Hyaluronan: from biomimetic to industrial business strategy

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State Key Laboratory of Natural and Biomimetic Drugs,

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Hyaluronan: From Biomimetic to Industrial Business

Strategy

Erminio Muranoa,b

, Danilo Perina, Riaz Khan

a and Massimo Bergamin

a,b

aPROTOS Research Institute, via Flavia 23/1c/o BIC Incubatori FVG, 34148, Trieste, Italy

bNEALYS srl, via Flavia 23/1c/o BIC Incubatori FVG, 34148, Trieste, Italy

[email protected]

Received: December 20th

, 2010; Accepted: February 2nd

, 2011

Hyaluronan (hyaluronic acid) is a naturally occurring polysaccharide of a linear repeating disaccharide unit consisting of β-(1→4)-linked

D-glucopyranuronic acid and β-(1→3)-linked 2-acetamido-2-deoxy-D-glucopyranose, which is present in extracellular matrices, the synovial

fluid of joints, and scaffolding that comprises cartilage.

In its mechanism of synthesis, its size, and its physico-chemical properties, hyaluronan is unique amongst other glycosaminoglycans. The

network-forming, viscoelastic and its charge characteristics are important to many biochemical properties of living tissues. It is an important

pericellular and cell surface constituent; its interaction with other macromolecules such as proteins, participates in regulating cell behavior

during numerous morphogenic, restorative, and pathological processes in the body. The knowledge of HA in diseases such as various forms

of cancers, arthritis and osteoporosis has led to new impetus in research and development in the preparation of biomaterials for surgical

implants and drug conjugates for targeted delivery.

A concise and focused review on hyaluronan is timely. This review will cover the following important aspects of hyaluronan: (i) biological

functions and synthesis in nature; (ii) current industrial production and potential biosynthetic processes of hyaluronan; (iii) chemical

modifications of hyaluronan leading to products of commercial significance; and (iv) and the global market position and manufacturers of

hyaluronan.

Keywords: Hyaluronan, hyaluronic acid, biosynthesis, tissue engineering, antitumor drugs, crosslinked HA, global markets.

Hyaluronan (also known as hyaluronic acid) (HA) is a

naturally occurring polysaccharide of a linear repeating

disaccharide unit consisting of β-(1→4)-linked D-

glucopyranuronic acid and β-(1→3)-linked 2-acetamido-2-

deoxy-D-glucopyranose, which is present in extracellular

matrices, the synovial fluid of joints, and scaffolding that

comprises cartilage. However, other organisms, such as

bacterial pathogens and virus infected micro algae, are able

to synthesis this polysaccharide. HA was first isolated

from the vitreous body of the eye by Mayer and Palmer [1]

and then found in all vertebrates tissues, from skin to

synovial fluid between joins, from umbilical cord to skin

and cartilage [2-5].

Despite its simplistic structure hyaluronan behaves quite

differently from other glycosaminoglycans in its

mechanism of synthesis, its size, and its physico-chemical

properties. The network-forming, viscoelastic and its

charge characteristics are important to many biochemical

properties of living tissues. It is an important pericellular

and cell surface constituent which, through interaction

with other macromolecules such as proteins, participates in

regulating cell behavior during numerous morphogenic,

restorative and pathological processes in the body. The

knowledge of such biological functions of hyaluronan has

led to the preparation of biomaterials for surgical implants

and drug conjugates for targeted delivery.

The interest in the role of hyaluronan diseases such as

various forms of cancers, arthritis and osteoporosis has led

to new impetus in research and development. Our

laboratory has been involved in the study of chemistry and

physico-chemical properties of hyaluronan with a view to

develop biomaterials and drugs conjugates.

As a general arrangement of the review the subject is

divided into: biological function and synthesis in nature;

industrial production of hyaluronan; chemical modification

of hyaluronan; and market position of hyaluronan.

Biological function and synthesis in nature

Hyaluronan was first isolated from the vitreous body of the

eye by Mayer and Palmer [1] and then found in all

vertebrates tissues, from skin to synovial fluid between

joins, from umbilical cord to skin and cartilage [2-9].

Hyaluronan is a glycosaminoglycan that has a number of

biological functions in vertebrates, being essential for

tissue and organ integrity and function. Hyaluronan

molecules are involved in the activation of signaling

NPC Natural Product Communications 2011

Vol. 6

No. 4

555 - 572

556 Natural Product Communications Vol. 6 (4) 2011 Murano et al.

pathways that control cell proliferation, differentiation,

adhesion and migration [10-20].

