Cell-free scaffold from jellyfish Cassiopea andromeda (Cnidaria; Scyphozoa) for skin tissue engineering

Materials Science & Engineering C 111 (2020) 110748

Contents lists available at ScienceDirect

Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec

Cell-free scaffold from jellyfish Cassiopea andromeda (Cnidaria; Scyphozoa) for skin tissue engineering

T

⁎

Irving Fernández-Cervantesa,b, Nayeli Rodríguez-Fuentesa,c, , Lorena V. León-Denizd, Luz E. Alcántara Quintanae, José M. Cervantes-Uca, Wilberth A. Herrera Kaoa, José D. Cerón-Espinosaf, Juan V. Cauich-Rodrígueza, Victor M. Castaño-Menesesg a

Centro de Investigación Científica de Yucatán, Unidad de Materiales, Calle 43 No. 130 x 32 y 34, Chuburná de Hidalgo, CP 97205 Mérida, Yucatán, Mexico Universidad Aeronáutica en Querétaro, Subdirección de Técnico Superior Universitario, Carretera estatal 200, Querétaro Tequisquiapan, No. 22154, CP 76270 Colón, Querétaro, Mexico c CONACYT-Centro de Investigación Científica de Yucatán, Unidad de Materiales, Calle 43 No. 130 x 32 y 34, Chuburná de Hidalgo, CP 97205 Mérida, Yucatán, Mexico d Universidad Autónoma de Yucatán, Biología Marina, Carretera Mérida-Xmatkuil Km. 15.5, Tizapán, 97100 Mérida, Yucatán, Mexico e CONACYT-Facultad de Enfermería y Nutrición, Universidad Autónoma de San Luis Potosí, Av. Niño Artillero 130 Zona Universitaria, 78240 San Luis Potosí, Mexico f Centro Dermatológico de Yucatán, Calle 66 548, Centro, 97000 Mérida, Yucatán, Mexico g Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, Juriquilla, Querétaro, Mexico b

A R T I C LE I N FO

A B S T R A C T

Keywords: Jellyfish Decellularization Skin tissue engineering Scaffolds Biomaterials

Disruption of the continuous cutaneous membrane in the integumentary system is considered a health problem of high cost for any nation. Several attempts have been made for developing skin substitutes in order to restore injured tissue including autologous implants and the use of scaffolds based on synthetic and natural materials. Current biomaterials used for skin tissue repair include several scaffold matrices types, synthetic or natural, absorbable, degradable or non-degradable polymers, porous or dense scaffolds, and cells capsulated in hydrogels or spheroids systems so forth. These materials have advantages and disadvantages and its use will depend on the desired application. Recently, marine organisms such as jellyfish have attracted renewed interest, because both its composition and structure resemble the architecture of human dermic tissue. In this context, the present study aims to generate scaffolds from Cassiopea andromeda (C. andromeda), with application in skin tissue engineering, using a decellularization process. The obtained scaffold was studied by infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), differential scanning calorimetry analysis (DSC), and scanning electron microscopy (SEM). Crystal violet staining and DNA quantification assessed decellularization effectiveness while the biocompatibility of scaffold was determined with human dermic fibroblasts. Results indicated that the decellularization process reduce native cell population leading to 70% reduction in DNA content. In addition, SEM showed that the macro and microstructure of the collagen I-based scaffold were preserved allowing good adhesion and proliferation of human dermic fibroblasts. The C. andromeda scaffold mimics human skin and therefore represents great potential for skin tissue engineering.

1. Introduction During the 20th century, the increase in the development of synthetic [1–6] and natural biomaterials for tissue engineering has been notorious [4,7,8]. The more recently biomaterials suitable for tissue engineering strategies include scaffolds designed by impression 3D [9–14] (due to its ability to control bulk geometry and internal structure), medical devices based on fibers at nanometric and micrometric scales (with functions such as real-time monitoring physiological

signals), delivering drugs, transplanting cells, in among [4,15–17]. Also, hydrogels, are scaffolds generally fabricated to mimic the intrinsic morphologies and functions of human tissues, because they can encapsulate cells [4,18] and acts as pharmaceutical repositories [19] and inclusive has antibacterial function [20]. In particular, biomaterials designed as promoters of the reparationregeneration of integumentary tissue have been widely developed [1,14,21–35]. However, the current challenge is that scaffolds imitate the physical, chemical and biological properties of host tissue. Some

⁎ Corresponding author at: CONACYT, Centro de Investigación Científica de Yucatán, A.C. Calle 43 No. 130 x 32 y 34, Chuburná de Hidalgo, 97205 Mérida, Yucatán, Mexico. E-mail addresses: [email protected], [email protected] (N. Rodríguez-Fuentes).

https://doi.org/10.1016/j.msec.2020.110748 Received 14 October 2019; Received in revised form 31 January 2020; Accepted 15 February 2020 Available online 19 February 2020 0928-4931/ © 2020 Published by Elsevier B.V.

Materials Science & Engineering C 111 (2020) 110748

I. Fernández-Cervantes, et al.

engineering. Similarly, decellularized marine tissues are seen as attractive alternatives due to their similarity to human tissues, reduced immune rejection, and less religious restrictions. In this context, has been demonstrated the effectivity of the decellularized tilapia skin, crosslinked electrospun tilapia collagen scaffold, on regeneration of rat calvarial defect [73]. Organisms belonging to the taxonomic class Scyphozoa, have an analogous structural organization in comparison with the structure of human skin; however, these organisms have not been used for the generation of scaffolds with potential application for skin tissue engineering. This study uses the manufacture of biomimetic scaffolds for the skin through decellularization, with the purpose of preserving the ECM structure present in the bell of jellyfish C. andromeda.

examples of these properties to imitate are: structural arrangement of the extracellular matrix (ECM), pore interconnectivity, chemical composition, mechanical functionality and structural stability, as well as biocompatibility and vascularization, in among other characteristics [21–24,36]. In this regard, a great diversity of scaffolds obtained from degradable polymers, both synthetic and natural, has been studied with great attention due to biocompatible properties, as well as the induction of cell proliferation. In this context, have been evaluated polymers such as polycaprolactone, poly (lactic-co-glycolic acid)/polyisoprene, polyurethanes, bacterial cellulose, collagen, proteoglycans, glycolipids, polysaccharides in among [25–33,36–40]. A large number of scaffolds, have been developed to support skin regeneration, frequently in combination with mesenchymal stem cells (MSCs), due to the MSC-based therapy combined with artificial scaffolds offers a promising strategy to promote wound healing [13,41–44]. The main polymer in this type of applications is collagen (CLG), one of the basic components ECM, being the most abundant protein present in mammals, representing 30% of the protein component of these organisms [45]. According to the medical potential that CLG represents, many different processes of extraction have been explored, from porcine, bovine, equine and marine sources [46]. All of these sources have different advantages related with its isolation facility, availability, however, is very important to consider that there are no diseases that are transmitted from these marine organisms to humans, and also, do not exhaust natural resources on the way. Moreover, the diversity and abundance of biomolecules present into marine environment, have allowed the development of studies for obtaining CLG from many marine organisms, such as sponges (Porifera), jellyfish (Cnidaria), octopus, squid (Mollusca) and fish, being Cnidaria class who has attracted attention due to their abundance and structural composition [38,45,47–55]. In particular, the isolation of biopolymers and/or scaffolds with application in skin tissue engineering from updown jellyfish C. andromeda is null, as well as the use of its structure and not only the composition of this organism. Under this scenario, this work focused in the evaluation of C. andromeda as source of natural scaffolds for its potential application for skin tissue engineering. C. andromeda is an upside-down jellyfish, recently re-classified taxonomically from C. xamachana in July 2019 [56]. This organism is a member of Cnidarians Phylum, and is native the eastern Mediterranean, but due to anthropogenic effects like eutrophication, global warming, shipping, overfishing, coastal developments and marine transports, invasively entered the coastal waters of Persian Gulf in the Nayband Lagoon, from Bushehr- Iran since 2014 [57,58], but nevertheless, currently also has an important presence in the Chelem Lagoon, located on the north coast of the state of Yucatan in the Gulf of Mexico. Investigations about this jellyfish have been focused in isolate some compounds with the hematologic, cytotoxic and anticancer properties [57,59]. However, the fabrication of scaffolds preserving the structure of C. andromeda is limited. One approach for the generation of scaffolds from C. andromeda is to imitate its structural distribution. In this context, has been promoted the development of experimental strategies such as the decellularization, this methodology allows the conservation of the micro-architecture of selected tissue, besides preserving a chemical composition suitable for its application in tissue engineering [60–63]. Decellularized scaffolds have promising applications in repair of wound defects. To date, diverse strategies have been developed and applied for skin coverage in animal models and clinical practice [64,65]. For example, a study conducted by Groeber F. et al., have demonstrated that vascularization of decellularized porcine jejunum gives rise to skin substitute with an architecture representative of the human dermis and epidermis, including skin annexes such as papillarylike structures [66]. Likewise, decellularized tissues such as small intestinal submucosa [67], human amniotic membrane [68], human foreskin [69], human placenta [70], and human dermal matrix [71,72] have been evaluated by the potential application in skin tissue

