Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : November 11, 2017
Accepted : January 10, 2018
Published online : March 31, 2018

Review article

Hyaluronic acid and articular cartilage
Mari Ogura 1, 2), Wakako Takabe 1), Masayuki Yagi 1), Yoshikazu Yonei 1)
1) Anti-Aging Medical Research Center and Glycative Stress Research Center, Faculty of Life and Medical Sciences,
Doshisha University, Kyoto, Japan2) Kyoto Bunkyo Junior College, Kyoto, Japan

Abstract

Osteoarthritis is a typical locomotive-syndrome-induced disease. Osteoarthritis (hereinafter referred as “OA”) is defined
as a progressive disease characterized by cartilage degeneration; due to the changes in regeneration and proliferation ofarticular cartilage and bone, and secondary synovitis induced by degeneration of articular cartilage and physical abrasion. It
has been clarified that aging, physical stress and oxidative stress induce the composition changes of cartilage matrix substrateand consequently, disorders of cartilage matrix are induced. This review outlines mechanism how disorders of cartilage
matrix are induced in an early stage of OA onset, focusing on hyaluronic acid (hereinafter referred as “HA”), which is acomponent of matrix. It has been reported that based on the early stages of OA patients and OA model animals, HA becomesfragmented, cartilaginous tissues migrate, and matrix-degrading enzymes are activated, which is normally restricted by HA.
As the result, disorders arise in articular cartilage substrate matrix. Administration of exogenous hyaluronic acid (HA) can
mitigate these reactions to some extent. The damaged articular cartilage releases proinflammatory cytokine and matrixdegrading enzymes such as HA degrading enzyme and aggrecan degrading enzyme are induced. It appears that exogenous
hyaluronic acid has a function of inhibitory against induction of matrix-degrading enzyme and mitigate migration of matrix
from cartilage (loss of HA and aggrecan). However, American Academy of Orthopaedic Surgeons (AAOS) announced apolicy that intra-articular administration of HA is not recommended. For prevention of OA, it is essential to clarify thedisorder mechanisms of cartilage matrix, identify initial symptoms in as early stage as possible. Therefore, countermeasuresare enabled to be conducted to prevent of onset and progression of OA in the early stage.

KEY WORDS: hyaluronic acid, hyaluronan, cartilage degeneration , osteoarthritis, aggrecan

Introduction

Our research center has participated in SIP (CrossMinisterial Strategic Innovation Promotion Program).
SIP promotes industry-academia collaboration to developfunctional agricultural, forest and fishery products andfood products for the next generation, which are effective tomaintain function for physical locomotion. With this goal,
multiple major food companies in Japan provide potential orpromising food ingredients, participate with their technology
and industry-academia research teams has been established.
The background of this project is that the elderly in this
rapidly increasing aging society, Japan, have compellingneeds to maintain and improve the quality of life (QOL),
which could result in the energy source of whole the society.
These days the elderly persons who have certifications ofContact Address: Professor Yoshikazu Yonei, MD, PhD
Glycative Stress Research Center,
Faculty of Life and Medical Sciences, Doshisha University
1-3, Tatara Miyakodani, Kyotanabe, Kyoto, 610-0394 Japan
Tel & FAX: +81- 774 - 65 - 6394 eMail: yyonei@mail.doshisha.ac.jp
Co-authors: Ogura M, m-ogura@po.kbu.ac.jp ;Takabe W, wtakabe@mail.doshisha.ac.jp ; Yagi M, yagi@yonei-labo.com

needed long-term-care have rapidly increased in number,
who have difficulties to live a self-supported life, as theirfunctions of daily life motion (physical locomotion)
are lowered. Thus, it is extremely urgent to tackle forcountermeasures for prevention.
A risk of locomotion syndrome is osteoarthritis (OA).
It enables to prevent of the onset and progression of OA
that signs of anomaly in articular cartilage substrate matrix
should be found at an early point. Furthermore, Hyaluronic
acid (HA) and glucosamine are effective as functional
food, which have been produced on a commercial basis
focusing OA-induced knee pain, arthralgia and lower backpain, lumbago. It has been reported recently that glycative
stress is involved in the mechanism of rheumatoid arthritis_ 12 _

Glycative Stress Research 2018; 5 (1): 012-020(c) Society for Glycative Stress ResearchGlycative Stress Research(hereinafter referred as “ RA” ) 1-5) and OA occurrence
mechanism. Further, it is suggested that HA is related tothe mechanism. This paper outlines the knowledge andinformation related to HA and OA. The tendency of the termof HA in Europe and the United States is to designate nothyaluronic acid but hyaluronan, which means scaffold. In thispaper, both of the terms are described as HA.

