Acta Biomaterialia xxx (2013) xxx–xxx

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Jellyfish collagen scaffolds for cartilage tissue engineering
Birgit Hoyer a,⇑, Anne Bernhardt a, Anja Lode a, Sascha Heinemann b, Judith Sewing c,d, Matthias Klinger e,
Holger Notbohm c, Michael Gelinsky a
a

University Hospital and Medical Faculty, Technische Universität Dresden, Centre for Translational Bone, Joint and Soft Tissue Research, Fetscher Str. 74, D-01307 Dresden, Germany
Max Bergmann Center of Biomaterials and Institute for Materials Science, Technische Universität Dresden, Budapester Str. 27, 01069 Dresden, Germany
c
Institute of Virology and Cell Biology, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany
d
CRM Coastal Research & Management GmbH, Tiessenkai 12, 24159 Kiel, Germany
e
Institute of Anatomy, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany
b

a r t i c l e

i n f o

Article history:
Received 27 May 2013
Received in revised form 28 August 2013
Accepted 22 October 2013
Available online xxxx
Keywords:
Marine collagen
Fibril formation
Jellyfish
Mesenchymal stem cells
Chondrogenic differentiation

a b s t r a c t
Porous scaffolds were engineered from refibrillized collagen of the jellyfish Rhopilema esculentum for
potential application in cartilage regeneration. The influence of collagen concentration, salinity and
temperature on fibril formation was evaluated by turbidity measurements and quantification of
fibrillized collagen. The formation of collagen fibrils with a typical banding pattern was confirmed by
atomic force microscopy and transmission electron microscopy analysis. Porous scaffolds from jellyfish
collagen, refibrillized under optimized conditions, were fabricated by freeze-drying and subsequent
chemical cross-linking. Scaffolds possessed an open porosity of 98.2%. The samples were stable under
cyclic compression and displayed an elastic behavior. Cytotoxicity tests with human mesenchymal stem
cells (hMSCs) did not reveal any cytotoxic effects of the material. Chondrogenic markers SOX9, collagen II
and aggrecan were upregulated in direct cultures of hMSCs upon chondrogenic stimulation. The formation of typical extracellular matrix components was further confirmed by quantification of sulfated
glycosaminoglycans.
Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction
Collagen is the prevailing component of extracellular matrices
in connective tissues. Due to its biocompatibility, biodegradability,
low immunogenicity and cell-adhesive properties, collagen is one
of the most frequently utilized materials in the field of tissue
engineering [1]. Collagen can be extracted from a variety of
organisms. Preferential sources of collagen for tissue engineering
applications are bovine skin and tendon as well as porcine skin.
However, collagen of bovine origin involves the risk of infection
with diseases such as bovine spongiform encephalopathy. Furthermore, mammalian collagens, especially of porcine origin, are
increasingly rejected for religious reasons [2]. Marine organisms
are an alternative natural source of collagen and, presumably, are
safer compared to mammals. Recent publications focus mainly
on the isolation and characterization of collagen from different fish
species, such as salmon [3], shark [4] or deep sea redfish [5] and
marine sponges [6]. Another attractive marine source for the
extraction of collagen is jellyfish. The global increase in jellyfish
population causes major problems in the ecological environment,
and their potential use in tissue engineering, next to food industry
⇑ Corresponding author. Tel.: +49 351 458 6694; fax: +49 351 458 7210.
E-mail address: birgit.hoyer@tu-dresden.de (B. Hoyer).

and medicine, may help to reduce their further expansion [7]. With
a collagen content of more than 60% [8], jellyfish has the potential
to become a significant source of collagen in biomedical
applications [9]. Isolation and molecular characterization of jellyfish collagen derived from Stomophulus nomurai has been reported
decades ago [10,11]. More recent investigations are concerned
with collagen from other jellyfish species, e.g. Rhopilema asamushi,
Stomolophus meleagris, Catostylus tagi and Rhizostoma pulmo [7–
9,12]. High collagen recovery rates have consistently been
reported. Amino acid analyses revealed a composition similar to
vertebrate collagen with, however, a lower content of hydroxyproline, which leads to relatively low denaturation temperatures
between 26 and 29.9 °C. Differences in the subunit composition
of collagens from different species are detectable in their respective electrophoretic pattern. Hence, it may be stated that collagens
of different jellyfish species show similarities to different vertebrate collagen types. Some jellyfish collagens are comparable to
vertebrate collagen IV or V [10,11], others seem to resemble vertebrate collagen I [7,12,13] and some show a unique structure with a
fourth a-chain [9]. Hsieh [14] postulates that jellyfish collagen of S.
meleagris is similar to vertebrate collagen type II according to the
molecular mobility, salting-out concentration, high content of
hydroxylysine, solubility properties, absence of disulfide bonds
and highly hygroscopic nature. Similar findings are described by

1742-7061/$ - see front matter Ó 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.actbio.2013.10.022

Please cite this article in press as: Hoyer B et al. Jellyfish collagen scaffolds for cartilage tissue engineering. Acta Biomater (2013), http://dx.doi.org/10.1016/
j.actbio.2013.10.022