2. Materials and methods 2.1. Materials Dulbecco's Modified Eagle Medium (DMEM), antibiotic–antimycotic solution, and trypsin–EDTA solution, fetal bovine serum (FBS), were obtained from Caisson Laboratories (Smithfield, UT, USA), whereas and CellTiter-Blue Viability Reagent was purchased from Promega Corporation (USA). TRIZOL Reagent was purchased by Invitrogen (California, USA). Acetic acid, ethanol, methanol, pepsin, NaCl, Triton X-100, glutaraldehyde was purchased by SIGMA (SIGMA ALDRICH). Crystal violet was purchased by HYCEL (HYCEL DE MEXICO). 2.2. Collection and pre-processing of C. andromeda Sixteen batches (seven organisms per batch) of jellyfish C. andromeda were collected in the northern coast of Yucatán in the Gulf of México (coordinates: 21° 15′–21° 17′ N and 89° 39′–89° 48′ O) (Permission to collect marine specimens: SGPA/DGVS/011043/17). Due to that factors such as living environment, age, presence of subspecies, etc., could affect the microstructure of scaffolds obtained from bell of jellyfish C. andromeda, adult specimens with similar size (15–20 cm diameter) and weight (150–200 g) were collected, under the guidance of the marine biology Lorena León, to guarantee the repeatability of experiments. Also, all specimens were obtained at a depth of 40 cm from ocean bed, during December 2017 to April 2018. These conditions were established in order to minimize the intraspecific phenotypic variability of jellyfish and maintain the reproducibility of the experiment in according to previously reported [74–76]. The bell (structure of the aboral zone) of specimens was separated from the gonads, tentacles, mouth, and oral arms (oral structures area), and used for obtaining integumentary scaffolds. The bell was transported in filtered seawater at 25 °C for subsequent analysis. 2.3. Scaffold decellularization Bell structures from C. andromeda were decellularized using ionic solutions and non-ionic detergents, in according to methodology proposed previously [47,77]. Briefly, each bell was rinsed with distilled water and freeze-dried at −50 °C in a sodium chloride (NaCl) solution (1 M) during 24 h in order to cause cell lysis. After, bells were stirred in a solution containing 0.5% (v/v) Triton X-100 and NaCl 1 M during 30 min., followed by a second treatment with a NaCl 1 M solution for 30 min. Finally, bleaching was conducted with hydrogen peroxide (3% v/v) for 10 min, followed by a rinsing with deionized water for 12 h with water changes every 4 h. Finally, the bell structure was dehydrated in ethanol series from 70%, 80%, 90% and 100% (v/v). The samples were stored at −4 °C for subsequent experiments. 2.4. Evaluation of the effectiveness of the decellularization process The effectiveness of the decellularization protocol was determined 2

Materials Science & Engineering C 111 (2020) 110748

I. Fernández-Cervantes, et al.

(+Dcell), a tensile test was done. A series of six specimens with dimensions of 60 mm × 11.4 mm × 0.6 mm were obtained and drying at 25 °C for 24 h. The scaffolds were subjected to tension tests in a Shimadzu universal AGS-X model testing machine with a 1 kN load cell. Maximum strength and the elastic modulus (E) were calculated.

qualitatively by crystal violet staining and by the quantification of the deoxyribonucleic acid (DNA) residual content in each decellularized (+Dcell) and non-decellularized (−Dcell) bells. 2.4.1. Crystal violet staining We used the crystal violet dye which binds to DNA, staining the cell nuclei of dark purple color; this technique has been used previously [78,79]. For crystal violet staining, 1 cm2 discs were stained with crystal violet in methanol (0.5%) for 5 min at 25 °C, and then, washed with PBS twice time for removing dye residues. Subsequently, the samples were observed in an optical microscopy at 100 μm of magnification (TCM-400, Labomed) to assess the presence/absence of jellyfish cells.

2.7. Cytotoxicity assay 2.7.1. Human dermic fibroblasts isolation Human fibroblasts were obtained from healthy donor voluntary in accordance with the World Medical Association's Declaration of Helsinki regarding ethical conduct of research involving humans and approved by the Research Ethic Board of the Hospital General “Dr. Ignacio Morones Prieto”, in San Luis Potosí, Mexico (CONBIOETICA-24CEI-001-20160427). The biopsies were twice washed with sterile phosphate-buffered saline (PBS), and the subcutaneous tissues were removed. The remaining dermal section was digested with 0.25% trypsin at 37 °C for 30 min. The no-digested fragments were eliminated with a Tweezers. Then, the solution resulting from the enzymatic digestion was centrifuged at 4 °C, 1200 rpm for 10 min, and the pellet obtained was re-suspended in high-glucose DMEM (H-DMEM), supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were seeded on 6 well culture plates, and maintained at 37 °C in a humidified atmosphere of 5% CO2 in air, at 37 °C for subsequent experiments. Culture medium was removed changed every third day.

2.4.2. Quantification of residual DNA DNA quantification was conducted by phenolic extraction with TRIZOL as proposed by Doyle et al. [61,80]. Then, the absorbance was read at 260 nm and at 280 nm in a spectrophotometer (Nanodrop 2000, Thermo Fisher Scientific Inc.) to measure the amount of DNA present (ng/mg) in +Dcell scaffolds and native tissue, −Dcell. The scaffolds used were freeze-drying for 12 h in Freeze Dryer (FreeZone 4.5 LABCONCO, Kansas City, MO). 2.5. Physicochemical characterization of the scaffold 2.5.1. Microstructure by SEM The microstructure and presence/absence of cells in the scaffolds was assessed by using a 6360 LV Jeol low vacuum scanning electron microscope. Scaffold was fixed in a glutaraldehyde solution (0.5%) for 30 min, and then, was gradually dehydrated in ethanol solution (from 10% to 100% v/v in dH2O). Subsequently, the samples were processed using supercritical carbon dioxide drying (scCO2, Tousimisi, model SAMDRI®-795) in according to Bown [81,82] at 1072 psi of pressure. Finally, sample were gold coated with a Denton Vacuum model Desk II equipment.

2.7.2. Cell assays For cell proliferation, 5 × 103 cells were seeded on UV sterilized scaffold discs (5 mm diameter) and pre-moistened in H-DMEM. After 0, 3 and 7 days of culture in a 96-well culture plates, cell viability was evaluated using CellTiter-Blue reagent following the instructions of the manufacturer. Briefly, the culture medium was discarded and fresh medium with 20% of CellTiter-Blue was added. The color turnover was quantified by measuring the absorbance of 200 μL of supernatant in a Cell Imaging Multi-Mode Reader Cytation3 (BioTek® Instruments, Inc.) at 570 nm and 600 nm. Experiments were repeated three times and using scaffold-free cultures as control). Fifth-passage fibroblasts were used for these experiments. The morphology and cell adhesion of the fibroblasts attached to the scaffolds was evaluated after 7 days in culture by SEM. Scaffolds were rinsed three times with PBS and fixed in 4% glutaraldehyde solution, dehydrated in ethanol series (70% to 100%) and prepared for SEM observation applying a thin layer of gold as mentioned above.