Aggrecan in articulation
Articular cartilage structure is shown in Fig. 1. Aggrecan,
which is a member of proteoglycan family, is a main
component of cartilage, filling intercellular space. Aggrecan
is a compound material of glucose and protein with amolecular weight of approximately several hundred thousand
Dalton and has a diversified functions as a biogenic
substances. Producing matrix with collagen and HA ( Fig. 2 ),aggrecan maintains cartilage and other articulation tissues( Fig. 3 ) . These components are functional components
to maintain and repair tissues, playing roles of systemconstitution and transmission material. The polysaccharide
moiety is called glycosaminoglycan, which is composedof chondroitin sulfate, heparan sulfate, keratan sulfate anddermatan sulfate 6-8).
As for distributions, HA are mainly is distributed insurface cell space and also present in connective tissue of ahollow space surrounding blood vessels. Chondroitin sulfate
is distributed in highly fibrotic parts and connective tissues
surrounding blood vessels. Dermatan sulfate is distributed insurface interstitial tissues and blood vessel endothelial celland heparan sulfate is distributed in blood vessel endothelial
cell 7).
Articular inflammation was induced by rheumatoid

arthritis (RA) and osteoarthritis (OA) and also aging-caused
regressive changes occur. Consequently, aggrecan and HA
are localized and change qualitatively and quantitatively.
Aggrecan comprises two groups; one contains chondroitin
sulfate affluently and the other contains keratin sulfate
affluently. The latter increases along with aging and changes
the association process state with HA 9).
It is evident that senescence-accelerated mouse (SAMP8
and SAMR1) researches shows decreased dyeability oftype Ⅰ collagen and HA-binding protein ( HABP) andincreased dyeability of type Ⅱ and type X collagen in thetemporomandibular articulationcaput mandibulae cartilage 10),which are induced by aging.
Degradation of agrin matrix is related to the process inarticular cartilage destruction of OA. Articular extracellularmatrix constructs a higher order structure by the interactionof HA-aggrecan network and type Ⅱ collagen fiber 11). HA
binds to aggrecan to protect articular cartilage from load
and to exert lubrication effect on the surface of cartilage 12).
In the articular cartilage destruction process, HA-aggrecan
network is degraded and then collagen fiber degradation
is proceeded. Extracellular matrix is degraded with MMP(matrix metalloproteinase) and ADAMTS (a disintegrin andmetalloproteinase with thrombospondin motifs) of a genefamily playing a significant role 11). In an early stage of thearticular cartilage destruction, ADAMTS4 and ADAMTS5,
which are called aggrecanase, play a leading part of aggrecan
degradation and HYBID (hyaluronan-binding protein
involved in hyaluronan depolymerization: KIAA1199) plays
an important role of HA degradation.
In this manner, metabolism of cartilage and bone issupported by the balance of generation and degradation of
matrix. Mechanical stress, oxidative stress and inflammationinduce degrading enzymes, which causes high turnover
metabolic bone deterioration.

Fig. 1. Schema of cartilage tissues.

Cartilage tissues areresilientfibrous connective tissues and consist of cartilage cells,
cartilage matrix of intercellular substances and perichondrium covering cartilage.

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Hyaluronic acid and articular cartilage

Fig. 2. Structure of hyaluronic acid.

HA has thestructure of bound disaccharide unit of N-acetylglucosamine and D-glucuronic acid, with a high molecularweight, of more than several hundred thousand, often reaching millions. HA, hyaluronic acid.

Fig. 3. Configuration of articular cartilage matrix..

Aggrecan is a cartilage-specific proteoglycan core protein or chondroitin sulfate proteoglycan/keratan sulfate proteoglycan
with a molecular weight of 2,500 kDa. Aggrecan is formed in a manner where core protein binds with keratan sulfate/chondroitin sulfate. Aggrecan bindswith hyaluronic acid through HAPLN1/Link protein1.Hyaluronic acid functions as
scaffoldingfor thematrix and binds to cartilage tissues through CD44. Hyaluronic acid, also through integrin and ICAM1, binds to cartilage tissues. Normal hyaline cartilage consists of type Ⅱ collagen microfibril and aggrecan. HAPLN1,
hyaluronan and proteoglycan link protein 1; ICAM-1, intercellular adhesion molecule-1.

