Sei sulla pagina 1di 11

Article

Cite This: ACS Biomater. Sci. Eng. 2018, 4, 2115−2125

Type I Collagen from Jellyfish Catostylus mosaicus for Biomaterial


Applications
Zahra Rastian,†,‡ Sabine Pütz,† YuJen Wang,† Sachin Kumar,† Frederik Fleissner,† Tobias Weidner,†,§
and Sapun H. Parekh*,†

Department of Molecular Spectroscopy, Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany

The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr
University of Medical Sciences, Moallem St., 7514633341 Bushehr, Iran
§
Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

*
Downloaded via UNIV NACIONAL DE COLOMBIA on September 11, 2018 at 15:32:16 (UTC).

S Supporting Information

ABSTRACT: Collagen is the predominant protein in animal


connective tissues and is widely used in tissue regeneration and
other industrial applications. Marine organisms have gained
interest as alternative, nonmammalian collagen sources for
biomaterial applications because of potential medical and
economic advantages. In this work, we present physicochem-
ical and biofunctionality studies of acid solubilized collagen
(ASC) from jellyfish Catostylus mosaicus (JASC), harvested
from the Persian Gulf, compared with ASC from rat tail
tendon (RASC), the industry-standard collagen used for
biomedical research. From the protein subunit (alpha chain)
pattern of JASC, we identified it as a type I collagen, and
extensive molecular spectroscopic analyses showed similar
triple helical molecular signatures for JASC and RASC. Atomic force microscopy of fibrillized JASC showed clear fibril
reassembly upon pH neutralization though with different temperature and concentration dependence compared with RASC.
Molecular (natively folded, nonfibrillized) JASC was shown to functionalize rigid substrates and promote MC3T3 preosteoblast
cell attachment and proliferation better than RASC over 6 days. On blended collagen−agarose scaffolds, both RASC and JASC
fibrils supported cell attachment and proliferation, and scaffolds with RASC fibrils showed more cell growth after 6 days
compared with those scaffolds with JASC fibrils. These results demonstrate the potential for this new type I collagen as a possible
alternative to mammalian type I collagen for biomaterial applications.
KEYWORDS: type I collagen, marine collagen, rat tail tendon collagen, collagen molecular structure, collagen biomaterials, jellyfish

■ INTRODUCTION
Collagen is the predominant protein in connective tissues of
(gelatin) form for various scaffolds such as sponges, sheets,
plugs, and pellets.9 The main sources of collagen for industrial
animals, constituting approximately 25−30% of the total uses have been the skin of animals such as pig and calf.
protein content (by mass) of the whole body in mammals.1 However, the risk of transferring diseases such as bovine
At least 27 different types of collagen, named type I to XXVII, spongiform encephalopathy (BSE), foot-and-mouth disease,
occur naturally and are categorized into different groups such as and avian influenza limits the application of land animal
basement, fibril forming, fibril associated, network forming, collagen for human application.10 Therefore, for human
anchoring fibrils, and transmembrane, of which fibril forming application, it is important to choose a collagen source that
collagens represent about 90% of the total collagen in the minimizes transmission of diseases to humans, in addition to
human body.2,3 Collagen molecules contain triple-helix economic and environmental considerations.11
structures made of three α chains. These chains contain the Marine organisms have become increasingly considered as
collagenous domain motif composed of Gly-Xaa-Yaa triplets alternative collagen sources in recent years12,13 as they are free
where Xaa and Yaa are often proline and hydroxyproline of zoonosis such as BSE, less immunogenic, and elicit minimal
residues.4 Collagen molecules form fibrils that have important inflammatory response.1 Genomic, molecular cloning, bio-
functions in the tissue mechanics and tissue repair.5,6 Thus, chemical, and structural studies have demonstrated similar
interest in collagen has become widespread among scientists
who investigate wound healing, fibrosis, neoplasia, and tissue Received: December 14, 2017
engineering, for example.6−8 Collagens for tissue engineering Accepted: April 24, 2018
applications are used in their native fibrillar or denatured Published: April 24, 2018

© 2018 American Chemical Society 2115 DOI: 10.1021/acsbiomaterials.7b00979


ACS Biomater. Sci. Eng. 2018, 4, 2115−2125
ACS Biomaterials Science & Engineering Article

characteristics between marine fibrillar collagens and human Nylon filter 0.45 μm (BD Falcon), and the supernatant was salted out
fibrillar collagen.11,14,15 Nevertheless, marine collagens show by adding NaCl to final concentration of 0.9 M. The resulting
some differences in physical and chemical properties compared precipitate was collected by centrifugation at 17 000g for 1 h. The
to animal collagens such as lower molecular weight and lower pellet was dissolved in a minimum volume of 0.5 N acetic acid,
dialyzed against 0.02 N acetic acid using a membrane with a MWCO
denaturing (melting) temperature.1,10,16 Collagen has been of 100 000 for 2 days with two buffer changes per day, and finally
extracted from marine vertebrates and invertebrates including stored at −20 °C for further experiments. This protein is heretofore
fish,17−19 squid,20,21 octopus,22 sponge,23 urchin;24 however, referred to as jellyfish acid solubilized collagen (JASC). All extraction
substantially less research has been done on their biocompat- steps were carried out at 4 °C until storage. Collagen concentrations
ibility and biomedical usage. Jellyfish is a particularly attractive were determined using the standard protocol of the modified Lowry
marine source for collagen for a variety of reasons.12,25 Some method.29 To obtain a series of calibration curves, a dilution series
species of jellyfish have been used as traditional food and ranging from 0.005 to 0.5 mg/mL of commercial rat collagen in 0.02 N
medicine in China for more than 1000 years, suggesting good acetic acid was used.
Fibril Formation. For fibril formation analysis, a protocol based on
biocompatibility of jellyfish constituent materials.16 Moreover, Hoyer et al.15 was used. Initially, 500 μL collagen solutions at different
in many countries, the increase in jellyfish population has concentrations were thoroughly mixed with 500 μL of 50 mM
caused problems in marine ecosystems.16,26,27 Using jellyfish as tris(hydroxymethyl)-aminomethl)-aminomethane (Tris) buffer (pH
a collagen source could help control the population and reduce 8) to reach a final pH 7.5−8 and incubated at different temperatures
the ecological impact from overpopulation while being for 4 h. The suspensions were centrifuged at 11 000 rpm (Heraeus
medically beneficial. Biofuge Pico, Thermo Fisher) for 5 min, and collagen content of
In this study, we report the extraction and characterization of supernatants was measured using modified Lowry method. Degree of
acid solubilized collagen from Catostylus mosaicus jellyfish fibril formation (%) was calculated on the basis of eq 1
harvested from the Bushehr coast in southern Iran and perform fibril formation (%) = [1 − ([collagen]sup /[collagen]init )] × 100
a comparative analysis with acid extracted collagen from rat tail (1)
tendon−the standard collagen research model. Our analytical
tests show that oral arm jellyfish collagen is a type I collagen, where [collagen]sup and [collagen]init are the supernatant and initial
concentrations of collagen, respectively.
maintains a triple helical structure both in the molecular and SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Elec-
fibril form, and supports preosteoblast cell growth similarly to trophoresis (SDS-PAGE) was performed using the NuPAGE Bis-Tris
rat tail tendon collagen I. Precast Mini Gel- 8% system. Twenty-five microliters of collagen