2.5.2. Thermal behavior/properties The thermal properties of the marine scaffold (+Dcell and −Dcell) were assessed by both differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Samples were freeze-dried at −50 °C in a Labconco FreeZone 1 Freeze Dryer. DSC thermograms were obtained by using an 8500 DSC from Perkin Elmer. For this, 10 mg of the scaffold were place in aluminum pans and heated from 25 °C to 600 °C at a heating rate of 5 °C/min under inert atmosphere. TGA thermograms were obtained with a TGA-7 from Perking Elmer by heating 10 mg of the samples from 30 °C to 600 °C at a heating rate of 10 °C/min under a nitrogen atmosphere as suggested by previous studies [28,82].

2.8. Statistical analysis Data are reported as the mean plus standard deviation. The statistical significance between groups was determined using one-way analysis of variance (ANOVA) (p < 0.05 was considered significant) using OriginPro 8.5.1 software.

2.5.3. Fourier Transform Infrared (ATR-FTIR) Spectroscopy IR spectra were recorded on Nicolet 8700 equipment (Thermo Scientific Inc.) using an average of 100 scans, a resolution of 4 cm−1 with ATR/ZnSe, in the 4000 to 750 cm−1 spectral range.

3. Results 3.1. Effectiveness of the jellyfish decellularization

2.5.4. EDX analysis Energy-dispersive X-ray spectroscopy (EDX) (Oxford Instruments, INCA Energy 200) coupled to the 6360 LV Jeol SEM assessed scaffold surface chemical composition. Similarly, after TGA experiments, the remaining residues were analyzed in order to verify that +Dcel and −Dcel, does not have any metal trace that can be toxic to human.

3.1.1. Jellyfish cells and scaffold structure The effect of decellularization on the structure and cell content of the jellyfish can be appreciated (Fig. 1). A decrease in cell number can be observed by crystal violet staining after decellularization i.e. more cells in −Dcell scaffolds (Fig. 1A) and less cells in +Dcell (Fig. 1B). SEM showed a further advantage of the decellularization protocol as micrographs did not show alterations in the typical arrangement of the jellyfish matrix (Fig. 1C, D). Images at higher magnification for −Dcell and +Dcell samples are displayed in Fig. 1E and F, respectively; as noted, porosity of scaffolds is clearly distinguished.

2.6. Mechanical resistance test 2.6.1. Tensile tests To determine the mechanical properties of decellularized scaffolds 3

Materials Science & Engineering C 111 (2020) 110748

I. Fernández-Cervantes, et al.

Fig. 1. Scaffold structure after decellularization process. A–B) Histological observation of the scaffold stained with violet crystal; violet cell nuclei are appreciated. C–F) SEM analysis of decellularized (+Dcell) and not decellularized (−Dcell) scaffolds; the porosity of the cell-free scaffold can be appreciated. Arrows indicate the jellyfish cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

stretching vibrations of CeN group (νCN) [28,37,39,40,50,77,83]. Finally, the fifth band was attributed to amide III at 1228 cm−1, and has been related to deformation vibration modes of NeH group (δNH) and stretching of the CeN group (νCN) out of plane [28]. Extraction of single-subcomponent bands is achieved using curve-fitting approaches to assign each subcomponent to a particular protein secondary structure of collagen type I present into scaffolds (Fig. 3B). Thus, deconvolution of the peaks in the 1720–1580 region showed β-sheet and α-helix arrangement in CLG, and four subcomponents of its secondary structure was obtained, belonging to the stretching of the β-sheets (ν β sheets) with vibration bands located at 1688 and 1631 cm−1, the stretching of the helix-α structure located at 1662 cm−1 and the stretching corresponding to the spiral-forming structure randomized at 1648 cm−1 [84]. With these results the presence of the general and detailed vibrational modes of the CLG-I is confirmed.

3.1.2. Residual DNA on scaffold In agreement with optical and SEM observations, a significant difference was observed in the genetic material content in +Dcell vs −Dcell scaffold (Fig. 2). Accordingly, decellularization process used in this work promoted a decreased of 70% of DNA content in +Dcell (2.05 ± 0.1 ng/mg dry weight) vs −Dcell (6.76 ± 1.29 ng/mg dry weight) scaffold. 3.2. Physicochemical properties of +Dcell scaffold 3.2.1. Composition by ATR-FTIR Spectroscopy FTIR spectra of jellyfish −Dcell and +Dcell scaffolds are shown in Fig. 3. Both spectra shown five typical absorptions of collagen type I (CLG-I) in agreement with previous reports [28,37,39,40,50,77,83]. Amide A, detected at 3278 cm−1, is associated with the asymmetric stretching vibrations of NeH bond (ν Asy NH) whereas amide B, located at 3058 cm−1, is related with the symmetrical stretching vibration of NeH bond (ν Sym NH). Amide I was identified at 1633 cm−1, it represents almost 80% of the stretching vibrations of the carbonyl group into peptides (νC]O) with some minimum stretching vibration of CN bonds (10%), also, the flexion movements of NH group (10%) as has previously reported [28]. Amide II was observed at 1547 cm−1, which corresponds to deformation vibrations of NeH group (δNH) and

3.2.2. Thermal properties DSC thermograms for −Dcell and +Dcell scaffolds are shown in Fig. 4A. A broad endothermic transition was detected at 123.63 °C and at 92 °C for −Dcell and +Dcell respectively and it was attributed to denaturation temperature of CLG-I as reported for other marine organisms [19,21,22]. Fig. 4B shows the TGA thermogram of −Dcell and +Dcell with the same drying treatments. In general, the main weight 4

Materials Science & Engineering C 111 (2020) 110748

I. Fernández-Cervantes, et al.

scaffolds experienced a maximum degradation in the temperature range of 584.92–693.18 °C (zone D), with degradation percentages of 70% and 30% for −Dcell and +Dcell respectively. The residual mass left after reaching 600 °C was relatively high for an organic material. Therefore, in order to corroborate the chemical composition of the remaining material EDX was used and the results summarized in Table 1. In general, carbon content was high for −Dcell and +Dcell (65–75 wt%) implying that a complete decomposition was not achieved because. In this regard, literature indicates that CLG thermodegradation process starts at 630 °C with a maximum at 800 °C [89]. Oxygen was the second element present in the residue, being as much as 9–17 wt%, attributing it to the possible moisture that absorption by carbonaceous char, binary salts or metal oxides remaining after calcination. Finally, elements such as sodium, magnesium, chlorine, potassium and calcium, commonly found as dissolved mono and diatomic ions in seawater [87] were detected. Fig. 5A and B shows the SEM micrographs of both −Dcell and +Dcell residues after TGA experiments. In general, both type of scaffolds showed polymorphic structures, whose crystalline growth of the cubic type is more evident in the ashes of −Dcell (Fig. 5A) compared to +Dcell (Fig. 5B). These crystals are similar to that of sodium chloride, which are stable up to 600 °C. The literature indicates, that the loss of mass of this compound begins around 657 °C when in potassium ions presence [90]. Fig. 5C and D show the EDX mapping performed on both ash remnants, in which it is clear that the crystalline residues correspond to sodium chloride. In addition, it is evident that oxygen is homogeneously distributed on the entire carbonized structure.