Hyaluronic acid
HA is widely distributed throughout the body tissues andorgans. HA is produced from articular synovia in articulationand is a main component of synovial fluid. Other than that,
HA is the component of articular cartilage aggrcan 13). Water
retentively 14-16), lubricant of articulation 12, 17-21), intercellularadhesion 22, 23) and immune regulatory activity 24) are significant
roles for HA to play.
HA exists in human blood and is clinically used as
an index for osteoarthritis (OA) and rheumatoid arthritis(RA) and also an index of hepatic fibrogenesis. Bloodconcentration of HAindicates as follows;
Healthy persons; 30.1 ± 16 ng/mL, RA patients; 220 ± 204
ng/mL, OA patients; 55.3 ± 31 ng/mL 25)

Healthy persons; 42.2 ± 45.6 ng/mL, RA patients; 372.5 ±
401.2 ng/mL 26)
Healthy persons; 33.7 ± 24.2 ng/mL, RA patients; 350.7 ±689.5 ng/mL 27)
RA patients showed higher values. Blood concentration of
HA has correlation with CRP, WBC and progression stage.
HA serum concentration increasesin OA along with pain
symptoms and progression of diseases and increases in RA
along with advancement of articulation destruction 13).
HA concentration of synovial fluid of healthy persons (atthe average age of 27.5) is 3.4 ng/mL 28). HA concentration ofsynovial fluid decreases along with aging in healthy persons.
However, HA concentration of synovial fluid of OA patients
does not show changes due to aging 29-31).
OA patients tend to have lower HA concentration of_ 14 _Glycative Stress Research

synovial fluid than healthy persons. When the amount ofsynovial fluid increases, HA concentration decreases 32, 33).
The reason is assumed that in OA, cartilage destruction
lowers HA production.
HA concentration of synovial fluid sometimes indicateslower in RA than in OA 34). HA concentration of synovial
tissue extracted solution is higher in RA than in OA, whichis induced by the HA production acceleration of synovial
tissues proliferated due to RA 35, 36). In the early and active
stage cases of RA patients’ synovial cells, the production of
hyaluronidase, which is an enzyme to cleave HA, is elevated.
In cases where hyaluronidase activities are high, HA isdegraded to be lowered in quantity and quality, which causes
low-viscosity synovial fluid 37).

