351
Tomonori Takagaki et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 351-358.

e- ISSN 0976 - 3651
Print ISSN 2229 - 7480

International Journal of Biological&Pharmaceutical Research
Journal homepage: www.ijbpr.com

IJBPR

INTERACTIONS BETWEEN JELLYFISH MUCIN AND
HYALURONAN IN HUMAN CHONDROCYTESTomonori Takagaki1, Masato Sato1, Tomoko Kawake1, Takayuki Baba2, Koji Kihira2,
Joji Mochida112Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, Japan.
Jellyfish Research Laboratories, Inc. KSP E501 Sakado 3-2-1, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan.

ABSTRACT
We previously investigated the effects of jellyfish mucin and hyaluronan on degeneration in the knee joints of whiterabbits. Here, the effects of jellyfish mucin and hyaluronan were examined in cultured human chondrocytes. We used a freezedried solid extract of jellyfish mucin from jellyfish mucin (from Nomura’s jellyfish, Nemopilema nomurai) that had been
purified using ion exchange chromatography. Knee articular chondrocytes obtained from operations were isolated
enzymatically and cultured with interleukin-1-alpha added when the cells were subconfluent. The cells were transferred to
culture medium with or without hyaluronan or jellyfish mucin. The cells were collected at various times, and the gene
expression levels of collagen types I and II, SOX9, matrix metalloproteinase-3, and aggrecan were evaluated using real-timepolymerase chain reaction (PCR). The concentrations of collagen type II, MMP-3, and transforming growth factorβ(TGFβ)beta secreted into the medium were measured using enzyme-linked immunosorbent assays (ELISAs). Real-time PCR
showed that the gene expression levels of collagen type II, SOX9, and aggrecan increased in cells exposed to both jellyfish
mucin and hyaluronan. Collagen type I gene expression was reduced by hyaluronan but was not affected by jellyfish mucin.
MMP-3 gene expression in the absence of hyaluronan was reduced by jellyfish mucin in a dose-dependent manner. ELISAs
showed that the concentration of transforming growth factor  with or without hyaluronan treatment was increased by jellyfish
mucin in a dose-dependent manner, but there was no effect of mucin or hyaluronan on the concentrations of collagen type II or
MMP-3. These results suggest that jellyfish mucin and hyaluronan may have beneficial effects on the physiology of cultured
human chondrocytes and might contribute to cartilage repair and regeneration.
Key Words: jellyfish mucin, hyaluronan, chondrocyte, cultured cells.
INTRODUCTION
Osteoarthritis (OA) is one of the most common of
the arthropathies and is characterized by cartilage
degeneration that progresses slowly over a long time. OA
appears most commonly in the load-bearing joints of thelower limbs, mainly the knees. There is a strong correlation
between the onset of OA and patient age, and it is a main
Corresponding Author
Masato Sato
Email: sato-m@is.icc.u-tokai.ac.jp

cause of pain and joint function disorder in the elderly.
(Felson, 1988) Moreover, young people often develop OA
after an injury such as a joint fracture or an injury to theknee cartilage, anterior cruciate ligament, or meniscus.
(Allen et al., 1984; Jacobsen, 1977)
Current conservative treatment of OA focuses
primarily on pain relief with systemic painkiller treatment
and local intra-articular treatments. Nonsteroidal antiinflammatory drugs (NSAIDs) are used widely as systemic
painkillers. (Hochberg et al., 2012) However, some studies
have reported that the use of NSAIDs might promote

