Oe et al.

Nutrition Journal (2016) 15:11

DOI 10.1186/s12937-016-0128-2

REVIEW Open Access

Oral hyaluronan relieves knee pain: a

review
Mariko Oe1 , Toshiyuki Tashiro2, Hideto Yoshida1, Hiroshi Nishiyama1, Yasunobu Masuda1*, Koh Maruyama3,
Takashi Koikeda4, Reiko Maruya5 and Naoshi Fukui6

Abstract
Hyaluronan (HA) is a component that is particularly abundant in the synovial fluid. Randomized, double-blinded,
placebo-controlled trials carried out between 2008 and 2015 have proven the effectiveness of HA for the treatment
of symptoms associated with synovitis, and particularly, knee pain, relief of synovial effusion or inflammation, and
improvement of muscular knee strength. The mechanism by which HA exerts its effects in the living body,
specifically receptor binding in the intestinal epithelia, has gradually been clarified. This review examines the effects
of HA upon knee pain as assessed in clinical trials, as well as the mechanism of these effects and the safety of HA.
Keywords: Hyaluronan, Hyaluronic acid, Dietary supplement, Knee, Joint, Osteoarthritis

Introduction [4]. This equals to 110 USD per month. In contrast, the
The number of patients with osteoarthritis (OA) is in- cost of effective HA dietary supplements for one month
creasing in developed countries. In the US, 43 million is 50 USD (Play Again Now®, Viscos LLC., USA). There
patients were estimated to have OA in 1997, and this have been no reports regarding the cost-effectiveness of
figure is projected to grow to more than 60 million by intra-articular injection versus dietary supplements.
2020 [1]. However, there are some advantages for consumers with
Severe OA is treated by osteotomy or artificial joint re- regard to HA dietary supplements, when one considers
placement, whereas conservative treatments include the potential risks and benefits.
intra-articular injection of hyaluronan (HA) [2]. The in- In 2004, Bucci and Turpin reported a review article on
jections improve symptoms, although they represent a the effectiveness of dietary supplements containing HA
mental burden and the risk of infection for patients who in the US; however, the report does not mention ran-
need to visit the hospital regularly to receive these pain- domized, double-blind, placebo-controlled trials [5].
ful injections. Therefore, this review discusses the efficacy of ingested
Meanwhile, dietary supplements such us HA, glucosa- HA in treating knee pain based on data from random-
mine, and chondroitin are sold as health foods, largely ized, double-blind, placebo-controlled trials as well as
targeting problems with the knee. The effects of low- the mechanism of action and safety of dietary HA.
dose HA treatment for knee pain has been the source of
much research (for example, a dose of HA not more
than 240 mg/day; Table 1) compared with glucosamine Characteristics of HA
(1500 mg/day) and chondroitin (675 mg/day) [3]. HA HA is a high molecular-weight polysaccharide composed
dietary supplements impose a small burden on patients. of repeating polymeric disaccharides of D-glucuronic acid
The average wholesale price for five vials of intra- and N-acetyl-D-glucosamine (Fig. 1) [6]. All vertebrates
articular HA injection (Hyalgen®, Fidia Farmaceutici and few microorganisms synthesize HA in vivo. In human
S.p.A., Italy) is 661 USD, which is effective for 6 months beings, HA is present in every connective tissue and organ
such as skin, synovial fluid, blood vessels, serum, brain,
* Correspondence: [email protected]
cartilage, heart valves, and the umbilical cord [7]. In par-
1
R&D Division, Kewpie Corporation, 2-5-7, Sengawa-cho, Chofu-shi, Tokyo, ticular, synovial fluid has the highest concentration of HA
Japan anywhere in the body at 3–4 mg/mL [8].
Full list of author information is available at the end of the article

© 2016 Oe et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Oe et al. Nutrition Journal (2016) 15:11 Page 2 of 10

Table 1 Summary of the knee pain-improving effects of ingested hyaluronan

Study designs Materials and Methods Subjects Results References
Randomized, double-blind, HA mixture at 630 mg (HA 60 mg; MW 24 patients with Significant improve of knee pain [34]
placebo-controlled trial <5 k) daily for 2 weeks knee pain (in Japan) and discomfort
Randomized, double-blind, HA mixture at 80 mg (HA 48 mg; MW 20 patients aged Significant improve from baseline [35]
placebo-controlled trial 1000 k) daily for 2 months ≥40 years with knee for bodily pain bodily pain subscale
OA (in USA) and physical component summary.
Randomized, double-blind, HA at 240 mg (MW 900 k) daily for 26 patients aged 50 Significant improve of knee pain [36]
placebo-controlled trial 8 weeks ~ 65 years with knee and stiffness
pain (in Japan)
Randomized, double-blind, HA at 200 mg (MW 900 k) daily for 25 patients with Significant improve of tatal WOMAC [37]
placebo-controlled trial 8 weeks knee OA [WOMAC score and activity of daily living
pain score > 10] (in
USA)
Retrospective cohort study, HA mixture at 80 mg (HA 48 mg; MW N/ 69 patients with Significant improve of synovial [38]
PCT-controlled trial A) daily for 6 months knee OA and effusion and knee pain
synovitis (in Spain)
Randomized, double-blind, HA mixture at 630 mg (HA 60 mg; MW 40 patients with Significant improve of pain/step-up [39]
placebo-controlled trial <5 k) daily for 4 months knee OA and and -down function and aggregate
synovitis (in Japan) total symptoms
Randomized, double-blind, HA mixture at 2520 mg (HA 72 mg; MW 29 patients with Significant improve of bone [40]
placebo-controlled trial <5 k) daily for 12 weeks knee OA and metabolism marker
synovitis (in Japan)
Randomized, double-blind, HA at 200 mg (MW 900 k) daily for 38 patients with Significant improve of Health [41]
placebo-controlled trial 12 months knee OA (in Japan) condition
21patients aged Significant improve of total JKOM
≦70 years with knee score, pain and stiffness in the knee
OA (in Japan) and general activities
Randomized, double-blind, HA mixture at 80 mg (HA 52 mg; MW N/ 40 healthy Significant improve of joint [42]
placebo-controlled trial A) daily for 90 days individuals with mild mechanics and muscle function
joint discomfort (in
Spain)
Meta-analysis included in two HA mixture at 80 mg (HA 48 mg; MW N/ 148 healthy Significant improve of muscle [43]
randomized, controlled, double- A) daily for 3 months individuals with mild function, synovial effusion and
blind, placebo-controlled trials knee pain (in Spain) reduces pain
Randomized, double-blind, HA mixture at 80 mg (HA 52 mg; MW N/ 68 healthy Significant improve of articular pain, [44]
placebo-controlled trial A) daily for 90 days individuals with mild synovial effusion and knee muscular
joint discomfort (in strength
Spain)
Randomized, double-blind, HA mixture at 80 mg (HA 56 mg; MW N/ 40 patients with Significant improve of total WOMAC [45]
placebo-controlled trial A) daily for 3 months knee OA (in USA) score and knee pain
Randomized, double-blind, HA mixture daily for 4 weeks (HA 225 mg 72 patients with Significant improve of knee pain [46]
placebo-controlled trial daily for first 2 weeks, HA 150 mg daily knee pain (in USA)
for last 2 weeks; MW 2500 k ~ 2800 k)