The primary structural function of HA is related to its

viscoelastic and hygroscopic properties. It acts as shock-

adsorber and offers lubrication to the joints, creates a

protective and flexible layer between tissues, influence cell

responses during a number of processes, inflammation and

cancer [21-28]. A wide range of different cell responses

are evoked by HA molecules which different molecular

weights [29]. In general, high molecular weight

hyaluronan inhibits cell differentiation and promotes cell

proliferation, being involved in tissue regeneration, wound

healing, epithelial integrity and embryogenesis. Lower

molecular weights HA are anti-angiogenic, immuno-

suppressive and hamper the differentiation by limiting the

cell-cell interactions or ligand access to cell receptors.

Finally, HA oligomers showed angiogenic and pro-

inflammatory activity, increase immune response and are

capable to slow down growth of a number of tumors in

vivo [30-37].

HA mediates his biological function through specific

protein receptors present on the different cell surface,

which include CD44, HARE, RHAMM, LYVE [29,38-43].

In nature, hyaluronan is synthesized by specific

glycosyltransferases (HA synthases or HAse) that

polymerizes the activated donor uridine diphospho- sugars

UDP-glucuronic acid and UDP-N-acetyl-glucosamine into

hyaluronan [44-47]. In nature, hyaluronan was shown to be

synthesized also by a small number of microbial pathogens

such us Pasteurella multocida and the Lancefield group A

and C streptococci among which Streptococcus equi,

the human pathogen Streptococcus pyogenes and the

bovine pathogen Streptococcus uberis. [48-58]. These

microorganisms adopted the strategy to encapsulate their

cells with hyaluronan in order to perfectly disguise from

the host animal defense system and allow the adhesion and

colonization. In Streptococcus and Pasteurella, the HA

capsules represent virulence factors affording to the

bacteria a stealth function against the recognition from the

host immune system [45,59-61]. On the contrary to other

bacterial capsule polysaccharides triggering the antibody

immune response, HA is not immunogenic because it is a

normal component of the host body conferring to these

pathogen bacteria an efficient molecular camouflage.

Moreover, the HA capsule provide a protection against the

reactive oxides released by leukocytes [62] and facilitate the

bacteria migration through epithelial layer into tissues [63].

In Streptococcus equi subsp. zooepidemicus hyaluronic

acid capsular material contributes to adherence properties

of the pathogen possibly conferring to the bacteria the

resistance to phagocytosis by macrophages [64-66].

The importance of the capsule in pharyngeal colonization

and other inflammatory syndromes caused by

Streptococcus pyogenes may reflect the role of hyaluronan

as a specific ligand for attachment of Streptococcus

pyogenes to the hyaluronan-binding protein CD44 on

pharyngeal epithelial cells [67,68].

Pasteurella multocida, a prevalent animal pathogen,

employs a hyaluronan polysaccharide capsule to avoid host

defenses. This pathogen is associated with various diseases

in many animal species; a number of studies suggested that

there is a correlation between the capsule and the virulence

of P. multocida, for example in the pathogenesis of fowl

cholera [52,69-74].

This biomimetic strategy of prokaryotic organisms

efficiently exploits the chemical identity of the bacterial

capsular HA molecules to that of the eukaryotic host

extracellular matrix. Such a biomimetic strategy adopted

by specialized prokaryotic cells to proliferate into an

animal host organism has the same goal as many types of

endogenous cells like tumor cells to invade and proliferate

into normal tissues. In fact, a large number of tumor cells

over express membrane receptors for hyaluronan in order

to more efficiently interact with this major component of

the extracellular matrix to easily spread and proliferate into

the tissues [75,76].

The existence of hyaluronan outside the animals and their

pathogens has been demonstrated in green microalgae

infected by specialized viruses. Chlorovirus is a group of

virus of Phycodnaviridae family that infects the eukaryotic

microalgae Chlorella [77,78]. Chlorovirus dsDNA genome

unusually encodes enzymes involved in sugar metabolism.

The 330-kilobase genome of the chlorovirus PBCV-1

encode other than fructose-6-phosfate aminotransferase

and UDP-glucose dehydrogenase also a HA synthase,

resulting in the production of an HA capsule surrounding

the Chlorella infected cells. However, some chlorovirus

contain both HA synthase and chitin synthase genes and

form both hyaluronan and chitin, an N-acetyl-glucosamine

based polysaccharide found in the crustaceans shell, in the

insect exoskeletons and fungi cell wall [79], on the surface

of their infected cells [80-82].

