Type I Collagen from Jellyfish Catostylus mosaicus for Biomaterial

Subscriber access provided by Kaohsiung Medical University

Bio-interactions and Biocompatibility

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 ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00979 • Publication Date (Web): 24 Apr 2018 Downloaded from http://pubs.acs.org on May 1, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Type I collagen from jellyfish Catostylus mosaicus for biomaterial applications

Zahra Rastian1,2 , Sabine Pütz1, YuJen Wang1, Sachin Kumar1, Frederik Fleissner1, Tobias Weidner1,3, Sapun H. Parekh1,* 1

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

Germany 2

The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute,

Bushehr University of Medical Sciences, Bushehr Province, Iran 3

Department of Chemistry, Aarhus University, Langelandsgade 140, 8000 Aarhus C, Denmark

*

Corresponding author: [email protected]

1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60

Page 2 of 40

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, non-mammalian collagen sources for biomaterial applications because of potential medical and economic advantages. In this work, we present physico-chemical and biofunctionality studies of acid solubilized collagen (ASC) from jellyfish Catostylus mosaicus (JASC), harvested from the Persian Gulf, compared to 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 to RASC. Molecular (natively folded, non-fibrillized) JASC was shown to functionalize rigid substrates and promote MC3T3 preosteoblast cell attachment and proliferation better than RASC over six 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 six days compared to 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


Page 3 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


Introduction Collagen is the predominant protein in connective tissues of animals, constituting approximately 25% – 30% of the total protein content (by mass) of the whole body in mammals.

1

At least 27 different types of collagen, named type I to XXVII occur naturally

and are categorized into different groups such as basement, fibril forming, fibril associated, network forming, anchoring fibrils, and transmembrane, of which fibril forming collagens represent about 90% of the total collagen in human body.

2-3

Collagen molecules contain

triple-helix structures made of three α chains. These chains contain the collagenous domain motif composed of Gly-Xaa-Yaa triplets where Xaa and Yaa are often proline and hydroxyproline residues.

4

Collagen molecules form fibrils that have important functions in

the tissue mechanics and tissue repair.

5-6

Thus, interest in collagen has become widespread

among scientists who investigate, e.g. wound healing, fibrosis, neoplasia, and tissue engineering. 6-8 Collagens for tissue engineering applications are used in their native fibrillar or denatured (gelatin) form for various scaffolds such as sponges, sheets, plugs and pellets. 9 The main sources of collagen for industrial uses have been the skin of animals such as pig and calf. However, the risk of transferring diseases such as bovine spongiform encephalopathy (BSE), foot-and-mouth disease, and avian influenza limits the application of land animal collagen for human application.

10

Therefore, for human application, it is

important to choose a collagen source that minimizes transmission of diseases to humans, in addition to economic and environmental considerations. 11 Marine organisms have become increasingly considered as alternative collagen sources in recent years

12-13

as they are free of zoonosis such as BSE, less immunogenic, and elicit

minimal inflammatory response.

1

Genomic, molecular cloning, biochemical, and structural

studies have demonstrated similar characteristics between marine fibrillar collagens and human fibrillar collagen.

11,14-15

Nevertheless, marine collagens show some differences in



Page 4 of 40

physical and chemical properties compared to animal collagens such as lower molecular weight and lower denaturing (melting) temperature.

1,10,16

marine vertebrates and invertebrates including fish,

17-19

urchin;

24

Collagen has been extracted from squid,

20-21

octopus,

22

sponge,

23

however, substantially less research has been done on their biocompatibility and

biomedical usage. Jellyfish is a particularly attractive marine source for collagen for a variety of reasons. 12, 25 Some species of jellyfish have been used as traditional food and medicine in China for more than 1000 years, suggesting good biocompatibility of jellyfish constituent materials.

16

Moreover, in many countries, the increase in jellyfish population has caused

problems in marine ecosystems. 16, 26-27 Using jellyfish as a collagen source could help control the population and reduce the ecological impact from overpopulation while being medically beneficial. In this study, we report the extraction and characterization of acid solubilized collagen from Catostylus mosaicus jellyfish harvested from Bushehr coast in southern Iran and perform a comparative analysis with acid extracted collagen from rat tail tendon – the standard collagen research model. Our analytical tests show that oral arm jellyfish collagen is a type I collagen, maintains a triple helical structure both in the molecular and fibril form, and supports preosteoblast cell growth similarly to rat tail tendon collagen I. Materials and methods Raw materials and chemical reagents Fresh Catostylus mosaicus jellyfish with creamy white color (0.2-2 kg) were obtained from the Persian Gulf in southwest Iran on Bushehr coast (Persian Gulf) and transported to the lab on ice. The jellyfish were first rinsed with chilled tap water and then with chilled distilled water for cleaning. Then, the umbrella and oral arm of 3 animals were cut to pieces separately, treated with 0.1 M NaOH in order to remove the non-collagenous proteins, and


Page 5 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


then washed by distilled water. These specimens were lyophilized and stored at -20 °C until further use. Rat tail collagen was prepared from Sprague-Dawley rats in compliance with the ethical guidelines of the Bushehr University of Medical Sciences. In total eight rat tails from 8 month-old rats were disinfected by submerging in 70% ethanol. The rat skin was removed and discarded, and the tendon was pared for acid extraction of collagen. Commercial type I collagen from rat tail tendon (also acid solubilized) was purchased from Sigma-Aldrich or from Ibidi GmbH as specified. Alpha Modified Eagle Medium (α-MEM) from Lonza and fetal bovine serum (FBS) from Gibco were used for cell culture. All other chemicals and reagents were of analytical grade. Preparation of acid soluble collagens (ASCs) Rat tail tendon collagen was extracted according to Chandrakasan et al.

28

with some

modifications as described below. Tendons were added to 0.5 M acetic acid (1g tendon per 250 ml). The mixture was left for 3 days with gentle stirring at 4 °C. To discard the undissolved part, centrifugation (5810 R, Eppendorf) at 1000 g (at 4 °C) was used for 30 min. Then 10% (w/v) NaCl was added to the supernatant to induce precipitation overnight at 4 °C, after which a 30 min centrifugation at 10000 g (at 4 °C) was applied to collect pelleted collagen, and the supernatant was discarded. The pellet was resuspended in 0.5M acetic acid at 4 °C and then dialyzed using a membrane for MWCO of 14,000 (Roth) against diluted glacial acetic acid (1:1000 v/v) at 4 °C for three days with two buffer changes per day. The dialyzed protein was then lyophilized for long-term storage at -20 °C. This protein is heretofore referred to as rat acid solubilized collagen (RASC). Jellyfish collagen was extracted according to Nagai

20

with slight modification. Jellyfish

pieces were extracted in 0.5 N acetic acid (5 mg/ml) for 3 days with gentle stirring. The extract was filtered through a Nylon filter 0.45 µm (BD Falcon), and the supernatant was



Page 6 of 40

salted out by adding NaCl to final concentration of 0.9 M. The resulting precipitate was collected by centrifugation at 17000 g for 1 h. The 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 of 100,000 for two days with two buffer changes per day, and finally stored at -20 °C for further experiments. This protein is heretofore referred to as jellyfish acid solubilized collagen (JASC). All extraction steps were carried out at 4 °C until storage. Collagen concentrations were determined using the standard protocol of the modified Lowry method.