Fig. 1 The structure of hyaluronan

Oe et al. Nutrition Journal (2016) 15:11 Page 3 of 10

HA is a polysaccharide with a mean molecular by oral HA which results in decreased pain. HA (MW,
weight (MW) ranging from several hundred to several 9 × 105; Hyabest® (J), Kewpie Corporation, Tokyo, Japan)
millions, and thus, it has high viscosity in water. The was administered orally to MRL-lpr/lpr mice, a Th-1-
viscosity of synovial fluid is attributable to HA and type autoimmune disease model. Oral HA binds to an
serves as a lubricant for joint movements, resulting in intestinal receptor (Toll-like receptor-4;TLR-4). Cyto-
a coefficient of friction of nearly zero in joint cartil- kine array analysis showed that HA enhanced the pro-
age [9]. It is known that patients with OA have di- duction of interleukin-10 (IL-10), an anti-inflammatory
minished HA concentrations in their synovial fluid cytokine. DNA array analysis of tissue from the large in-
[10]. To restore the decreased levels of HA and treat testine showed that HA up-regulates suppressor of cyto-
OA, HA intra-articular injections are widely used. kine signaling 3 (SOCS3) expression and down-regulates
The functions of HA include preventing cartilage de- pleiotrophin expression. These results suggest that the
naturation [11, 12], protecting the outer layer of car- binding of HA to TLR-4 promotes IL-10 and SOCS3 ex-
tilage [13–20], blocking synovial inflammation [21, pression and suppresses pleiotrophin expression leading
22], increasing chondrocyte density [23], promoting to anti-inflammation of arthritis (Fig. 2).
synovium metabolism [24], normalizing synovial fluid
[25], and treating sharp pain [26]. The mechanism by Clinical trials of oral HA for the treatment of knee
which HA diminishes pain has been reported. HA pain
intra-articular injections decrease the levels of inflam- We searched the databases up to October 29, 2015;
matory substances such as prostaglandin E2 resulting PubMed, the Cochrane Library, Scopus, UMIN-CTR,
in reduced pain [27, 28]. Therefore, HA is known to JDreamIII (in Japanese), and Ichushi Web (in Japanese).
have a strong relationship with knee joint health. The search words used for all databases contained the
terms; hyaluronan, intake and knee pain. We selected fol-
Mechanisms of oral HA treatment for knee pain lowing 13 relevant reports after full text review [31–43].
It is generally believed that it is difficult for the body to Many randomized, double-blind, placebo-controlled
absorb a polysaccharide. HA is not absorbed into the trials have demonstrated the effectiveness of dietary HA
body as a high-molecular-weight polymer after ingestion. in alleviating knee pain since 2008 in the US, EU, and
A test using intestinal epithelia model cells (Caco-2 Asia (Table 1).
cells) revealed that HA with a MW exceeding 1 × 105 is In 2008, a dietary supplement containing HA as the
rarely absorbed. On the contrary, the amount of HA principal ingredient was reported following two clinical
absorbed by Caco-2 cells increases as the MW of HA trials. As the supplement contains multiple components,
decreases to 7 × 104, 2 × 104, or 5 × 103 [29]. Kurihara et the potential effects of ingredients other than HA on
al. reported that HA is decomposed into 2–6-membered knee pain cannot be denied but the effects of the supple-
polysaccharides by enteric bacteria, and these polysac- ment on knee pain were confirmed.
charides are partially absorbed into the body by the Hatayama et al. [34] treated 24 Japanese patients with
small intestine [30]. Following the decomposition of HA chronic knee pain (HA group, n = 13; placebo group, n
by enteric bacteria to a low MW form, free polysaccha- = 11, mean age, 47.5) with an HA mixture at a dose of
rides are known to migrate into the joints and other tis- 1800 mg/day (HA content, 60 mg/day) or placebo for
sues. Lactobacillus and Bzfidobacterirn have been 2 weeks. The HA group displayed a significant improve-
reported as examples of enteric bacteria that play a crit- ment in knee pain and discomfort compared with the
ical role in HA absorption [31]. Balogh et al. reported placebo group (p < 0.05).
that 99 mtechnetium-labeled HA (MW, 1 × 106) is accu- In the US, Kalman et al. [35] treated 20 subjects aged
mulated by tissues such as joints after oral administra- ≥40 years with knee OA (HA group, n = 11; placebo
tion in rats and dogs [32]. The pattern of tissue uptake group, n = 9, mean age, 56.3) with an HA mixture at a
of radioactivity for 99mtechnetium-labeled HA did not dose of 80 mg/day (HA content, 48 mg/day) or placebo
resemble that for 99mtechnetium-labeled pertechnetate for 2 months and their outcomes were evaluated using
(PCT), which was used as a control; this implies that the index for knee joint pain; the Western Ontario and
99m
technetium-labeled HA did not release 99mtechne- McMaster Universities Osteoarthritis Index (WOMAC),
tium in tissues. and the Short Form-36 Acute US Version 2 (SF-36v2) as
These studies clearly suggest that HA is absorbed by a quality of life (QOL) index. [REMARK 4] The
the body; however further research is needed to clarify WOMAC score in both groups revealed significant im-
the effects of the route of absorption on knee pain relief. provements in knee pain compared with baseline (p <
Another mechanism was clarified by Asari et al. in 0.05) but the physical function and total symptom scores
2010 [33]. This report identified a signaling cascade in of the HA group displayed a more drastic improvement
which receptors on intestinal epithelial cells are activated than those of the placebo group. On the SF-36 v2, the
Oe et al. Nutrition Journal (2016) 15:11 Page 4 of 10