Joint surfaces are covered by hyaline cartilage. Cartilage repair is very challenging since all three types of cartilage tissue are nonvascularized and have therefore a poor intrinsic regeneration capacity. Absorbance was measured after 15 min at 590 nm using a microplate reader Infinite M200Pro (Tecan.actbio. Following 60 h of pepsin digestion at 4 °C. both in the shape of either hydrogels [26. It was stored at 20 °C and lyophilized only when required to avoid unwanted cross-linking. followed by collagen XI and IX [22.4.05% acetic acid.10. the ratio of fibrillized to total initial collagen was calculated. http://dx. Tubular porous scaffolds from jellyfish collagen reinforced with poly(lactic-co-glycolic) acid fibers were developed by freeze-drying and electrospinning techniques [19. when seeded with endothelial cells and smooth muscle cells in a perfusion system.05% acetic acid and dialyzed against 0.3. 3. Finally. Germany). pieces of jellyfish were homogenized. Germany). Please cite this article in press as: Hoyer B et al. the solution was centrifuged for cleaning. Materials for scaffolds in cartilage tissue engineering consist mainly of natural or synthetic polymers [26]. Hoyer et al.4. Furthermore we wanted to evaluate the suitability of the scaffolds for potential usage in cartilage tissue engineering. In clinical practice autologous chondrocytes are used for this purpose. the pellet was resuspended in a small amount of supernantant and transferred to 96-well cell culture dishes.1016/ j. [17] generated porous scaffolds by freeze-drying and subsequent chemical cross-linking of acidsolubilized jellyfish collagen.022 . There are. / Acta Biomaterialia xxx (2013) xxx–xxx Bermueller et al. For calibration. Materials and methods 2. and once more in deionized water. Germany).01. as the main component of cartilage extracellular matrix. which has properties of both connective tissues and hyaline cartilage. jellyfish collagen derived from R. Collagen was then precipitated from the supernatant by adding NaH2PO4 and KCl at pH 7 for 12 h. Germany). being similar to other collagen sources. There are three different types of cartilage: hyaline. The main difference to the other two types is elastin and a higher number of cells [21]. such as bone marrow or adipose tissue [25]. silk. 2. esculentum (LiroyBV. Song et al.1. collagen type I and fewer proteoglycans are present [24]. Switzerland). 2. 60. limitations to this procedure due to the induction of morbidity at the donor site and instability in monolayer cultivation [25]. combining jellyfish collagen and hyaluronic acid [18]. 2. One approach to the regeneration of cartilage defects is the implantation of a tissue-engineered scaffold colonized with cells. these constructs were cultivated to test the influence of electrospinning parameters on cell adhesion and proliferation.01 M HCl. The Netherlands) as described before [33]. porous scaffolds were obtained after freezing at a speed of 1 K min1 and subsequent freeze-drying (Alpha 1–2.23]. All cartilage types consist of chondrocytes and an extracellular matrix with collagens. collagen. upon completion of the fibril formation process over 4 h at 4 or 25 °C. which consists of only one type of a-chain and shows a degree of glycosylation similar to that of vertebrate collagen type II [15].2.35]. fibrocartilaginous and elastic. Fibril formation was quantified indirectly by measuring the collagen concentration in the supernantant using the modified Bradford assay.doi. the suspension was centrifuged for 15 min at 10. Scaffold preparation Based on the results of the fibril formation studies we developed the following protocol for scaffold preparation. USA) containing 0. After equilibration in 0.000g. ChondroFillerÒ (Amedrix.28] or porous matrices [27. Mesenchymal stem cells from various tissues. Chondro-GideÒ (Geistlich Pharma AG. NovocartÒ 3D (TETECÒ Tissue Engineering Technologies AG. CartimaixÒ (Matricel GmbH. a graded series of jellyfish collagen stock solution was used. Atomic force microscopy (AFM) Droplets of refibrillized jellyfish collagen suspended in 50 mM Tris buffer. The scaffolds were chemically crosslinked in a 1 wt.27]. The graphs show the mean ± standard deviation (n = 6).g.% solution of N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC. 2 and 1 mg ml1 were thoroughly mixed with 500 ll of 50 mM tris-(hydroxymethyl)-aminomethane (Tris) buffer containing graded sodium chloride concentrations of 20.g. mammalian collagen is already used in the form of membranes. Switzerland). Since collagen II. however. as described previously [34. in fibrocartilage (in knee joint menisci and intervertebral discs). The aim of the present study was to characterize fibril formation parameters of jellyfish collagen to find optimal parameters for the fabrication of stable porous scaffolds from refibrillized jellyfish collagen. 4.29.% ethanol for 2 h. differing in protein types and proportions [21].org/10. salted jellyfish was cut into pieces and extensively rinsed with cold water until salinity was 60.2 B. So far only a few publications have reported on the preparation of jellyfish collagen-based scaffolds for tissue engineering applications. esculentum might be a predominant prospective material for the preparation of scaffolds in cartilage tissue engineering. represent an alternative source of superior availability and are for that reason the object of research in cartilage tissue engineering [26. Analogous to hyaline cartilage. Collagen Collagen was extracted by pepsin digestion from cured jellyfish R. elastic cartilage is mainly composed of collagen type II. for jellyfish collagen of Rhopilema esculentum. CaReS (Arthro Kinetics AG. 80 and 100 mM. Turbidity was measured at 313 nm over 60 min at 4 °C or 25 °C using a UV– Vis spectrophotometer Cary 50 Bio (Varian. e. revealing the degree of fibril formation. Christ. pH 8. Seeded with osteosarcoma cells.2013. Germany) or hydrogels. Briefly. A 5 mg ml1 stock solution of jellyfish collagen was merged 1:1 with 50 mM Tris buffer. Acta Biomater (2013). e. After careful rinsing in deionized water.5 M acetic acid for at least 30 min. 40. In brief. Fibril formation For reassembly analysis. which is predominantly collagen type II (90–95%). or.% glycine solution. in 1 wt. On the contrary.035 mg ml1 SDS (Sigma–Aldrich). has been shown to support the chondrogenic differentiation and maintenance of chondrogenic phenotype to a higher extent compared to collagen I [16]. to generate vascular grafts. In the clinical environment.30–32]. Prior to use. Another type of porous scaffold was established by the same group. at 4 °C. were transferred onto the surface of mica discs. pH 8. Germany) for 24 h. a final freeze-drying step concluded the procedure.30]. 5 ll of the supernantant were mixed with 250 ll Bradford reagent (Sigma–Aldrich. Jellyfish collagen scaffolds for cartilage tissue engineering. This preparation with a resulting pH 7. Natural polymers include agarose. Three-dimensional (3D) sponge-like. Biocompatibility investigations comprised the attachment of human fibroblasts as well as the immune response after implantation of the scaffolds in vivo. alginate or chitosan [28. 2.20].4 was stirred for 12 h at 4 °C. Germany) in 80 vol. Upon centrifugation at 5000g and 4 °C. Rotterdam. After another centrifugation step the collected collagen was redissolved in 0. a stock solution was generated under constant stirring at 4 °C with a concentration of 5 mg ml1 lyophilized collagen dissolved in 0. proteoglycans and water. final pH was adjusted to 7. Fluka. 500 ll collagen stock solution in graded concentrations of 5.