29

To obtain a

series of calibration curves, a dilution series ranging from 0.005 to 0.5 mg/ml of commercial rat collagen in 0.02 N acetic acid was used. Fibril formation For fibril formation analysis, a protocol based on Hoyer et al.

15

was used. 500 µl collagen

solutions at different concentrations were thoroughly mixed with 500 µl of 50 mM tris(hydroxymethyl)-aminomethl)-aminomethane (Tris) buffer (pH 8) to reach a final pH 7.5-8 and incubated at different temperatures for 4h. The suspensions were centrifuged at 11,000 rpm (Heraeus Biofuge Pico, Thermo Fisher) for 5 min, and collagen content of supernatants was measured using modified Lowry method. Degree of fibril formation (%) was calculated based the Eq.1 fibril formation % = [ 1 −

[] ! [] ] × 100 (1)

where [collagen]sup and [collagen]init

are the supernatant and initial concentrations of

collagen, respectively. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)


Page 7 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


Electrophoresis (SDS-PAGE) was performed using the NuPAGE® Bis-Tris Precast Mini Gel- 8 % system. 25 µL collagen solution containing 5-10 µg collagen in NuPAGE® LDS sample buffer and reducing agent (Invitrogen) was prepared according to the manufacturer instructions and was loaded per well. Commercial rat tail collagen type I was also prepared and loaded as an internal standard collagen. 10 µL of a high molecular weight marker: ranging from 10-225 kDa (Novagen) was loaded as well. Electrophoresis was performed in a mini dual vertical electrophoresis unit (Novex) using NuPAGE® MES SDS running buffer (Invitrogen). The gel was stained using a Silver Quest staining kit (Thermo Fisher). Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy Extracted collagen samples were dialyzed in Milli-Q water and lyophilized for ATR-FTIR analysis. FTIR spectra of collagen samples were obtained using a TENSOR II IR spectrometer (Bruker) equipped with the DTGS detector and single reflection diamond crystal. Infrared spectra were recorded in the range of 600-4000 cm-1 by averaging 50 measurements for each sample. Circular dichroism spectroscopy Circular dichroism (CD) spectroscopy in the far ultraviolet region is sensitive to the secondary structure of proteins in solution, which can be used for identifying the triple helical structure of collagen.

30

CD spectra were obtained experimentally using Jasco J-815 CD

Spectrometer (Easton). Collagen solution was prepared with concentration of 200-300 µg/ml in 0.02 N acetic acid for RASC, JASC, and commercial rat tail collagen as a standard. All samples were placed into quartz cell with a path length of 1 mm where optical activity was measured between 250-185 nm at scan speed of 50 nm/min with an interval of 0.5 nm at 4 °C. The response time and data accumulation were four seconds and four times, respectively.



Page 8 of 40

Protein melting point and amino acid analysis Protein thermal stability was measured in a label-free fluorometric analysis using the Prometheus NT.48 (NanoTemper Technologies GmbH). Briefly, the shift of intrinsic tryptophan fluorescence of proteins upon temperature-induced unfolding was monitored by detecting the emitted fluorescence at 330 and 350 nm. Thermal unfolding was performed for collagen samples in 0.02 N acetic acid (1 mg/ml) in nano differential scanning fluorimetry (nanoDSF) grade high-sensitivity glass capillaries at a heating rate of 1 °C per minute. Protein melting points (Tm) were calculated from the first derivative of the ratio of tryptophan emission intensities at 330 and 350 nm from 20-50 °C. The JASC sample was lyophilized and subjected to amino acid analysis Genaxxon Bioscience. 31 Coherent anti-Stokes Raman scattering spectroscopy Broadband coherent anti-Stokes Raman scattering (BCARS) spectra were acquired for fibrillized collagen samples, (JASC at room temperature and RASC at 37 °C, pH 7.5 and fibrillized overnight) all of which were previously lyophilized. The experimental setup has been described extensively elsewhere. 32 The fibrillized sample was sandwiched between two coverslips with a double-sided tape as a spacer of ~ 80 µm. 21 x 21 hyperspectral images were taken with a step size of 0.5 µm between pixels and with an exposure time of 300 ms per spectrum. To obtain a quantitative Raman-like spectrum, the resonant component of the CARS spectra was extracted by employing a Kramer-Kronig transform on the raw data.

33-34

Spectra shown are averages from 21 x 21 spatial pixels. AFM imaging


Page 9 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


JASC collagen solution was fibrillized (in Tris buffer, pH 7.5-8) overnight at 4 ˚C or at room temperature as indicated. 20 µl suspensions of fibrillized collagen were added to freshly cleaved mica and spin coated for 30 sec at 30 rpm, after which the sample was rinsed with Milli-Q water during spinning for less than 10 seconds to remove salt. Samples were then dried under nitrogen flow. Using this protocol increased the amount of collagen and improved fibril spreading on the mica surface compared to drop-casting the collagen and allowing it to dry at room temperature. AFM measurements were carried out in AC mode in air using a Cypher (Asylum Research) AFM with the ARC2 SPM controller and PPPNCHAuD cantilever with imaging speeds of 1.95 Hz, scanning 256 lines. Imaging fields were usually chosen to be 20 × 20 µm2 or 5 × 5 µm2. Cell culture and on collagen-coated polystyrene Non-treated petri dishes (Greiner) were coated with 10 µg/ mL molecular (non-fibrillized) RASC (at 37 °C) or JASC (at room temperature or 37 °C) overnight. Wells were washed once in PBS, and 1000 cells/cm2 of MC3T3-E1 murine preosteoblast cells (DSMZ, ACC210) were added to each well. Cells were cultured in α-MEM, containing 10% v/v FBS with Penicillin-Streptomycin antibiotics (Gibco), and incubated in 5% CO2 with 95% humidity at 37 °C cells for six days. Control experiments were performed with cells in uncoated wells. Cells were counted from five different wells for each condition at Day 6 using a hemocytometer to determine the cell density. Passage two MC3T3-E1cells were used for all experiments. Cell culture and preparation of collagen-agarose scaffolds Based on Ulrich et al.

35

with some modification, hybrid collagen-agarose scaffolds

containing 1% (w/w) agarose and 3 mg/ml collagen fibrils were prepared from RASC and JASC. To prepare fibrillized collagen, 18 µl HEPES (300 mM) and enough NaOH (1M) for



Page 10 of 40

neutralization were added to 800 µl of collagen stock solution (5 mg/mL) after which αMEM was added to make a final volume of 1 ml (4 mg/ml collagen). RASC solution was incubated at 37 ˚C, and JASC was incubated at room temperature or 37 ˚C; all samples were incubated overnight. A stock solution of 4% w/w of low gelling temperature agarose (Sigma) in phosphate buffer solution (PBS) was prepared and autoclaved. 250 µl of agarose stock solution added to 750 µl collagen fibrils suspension, mixed carefully, and then added to wells in a non-treated (but sterile) 48-well plate (Greiner). These solutions were allowed to gel for 1h at room temperature in the tissue culture hood before cell seeding. 15,000 cells/cm2 of MC3T3-E1 cells were added to each well with media (α-MEM containing 10% v/v FBS with Penicillin-Streptomycin antibiotics). Cells were incubated at 37 ˚C for six days to measure viability and growth, assayed by live / dead staining. Control experiments were done with pure 1% agarose or seeding cells directly on the well plate surface. Passage two MC3T3-E1 cells were used for all experiments. Live and dead staining Each well of the cell culture plate was gently washed by PBS and incubated in PBS containing 2 µM calcein (Invitrogen, Thermo Fisher) and 4 µM ethidium homodimer-1 (Sigma).