The function of a HA capsule in chlorovirus infected

chlorella cells is still unknown; however, since the genes

encoding for HA synthesis are transcribed early in

PBCV-1 infection and HA accumulates on the external

surface of the infected chlorella cells, hyaluronan synthesis

might be related to the virus life cycle and the recognition

of infected cells. The distribution of hyaluronan synthases

from vertebrates to bacterial pathogens and algal viruses

makes these fascinating glycosyltransferases an interesting

evolutionary case which can advance the knowledge on the

biological functions of a typical natural molecule like

hyaluronan.

Industrial production of hyaluronan

HA has been traditionally produced by extracting from

rooster combs and bovine vitreous humor The process is

difficult, particularly to ensure the biological safety and to

Review on hyaluronan Natural Product Communications Vol. 6 (4) 2011 557

control the molecular weight of the product [3]. Hence,

since 1980‘s, the commercial production of HA has moved

to the fermentation process using streptococcal bacteria.

The most used strains are S. equi subsp. equi and S. equi

subsp. zooepidemicus that naturally achieve good HA

production rate.

Improved HA producer streptococcal strain has been

obtained by random mutagenesis followed by a serial

selection of the mutant clones. The aim of the strain

selection was mainly oriented to the reduction of the HA-

depolymerising enzyme (hyaluronidase-negative strains)

and other extracellular proteins such as the virulence factor

streptolysins, the exotoxins responsible for the β-hemolysis

(non-hemolytic strains) [83] as well as the production of

high molecular weight HA [84,85]. Several studies were

performed in order to improve the HA yield, molecular

weight and reduce the polydispersity by an accurate

control of the growing conditions. Unfortunately,

Streptococci are lactic acid bacteria that demand expensive

complex growth medium containing yeast or animal

extract, peptone and serum supplemented with an high

glucose content [83,86,87].

The HA biosynthesis require a large amount of energy and

compete with the cell growth because of the large

pathways overlapping with the cell wall biosynthesis

(Figure 1). Indeed, when unlimited resource are available,

the optimal growth conditions is observed at 37°C and

around pH 7 while, the highest HA productivity and

molecular weight is observed at suboptimal growing

conditions. Limiting the resources available, in particular

glucose, first occurs a decline in HA productivity

followed by the HA molecular weight [55]. Streptococci

are facultative anaerobes incapable to oxidative

phosphorylation however, aerobic culturing condition it is

known to greatly improve the HA production. These

effects are probably the results of improved energy

efficiency when the NADH oxidase (NOX) removes the

excess of NADH in presence of oxygen [88].

Metabolically engineered Streptococcus strain with an

increased NOX activity resulted in 33% improvement of

ATP yield leading to a 15% improvement in biomass.

Unfortunately, any HA yield or molecular weight

improvement was not observed [89].

Even under the best conditions, and the use of continuous

cultivation, the HA yield does not exceed the practical

limit of 5-10 g/L. Further improvement requires genetic

engineering of the producer microorganism. Partial or

complete genome sequence of the most relevant HA

producing microorganisms is now available including S.

equi [90,91], S. pyogenes [92,93] and Pasteurella

multocida [94]. These information, combined with the

advanced vectors developed for a wide number of hosting

cell, allows the metabolic engineering of the HA pathway.

Moreover, it is possible to transfer the HA synthesis

capability from natural producer to non-producer hosting

cells. Even though the metabolic engineering and genomic

insertion for the improvement of HA production is

possible in streptococci, the same result was easier

achieved heterologus host microorganisms. Other

advantages of heterologus HA synthesis reside in the

exploitation of microorganism generally considered as safe

for human health and the easier culture preparation and

processing.

The enzymes involved in the HA pathway in streptococci

are encoded in the bacterial genome as a monocistronic

has operon. The has operon includes three genes: hasA

that encodes for the HA synthetase [95], hasB that

encode for UDP-glucose dehydrogenase [96] and hasC that

Figure 1: Schematic representation of the streptococci HA synthesis pathway. The enzymes and well as the correspondent gene locus is in italic; the has operon genes

(hasA, hasB and hasC) is underlined.


encodes for UDP-glucose pyrophosphorylase [97].