solution containing 5−10 μg collagen in NuPAGE LDS sample buffer
MATERIALS AND METHODS and reducing agent (Invitrogen) was prepared according to the
manufacturer instructions and was loaded per well. Commercial rat tail
Raw Materials and Chemical Reagents. Fresh Catostylus collagen type I was also prepared and loaded as an internal standard
mosaicus jellyfish with a creamy white color (0.2−2 kg) were obtained for collagen. Ten microliters of a high-molecular-weight marker:
from the Persian Gulf in southwest Iran on Bushehr coast and ranging from 10 to 225 kDa (Novagen) was loaded as well.
transported to the lab on ice. The jellyfish were first rinsed with chilled Electrophoresis was performed in a mini dual vertical electrophoresis
tap water and then with chilled distilled water for cleaning. Then, the unit (Novex) using NuPAGE MES SDS running buffer (Invitrogen).
umbrella and oral arm of three animals were cut to pieces separately, The gel was stained using a Silver Quest staining kit (Thermo Fisher).
treated with 0.1 M NaOH in order to remove the noncollagenous Attenuated Total Reflectance Fourier Transform Infrared
proteins, and then washed by distilled water. These specimens were (ATR-FTIR) Spectroscopy. Extracted collagen samples were dialyzed
lyophilized and stored at −20 °C until further use. Rat tail collagen was in Milli-Q water and lyophilized for ATR-FTIR analysis. FTIR spectra
prepared from Sprague−Dawley rats in compliance with the ethical of collagen samples were obtained using a TENSOR II IR
guidelines of the Bushehr University of Medical Sciences. In total, spectrometer (Bruker) equipped with the DTGS detector and single
eight rat tails from 8 month-old rats were disinfected by submerging in reflection diamond crystal. Infrared spectra were recorded in the range
70% ethanol. The rat skin was removed and discarded, and the tendon of 600−4000 cm−1 by averaging 50 measurements for each sample.
was pared for acid extraction of collagen. Circular Dichroism Spectroscopy. Circular dichroism (CD)
Commercial type I collagen from rat tail tendon (also acid spectroscopy in the far ultraviolet region is sensitive to the secondary
solubilized) was purchased from Sigma-Aldrich or from Ibidi GmbH as structure of proteins in solution, which can be used for identifying the
specified. Alpha Modified Eagle Medium (α-MEM) from Lonza and triple helical structure of collagen.30 CD spectra were obtained
fetal bovine serum (FBS) from Gibco were used for cell culture. All experimentally using Jasco J-815 CD Spectrometer (Easton). Collagen
other chemicals and reagents were of analytical grade. solution was prepared with concentration of 200−300 μg/mL in 0.02
Preparation of Acid-Soluble Collagens (ASCs). Rat tail tendon N acetic acid for RASC, JASC, and commercial rat tail collagen as a
collagen was extracted according to Chandrakasan et al.28 with some standard. All samples were placed into quartz cell with a path length of
modifications as described below. Tendons were added to 0.5 M acetic 1 mm where optical activity was measured between 250 and 185 nm at
acid (1 g tendon per 250 mL). The mixture was left for 3 days with scan speed of 50 nm/min with an interval of 0.5 nm at 4 °C. The
gentle stirring at 4 °C. To discard the undissolved part, centrifugation response time and data accumulation were four seconds and four
(5810 R, Eppendorf) at 1000g (at 4 °C) was used for 30 min. Then times, respectively.
10% (w/v) NaCl was added to the supernatant to induce precipitation Protein Melting Point and Amino Acid Analysis. Protein
overnight at 4 °C, after which a 30 min centrifugation at 10 000g (at 4 thermal stability was measured in a label-free fluorometric analysis
°C) was applied to collect pelleted collagen, and the supernatant was using the Prometheus NT.48 (NanoTemper Technologies GmbH).
discarded. The pellet was resuspended in 0.5 M acetic acid at 4 °C and Briefly, the shift of intrinsic tryptophan fluorescence of proteins upon
then dialyzed using a membrane for MWCO of 14 000 (Roth) against temperature-induced unfolding was monitored by detecting the
diluted glacial acetic acid (1:1000 v/v) at 4 °C for 3 days with two emitted fluorescence at 330 and 350 nm. Thermal unfolding was
buffer changes per day. The dialyzed protein was then lyophilized for performed for collagen samples in 0.02 N acetic acid (1 mg/mL) in
long-term storage at −20 °C. This protein is heretofore referred to as nano differential scanning fluorimetry (nanoDSF) grade high-
rat acid solubilized collagen (RASC). sensitivity glass capillaries at a heating rate of 1 °C per minute.
Jellyfish collagen was extracted according to Nagai20 with slight Protein melting points (Tm) were calculated from the first derivative
modification. Jellyfish pieces were extracted in 0.5 N acetic acid (5 mg/ of the ratio of tryptophan emission intensities at 330 and 350 nm from
mL) for 3 days with gentle stirring. The extract was filtered through a 20 to 50 °C.

2116 DOI: 10.1021/acsbiomaterials.7b00979


ACS Biomater. Sci. Eng. 2018, 4, 2115−2125
ACS Biomaterials Science & Engineering Article

Figure 1. (A) SDS-PAGE patterns of acid-soluble collagens. Lane (1) high-molecular-weight protein marker, lane (2) type I collagen from rat tail
(Sigma), lane (3) extracted collagen from rat tail tendon (RASC), and lane (4) extracted collagen from jellyfish oral arm (JASC). (B) Circular
dichroism (CD) spectra recorded at 4 °C for RASC, JASC, and commercial rat tail collagen (Ibidi). Vertical lines mark peaks (maxima and minima)
of commercial rat tail collagen for reference.

The JASC sample was lyophilized and subjected to amino acid neutralization were added to 800 μL of collagen stock solution (5 mg/
analysis Genaxxon Bioscience.31 mL) after which α-MEM was added to make a final volume of 1 mL (4
Coherent Anti-Stokes Raman Scattering Spectroscopy. mg/mL collagen). RASC solution was incubated at 37 °C, and JASC
Broadband coherent anti-Stokes Raman scattering (BCARS) spectra was incubated at room temperature or 37 °C; all samples were
were acquired for fibrillized collagen samples (JASC at room incubated overnight. A stock solution of 4% w/w of low gelling
temperature and RASC at 37 °C, pH 7.5, and fibrillized overnight), temperature agarose (Sigma) in phosphate buffer solution (PBS) was
all of which were previously lyophilized. The experimental setup has prepared and autoclaved. Agarose stock solution (250 μL) was added
been described extensively elsewhere.32 The fibrillized sample was to 750 μL of collagen-fibril suspension, mixed carefully, and then
sandwiched between two coverslips with a double-sided tape as a added to wells in a nontreated (but sterile) 48-well plate (Greiner).
spacer of ∼80 μm. Hyperspectral images (21 × 21) were taken with a These solutions were allowed to gel for 1 h at room temperature in the
step size of 0.5 μm between pixels and with an exposure time of 300 tissue culture hood before cell seeding. MC3T3-E1 cells (15 000 cells/
ms per spectrum. To obtain a quantitative Raman-like spectrum, the cm2) were added to each well with media (α-MEM containing 10% v/
resonant component of the CARS spectra was extracted by employing v FBS with penicillin−streptomycin antibiotics). Cells were incubated
a Kramer-Kronig transform on the raw data.33,34 Spectra shown are at 37 °C for 6 days to measure viability and growth, assayed by live/
averages from 21 × 21 spatial pixels. dead staining. Control experiments were done with pure 1% agarose or
AFM Imaging. JASC collagen solution was fibrillized (in Tris seeding cells directly on the well plate surface. Passage two MC3T3-E1
buffer, pH 7.5−8) overnight at 4 °C or at room temperature as cells were used for all experiments.
indicated. Twenty microliter suspensions of fibrillized collagen were Live and Dead Staining. Each well of the cell culture plate was
added to freshly cleaved mica and spin coated for 30 s at 30 rpm, after gently washed by PBS and incubated in PBS containing 2 μM calcein
which the sample was rinsed with Milli-Q water during spinning for (Invitrogen, Thermo Fisher) and 4 μM ethidium homodimer-1
less than 10 s to remove salt. Samples were then dried under nitrogen (Sigma).36 Stained cells were imaged using an inverted fluorescence
flow. Using this protocol increased the amount of collagen and microscope (Olympus IX81, Japan) with a 10×, 0.3 NA (UPlanFl,
improved fibril spreading on the mica surface compared to drop- Olympus) to image both the live and dead fluorescence stain for all
casting the collagen and allowing it to dry at room temperature. AFM samples using a standard eGFP and TRITC filter sets (Chroma). At
measurements were carried out in AC mode in air using a Cypher least three fields of view were imaged for both live and dead channels
(Asylum Research) AFM with the ARC2 SPM controller and PPP- for each sample, and the same exposure time was used for both
NCHAuD cantilever with imaging speeds of 1.95 Hz, scanning 256 channels. No additional contrast enhancement was employed.
lines. Imaging fields were usually chosen to be 20 × 20 μm2 or 5 × 5 Mechanical Characterization. The viscoelastic properties of
μm2. fibrillized RASC, fibrillized JASC, agarose gel (1% w/w), and
Cell Culture and on Collagen-Coated Polystyrene. Non- collagen−agarose scaffolds from RASC and JASC (final concen-
treated Petri dishes (Greiner) were coated with 10 μg/mL molecular trations: 3 mg/mL collagen, 1% (w/w) agarose) were measured by
(nonfibrillized) RASC (at 37 °C) or JASC (at room temperature or 37 rheology. Pure RASC and JASC hydrogels (2.5 mg/mL collagen
°C) overnight. Wells were washed once in PBS, and 1000 cells/cm2 of concentration) were rheologically characterized after overnight
MC3T3-E1 murine preosteoblast cells (DSMZ, ACC-210) were added fibrillization at 37 °C and 95% relative humidity directly in the
to each well. Cells were cultured in α-MEM, containing 10% v/v FBS measurement cell. Rheology was done on a TA-hybrid DHR-2
with Penicillin-Streptomycin antibiotics (Gibco), and incubated in 5% rheometer at 37 °C in the parallel plate geometry with 25 mm plates
CO2 with 95% humidity at 37 °C cells for 6 days. Control experiments with a gap size between 0.24 and 0.49 mm. A frequency sweep (0.1−
were performed with cells in uncoated wells. Cells were counted from 100 rad/s) was applied to the top plate with strains always less than
five different wells for each condition at Day 6 using a hemocytometer 1%. The software from the instrument directly calculated the storage
to determine the cell density. Passage two MC3T3-E1cells were used modulus (G′) and loss modulus (G″) as a function of frequency.
for all experiments. For collagen−agarose scaffold rheology, RASC and JASC were
Cell Culture and Preparation of Collagen-Agarose Scaffolds. fibrillized overnight at 37 °C at 95% relative humidity followed by
Based on Ulrich et al.35 with some modification, hybrid collagen− mixing with low-melt agarose at room temperature. Mixed solutions
agarose scaffolds containing 1% (w/w) agarose and 3 mg/mL collagen were allowed to set for 1 h directly in the measurement cell. Rheology
fibrils were prepared from RASC and JASC. To prepare fibrillized was measured with an Anton Paar model Physica MCR 301 rheometer
collagen, 18 μL HEPES (300 mM) and enough NaOH (1 M) for in the parallel plate geometry with 25 mm plates at 37 °C with a gap