36

Stained cells were imaged using an inverted fluorescence microscope (Olympus

IX81, Japan) with a 10X, 0.3 NA (UPlanFl, Olympus) to image both the live and dead fluorescence stain for all samples using a standard eGFP and TRITC filter sets (Chroma). At least three fields of view were imaged for both live and dead channels for each sample, and the same exposure time was used for both channels. No additional contrast enhancement was employed. Mechanical characterization


Page 11 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


The viscoelastic properties of fibrillized RASC, fibrillized JASC, agarose gel (1% w/w), and collagen-agarose scaffolds from RASC and JASC (final concentrations: 3 mg/mL collagen, 1% (w/w) agarose) were measured by rheology. Pure RASC and JASC hydrogels (2.5 mg/mL collagen concentration) were rheologically characterized after overnight fibrillization at 37 °C and 95% relative humidity directly in the measurement cell. Rheology was done on a TA-hybrid DHR-2 rheometer at 37 °C in the parallel plate geometry with 25 mm plates with a gap size between 0.24 and 0.49 mm. A frequency sweep (0.1 – 100 rad/s) was applied to the top plate with strains always less than 1%. The software from the instrument directly calculated the storage modulus (G’) and loss modulus (G”) as a function of frequency. For collagen-agarose scaffold rheology, RASC and JASC were fibrillized overnight at 37 °C at 95% relative humidity followed by mixing with low-melt agarose at room temperature. Mixed solutions were allowed to set for 1 hour directly in the measurement cell. Rheology was measured with an Anton Paar model Physica MCR 301 rheometer in the parallel plate geometry with 25 mm plates at 37 °C with a gap size fixed to 1 mm. A frequency sweep (0.1 – 100 rad/s) was applied to the top plate with strains always less than 1%. Data processing was performed using Rheoplus software to calculate G’ and G” as a 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



Page 12 of 40

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 arms, respectively, 0.453 and 1.94 (mg/g wet tissue) for 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 Cyanea nozakii Kishinouye from the

Yellow sea was 13.0% (dry weight).

37

In light of these studies, we surmise that our finding

of greater collagen content in the oral arm compared to umbrella of C. mosaicus is consistent with previous work, and the amount of ASC extracted collagen is species-dependent. As the oral arms of the jellyfish used here provided the highest yield, JASC from this tissue was used for rest of the studies in this work. Molecular characterization of collagen molecules Collagen type identification and molecular structure Figure 1A shows the SDS-PAGE pattern of RASC and JASC. Rat tail tendon collagen from Ibidi or Sigma (also ASCs) were used as standard type I collagen. Type I collagen contains two α1 chains and one α2 chain as well as β dimers. The electrophoretic patterns of extracted rat tail collagen and jellyfish collagen are qualitatively similar to the commercial sample. Slight shifts in the position of the α chain bands observed for jellyfish collagen could be caused by small differences in the amino acid sequences and molecular weight.

16,38

Circular

dichroism (CD) spectra of extracted collagen samples from rat and jellyfish are shown in


Page 13 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


Figure 1B. The collagen molecule, having a triple helix structure, exhibits a unique CD spectrum with a small positive peak between 220 and 225 nm and a large negative at 197 nm. 39

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.

RASC showed a maximum at 222 nm and minimum at 198 nm; JASC showed a maximum at 220 nm and minimum at 197 nm, and commercial collagen (Ibidi) showed a maximum at 222 nm and minimum at 198 nm. The location of the maximum peak in all collagens samples is characteristic of the collagen triple helix while the minimum centered at 197 nm is characteristic of a random coil. 30,40 Since our extracted RASC and commercial rat tail tendon collagen exhibit almost identical SDS-PAGE patterns and CD spectra, they are collectively referred to as RASC for the remainder of this work. Infrared (IR) spectroscopy is an analytical technique that depicts the vibrational characteristics of chemical functional groups and provides information about protein



secondary structure.

41

Page 14 of 40

Attenuated total internal reflection (ATR) Fourier transform infrared

spectroscopy (FTIR) was used to study the secondary structure and functional groups of JASC and compare it with RASC (Fig. 2). Nine normal modes for amide bands named A, B, and I-VII are allowed for proteins.

42

The main absorption bands in JASC were Amide A,

Amide B, Amide I, Amide II and Amide III, which are typically in the range from 3200-3440 cm-1, 3100 cm-1, 1600-1700 cm-1, 1510-1580 cm-1, and 1200-1300 cm-1,

43-44

respectively.

The Amide A is due to the N-H stretching vibration, and this mode does not depend on the backbone conformation but is very sensitive to the strength of hydrogen bonds (N-H...C=O). 45

The stretching vibrations of N-H group, corresponding to Amide A, occur commonly in the

range of 3280–3300 cm-1

46

however free N-H stretching vibration commonly occurs in the

range of 3400-3440 cm-1. 44 In RASC and JASC, the Amide A vibration peaked at 3296 cm-1

and 3292 cm-1, respectively, indicating that the N-H group is involved in hydrogen bonding, which is known to stabilize the triple helical structure of collagen.

16,47

Collagen from calf

skin and bamboo shark exhibit similar Amide A bands. 43-44,46 Figure 2. ATR-FTIR spectra of JASC and RASC as lyophilized powders.


Page 15 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


The Amide I band is the most intense structure-revealing band in proteins, so this band is a useful marker for the analysis of secondary structure of proteins in FTIR.

47

It is associated

with C=O stretching vibration coupled with the N-H bending vibration along the polypeptide backbone or with hydrogen bonding coupled with COO-, CN stretching and CCN deformation.

43-44,48

Bands near 1630 cm-1 indicate imide residues, and bands at 1660 cm-1

and 1675 cm-1 are assigned to intermolecular cross-links and β-turns, respectively.

47

The

Amide I bands at 1636 cm-1 for RASC and 1641 cm-1 for JASC reflect the presence of imides. Similar frequencies have been seen for calf skin collagen.

16,46

The Amide II band is

associated with the N-H bending vibration coupled with the C-N stretching vibration. 46 This amide was found in RASC and JASC at 1541 cm-1 and 1538 cm-1, respectively, again similar to collagen extracted from calf skin and bamboo shark.

16

Lower frequencies in this region

indicate that the N-H group is involved in bonding with α chain 46 and that hydrogen bonding in collagen is present. 49 The Amide III, which is referred to as the “collagen fingerprint” was found in RASC and JASC at 1235 cm-1 and 1234 cm-1, respectively. This band has been seen at 1235 cm-1 for calf skin collagen and higher frequency for collagen from other marine sources. 16,46 The peaks in the lower fingerprint region at 1060 cm-1 for RASC and 1030 cm-1 for JASC could belong to carbohydrate moieties.

