Jellyfish mucin may have potential disease-modifying effects on osteoarthritis
© Ohta et al; licensee BioMed Central Ltd. 2009
Received: 7 April 2009
Accepted: 8 December 2009
Published: 8 December 2009
We aimed to study the effects of intra-articular injection of jellyfish mucin (qniumucin) on articular cartilage degeneration in a model of osteoarthritis (OA) created in rabbit knees by resection of the anterior cruciate ligament. Qniumucin was extracted from Aurelia aurita (moon jellyfish) and Stomolophus nomurai (Nomura's jellyfish) and purified by ion exchange chromatography. The OA model used 36 knees in 18 Japanese white rabbits. Purified qniumucin extracts from S. nomurai or A. aurita were used at 1 mg/ml. Rabbits were divided into four groups: a control (C) group injected with saline; a hyaluronic acid (HA)-only group (H group); two qniumucin-only groups (M groups); and two qniumucin + HA groups (MH groups). One milligram of each solution was injected intra-articularly once a week for 5 consecutive weeks, starting from 4 weeks after surgery. Ten weeks after surgery, the articular cartilage was evaluated macroscopically and histologically.
In the C and M groups, macroscopic cartilage defects extended to the subchondral bone medially and laterally. When the H and both MH groups were compared, only minor cartilage degeneration was observed in groups treated with qniumucin in contrast to the group without qniumucin. Histologically, densely safranin-O-stained cartilage layers were observed in the H and two MH groups, but cartilage was strongly maintained in both MH groups.
At the concentrations of qniumucin used in this study, injection together with HA inhibited articular cartilage degeneration in this model of OA.
Osteoarthritis (OA) is one of the most common joint diseases and is characterized by the gradual degeneration of cartilage over a long time (regressive degeneration). This disease commonly develops in the weight-bearing joints of the lower limbs, such as the knee and hip joints, and onset shows a close correlation closely with age. OA is thus one of the main causes of pain and joint dysfunction among the elderly, and is also often seen in young people after traumas such as a fracture, anterior cruciate ligament transection (ACL-T), meniscus injury or in the presence of an underlying disease such as hemophilia . Currently, pharmacotherapies for OA focus mainly on the alleviation of pain and consist of systemic analgesic therapies and local intra-articular treatments. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used as systemic analgesic therapies . However, pathological progression of OA can be accelerated by the use of NSAIDs [3–6]. Similarly, hyaluronan (HA) injection therapy is a widely recognized part of local intra-articular treatments, inhibiting the destruction of articular cartilage by increasing the viscosity of synovial fluid [7–10].
The presence of a thin membrane layer on the articular cartilage surface is believed to protect against external impact and reduce friction. This membrane is formed from glycoproteins with a mucin-type domain, some of which have been identified in humans, including tribonectin and lubricin [11, 12]. These glycoproteins in the joints show tandem repeat regions composed of 7-8 amino acids in the mucin domain; more than 90% of the threonines and serines can form O-glycosyl bonds and are glycosylated. The sugar chains are short, consisting of 2-3 monosaccharides (including sialic acid), with very little diversity (glycoforms) in the sugar chains. These mucins display characteristics very similar to those of qniumucin, the jellyfish mucin analyzed in this study.
At present, no methods have been established to produce mucins artificially on a sufficiently large scale for therapeutic use. Chemical synthesis is not very practical, as the cost of producing a mucin-type polymer is high, even when the structure is very simple. Although O-glycosylation is a typical posttranslational modification in biological systems, occurring in the Golgi apparatus, synthetic versions of this procedure, in which sugar chains are attached after the expression of core proteins, can only be performed in a limited manner. Under these circumstances, extraction of natural abundant mucins from organisms is most often used. In the industrial production of mucins, only extraction from the gastric juices or saliva of domestic animals has proven commercially successful. However, the purity and homogeneity of these mucins are insufficient for use as a single substance and they have been further avoided since the discovery of bovine spongiform encephalopathy (BSE) . Mucins have also been extracted from marine creatures, such as starfish  and squid , but this technique is also ineffective in terms of efficiency and cost.