2117 DOI: 10.1021/acsbiomaterials.7b00979


ACS Biomater. Sci. Eng. 2018, 4, 2115−2125
ACS Biomaterials Science & Engineering Article

size fixed to 1 mm. A frequency sweep (0.1−100 rad/s) was applied to transform infrared spectroscopy (FTIR) was used to study the
the top plate with strains always less than 1%. Data processing was secondary structure and functional groups of JASC and
performed using Rheoplus software to calculate G′ and G″ as a compare it with RASC (Figure 2). Nine normal modes for
function of frequency. All mechanical data were plotted in Igor Pro.
6.34.
Statistics. Mean and standard deviation are shown in all figures.
The difference in the collagen fibril formation within each parameter
was statistically evaluated with one-way ANOVA and Tukey’s test via
OriginPro 9.

■ RESULTS
Preparation of Acid-Soluble Collagens. Collagen from
the oral arm and umbrella tissues of Persian Gulf Catostylus
mosaicus jellyfish were extracted separately via acid extraction as
detailed in the Materials and Methods. By measuring the
amount of extracted collagen, the yield of C. mosaicus jellyfish
acid solubilized collagen (JASC), based on lyophilized dry
weight, from umbrella and oral arm tissues were found to be
14.61 ± 0.57 and 22.47 ± 1.25 mg/g dry weight (1.46% and
2.24%), respectively. The ASC yield of other jellyfishes from
Tunisian Mediterranean coast were 0.83−3.15 and 2.61−10.3
(mg/g wet tissue) for Rhizostoma pulmo umbrella and oral Figure 2. ATR-FTIR spectra of JASC and RASC as lyophilized
arms, respectively, 0.453 and 1.94 (mg/g wet tissue) for powders.
Cotylorhiza tuberculate umbrella and oral arms, respectively,
0.074 and 0.0079 (mg/g wet tissue) for whole body Pelagia
noctiluca and Aurelia aurita, respectively.11 The ASC yield of amide bands named A, B, and I−VII are allowed for proteins.42
Cyanea nozakii Kishinouye from the Yellow sea was 13.0% (dry The main absorption bands in JASC were Amide A, Amide B,
weight).37 In light of these studies, we surmise that our finding Amide I, Amide II, and Amide III, which are typically in the
of greater collagen content in the oral arm compared with the range from 3200 to 3440 cm−1, 3100 cm−1, 1600−1700 cm−1,
umbrella of C. mosaicus is consistent with previous work, and 1510−1580 cm−1, and 1200−1300 cm−1,43,44 respectively. The
the amount of ASC extracted collagen is species-dependent. As Amide A is due to the N−H stretching vibration, and this mode
the oral arms of the jellyfish used here provided the highest does not depend on the backbone conformation but is very
yield, JASC from this tissue was used for rest of the studies in sensitive to the strength of hydrogen bonds (N−H···CO).45
this work. The stretching vibration of hydrogen bonded N−H group
Molecular Characterization of Collagen Molecules. occur commonly in the range of 3200−3300 cm−1;46 however,
Collagen Type Identification and Molecular Structure. Figure free N−H stretching vibration commonly occurs in the range of
1A shows the SDS-PAGE pattern of RASC and JASC. Rat tail 3400−3440 cm−1.44 In RASC and JASC, the Amide A vibration
tendon collagen from Ibidi or Sigma (also ASCs) were used as peaked at 3296 and 3292 cm−1, respectively, indicating that the
standard type I collagen for comparison. Type I collagen N−H group is involved in hydrogen bonding, which is known
contains two α1 chains and one α2 chain as well as β dimers. to stabilize the triple helical structure of collagen.16,47 Collagen
The electrophoretic patterns of extracted rat tail collagen and from calf skin and bamboo shark exhibit similar Amide A
jellyfish collagen are qualitatively similar to the commercial bands.43,44,46
sample. Slight shifts in the position of the α chain bands The Amide I band is the most intense structure-revealing
observed for jellyfish collagen could be caused by small band in proteins, so this band is a useful marker for the analysis
differences in the amino acid sequences and molecular of secondary structure of proteins in FTIR.47 It is associated
weight.16,38 Circular dichroism (CD) spectra of extracted with CO stretching vibration coupled with the N−H
collagen samples from rat and jellyfish are shown in Figure 1B. bending vibration along the polypeptide backbone or with
The collagen molecule, having a triple helix structure, exhibits a hydrogen bonding coupled with COO−, CN stretching, and
unique CD spectrum with a small positive peak between 220 CCN deformation.43,44,48 Bands near 1630 cm−1 indicate imide
and 225 nm and a large negative at 197 nm.39 residues, and bands at 1660 and 1675 cm−1 are assigned to
RASC showed a maximum at 222 nm and minimum at 198 intermolecular cross-links and β-turns, respectively.47 The
nm; JASC showed a maximum at 220 nm and minimum at 197 Amide I bands at 1636 cm−1 for RASC and 1641 cm−1 for
nm, and commercial collagen (Ibidi) showed a maximum at JASC reflect the presence of imides. Similar frequencies have
222 nm and minimum at 198 nm. The location of the been seen for calf skin collagen.16,46 The Amide II band is
maximum peak in all collagens samples is characteristic of the associated with the N−H bending vibration coupled with the
collagen triple helix while the minimum centered at 197 nm is C−N stretching vibration.46 This amide was found in RASC
characteristic of a random coil.30,40 Since our extracted RASC and JASC at 1541 and 1538 cm−1, respectively, again similar to
and commercial rat tail tendon collagen exhibit almost identical collagen extracted from calf skin and bamboo shark.16 Lower
SDS-PAGE patterns and CD spectra, they are collectively frequencies in this region indicate that the N−H group is
referred to as RASC for the remainder of this work. involved in bonding with α chain46 and that hydrogen bonding
Infrared (IR) spectroscopy is an analytical technique that in collagen is present.49 The Amide III, which is referred to as
depicts the vibrational characteristics of chemical functional the “collagen fingerprint” was found in RASC and JASC at
groups and provides information about protein secondary 1235 and 1234 cm−1, respectively. This band has been seen at
structure.41 Attenuated total internal reflection (ATR) Fourier 1235 cm−1 for calf skin collagen and higher frequency for
2118 DOI: 10.1021/acsbiomaterials.7b00979
ACS Biomater. Sci. Eng. 2018, 4, 2115−2125
ACS Biomaterials Science & Engineering Article