50

Table 1 summarizes the band positions

and their assignments for the FTIR spectra from RASC and JASC. Table 1: FTIR peak locations (in cm-1) and assignments for jellyfish and rat tail collagen

Vibration

JASC loc.

RASC loc. Nominal location 41-42 Assignment

Amide A

3292 cm-1

3296 cm-1

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

Page 16 of 40

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

Amino acid analysis and protein melting point Collagen molecules are characterized by triplets of glycine and two other amino acids (GlyXaa-Yaa), where proline and hydroxyproline are the most common amino acids.

38

The

collagen triple helix is a super-coiled, right-handed structure comprised of three parallel α chains in which glycine is the key for super-coiling of α chains. compositions of freeze dried JASC and rat tail collagen

53

51-52

The amino acid

are presented in Table S1. Amino

acid composition was expressed as residues per 100 residues. JASC was rich in glycine (26.86 residues/100 residues), similar to other collagens. The imino acid content (hydroxyproline and proline) of JASC was 12.43 residues/100 residues, which is slightly more than that of C. nozakii jellyfish (11.9 residues/100 residues), (12.2 residues/100 residues),

25

37

similar to S. meleagris

and less than cod skin collagen (15.4 residues/100 residues)

or carp skin collagen (19.2 residues/100 residues). 49 The melting temperature (the temperature at which the folded and unfolded states are equally populated at equilibrium) of RASC and JASC molecules was measured by nanoDSF of tryptophan fluorescence. This measurement showed that RASC had a melting point of 36.2 ± 0.7 °C, and JASC had a melting point of 31.9 ± 1.1 °C (n = 3 for both). The melting point of JASC was higher than for S. meleagris (26 °C) and C. nozakii (23.8 °C).

25,35

Generally,

imino acid content and degree of proline hydroxylation are related to collagen thermo-


Page 17 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


stability.

35

Moreover, higher content of proline and position of the imino acids play an

important role in stabilization of the collagen triple helix.

54

The imino acid content and

degree of hydroxylation in rat tail collagen is 17.95 residues/100 residues and 38.16%, respectively, more than the imino acid content (12.43 residues/100 residues) and degree of hydroxylation (30.73%) in JASC. Cysteines, which form disulfide bridges between α chains chains of collagen triple helix,

55

were not found in JASC further contributing to its lower

melting temperature, similar to other marine collagens. 13, 56-57 Fibrillization, fibril morphology, fibril molecular structural characterization Type I collagen molecules obtained from collagen fibers in tissues can undergo in vitro reassembly into fibrils. Collagen fibrillization depends on electrostatic and hydrophobic interactions.

15,41,57-60

Electrostatic interactions between amino acid residues are regulated by

factors such as pH and ionic strength. The formation of net charges between collagen molecules of neighboring fibrils could create electrostatic attractions that can stabilize or destabilize fibers.

59-63

Moreover, temperature affects the molecular hydrophobic interactions

and therefore the kinetics of collagen fibrillization, in addition to pH and ionic strength.

64

The degree of fibrillization of RASC and JASC were measured by neutralizing the pH as a function of initial concentration and incubation temperature as described in the Materials and Methods. RASC fibrillization increased with initial concentration of collagen (Fig. 3A); however, the same graph shows that JASC did not show a trend with increasing collagen concentration and showed the same degree of fibrillization at the lowest and highest initial collagen concentrations. Statistical analysis showed that initial concentration of collagen had significant influence (p< 0.05) on RASC fibril formation, but no significant influence (p< 0.05) on JASC (Figure 3A, inset). Figures 3B and C show that raising the temperature resulted in more fibrillization of RASC but had a negative effect on JASC fibrillization (both of which were statistically significant) consistent with the finding that the melting point of



Page 18 of 40

molecular JASC was almost five degrees lower than that of molecular RASC. Similar trends of fibril formation of jellyfish R. esculentum as function of initial concentration and temperature have been seen by Hoyer et al. 15

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 based on initial concentration as stated in the methods. * indicate p < 0.05 by ANOVA with Tukey’s.

The morphology of the reassembled JASC collagen fibrils was assessed by AFM imaging. Fibrils aggregated during reassembly at room temperature and separated fibrils (or fibers) were rarely seen (Fig. 4A-C). Line profiles of separated JASC fibrils showed a width of 2578 nm (Fig. 4D). The morphology of fibrils at 4 °C was also visualized by AFM (Fig. 4E-F), 18 ACS Paragon Plus Environment

Page 19 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


which showed that cold temperature resulted in shorter and broader fibrils (46-50 nm width). Increase in fibril width with lowering temperature has been previously reported for calf skin collagen. 59-60 AFM images RASC fibrils are given in Figure S1. 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.

In order to characterize the molecular structure of fibrillized collagen from both sources in solution, we used BCARS spectroscopy, a coherent analog of spontaneous Raman spectroscopy, to avoid the strong water absorption in FTIR spectroscopy and eliminate photo damage observed in spontaneous Raman. Raman-like spectra of RASC and JASC are shown in Figure 5. The Amide I band is located at 1653 cm-1 and 1659 cm-1 for JASC and RASC, respectively. The slight frequency shift of the Amide I band possibly arises from a different



amino acid composition between the two collagens. samples belong to CH2 bending.

66

65

Page 20 of 40

The bands at 1443 cm-1 for both

The amide III region at 1230-1330 cm-1 shows similar

pattern in both collagen samples. As mentioned above in the FTIR results, the strong Amide III vibration is characteristic of the triple helix structure in collagen. The vibrations at 1246 cm-1 and 1271 cm-1 are assigned to proline-rich and proline-poor regions, respectively.

65

These peaks have been seen at 1248 cm-1 and 1271 cm-1 for bovine Achilles tendon and calf skin gelatin.

66

The vibrational spectroscopy of fibrillized RASC and JASC shows that their

molecular structures are nearly identical. Interestingly, after fibrillizing JASC and RASC, we re-tested for fibril thermal transitions and found that RASC and JASC fibrils had thermal

transitions (presumably thermal denaturation) at ~ 53 °C and ~ 55 °C, respectively, again suggesting their molecular structures – at the fibril level – are identical. We note that the fibrillization temperature (at room temperature or 37 ˚C) for JASC did not affect the thermal transition. Figure 5. Raman-like spectra of in situ RASC and JASC fibrils. For comparison, spectra were normalized on the maximum of the Amide I band.