Hoyer et al. Medical Clinic I.2013. Please cite this article in press as: Hoyer B et al.3 mm s1 was used. samples were sealed in 50 ll melting pans of aluminum (BO14-3003 and BO 14-3017. AFM imaging was performed in tapping mode in air using a NanoscopeÒ IIIa Bioscope (Digital Instruments/Veeco. samples were rinsed five times with PBS. each scaffold was seeded with 1. 2. with a heating rate of 2 K min1.actbio. height: 3 mm) were incubated in 1 ml cell culture medium (DMEM containing 10% fetal calf serum. / Acta Biomaterialia xxx (2013) xxx–xxx The collagenous material was allowed to adsorb for 30 min followed by rinsing with deionized water and drying in air.5-diphenyltetrazolium bromide (MTT. 8  103 hMSCs were seeded in 48-well tissue culture plates and cultivated for 1. Quantachrome Instruments. Perkin Elmer.5.2 Hz.1016/ j. The CytoTox 96Ò non-radioactive cytotoxicity assay (Promega. in which several sections of the widest fibrils were measured.8.6. Graphs show mean ± standard deviation (n = 3). Differential scanning calorimetry (DSC) After thoroughly rinsing in deionized water. Height. 5% CO2/95% air incubator. Samples for gene expression analysis (n = 3) were taken after 1 and 21 days of cultivation. Dresden University Hospital Carl Gustav Carus) were isolated from bone marrow aspirate of two healthy male donors (age 30 and 32) after providing written informed consent. Germany) and 35 lM proline (Sigma–Aldrich). software version 9. MTT staining Uniaxial compressive tests were conducted using an Instron 5566 testing machine (Instron GmbH.2 mM 3-(4. cylindrical. An empty melting pan was used as reference sample. 2. The cytoskeleton of the cells was stained using Alexa Fluor 488Ò phalloidin (Invitrogen. a velocity of 0. Jellyfish collagen scaffolds for cartilage tissue engineering. USA) and finally measured in a Pyris 6 DSC.9.5 lg ml1 transferrin. A Philips XL 30/ESEM with field emission gun operated in SEM mode was used.1 software (Zeiss. containing 10% fetal calf serum.11. On completing the respective cultivation period. Upon removal of excess liquid.5% glutaraldehyde in phosphate-buffered saline (PBS). Germany). Samples for biochemical analysis (n = 3) were washed twice with PBS and frozen at 80 °C in 2 ml Nalgene tubes containing six ceramic beads (Peqlab. Both deflection and height images were captured simultaneously at imaging speeds of 1. Cell culture Human mesenchymal stem cells (hMSCs) (kindly provided by Professor Martin Bornhäuser and co-workers. a cell-seeded and a non-seeded scaffold were incubated in culture medium with 1.10. http://dx. Absorbance was read at 492 nm in a microplate reader.10. 2. 100 U ml1 penicillin and 100 lg ml1 streptomycin) at 37 °C. Sigma–Aldrich) at 37 °C for 4 h. Acta Biomater (2013).2 mM ascorbic acid-2-phosphate (Sigma–Aldrich).doi. height: 3 mm) were incubated in cell culture expansion medium for 24 h.3 mm. During lysis. 10 ng ml1 TGF-b3 (Milteny Biotec.5% glutaraldehyde in PBS. samples were lysed with 1% Triton X-100 in PBS for 50 min on ice.13. Gibco. respectively. 3 2. 2. Mechanical measurements 2. For SEM investigations. USA).5dimethylthiazol-2-yl)-2. Cytotoxicity test Cytotoxicity of jellyfish collagen scaffolds was tested by indirect cultivation of cells with scaffold extracts. gamma-sterilized jellyfish collagen scaffolds (diameter: 6 mm. Ten cylindrical samples (diameter 10. Germany). After 24 h the culture medium was replaced with chondrogenic medium containing 100 U ml1 penicillin and 100 lg ml1 streptomycin. samples were washed with PBS and fixed in 2. samples were incubated for 10 min in an ice-cooled ultrasonic bath. 10 lg ml1 insulin. either with scaffoldconditioned medium or normal medium. Cells were expanded in Dulbecco’s modified Eagle’s medium (DMEM) low glucose (Biochrom.1 (Perkin Elmer). Confocal laser scanning microscopy (cLSM) Cell-seeded scaffolds were fixed with 3.12. Germany). Samples were imaged using a Zeiss cLSM 510.7% formaldehyde in PBS. USA) and aluminum reflex coated silicon tips (force constant 40 N m1). samples were washed twice in PBS and frozen at 80 °C for later analysis. 2. 0. Wet samples were compressed to 50% of their initial height. After 21 days of cultivation. The first medium change was 1 day after seeding and later twice a week over 21 days. After thawing.2 lg ml1 ethanolamine (ITS-X mix. Germany). scanning 512 lines. All samples were fixed on carbon pads and sputter-coated with gold. Grids were examined with a Philips TEM 400 at 60 kV. USA) was used for determination of the lactate dehydrogenase (LDH) activity according to the manufacturer’s instructions. Scanning electron microscopy (SEM) Cell-seeded scaffolds were fixed with 2. 6. Prior to cell seeding. and allowed to dry.1 mm s1 and for cyclic tests (50 cycles) a velocity of 0. Fibril diameter was measured semiquantitatively from two pictures with Axio Vision 3. For static tests. 100 U ml1 penicillin and 100 lg ml1 streptomycin (Biochrom) at 37 °C in a humidified. The application of hMSCs for in vitro experiments was approved by the ethics committee of the Medical Faculty of Technische Universität Dresden.2% Triton X-100 in PBS for 5 min. 5. Intracellular conversion of MTT into a dark blue formazan derivative was macroscopically evaluated. negatively stained with 1% phosphotungstic acid in distilled water.7 ng ml1 selenium.org/10.2  106 cells in expansion medium. 2. Germany).3 ± 0.022 . followed by dehydration in graded series of ethanol and critical point drying (BAL-TEC CPD 30. USA) and nuclei with DAPI (Sigma–Aldrich). Plano GmbH. Liechtenstein). washed with PBS and immediately subjected to RNA isolation as described below. 2. LDH activity of the samples was correlated with the number of cells using a calibration line of defined cell numbers. 7 or 14 days. Transmission electron microscopy (TEM) 5 ll graded dilutions of refibrillized jellyfish collagen were applied to formvar-coated copper grids (SF 162-3. Measurements were taken from height images using the Nanoscope software. diameter and weight were measured to calculate porosity. Porosity The real volume of freeze-dried jellyfish collagen sponges was evaluated in a helium pycnometer (Ultrapyc1200e.2 mm) were incubated in modified simulated body fluid (SBF) [36] for 24 h prior to measurements.7.7 ± 0. 107 M dexamethasone (Sigma–Aldrich). 0. Germany). followed by blocking of autofluorescence with 3% bovine serum albumin (Sigma–Aldrich) in PBS. After treatment with 0.B. height 10. Jellyfish collagen scaffolds (diameter: 6 mm.