In this study, we discuss the effects of intra-articular injection of qniumucin on cartilage degeneration in a rabbit model of OA.
Results of post hoc testing (Scheffé's method).
Difference of averages (I-J)
This study examined the effects of intra-articular injection of qniumucin in a rabbit model of OA using resection of the anterior cruciate ligament. The results were as follows. First, when qniumucin alone was injected, cartilage degeneration did not differ from that in the control group. Second, only minor cartilage degeneration was observed when a mixture of HA and qniumucin was injected compared with the degeneration present after injection of HA only. Third, no significant difference in the effects of qniumucin isolated from different species of jellyfish was apparent in this study.
HA is responsible for the viscosity and elasticity of synovial fluid and thus plays roles in lubrication and shock-absorption. An increase in low molecular weight HA reportedly reduces the viscosity and elasticity of synovial fluid under inflammatory conditions such as OA . However, various other effects have been attributed to HA, including anti-inflammatory and analgesic effects, inhibition of cartilage degeneration and an ability to enhance damage repair .
Safranin-O staining reflects the accumulation of proteoglycan and acts as an index of cartilage degeneration in tissues. Although a reduction in safranin-O staining of the extracellular matrix was seen in our study, cartilage degeneration was clearly inhibited in groups H and MH compared with the degeneration observed in group C.
In recent years, a glycoprotein with a mucin region called lubricin (a superficial zone protein and member of the tribonectin family) has been identified in the synovial fluid and on the articular surface. The presence of lubricin in both areas contributes to reductions in articular friction. This substance is characterized by a mucin-type region in which O-glycans are connected to the protein backbone, with nonmucin-type sequences at both ends. This nonmucin region has been suggested to interact with the cartilage surface to facilitate the adherence of lubricin. The mucin and nonmucin regions are assumed to play different roles, with the former extending the sugar chains outward like a brush to reduce friction and the latter promoting adhesion of lubricin to the cartilage surface. This is referred to as the "brushing model", based on the inferred shape of the molecule . No such mechanism has been suggested for tribonectin, but this substance shares some common characteristics with lubricin insofar as it also displays a mucin-type sequence that reduces friction together with a nonmucin region. The mucin region is believed to adsorb densely to the cartilage tissue surface (perhaps as a film) to reduce friction. However, adhesion of these mucin-type glycoproteins to the articular surface after injection has yet to be directly confirmed.
Mucin or mucin-like substances are likely to form a self-assembled film (SAF) on both hydrophobic and hydrophilic surfaces. One significant example of a SAF in biological systems is the mucin film on the ocular surface that protects the eyeball . Although the bond created by each single sugar chain makes only a small contribution to the total adsorption energy, cooperative interaction of many glycan chains concentrated in a small area provides a sufficient gain in free energy (and a reduction in entropy) to immobilize the polymer chain of the mucin. Adhesion and SAF formation on the surface are possible without any selective interaction, such as that suggested for the nonmucin sequence of lubricin, which may be the first trigger of adhesion. Therefore, if the goal of treatment is to reduce friction, any kind of mucin that lacks a nonmucin region could be used instead of lubricin or tribonectin. Jay mentioned the synergic effects of lubricin and HA in an in vitro study . Mucin may have potential synergic effects with HA, as mucin is one component of lubricin. In this study, we first demonstrated that exogenous mucin derived from natural jellyfish showed synergic effects with HA using an in vivo animal model. These effects might be induced by improving the viscosity and friction properties of synovial fluid and enhancing the self-assembly capacity of cartilage.
Many different kinds of mucins are known. As no large-scale production of artificial polymeric mucins has been achieved with biotechnology or chemical synthesis, extraction from the natural environment remains the most appropriate method for provision as commercial substances. Mucins are widely distributed in animals and plants as components of mucus. For example, in plants mucins are found in extracts of lotuses, okra and yams. However, the structure of plant mucins is completely different from that of animal mucins, with a short peptide connected to long sugar chains, such as galactan and mannan . We have therefore focused on animal mucins as candidate materials for therapeutic injection.