Cell attachment and viability on collagen-agarose scaffolds


Page 21 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


Bone is a type I collagen-rich organ and collagen-based biomaterials have been extensively studied for bone tissue engineering. As JASC is a type I collagen from a new source, it is necessary to demonstrate its ability to support cell 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 MC3T3-E1 preosteoblasts on (molecular) collagen-coated (non-treated) polystyrene petri dishes. Parallel experiments done with RASC coated wells show that cells not only attach, but also proliferate, on both types of collagencoated wells whereas the cell solution applied to petri dishes hardly wetted the surface on uncoated dishes. Statistical analysis showed that cell proliferation over six days on JASC coated wells surpassed that on RASC for substrates coated with the same solution concentration of natively folded, non-fibrillized collagen molecules (Fig. 6A). We note that coating dishes with JASC at room temperature or 37 °C resulted in statistically identical proliferation. 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) (Fig. S2), which is still very soft compared to, e.g., 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



Page 22 of 40

made collagen-agarose hybrid scaffolds. All scaffolds had 1% (w/w) agarose, and the collagen-agarose hybrid scaffolds contained a final concentration of collagen of 3 mg/mL that was fibrillized (overnight at 37 °C) before blending the two polymers. The mechanical properties of the collagen-agarose scaffolds were measured using parallel plate rheology (Fig. 6B). We found that the storage modulus (G') and loss modulus (G'') as a function of frequency for 1% (w/w) agarose gel were only mildly affected by incorporation of either RASC or JASC over the range of frequencies measured. The incorporation of collagen into agarose resulted in a slightly increased storage modulus compared to agarose alone; however, the loss moduli appeared unchanged by incorporation of collagen into the agarose matrix. Compared to our measured moduli for pure JASC and RASC hydrogels (Fig. S2), these scaffolds were nearly 50-fold stiffer. Figure 6C shows the attachment and viability of MC3T3-E1 cells to collagen-agarose scaffolds. From the images in Figure 6C and Figure S3, it is clear that scaffolds incorporating RASC and JASC fibrils showed much improved cell attachment (at Day 1) compared to agarose alone. Cells did not attach nearly as well to the (non-treated) polystyrene well-plate – similar to what was observed for cells on pure agarose (Fig. S3). Both of these substrates showed almost no cell growth over the six day incubation period (Fig. S3). The number of viable MC3T3-E1 cells on RASC-agarose and JASC-agarose increased by ~ 10- and ~ 4fold, respectively, over the six day incubation period. Cell viability was almost 100% on both JASC and RASC collagen-agarose scaffolds. Taken together, these experiments show that JASC exhibits similar ability as RASC to support cell proliferation and functionalize agarose and rigid substrates.


Page 23 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


Figure 6. (A) MC3T3-E1 proliferation measured on JASC or RASC coated (non-treated) 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) one hour 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 one day or six 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 15000 cells/cm2. * indicates p < 0.05 between the marked samples by ANOVA with Tukey’s.



Page 24 of 40

Discussion Pathological risks of non-human, mammalian collagens for human application underscore the importance of research for alternative collagen sources, such as marine collagen. As reported here and elsewhere, there are clear differences between marine and mammalian collagen such as lower imino acid content, lower melting point, and lower viscosity in marine collagen. 13,72 SDS-PAGE, CD spectra and FTIR were used for molecular characterization of JASC and RASC (as a prototypical type I collagen molecule). JASC's SDS-PAGE showed α1 and α2 chains, as well as β dimers, with qualitatively more α1 chains – characteristic of the composition of type I collagen. The same maxima and minima of CD spectra were seen for our extracted RASC and JASC, as well as commercial rat tail tendon collagen – which is presumed to contain the characteristic triple helical collagen structure. Moreover, JASC and RASC had nearly identical vibrational FTIR and BCARS spectra, which demonstrates similarities in both the molecular collagen and fibrillar collagen secondary structures, respectively. The FTIR (and BCARS) spectra show the characteristic “triple helix” peak in the Amide III, and a lower frequency of Amide A in FTIR. This lower frequency is commonly seen for collagen molecules due to the hydrogen bonding that stabilizes the triple helical structure of collagen. Similar frequencies for Amide I, II, and III vibrations were seen in RASC and JASC, consistent with other animal based and marine collagens. Based on the function of collagen as a biological scaffolding protein, one might expect that its physical-chemical properties depend on the habitat and amino acid composition of the species from which it is extracted. This is indeed true for marine collagen, e.g. with melting temperature. Ribbon jellyfish taken from the warm coast of Penang Island had higher melting temperature (37.38 ˚C)

16

compared to jellyfish from the Chinese Yellow Sea (C. nozakii

Kishinouye, 23.8 ˚C), 37 Tunisian Mediterranean coast (R. pulmo, 28.9 ˚C), 11 Senzaki Bay of Japan (R.asamushi, 28.8 ˚C), 12 or Persian Gulf (C.mosaicus, JASC, 31.9 ˚C) studied here.


Page 25 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


Similar fibril reassembly in vitro was seen for JASC and R. esculentum jellyfish.

15

JASC

showed similar features to rat tail tendon collagen in terms of structural and amino acid content (save for amount of imino acids and cysteines) and fibril morphology. JASC molecules had a melting temperature ~ five degrees lower than that of RASC molecules; however, JASC and RASC fibrils showed thermal transition temperatures that were very close (~ 55 and 53 °C), further supporting the idea that these two collagens form structurally similar fibrils. Both JASC and RASC formed soft hydrogels (~10 Pa), as indicated by their rheological properties. Therefore, to prepare more rigid biomaterials for potential tissue engineering applications, agarose was blended with JASC (or RASC) fibrils. This is a common strategy for scaffold production in biomaterials research. 35 With these scaffolds, we found that RASC and JASC (both fibrillized at 37 °C) promoted cell attachment and proliferation when blended with agarose. RASC-agarose showed 2.5-fold more cell growth after six days. As the transition temperature for JASC and RASC fibrils was essentially the same, we speculate that the amount of fibrils was 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 molecular structure of JASC fibrils is different when formed at room temperature versus 37 °C; however, we have no evidence for this at this time. Nevertheless, both RASC and JASC fibrils showed the capacity to functionalize agarose scaffolds for cell growth and proliferation, consistent to what has been seen for other jellyfish species as well. 11,15,73 As a final note, we observed that when a rigid substrate (polystyrene) was coated with molecular (natively folded, non-fibrillized) JASC or RASC collagen, as is often done for tissue culture substrates, JASC-coated substrates promoted more cell growth than RASC, independent of the temperature that was used for collagen coating. This finding is similar to what has been



Page 26 of 40

observed by coating rigid substrates with molecular jellyfish collagens from the Mediterranean, where denaturing the collagen had almost no impact on cell attachment.

11

Moreover, gelatin (thermally denatured mammalian collagen) has been shown to functionalize surfaces and support cell growth similarly (or even better) than molecular collagen.

74,75

Apparently, the folded structure of molecular collagen is not a requirement to

promote cell attachment and growth, as collagen denaturing can result in revealing cryptic binding sites of, e.g. αvβ3 integrins, even as other integrin binding sites become unavailable. 75,76

Conclusions Acid soluble collagen was successfully extracted from Catostylus mosaicus jellyfish (JASC) and identified as type I collagen. Molecular spectroscopy (CD, IR and Raman) of JASC confirmed retention of the triple helical structure in extracted and purified collagen molecules and reassembled fibrils. These spectroscopies further showed nearly identical signatures compared to prototypical acid solubilized type 1 collagen from rat tail tendon (RASC). JASC molecules (but not fibrils) had a lower melting temperature compared to RASC, which was expected from the lower imino acid composition. Collagen fibril reassembly was verified biochemically and with AFM imaging where JASC and RASC fibers showed similar widths. JASC, being a type I collagen, was shown to support preosteoblast attachment and growth was on blended JASC-agarose biomaterials similar to that seen on RASC-agarose scaffolds, demonstrating its potential in bone tissue engineering applications. This work shows that C. mosaicus collagen is a promising alternative collagen source in fibrillar or non-fibrillar form for tissue engineering studies or industrial use. The next step in applying JASC to biomaterials research is long-term osteoblast or bone marrow stromal cell culture on scaffolds to determine to what extent osteogenesis is supported.