352
Tomonori Takagaki et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 351-358.

further pathological conditions. (Huskission et al., 1995;
Rashad et al., 1989; Dougados et al., 1996; Reijman et al.,2005) A potential positive effect of the intra-articular
injection of hyaluronan—a widely used local treatment—isthat it increases the viscosity of the synovial fluid andinhibits destruction of the articular cartilage. (Petrella etal., 2002; Dougados et al., 1993; Salk et al., 2006;
Kobayashi et al., 1994)
A thin membrane on the surface of articularcartilage is involved in protecting and reducing friction on
the surface. The membrane comprises glycoproteins with amucin-type structure similar to tribonectin and lubricin.
(Rhee et al., 2005; Jay et al., 2007) Human glycoproteinsin joint have tandem repeat regions of 7–8 amino acids inthe mucin domain, and more than 90% of the mucin-typesugar chain with binding affinity to amino acids is subject
to glycosylation. The sugar chain is short (glycoform) withas few as 2–3 glucose molecules and includes sialic acidresidues. The characteristics are very similar to those ofjellyfish mucin, which was used in this study. (Masuda etal., 2007)
Figure 1 shows a swarm of Nomura’s jellyfish(Nemopilema nomurai). Takayuki Baba, a collaborator inthis study, discovered jellyfish mucin in many jellyfish,
including Nomura’s jellyfish, and purified it successfully.
(Masuda et al., 2007) Its purity is so high thathomogeneous products can be prepared on an industrial
scale. In our previous studies, we reported that jellyfish
mucin and hyaluronan administration reduced articularcartilage degeneration in the knee joints of Japanese whiterabbits. (Ohta et al., 2009) Here, the interaction between
jellyfish mucin and hyaluronan was examined in cultured
human chondrocytes.
METHODS
Human articular chondrocytes
The Institutional Review Board of Tokai
University School of Medicine approved this study. Afterobtaining informed consent. Human articular chondrocytes
were obtained during total knee replacement in theaffiliated hospital of Tokai University School of Medicine
and were isolated by enzymatic treatment. The patientswere one man and two women with an average age of 70.3
(range 68–73) years.
Reagents
Artz® (Kaken Pharmaceutical Co., Ltd., Tokyo,
Japan) was used as a high-polymer hyaluronan. The mean
molecular weight of this hyaluronan is approximately
800,000 Da. It was used at a concentration of 25 mg/2.5
mL. Jellyfish mucin was prepared by dissolving the extract
derived from Nomura’s jellyfish in saline at 1 mg/mL.
Extraction and purification of jellyfish mucin
The mesoglea, the major part of the umbrella in
Nomura’s jellyfish, was cut into small pieces andsuspended in water. After removing insoluble material bycentrifugation at 10,000  g, ethanol was added to thesupernatant at one-third of the volume of the pellet. The
resulting precipitate was harvested by centrifugation at
10,000  g and dissolved in water. The supernatant wascollected by centrifugation at 10,000  g, dialyzed against
water, and lyophilized. The lyophilized material was thendissolved in phosphate-buffered saline (PBS) andincubated with anion-exchange gel beads derivatized with
diethylaminoethyl (DEAE) resin (Toyopearl DEAE-650M;
Tosoh, Tokyo, Japan) for 1 h. The beads were washed wellwith phosphate buffer, and the bound proteins were eluted
with elution buffer (phosphate buffer plus 0.5 M NaCl).
The eluent was collected by filtration, dialyzed against
water, and lyophilized.
Cell culture
Cartilage samples from patients were cut into
slices about 1 mm thick and were treated enzymatically in
0.4% (w/v) of Actinase E (Kaken Pharmaceutical Co.,
Ltd.) in Dulbecco’s modified Eagle’s medium/F12 medium(DMEM/F12; Gibco, Carlsbad, CA, USA) for 1 h.
Additional enzymatic treatment continued for 6 h in
0.016% (w/v) bacterial collagenase P (Roche Diagnostics
GmbH, Mannheim, Germany) in DMEM/F12 medium.
After enzymatic treatment, the cartilage tissues were
filtered through a 100-m cell strainer (Becton Dickinson
Labware Co. Ltd, Franklin Lakes, NJ, USA), seeded at 1 
104 cells per 100-mm dish, and cultured. Monolayer
primary cultured cells (P0) were treated with 0.05%
trypsin/EDTA (Gibco) at 37°C for about 10 min. The
obtained cells were washed in PBS, suspended in culture
medium, and subcultured at 1  104 cells per dish. Passage
1 cells were seeded at 1.0  104 cells per 100-mm dish andcultured in the AA+AB+/DMEM F12 culture medium withor without hyaluronan or jellyfish mucin at theconcentrations shown in Table I (n = 6).
For real-time polymerase chain reaction (PCR)
and enzyme-linked immunosorbent assay (ELISA)
procedures, passage 1 cells were seeded at 1  104 cells perdish and cultured. When the cells became subconfluent,
they were transferred into culture medium containing
interleukin 1 (IL-1) (Perotech Inc., Rocky Hill, NJ,
USA)
with
20%
fetal
bovineserum/AA+/
AB+/DMEM/F12. After 12 h, the culture medium wasremoved, and the cells were washed twice in PBS. The
cells were assigned to six testing groups: a control
group(A), 100 g/mL of jellyfish mucin only group (B),300 g/mL of jellyfish mucin only group(C), hyaluronan
only group(D), hyaluronan + 100 g/mL of jellyfish mucingroup(E) and hyaluronan + 300 g/mL of jellyfish mucingroup(F) (Table I). For real-time PCR (n = 6), the cells ineach group were collected at 0, 6, 12, 24, and 48 h. The
cells were washed twice in PBS and then dissolved in 1 mlof TRIzol reagent (Life Technologies Gaithersburg, MD,
USA). For ELISAs (n = 4), the culture media for each