Animal mucins are produced and retained as components of mucus by all living animals, regardless of taxonomy. Huge potential variations exist in components such as the core peptide sequence, nonmucin domain sequence and structure of the sugar chains (glycoforms) according to the animal species. However, there are very few examples of animal mucins that are mass produced by domestic animals . These are broadly classified as gastric mucins  and submaxillary salivary gland mucins [23, 24]. Gastric mucins constitute a mixture harvested from the lavage fluids of internal organs as low-purity materials. Only total monosaccharide (for example, sialic acid) and amino acid analyses have been performed, with no further characterization, so gastric mucins constitute an inexpensive material suitable for mass production. In contrast, submaxillary mucins are very pure and detailed structural analyses of their amino acid sequences and saccharide compositions have been performed . A monoclonal antibody directed against submaxillary mucins known as sialyl Tn antigen has been produced and used as a tumor marker . However, these animal mucins risk contamination with foreign substances unless thorough purification is performed, so use tends to be avoided. For example, prions causing BSE cannot be eliminated. Mucins from snails , starfish  and squid  are currently available on the market, but production volumes are limited. Among these mucin alternatives, qniumucin, which is harvested from jellyfish, was discovered in recent years by our colleagues . As mass production is inexpensive, qniumucin is a candidate mucin for wide application in many patients as a treatment for OA. Qniumucin is characteristically an almost pure monotonously repeated sequence of short mucin regions called "tandem repeats". The tandem repeat unit consists of eight amino acids (sequence VVETTAAP or VIETTAAP) and the sugar chains are short (usually only 1-3 sugars), with only a few types of monosaccharides and no sialic acid. Since qniumucin seems to have very few peptide sequences other than the mucin portion, only mild biological reactions are expected from the immune system in the form of allergies. This low potential for biological rejection is a further advantage of the use of this mucin. The risks involved in providing a mass product on an industrial scale are thus significantly reduced.
In a preliminary experiment to test the effects of injection with qniumucin, no elevation of the blood cell count or C-reactive protein and no swelling of the joints were observed (unpublished results). No histological findings of synovium have been detected among normal and injected joints (unpublished results). The possibility that an endogenous endotoxin, identified in this preliminary experiment, might cause adverse effects is a concern, but no such effects have yet been observed. After the purification method was improved to preclude any contamination with the solvent from high-performance liquid chromatography, the concentration of endotoxin in qniumucin purified by ion-exchange chromatography and in unpurified qniumucin were both <10 EU/ml when characterized by Endosafe-PTS (Charles River Laboratories Japan, Kanagawa, Japan). Based on these considerations, we proceeded with the intra-articular injection of this qniumucin, expecting a friction-reducing effect. The present results represent a promising step in the development of a new treatment for cartilage degeneration.
After injecting a mixture of mucin and HA, cartilage degeneration was significantly inhibited compared with that in rabbits injected with HA alone. This effect might be induced by improving the viscosity and friction properties of the synovial fluid and enhancing the self-assembly capacity of the cartilage.
All procedures using animals in this study were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) published by the National Institutes of Health, USA, and the Guidelines of Tokai University on Animal Use.
Artz® (Kaken Pharmaceutical, Tokyo, Japan) was used in making the solution of polymeric HA. The average molecular weight of this HA is approximately 800,000 and the concentration used was 25 mg/2.5 ml. Qniumucin from S. nomurai or A. aurita was dissolved in saline at a concentration of 1 mg/ml to create stock solutions.