Page 27 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


Associated content Supporting information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. Figure S1 - S3 and Table S1 (PDF). Author contributions Z.R. and S.H.P. designed the study. Z.R. carried out collagen extraction and molecular and fibrillar characterization. Y.W. performed the rheology measurements; F.F. performed BCARS spectroscopy. S.P. and S.K. carried out cell culture and fluorescence imaging experiments. Z.R., F.F., T.W., and S.H.P and wrote the article. Acknowledgements Z.R acknowledges a Fellowship from National Elites Foundation of Iran for her postdoctoral research contract No. PD 20/67/19651. F.F. was supported by a PhD Fellowship from the Max Planck Graduate Center. S.K. thanks the Alexander von Humboldt Foundation for financial support. T.W. thanks the Marie Curie Program of the European Union for support of this work (CIG grant #322124). S.H.P. acknowledges funding from the DFG #PA252611-1. The 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 AFM imaging, Dr. Svenja Winzen (MPIP) for DSF analysis, Dr. Angelika Kühnle (Johannes Gutenberg Universtät Mainz) for AFM imaging, Andreas Hanewald for rheology measurements, and Dr. Dirk Schneider and Dr. Stephan Hobe (Johannes Gutenberg Universtät Mainz) for CD spectroscopy measurements. We also thank Mischa Schwendy for providing commercial collagen samples.



Page 28 of 40

Conflicts of Interest The authors declare no conflict of interest.


Page 29 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


References

(1) Silvipriya, K.S.; Kumar, K.K.; Bhat, A.R.; Kumar, B.D.; John A. Collagen: Animal Sources and Biomedical Application. J.App. Sci. Pharm. 2015, 5, 123-127. DOI:10.7324/JAPS.2015.50322 (2) Birk, D.E.; Bruckner, P. Collagen Suprastructures, in: J. Brinckmann, H. Notbohm, P.K. Müller (Eds.), Collagen Prim. Struct. Process. Assem. Springer Berlin Heidelberg. Berlin, Heidelberg. 2005, 185–205. DOI:10.1007/b103823. (3) Gelse, K.; Pöschl, E.; Aigner, T. Collagens—structure, function, and biosynthesis. Adv. Drug Deliv. Rev. 2003, 55, 1531–1546. DOI:10.1016/j.addr.2003.08.002 (4) Kadler, K.E.; Holmes, D.F.; Trotter, J.A.; Chapman, J.A. Collagen fibril formation. Biochem. J. 1996, 316, 1–11. (5) Ramshaw, J.A.M.; Peng, Y.Y.; Glattauer, V.; Werkmeister, J.A. Collagens as biomaterials. J. Mater. Sci. Mater. Med. 2009, 20, 3–8. DOI:10.1007/s10856-008-3415-4. (6) Parmar, P.A.; Chow, L.W.; St-Pierre, J.P.; Horejs, C.M.; Peng, Y.Y.; Werkmeister, J.A.; Ramshaw, J.A.M.; Stevens, M.M. Collagen-mimetic peptide-modifiable hydrogels for articular

cartilage

regeneration.

Biomaterials.

2015,

54,

213–225.

DOI:10.1016/j.biomaterials.2015.02.079. (7) Hsieh, Y.H.P.; Rudloe, J. Potential of utilizing jellyfish as food in Western countries. Trends Food Sci. Technol. 1994, 5, 225–229. 10.1016/0924-2244(94)90253-4. (8) He, J.; Su, Y.; Huang, T.; Jiang, B.; Wu, F.; Gu, Z. Effects of material and surface functional group on collagen self-assembly and subsequent cell adhesion behaviors. Colloids Surfaces B Biointerfaces. 2014, 116, 303–308. DOI:10.1016/j.colsurfb.2014.01.009.



Page 30 of 40

(9) Cen, L.; Liu, W.; Cui, L.; Zhang, W.; Cao, Y. Collagen tissue engineering: Development of

novel

biomaterials

and

applications.

Pediatr.

Res.

2008.

63,

492–496.1

DOI:0.1203/PDR.0b013e31816c5bc3. (10) Nagai, T. Characterization of acid-soluble collagen from skins of surf smelt (Hypomesus pretiosus japonicus Brevoort). Food Nutr. Sci. 2010, 1, 59. DOI:10.4236/fns.2010.12010 (11) Addad, S., Exposito, J.-Y.; Faye, C.; Ricard-Blum, S.; Lethias, C. Isolation, characterization and biological evaluation of jellyfish collagen for use in biomedical applications. Mar. Drugs. 2011, 9, 967–983. DOI:10.3390/md9060967. (12) Nagai, T.; Worawattanamateekul,W.; Suzuki, N.; Nakamura, T.; Ito, T.; Fujiki, K.; Nakao M.; Yano, T. Isolation and characterization of collagen from rhizostomous jellyfish (Rhopilema

asamushi),

Food

Chem.

2000,

70,

205–208.

DOI:10.1016/S0308-

8146(00)00081-9. (13) Subhan, F.; Ikram, M.; Shehzad, A.; Ghafoor, A. Marine collagen: an emerging player in biomedical applications. J. Food Sci. Technol. 2015, 52, 4703–4707. DOI:10.1007/s13197014-1652-8. Epub 2014 Dec 23. (14) Exposito, J.Y.; Valcourt, U.; Cluzel, C.; Lethias, C. The fibrillar collagen family. Int. J. Mol. Sci. 2010, 11, 407–426. DOI:10.3390/ijms11020407 (15) Hoyer, B.; Bernhardt, A.; Lode, A.; Heinemann, S.; Sewing, J.; Klinger, M.; Notbohm, H.; Gelinsky, M. Jellyfish collagen scaffolds for cartilage tissue engineering. Acta Biomater. 2014, 10, 2, 883–892. DOI:10.1016/j.actbio.2013.10.022. (16) Barzideh, Z.; Latiff, A.A.; Gan, C.; Benjakul, S.; Karim, A.A. Isolation and characterisation of collagen from the ribbon jellyfish (Chrysaora sp.). Int. J. Food Sci. Technol. 2014, 49, 1490–1499. DOI:10.1111/ijfs.12464