353
Tomonori Takagaki et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 351-358.

group were obtained at 0, 12, 24, 48, and 72 h, and storedin sample tubes at −20°C.
RNA isolation and cDNA synthesis
Total RNA was extracted using RNeasy Mini kits
(Qiagen Inc., Valencia, CA, USA) according to the
manufacturer’s instructions. RNA quality from eachsample was determined using the A260/280 absorbanceratio and by electrophoresis on 1.2% agarose formaldehydegels. Aliquots of 1.0–2.0 g of total RNA were reverse
transcribed into single-strand cDNA using MuLV reverse
transcriptase (Applied Biosystems, Foster City, CA, USA).
The reverse transcription reaction was performed in athermocycler at 42°C for 60 min and then at 95°C for 5
min.
Primer design and real-time PCR
In this study, the cells in the six groups shown in
Table I were collected at 0, 6, 12, 24, and 48 h, washedtwice in PBS, and then dissolved in 1 ml of TRIzol. All
oligonucleotide primer sets were designed based upon the
published mRNA sequences. The expected ampliconlengths ranged from 70 to 200 bp. The oligonucleotide
primers used in this study are listed in Table II. Real-time
PCR was performed in a SmartCycler II (Cepheid,
Sunnyvale, CA, USA) with SYBR® Green PCR MasterMix (Applied Biosystems). Aliquots of 2–2.5 l of cDNA
template were used for real-time PCR in a final volume of
25 l. cDNA was amplified according to the following
condition: 95°C for 15 s and 60°C for 60 s for 35–45
amplification cycles.
Fluorescence changes of SYBR Green weremonitored after every cycle. A melting curve analysis was
performed (0.5°C/s increase from 55°C to 95°C with
continuous fluorescence monitoring) at the end of cycling
to ensure that single PCR products were obtained. The
amplicon size and reaction specificity were confirmed by
2.5% agarose gel electrophoresis. All reactions wererepeated in six separate PCR runs using RNA isolated fromfour sets of human samples. The results were evaluatedusing SmartCycler II software. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) primers were used tonormalize samples. To monitor any crossover
contaminations of PCR, RNase-free water (Qiagen Inc.)
was included in the RNA extraction as a negative control.
To ensure the quality of data, a negative control wasalways applied in each run.
Evaliations of humoral facters
The culture media from the six groups shown in
Table I were obtained at 0, 12, 24, 48, and 72 h. For
collagen type II, matrix metalloproteinase-3 (MMP-3), andtransforming growth factor-1 (TGF-1), the supernatantsfrom each group were assayed by ELISA according to theprotocol in each kit (Collagen Type II ELISA Kit,
Catalogue No. M036000, Whittaker-MA Bioproducts,