Heterologus expression of HA synthetatse (hasA gene in

streptococci) in host bacteria such us Escherichia coli,

Enterococcus faecalis or Bacillus subtilis, has shown to be

sufficient to direct the production of HA [70,98]. However,

since HA and cell wall biosynthesis has partially common

pathway (Figure 1), the depletion of sugar precursor

caused by the high activity of HA synthetase would stop

the cell from growing. To overcome this problem, at least

a second gene hasB or its homologue must to be

introduced at the same time [40,98].

Several microorganisms have been tried for the

transformation into hosting cells for HA synthesis:

Escherichia coli, Bacillus subtilis, Lactococcus lactis and

Agrobacterium sp. The initial genetic modifications were

performed on E. coli by the cloning and expression of the

hasA gene from S. equi encoding for HA synthetase.

However, the experiments demonstrated that, over

expression of HA synthetase itself severely compromise

the cells ability to growth [95]. More recently, a better

result in the E. coli HA production was achieved by the co-

expression of HA synthetase hasA gene (derived from P.

multocida or S. pyogenes) respectively with hasB

homologue (UDP-glucose dehydrogenase) or hasB + hasC

homologues (UDP-glucose dehydrogenase + UDP-glucose

pyrophosphorylase) from E. coli. The aim was to improve

the intracellular availability of sugar precursor and prevent

the inhibition of cell growing. The genes were organized

into an artificial operon designed and constructed onto the

backbone of proper plasmidic vectors. The HA yield was

about 2 g/L but raised up to 3.8 g/L when the culture

media was supplemented with glucosamine, suggesting

close connection between energy metabolism and

precursor supply. Even if the HA production was lower

than the streptococci strains or others recombinant

microorganisms, this work suggests suggest engineering

routs to be exploited and the potential use of E. coli for

industrial HA production [99,100].

Interesting was the works performed onto food grade

microorganisms Agrobacterium sp. and Lactococcus lactis

[101,102]. Agrobacterium is already used in the production

of curdlan for food applications and is generally

considered as safe. In 2007, Mao and Chen, successfully

transfered HA synthetase gene (hasA) from P. multocida

and an UDP-glucose dehydrogenase homologue gene

(hasB) from E. coli into Agrobacterium host. The co-

expression of the two enzymes enables the host

microorganism to produce HA up to 0.3 g/L. Even if the

HA production yield was considerably lower than standard

streptococci, the recombinant synthesis of HA in

Agrobacterium specie was the first example of HA

production into Gram-negative bacteria that naturally

produce a food product [102].

A similar result was obtained by Chien and Lee [101] on

Lactococcus lactis, a food grade Gram-positive bacterium

extensively used in the production of buttermilk and

cheese. HA synthetase gene hasA from S. equi subsp.

zooepidemicus was expressed alone or on combination

with hasB gene (UDP-glucose dehydrogenase). The

authors use a nisin-controlled expression vector (NICE) in

order to separate the cell expansion from the HA

production. When the desired rate cell density was

reached, nisin was added to the culture to induce the

expression of the foreign genes and finally, the HA

production. The HA yield obtained was 0.08 g/L and 0.65

g/L respectively when HA synthetase (hasA) was

expressed alone or in combination with UDP-glucose

dehydrogenase (hasB). Even the production yield needs

improvement, because the host microorganism is a food-

grade lactic acid bacteria, the HA production has high

potential to applied in the field of food and biomedical

industry [101].