Fig. 2. Residual DNA from decellularization process. DNA content in decellularized scaffold (+Dcell) decreased compared with scaffold before decellularization process (−Dcell). DNA content is expressed as ng/mg of dry weight of material. Mean ± SD. n = 7. *p-Value < 0.05.

loss occurs in four different temperature ranges given by zone A (< 150 °C), zone B (150–250 °C), zone C (250–500 °C) and zone D (> 500 °C). Zone A corresponds to the loss of physiosorbed water, where this account for a mass loss between 1.1 and 3.8% for −Dcell and +Dcell, and occurs in the 58.3–61.0 °C range in both cases. This is consistent with the literature, which indicates that organisms belonging to the sub-phylum Medusozoa have an abundant constitution of seawater [55,87]. The next thermal degradation, was observed in the temperature range from 200 °C to 350 °C (zones B and C), and it was related to the degradation of CLG as reported previously [28,85,88]. However, there was a difference in mass loss as +Dcell showed 9.08% while −Dcell showed a mass loss of 32.15%. The difference in mass loss can be explained due to their different initial water content which in the case of +Dcell scaffold was less before carrying out the decellularization processing. After the second decomposition, the next mass loss occurred between 383.9 and 390.7 °C being close to 16.46% for both samples which is mainly attributed to total decomposition of charred collagen, according to the literature [28,85,88]. Finally, both

3.3. Mechanical properties of +Dcell scaffold Stress-strain curves for decellularized tissue (+Dcell) show the typical behavior of tissue composed by collagen fibers [17,24,91,92]. The maximum strength obtained was 6.64 ± 1.32 MPa and the elastic modulus reached the value of 4.52 ± 0.99 MPa. Variations in values could be due to different kinds of fibrillar collagen distribution on decellularized tissue (+Dcell) and/or for decellularized process [93,94]. 3.4. Biocompatibility of scaffold 3.4.1. Cell Adhesion onto scaffold The electronic micrographs show that +Dcell scaffold is suitable for the fibroblast adhesion. Cells seeded onto the scaffold during 7 days showed good adhesion on jellyfish surface (Fig. 6A). A close inspection at higher magnification showed fibroblasts spreading on surface of the

Fig. 3. ATR-FTIR spectra of decelullarized (+Dcell) and non-decellularized (−Dcell) jellyfish scaffold. A) The typical collagen-I bands are showed in the infrared spectrum. B) Extraction of single-subcomponent bands is achieved using curve-fitting approaches to amide I band with its underlying band subcomponents: alphahelix, beta-sheet, random coil. r = 0.9. 5

Materials Science & Engineering C 111 (2020) 110748

I. Fernández-Cervantes, et al.

Fig. 4. Thermal behavior of −Dcell and +Dcell scaffolds. A) DSC thermogram exhibiting a main peak related to collagen thermal denaturation at 92 °C and 123.63 °C for −Dcell and +Dcell respectively. B) TGA analysis showing the main weight loss related at loss of physiosorbed water and chemical combustion. The dotted black and red lines correspond to the first derivative (ΔWeight/ΔTemperature). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(Cnidaria) [38,45,47–55]. However, this work focused not on obtaining jellyfish collagen, but on taking advantage of its three-dimensional distribution of CLG in this marine organism through decellularization process from up-down jellyfish C. andromeda. The decellularization process followed in our study for obtaining CLG scaffolds from jellyfish allowed a 70% reduction of native cells that correspond to 2.05 ± 0.1 ng/mg dry weight for cell-free scaffold (Fig. 2). It has been reported [99] that a lower concentration of < 50 ng/mg implies that the matrix can be considered as decellularized; thus, the decellularized jellyfish scaffolds could be considered as decellularized material as the DNA content obtained was of only 2.05 ± 0.1 ng/mg dry weight. This result is similar to other decellularized tissues such as human dermis (1.67–2.78 ng/mg wet weight) [60]. Also, there are some commercially available decellularized human tissues such as AlloDermTM and GraftJacketTM that exhibit higher values (272.8 ± 168.8 ng/mg tissue, and 134.6 ± 44.0 ng/mg dry weight respectively) [60,100,101], than those obtained in our material. Interestingly, the decellularization process does not affect the threedimensional structure of the material, yielding porous scaffolds that bio-mimic the micro, macro and chemical composition of human skin (Fig. 1). In this regard, it has been reported that several decellularized tissues such as porcine jejunum [66], small intestinal submucosa [67], human amniotic membrane [68], human foreskin [69], human placenta [70], human dermal matrix [71,72], and tilapia skin [83] exhibit physicochemical, mechanical and biological properties that make them excellent candidates for skin tissue engineering. However, the cell-free scaffold obtained in this work has advantages, in comparison with the other mentioned materials, such as its availability, is a renewable resource that offers a zero impact on human feed supplies and can be collected and produced in a sustainable way without affecting the ecosystems and without depleting the resource on the way, further it does not present risk of diseases transmission to humans. The physicochemical properties analysis of cell-free scaffold through FTIR assay, allowed the identification of collagen present in C. andromeda. This analysis revealed that +Dcell and −Dcell, consisting of CLG-I as previously reported [28,51,86,88,102]. The spectra showed consistent vibration peaks in both cases, demonstrating the similarity between the native jellyfish tissue (−Dcell) and the scaffold obtained post-processing (+Dcell), maintaining the characteristic peak of “triple helix” in Amide III, and a lower frequency of Amide A. Similar frequencies were observed for Amide I, II and III vibrations in +Dcell and −Dcell, consistent with other sources of CLG reported in the literature

Table 1 Mass percentage of the elements presents in the ashes. Element

Symbol

−Dcell Weight (%)

+Dcell Weight (%)

Carbon Oxygen Sodium Magnesium Chlorine Potassium Calcium

C O Na Mg Cl K Ca

67.64 ± 5.8 9.06 ± 0.4 6.39 ± 2.7 1.97 ± 0.8 13.06 ± 3.7 0.6 ± 0.09 0.68 ± 0.2

75.44 ± 14.6 17.82 ± 11.5 1.945 ± 0.2 0.12 ± 0.03 1.725 ± 0.6 0.12 ± 0.01 0.3 ± 0.08

scaffold (Fig. 7B). 3.4.2. Proliferation of human dermal fibroblast on scaffold Cell proliferation increased with time up to 7 days in cell culture in the absence of a scaffold (−scaffold) and in cells cultured in contact with +Dcell scaffold. However, the −Dcell scaffold showed an increase in cell proliferation up to 3 days and then exhibited a reduction in cell proliferation after 7 days. This means that the proliferation of human dermic fibroblasts in +Dcell and −Dcell, was similar at 0 and 3 days, however at seven day of cell culture, +Dcell showed a significant increase in cell proliferation (3.03 ± 0.3 RAU) in comparison with −Dcell (1.70 ± 0.2 RAU) as show in Fig. 7. 4. Discussion The main polymer used for skin tissue engineering is CLG, due to its physical and biological properties. Genetic and protein homology of this natural biopolymer between different species with the human has promoted its isolation principally from porcine, bovine and equine sources [37,46,85,95–98]. All of these sources have different advantages related to ease of isolation and availability, however, is very important to consider some other aspects. Collagen obtained from cow and pigs raise some concern related to the transmission of diseases such as bovine spongiform encephalopathy and foot and mouth disease. For this reason, many groups and companies are working with other types of collagens with the advantage of non-transmitting diseases being hypoallergenic and being environment sustainable in contrast to the meat industry based on cattle. In this context, the biomolecules present into marine environment, have allowed the development of studies for obtaining CLG from many marine organisms, such as jellyfish 6

Materials Science & Engineering C 111 (2020) 110748

I. Fernández-Cervantes, et al.

Fig. 5. SEM and EDX analysis of −Dcell and +Dcell jellyfish scaffolds residues of after thermal degradation. A–B) Microtopography of the residual ashes from native tissue (−Dcell) and decellularized tissue (+Dcell) by SEM. Crystalline structures are observed in –Dcell. C–D) Chemical mapping (EDX) of the residual ashes. It is visualized with hue: reddish to oxygen, green to sodium and blue to chlorine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

degradation of CLG, according to previous reports [28,85,88]. The third weight loss (383.9–390.7 °C), was common for −Dcell and +Dcell and can be attributed to combustion phenomena, according to the literature [28,85,88]. Finally, all scaffolds exhibited four weight losses attributed to the maximum degradation. EDX analysis of collagen residues after the TGA experiments showed that large amounts NaCl remained in native scaffold but there was no evidence of the presence of toxic elements or heavy metals (Fig. 5). Tensile mechanical properties obtained for +Dcell scaffold were of 4.52 ± 0.99 MPa, 6.64 ± 1.32 MPa for maximum strength and elastic modulus, respectively. These values were similar to other collagenous tissues and slight higher to reported by human skin whose elastic modulus are ranged between 0.42 MPa and 0.85 MPa [105]. Besides, stress–strain curves yielded a typically S-shaped behavior, which is comparable with collagenous tissues according to reported previously