Walkersville, MD, USA; Human Total MMP-3 Kit,
Catalogue No. DMP300, R&D Systems. Minneapolis, MN,
USA; Human TGF-1 Kit, Catalogue No. DB100B, R&D
Systems). The absorbance values of the ELISA samples
were measured at 450 nm with a spectrophotometer andwere compared with the standard curves provided in thecollagen type II, MMP-3, and TGF-1 kits.
Cell proliferation activity
Proliferation was measured using a 3-(4,5dimethyl-2-thiazolyl) -2,5- diphenyl-2 H - tetrazolium
bromide (MTT; Dojindo, Kumamoto, Japan) assay on
culture days 3, 5, 7, 9, 11, and 14. There were six replicates
per experimental condition. The cells were collected ondays 3, 5, 7, 9, 11, and 14, and washed twice in PBS; 100
l of MTT solution was added, and the cells wereincubated in air with 5% CO2 at 37°C for 2 h. The
supernatant was absorbed, suspended in 100 l of dimethyl
sulfoxide, and moved to a round plate to measure lightabsorbance (optical density at 590 nm wavelength) using aplate reader (SoftMax Pro; Molecular Devices Corp.,
Sunnyvale, CA, USA). The culture medium was changed
every 2–3 days.
Statistical analysis
All data are expressed as the mean ± standard
error. Factorial analysis of variance (ANOVA) was applied
for comparisons. When an ANOVA was significant,individual differences between treatments were tested by
the multiple-comparison method of Scheffe. P < 0.05 wasconsidered significant.
RESULTS
Real-time PCR results
Genes important for maintaining characteristics ofarticular cartilage such as COL2, SOX9, AGC wereexpressed in a different manner. COL1 and MMP-3 werealso expressed uniquely.
Collagen type I (COL1)
In the presence of hyaluronan (Groups D, E, andF), the overall expression of COL1 was reduced. There
was no effect of jellyfish mucin concentration (Figure 2A).
Collagen type II (COL2)
In the absence of hyaluronan (Groups A, B, andC), the expression of COL2 was unchanged, and there wasno effect of jellyfish mucin concentration. However, theexpression of COL2 increased significantly in the presenceof hyaluronan (Groups E and F). A marked increase wasseen in Group E (100 g/mL of jellyfish mucin; Figure
2B).
SRY-related HMG Box 9 (SOX9)
In the absence of hyaluronan (Groups A, B, andC), the expression of SOX9 increased significantly only in354
Tomonori Takagaki et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 351-358.

Group B, in which 100 g/mL of jellyfish mucin wasadded. However, in the presence of hyaluronan (Groups D,
E, and F), the expression of SOX9 increased with added
mucin in a dose-dependent manner (Figure 2C).
Matrix metalloproteinase-3 (MMP-3)
In the absence of hyaluronan (Groups A, B, andC), the expression of MMP-3 was reduced by the added
jellyfish mucin in a dose-dependent manner. However, inthe presence of hyaluronan (Groups D, E, and F), theexpression of MMP-3 decreased both with and without
added mucin (Figure 2D).
Aggrecan (AGC)
In the absence of hyaluronan (Groups A, B, andC), the expression of AGC was reduced by the addition ofjellyfish mucin. However, in the presence of hyaluronan(Groups D, E, and F), the expression of AGC increased
significantly following the addition of 300 g/mL mucin(Figure 2E).
Analysis of humoral factors by ELISA
Collagen type II
There were no appreciable changes in the quantityof collagen type II secreted from the cells over time, withor without hyaluronan or jellyfish mucin (Figure 3A).

MMP-3
MMP-3 secretion was not influenced by treatment
with hyaluronan or jellyfish mucin, and the amount of
MMP-3 secreted reached a steady state at 24 h(From22.16±1.42 ng/ml (group F of 24hours) to 26.73±0.50
ng/ml (group D of 72hours) (Figure 3B).
TGF-The amount of secreted TGF- was not influenced
by the presence or absence of hyaluronan, but it increased

over time with added jellyfish mucin in a dose-dependent
manner (Figure 3C).
Cell proliferation rates
Cell proliferation increased following the
introduction of hyaluronan and mucin. However, treatment
with jellyfish mucin increased the cell proliferation activity
more than did hyaluronan. Cell proliferation increased
further when both agents were added (Figure 4).
DISCUSSION
Hyaluronan in the synovial fluid contributes to theconsistency and elasticity of synovial fluid, which serves
as a lubricant and shock absorber. Increasing content of
low-molecular-weight hyaluronan in the joints decreases
the consistency and elasticity of the synovial fluid in
inflammatory states such as OA. (Pritzker et al., 2006)
Hyaluronan is involved in anti-inflammatory actions,analgesia, reduction in cartilage degeneration, and healing.
(Konttinen et al., 1991; Schiavinato et al., 1989)
Lubricin, which is included in the mucincontaining glycoprotein/tribonectin group of molecules(superficial zone protein), has been recently identified as acomponent of synovial fluid and the joint surface. (Changet al., 2008) Lubricin controls lubrication in the articularcartilage. Its coefficient of friction is very low at 0.005–
0.03. It has been hypothesized that lubricin contributes toreducing joint friction. (Chang et al., 2008) The effects of
mucins in the human body go beyond surface protection
and reducing friction over the articular cartilage. Thesemolecules provide various functions, such as protection of
the ocular surface by a mucin film, (Davidson et al., 2004)
protection against infection by Helicobacter pylori in thegastric mucosa, (Kawakubo et al., 2004) and protection of
the early embryo in the Fallopian tube. (Lapensee et al.,1997).