Cell culture Experiments
Researches which has been reported are expoundedin three categories, cell culture experiments, animal
experiments and clinical trials. Cartilage matrix degradation
product is called matrikine, which induces matrixdegrading enzyme to destruct cartilage. As a result, articulardisorders are induced. The macromolecular hyaluronic
acid infiltrates damaged cartilage of OA and RA and binds
with HA receptor on the surface of cartilage cells andinhibits cytokine and matrikines from catabolism. HA has apharmacological effect to prevent cartilage destruction 38).
Experiments have been conducted to stimulate cultured
cartilage cells and to reproduce OA. Cartilage cells shows
varied cell responses in being stimulated by oxidative stress(i. e.oxygen-derived free radical [ODFR], inflammation,
inflammatory cytokine (IL-1α, IL-1β, TNFα), collagenfragment derived from cell destruction and RA synovial
fluid. The stimulus activates p38MAP kinase, activates
nuclear factor-kappa B (NF-κB), promotes phosphorylationand nuclear translocation, produces MMP (MMP-1, MMP-3,
and MMP-13) and increases secretion extracellular 39-41). A
kind of C-C chemokine, RANTES 42), ADAMTS4 43), which
has degradation activity of aggrecan and prostromelysin 44),are materials which are produced by stimulus of IL-1α. The
production of TIMP-1, which is a tissue inhibitor and has afunction to protect cartilage, is inhibited 44).
IL-1β promotes the destruction of cartilage matrix.
Furthermore, IL-1β upregulates the expression of RHAMM
mRNA of cartilage tissues 45), and decreases the expressionof α2(VI), α1(II), α1(IX) and α2(XI) collagen genes, which
involve in the production of collagen 46,47). IL-1β affects
cartilage tissues and increases the expression of CD44 48, 49).
In cartilage tissue, inductive NO synthetic enzyme(iNOS) is induced by the stimuli of fibronectin fragment,
which contains binding-site of C-terminal heparin and the
production of NO increases 50). Changes in concentration of
intracellular calcium ([Ca2+]i) involves theses reactions 51).
HA acts inhibitively these reactions.
In normal cartilage tissues, HA has a high affinity
with cartilage surfaces and cannot be absorbed; HA can beabsorbed into the deep zone of OA cartilage 52).
HA has very little effect on the synthesis of aggrecan in
cartilage tissues 53, 54), but inhibits the migration of aggrecan
from cartilage tissues 52, 54, 55). Rooster comb-derived HA isalso recognized to inhibit the migration of aggrecan 54).
HA inhibits the migration of aggrecan and the productionof MMP through CD44, which is a main receptor on thesurface of cartilage tissues 39). In cartilage tissues, celladhesion molecules (ICAM-1) are regarded as HA receptors.
HA inhibitory effects against the production of MMP, whichis collagen-fragment induced, are exerted through ICAM-1 56).
HA promotes the production of the tissue inhibitors
metalloproteinase-1 and TIMP-1 in cartilage tissues 57). HA
increases stromelysin activity but decreases the fractionof stromelysin/TIMP-1 57). HA inhibits the fibronectin
segregation from cartilage tissues 58).
It has been reported that exogenous hyaluronic acidinhibits the production of ADAMTS4 due to cartilage
tissue IL-1α stimuli 43), and inhibits the decline of the gene
expression of collagen alpha-2(VI) due to IL-1α stimuli 46, 47).
Comparing normal cartilage and OA cartilage, in
OA, the production of NO by HBFN-f is exacerbated andCD44 is upregulated 59). HA inhibits the acceleration of NO
production through the coaction with CD44.
The expression of RHAMM mRNA is upregulated by
IL-1β and TNF-α in cartilage tissues 60). In an examinationof HA receptors (CD44) and receptors for hyaluronanmediated motility (RHAMM) in knee synovial membranetissues of OA patients, the results showed that progressive
OA patients had more strongly dyed cells than healthy
subjects. Furthermore, expressions of both CD44 andRHAMM were stronger in OA patients than healthy
subjects, using Western Blotting Methods 61). This finding
revealed that the onset and progression of OA involved thechanges in the level of hyaluronan-binding protein.
HA also affects the differentiation and maturity ofcartilage cells 62). An appropriate amount of HA promotes
the differentiation of cartilage, but adding high concentrationof HA in other conditions inhibits the differentiation of
cartilage. In the differentiation processfrom cartilage stem
cell to cartilage cell, gene expressions related to chondrocyte
differentiation, aggrecan and Sox9, are elevated, and gene
expressions of CD44, TGF-β1 and hyaluronic acid synthetic
enzyme 2 are elevated. The quantity of HA and GAG in culture
supernatant are increased 63). It is evident that exogenous
hyaluronic acid promotes the production of HA by themechanism of autocrine and paracrine and also promotably
affects chondrocyte differentiation.
Depolymerization of hyaluronic acid by a hyaluronidase
treatment accompanies the increase in MMP expression
and upregulation of CD44, and induces the destruction of
cartilage 64). HA depolymerizationis induced by hydroxyl
radical, which is reactive oxygen species 65). It is assumed
that in the case where conditions of strong oxidative stressare continuously present, or in the case of hyaluronidase
activity acceleration, exogenous hyaluronic acid fragments
could induce adverse events.
HA is produced mainly by synovial membrane cells. In
OA and RA, cytokine in synovial membrane cells increases 38).
Periostin, which isone of the synovial-membrane-cell-derived
cytokines, is a key molecule of inflammation and cartilage
degeneration in OA. Periostin promotes the acceleration ofNO generation to cells of articular tissue and elevates theexpression of inflammatory cytokine and MMP. Periostindependent NO generation is inhibited concentrationdependently by HA 66). I L-13 stimuli increases the
productivity of periostin in synovial membrane cells, and
HA has an inhibitory effect on this 67).