Table 1. FTIR Peak Locations (in cm−1) and Assignments for Jellyfish and Rat Tail Collagen
vibration JASC loc. RASC loc. nominal location41,42 assignment
−1 −1
Amide A 3292 cm 3296 cm 3200−3440 cm−1 N−H stretch and hydrogen bond
Amide B 2921 cm−1 2927 cm−1 3100 cm−1 CH2 asymmetrical stretch
Amide I 1641 cm−1 1636 cm−1 1600−1700 cm−1 CO stretch coupled with hydrogen bond
Amide II 1538 cm−1 1541 cm−1 1510−1580 cm−1 N−H bend and stretch
Amide III 1234 cm−1 1235 cm−1 1200−1300 cm−1 N−H bend and stretch, C−O stretch, collagen fingerprint

Figure 3. Fibril formation of RASC and JASC. (A) Effect of collagen initial concentration on degree of fibril formation of RASC and JASC at room
temperature. Inset is the statistical analysis for RASC where green boxes mark statistically significant differences according to p < 0.05 by ANOVA
with Tukey’s. (B,C) Effect of temperature on degree of fibril formation of 0.75 mg/mL RASC (B) and 0.75 mg/mL JASC (C). The percentage was
calculated on the basis of initial concentration as stated in the methods. * indicate p < 0.05 by ANOVA with Tukey’s post-hoc test.

collagen from other marine sources.16,46 The peaks in the lower composition was expressed as residues per 100 residues. JASC
fingerprint region at 1060 cm−1 for RASC and 1030 cm−1 for was rich in glycine (26.86 residues/100 residues), similar to
JASC could belong to carbohydrate moieties.50 Table 1 other collagens. The imino acid content (hydroxyproline and
summarizes the band positions and their assignments for the proline) of JASC was 12.43 residues/100 residues, which is
FTIR spectra from RASC and JASC. slightly more than that of C. nozakii jellyfish (11.9 residues/100
Amino Acid Analysis and Protein Melting Point. Collagen residues), 37 similar to S. meleagris (12.2 residues/100
molecules are characterized by triplets of glycine and two other residues),25 and less than cod skin collagen (15.4 residues/
amino acids (Gly-Xaa-Yaa), where proline and hydroxyproline 100 residues) or carp skin collagen (19.2 residues/100
are the most common amino acids.38 The collagen triple helix is residues).49
a supercoiled, right-handed structure composed of three parallel The melting temperature (the temperature at which the
α chains in which glycine is the key for supercoiling of α folded and unfolded states are equally populated at
chains.51,52 The amino acid compositions of freeze-dried JASC equilibrium) of RASC and JASC molecules was measured by
and rat tail collagen53 are presented in Table S1. Amino acid nanoDSF of tryptophan fluorescence. This measurement
2119 DOI: 10.1021/acsbiomaterials.7b00979
ACS Biomater. Sci. Eng. 2018, 4, 2115−2125
ACS Biomaterials Science & Engineering Article

Figure 4. AFM images of fibrillized JASC at room temperature. (A) height image and (B,C) amplitude image. (D) Height profile over region marked
by white line in (C). (E) and (F) AFM images of fibrillized JASC at 4 °C.

showed that RASC had a melting point of 36.2 ± 0.7 °C, and incubation temperature as described in the Materials and
JASC had a melting point of 31.9 ± 1.1 °C (n = 3 for both). Methods. RASC fibrillization increased with initial concen-
The melting point of JASC was higher than for S. meleagris (26 tration of collagen (Figure 3A); however, the same graph shows
°C) and C. nozakii (23.8 °C).25,35 Generally, imino acid content that JASC did not show a trend with increasing collagen
and degree of proline hydroxylation are related to collagen concentration but showed the same degree of fibrillization at
thermo-stability.35 Moreover, higher content of proline and the lowest and highest initial collagen concentrations. Statistical
position of the imino acids play an important role in analysis showed that initial concentration of collagen had a
stabilization of the collagen triple helix.54 The imino acid significant influence (p < 0.05) on RASC fibril formation, but
content and degree of hydroxylation in rat tail collagen is 17.95 no significant influence (p < 0.05) on JASC (Figure 3A, inset).
residues/100 residues and 38.16%, respectively, more than the Figure 3B,C show that raising the temperature resulted in more
imino acid content (12.43 residues/100 residues) and degree of fibrillization of RASC but had a negative effect on JASC
hydroxylation (30.73%) in JASC. Cysteines, which form fibrillization (both of which were statistically significant)
disulfide bridges between α chains chains of collagen triple consistent with the finding that the melting point of molecular
helix,55 were not found in JASC further contributing to its JASC was almost five degrees lower than that of molecular
lower melting temperature, similar to other marine colla- RASC. Similar trends of fibril formation of jellyfish R.
gens.13,56,57 esculentum as a function of initial concentration and temper-
Fibrillization, Fibril Morphology, Fibril Molecular ature have been seen by Hoyer et al.15
Structural Characterization. Type I collagen molecules The morphology of the reassembled JASC collagen fibrils
obtained from collagen fibers in tissues can undergo in vitro was assessed by AFM imaging. Fibrils aggregated during
reassembly into fibrils. Collagen fibrillization depends on reassembly at room temperature and separated fibrils (or
electrostatic and hydrophobic interactions.15,41,57−60 Electro- fibers) were rarely seen (Figure 4A−C). Line profiles of
static interactions between amino acid residues are regulated by separated JASC fibrils showed a width of 25−78 nm (Figure
factors such as pH and ionic strength. The formation of net 4D). The morphology of fibrils at 4 °C was also visualized by
charges between collagen molecules of neighboring fibrils could AFM (Figure 4E,F), which showed that cold temperature
create electrostatic attractions that can stabilize or destabilize resulted in shorter and broader fibrils (46−50 nm width).
fibers.59−63 Moreover, temperature affects the molecular Increase in fibril width with lowering temperature has been
hydrophobic interactions and therefore the kinetics of collagen previously reported for calf skin collagen.59,60 AFM images
fibrillization, in addition to pH and ionic strength.64 The degree RASC fibrils are given in Figure S1.
of fibrillization of RASC and JASC were measured by In order to characterize the molecular structure of fibrillized
neutralizing the pH as a function of initial concentration and collagen from both sources in solution, we used BCARS
2120 DOI: 10.1021/acsbiomaterials.7b00979
ACS Biomater. Sci. Eng. 2018, 4, 2115−2125
ACS Biomaterials Science & Engineering Article