Fig. 2 Mechanism of improve the arthritis. Oral administration of hyaluronan modulates inflammation by upregulating suppressor of cytokine
signaling-3 expression and down-regulating pleiotrophin expression via Toll-like receptor-4 in intestinal epithelial cells

scores of the HA group were significantly improved ver- = 17, mean age, 70.8) were orally administered 200 mg/
sus baseline (p < 0.05) for bodily pain and the physical day HA or placebo for 8 weeks with the findings il-
component summary, and the scores were more signifi- lustrating that the WOMAC score was significantly
cantly improved in the HA group than in the placebo improved in both groups versus baseline (p < 0.05),
group. particularly among the 25 patients with WOMAC
In 2009, two clinical trials using highly pure HA (more pain scores of more than 10 (HA group, n = 13; pla-
than 98 % pure) were reported. These trials revealed that cebo group, n = 12). In addition, the WOMAC total
the effects of HA-containing supplements on knee pain score and activity of daily living score were signifi-
were attributable to HA. cantly improved in the HA group compared with the
Iwaso et al. [36] reported that 33 Japanese patients placebo group (p < 0.05).
with knee pain (HA group, n = 16; placebo group, n = 17, In the same year, Möller et al. [38] conducted a retro-
mean age, 58.3) were administered HA at 240 mg/day or spective cohort study in Spain in which HA was com-
placebo for 8 weeks and outcomes were evaluated using pared with the analgesic drug paracetamol (PCT).
the Japanese Knee Osteoarthritis Measure (JKOM). The Sixty-nine patients (mean age, N/A) with knee OA and
JKOM score was significantly improved compared with synovitis were administered an HA mixture at 80 mg/
baseline in both groups (p < 0.01), particularly among 26 day (HA content, 48 mg/day) or PCT for 6 months.
patients aged 50–65 years (HA group, n = 13; placebo Ultrasonography showed that the course of synovitis in
group, n = 13). Significant improvements in knee pain the suprapatellar recess was significantly reduced in the
and stiffness were recorded in the HA group compared HA group compared with the PCT group (p < 0.0001),
with the placebo group (p < 0.05). and the number of severe synovial effusion cases were
Sato et al. [37] reported a study in which 37 Ameri- significantly reduced in the HA group compared with
cans with knee OA (HA group, n = 20; placebo group, n the PCT group (p < 0.001).
Oe et al. Nutrition Journal (2016) 15:11 Page 5 of 10

Between 2010 and 2015, eight clinical trials were re-

ported indicating that interest in HA intake was becom-
ing more intense in developed nations.
In 2010, Nagaoka et al. [39] reported that 40 Japanese
patients with knee OA (HA group, n = 19; placebo
group, n = 21, mean age, 62.9) were administered an oral
HA mixture at 1800 mg/day (HA content, 60 mg/day)
or placebo for 4 months and the result illustrated that
pain/step-up and step-down function and the aggregate
total symptoms of the Japanese Orthopaedic Association
clinical trials response criteria were significantly im-
proved in the HA group compared with the placebo
group (p < 0.05) indicating relief of knee pain.
In 2012, Yoshimura et al. [40] reported that 29 Japa-
nese athletes (HA group, n = 14; placebo group, n = 15,
mean age, 20.0) were administered an HA mixture at
4800 mg/day (HA content, 72 mg/day) or placebo for
12 weeks. The urine levels of the bone metabolic
markers N-terminal telopeptides of bone-specific type I
collagen were significantly lower in the HA group (p <
0.05). This result indicates that oral HA influences knee
joint health.
Tashiro et al. [41] reported a study in which 38 Japa-
nese patients with knee OA (HA group, n = 18; placebo Fig. 3 Oral hyaluronan improve knee osteoarthritis: a randomized,
group, n = 20, mean age, 69.9) were administered highly double-blind, placebo-controlled trial. Twenty-one subjects (≤70 years
pure HA (more than 97 %) at 200 mg/day or placebo for of age) were randomly divided into two groups (hyaluronan group, n
12 months. During the trial, the patients were requested = 11; placebo group, n = 10). Variations in the total Japanese Knee
Osteoarthritis Measure score relative to baseline are shown. □
to conduct quadriceps-strengthening exercise daily as
hyaluronan; ■ placebo. Values are presented as the mean ± SE.
part of the treatment. The result reveals that quality of p < 0.05 vs. baseline; #p < 0.05 vs. placebo group
life according to the JKOM score was significantly im-
proved in the HA group versus the placebo group (p <
0.05) and this trend was more obvious among patients pain (VAS between 30 and 50 mm, aged from 20 to 75).
aged ≤70 years (n = 21). For these relatively younger pa- In the studies, patients were administered an oral HA
tients in the HA group, the total JKOM score was sig- mixture at 80 mg/day (HA content, 48 mg/day) or pla-
nificantly better than that in the placebo group (p < cebo for 3 months and outcomes were evaluated using
0.05). Thus, HA intake and quadriceps-strengthening ex- an isokinetic dynamometer. The analysis indicated that
ercise affectively alleviate knee pain particularly in pa- the affected joint displayed greater flexion in the HA
tients aged ≤70 years (Fig. 3). group than in the placebo group (p = 0.039). The result
In 2013, Martinez-Puig et al. [42] reported that 50 of ultrasound examination for synovial effusion illus-
healthy subjects with joint discomfort (VAS between 10 trated that synovial effusion was significantly reduced in
and 40 mm; HA group, n = 20; placebo group, n = 20, the HA group (p = 0.029). An evaluation using a VAS in-
mean age, 59.6) were administered an HA mixture at dicated that knee pain was additionally improved in the
80 mg/day (HA content, 48 mg/day) or placebo for HA group compared with the findings in the placebo
90 days and outcomes were evaluated using an isokinetic group (p = 0.0036).
dynamometer. Maximum peak torque was significantly In 2014, Sánchez et al. [44] reported that 68 patients
greater in the HA group than in the placebo group (p < with mild knee pain (VAS between 30 and 50 mm; HA
0.05). In the HA group, the total knee extension and group, n = 34; placebo group, n = 34, mean age, 69.5)
mean power of knee extension were also significantly were administered an oral HA mixture at 80 mg/day
higher than the values in the placebo group (p < 0.05). (HA content, 48 mg/day) or placebo for 90 days. A sig-
This trial demonstrated that oral HA effectively im- nificant improvement in knee pain according to the VAS
proves the ability of the knee joint to bend and stretch. was recorded in the HA group compared with the pla-
Moriña et al. [43] reported a meta-analysis of two ran- cebo group (p = 0.0005). An ultrasound examination in-
domized, controlled, double-blind, placebo-controlled dicated that synovial effusion was reduced in the HA
trials involving a total of 148 patients with mild knee group (p = 0.041). The result using an isokinetic
Oe et al. Nutrition Journal (2016) 15:11 Page 6 of 10