Figure 1. Nomura’s jellyfish. The world’s largest jellyfish grows to over 1 m in diameter and weighs more than 200 kg(photo by Mr. Ryoichi Kanzaki; SEA EGG DIVERS).

355
Tomonori Takagaki et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 351-358.

Figure 2. Real-time PCR. The overall gene expression of COL1 was reduced in the presence of hyaluronan (A). In theabsence of hyaluronan, COL2 gene expression was almost constant and was unrelated to the concentration of added jellyfish
mucin. However, in the presence of hyaluronan, COL2 gene expression increased significantly with the addition of mucin;
the percentage increase was greatest at 100 g/mL mucin (B). In the presence of hyaluronan, SOX9 gene expression
increased in a dose-dependent manner with added mucin and increased significantly with 100 g/mL of mucin. (C). In theabsence of hyaluronan, MMP-3 gene expression was reduced by mucin in a dose-dependent manner. However, in thepresence of hyaluronan, MMP-3 gene expression was not influenced by the presence or absence of mucin (D). In the absenceof hyaluronan, adding jellyfish mucin reduced AGC gene expression. However, AGC gene expression was significantly
increased by the addition of 300 g/mL of mucin in the presence of hyaluronan (E).

Figure 3. ELISA. The quantity of collagen II secreted by the cells did not change over time with or without hyaluronan or
mucin treatment (A). MMP-3 secretion was not influenced by hyaluronan or mucin, and its secretion level reached a steadystate at 24 h. At 24 h, significant difference were found between group C and F (*) (P < 0.05)(B).
TGF- secretion was not influenced by the presence or absence of hyaluronan. The level of TGF- secretion increased withadded mucin in a dose-dependent manner and over time. At 24, 48, and 72 h, significant differences were found between
groups A, B, C, E, and F (*), between groups B, C, and F (†), between groups D, E, and F (§), and between groups E and F(‡) (all P < 0.0001)(C).

356
Tomonori Takagaki et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 351-358.

Figure 4. MTT assay. Adding hyaluronan and mucin increased chondrocyte proliferation, although mucin treatment
increased cell proliferation more than did hyaluronan. Cell proliferation was increased further by adding both hyaluronan andmucin. Significant differences were found between groups A, B, C, E, and F (*P < 0.01) on day 3. On day 5, significant
differences were found between groups A and E (†P < 0.01), between groups B and E (‡P < 0.01), and between groups D, E
and F (§P < 0.001).

Table 1. Experimental Groups
A
Hyaluronan (500 g/mL)
Mucin (g/mL)––

Table 2. List of Primers Used in Real-time PCR
Primer ID
Accession No.
COL 1-F
NM_000088
COL 1-R
COL 2-F
NM_033150
COL 2-R
SOX 9-F
NM_000346
SOX 9-R
MMP-3-F
NM_002422
MMP-3-R
Aggrecan-F
NM_001135
Aggrecan-R
GAPDH-F
NM_002046
GAPGH-R

B

C

–
100

–
300

D
+
–

Sequence
AAG GGT GAG ACA GGC GAA CAATTG CCA GGA GAA CCA GCA AGAGGA CTT TTC TTC CCT CTC T
GAC CCG AAG GGT CTT ACA GGA
AAC GCC GAG CTC AGC AAG A
CCG CGG CTG GTA CTT GTA ATCATT CCA TGG AGC CAG GCT TTC
CAT TTG GGT CAA ACT CCA ACT GTG
TCG AGG ACA GCG AGG CC
TCG AGG GTG TAG GCG TGT AGAGAGCA CCG TCA AGG CTG AGA ACATG GTG GTG AAG ACG CCA GT