spectroscopy, a coherent analog of spontaneous Raman concentration of natively folded, nonfibrillized collagen
spectroscopy, to avoid the strong water absorption in FTIR molecules (Figure 6A). We note that coating dishes with
spectroscopy and eliminate photodamage observed in sponta- JASC at room temperature or 37 °C resulted in statistically
neous Raman. Raman-like spectra of RASC and JASC are identical proliferation.
shown in Figure 5. The Amide I band is located at 1653 and As a next step, we attempted to use JASC in biomaterial
scaffolds. Similar to previous studies (for collagen concentration
of 2−3 mg/mL),67−70 we found that RASC formed hydrogels
with shear storage moduli (G′) of order 10 Pa. JASC hydrogels
were slightly stiffer (G′ ∼ 20 Pa) (Figure S2), which is still very
soft compared to, for example, bone, in which type I collagen is
a large component. Therefore, we employed a strategy to
functionalize a stiffer host scaffold, in this case agarose. Agarose
is a polysaccharide polymer that forms hydrogels and is often
used in biomaterial and tissue engineering applications because
its mechanical properties can be tuned to values appropriate for
many different tissues. However, it is necessary to functionalize
agarose materials such that they support cell growth and
attachment. One common strategy to functionalize agarose is
by incorporating cell adhesion peptides such as arginine-
glycine-aspartic acid (RGD); another strategy is incorporating
collagen fibrils themselves into the agarose matrix.35,71 We
employed the latter strategy in this work and made collagen−
Figure 5. Raman-like spectra of in situ RASC and JASC fibrils. For agarose hybrid scaffolds. All scaffolds had 1% (w/w) agarose,
comparison, spectra were normalized on the maximum of the Amide I and the collagen−agarose hybrid scaffolds contained a final
band. concentration of collagen of 3 mg/mL that was fibrillized
(overnight at 37 °C) before blending the two polymers. The
1659 cm−1 for JASC and RASC, respectively. The slight mechanical properties of the collagen−agarose scaffolds were
frequency shift of the Amide I band possibly arises from a measured using parallel plate rheology (Figure 6B). We found
different amino acid composition between the two collagens.65 that the storage modulus (G′) and loss modulus (G″) as a
The bands at 1443 cm−1 for both samples belong to CH2 function of frequency for 1% (w/w) agarose gel were only
bending.66 The Amide III region at 1230−1330 cm−1 shows mildly affected by incorporation of either RASC or JASC over
similar pattern in both collagen samples. As mentioned above the range of frequencies measured. The incorporation of
in the FTIR results, the strong Amide III vibration is collagen into agarose resulted in a slightly increased storage
characteristic of the triple helix structure in collagen. The modulus compared to agarose alone; however, the loss moduli
vibrations at 1246 and 1271 cm−1 are assigned to proline-rich appeared unchanged by incorporation of collagen into the
and proline-poor regions, respectively.65 These peaks have been agarose matrix. Compared with our measured moduli for pure
seen at 1248 and 1271 cm−1 for bovine Achilles tendon and calf JASC and RASC hydrogels (Figure S2), these scaffolds were
skin gelatin.66 The vibrational spectroscopy of fibrillized RASC nearly 50-fold stiffer.
and JASC shows that their molecular structures are nearly Figure 6C shows the attachment and viability of MC3T3-E1
identical. Interestingly, after fibrillizing JASC and RASC, we cells to collagen−agarose scaffolds. From the images in Figure
retested for fibril thermal transitions and found that RASC and 6C and Figure S3, it is clear that scaffolds incorporating RASC
JASC fibrils had thermal transitions (presumably thermal and JASC fibrils showed much improved cell attachment (at
denaturation) at ∼53 °C and ∼55 °C, respectively, again Day 1) compared to agarose alone. Cells did not attach nearly
suggesting their molecular structuresat the fibril levelare as well to the (nontreated) polystyrene well-platesimilar to
identical. We note that the fibrillization temperature (at room what was observed for cells on pure agarose (Figure S3). Both
temperature or 37 °C) for JASC did not affect the thermal of these substrates showed almost no cell growth over the 6-day
transition. incubation period (Figure S3). The number of viable MC3T3-
Cell Attachment and Viability on Collagen-Agarose E1 cells on RASC-agarose and JASC-agarose increased by ∼10-
Scaffolds. Bone is a type I collagen-rich organ, and collagen- and ∼4-fold, respectively, over the 6-day incubation period.
based biomaterials have been extensively studied for bone tissue Cell viability was almost 100% on both JASC and RASC
engineering. As JASC is a type I collagen from a new source, it collagen−agarose scaffolds. Taken together, these experiments
is necessary to demonstrate its ability to support cell show that JASC exhibits similar ability as RASC to support cell
proliferation and functionalize agarose and rigid substrates.


attachment and proliferation as a basic requirement in
biomaterials research. In order to ascertain if JASC supported
cell attachment and promoted cellular proliferation, we cultured DISCUSSION
MC3T3-E1 preosteoblasts on (molecular) collagen-coated Pathological risks of nonhuman, mammalian collagens for
(nontreated) polystyrene Petri dishes. Parallel experiments human application underscore the importance of research for
done with RASC coated wells show that cells not only attach alternative collagen sources, such as marine collagen. As
but also proliferate on both types of collagen-coated wells, reported here and elsewhere, there are clear differences
whereas the cell solution applied to Petri dishes hardly wetted between marine and mammalian collagen such as lower
the surface on uncoated dishes. Statistical analysis showed that imino acid content, lower melting point, and lower viscosity
cell proliferation over 6 days on JASC-coated wells surpassed in marine collagen.13,72 SDS-PAGE, CD spectra, and FTIR
that on RASC for substrates coated with the same solution were used for molecular characterization of JASC and RASC
2121 DOI: 10.1021/acsbiomaterials.7b00979
ACS Biomater. Sci. Eng. 2018, 4, 2115−2125
ACS Biomaterials Science & Engineering Article

Figure 6. (A) MC3T3-E1 proliferation measured on JASC or RASC coated (nontreated) polystyrene dishes. The seeding density was 1000 cells/
cm2. The results are shown as mean with standard deviation as error bars from N = 5 dishes. (B) Mechanics of hybrid collagen−agarose scaffolds.
Parallel plate rheology of different agarose scaffolds was measured (at 37 °C) 1 h after agarose gelation at room temperature. Symbols are an average
of three samples for collagen−agarose scaffolds, whereas only a single scaffold was measured for agarose. (C) Fluorescence images of MC3T3-E1
cells after 1 day or 6 days of culture on collagen−agarose hydrogels as stated in the figure. Collagen was fibrillized at 37 °C for both RASC and JASC
before blending with agarose. Green shows living cells while red shows dead cells. The graph shows results as mean with standard deviation as error
bars from N > 3 dishes per condition and day. The seeding density was 15 000 cells/cm2. * indicates p < 0.05 between the marked samples by
ANOVA with Tukey’s post-hoc test.

(as a prototypical type I collagen molecule). JASC’s SDS-PAGE depend on the habitat and amino acid composition of the
showed α1 and α2 chains, as well as β dimers, with qualitatively species from which it is extracted. This is indeed true for marine
more α1 chainscharacteristic of the composition of type I collagen (e.g., with melting temperature). Ribbon jellyfish taken
collagen. The same maxima and minima of CD spectra were from the warm coast of Penang Island had a higher melting
seen for our extracted RASC and JASC, as well as commercial temperature (37.38 °C)16 compared with jellyfish from the
rat tail tendon collagenwhich is presumed to contain the Chinese Yellow Sea (C. nozakii Kishinouye, 23.8 °C),37
characteristic triple helical collagen structure. Moreover, JASC Tunisian Mediterranean coast (R. pulmo, 28.9 °C),11 Senzaki
and RASC had nearly identical vibrational FTIR and BCARS Bay of Japan (R. asamushi, 28.8 °C),12 or Persian Gulf (C.
spectra, which demonstrates similarities in both the native
mosaicus, JASC, 31.9 °C) studied here.
collagen and fibrillar collagen secondary structures, respectively.
Similar fibril reassembly in vitro was seen for JASC and R.
The FTIR (and BCARS) spectra show the characteristic “triple
esculentum jellyfish.15 JASC showed similar features to rat tail
helix” peak in the Amide III, and a lower frequency of Amide A
in FTIR. This lower frequency is commonly seen for collagen tendon collagen in terms of structural and amino acid content
molecules because of the hydrogen bonding that stabilizes the (save for amount of imino acids and cysteines) and fibril
triple helical structure of collagen. Similar frequencies for morphology. JASC molecules had a melting temperature of ∼5
Amide I, II, and III vibrations were seen in RASC and JASC, degrees lower than that of RASC molecules; however, JASC
consistent with other animal-based and marine collagens. and RASC fibrils showed thermal transition temperatures that
Based on the function of collagen as a biological scaffolding were very close (∼55 and 53 °C), further supporting the idea
protein, one might expect that its physical-chemical properties that these two collagens form structurally similar fibrils.
2122 DOI: 10.1021/acsbiomaterials.7b00979
ACS Biomater. Sci. Eng. 2018, 4, 2115−2125
ACS Biomaterials Science & Engineering Article