[48,102]. Moreover, the secondary structure of the CLG type I contained in both cases was determined, identifying the α-helix, β-sheet, random coil chains respectively (Fig. 3). These results are consistent with previously the collagen isolated from some marine invertebrate animals and scales and skin of fish [1,6,10,16,22]. The thermal behavior of the −Dcell and +Dcell scaffolds analyzed by DSC and TGA approach (Fig. 4), shown a main peak related to denaturation temperature of CLG-I, these results are consistent with the denaturation parameters previously reported for CLG from other marine organisms [19,21,22]. Thermogravimetric analysis by TGA exposed four different temperature ranges given related with weight loss. The first loss is attributed to the loss of physiosorbed water, which is consistent with the literature, about the high water content of subphylum Medusozoa [55,87]. The following thermal degradation, corresponding to the weight loss of organic molecules, related to the

Fig. 6. Fibroblast adhesion on +Dcell scaffold. The SEM micrographs showed the spreading morphology of dermic fibroblast (F) on surface of jellyfish scaffold, which exhibit cytoplasmic projection as filopodial at 7 days of cell culture. A) Panoramic vision at 100 μm, B) magnification at 20 μm. 7

Materials Science & Engineering C 111 (2020) 110748

I. Fernández-Cervantes, et al.

[2] A. Trbakovic, P. Hedenqvist, T. Mellgren, C. Ley, J. Hilborn, D. Ossipov, S. Ekman, C.B. Johansson, M. Jensen-Waern, A. Thor, A new synthetic granular calcium phosphate compound induces new bone in a sinus lift rabbit model, J. Dent. 70 (2018) 31–39. [3] E. Jung, S.-W. Kim, A. Cho, Y.-J. Kim, G.-J. Jeong, J. Kim, S.H. Bhang, T. Yu, Synthesis of sub 3 nm-sized uniform magnetite nanoparticles using reverse micelle method for biomedical application, Materials (Basel) 12 (23) (2019), https://doi. org/10.3390/ma12233850 pii: E3850. [4] Y. Zhang, J. Ding, B. Qi, W. Tao, J. Wang, C. Zhao, H. Peng, J. Shi, Multifunctional fibers to shape future biomedical devices, Adv. Funct. Mater. 29 (2019), https:// doi.org/10.1002/adfm.201902834. [5] F.J. Aguilar-Pérez, R.F. Vargas-Coronado, J.M. Cervantes-Uc, J.V. CauichRodríguez, R. Rosales-Ibañez, J.A. Rodríguez-Ortiz, Y. Torres-Hernández, Titanium-castor oil based polyurethane composite foams for bone tissue engineering, J. Biomater. Sci. Polym. Ed. 30 (2019) 1415–1432. [6] O. Castillo-Cruz, F. Avilés, R. Vargas-Coronado, J.V. Cauich-Rodríguez, L.H. ChanChan, V. Sessini, L. Peponi, Mechanical properties of L-lysine based segmented polyurethane vascular grafts and their shape memory potential, Mater. Sci. Eng. C Mater. Biol. Appl. 102 (2019) 887–895. [7] J. Huang, K. Huang, X. You, G. Liu, G. Hollett, Y. Kang, Z. Gu, J. Wu, Evaluation of tofu as a potential tissue engineering scaffold, J. Mater. Chem. B 6 (2018) 1328–1334. [8] K. Huang, J. Wu, Z. Gu, Black phosphorus hydrogel scaffolds enhance bone regeneration via a sustained supply of calcium-free phosphorus, ACS Appl. Mater. Interfaces 11 (2019) 2908–2916. [9] K.K. Gómez-Lizárraga, C. Flores-Morales, M.L. Del Prado-Audelo, M.A. ÁlvarezPérez, M.C. Piña-Barba, C. Escobedo, Polycaprolactone- and polycaprolactone/ ceramic-based 3D-bioplotted porous scaffolds for bone regeneration: a comparative study, Mater. Sci. Eng. C Mater. Biol. Appl. 79 (2017) 326–335. [10] A. Lapomarda, A. De Acutis, I. Chiesa, G.M. Fortunato, F. Montemurro, C. De Maria, M. Mattioli Belmonte, R. Gottardi, G. Vozzi, Pectin-GPTMS-based biomaterial: toward a sustainable bioprinting of 3D scaffolds for tissue engineering application, Biomacromolecules (2019), https://doi.org/10.1021/acs.biomac. 9b01332. [11] K.S. Hamid, S.G. Parekh, S.B. Adams, Salvage of severe foot and ankle trauma with a 3D printed scaffold, Foot Ankle Int 37 (2016) 433–439. [12] L. Zhang, G. Yang, B.N. Johnson, X. Jia, Three-dimensional (3D) printed scaffold and material selection for bone repair, Acta Biomater. 84 (2019) 16–33. [13] B.N. Blackstone, M.M. Malara, M.E. Baumann, K.L. McFarland, D.M. Supp, H.M. Powell, Fractional CO2 laser micropatterning of cell-seeded electrospun collagen scaffolds enables rete ridge formation in 3D engineered skin, Acta Biomater. 15 (102) (2019) 287–297, https://doi.org/10.1016/j.actbio.2019.11. 051. [14] W. LaBarge, A. Morales, D. Pretorius, A.M. Kahn-Krell, R. Kannappan, J. Zhang, Scaffold-free bioprinter utilizing layer-by-layer printing of cellular spheroids, Micromachines (Basel) 10 (2019), https://doi.org/10.3390/mi10090570. [15] J. Ding, J. Zhang, J. Li, D. Li, C. Xiao, H. Xiao, H. Yang, X. Zhuang, X. Chen, Electrospun polymer biomaterials, Prog. Polym. Sci. 90 (2019) 1–34. [16] C. Wang, J. Wang, L. Zeng, Z. Qiao, X. Liu, H. Liu, J. Zhang, J. Ding, Fabrication of electrospun polymer nanofibers with diverse morphologies, Molecules 24 (2019) 834, https://doi.org/10.3390/molecules24050834. [17] A. Sensini, L. Cristofolini, Biofabrication of electrospun scaffolds for the regeneration of tendons and ligaments, Materials (Basel) 11 (2018), https://doi.org/ 10.3390/ma11101963. [18] A. Tamayol, M. Akbari, Y. Zilberman, M. Comotto, E. Lesha, L. Serex, S. Bagherifard, Y. Chen, G. Fu, S.K. Ameri, W. Ruan, E.L. Miller, M.R. Dokmeci, S. Sonkusale, A. Khademhosseini, Flexible pH-sensing hydrogel fibers for epidermal applications, Adv Healthc Mater 5 (2016) 711–719. [19] X. Feng, J. Li, X. Zhang, T. Liu, J. Ding, X. Chen, Electrospun polymer micro/ nanofibers as pharmaceutical repositories for healthcare, J. Control. Release 302 (2019) 19–41. [20] S. Li, S. Dong, W. Xu, S. Tu, L. Yan, C. Zhao, J. Ding, X. Chen, Antibacterial hydrogels, Adv Sci (Weinh) 5 (2018), https://doi.org/10.1002/advs.201700527. [21] K. Ghosal, C. Agatemor, Z. Špitálsky, S. Thomas, E. Kny, Electrospinning tissue engineering and wound dressing scaffolds from polymer-titanium dioxide nanocomposites, Chem. Eng. J. 358 (2019) 1262–1278. [22] S. Wang, Y. Xiong, J. Chen, A. Ghanem, Y. Wang, J. Yang, B. Sun, Three dimensional printing bilayer membrane scaffold promotes wound healing, Front. Bioeng. Biotechnol. 7 (2019), https://doi.org/10.3389/fbioe.2019.00348. [23] F. Urciuolo, C. Casale, G. Imparato, P.A. Netti, Bioengineered skin substitutes: the role of extracellular matrix and vascularization in the healing of deep wounds, J. Clin. Med. 8 (2019), https://doi.org/10.3390/jcm8122083. [24] K. Ghosal, A. Chandra, G. Praveen, S. Snigdha, S. Roy, C. Agatemor, S. Thomas, I. Provaznik, Electrospinning over solvent casting: tuning of mechanical properties of membranes, Sci. Rep. 8 (2018) 1–9. [25] K. Ghosal, S. Thomas, N. Kalarikkal, A. Gnanamani, Collagen coated electrospun polycaprolactone (PCL) with titanium dioxide (TiO2) from an environmentally benign solvent: preliminary physico-chemical studies for skin substitute, J. Polym. Res. 21 (2014), https://doi.org/10.1007/s10965-014-0410-y. [26] H. Yu, X. Chen, J. Cai, D. Ye, Y. Wu, L. Fan, P. Liu, Novel porous three-dimensional nanofibrous scaffolds for accelerating wound healing, Chem. Eng. J. 369 (2019) 253–262. [27] S. Arasteh, S. Khanjani, H. Golshahi, S. Mobini, M.T. Jahed, H. Heidari-Vala, H. Edalatkhah, S. Kazemnejad, Efficient wound healing using a synthetic nanofibrous bilayer skin substitute in murine model, J. Surg. Res. 245 (2020) 31–44. [28] M.L. Del Prado Audelo, K.K. Gómez Lizárraga, D.M. Giraldo Gómez, H. Martínez