Many types of glycoprotein mucins appear as
ingredients of mucus and are widely distributed in animals
and plants. Although various forms of mucins are found inplants such as lotus, okra, and yam, many such molecules
have a short peptide chain or a long sugar chain, whichplaces them in a group of materials very different fromanimal-derived mucins. By contrast, all animals maintainmucins as ingredients of mucus and digestive fluids. The
sequence of the core peptide and the structure of sugar
chains and non-mucin-type domains vary between different
animals. Mucins are commonly derived from domestic
animals and can be mass-produced but, currently, there arefew examples of animal-derived mucins that are massproduced as materials. However, mass production of
mucins tends to be avoided because it is impossible to

E

F

+
100

+
300

Expected Size(bp)
170
113
138
138
94
142

prevent completely contamination by prions, which cause
bovine spongiform encephalopathy. Mucins derived fromsnails, starfish, sponges, squids, and other species are
distributed commercially (Grundy et al., 2000; Kimura etal., 2003; Adikwn et al., 2007) but in limited amounts.
Organic chemical construction methods to obtain mucintype high polymers with a simple structure require
substantial costs and are not practical.
Nomura’s jellyfish can interfere with fishing in
the Japanese Sea by swamping fishing nets. These jellyfish
also accumulate in the waterways from which seawater ispiped for cooling power stations located on the coast,
thereby causing water flow reductions that can cause plant
shutdowns. Removal of these jellyfish is routine work at
power stations, industrial facilities, and ports located in357
Tomonori Takagaki et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 351-358.

coastal areas. It is difficult to deal with the large numbersof jellyfish that are collected and then need to be discarded.
Jellyfish mucin has a simple structure comprisingalmost solely mucin sequences and is economically viable
for mass production. (Matsuda et al., 2007) It has been
confirmed that the protein structure of jellyfish mucin issimilar to that of typical human mucin (MUC5AC).
Consequently, it is has potential to be used widely inmaterials for therapeutic treatments. The tandem repeat
structure comprises only eight amino acids (sequence
VVETTAAP or VIETTAAP). Moreover, the sugar chainsare short, there are few kinds of constituent sugars andsialic acids, and there are no peptide sequences present
except for that of the mucin. Therefore, it is expected thatjellyfish mucin’s antigenicity and allergenicity will be mildwhen used therapeutically. The effects of jellyfish mucin
on friction reduction were as expected, and we havereported that administration of jellyfish mucin along withhyaluronan reduces articular cartilage degeneration inwhite rabbit knee joints. (Ohta et al., 2009)
In the present study, the effects were examined invitro in human cultured chondrocytes. Real-time PCR
showed that the gene expression of COL2, SOX9, andAGC was increased in the presence of both mucin andhyaluronan. Hyaluronan reduced COL1 gene expression,
but jellyfish mucin had no effect on this gene. In theabsence of hyaluronan, MMP-3 gene expression was
reduced by mucin in a dose-dependent manner. The ELISA
results showed that, although hyaluronan and mucin did
not affect COL2 and MMP-3 concentrations, theconcentration of TGF- was increased by jellyfish mucin
in a dose-dependent manner with or without hyaluronan. In
addition, chondrocyte proliferation increased significantlyin the presence of both hyaluronan and jellyfish mucin.
Therefore, we hypothesize that the ability of jellyfish
mucin to reduce cartilage degeneration might reflect thepromotion of the effects of an anabolic factor. The effects
of mucin were increased further when administered in
combination with hyaluronan. As the effect of catabolic
factors, the expression of MMP-3 was reduced, but there
was no effect on COL1 expression, although an effect ofhyaluronan was found. Hyaluronan had no effect on TGF, but its expression was increased by jellyfish mucin in adose-dependent manner. This suggests that the growth-