Page 31 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


(17) Nagai, T.; Araki, Y.; Suzuki, N. Collagen of the skin of ocellate puffer fish (Takifugu rubripes). Food Chem. 2002, 78, 173–177. DOI:10.1016/S0308-8146(01)00396-X (18) Liu, H.; Zhao, L.; Guo, S.; Xia,Y.; Zhou, P. Modification of fish skin collagen film and absorption property of tannic acid. J. Food Sci. Technol. 2014, 51, 1102–1109. DOI:10.1007/s13197-011-0599-2. (19) Wang, L., An, X.; Xin, Z.; Zhao, L.; Hu, Q. Isolation and Characterization of Collagen from the Skin of Deep‐Sea Redfish (Sebastes mentella). J. Food Sci. 2007, 72, 450-455. DOI:10.1111/j.1750-3841.2007.00478.x (20) Nagai, T. Collagen from diamondback squid (Thysanoteuthis rhombus) outer skin. Zeitschrift Für Naturforsch. C. 2004, 59, 271–275. DOI:10.1515/znc-2004-3-426 (21) Ando, M.; Makino, M.; Tsukamasa,Y.; Makinodan, Y.; Miyosh, M. Interdependence between heat solubility and pyridinoline contents of squid mantle collagen. J. Food Sci. 2001, 66, 265–269. DOI:10.1111/j.1365-2621.2001.tb11329.x (22) Nagai, T.; Nagamori, K.; Yamashita, E.; Suzuki, N. Collagen of octopus Callistoctopus arakawai arm. Int. J. Food Sci. Technol. 2002, 37, 285–289. DOI:10.1046/j.13652621.2002.00568.x (23) Lin, Z.; Solomon, K.L.; Zhang, X.; Pavlos, N.J.; Abel, T.; Willers, C.; Dai, K.; Xu, J.; Zheng, Q.; Zheng, M. In vitro evaluation of natural marine sponge collagen as a scaffold for bone tissue engineering. Int. J. Biol. Sci. 2011, 7. 968-977. DOI:10.7150/ijbs.7.968 (24) Nagai, T.; Suzuki, N. Partial characterization of collagen from purple sea urchin (Anthocidaris crassispina) test. Int. J. Food Sci. Technol. 2000, 35, 497–501. DOI:10.1046/j.1365-2621.2000.00406.x.



Page 32 of 40

(25) Nagai, T.; Ogawa, T.; Nakamura, T.; Ito, T.; Nakagawa, H.; Fujiki, K.; Nakao, M.; Yano, T. Collagen of edible jellyfish exumbrella. J. Sci. Food Agric. 1999, 79, 855–858. (26) Dehghan, M.S.; Koochaknejad, E.; Mousavi Dehmourdi, L.; Zarshenas, A.; Mayahi, M. .Jellyfish of Khuzestan coastal waters and their impact on fish larvae populations. Iran. J. Fish. Sci. 2017, 16, 422–430. (27) Daryanabard, R.; Dawson, M.N. Jellyfish blooms: Crambionella orsini (Scyphozoa: Rhizostomeae) in the Gulf of Oman, Iran, 2002–2003. J. Mar. Biol. Assoc. United Kingdom. 2008, 88, 477–483. DOI:10.1017/S0025315408000945. (28) Chandrakasan, G., Torchia, D.A. Piez, K.A. Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the nonhelical ends in solution. J. Biol. Chem.1976, 251, 6062–6067. (29) Komsa-Penkova, R., R. Spirova, and B. Bechev. Modification of Lowry’s method for collagen concentration measurement. J. Biochem. Biophys. Methods. 1996, 32, 33–43. DOI:10.1016/0165-022X(95)00046-T. (30) Pelc, D., Marion, S.; Požek, M.; Basletić, M. Role of microscopic phase separation in gelation

of

aqueous

gelatin

solutions.

Soft

Matter.

2014,

10,

348–356.

DOI:10.1039/C3SM52542B. (31)

http://www.genaxxon.com/shop/en/shop-all-products/bioscience-services/amino-acid-

analysis/, (32) Billecke, N., Rago, G.; Bosma, M.; Eijkel, G.; Gemmink, A.; Leproux, P.; Huss, G.; Schrauwen, P.; Hesselink, M.K.C.; Bonn, M. Chemical imaging of lipid droplets in muscle


Page 33 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


tissues using hyperspectral coherent Raman microscopy. Histochem. Cell Biol. 2014,141, 263–273. DOI:10.1007/s00418-013-1161-2. Epub 2013 Nov 2. (33) Parekh, S.H.; Lee, Y.J.; Aamer, K.A.; Cicerone, M.T. Label-free cellular imaging by broadband coherent anti-Stokes Raman scattering microscopy. Biophys. J. 2010, 99, 2695– 2704. DOI:10.1016/j.bpj.2010.08.009. (34) Liu, Y., Lee, Y.J.; Cicerone, M.T. Broadband CARS spectral phase retrieval using a time-domain

Kramers–Kronig

transform.

Opt.

Lett.

2009,

34,

1363–1365.

DOI:10.1364/OL.34.001363 (35) Ulrich, T.A.; Jain, A.K.; Tanner, J. MacKay, L.; Kumar, S. Probing cellular mechanobiology in three-dimensional culture with collagen–agarose matrices. Biomaterials. 2010. 31, 1875–1884. DOI:10.1016/j.biomaterials.2009.10.047. (36) Chatterjee, K.; Lin-Gibson, S.; Wallace, W.E.; Parekh, S.H.; Lee, Y.J.; Cicerone, M.T.; Young, M.F.; Simon, C.G. The effect of 3D hydrogel scaffold modulus on osteoblast differentiation and mineralization revealed by combinatorial screening. Biomaterials. 2010, 31, 5051–5062. DOI:10.1016/j.biomaterials.2010.03.024. (37) Zhang, J., Duan, R.; Huang, L.; Song, Y.; Regenstein, J.M. Characterisation of acidsoluble and pepsin-solubilised collagen from jellyfish (Cyanea nozakii Kishinouye). Food Chem. 2014, 150, 22–26. DOI:10.1016/j.foodchem.2013.10.116. (38) Silva, T.H.; Moreira-Silva, J.; Marques, A.L.P.; Domingues, A.; Bayon,Y.; Reis, R.L. Marine origin collagens and its potential applications. Mar. Drugs. 2014, 12, 5881–5901. DOI:10.3390/md12125881.



Page 34 of 40

(39) Mitra, T.; Sailakshmi, G.; Gnanamani, A.; Mandal, and A.B. Di-carboxylic acid crosslinking interactions improves thermal stability and mechanical strength of reconstituted type I collagen. J. Therm. Anal. Calorim. 2011, 325–330. DOI:105, 10.1007/s10973-011-1472-2. (40) Nishio, T.; Hayashi, R. Regeneration of a collagen-like circular dichroism spectrum from

industrial

gelatin.

Agric.

Biol.

Chem.

1985,

49,

1675–1682.

DOI:10.1080/00021369.1985.10866972. (41) Vidal Bde, C., Mello M.L. Collagen type I amide I band infrared spectroscopy. Micron. 2011, 42, 283–289. DOI:10.1016/j.micron.2010.09.010. (42) Pelton, J.T.; McLean, L.R. Spectroscopic methods for analysis of protein secondary structure. Anal. Biochem. 2000, 277, 167–176. DOI:10.1006/abio.1999.4320. (43) Kittiphattanabawon, P., Benjakul, S. Visessanguan, W.; Kishimura, H.; Shahidi, F.; Isolation and characterisation of collagen from the skin of brownbanded bamboo shark (Chiloscyllium

punctatum).