annealing temperatures and amplicon sizes for each gene are summarized in Table 1. Determination of sulfated glycosaminoglycan (sGAG) and DNA content 1000 ll papain digestion solution (125 lg ml1 papain. 1 ll cDNA in 20 ll reaction mixtures containing specific primer pairs were used for amplification in PCR analysis to detect transcripts of collagen I. A similar tendency was observed with different TRIS buffer concentration (data not shown). 3. 5) revealed also a banding pattern. were observed. 50 ll of the samples were applied for quantification using the sGAG assay (Kamiya Biomedical Company. collagen IIa.actbio. Reverse transcriptase PCR (RT-PCR) RNA was extracted from cell-seeded scaffolds using the peqGOLD Micro Spin Total RNA Kit (Peqlab) according to the manufacturer’s instructions.5–2. 4B and C) exhibited irregular banding patterns of the fibrils with 30–40 nm gaps between overlap regions occurring most frequently. 3A) revealed slightly lower turbidity plateaus at 25 °C. collagen X. 2A and B) in the suspension.5 nm and a length of 4871 ± 683 nm. Primer sequences (Eurofins MWG Operon). Sodium chloride had a significant influence (p < 0. 1C). Absorbance was assessed at 610 nm in a microplate reader.001) on the degree of fibril formation (Fig. Nevertheless.1. Morphology of jellyfish collagen fibrils The morphology of reassembled jellyfish collagen fibrils was visualized by AFM.1 ± 0.14. The difference in the proportion of reassembled collagen within each parameter was statistically evaluated with two-way ANOVA and post hoc Tukey test via Origin 8. Thick representatives of jellyfish collagen fibrils had a width of 53.1. Results 3. As shown in Fig.10.2.org/10. Influence of NaCl concentration With an increasing amount of sodium chloride from 0 to 100 mM (Fig. RUNX2 and b-actin. large fibrils exhibited a width of 49 ± 6 nm. Higher magnification (Fig. Investigations of varying sodium chloride concentration did not show such a difference between the two tested temperatures. Increase of the salinity over 20 mM reduced the amount of jellyfish collagen fibrils. however. here a period of 67 nm was observed. Influence of temperature Comparing the collagen concentrations of 2. 100 ng of total RNA were reverse transcribed into cDNA in a 20 ll reaction mixture containing 200 U of Superscript II Reverse Transcriptase (Invitrogen. Influence of collagen concentration.1016/ j. Subsequently. The samples were homogenized (2  10 s at 5900 rpm) using a PrecellysÒ 24 apparatus (Peqlab) and were then incubated at 60 °C for 24 h. http://dx. Hoyer et al.1. The degree of fibril formation was not significantly influenced by the collagen concentration. Eppendorf. Acta Biomater (2013). Germany) and the resulting PCR products were visualized using the FlashGel™ Dock system (Cambrex Bio Science. 0. Table 1 Primers for RT-PCR. The small fibrils measured 32 ± 5 nm in width.6 nm. 3. a clear difference in the turbidity as a function of the collagen concentration could be observed in the plateau phases. 3. branched fibrils. The intensity of fluorescence was measured at excitation/emission wavelengths of 485/535 nm. Another aliquot of the cell lysate was mixed with the Quant-iT™ PicoGreenÒ dsDNA reagent (Molecular Probes. On the other hand. varying in thickness and length.3 nm in height and 469 ± 103 nm in length. 5 mM cystein in deionized water) was added to the frozen cell-seeded scaffolds. respectively. 1 mM EDTA) and incubated for 5 min in the dark. Germany) and 40 U of RNase inhibitor RNase OUT (Invitrogen).1. 1.5 ± 0. the turbidity plateau decreased. At 25 °C the amount of reassembled collagen was significantly (p < 0.3. Relative fluorescence units were correlated to the amount of DNA using a calf thymus DNA calibration line. USA) according to the manufacturer’s instructions. 12.6. Marker Collagen I Collagen IIa Collagen X Aggrecan SOX9 b-Actin RUNX2 Primer sequences 0 Tannealing (°C) 0 For:5 -CCAGAAGAACTGGTACATCA-3 rev: 50 -CCGCCATACTCGAACTGGAA-30 For: 50 -GAACATCACCTACCACTGCAAG-30 rev: 50 -GCAGAGTCCTAGAGTGACTGAG-30 For: 50 -GCCCACTACCCAACACCAAGAC-30 rev: 50 -CCTGGCAACCCTGGCTCTC-30 For: 50 -ACCACCGAGCCAGAAAACCAGAC-30 rev: 50 -CCTCTGAGGGGAACAGCTCCAC-30 For: 50 -GTGCTCAAAGGCTACGACTG-30 rev: 50 -CGTTCTTCACCGACTTCCTC-30 For: 50 -GGACTTCGAGCAAGAGATGG-30 rev: 50 -AGCACTGTGTTGGCGTACAG-30 For: 50 -GGTAACGATGAAAATTATTCTGCTG-30 rev: 50 -CCGAGGTCCATCTACTGTAAC-30 Amplicon size (bp) 60 96 60 488 50 196 64 268 62 316 55 234 55 201 Please cite this article in press as: Hoyer B et al.15. SOX9. 5 mM ethylenediaminetetraacetic acid (EDTA). Germany). 2. / Acta Biomaterialia xxx (2013) xxx–xxx 2.5 mM dNTPs (Invitrogen). During the RNA isolation procedure. 2. Statistics Mean and standard deviation are shown in the figures. 100 mM Na2HPO4. Graphs of sGAG concentration in relation to DNA content show mean ± standard deviation (n = 6).5 ng ll1 random hexamers (Eurofins MWG Operon.4 ± 8.5 mg ml1 at 4 and at 25 °C (Fig. Influence of final collagen concentration The influence of the final collagen concentration on fibril formation was investigated in the range of 0.5 mg ml1 by monitoring the change of turbidity at 313 nm (Fig 1A and B) as well as by indirect evaluation of the degree of fibril formation via modified Bradford assay (Fig. aggrecan. USA). 3. Turbidity started to rise shortly after mixing the solutions and was too fast to be monitored in the spectrometer.022 .doi. 2C).16.1.05) diminished (Fig.2013. Jellyfish collagen scaffolds for cartilage tissue engineering. The PCR experiments were carried out in a Thermocycler (Vapo-Protect Mastercycler Pro. a height of 3. diluted 1:800 in TE buffer (10 mM TRIS. respectively. 4A. USA). 3B).4 B.2. ionic strength and temperature on fibril formation 3. cell lysates of three samples cultured under identical conditions were pooled. TEM analysis of fibrils from jellyfish collagen (Fig.