Table 2 Safety tests of hyaluronan

Test procedures Subjects Route Results References
Randmized, double-blind, Human Oral administration, 60 mg/day for 2 weeks No adverse event [34]
placebo-controlled trial related to hyaluronan.
Human Oral administration, 48 mg/day for 2 months No adverse event [35]
related to hyaluronan.
Human Oral administration, 240 mg/day for 8 weeks No adverse event [36]
related to hyaluronan.
Human Oral administration, 200 mg/day for 8 weeks No adverse event [37]
related to hyaluronan.
Human Oral administration, 60 mg/day for 4 months No adverse event [39]
related to hyaluronan.
Human Oral administration, 200 mg/day for 12 months No adverse event [41]
related to hyaluronan.
Human Oral administration, 52 mg/day for 3 months No adverse event [42]
related to hyaluronan.
Human Oral administration, 52 mg/day for 3 months No adverse event [43]
related to hyaluronan.
Human Oral administration for 4 weeks (225 mg/day for first No adverse event [46]
2 weeks, 150 mg/day for last 2 weeks) related to hyaluronan.
single-dose toxicity study Mouse Oral administration LD50 (mg/kg) > 2400 [48]
Mouse Oral administration LD50 (mg/kg) > 500 [49]
Rat Oral administration LD50 (mg/kg) > 800 [48]
Rat Oral administration LD50 (mg/kg) > 200 [50]
Rabbit Oral administration LD50 (mg/kg) > 1000 [48]
Repeated-dose toxicity study Rat Subcutaneous administration for 13 weeks with 4 weeks NOAEL 50 mg/kg/day [51]
recovery test
Beagle dog Subcutaneous administration for 13 weeks with 4 weeks NOAEL 10 mg/kg/day [52]
recovery test
Rat Oral administration for 30 days NOAEL 1500 mg/kg/day [53]
Rat Oral administration for 90 days NOAEL 1333 mg/kg/day [54]
Rat Oral administration for 90 days NOAEL 1000 mg/kg/day [55]
Rat Oral administration 28 days NOAEL 3500 mg/kg/day [56]
Rat Intraperitoneal administration 90 days NOAEL 9 mg/kg/day [57]
Rat Intraperitoneal administration for 3 months NOAEL 60 mg/kg/day [58]
Rat Oral administration for 13 weeks NOAEL 12.5 mg/kg/day [59]
Rat Oral administration for 90 days with 28 days recovery test NOAEL 48 mg/kg/day [60]
Beagle dog Intra-articular administration for 6 months NOAEL 12 mg/kg/day [61]
Reproductive and developmental Rat Subcutaneous administration NOAEL 50 mg/kg/day [62–64]
toxicity studies
Rat Oral administration NOAEL 670 mg/kg/day [65]
Rat Subcutaneous administration NOAEL 50 mg/kg/day [66–68]
Rabbit Subcutaneous administration NOAEL 50 mg/kg/day [69]
Mutagenicity Reverse mutation Bacteria(Ames 1000 μg/plate Negative [70]
test test test)
Chromosomal Mammalian 1.00 mg/mL Negative [71]
aberration test cultured cell
Mammalian 1000 μg/plate Negative [72]
cultured cell
Micronucleus test Mouse 300 mg/kg Negative [73]
Mouse Intraperitoneal administration, 30 mg/kg Negative [74]
Antigenicity study Mouse, Rat Intraperitoneal administration, 100 μg/個体 Negative [75]
Oe et al. Nutrition Journal (2016) 15:11 Page 7 of 10

Table 2 Safety tests of hyaluronan (Continued)

Guinea pig Negative
Rabbit Intramuscularl administration, 30 mg/kg Negative [76]
Influence on cancer cell Mouse, cell Oral administration, 200 mg/day No influence on cancer [77]
cell