Food

Chem.

2010,

119,

1519–1526.

DOI:10.1016/j.foodchem.2009.09.037. (44) Kittiphattanabawon, P., Benjakul, S., Visessanguan, W.,

Shahidi, F. Isolation and

characterization of collagen from the cartilages of brownbanded bamboo shark (Chiloscyllium punctatum) and blacktip shark (Carcharhinus limbatus). LWT-Food Sci. Technol. 2010, 43, 792–800. DOI:10.1007/s13197-014-1520-6. (45) Krimm, S.; Bandekar, J. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv. Protein Chem. 1986, 38, 181–364. DOI:10.1016/S00653233(08)60528-8


Page 35 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


(46) Ahmad, M.; Benjakul, S.. 2010. Extraction and characterisation of pepsin-solubilised collagen from the skin of unicorn leatherjacket (Aluterus monocerous). Food Chem. 2010, 120, 817–824. DOI:10.1016/j.foodchem.2009.11.019. (47) Cao, H.; Xu, S.-Y. Purification and characterization of type II collagen from chick sternal cartilage. Food Chem. 2008, 108, 439–445. DOI:10.1016/j.foodchem.2007.09.022. (48) Pati, F.; Adhikari, B.; Dhara, S. Isolation and characterization of fish scale collagen of higher

thermal

stability.

Bioresour.

Technol.

2010,

101,

3737–3742.

DOI:10.1016/j.biortech.2009.12.133. (49) Duan, R., Zhang, J.; Du, X.; Yao, X.; Konno, K. Properties of collagen from skin, scale and

bone

of

carp

(Cyprinus

carpio).

Food

Chem.

2009,

112,

702–706.

DOI:10.1016/j.foodchem.2008.06.020 (50) Belbachir, K.; Noreen, R.; Gouspillou, G.; Petibois, C. Collagen types analysis and differentiation by FTIR spectroscopy. Anal. Bioanal. Chem. 2009, 395, 829–837. DOI:10.1007/s00216-009-3019-y. (51) Muiznieks, L.D., Keeley, F.W. 2013. Molecular assembly and mechanical properties of the extracellular matrix: a fibrous protein perspective. Biochim. Biophys. Acta (BBA)Molecular Basis Dis. 2013, 1832, 866–875. DOI:10.1016/j.bbadis.2012.11.022. (52)

Adams,

E.

Invertebrate

collagens.

Science,.

1978,

202,

591–598.

DOI:10.1126/science.212833 (53) Mayne, J.; Robinson, J.J. Comparative analysis of the structure and thermal stability of sea urchin peristome and rat tail tendon collagen. J. Cell. Biochem. 2002, 84, 567–574. DOI:10.1002/jcb.10048



Page 36 of 40

(54) Brown, F.R.; Hopfinger, A.J.; Blout, E.R. The collagen-like triple helix to random-chain transition: Experiment and theory. J. Mol. Biol. 1972, 63, 101–115. DOI:10.1016/00222836(72)90524-4. (55) Kotch, F.W.; Raines, R.T. Self-assembly of synthetic collagen triple helices. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 3028–3033. DOI:10.1073/pnas.0508783103. (56) Muralidharan, N.; Shakila, R.J.; Sukumar, D.; Jeyasekaran, G. 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. DOI:10.1007/s13197-011-0440y. Epub 2011 Aug 6. (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. DOI:10.3390/md15040102 (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. DOI:10.1016/j.bpj.2008.10.063. (59) Keech, M.K. The Formation of Fibrils From Collagen Solutions. J. Biophys. Biochem. Cytol. 1961, 9, 193–209. DOI:10.1083/jcb.9.1.193. (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. DOI:10.1042/bj0750588. (61) Rosenblatt, J., Devereux, B., Wallace, D.G. Injectable collagen as a pH-sensitive hydrogel. Biomaterials. 1994, 15, 985–995. DOI:10.1016/0142-9612(94)90079-5


Page 37 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


(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. DOI:10.1115/1.1449904 (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. DOI:10.1016/j.jmb.2007.12.047. (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. http://www.jbc.org/content/253/18/6578.citation (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. J. Spectrosc. 2012, 27, 421–427. DOI:10.1155/2012/686183. (66) Frushour, B.G.; Koenig, J.L. Raman scattering of collagen, gelatin, and elastin. Biopolymers. 1975, 14, 379–391. DOI:10.1002/bip.1975.360140211. (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. DOI: 10.1529/biophysj.106.097998.



Page 38 of 40

(68) Stein, A.M; Vader, D.A.; Weitz, D.A.; Sander, L.M. The micromechanics of threedimensional collagen-I gels. Complixity. 2010, 16(4), 22-28. DOI: 10.1002/cplx.20332. (69) Shayegan, M.; Frode, N.R. Microrheological Characterization of Collagen Systems: From Molecular Solutions to Fibrillar Gels. PLOS one. 2014, 9(7), e102938. DOI: 10.1371/journal.pone.0102938. (70) Hoyer, B.; Bernhardt, A.; Lode, 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. DOI: 10.1021/bm201776r. (71) Verma, V.; Verma, P.; Kar, S.; Ray, P.; Ray, A.R. Fabrication of agar‐gelatin hybrid scaffolds using a novel entrapment method for in vitro tissue engineering applications. Biotechnol. Bioeng. 2007, 96, 392–400. DOI:10.1002/bit.21111. (72) Izzati, H.; Zainol I.; Hanim, H. Low Molecular Weight Collagen from Tilapia Fish Scales for Potential Cosmetic Application. Der Pharma Chemica. 2017, 9, 108–114. http://www.derpharmachemica.com/archive.html (73) Song, E.; Kim, S.Y.; Chun, T.; Byun, H.J.; Lee, Y.M. Collagen scaffolds derived from a marine source and their biocompatibility. Biomaterials. 2006, 27, 2951–2961. DOI:10.1016/j.biomaterials.2006.01.015. (74) Ratanavaraporn, J.; Damrongsakkul1, S.; Sanchavanakit, N.; Banaprasert, T.; Kanokpanont, S. Comparison of Gelatin and Collagen Scaffolds for Fibroblast Cell Culture, J.M.M.M. 2006, 16(1),31-36. (75) Davidenko,N.; Schuster, C.F.; Bax, D.V.; Farndale, R.W.; Hamaia, S.; Best, S.M.; Cameron, R.E.; Evaluation of cell binding to collagen and gelatin: a study of the effect of 2D and 3D architecture and surface chemistry, J Mater Sci Mater Med. 2016; 27(10): 148.


Page 39 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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 55 56 57 58 59 60


(76) David, G.E.; Affinity of integrins for damaged extracellular matrix: αvβ3 binds to denatured collagen type I through RGD sites, Biochem and Biophys Res Comm. 1992; 182(3): 1025-1031.



TOC graphic 185x100mm (72 x 72 DPI)

ACS Paragon Plus Environment

Page 40 of 40

Type I Collagen from Jellyfish Catostylus mosaicus for Biomaterial

Recommend Documents