Human mesenchymal stem cells were seeded on porous jellyfish collagen scaffolds and cultivated under chondrogenic stimulation for 21 days.doi. 11A).B.4. 6A) by means of lyophilization. 3. The compressive stress at 20% compression was 1. with scaffolds incubated in SBF. The staining showed viable cells throughout the whole scaffold. 3. An increase of 12 K of the peak temperature was achieved by chemical cross-linking of jellyfish collagen with 1% EDC. 6B) revealed an open. Influence of the NaCl concentration on fibril reassembly of jellyfish collagen: turbidity measurement at 4 °C (A) and 25 °C (B). / Acta Biomaterialia xxx (2013) xxx–xxx 5 Fig.3. Viability and chondrogenic differentiation of hMSC in scaffolds from jellyfish collagen Initially.4% was assessed by measuring the pore volume with a helium pycnometer. Cell distribution was evaluated macroscopically by MTT staining (Fig. Hoyer et al. collagen IIa.1016/ j.2013. In static tests (Fig. 9). Fig. Only viable cells metabolize the yellowish MTT into a dark blue formazan derivative. and degree of fibril formation measured by Bradford assay (C).org/10. and degree of fibril formation measured by Bradford assay (C). Acta Biomater (2013).10. http://dx. DSC (Fig.93 kPa for 3-D jellyfish collagen scaffolds was measured. A porosity of 98.98 ± 0.20 kPa. The housekeeping gene b-actin and collagen I were expressed at similar levels on Please cite this article in press as: Hoyer B et al. 10).5 mg ml1: turbidity measurement (A) and degree of fibril formation measured by Bradford assay (B). Gene expression of b-actin. 3. the cytotoxicity of jellyfish collagen was tested. Cyclic tests (Fig. a compressive modulus of 9. 8A). Influence of the reaction temperature on the fibril reassembly of jellyfish collagen exemplary for a final collagen concentration of 2. aggrecan. 8B) revealed a highly elastic behavior of the jellyfish collagen scaffolds. Fig. Influence of the final collagen concentration on fibril reassembly of jellyfish collagen: turbidity measurement at 4 °C (A) and 25 °C (B). SEM analysis (Fig. Jellyfish collagen scaffolds for cartilage tissue engineering. Characterization of porous jellyfish collagen scaffolds Porous 3-D jellyfish collagen scaffolds were engineered in different sizes (Fig. 7) was used to determine the effect of chemical crosslinking on the denaturation temperature of jellyfish collagen. There was no significant difference in the number of cells in both examined media. showing that jellyfish collagen did not release cytotoxic substances.022 .79 ± 0. collagen I. Cells on polystyrene were cultivated with either scaffold extract medium or fresh medium (Fig. 1. interconnected pore structure. collagen X.2 ± 0. 2. RUNX2 and SOX9 was investigated (Fig. Mechanical properties were evaluated under wet conditions.actbio.

