and toughness U (J m−3) were calculated.
Regarding electrospun structures, to evaluate how variations in the process parameters affect the mechanical properties, four different cases (
Combination of different electrospinning parameters analyzed to evaluate their effect on mechanical properties of keratin-based structures.
Case 1 | V1 | 1:2 | 20 | 1 | 2 | |
V2 | 1:2 | 20 | 1 | 2 | ||
Case 2 | T1 | 1:1 | 20 | 3 | 30 | |
T2 | 1:1 | 20 | 3 | 30 | ||
T3 | 1:1 | 20 | 3 | 30 | ||
Case 3 | F1 | 1:1 | 20 | 30 | 2 | |
F2 | 1:1 | 20 | 30 | 2 | ||
Case 4 | C1 | 20 | 1 | 30 | 2 | |
C2 | 20 | 1 | 30 | 2 |
The microscopic structure was analyzed by the scanning electron microscopy (SEM), focusing attention on diameter and distribution of the fibers. Three images were acquired for each sample at 300×, 500×, and 1,000× magnification. A qualitative diameter analysis was carried out by randomly measuring fibers in acquired images (at 1,000× magnification) by ImageJ software.
Cell culture was performed on K:G 1:1 electrospun samples given their mechanical properties more similar to biological tissues (see section Results), and the following electrospinning parameters were selected: 30 kV, 20 cm, 1 ml/h, 1 h. Human epithelial HEK-293T cells, rat neuronal RT4-P6D2T cells and human primary skin fibroblasts were plated at density of 2 × 104 cells/well in a 12-well plate on keratin-based structure and cultured in DMEM-10%FBS (Invitrogen) and evaluated at three time points (1, 2, and 4 days).
For proliferation assay, 5-ethynyl-2′-deoxyuridine (EdU, 10 μM in PBS, Invitrogen) was added in the medium 12 h before the end of the experiment. To monitor cell vitality, CellTracker ™Green CMFDA (5 μM in PBS, Invitrogen) was added for 1.5 h before fixation. Cells were fixed in 4% PFA for 5 min, washed twice in PBS for 5 min and then stained for EdU with Click-IT EdU Alexa Fluor 555 Imaging Kit (Life Technologies). Hoechst 33258 (Sigma Aldrich) was used as nuclear counterstain. Immunofluorescence acquisition was performed using Zeiss LSM 700 confocal microscopy.
A Shapiro-Wilk test was performed to evaluate if data distributions were normal. Statistical differences between Gaussian groups were evaluated by one-way ANOVA. Having three groups, in case of the existence of a significant difference, a
Films presented a large initial (within the first hour) swelling, which increases with the keratin concentration presenting a weight increase of 400% in the case of K:G 1:2 and 700% in case of K:G 2:1, before reaching a plateau after 6 h (
Samples with higher keratin content present a higher weight loss (around 80%) respect to samples with a lower content of keratin (60%) (
Both hydrated and dried samples present an high hydrophilicity, with a low contact angle (<40° for dry samples and <8° for wet samples). The influence of keratin content is significant only in hydrated sample (
The measure of the surface charge density shows negative values where the keratin content prevails, regardless the type of mathematical model used for analyzing data (
The keratin and gelatin based solutions resulted suitable for electrospinning, forming 3D structures with variable thickness depending on set parameters: for example, an increase in deposition time or flow rate increases the thickness. The diameter of the fibers was instead evaluated by SEM images analysis. The examined samples were electrospun at the same flow rate and deposition time, and thus these parameters were excluded from the analysis of the results.
Samples show a decrease in the diameter of the fibers as the applied voltage increases (
SEM images for different electrospun samples (1 h deposition at 1 ml/h):
Mechanical characterization of casted wet samples showed that ( the elastic modulus and maximum stress increases as the gelatin content increases (one-way ANOVA on elastic modulus data: the failure strain decreases as the gelatin content increases (
These statistically significant differences indicate that the increase of gelatin content creates stiffer but more fragile biomaterials. Mechanical properties of electrospun films were evaluated according to different cases showed in
Stress strain curves for electrospun films (in each graph εr and σmax are showed):
To test the efficacy of keratin-based structure in sustaining vitality and proliferation of cells we adopted different approaches.