Both JASC and RASC formed soft hydrogels (∼10 Pa), as


indicated by their rheological properties. Therefore, to prepare

*
ASSOCIATED CONTENT
S Supporting Information
more rigid biomaterials for potential tissue engineering The Supporting Information is available free of charge on the
applications, agarose was blended with JASC (or RASC) fibrils. ACS Publications website at DOI: 10.1021/acsbiomater-
This is a common strategy for scaffold production in ials.7b00979.
biomaterials research.35 With these scaffolds, we found that
Figures S1−S3: AFM images of fibrillized RASC,
RASC and JASC (both fibrillized at 37 °C) promoted cell
mechanics of RASC and JASC hydrogels, fluorescence
attachment and proliferation when blended with agarose. images of MC3T3 cells and a graph depicting cell
RASC-agarose showed 2.5-fold more cell growth after 6 days. density; and Table S1: amino acid composition (number
As the transition temperature for JASC and RASC fibrils was per 100 amino acids) of JASC and rat tail collagen
essentially the same, we speculate that the amount of fibrils was (PDF)
lower in the JASC-agarose gels (due to reduced fibrillization of
JASC at 37 °C). Reduced amount of JASC fibrils in the hybrid
scaffolds compared to that in the RASC-agarose scaffolds could
alter the cell attachment and growth. It is also possible that the
■ AUTHOR INFORMATION
Corresponding Author
molecular structure of JASC fibrils is different when formed at *E-mail: parekh@mpip-mainz.mpg.de.
room temperature versus 37 °C; however, we have no evidence ORCID
for this at this time. Nevertheless, both RASC and JASC fibrils Sapun H. Parekh: 0000-0001-8522-1854
showed the capacity to functionalize agarose scaffolds for cell Author Contributions
growth and proliferation, consistent to what has been seen for Z.R. and S.H.P. designed the study. Z.R. carried out collagen
other jellyfish species as well.11,15,73 As a final note, we observed extraction and molecular and fibrillar characterization. Y.W.
that when a rigid substrate (polystyrene) was coated with performed the rheology measurements; F.F. performed BCARS
molecular (natively folded, nonfibrillized) JASC or RASC spectroscopy. S.P. and S.K. carried out cell culture and
collagen, as is often done for tissue culture substrates, JASC- fluorescence imaging experiments. Z.R., F.F., T.W., and S.H.P
coated substrates promoted more cell growth than RASC, wrote the article.
independent of the temperature that was used for collagen Notes
coating. This finding is similar to what has been observed by The authors declare no competing financial interest.


coating rigid substrates with molecular jellyfish collagens from
the Mediterranean, where denaturing the collagen had almost ACKNOWLEDGMENTS
no impact on cell attachment.11 Moreover, gelatin (thermally
Z.R acknowledges a Fellowship from National Elites
denatured mammalian collagen) has been shown to function-
Foundation of Iran for her postdoctoral research contract No.
alize surfaces and support cell growth similarly (or even better) PD 20/67/19651. F.F. was supported by a PhD Fellowship
than native collagen.74,75 Apparently, the folded structure of from the Max Planck Graduate Center. S.K. thanks the
native collagen is not a requirement to promote cell attachment Alexander von Humboldt Foundation for financial support.
and growth, as collagen denaturing can result in revealing T.W. thanks the Marie Curie Program of the European Union
cryptic binding sites of αvβ3 integrins, for example, even as for support of this work (CIG grant #322124). S.H.P.
other integrin binding sites become unavailable.75,76 acknowledges funding from the DFG #PA252611-1. The

■ CONCLUSIONS
Acid-soluble collagen was successfully extracted from Catostylus
authors wish to thank Dr. Iraj Nabipour (Bushehr University of
Medical Sciences) for advice and assistance in acquiring the
Catostylus mosaicus samples, Dr. Rüdiger Berger (MPIP) for
mosaicus jellyfish (JASC) and identified as type I collagen. AFM imaging, Dr. Svenja Winzen (MPIP) for DSF analysis, Dr.
Molecular spectroscopy (CD, IR and Raman) of JASC Angelika Kühnle (Johannes Gutenberg Universtät Mainz) for
AFM imaging, Andreas Hanewald (MPIP) for rheology
confirmed retention of the triple helical structure in extracted
measurements, and Dr. Dirk Schneider and Dr. Stephan
and purified collagen molecules and reassembled fibrils. These
Hobe (Johannes Gutenberg Universtät Mainz) for CD
spectroscopies further showed nearly identical signatures
spectroscopy measurements. We also thank Mischa Schwendy
compared to prototypical acid solubilized type 1 collagen for providing commercial collagen samples.


from rat tail tendon (RASC). JASC molecules (but not fibrils)
had a lower melting temperature compared with RASC, which REFERENCES
was expected from the lower imino acid composition. Collagen
(1) Silvipriya, K. S.; Kumar, K. K.; Bhat, A. R.; Kumar, B. D.; John,
fibril reassembly was verified biochemically and with AFM A.; Lakshmanan, P. Collagen: Animal Sources and Biomedical
imaging where JASC and RASC fibers showed similar widths. Application. J. Appl. Pharm. Sci. 2015, 5, 123−127.
JASC, being a type I collagen, was shown to support (2) Birk, D. E.; Bruckner, P. Collagen Suprastructures. In Collagen
preosteoblast attachment and growth on blended JASC-agarose Prim. Struct. Process. Assem.; Brinckmann, J., Notbohm, H., Müller, P.
biomaterials similar to that seen on RASC-agarose scaffolds, K., Eds.; Springer: Berlin, 2005; pp 185−205. DOI:10.1007/b103823.
demonstrating its potential in bone tissue engineering (3) Gelse, K.; Pöschl, E.; Aigner, T. Collagensstructure, function,
applications. This work shows that C. mosaicus collagen is a and biosynthesis. Adv. Drug Delivery Rev. 2003, 55, 1531−1546.
(4) Kadler, K. E.; Holmes, D. F.; Trotter, J. A.; Chapman, J. A.
promising alternative collagen source in fibrillar or nonfibrillar
Collagen fibril formation. Biochem. J. 1996, 316, 1−11.
form for tissue engineering studies or industrial use. The next (5) Ramshaw, J. A. M.; Peng, Y. Y.; Glattauer, V.; Werkmeister, J. A.
step in applying JASC to biomaterials research is long-term Collagens as biomaterials. J. Mater. Sci.: Mater. Med. 2009, 20, 3−8.
osteoblast or bone marrow stromal cell culture on scaffolds to (6) Parmar, P. A.; Chow, L. W.; St-Pierre, J. P.; Horejs, C. M.; Peng,
determine to what extent osteogenesis is supported. Y. Y.; Werkmeister, J. A.; Ramshaw, J. A. M.; Stevens, M. M. Collagen-