Fig. Genes coding for collagen II.001) from day 1 to day 21 (Fig. showing a significant increase (p < 0. Discussion Fig. In this study.10. cells of a more spherical shape (indicated by arrows) were discovered which resemble the chondrogenic phenotype. Bar shows 200 nm. collagen X. AFM images (left: height image.doi. Porous Please cite this article in press as: Hoyer B et al. RUNX2 and SOX9 were upregulated. right: amplitude image) of fibrillized jellyfish collagen. Although fibril formation of collagen has been extensively examined. Acta Biomater (2013). similar to fibroblast phenotype.actbio.022 . 5. 12D shows the opposing seeding side of the jellyfish collagen scaffold. According to SEM and cLSM images after 21 days of cultivation (Fig. a large number of cells were found on the seeding side of the scaffolds. The development of suitable matrices for hosting chondrogenically stimulated cells is a prerequisite for this purpose.1016/ j. it is still not common to use fibrillized collagen for scaffold fabrication. / Acta Biomaterialia xxx (2013) xxx–xxx Fig. Furthermore. sGAG concentration was biochemically quantified. we describe the manufacture of fibrillized jellyfish collagen to be used as a biomimetic matrix for cartilage tissue engineering.org/10. Collagen is a well-investigated biomaterial that has been studied since the beginning of the last century. 12A–C). TEM image of stained jellyfish collagen fibrils. Hoyer et al. One strategy to meet this challenge is tissue engineering. 4. Insets in A and B represent the height scale. day 1 and day 21. arranged in small clusters. 11B).2013. aggrecan. Panel C is a magnification of the marked section in B and was used for the section analysis. which spread over the pores.6 B. Yet in between. Here only spherical cells are found. Jellyfish collagen scaffolds for cartilage tissue engineering. http://dx. 4. Sufficient regeneration of cartilage defects is still one of the major challenges in orthopedics.