Firstly, we used the CellTracker Green dye to test if different types of cells are vital after 4 days of culture in the keratin based structure. This fluorescent probe was designed to freely pass through cell membranes, but it is transformed in a cell-impermeant reaction product only in vital cells. As shown in
Different cell lines are vital and proliferate in keratin based structure.
Representative image sequence for HEK-293T and RT4-P6D2T nuclei after 4 days of culture, showing cells growing on different planes. Red arrows indicate some nuclei belonging to cells grew on different planes in keratin bases structure. Hoechst was used for nuclear staining. Scale bar is 50 μm.
In this work we presented a method for the use of a waste material such as keratin extracted from poultry feathers and its potential applications in tissue engineering field.
Results from casted samples characterization indicates that we can tune the chemical and mechanical properties of keratin-based biomaterials by varying the ratio between gelatin and keratin content, keeping the GPTMS content constant. Samples with higher keratin content present a better ability to adsorb water, which can be addressed to a less dense mesh of intermolecular bonds, while increasing the gelatin content let to a more dense molecular mesh with a lower weight loss and a lower swelling. These conclusions are also confirmed by mechanical testing, which indicates more rigid samples with increased gelatin content. The surface charge density test highlighted a difference between the different types of solutions. In particular, as the keratin content increases the surface charge density becomes more negative due to the isoelectric point of the two materials as explained in the section Results.
With regard to the electrospun structures, the main results obtained from the mechanical characterization can be summarized as follows:
as the flow rate increases and the duration of the electrospinning process is extended, stiffer but considerably more brittle structures are obtained; with a higher percentage of gelatin, there is an increase in the strength and toughness of the fiber matrix, as showed by casted films; the increase in voltage leads to embrittlement and lower strength of the fibers; the elastic modulus of the nanofiber matrix is directly proportional to the electrospinning time and the flow rate, while it is not influenced by the voltage and the gelatin percentage of the solution; the maximum stress is inversely proportional to the voltage and directly proportional to the percentage of gelatin present in the solution; the failure strain is influenced by all the parameters of the electrospinning process. In particular, it is inversely proportional to the voltage, the duration of the test and the flow rate, while it increases as the percentage of gelatin increases; the energy per unit of volume stored by matrix (i.e., toughness) decreases with increasing voltage and flow rate.
Main results of mechanical characterization are summarized in
Mechanical characterization on electrospun samples results: ↑means direct proportionality, ↓means inverse proportionality, – means that there is no correlation between parameters.
Voltage | ||||
% gelatin | ||||
Electrospinning duration | ||||
Flow |
Furthermore, based on the effects that each parameter of the electrospinning process has on the mechanical properties of the nanofiber matrices, it is possible to choose which combination of parameter is the best according to different applications. For example, when regenerating biological tissues, it is preferable that the mechanical characteristics of the electrospun structures are as close as possible to those of the tissue to reproduce. In particular, the results obtained from the mechanical characterization are comparable to two specific soft tissues, such as nerve tissue and skin. Comparing skin values (Dunn and Silver,
Considering, instead, the nervous tissue and its mechanical properties (Kamra et al.,
In this paper we showed that waste materials such as keratin hydrolizates derived from chicken feathers can be reused to develop a novel biomaterial, whose chemical, physical and mechanical properties can be tuned varying the gelatin content. Moreover, these materials can be processed by electrospinning system and the mechanical and biological tests showed that they could have promising applications in the tissue engineering, regenerative medicine, and biofabrication areas.
The datasets generated for this study are available on request to the corresponding author.
GF, FD, and CD: writing—original draft preparation. GF, FD, SB, AD, FB, AL, DB, and PB: investigation. CM, GF, CD, and FM: data analysis. PB and GV: conceptualization and funding acquisition.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank Po.Te.Co s.c.r.l (Santa Croce sull'Arno, Pisa) for kindly performing scanning electron microscopy.