dynamometer illustrated that muscular strength (peak symptoms associated with synovitis, mainly pain [33–37,
torque) was significantly greater among patients in the 39, 41, 46]; four involved measures taken to relieve syn-
HA group than among those in the placebo group (p = ovial effusion or inflammation [38, 43–45]; and three in-
0.0324). Blood tests for RNA expression were conducted volved measures taken to improve knee muscular
in 20 patients (HA group, n = 10; placebo group, n = 10). strength [42–44].
The results demonstrated that the expression of genes The studies that used a mixture containing HA and
related to glycosaminoglycan metabolism and extracellu- other components could not rule out the possibility that
lar matrix dynamics differed between the HA and pla- the other components had effects on knee pain. How-
cebo groups. It was concluded that differences in gene ever, studies that used highly pure HA (more than 97 %)
expression explained the improvements in knee pain also reported a beneficial effect on knee pain. Thus, oral
and muscular strength in the HA group. HA can effectively improve knee pain.
In 2015, Nelson et al. [45] treated 40 patients with It is apparent that there were slightly more female sub-
knee OA (VAS > 50 mm; HA group, n = 21; placebo jects than males investigated in the above-mentioned
group, n = 19, mean age, 61.0) with an oral HA mixture clinical studies (mean ratio: males 43 %, females 57 %).
at 80 mg/day (HA content, 56 mg/day) or placebo for However, each study involved the same gender ratio for
3 months. Significant improvements in the VAS, HA and placebo groups. In general, the number of fe-
WOMAC total score, and WOMAC pain score were ob- male OA patients was larger than the number of males.
served in the HA group compared with the placebo This is probably because females have lesser muscles
group (p < 0.05). An analysis of serum and synovial fluid than males and therefore, the burden on the knee in-
illustrated that inflammatory cytokine levels were signifi- creases. It has been reported that HA is regulated by
cantly increased in the placebo group (p < 0.05) and sig- ovarian steroids [47]; however, it is not yet clear whether
nificantly decreased in the HA group (p < 0.05). the oral intake of HA influences hormonal balance. This
Regarding bradykinin, which related to inflammation needs further research.
and pain, and leptin, which is related to OA caused by HA intake differs among the respective studies, such
obesity, the levels of both substances were significantly as 48–240 mg/day for 2 weeks–12 months. Thus, further
lower in the HA group than in the placebo group (p < research is needed to clarify the minimal effective dose
0.05). Ten patients in each group drank large amounts and the minimum intake period of HA.
of water to examine the turnover of HA in synovial fluid.
The turnover rate was significantly decreased in the HA Safety of HA
group compared with that in the placebo group (p = Chicken comb, which contains highly concentrated HA
0.046). Consequently, this report demonstrated that the has long been consumed in Germany, France, and
oral intake of HA is useful for treating obese patients China. In Japan, HA dietary supplements have been
with OA. available since 1992. HA is approved as a healthy raw
In 2015, Jensen et al. [46] conducted a study in which material of new resource food in China, as a food addi-
72 patients with knee OA (HA group, n = 37; placebo tive and healthy functional food in Korea, and as a food
group, n = 35, mean age, 47.2) were administered a li- additive in Japan. In addition, HA is sold globally in
quid HA mixture or placebo for 4 weeks. For the first countries such as the US, Canada, Italy, and Belgium
2 weeks, the HA mixture was given at 45 mL/day (HA and no adverse event caused by HA has been reported.
content, 225 mg/day) and it was given at 30 mL/day Several safety studies of HA have been conducted
(HA content, 150 mg/day) for the next 2 weeks. After (Table 2). No toxicity was observed in single-dose tox-
2 weeks of intake, knee pain as evaluated using a VAS icity studies [48–50], repeated-dose toxicity studies [51–
was significantly improved in the HA group compared 61], reproductive and developmental toxicity studies
to baseline (p < 0.05). On the contrary, no improvement [62–69], mutagenicity tests [70–74], and antigenicity
in knee pain was recorded in the placebo group. Thus, studies [75, 76]. It is reported that exogenous HA such
oral HA can relieve knee pain. us that administered orally is not harmful to human can-
Among all the studies on clinical trials from 2008 to cer cells [77]. A 12 month clinical study identified no ad-
2015, seven studies involved the treatment of the verse event attributable to HA [41]. Orally administrated
Oe et al. Nutrition Journal (2016) 15:11 Page 8 of 10