2123 DOI: 10.1021/acsbiomaterials.7b00979


ACS Biomater. Sci. Eng. 2018, 4, 2115−2125
ACS Biomaterials Science & Engineering Article

mimetic peptide-modifiable hydrogels for articular cartilage regener- structure of the nonhelical ends in solution. J. Biol. Chem. 1976, 251,
ation. Biomaterials 2015, 54, 213−225. 6062−6067.
(7) Hsieh, Y. H. P.; Rudloe, J. Potential of utilizing jellyfish as food in (29) Komsa-Penkova, R.; Spirova, R.; Bechev, B. Modification of
Western countries. Trends Food Sci. Technol. 1994, 5, 225−229. Lowry’s method for collagen concentration measurement. J. Biochem.
(8) He, J.; Su, Y.; Huang, T.; Jiang, B.; Wu, F.; Gu, Z. Effects of Biophys. Methods 1996, 32, 33−43.
material and surface functional group on collagen self-assembly and (30) Pelc, D.; Marion, S.; Požek, M.; Basletić, M. Role of microscopic
subsequent cell adhesion behaviors. Colloids Surf., B 2014, 116, 303− phase separation in gelation of aqueous gelatin solutions. Soft Matter
308. 2014, 10, 348−356.
(9) Cen, L.; Liu, W.; Cui, L.; Zhang, W.; Cao, Y. Collagen tissue (31) Amino Acid Analysis. See the following: http://www.genaxxon.
engineering: Development of novel biomaterials and applications. com/shop/en/shop-all-products/bioscience-services/amino-acid-
Pediatr. Res. 2008, 63, 492−496. analysis/.
(10) Nagai, T. Characterization of acid-soluble collagen from skins of (32) Billecke, N.; Rago, G.; Bosma, M.; Eijkel, G.; Gemmink, A.;
surf smelt (Hypomesus pretiosus japonicus Brevoort). Food Nutr. Sci. Leproux, P.; Huss, G.; Schrauwen, P.; Hesselink, M. K. C.; Bonn, M.;
2010, 01, 59. Parekh, S. H. Chemical imaging of lipid droplets in muscle tissues
(11) Addad, S.; Exposito, J.-Y.; Faye, C.; Ricard-Blum, S.; Lethias, C. using hyperspectral coherent Raman microscopy. Histochem. Cell Biol.
Isolation, characterization and biological evaluation of jellyfish collagen 2014, 141, 263−273.
for use in biomedical applications. Mar. Drugs 2011, 9, 967−983. (33) Parekh, S. H.; Lee, Y. J.; Aamer, K. A.; Cicerone, M. T. Label-
(12) Nagai, T.; Worawattanamateekul, W.; Suzuki, N.; Nakamura, T.; free cellular imaging by broadband coherent anti-Stokes Raman
Ito, T.; Fujiki, K.; Nakao, M.; Yano, T. Isolation and characterization scattering microscopy. Biophys. J. 2010, 99, 2695−2704.
of collagen from rhizostomous jellyfish (Rhopilema asamushi). Food (34) Liu, Y.; Lee, Y. J.; Cicerone, M. T. Broadband CARS spectral
Chem. 2000, 70, 205−208. phase retrieval using a time-domain Kramers−Kronig transform. Opt.
(13) Subhan, F.; Ikram, M.; Shehzad, A.; Ghafoor, A. Marine Lett. 2009, 34, 1363−1365.
collagen: an emerging player in biomedical applications. J. Food Sci. (35) Ulrich, T. A.; Jain, A. K.; Tanner, J.; MacKay, L.; Kumar, S.
Technol. 2015, 52, 4703−4707. Probing cellular mechanobiology in three-dimensional culture with
(14) Exposito, J. Y.; Valcourt, U.; Cluzel, C.; Lethias, C. The fibrillar collagen−agarose matrices. Biomaterials 2010, 31, 1875−1884.
collagen family. Int. J. Mol. Sci. 2010, 11, 407−426. (36) Chatterjee, K.; Lin-Gibson, S.; Wallace, W. E.; Parekh, S. H.;
(15) Hoyer, B.; Bernhardt, A.; Lode, A.; Heinemann, S.; Sewing, J.; Lee, Y. J.; Cicerone, M. T.; Young, M. F.; Simon, C. G. The effect of
Klinger, M.; Notbohm, H.; Gelinsky, M. Jellyfish collagen scaffolds for 3D hydrogel scaffold modulus on osteoblast differentiation and
cartilage tissue engineering. Acta Biomater. 2014, 10 (2), 883−892.
mineralization revealed by combinatorial screening. Biomaterials 2010,
(16) Barzideh, Z.; Latiff, A. A.; Gan, C.; Benjakul, S.; Karim, A. A.
31, 5051−5062.
Isolation and characterisation of collagen from the ribbon jellyfish
(37) Zhang, J.; Duan, R.; Huang, L.; Song, Y.; Regenstein, J. M.
(Chrysaora sp.). Int. J. Food Sci. Technol. 2014, 49, 1490−1499.
Characterisation of acid-soluble and pepsin-solubilised collagen from
(17) Nagai, T.; Araki, Y.; Suzuki, N. Collagen of the skin of ocellate
jellyfish (Cyanea nozakii Kishinouye). Food Chem. 2014, 150, 22−26.
puffer fish (Takifugu rubripes). Food Chem. 2002, 78, 173−177.
(38) Silva, T. H.; Moreira-Silva, J.; Marques, A. L. P.; Domingues, A.;
(18) Liu, H.; Zhao, L.; Guo, S.; Xia, Y.; Zhou, P. Modification of fish
Bayon, Y.; Reis, R. L. Marine origin collagens and its potential
skin collagen film and absorption property of tannic acid. J. Food Sci.
Technol. 2014, 51, 1102−1109. applications. Mar. Drugs 2014, 12, 5881−5901.
(19) Wang, L.; An, X.; Xin, Z.; Zhao, L.; Hu, Q. Isolation and (39) Mitra, T.; Sailakshmi, G.; Gnanamani, A.; Mandal, A. B. Di-
Characterization of Collagen from the Skin of Deep-Sea Redfish carboxylic acid cross-linking interactions improves thermal stability
(Sebastes mentella). J. Food Sci. 2007, 72, E450−E455. and mechanical strength of reconstituted type I collagen. J. Therm.
(20) Nagai, T. Collagen from diamondback squid (Thysanoteuthis Anal. Calorim. 2011, 105, 325−330.
rhombus) outer skin. Z. Naturforsch., C: J. Biosci. 2004, 59, 271−275. (40) Nishio, T.; Hayashi, R. Regeneration of a collagen-like circular
(21) Ando, M.; Ando, M.; Makino, M.; Tsukamasa, Y.; Makinodan, dichroism spectrum from industrial gelatin. Agric. Biol. Chem. 1985, 49,
Y.; Miyosh, M. Interdependence between heat solubility and 1675−1682.
pyridinoline contents of squid mantle collagen. J. Food Sci. 2001, 66, (41) de Campos Vidal, B.; Mello, M. L. Collagen type I amide I band
265−269. infrared spectroscopy. Micron 2011, 42, 283−289.
(22) Nagai, T.; Nagamori, K.; Yamashita, E.; Suzuki, N. Collagen of (42) Pelton, J. T.; McLean, L. R. Spectroscopic methods for analysis
octopus Callistoctopus arakawai arm. Int. J. Food Sci. Technol. 2002, 37, of protein secondary structure. Anal. Biochem. 2000, 277, 167−176.
285−289. (43) Kittiphattanabawon, P.; Benjakul, S.; Visessanguan, W.;
(23) Lin, Z.; Solomon, K. L.; Zhang, X.; Pavlos, N. J.; Abel, T.; Kishimura, H.; Shahidi, F. Isolation and characterisation of collagen
Willers, C.; Dai, K.; Xu, J.; Zheng, Q.; Zheng, M. In vitro evaluation of from the skin of brownbanded bamboo shark (Chiloscyllium
natural marine sponge collagen as a scaffold for bone tissue punctatum). Food Chem. 2010, 119, 1519−1526.
engineering. Int. J. Biol. Sci. 2011, 7, 968−977. (44) Kittiphattanabawon, P.; Benjakul, S.; Visessanguan, W.; Shahidi,
(24) Nagai, T.; Suzuki, N. Partial characterization of collagen from F. Isolation and characterization of collagen from the cartilages of
purple sea urchin (Anthocidaris crassispina) test. Int. J. Food Sci. brownbanded bamboo shark (Chiloscyllium punctatum) and blacktip
Technol. 2000, 35, 497−501. shark (Carcharhinus limbatus). LWT-Food Sci. Technol. 2010, 43,
(25) Nagai, T.; Ogawa, T.; Nakamura, T.; Ito, T.; Nakagawa, H.; 792−800.
Fujiki, K.; Nakao, M.; Yano, T. Collagen of edible jellyfish exumbrella. (45) Krimm, S.; Bandekar, J. Vibrational spectroscopy and
J. Sci. Food Agric. 1999, 79, 855−858. conformation of peptides, polypeptides, and proteins. Adv. Protein
(26) Dehghan, M. S.; Koochaknejad, E.; Mousavi Dehmourdi, L.; Chem. 1986, 38, 181−364.
Zarshenas, A.; Mayahi, M. Jellyfish of Khuzestan coastal waters and (46) Ahmad, M.; Benjakul, S. 2010. Extraction and characterisation of
their impact on fish larvae populations. Iran. J. Fish. Sci. 2017, 16, 422− pepsin-solubilised collagen from the skin of unicorn leatherjacket
430. (Aluterus monocerous). Food Chem. 2010, 120, 817−824.
(27) Daryanabard, R.; Dawson, M. N. Jellyfish blooms: Crambionella (47) Cao, H.; Xu, S.-Y. Purification and characterization of type II
orsini (Scyphozoa: Rhizostomeae) in the Gulf of Oman, Iran, 2002− collagen from chick sternal cartilage. Food Chem. 2008, 108, 439−445.
2003. J. Mar. Biol. Assoc. U. K. 2008, 88, 477−483. (48) Pati, F.; Adhikari, B.; Dhara, S. Isolation and characterization of
(28) Chandrakasan, G.; Torchia, D. A.; Piez, K. A. Preparation of fish scale collagen of higher thermal stability. Bioresour. Technol. 2010,
intact monomeric collagen from rat tail tendon and skin and the 101, 3737−3742.