stimulating effects of TGF- on chondrocytes might havebeen a side effect of the jellyfish mucin used in this study.
TGF- is a multifunctional cytokine (Sporn et al., 1988)
that controls the growth and differentiation of cells, andprotein synthesis in the extracellular matrix. (Madri et al.,1988) TGF- inhibits the growth of osteoclast-like cells,
stimulates endochondral ossification, and promotes
osteogenesis. (Chenu et al., 1988; Critchlow et al., 1995)
TGF--induced osteogenesis is mediated by stimulation ofsubperiosteal pluripotent cells, which can differentiate into
chondrogenic cells. (Joyce et al., 1990; Miettinen et al.,1994) Recent reports on the in vivo effects of TGF- foundthat its activity is closely associated with mesenchymal
tissues such as connective tissue, cartilage, and bone.
(Andrew et al,. 1993; Bourque et al., 1993) There is one
report of the potential of TGF- to promote fracture
healing, (Critchlow et al., 1995) but this effect was shortterm, and its high cost limits its clinical application.
Therefore, a method to prolong the effect of endogenous
TGF- for a longer time is required for articular cartilage
repair. Jellyfish mucin, which was used in this study, might
become one of the solutions to this problem.
CONCLUSIONS
Jellyfish mucin and hyaluronan appear toinfluence the physiology of cultured human chondrocytes
and may contribute to cartilage repair and regeneration.
COMPETING INTERESTS
The authors declare that they have no competinginterests.
AUTHORS’ CONTRIBUTIONS
TT and TK performed most of the experiments.
TT, TK, and TB performed the PCR analyses. KK and YK
performed the statistical analyses. MS and JM designed
and coordinated the study, and helped draft the manuscript.
All authors approved the final manuscript.
ACKNOWLEDGMENTS
The
authors
thankJellyfish
Laboratories, Inc. for providing jellyfish mucin.

Research

REFERENCES
Adikwu MU, Alozie BU. Application of snail mucin dispersed in detarium gum gel in wound healing. Sci Res Essay. 2007; 2:
195–198.
Allen PR, Denham RA, Swan AV. Late degenerative changese after menisectomy. JBJS. 1984: 66B: 666-671
Andrew JG, Hoyland JA, Andrew SM et al. Demonstration of TGF-1 mRNA by in situ hybridization in normal humanfracture healing. Calcif Tissue Int. 1993; 52: 74–78.
Bourque WT, Gross M, Hall BK. Expression of four growth factors during fracture repair. Int J Dev Biol. 1993; 37: 573–579
Chang DP, Abu-Lail NI, Guilak F et al. Conformational mechanics, adsorption, and normal force interactions of lubricin andhyaluronic acid on model surfaces. Langmuir. 2008; 24: 1183–1193.
Chenu C, Pfeilschifter J, Mundy GR et al. Transforming growth factor 13 inhibits formation of osteoclast-like cells in long-term
human marrow cultures. Proc Natl Acad Sci U S A. 1988; 85: 5683–5687.