Fig. 7. Fibroblast proliferation on scaffold. Viability and cell proliferation were evaluated by chemical rezafurin reduction by mitochondrial activity during 7 days of cell culture. There is a significant difference between +Dcell and −Dcell at 7 day of culture. n = 9, *p-value < 0.05. −scaffold, represent the experimental condition without scaffold in contact with cell culture.

[103,104]. Cytotoxicity tests demonstrate that the decellularized scaffolds are suitable for cell adhesion (Fig. 6) and proliferation (Fig. 7) of human fibroblast. Therefore, the cell-free scaffold from C. andromeda obtained after decellularization process can be potentially used for skin tissue engineering. Additional studies are necessary to determine the degradation rate as well as the immunologic response by +Dcell scaffold. 5. Conclusions The results of the physicochemical and biological characterization of the cell-free scaffold from jellyfish C. andromeda (Cnidaria; Scyphozoa) indicate that decellularization process has not significantly altered the morphology of scaffolds, and the structural stability and thermal properties were preserved, therefore, the cell-free scaffold obtained in this work can be potentially used for skin tissue engineering. More studies are necessary for determinate the degradation rate as well as the in vivo behavior by +Dcell scaffold. Acknowledgments Thanks to Santiago Duarte Aranda from CICY, by technical assistance in physicochemical processing. The authors acknowledges to CONACYT for the MSc scholarship (number 762853) and for the financial support through 283972 project (Ciencia Básica) and 248378 (Atención a Problemas Nacionales). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References [1] A. Vijayan, P.P. James, C.K. Nanditha, G.S.V. Kumar, Multiple cargo deliveries of growth factors and antimicrobial peptide using biodegradable nanopolymer as a potential wound healing system, Int. J. Nanomedicine 14 (2019) 2253–2263.

8

Materials Science & Engineering C 111 (2020) 110748

I. Fernández-Cervantes, et al.

[29]

[30]

[31]

[32]

[33] [34]

[35]

[36]

[37]

[38]

[39]

[40] [41]

[42]

[43] [44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53] [54]

Hernández, N. Rodríguez-Fuentes, A.E. Castell Rodríguez, B.E. Montufar, M.C. Piña Barba, Development of collagen-EDC scaffolds for skin tissue engineering: physicochemical and biological characterization, IJOER Engineering Journal 2 (2016) 73–83. D.R. Marques, L.A.L. Dos Santos, M.A. O'Brien, S.H. Cartmell, J.E. Gough, In vitro evaluation of poly (lactic-co-glycolic acid)/polyisoprene fibers for soft tissue engineering, J. Biomed. Mater. Res. Part B Appl. Biomater 105 (2017) 2581–2591. B.S. Gregorí Valdés, C.S.B. Gomes, P.T. Gomes, J.R. Ascenso, H.P. Diogo, L.M. Gonçalves, R. Galhano dos Santos, H.M. Ribeiro, J.C. Bordado, Synthesis and characterization of Isosorbide-based polyurethanes exhibiting low cytotoxicity towards HaCaT human skin cells, Polymers (Basel) (2018) 10, https://doi.org/10. 3390/polym10101170. P. Król, Ł. Uram, B. Król, K. Pielichowska, M. Walczak, Study of chemical, physicomechanical and biological properties of 4,4′-methylenebis(cyclohexyl isocyanate)based polyurethane films, Mater. Sci. Eng. C Mater. Biol. Appl. 93 (2018) 483–494. Meng E, Chen C-L, Liu C-C, Liu C-C, Chang S-J, Cherng J-H, Wang H-H and Wu S-T 2019 Bioapplications of bacterial cellulose polymers conjugated with resveratrol for epithelial defect regeneration Polymers (Basel) 11 doi: https://doi.org/10. 3390/polym11061048. G.D. Mogoşanu, A.M. Grumezescu, Natural and synthetic polymers for wounds and burns dressing, Int. J. Pharm. 463 (2014) 127–136. S. Zhang, Q. Ou, P. Xin, Q. Yuan, Y. Wang, J. Wu, Polydopamine/puerarin nanoparticle-incorporated hybrid hydrogels for enhanced wound healing, Biomater. Sci. 7 (2019) 4230–4236. K. Ghosal, A. Manakhov, L. Zajíčková, S. Thomas, Structural and surface compatibility study of modified electrospun poly(ε-caprolactone) (PCL) composites for skin tissue engineering, AAPS PharmSciTech 18 (2017) 72–81. Chaudhari AA, Vig K, Baganizi DR, Sahu R, Dixit S, Dennis V, Singh SR and Pillai S R 2016 Future prospects for scaffolding methods and biomaterials in skin tissue engineering: a review Int. J. Mol. Sci. 17 https://doi.org/10.3390/ijms17121974. S. Sharif, J. Ai, M. Azami, J. Verdi, M.A. Atlasi, S. Shirian, A. Samadikuchaksaraei, Collagen-coated nano-electrospun PCL seeded with human endometrial stem cells for skin tissue engineering applications, J. Biomed. Mater. Res. Part B Appl. Biomater 106 (2017) 1578–1586, https://doi.org/10.1002/jbm.b.33966. Y. Zhuang, H. Hou, X. Zhao, Z. Zhang, B. Li, Effects of collagen and collagen hydrolysate from jellyfish (Rhopilema esculentum) on mice skin photoaging induced by UV irradiation, J. Food Sci. 74 (2009) H183–H188. S. Intaraprasit, A. Faikrua, A. Sittichokechaiwut, J. Viyoch, Efficacy evaluation of the fibroblast-seeded collagen/chitosan scaffold on application in skin tissue engineering, Science Asia 38 (2012) 268–277. M.A. Rizk, N.Y. Mostafa, Extraction and characterization of collagen from buffalo skin for biomedical applications, Orient. J. Chem. 32 (2016) 1601–1609. K.C. Murphy, J. Whitehead, D. Zhou, S.S. Ho, J.K. Leach, Engineering fibrin hydrogels to promote the wound healing potential of mesenchymal stem cell spheroids, Acta Biomater. 64 (2017) 176–186. C. Qi, L. Xu, Y. Deng, G. Wang, Z. Wang, L. Wang, Sericin hydrogels promote skin wound healing with effective regeneration of hair follicles and sebaceous glands after complete loss of epidermis and dermis, Biomater Sci 6 (2018) 2859–2870. Y. Han, X. Li, Y. Zhang, Y. Han, F. Chang, J. Ding, Mesenchymal stem cells for regenerative medicine, Cells (2019) 8, https://doi.org/10.3390/cells8080886. K.-C. Tang, K.-C. Yang, C.-W. Lin, Y.-K. Chen, T.-Y. Lu, H.-Y. Chen, N.-C. Cheng, J. Yu, Human adipose-derived stem cell secreted extracellular matrix incorporated into electrospun poly(lactic-co-glycolic acid) nanofibrous dressing for enhancing wound healing, Polymers (Basel) 11 (2019), https://doi.org/10.3390/ polym11101609. N.M.H. Khong, F.M. Yusoff, B. Jamilah, M. Basri, I. Maznah, K.W. Chan, N. Armania, J. Nishikawa, Improved collagen extraction from jellyfish (Acromitus hardenbergi) with increased physical-induced solubilization processes, Food Chem. 251 (2018) 41–50. J.F. Burke, I.V. Yannas, W.C. Quinby, C.C. Bondoc, W.K. Jung, Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury, Ann. Surg. 194 (1981) 413–428. E. Song, S. Yeon Kim, T. Chun, H.-J. Byun, Y.M. Lee, Collagen scaffolds derived from a marine source and their biocompatibility, Biomaterials 27 (2006) 2951–2961. Y.E. Arslan, T. Sezgin Arslan, B. Derkus, E. Emregul, K.C. Emregul, Fabrication of human hair keratin/jellyfish collagen/eggshell-derived hydroxyapatite osteoinductive biocomposite scaffolds for bone tissue engineering: from waste to regenerative medicine products, Colloids Surf B Biointerfaces 154 (2017) 160–170. J.P. Widdowson, A.J. Picton, V. Vince, C.J. Wright, A. Mearns-Spragg, In vivo comparison of jellyfish and bovine collagen sponges as prototype medical devices, J. Biomed. Mater. Res. Part B Appl. Biomater 106 (2018) 1524–1533. X. Cheng, Z. Shao, C. Li, L. Yu, M.A. Raja, C. Liu, Isolation, characterization and evaluation of collagen from jellyfish Rhopilema esculentum Kishinouye for use in hemostatic applications, PLoS One 12 (2017) e0169731. W. Pustlauk, B. Paul, M. Gelinsky, A. Bernhardt, Jellyfish collagen and alginate: combined marine materials for superior chondrogenesis of hMSC, Mater. Sci. Eng. C Mater. Biol. Appl. 64 (2016) 190–198. Jellagen BioNovus Life Sciences, Purified jellyfish collagen for tissue engineering research, cell culture & biochemistry, BioNovus Life Sciences, https://www. bionovuslifesciences.com.au/jellagen/, (2019) , Accessed date: 1 October 2019. C. Gambini, B. Abou, A. Ponton, A.J.M. Cornelissen, Micro- and macrorheology of jellyfish extracellular matrix, Biophys. J. 102 (2012) 1–9. W. Pustlauk, B. Paul, S. Brueggemeier, M. Gelinsky, A. Bernhardt, Modulation of