_ 15 _

Hyaluronic acid and articular cartilage

Animal experiment

Clinical study

It has been reported that in animal experiments, intraarticular administration of macromolecule HA has an effectof pain relief 68-75). It is assumed that molecular conformation
characterized by macromolecule HA greatly contributes to it.
Mechanisms of pain relief are assumed as follows 68);
1) HA coats pain receptors of tissues, such as the synovial
membrane.
2) Endogenous pain producing substance is captured.
In articulation of a cow model for OA with pressure
stress, the following are shown; ROS generation increases,
gene expressions of type Ⅱ collagen and aggrecan are
inhibited 76), inhibitory HA exerts an anti-oxidative effect andinhibits the generation of reactive oxygen species (ROS) 77),
matrix synthesis control element (SOX9) is decreased in theexpression by pressure load, and adding HA restores it 76).
Also, phosphorylated P38 and MMP-13 increase in expression
by pressure load, and adding HA inhibits it 76). Mechanicalstress to cartilage cells induces ROS synthesis acceleration
and P38 MAPK phosphorylation. Finally, articular cartilage
degeneration is induced through the inhibition of matrix
synthesis and the promotion of MMP13 generation.
Contrarily, it is assumed that HA has a mechanism to exert
effects of protection of cartilage by the inhibition of ROS
production and P38 MAPK phosphorylation through thereceptor, CD44 76).
It has been reported that in experiments with a Rabbit
model of OA, the expressions of VEGF and VEGFR-2mRNA increase. However, HA intra-articular administration
has small effects on the expression of VEGF-2 mRNA,
while inhibiting the expression of VEGFR-2 mRNA 78). HA
administration inhibits the generation of PGE 2 and MMP 79).
Proteoglycan, type Ⅱ collagen and a residual quantity of HA
result in a decrease, and a positive rate of apoptotic celland degradation product of aggrecan results in an increase.
However, macromolecule HA administration to a joint cavity
corrects these changes 80).
Iodoacetic-acid-induced OA rats were examined toanalyze HA response genes of DNA microarray in cartilage
tissues. Results showed that the gene expression of typeIV, IX, XI collagen and adrenomedullin decreased due to
OA and macromolecule HA administration intra-articularadministration restored them. Inf lammation-related
factors such as phospholipase A2 and Toll-like receptor
8 were accelerated in gene expression due to OA, andmacromolecule HA administration inhibited those 81).
It was suggested that macromolecule HA intra-articularadministration affects clinical conditions to control thegene expression of collagen, anti-inflammation factors andinflammation-related factors 81).
It is evident that HA action mechanism of the inhibition
of OA progression is partly related to the inhibition of VEGF
mRNA expression 79).
In spite of the results of these researches, the American
Academy of Orthopaedic Surgeons, AAOS, released arevised guideline of knee osteoarthritis in June of 2013
and announced that intra-articular HA administration
was no longer recommended as a method of treatment
for patients with symptomatic osteoarthritis of the knee(recommendation grade: strong). Also, the Osteoarthritis
Research Society International (OARSI) has a negative
advocacy piece against intra-articular HA administration.

Some clinical studies have revealed the pain relief effects
of HA oral ingestion. Processed food from rooster comb
extracts containing low molecular HA was examined in arandomized double blind comparative trial; including low
molecular HA, and HA degraded to a low molecular weight.
Subjects were 40 patients with knee osteoarthritis (OA). In
Japanese Orthopaedic Association (JOA) Evaluation Criteria,
2 of 5 subscales and total scores were significantly improvedfor “pain and ability on walking” and “pain and ability on
ascending and descending stairs” 82, 83). Furthermore, in arandomized double blind comparative trial examining rooster
comb extracted processed food containing low molecular HA
with 66 soccer athletes, a tendency toward improvement was
shown in “pain when placing pressure on foot joint,” and “painof articulation coxae while exercising” 84). An examinationof prepared soybean milk mixed with N-acetylglucosamine
was conducted in a randomized double blind comparative
trial, where 67 subjects with mild pain, stiffness anddiscomfort pain in the knee joint showed that “pain in
the knee joint when ascending and descending stairs andduring resting period” were significantly improved 85). In aclinical study onthe ingestion of glucosamine-chondroitinquercetinglucoside with 46 OA patients and 22 RA patients,
OA patients showed a reduction of pain but RA patients did
not show a reduction of pain 86).
These results of clinical studies have indicated thepossibilities that HA with a relatively small molecular weight
are effective on OA-derived arthralgia. There are a certain
amount of unsolved questions remaining regarding action
mechanisms for how low molecular HA is digested, degradedand absorbed after oral ingestion, and also how to mitigate
pain.
As for influences on the range of joint motion by HA,
research of OA rabbit models with knee arthrodesis has beenreported. Macromolecule HA administration into a joint
cavity mitigates the lowered range of knee joint motion 87).
HA with a molecular weight of 2.02 million was more
effective than HA with a molecular weight of 0.95 million.
There has been no research reported regarding a low molecular
HA like this specimen. The HA mechanism has not yet been
completely clarified. However, some mechanisms have been
assumed such as, HA has an inhibitory effect against fibrosis,
involves water retaining action 88), elevates the fluidity andrestores the function of synovial fluid 89), and at the sametime, inhibits the separation of glycosaminoglycan, which is
a component of cartilage tissue and restores the quantity ofglycosaminoglycan in the cartilage, suppressing the progressof cartilage degeneration 90-92).
In articular cartilage tissues stimulated by adding IL-1
and RA, aggrecan is promoted to free to extracellular and thesynthesis and secretion of MMP is induced. These reactions
are forced by HA 39). It has been confirmed in research ofarticular cartilage cultured cells of rabbit models that HA
had little effect on the synthesis of aggrecan but inhibited
aggrecan to separate from the substrate 53).
A clinical study of a soymilk beverage containing
N-acetyl glucosamine on OA in a double blind parallelcomparative trial revealed that the range of joint motion was
significantly improved after 8 weeks from the ingestion 85).