HA is used by body tissues and approximately 90 % of 6. Fraser JR, Laurent TC, Laurent UB. Hyaluronan: its nature, distribution,
exogenous HA is metabolized and released in expiration functions and turnover. J Intern Med. 1997;242:27–33.
7. Laurent TC, Fraser JR. Hyaluronan. FASEB J. 1992;6:2397–402.
and urine; thus, the polysaccharide does not accumulate 8. Balazs EA, Watson D, Duff IF, Roseman S. Hyaluronic acid in synovial fluid. I.
at excessive levels in the body [78]. There were no ad- Molecular parameters of hyaluronic acid in normal and arthritic human
verse events attributable to HA in any of the aforemen- fluids. Arthritis Rheum. 1967;10:357–76.
9. Seror J, Merkher Y, Kampf N, Collinson L, Day AJ, Maroudas A, et al. Articular
tioned clinical trials [34–44]. Based on these factors, HA cartilage proteoglycans as boundary lubricants: structure and frictional
can be considered as a safe food material. interaction of surface-attached hyaluronan and hyaluronan–aggrecan
complexes. Biomacromolecules. 2011;12:3432–43.
10. Bagga H, Burkhardt D, Sambrook P, March L. Longterm effects of
Conclusion intraarticular hyaluronan on synovial fluid in osteoarthritis of the knee. J
The population is aging rapidly around the world. In Rheumatol. 2006;33:946–50.
addition to medical treatment, self-medication is more 11. Shimizu C, Kubo T, Hirasawa Y, Coutts RD, Amiel D. Histomorphometric and
biochemical effect of various hyaluronans on early osteoarthritis. J
commonly practiced to reduce patient burden and en- Rheumatol. 1998;25:1813–9.
hance QOL. HA is a safe raw material and the efficacy 12. Listrat V, Ayral X, Patarnello F, Bonvarlet JP, Simonnet J, Amor B, et al.
of oral HA in relieving knee pain was demonstrated in Arthroscopic evaluation of potential structure modifying activity of
hyaluronan (Hyalgan) in osteoarthritis of the knee. Osteoarthritis Cartilage.
several clinical trials. HA as a dietary supplement ex- 1997;5:153–60.
hibits mild efficacy and no side effects. By utilizing these 13. Ghosh P, Holbert C, Read R, Armstrong S. Hyaluronic acid (hyaluronan) in
characteristics, HA dietary supplements provide at least experimental osteoarthritis. J Rheumatol Suppl. 1995;43:155–7.
14. Schiavinato A, Lini E, Guidolin D, Pezzoli G, Botti P, Martelli M, et al.
some possibility for the treatment and prevention of ser- Intraarticular sodium hyaluronate injections in the Pond-Nuki experimental
ious conditions in patients with OA exhibiting mild knee model of osteoarthritis in dogs. II. Morphological findings. Clin Orthop Relat
pain. This review may improve the understanding of HA Res. 1989;241:286–99.
15. Yoshimi T, Kikuchi T, Obara T, Yamaguchi T, Sakakibara Y, Itoh H, et al.
dietary supplements and it is expected that HA will
Effects of high-molecular-weight sodium hyaluronate on experimental
emerge as a modality for treating knee pain that can be osteoarthrosis induced by the resection of rabbit anterior cruciate ligament.
safely used by patients. Clin Orthop Relat Res. 1994;298:296–304.
16. Sakakibara Y, Miura T, Iwata H, Kikuchi T, Yamaguchi T, Yoshimi T, et al.
Abbreviations Effect of high-molecular-weight sodium hyaluronate on immobilized rabbit
HA: hyaluronan; IL-10: interleukin-10; JKOM: the Japanese Knee Osteoarthritis knee. Clin Orthop Rel Res. 1994;299:282–92.
Measure; LD50: lethal dose 50 %; MW: mean molecular weight; N/A: Not 17. Fu LL, Maffulli N, Chan KM. Intra-articular hyaluronic acid following knee
available; NOAEL: No Observed Adverse Effect Level; OA: osteoarthritis; immobilisation for 6 weeks in rabbits. Clin Rheumatol. 2001;20(2):98–103.
PCT: paracetamol; SF-36v2: the Short Form-36 Acute US Version 2; 18. Yoshioka M, Shimizu C, Harwood FL, Coutts RD, Amiel D. The effects of
SOCS3: Suppresor Of Cytokine Signaling3; TLR-4: Toll-Like Recepter 4; hyaluronan during the development of osteoarthritis. Osteoarthritis
WOMAC: The Western Ontario and McMaster Universities Osteoarthritis Cartilage. 1997;5:251–60.
Index. 19. Shimizu C, Yoshioka M, Coutts RD, Harwood FL, Kubo T, Hirasawa Y, et al.
Long-term effects of hyaluronan on experimental osteoarthritis in the rabbit
Competing interests knee. Osteoarthritis Cartilage. 1998;6:1–9.
The authors declare that they have no competing interests. 20. Wenz W, Breusch SJ, Graf J, Stratmann U. Ultrastructural findings after
intraarticular application of hyaluronan in a canine model of arthropathy. J
Authors’ contributions Orthop Res. 2000;18:604–12.
All authors managed the literature searches, formulated the hypothesis, and 21. Frizziero L, Govoni E, Bacchini P. Intra-articular hyaluronic acid in the
contributed to the discussion and conclusions. All authors read and treatment of osteoarthritis of the knee: clinical and morphological study.
approved the final manuscript. Clin ExpRheumatol. 1998;16:441–9.
22. Pasquali Ronchetti I, Guerra D, Taparelli F, Boraldi F, Bergamini G, Mori G, et
Author details al. Morphological analysis of knee synovial membrane biopsies from a
1
R&D Division, Kewpie Corporation, 2-5-7, Sengawa-cho, Chofu-shi, Tokyo, randomized controlled clinical study comparing the effects of sodium
Japan. 2Tokyo Yamate Medical Center, 3-22-1 Hyakunin-cho, Shinjyuku-ku, hyaluronate (Hyalgan) and methylprednisolone acetate (Depomedrol) in
Tokyo, Japan. 3Gate Town Hospital, 1-6-19, Sekimachi-kita, Nerima-ku, Tokyo, osteoarthritis. Rheumatology. 2001;40:158–69.
Japan. 4Shiba Palace Clinic, 1-9-10 Hamamatsucho, Minato-ku, Tokyo, Japan. 23. Guidolin DD, Ronchetti IP, Lini E, Guerra D, Frizziero L. Morphological
5
SOUKEN Corporation, 1-9-10, Hamamatsu-cho, Minato-ku, Tokyo, Japan. analysis of articular cartilage biopsies from a randomized, clinical study
6
Department of Life Science, Graduate School of Arts and Sciences, The comparing the effects of 500–730 kDa sodium hyaluronate (Hyalgan) and
University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo, Japan. methylprednisolone acetate on primary osteoarthritis of the knee.
Osteoarthritis Cartilage. 2001;9:371–81.
Received: 9 November 2015 Accepted: 19 January 2016 24. Maniwa S, Ochi M, Motomura T, Nishikori T, Chen J, Naora H. Effects
of hyaluronic acid and basic fibroblast growth factor on motility of
chondrocytes and synovial cells in culture. Acta Orthop Scand.
References 2001;72:299–303.
1. Centers for disease control and prevention. Prevalence of arthritis-United 25. Asari A, Miyauchi S, Matsuzaka S, Ito T, Kominami E, Uchiyama Y. Molecular
States, 1997. MMWR Morb Mortal Wkly Rep. 2001;50:334–6. eight-dependent effects of hyaluronate on the arthritic synovium. Arch
2. Peyron JG, Balazs EA. Preliminary clinical assessment of Na-hyaluronate Histol Cytol. 1998;61:125–35.
injection into human arthritic joints. Pathol Biol. 1974;22:731–6. 26. Day R, Brooks P, Conaghan PG, Petersen M. A double blind,
3. Nakamura H, Nishioka K. Effects of glucosamine/chondroitin supplement on randomized, multicenter, parallel group study of the effectiveness and
osteoarthritis: involvement of PGE2 and YKL-40. Jpn J Joint Dis. 2002;11: tolerance of intraarticular hyaluronan in osteoarthritis of the knee. J
175–84. Rheumatol. 2004;31:775–82.
4. Dennis Y, Wen MD. Intra-articular hyaluronic acid injections for knee 27. Santangelo KS, Johnson AL, Ruppert AS, Bertone AL. Effects of hyaluronan
osteoarthritis. Am Fam Physician. 2000;62:565–70. treatment on lipopolysaccharide-challenged fibroblast-like synovial cells.
5. Bucci RL, Turpin AA. Will the real hyaluronan please stand up. JAN. 2004;54 Arthritis Res Ther. 2007;9:R1.
Oe et al. Nutrition Journal (2016) 15:11 Page 9 of 10