2124 DOI: 10.1021/acsbiomaterials.7b00979


ACS Biomater. Sci. Eng. 2018, 4, 2115−2125
ACS Biomaterials Science & Engineering Article

(49) Duan, R.; Zhang, J.; Du, X.; Yao, X.; Konno, K. Properties of (71) Verma, V.; Verma, P.; Kar, S.; Ray, P.; Ray, A. R. Fabrication of
collagen from skin, scale and bone of carp (Cyprinus carpio). Food agar-gelatin hybrid scaffolds using a novel entrapment method for in
Chem. 2009, 112, 702−706. vitro tissue engineering applications. Biotechnol. Bioeng. 2007, 96, 392−
(50) Belbachir, K.; Noreen, R.; Gouspillou, G.; Petibois, C. Collagen 400.
types analysis and differentiation by FTIR spectroscopy. Anal. Bioanal. (72) Izzati, H.; Zainol, I.; Hanim, H. Low Molecular Weight Collagen
Chem. 2009, 395, 829−837. from Tilapia Fish Scales for Potential Cosmetic Application. Der
(51) Muiznieks, L. D.; Keeley, F. W. 2013. Molecular assembly and Pharma Chemica 2017, 9, 108−114.
mechanical properties of the extracellular matrix: a fibrous protein (73) Song, E.; Yeon Kim, S.; Chun, T.; Byun, H. J.; Lee, Y. M.
perspective. Biochim. Biophys. Acta, Mol. Basis Dis. 2013, 1832, 866− Collagen scaffolds derived from a marine source and their
875. biocompatibility. Biomaterials 2006, 27, 2951−2961.
(52) Adams, E. Invertebrate collagens. Science 1978, 202, 591−598. (74) Ratanavaraporn, J.; Damrongsakkul, S.; Sanchavanakit, N.;
(53) Mayne, J.; Robinson, J. J. Comparative analysis of the structure Banaprasert, T.; Kanokpanont, S. Comparison of Gelatin and Collagen
and thermal stability of sea urchin peristome and rat tail tendon Scaffolds for Fibroblast Cell Culture. J. Met., Mater. Miner. 2006, 16
collagen. J. Cell. Biochem. 2002, 84, 567−574. (1), 31−36.
(54) Brown, F. R.; Hopfinger, A. J.; Blout, E. R. The collagen-like (75) Davidenko, N.; Schuster, C. F.; Bax, D. V.; Farndale, R. W.;
triple helix to random-chain transition: Experiment and theory. J. Mol. Hamaia, S.; Best, S. M.; Cameron, R. E. Evaluation of cell binding to
Biol. 1972, 63, 101−115. collagen and gelatin: a study of the effect of 2D and 3D architecture
(55) Kotch, F. W.; Raines, R. T. Self-assembly of synthetic collagen and surface chemistry. J. Mater. Sci.: Mater. Med. 2016, 27 (10), 148.
triple helices. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 3028−3033. (76) David, G. E. Affinity of integrins for damaged extracellular
(56) Muralidharan, N.; Jeya Shakila, R.; Sukumar, D.; Jeyasekaran, G. matrix: αvβ3 binds to denatured collagen type I through RGD sites.
Biochem. Biophys. Res. Commun. 1992, 182 (3), 1025−1031.
Skin, bone and muscle collagen extraction from the trash fish, leather
jacket (Odonus niger) and their characterization. J. Food Sci. Technol.
2013, 50, 1106−1113.
(57) Hu, Z.; Yang, P.; Zhou, C.; Li, S.; Hong, P. Marine Collagen
Peptides from the Skin of Nile Tilapia (Oreochromis niloticus):
Characterization and Wound Healing Evaluation. Mar. Drugs 2017, 15,
102.
(58) Yang, Y.; Kaufman, L. J. Rheology and confocal reflectance
microscopy as probes of mechanical properties and structure during
collagen and collagen/hyaluronan self-assembly. Biophys. J. 2009, 96,
1566−1585.
(59) Keech, M. K. The Formation of Fibrils From Collagen
Solutions. J. Cell Biol. 1961, 9, 193−209.
(60) Wood, G. C.; Keech, M. K. The formation of fibrils from
collagen solutions 1. The effect of experimental conditions: kinetic and
electron-microscope studies. Biochem. J. 1960, 75, 588−598.
(61) Rosenblatt, J.; Devereux, B.; Wallace, D. G. Injectable collagen
as a pH-sensitive hydrogel. Biomaterials 1994, 15, 985−995.
(62) Roeder, B. A.; Kokini, K.; Sturgis, J. E.; Robinson, J. P.; Voytik-
Harbin, S. L. Tensile mechanical properties of three-dimensional type I
collagen extracellular matrices with varied microstructure. J. Biomech.
Eng. 2002, 124, 214−222.
(63) Gobeaux, F.; Mosser, G.; Anglo, A.; Panine, P.; Davidson, P.;
Giraud-Guille, M.-M.; Belamie, E. Fibrillogenesis in dense collagen
solutions: a physicochemical study. J. Mol. Biol. 2008, 376, 1509−1522.
(64) Williams, B. R.; Gelman, R. A.; Poppke, D. C.; Piez, K. A.
Collagen fibril formation. Optimal in vitro conditions and preliminary
kinetic results. J. Biol. Chem. 1978, 253, 6578−6585.
(65) Nguyen, T. T.; Gobinet, C.; Feru, J.; Pasco, S. B.; Manfait, M.;
Piot, O. Characterization of type I and IV collagens by Raman
microspectroscopy: Identification of spectral markers of the dermo-
epidermal junction. Spectroscopy 2012, 27, 421−427.
(66) Frushour, B. G.; Koenig, J. L. Raman scattering of collagen,
gelatin, and elastin. Biopolymers 1975, 14, 379−391.
(67) Raub, C. B.; Suresh, V.; Krasieva, T.; Lyubovitsky, J.; Mih, J. D.;
Putnam, A. J.; Tromberg, B. J.; George, S. C. Noninvasive Assessment
of Collagen Gel Microstructure and Mechanics Using Multiphoton
Microscopy. Biophys. J. 2007, 92 (6), 2212−2222.
(68) Stein, A. M.; Vader, D. A.; Weitz, D. A.; Sander, L. M. The
micromechanics of three-dimensional collagen-I gels. Complixity 2011,
16 (4), 22−28.
(69) Shayegan, M.; Frode, N. R. Microrheological Characterization of
Collagen Systems: From Molecular Solutions to Fibrillar Gels. PLoS
One 2014, 9 (7), e102938.
(70) Hoyer, B.; Bernhardt, A.; Heinemann, S.; Stachel, I.; Meyer, M.;
Gelinsky, M. Biomimetically Mineralized Salmon Collagen Scaffolds
for Application in Bone Tissue Engineering. Biomacromolecules 2012,
13 (4), 1059−1066.

2125 DOI: 10.1021/acsbiomaterials.7b00979


ACS Biomater. Sci. Eng. 2018, 4, 2115−2125

Potrebbero piacerti anche