Number of viable cells was calculated from LDH activity after total lysis. 6.2–2. 7.42–44].doi. Compressive stress–strain curve of 3-D jellyfish collagen scaffolds: static compression (A) and cyclic compression (B) of wet samples. respectively. Viability of human mesenchymal stem cells cultured with scaffold extract (scaffolds) or fresh medium (control).022 . 1) in the range of 0.2–0. Jellyfish collagen scaffolds for cartilage tissue engineering. four times higher than the highest concentration investigated in this study.05) at 4 °C compared to 25 °C. Scaffolds made of fibrillized marine collagens derived from salmon skin have been reported only by a Japanese research group [45] and recently by us [34]. extent of fibril formation (Fig. This was a surprising result since sea water contains 455 mM sodium chloride [49]. 9. / Acta Biomaterialia xxx (2013) xxx–xxx 7 Fig. Fibril formation of jellyfish collagen was further influenced by ionic strength (Fig. DSC measurement of jellyfish collagen without and with 1% EDC crosslinking. both temperatures being below the denaturation temperature of jellyfish collagen. we conducted the fibril formation studies at 4 and 25 °C. collagen-based scaffolds are more frequently prepared by freezedrying solubilized collagen monomers.1016/ j.10. Hoyer et al.41]. A challenge in the handling of marine collagens is their lower denaturation temperature compared to collagens from mammalian sources. Changes in the collagen concentration (Fig. In contrast to structures of freeze-dried monomeric collagen.org/10. Consequently. 3).5 mg ml1 did not significantly influence the percentage of fibrillized collagen. With increasing sodium chloride concentration (0–100 mM) the absorbance values of the turbidity plateaus decreased.38]. For collagen derived from R.5–2. http://dx. structures of fibrillized collagen mimic the natural extracellular matrix and can provide additional mechanical strength [46]. hydrogels [28. In the field of bone tissue engineering. Wood and Keech [47] reported a similar temperature effect on the fibril formation of collagen from calf skin caused by different fibril widths of 500 and 1000 nm. and consequently the amount of fibrillized collagen was reduced. some studies focus on mineralization of fibrillized collagen to produce a biomaterial which mimics the mineralized bone matrix [35. films [39] or as coatings [40. 2).B. esculentum we determined a denaturation temperature of 32 °C by means of circular dichroism (data not shown).5 mg ml1 [48]. 8. Increasing turbidity values of the plateau phase were also documented for salmon collagen type I in the range of 0. which was significantly increased (p < 0. (A) Scaffolds of different sizes and (B) SEM image of a 3-D jellyfish collagen scaffold. Fig.actbio. There are a few reports on fibrillized collagen matrices in the form of lyophilized sponges [37]. Please cite this article in press as: Hoyer B et al.2013. Acta Biomater (2013). The reaction temperature had a considerable influence on the Fig. respectively. while the absorbance values of the turbidity plateaus increased with increasing collagen concentration.5 mg ml1 [34] and bovine collagen type II in the range of 0. Based on those findings a Fig.