28. Ikeda K. Changes in synovial fluid markers and clinical effects after intra- 47. Raheem KA, Marei WF, Mifsud K, Khalid M, Wathes DC, Fouladi-Nashta AA.
articular injection of sodium hyaluronate : with particular reference to the Regulation of the hyaluronan system in ovine endometrium by ovarian
anti-inflammatory effect in terms of prostaglandin E2 concentration. Tokyo steroids. Reproduction. 2013;145:491–504.
Women's Medical University. 1998;68:22–36 (in Japanese). 48. Nagano K, Goto S, Abe Y, Yamaguchi T. Acute toxicity test of sodium
29. Hisada N, Satsu H, Mori A, Totsuka M, Kamei J, Nozawa T, et al. Low- hyaluronate. Jpn Pharmacol Ther. 1984;12:37–45 (in Japanese).
Molecular-Weight Hyaluronan Permeates through Human Intestinal Caco-2 49. Wakizawa Y, Eiro H, Matsumoto H, Yamagi M, Yoshihara K, Kuramoto M, et
Cell Monolayers via the Paracellular Pathway. Biosci Biotechnol Biochem. al. Acute toxicity study of sodium yaluronate(SL-1010) in mice. Jpn
2008;72:1111–4. Pharmacol Ther. 1991;19:7–12 (in Japanese).
30. Kurihara H, Kawada C, Yoshida H, Kimura M, Kanemitsu T, Masuda Y, et al. 50. Morita H, Kawakami Y, Shimomura K, Sunaga M. Acute toxicity study of
Absorption and effect on the skin of oral hyaluronan, 14th Scientific sodium yaluronate(SL-1010) in rats and dogs. Jpn Pharmacol Ther. 1991;19:
meeting of Japanese society of anti-aging medicine. 2014 (in Japanese). 13–8 (in Japanese).
31. Ishibashi G, Yamagata T, Rikitake S, Takiguchi Y. Digestion and fermentation 51. Morita H, Kawakami Y, Suzuki S, Hirata M, Koizumi H. Thirteen-week
of hyaluronic acid. Journal for the integrated study of dietary habits. 2002; subcutoneous toxicity study on sodium hyaluronate(SL-1010) with 4-week
13(2):107–11 (in Japanese). recovery test in rats. Jpn Pharmacol Ther. 1991;19:19–52 (in Japanese).
32. Balogh L, Polyak A, Mathe D, Kiraly R, Thuroczy J, Terez M, et al. Absorption, 52. Morita H, Shimomura K, Suzuki S, Hirata M, Koizumi H. Thirteen-week
uptake and tissue affinity of high-molecular-weight hyaluronan after oral subcutoneous toxicity study on sodium hyaluronate(SL-1010) with 4-week
administration in rats and dogs,”. J Agric Food Chem. 2008;56:10582–93. recovery test in dogs. Jpn Pharmacol Ther. 1991;19:53–80 (in Japanese).
33. Asari A, Kanemitsu T, Kurihara H. Oral administration of high molecular 53. Yang G, Zhang T, Guo X, Qu B, Song Y. Toxicological test on calcium
weight hyaluronan (900 kDa) controls immune system via Toll-like receptor hyaluronate. Food and Drug. 2009;11:46–8 (in Chinese).
4 in the intestinal epithelium. J Biol Chem. 2010;285:24751–8. 54. Tan H, Zhao P, Ma Y, Li F, Fu W, He L, et al. Study on toxicology of sodium
34. Hatayama T, Nagano M, Yamaguchi N, Kumagai S, Ohnuki K. The effect of a hyaluronate. Applied Prev Med. 2008;14:296–9 (in Chinese).
supplement on knee pain and discomfort evaluated by visual analogue 55. Guo F, Gao P, Geng G, Wang H, Liu H, Zhi Y, et al. Study on toxicology of
scale(VAS): a randomized, double-blind, placebo-controlled study. Kenko- sodium hyaluronate. Chinese J Biochem Pharm. 2010;31:316–9 (in Chinese).
shien. 2008;10:13–7 (in Japanese). 56. Oe M, Yoshida T, Kanemitsu T, Matsuoka R, Masuda Y. Repeated 28-day oral
35. Kalman DS, Heimer M, Valdeon A, Schwartz H, Sheldon E. Effect of a natural toxicological study of hyaluronic acid in rats. Pharmacometrics. 2011;81:11–
extract of chicken combs with a high content of hyaluronic acid (Hyal- 21 (in Japanese).
Joint®) on pain relief and quality of life in subjects with knee osteoarthritis: a 57. Takahashi J, Seto A, Ishizuka T, Kimura K, Koyama M, Kashiwabara J, et al.
pilot randomized double-blind placebo-controlled trial. Nutr. J.. 2008;7 Repeated 90-day oral toxicological study of hyaluronic acid in rats. Jpn J
36. Iwaso H, Sato T. Examination of the efficacy and safety of oral Med Pharm Sci. 1985;13:1458–79 (in Japanese).
administration of Hyabest® (J)-highly-pure hyaluronic acid for knee joint 58. Hasegawa T, Miyoshi K, Nomura A, Nakazawa M. Subacute toxicity test on
pain. J Jpn Soc Clin Sports Med. 2009;58:566–72 (in Japanese). sodium hyaluronate(SPH) in rats by intraperitoneal Administration for
37. Sato T, Iwaso H. An Effectiveness study of hyaluronic acid Hyabest® (J) in 3 months and recovery test. Pharmacometrics. 1984;28:1021–40 (in
the treatment of osteoarthritis of the knee on the patients in the United Japanese).
State. J New Rem & Clin. 2009;58:551–8. 59. Kato T, Nakajima S, Kurihara H, Asari A, Sekiguchi T, Sunose A. Preliminary
38. Möller I, Martinez-Puig D, Chetrit C. Oral administration of a natural extract study for the toxicity study on sodium hyaluronate(Na-HA) in rats by
rich in hyaluronic acid for the treatment of knee OA with synovitis: a repeated oral administration for 13 weeks. The Clinical Report. 1993;27:
retrospective cohort study. Clin Nutr Suppl. 2009;4:171–2. 5809–30.
39. Nagaoka I, Nabeshima K, Murakami S, Yamamoto T, Watanabe K, Tomonaga 60. Ishihara M, Inoue T, Ishiyama Y, Sakata T, Ichikawa A, Funahashi N, et al.
A, et al. Evaluation of the effects of a supplementary diet containing Toxicity study on sodium hyaluronate(Na-HA) in Rats by repeated oral
chicken comb extract on symptoms and cartilage metabolism in patients administration for 90 days followed by 28-day recovery study.
with knee osteoarthritis. Exp Ther Med. 2010;1:817–27. Pharmacometrics. 1996;51:97–113.
40. Yoshimura M, Aoba Y, Watari T, Momomura R, Watanabe K, Tomonaga A, et 61. Miyoshi K, Hasegawa T, Nakazawa M. Chronic toxicity test on sodium
al. Evaluation of the effect of a chicken comb extract-containing hyaluronate(SPH) in beagle dogs by intra-articular administration for
supplement on cartilage and bone metabolism in athletes. Exp Ther Med. 6 months and recovery test (1) General findings. Pharmacometrics. 1985;29:
2012;4:577–80. 49–81 (in Japanese).
41. Tashiro T, Seino S, Sato T, Matsuoka R, Masuda Y, Fukui N. Oral 62. Ono C, Iwama A, Nakajima Y, Kitsuya A, Nakamura T. Reproductive and
administration of polymer hyaluronic acid alleviates symptoms of knee developmental toxicity study on sodium hyaluronate(SH)-(1)Study on
osteoarthritis: a double-blind, placebo-controlled study over a 12-month subcutaneous administration to rats during the period of organogenesis-.
period. Sci. World J.. 2012;doi:10.1100/2012/167928. Jpn Pharmacol Ther. 1992;20:11–26 (in Japanese).
42. Martinez-Puig D, Möller I, Fernández C, Chetrit C. Efficacy of oral 63. Ono C, Fujiwara Y, Koura S, Tsuchida H, Nakamura T. Reproductive and
administration of yoghurt supplemented with a preparation containing developmental toxicity study on sodium hyaluronate(SH)-(2)Study on
hyaluronic acid (Mobilee™) in adults with mild joint discomfort: a subcutaneous administration to rats prior to and in the early stages of
randomized, double-blind, placebo controlled intervention study. Mediterr J pregnancy-. Jpn Pharmacol Ther. 1992;20:27–35 (in Japanese).
Nutr Metab. 2013;6:63–8. 64. Ono C, Ishitobi H, Kuzuoka K, Konagai S, Nakamura T. Reproductive and
43. Moriña D, Solà R, Valls RM, López de Frutos V, Montero M, Giralt M, et al. developmental toxicity study on sodium hyaluronate(SH)-(3)Study on
Efficacy of a Low-fat Yogurt Supplemented with a Rooster Comb Extract on subcutaneous administration to rats during the perinatal and lactation
Joint Function in Mild Knee Pain Patients: A Subject-level Meta-analysis. Ann period-. Jpn Pharmacol Ther. 1992;20:37–50 (in Japanese).
Nutr Metab. 2013;63:1386. 65. Guo F, Geng G, Wang H, Liu H, Zhi Y. Teratogenicity test of sodium
44. Sánchez J, Bonet ML, Keijer J, van Schothorst EM, Mölller I, Chetrit C, et al. hyaluronate. Food Drug. 2010;12:321–3 (in Chinese).
Blood cells transcriptomics as source of potential biomarkers of articular 66. Tanaka C, Sasa H, Hirama S, Inaba T, Tokunaga S, Eiro H, et al. Reproductive
health improvement: effects of oral intake of a rooster combs extract rich in and divelopmental toxicity studies of sodium hyaluronate(SL-1010)(I)-
hyaluronic acid. Genes Nutr. 2014;9:417. Fertility study in rats-. Jpn Pharmacol Ther. 1991;19:81–92 (in Japanese).
45. Nelson FR, Zvirbulis RA, Zonca B, Li KW, Turner SM, Pasierb M, et al. The 67. Tanaka C, Sasa H, Hirama S, Inaba T, Tokunaga S, Eiro H, et al. Reproductive
effects of an oral preparation containing hyaluronic acid (Oralvisc®) on and divelopmental toxicity studies of sodium hyaluronate(SL-1010)(II)-
obese knee osteoarthritis patients determined by pain, function, bradykinin, Teratogenicity study in rats-. Jpn Pharmacol Ther. 1991;19:93–110 (in
leptin, inflammatory cytokines, and heavy water analyses. Rheumatol Int. Japanese).
2015;35:43–52. 68. Wada K, Hashimato Y, Mizutani M, Chiaki T. Reproductive and divelopmental
46. Jensen GS, Attridge VL, Lenninger MR, Benson KF. Oral intake of a liquid toxicity studies of sodium hyaluronate(SL-1010)(III)-Teratogenicity study in
high-molecular-weight hyaluronan associated with relief of chronic pain rabbits-. Jpn Pharmacol Ther. 1991;19:111–9 (in Japanese).
and reduced use of pain medication: results of a randomized, placebo- 69. Tateda C, Nagaoka S, Nagai T, Nakamura T. Reproductive and
controlled double-blind pilot study. J Med Food. 2015;18:95–101. developmental toxicity study on sodium hyaluronate(SH)-(4)Study on
Oe et al. Nutrition Journal (2016) 15:11 Page 10 of 10