Extraction and purification of qniumucin
The mesogloea, the major part of the umbrella in jellyfish, was cut into small pieces and suspended in water. After removing insoluble material by centrifugation at 10,000 × g, one third of the volume of ethanol was added to the supernatant. The resulting precipitate was harvested by centrifugation at 10,000 × g and dissolved in water. Supernatant was collected by centrifugation at 10,000 × g, dialyzed against water, and lyophilized. The lyophilized material was then dissolved in phosphate buffer and incubated with anion-exchange gel beads (Diethylaminoethyl (DEAE) - resin, Toyopearl DEAE-650M; Tosoh, Tokyo, Japan) for 1 h. The beads were washed well with phosphate buffer and the bound proteins were eluted with elution buffer (phosphate buffer, 0.5 M NaCl). The eluent was collected by filtration, dialyzed against water, and lyophilized.
Japanese white rabbits (females weighing 3 kg) were purchased from Tokyo Laboratory Animals Science (Tokyo, Japan). The rabbits were kept individually and reared in a fiber-reinforced polymer cage (width 450 mm × height 450 mm × depth 900 mm).
An ACL-T model [28–34] was prepared for use as the OA model. With the rabbits under inhalation anesthesia with isoflurane (Forane®; Abbott Japan, Tokyo, Japan), a 3-cm incision was made aseptically on the medial side of both knees to expose the patella and patellar tendon, and the articular capsule was incised. The patella was then dislocated outwardly to an extended position and the knee joint was bent for macroscopic resection of the anterior cruciate ligament. The patella was then repositioned and the subdermal muscular layer and skin were sutured with nylon thread.
Macroscopic and histological evaluation of the articular cartilage
OA cartilage histopathology grade assessment; grading methodology
Grade (key feature)
Associated criteria (tissue reaction
Grade 1: surface intact
Matrix: superficial zone intact, oedema and/or superficial fibrillation (abrasion), focal superficial matrix condensation
Cells: death, proliferation (clusters), hypertrophy, superficial zone Reaction must be more than superficial fibrillation only
Grade 2: surface discontinuity
+ Matrix discontinuity at superficial zone (deep fibrillation)
± Cationic stain matrix depletion (Safranin O or Toluidine Blue) upper 1/3 of cartilage
± Focal perichondronal increased stain (mid zone)
± Disorientation of chondron columns
Cells: death, proliferation (clusters), hypertrophy
Grade 3: vertical fissures (clefts)
Matrix vertical fissures into mid zone, branched fissures
± Cationic stain depletion (Safranin O or Toluidine Blue) into lower 2/3 of cartilage (deep zone)
± New collagen formation (polarized light microscopy, Picro Sirius Red stain)
Cells: death, regeneration (clusters), hypertrophy, cartilage domains adjacent to fissures
Grade 4: erosion
Cartilage matrix loss: delamination of superficial layer, mid layer cyst formation
Excavation: matrix loss superficial layer and mid zone
Grade 5: denudation
Surface: sclerotic bone or reparative tissue including fibrocartilage within denuded surface. Microfracture with repair limited to bone surface
Grade 6: deformation
Bone remodelling (more than osteophyte formation only). Includes: microfracturewith fibrocartilaginous and osseous repair extending above the previous surface
OA score; semi-quantitative method
Stage % Involvement (surface, area, volume)
Grade (key feature)
Stage 1 <10%
Stage 2 10-25%
Stage 3 25-50%
Stage 4 >50%
Grade 1(surface intact)
Grade 2 (surface discontinuity)
Grade 3 (vertica fissures, clefts)
Grade 4 (erosion)
After analysis of variance, the least significant differences were used for post hoc testing (Scheffé's method). Average OA scores were compared and values of P < 0.05 were considered significant.
List of abbreviations
nonsteroidal anti-inflammatory drugs
bovine spongiform encephalopathy
anterior cruciate ligament transaction
ethylene diaminetetraacetic acid
This research was partly supported by Grants-In-Aid for Scientific Research No. 17034067 in the Priority Area of "Molecular Nano Dynamics" and No. 17300166 and the High-Tech Research Center Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The research was also supported by the project to develop "innovative seeds" (Creation and Support Program for Start-ups from Universities) of the Japanese Science and Technology Agency and the General Insurance Association of Japan, Mitsui Sumitomo Insurance Welfare Foundation.
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