which is also synthesized in hypertrophic chondrocytes and is therefore associated with hypertrophic induction [57]. [59]. owing to the high porosity of 98% and the use of a natural protein. 11. 11A). 8) of jellyfish collagen scaffolds was increased 13-fold compared to salmon collagen scaffolds [34]. jellyfish collagen is a fibrilforming collagen with a distinct banding pattern. Bars show 50 lm. 10) and upregulation of chondrogenic markers on mRNA level (Fig. Additionally. Bars show 3 mm. we found collagen type X to be upregulated. stained jellyfish collagen fibrils showed a periodic banding of 67 nm. leading to fibrils with a diameter of 10–30 nm [11].4. Direct cultivation of hMSCs under chondrogenic stimulation revealed viable cells (Fig. Hoyer et al.actbio. Both research groups reported in accordance a substantial increase of collagen type II and aggrecan mRNA after 3 weeks of cultivation. Similar non-cytotoxic effects on human fibroblasts and smooth muscle cells incubated with extracts of collagen of Stomolophus nomurai meleagris were obtained by Song et al. RT-PCR expression analysis of different genes (A) and biochemical quantification of sulfated glycosaminoglycan concentration (B). SEM images (A. 4) and TEM (Fig. protocol was established for fibril formation of jellyfish collagen at a temperature of 4 °C: 5 mg ml1 collagen dissolved in hydrochloric acid was mixed 1:1 with 50 mM Tris buffer at pH 8. This was already reported for jellyfish collagen with a glycosylation grade of 23–25 residues/1000 amino acids. Cytotoxicity of jellyfish collagen (Fig. which could explain the slightly thicker fibrils with diameters between 30 and 60 nm. To sum up. compared to other materials. [58] detected an increase of collagen type X mRNA earlier than collagen II mRNA in chondrogenically stimulated pellet cultures of hMSCs.org/10. However. The issue of the denaturation temperature being below 37 °C was solved by cross-linking with a carbodiimid derivative verified with DSC measurements (Fig. yielding a solution with the resultant pH of 7.022 . C. which is typical for collagens of type I and II [53. limited mechanical properties. 7). banding of jellyfish collagen fibrils was less distinct. 5) images showing the typical banding of collagen fibrils. who studied the chondrogenic differentiation of rabbit MSCs in porous scaffolds from bovine collagen type II. MTT staining of viable human mesenchymal stem cells cultured with chondrogenic supplements after 21 days of cultivation. and cross-section of a cell-seeded scaffold (B).doi. which are in the range of native mammalian cartilage collagen type II fibrils (5–120 nm) [51]. Similar results were reported by Jakobsen et al. http://dx. and by Chen and co-workers [56]. / Acta Biomaterialia xxx (2013) xxx–xxx Fig. No significant difference in the proliferation rate of hMSCs cultivated with scaffold extracts or fresh medium was observed.10. some researchers express doubts as to whether an increased collagen type X transcript level in chondrogenically stimulated MSC cultures is really associated with hypertrophy. Compressive modulus (Fig. esculentum showed a lower glycosylation grade of 9 residues/1000 amino acids.1016/ j. [52] demonstrated a banding pattern of 48. resulting in 98% porosity comparable to bovine collagen scaffolds produced by Dawson et al. 12. However. 6) made of fibrillized jellyfish collagen were prepared by a freeze-drying technique.6 nm in porcine cartilage collagen. (A–C) Cell-seeding side and (D) opposite side of scaffold. [28]. indicating that fibril association was slightly disordered. This is a well-known phenomenon of collagen fibrils with diameters below 30 nm [51]. For example. Jellyfish collagen scaffolds for cartilage tissue engineering. [55]. collagen scaffolds generally have. Fibril formation was verified with AFM (Fig. The D-periodic banding pattern of 67 nm could not be clearly identified in AFM for jellyfish collagen. Tsai et al. Nevertheless. Other studies have demonstrated chondrogenic differentiation of MSCs in the 3-D environment of scaffolds. 3-D porous scaffolds (Fig. Jellyfish collagen scaffolds without (left) and with (right) cells (A). who cultivated bovine MSCs in collagen type I and II gels.54]. Mwale et al. 9) was indirectly assessed by cultivation of cells with scaffold extracts. indicating the absence of cytotoxic effects of the scaffold extracts. Please cite this article in press as: Hoyer B et al. however. [17]. who observed a simultaneous upregulation of the chondrogenic markers SOX9 and aggrecan on the one hand and collagen type X on the Fig. Acta Biomater (2013).2013. 10. suggesting that collagen type X is no reliable hypertrophy marker in stem cell differentiation. D) and cLSM image (B) of chondrogenically stimulated hMSCs cultivated on jellyfish collagen scaffolds for 21 days.8 B. Collagen from R. Collagen-based matrices were applied in particular by Bosnakovski et al. It is well known that the glycosylation grade is inversely related to the fibril diameter [50]. Analysis of unstimulated hMSCs on jellyfish collagen scaffolds after 1 day of cultivation and chondrogenically stimulated hMSCs on jellyfish collagen scaffolds after 21 days of cultivation. In TEM. Fig.

00478. Worch H. In our experiments a tendency to hypertrophic development is furthermore indicated by the upregulation of RUNX2. Isolation and characterization of collagen from rhizostomous jellyfish (Rhophilema asamushi).org/10. Please cite this article in press as: Hoyer B et al.02. [12] Calejo MT.1750-3841.org/10. http://dx. We successfully engineered a cytocompatible matrix with the potential to support and maintain chondrogenic stimulation of human mesenchymal stem cells. Arthritis Res 2002. http://dx.20:2073–87. [20] Park JS. J Biomed Mater Res 1997. Minas T. Worawattanamateekul W. Ogawa T.1016/j.org/10.doi.actbio. 01GN0962) and Prof.007.1111/j. Tissue Eng 2003.171:1–15.2012. [19] Jeong SI.70:205–8. In vitro chondrogenesis of bone marrow-derived mesenchymal stem cells in a photopolymerizing hydrogel.63].2011. Giménez B.doi.015.003.0. [21] Wachsmuth L. Kim H. Lai JH.2006.21:431–40. as confirmed by immunofluorescent staining and gene expression analysis [61].tea. An X. 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