_ 16 _Glycative Stress Research

Relationshipbetween glycative stress and HA

Conclusion

The relationship between HA and glycative stress havenot been paid a full attention to so far. However, it has
become known that glycative stress involves the mechanismof pathogenesis of OA 93-96). Mechanisms are assumed to be
as follows; DAMPs (damage-associated molecular patterns)and HMGB-1, which are released from impaired cells intoa joint cavity, promote the generation of inflammatory
cytokine, through the mediums of TLRs (toll-like receptors)
and RAGE (receptor for advanced glycation end products)
on immunocompetent cells. The induced inflammatory
tissue injury leads to the onset and progression of OA 96). In
an OA joint cavity, AGEs (advanced glycation end products)
induce inflammation through the mediums of RAGE andother scavenger receptors 95). An examination of serum
malondialdehyde concentration of a rabbit OA model showed
as follows; control group (untreated); 2.05 ± 0.37 nmol/
mL, hyaluronan group; 1.94 ± 0.54 nmol/mL, cortisonegroup; 1.98 ± 0.37 nmol/mL and hyaluronan and cortisone
combination group; 1.55 ± 0.41 nmol/mL. The combinationgroup was significantly lower than the untreated group 97).
Malondialdehyde acts as an intermediate in the process ofthe formation of AGEs and has an effect of accelerating theformation of AGEs. Therefore, this view is striking in termsof relating HA to glycative stress.
An experiment of administering feed containing HA torats revealed that the ingestion of HA exerted thebeneficial
influences to intestinal bacterium flora and serum cholesterol
metabolism 98). The data proved a significant decrease of
TC in this test. The relation between HA and glycolipid
metabolism is of great interest, and further research is
expected for the future development.

The cartilage matrix plays an essential role to consider
OA pathophysiology. Focusing on HA, which is a matrix
component, it is concluded that mechanical stress, oxidative
stress and glycative stress induce HA fragmentation and themigration of cartilage tissues in early stages of OA, and thedecline of HA induces the activation of a matrix degrading
enzyme, which is normally suppressed by HA. These
processes are factors of a matrix disorder. Exogenous HA canact suppressively on the enzyme induction, and as a result,
reduces the matrix separation (the loss of HA and aggrecan)from cartilage. However, differences in effects, dependingon the molecular size, have not been clarified. It is necessary
for the prevention of OA to elucidate the mechanisms ofcartilage matrix disorders and to enforce countermeasures
for the prevention of OA progression, identifying indicationsof early symptoms.

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Acknowledgement
This work was partially supported by the JapaneseCouncil for Science, Technology and Innovation, SIP(Project ID 14533567 ), “Technologies for creating nextgeneration agriculture, forestry and fisheries” (funding
agency: Bio-oriented Technology Research Advancement
Institution, NARO.

Conflict of interest statement
There are no items deemed to be conflicts of interest in
this research.

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