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64] [65]

[66]

[67]

[68]

[69]

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78] [79]

9

chondrogenic differentiation of human mesenchymal stem cells in jellyfish collagen scaffolds by cell density and culture medium, J. Tissue Eng. Regen. Med. 11 (2017) 1710–1722. Hernández JMA, Couoh-Concha A del J, Loman-Ramos L and Leon-Deniz LV 2019 Range expansion of two box jellyfish (Cnidaria, Cubozoa) in southern Gulf of Mexico Check List 15 405–10. A.G. Collins, G. Jarms, A.C. Morandini, World list of Scyphozoa. Cassiopea andromeda (Forsskål, 1775), Accessed through: World Register of Marine Species at, 2019. http://www.marinespecies.org/aphia.php?p=taxdetails&id=135295. M.R. Mirshamsi, R. Omranipour, A. Vazirizadeh, A. Fakhri, F. Zangeneh, G.H. Mohebbi, R. Seyedian, J. Pourahmad, Persian Gulf jellyfish (Cassiopea andromeda) venom fractions induce selective injury and cytochrome C release in mitochondria obtained from breast adenocarcinoma patients, Asian Pac. J. Cancer Prev. 18 (2017) 277–286. I. Nabipour, M. Moradi, G.H. Mohebbi, A first record on population of the alien venomous jellyfish, Cassiopea andromeda (Forsskål, 1775) (Cnidaria: Scyphozoa: Rhizostomea) in the Nayband Lagoon from Bushehr-Iran (Persian Gulf), J. Chem. Pharm. Res. 0975-7384, 7 (3) (2015) 1710–1713. F.F. Radwan, J.W. Burnett, D.A. Bloom, T. Coliano, M.E. Eldefrawi, H. Erderly, L. Aurelian, M. Torres, E.P. Heimer-de la Cotera, A comparison of the toxinological characteristics of two Cassiopea and Aurelia species, Toxicon 39 (2001) 245–257. M.A. Moore, B. Samsell, G. Wallis, S. Triplett, S. Chen, A.L. Jones, X. Qin, Decellularization of human dermis using non-denaturing anionic detergent and endonuclease: a review, Cell Tissue Bank, vol. 16, 2015, pp. 249–259. G. Benítez-Arvizu, G. Gutierrez-Iglesias, M. Cerbón-Cervantes, N. RodríguezFuentes, J. Tapia-Ramirez, L. Alcántara-Quintana, Skin regeneration after scalp trauma through autologous transplant of acellular dermis: a Mexican case study, International Journal of Recent Scientific Research (IJRSR) 7 (2016) 8789–8793. Y. Takami, R. Yamaguchi, Y. Matsuda, Method of Preparing Isolated Cell-free Skin, Cell-free Dermal Matrix, Method of Producing the Same and Composite Cultured Skin with The Use of the Cell-free Dermal Matrix WO2005063315A1, (2007). A.H. Morris, J. Chang, T.R. Kyriakides, Inadequate processing of decellularized dermal matrix reduces cell viability in vitro and increases apoptosis and acute inflammation in vivo, Biores Open Access 5 (2016) 177–187. H. Cui, Y. Chai, Y. Yu, Progress in developing decellularized bioscaffolds for enhancing skin construction, J. Biomed. Mater. Res. A 107 (2019) 1849–1859. J.R. Yu, J. Navarro, J.C. Coburn, B. Mahadik, J. Molnar, J.H. Holmes, A.J. Nam, J.P. Fisher, Current and future perspectives on skin tissue engineering: key features of biomedical research, translational assessment, and clinical application, Adv Healthc Mater 8 (2019), https://doi.org/10.1002/adhm.201801471. F. Groeber, L. Engelhardt, J. Lange, S. Kurdyn, F.F. Schmid, C. Rücker, S. Mielke, H. Walles, J. Hansmann, A first vascularized skin equivalent as an alternative to animal experimentation, ALTEX 33 (2016) 415–422. L. Shi, Y. Hu, M.W. Ullah, I. Ullah, H. Ou, W. Zhang, L. Xiong, X. Zhang, Cryogenic free-form extrusion bioprinting of decellularized small intestinal submucosa for potential applications in skin tissue engineering, Biofabrication (2019) 11, https:// doi.org/10.1088/1758-5090/ab15a9. Z. Kakabadze, D. Chakhunashvili, K. Gogilashvili, K. Ediberidze, K. Chakhunashvili, K. Kalandarishvili, L. Karalashvili, Bone marrow stem cell and decellularized human amniotic membrane for the treatment of nonhealing wound after radiation therapy, Exp. Clin. Transplant. 17 (2019) 92–98. V. Purpura, E. Bondioli, E.J. Cunningham, G. De Luca, D. Capirossi, E. Nigrisoli, T. Drozd, M. Serody, V. Aiello, D. Melandri, The development of a decellularized extracellular matrix-based biomaterial scaffold derived from human foreskin for the purpose of foreskin reconstruction in circumcised males, J Tissue Eng 9 (2018), https://doi.org/10.1177/2041731418812613. Ö.S. Somuncu, Y. Coşkun, B. Ballica, A.F. Temiz, D. Somuncu, In vitro artificial skin engineering by decellularized placental scaffold for secondary skin problems of meningomyelocele, J. Clin. Neurosci. 59 (2019) 291–297. S. Cazzell, A randomized controlled trial comparing a human acellular dermal matrix versus conventional care for the treatment of venous leg ulcers, Wounds 31 (2019) 68–74. C.M. Zelen, D.P. Orgill, T.E. Serena, R.E. Galiano, M.J. Carter, L.A. DiDomenico, J. Keller, J.P. Kaufman, W.W. Li, An aseptically processed, acellular, reticular, allogenic human dermis improves healing in diabetic foot ulcers: a prospective, randomised, controlled, multicentre follow-up trial, Int. Wound J. 15 (2018) 731–739. Lau CS, Hassanbhai A, Wen F, Wang D, Chanchareonsook N, Goh BT, Yu N and Teoh S-H 2019 Evaluation of decellularized tilapia skin as a tissue engineering scaffold J. Tissue Eng. Regen. Med. 13 1779–91. N.J. Colley, R.K. Trench, Selectivity in phagocytosis and persistence of symbiotic algae in the scyphistoma stage of the jellyfish Cassiopeia xamachana, Proc. R. Soc. Lond. B Biol. Sci. 219 (1983) 61–82. P. Cabrales-Arellano, T. Islas-Flores, P.E. Thomé, M.A. Villanueva, Indomethacin reproducibly induces metamorphosis in Cassiopea xamachana scyphistomae, PeerJ (2017) 5, https://doi.org/10.7717/peerj.2979. R. Payne Bigelow, The Anatomy and Development of Cassiopea xamachana, vol 5, Boston Society of Natural History, Boston, 1900, https://doi.org/10.5962/bhl. title.31420 Pub. by the. T. Nagai, Characterization of acid-soluble collagen from skins of surf smelt (Hypomesus pretiosus japonicus Brevoort), Food Nutr. Sci. 01 (2010) 59, https:// doi.org/10.4236/fns.2010.12010. M.K. Dutt, Basic dyes for the staining of DNA in mammalian tissues and absorption spectra of stained nuclei in the visible light, Microsc Acta 86 (1982) 59–68. M.K. Dutt, Staining of depolymerised DNA in mammalian tissues with methyl violet 6B and crystal violet, Folia Histochem Cytochem (Krakow) 18 (1980)