subcutaneous administration to rabbits during the period of

organogenesis-. Jpn Pharmacol Ther. 1992;20:51–8 (in Japanese).
70. Sugiyama C, Yagame O. Mutagenicity tests on sodium hyaluronate(SL-1010)(I)-
Reverse mutation test in bacteria-. Jpn Pharmacol Ther. 1991;19:177–81
(in Japanese).
71. Sugiyama C, Kobayashi H. Mutagenicity tests on sodium hyaluronate(SL-1010)(II)-
In vitro cytogenetic test-. Jpn Pharmacol Ther. 1991;19:183–91 (in Japanese).
72. Onishi M, Nagata T, Saigou K, Sameshima H, Nagata R. Mutagenicity studies
of sodium hyaluronate(SH). Jpn Pharmacol Ther. 1992;20:65–72 (in
Japanese).
73. Hara T, Horiya N, Katoh M, Shibuya T. Micronucleus test in mice on sodium
hyaluronate(SL-1010). Jpn Pharmacol Ther. 1991;19:193–7 (in Japanese).
74. Aruga F, Miwa Y, Fuzimura T, Ohta S. Micronucleus test of sodium
hyaluronate(SH) with mice. Jpn Pharmacol Ther. 1992;20:73–5 (in Japanese).
75. Takemoto M, Ohzone Y, Asahi K. Antigenicity test of sodium
hyaluronate(SH). Jpn Pharmacol Ther. 1992;20:59–64 (in Japanese).
76. Kameji R, Itokawa S, Yamawaki C, Tsujita K, Hara Y, Shukunobe K.
Antigenicity tests on sodium hyaluronate(SL-1010) in rabbit. Jpn Pharmacol
Ther. 1991;19:159–75 (in Japanese).
77. Seino S, Takashita F, Asari A, Masuda Y, Kunou M, Ochiya T. No Influence of
exogenous hyaluronan on the behavior of human cancer cells or
endothelial cell capillary formation. J Food Sci. 2014;79:1469–75.
78. Oe M, Mitsugi K, Odanaka W. Orally Administered Hyaluronic Acid Migrates
into the Skin of Rats. Sci World J. 2014;2014:378024.

Submit your next manuscript to BioMed Central

and we will help you at every step:
• We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
• We provide round the clock customer support
• Convenient online submission
• Thorough peer review
• Inclusion in PubMed and all major indexing services
• Maximum visibility for your research

Submit your manuscript at

www.biomedcentral.com/submit