358
Tomonori Takagaki et al. / International Journal of Biological & Pharmaceutical Research. 2015; 6(5): 351-358.

Chenu C, Pfeilschifter J, Mundy GR et al. Transforming growth factor 13 inhibits formation of osteoclast-like cells in long-term
human marrow cultures. Proc Natl Acad Sci U S A. 1988; 85: 5683–5687.
Davidson HJ, Kuonen VJ. The tear film and ocular mucins. Vet Ophthalmol. 2004; 7: 71–77.
Dougados M, Gueguen A, Nguyen M et al. Radiological progression of hip osteoarthritis: Definition, risk factors andcorrelations with clinical status. Ann Rheum Dis. 1996; 55: 356–362.
Dougados M, Nguyen M, Listrat V et al. High molecular weight sodium hyaluronate (hyalectin) in osteoarthritis of the knee: A
1 year placebo-controlled trial. Osteoarthr Cartilage. 1993; 1: 97–103.
Dougados M, Nguyen M, Listrat V et al. High molecular weight sodium hyaluronate (hyalectin) in osteoarthritis of the knee: A
1 year placebo-controlled trial. Osteoarthr Cartilage. 1993; 1: 97–103.
Grundy MM, McKenzie JD, Richardson NV et al. Product from Starfish. US Patent No. 6991810 2000.
Hochberg MC, Altman RD, April KT et al. American College of Rheumatology 2012 Recommendations for use ofnonpharmacologic and pharmacologic therapies in Osteoarthritis of the hand, hip, and knee. Arthritis care & Research.
2012: 64: 465-474
Huskisson EC, Berry H, Gishen P et al. Effects of antiinflammatory drugs on the progression of osteoarthritis of the knee.
LINK study group. Longitudinal investigation of nonsteroidal antiinflammatory drugs in knee osteoarthritis. J
Rheumatol. 1995; 22: 1941–1946.
Jacobsen K: Osteoarthritis following insufficiency of cruciate ligament in man. Acta Orthop Scand. 1977: 48: 520-526
Jay GD, Torres JR, Warman ML et al. The role of lubricin in the mechanical behavior of synovial fluid. Proc Natl Acad Sci U S
A. 2007; 104: 6194–6199.
Joyce ME, Roberts AB, Sporn MB et al. Transforming growth factor-beta and the initiation of chondrogenesis and osteogenesis
in the rat femur. J Cell Biology. 1990; 110: 2195–2207.
Joyce ME, Roberts AB, Sporn MB et al. Transforming growth factor-beta and the initiation of chondrogenesis and osteogenesis
in the rat femur. J Cell Biology. 1990; 110: 2195–2207.
Kimura S, Gohda T, Sakurai Y. Characterization of nidamental mucin from Japanese common squid Todarodes pacificus. J
Tokyo Univ Fish. 2003; 89: 7–13.
Kobayashi Y, Okamoto A, Nishinari K. Viscoelasticity of hyaluronic acid with different molecular weights. Biorheology. 1994;
31: 235–244.
Konttinen YT, Saari H, Nordstrom DC. Effect of interleukin-1 on hyaluronate synthesis by synovial fibroblastic cells. Clin
Rheumatol. 1991; 10: 151–154.
Lapensée L, Paquette Y, Bleau G. Allelic polymorphism and chromosomal localization of the human oviductin gene (MUC9).
Fertil Steril. 1997; 68: 702–708.
Madri JA, Pratt BM, Tucher AM. Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon
the composition and organization of the extracellular matrix. J Cell Biol. 1988; 106: 1375–1384.
Masuda A, Baba T, Dohmae N et al. Mucin (qniumucin), a glycoprotein from jellyfish and determination of its main chain
structure. J Nat Prod. 2007; 70: 1089–1092.
Miettinen PJ, Ebner R, Lopez AR et al. TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal
cells: Involvement of type I receptors. J Cell Biology. 1994; 127(6 Pt 2): 2021–2036.
Ohta N, Sato M, Ushida K et al. Jellyfish mucin may have potential disease-modifying effects on osteoarthritis. BMC
Biotechnology. 2009; 9: 98.
Petrella RJ, DiSilvestro MD, Hildebrand C. Effects of hyaluronate sodium on pain and physical functioning in osteoarthritis of
the knee. A randomized, double-blind, placebo-controlled clinical trial. Arch Intern Med. 2002; 62: 292–298.
Pritzker KP, Gay S, Jimenez SA et al. Osteoarthritis cartilage histopathology: Grading and staging. Osteoarthr Cartilage. 2006;
14: 13–29.
Rashad S, Revell P, Hemingway A et al. Effect of non-steroidal anti-inflammatory drugs on the course of osteoarthritis. Lancet.
1989; 2: 519–522.
Reijman M, Bierma-Zeinstra SM, Pols HA et al. Is there an association between the use of different types of nonsteroidal antiinflammatory drugs and radiologic progression of osteoarthritis? The Rotterdam Study. Arthritis Rheum. 2005; 52:
3137–3142.
Rhee DK, Marcelino J, Baker M et al. The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell
overgrowth. J Clin Invest. 2005; 115: 622–631.
Salk RS, Chang TJ, D’Costa WF et al. Sodium hyaluronate in the treatment of osteoarthritis of the ankle: A controlled,
randomized, double-blind pilot study. J Bone Joint Surg Am. 2006; 88: 295–302.
Schiavinato A, Guidolin D, Pezzoli G et al. Intra-articular sodium hyaluronate injections in the pond-nuki experimental modelof osteoarthritis in dogs. Clin Orthop. 1989; 241: 286–299.
Sporn MB, Robert AB. Peptide growth factors are multifunctional. Nature. 1988; 332: 217–219.