Materials Science & Engineering C 111 (2020) 110748

I. Fernández-Cervantes, et al.

[92] B. Lee, X. Zhou, K. Riching, K.W. Eliceiri, P.J. Keely, S.A. Guelcher, A.M. Weaver, Y. Jiang, A three-dimensional computational model of collagen network mechanics, PLoS One 9 (2014), https://doi.org/10.1371/journal.pone.0111896. [93] B.A. Roeder, K. Kokini, J.E. Sturgis, J.P. Robinson, S.L. Voytik-Harbin, Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure, J. Biomech. Eng. 124 (2002) 214–222. [94] M. Terzini, C. Bignardi, C. Castagnoli, I. Cambieri, E.M. Zanetti, A.L. Audenino, Ex vivo dermis mechanical behavior in relation to decellularization treatment length, Open Biomed Eng J 10 (2016) 34–42. [95] J.F. Burke, I.V. Yannas, W.C. Quinby, C.C. Bondoc, W.K. Jung, Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury, Ann. Surg. 194 (4) (1981) 413–428. [96] W.H. Eaglstein, V. Falanga, Tissue engineering and the development of Apligraf®, a human skin equivalent, Clin. Ther. 19 (1997) 894–905. [97] W. Friess, Collagen—biomaterial for drug delivery, Eur. J. Pharm. Biopharm. 45 (1998) 113–136. [98] R. Parenteau-Bareil, R. Gauvin, F. Berthod, Collagen-based biomaterials for tissue engineering applications, Materials (Basel) 3 (2010) 1863–1887. [99] P.M. Crapo, T.W. Gilbert, S.F. Badylak, An overview of tissue and whole organ decellularization processes, Biomaterials 32 (2011) 3233–3243. [100] K.A. Derwin, A.R. Baker, R.K. Spragg, D.R. Leigh, J.P. Iannotti, Commercial extracellular matrix scaffolds for rotator cuff tendon repair. Biomechanical, biochemical, and cellular properties, J. Bone Joint Surg. Am. 88 (2006) 2665–2672. [101] J.M. Choe, T. Bell, Genetic material is present in cadaveric dermis and cadaveric fascia lata, J. Urol. 166 (2001) 122–124. [102] Z. Rastian, S. Pütz, Y. Wang, S. Kumar, F. Fleissner, T. Weidner, S.H. Parekh, Type I collagen from jellyfish Catostylus mosaicus for biomaterial applications, ACS Biomater. Sci. Eng. 4 (2018) 2115–2125. [103] M. Meyer, Processing of collagen based biomaterials and the resulting materials properties, Biomed. Eng. Online (2019) 18, https://doi.org/10.1186/s12938-0190647-0. [104] P. Fratzl, K. Misof, I. Zizak, G. Rapp, H. Amenitsch, S. Bernstorff, Fibrillar structure and mechanical properties of collagen, J. Struct. Biol. 122 (1998) 119–122. [105] M. Pawlaczyk, M. Lelonkiewicz, M. Wieczorowski, Age-dependent biomechanical properties of the skin, Postepy Dermatol Alergol 30 (2013) 302–306.

79–83. [80] J. Doyle, J. Doyle, A rapid DNA isolation procedure for small quantities of fresh leaf tissue, Phytochemical Bulletin 19 (1987) 11–15. [81] Z.K. Brown, P.J. Fryer, I.T. Norton, R.H. Bridson, Drying of agar gels using supercritical carbon dioxide, J. Supercrit. Fluids 54 (2010) 89–95. [82] N. Rodríguez-Fuentes, A.G. Rodríguez-Hernández, J. Enríquez-Jiménez, L.E. Alcántara-Quintana, L. Fuentes-Mera, M.C. Piña-Barba, A. Zepeda-Rodríguez, J.R. Ambrosio, Nukbone® promotes proliferation and osteoblastic differentiation of mesenchymal stem cells from human amniotic membrane, Biochem. Biophys. Res. Commun. 434 (2013) 676–680. [83] Z. Hu, P. Yang, C. Zhou, S. Li, P. Hong, Marine collagen peptides from the skin of Nile tilapia (Oreochromis niloticus): characterization and wound healing evaluation, Mar Drugs 15 (2017), https://doi.org/10.3390/md15040102. [84] K. Belbachir, R. Noreen, G. Gouspillou, C. Petibois, Collagen types analysis and differentiation by FTIR spectroscopy, Anal. Bioanal. Chem. 395 (2009) 829–837. [85] V. Samouillan, F. Delaunay, J. Dandurand, N. Merbahi, J.-P. Gardou, M. Yousfi, A. Gandaglia, M. Spina, C. Lacabanne, The use of thermal techniques for the characterization and selection of natural biomaterials, J Funct Biomater 2 (2011) 230–248. [86] G. Leyva-Gómez, E. Lima, G. Krötzsch, R. Pacheco-Marín, N. Rodríguez-Fuentes, D. Quintanar-Guerrero, E. Krötzsch, Physicochemical and functional characterization of the collagen-polyvinylpyrrolidone copolymer, J. Phys. Chem. B 118 (2014) 9272–9283. [87] R.C. Brusca, G.J. Brusca, Invertebrates, Second ed., Sinauer Associates, Sunderland, Massachusetts, 2003. [88] B.H. León-Mancilla, M.A. Araiza-Téllez, J.O. Flores-Flores, M.C. Piña-Barba, Physico-chemical characterization of collagen scaffolds for tissue engineering, Journal of Applied Research and Technology 14 (2016) 77–85. [89] M.A. Gómez, C.C. Alzate, A. Márquez, J.W. Restrepo, F. Jaramillo, Effect of temperature on the tensile strength of some fiber reinforcements for ceramic or polymer matrices, Revista EIA 1 (2009) 65–75. [90] M. Broström, S. Enestam, R. Backman, K. Mäkelä, Condensation in the KCl–NaCl system, Fuel Process. Technol. 105 (2013) 142–148. [91] P. Gaur, A. Chawla, K. Verma, S. Mukherjee, S. Lalvani, R. Malhotra, C. Mayer, Characterisation of human diaphragm at high strain rate loading, J. Mech. Behav. Biomed. Mater. 60 (2016) 603–616.

10