Novozymes obtained a successful result in the recombinant

production of HA in Bacillus subtilis [103-105]. The

microorganism is a well known non-pathogenic, Gram-

positive bacterium that was extensively studied and with a

wide range of vector available that make it an obvious

potential host for HA production. The authors produce a

series of expression cassette containing the gene hasA (HA

synthetase from S. equisiilis) alone or in combination with

B. subtilis homologues of hasB (tuaD in Bacillus,

encoding for UDP-glucose dehydrogenase), hasC (gtaB in

Bacillus, encoding for UDP-glucose pyrophosphorylase),

hasD (gcaD in Bacillus, encoding for Uridyltransferase/N-

acetyltransferase bifunctional enzyme). The expression

cassettes were cloned into appropriate vectors to construct

artificial operons that were stable introduced into a specific

locus of Bacillus subtilis host genome. The production

yield obtained by the transformed Bacillus strain resulted

to be comparable with the streptococci strains when hasA

and tuaD genes were co-expressed by the artificial operon

[104]. Interestingly, the overexpression of hasA alone does

not seem to hinder the cell growth. Indeed, the authors

report that, in contrast to E. coli, the Bacillus strain

expressing HA synthetase alone resulted in HA production

even if lower than others transformed strains. The HA

production was dramatically improved by the co-

expression of hasA and tuaD into the same operon. The

meaning of the tuaD needing reside in the evidence that,

endogenous tuaD gene is expressed only under phosphate

starvation [55]. B. subtilis is inherently more metabolic

robust than E. coli, at least regarding the HA synthesis,

allowing a simultaneous support of both cell growing and

HA synthesis [105]. In spite of no benefit in terms of HA

yield and molecular weight compared to streptococci

fermentation, recovery from B. subtilis is much easier

because HA is not cell-associated and there is no

contamination by streptococcal pathogenic factors [40].

As an alternative to the prokaryotic HA production, it is

possible to mention the HA synthesis occurring Chlorella

cells, a unicellular green algae, when infected by

Chlorovirus [106]. Experimentally, approximately 0.5-1

g/L of HA was recovered from infected Chlorella cells


after 4 hours of infection [107]. The yield is lower than typically ones achieved in streptococcal fermentation but further implementation is possible to improve it [40].

Finally, it is interesting the work of Takeo and colleagues [108] on the cloning and expression of mammalian HA synthetase. The authors genetically modify the genome of the fruit fly Drosophila melanogaster, a non-HA-synthesizing animal, by introduction of the single gene encoding mouse HA synthetase. The expression of HA synthetase in vivo resulted in massive HA accumulation in the extracellular space of the Drosophila tissues and caused various morphological defect. The original aim of the work was the development of an in vivo model to study the biosynthetic machinery of the polysaccharides besides, this could represent an alternative route for the HA production in eukaryotic cells [108].

Chemical modification of hyaluronan

The interest in the role of hyaluronic acid in diseases such as various forms of cancers, arthritis and osteoporosis has led to new impetus in research and development. Our laboratory has been involved in the study of chemistry and physico-chemical properties of hyaluronic acid with a view to develop biomaterials and drugs conjugates [109].

The repeating disaccharide unit, β-(1→4)-linked D-gluco-pyranuronic acid and β-(1→3)-linked 2-acetamido-2-deoxy-D-glucopyranose, of hyaluronic acid contains two free hydroxyl groups at C-4,6 positions in the β-D-GlcpNAc moiety and in the β-D-GlcpA a carboxylic group at C-5’ and two free hydroxyl groups at C-2’,3’ positions, as shown in Figure 2. The chemical modifications on the hyaluronic acid backbone occur in these positions. In our laboratory, we have focused our research on regioselective modifications of the hydroxyl groups of HA. In particular, reactions at the 4- and 6-OH groups of the GlcNAc residues of the HA have led to a large number of well characterized derivatives.

Figure 2: Chemical structure of the repeating unit β-(1→4)-linked D-glucopyranuronic acid and β-(1→3)-linked 2-acetamido-2-deoxy-D-glucopyranose

Unlike simple carbohydrates, chemical reactions of hyaluronic acid usually do not result in discrete compounds; a certain proportion of the molecule may be partially substituted or unsubstituted.

High-resolution NMR experiments have been used to determine the position and the degree of substitution and in some cases to study on a qualitative basis the effect of specific substitution on the conformation of the molecule. Molecular weight and molecular weight distribution determination, using High Performance Size Exclusion Chromatography (HP-SEC), has been performed to ascertain the molecular integrity of the reaction products. The low solubility of hyaluronic acid in organic solvents has been overcome by converting it into different organic counterions such as tetrabutylammonium, pyridinium and collidinium salts [110]. Such a derivatization imparts complete solubility in organic solvents and allows the chemical reaction to occur.

6-halodeoxy-hyaluronic acid: Recently, HA was used as sodium salt without any derivatization in organic solvent (DMF) and in presence of methanesulfonyl chloride to generate regioselective 6-deoxy-6-chloro derivatives [111]. The reaction occurred in heterogeneous phase yielding halogenated HA derivatives suitable for further derivatization with drugs bearing nucleophilic groups. The complex methanesulfonyl chloride-DMF complex formed during the reaction permits selective replace of primary hydroxyl groups by chlorine [112].

Alkyl and Aryl Esters derivatives of HA: The carboxyl group of hyaluronic acid has been esterified by treatment with tetrabutylammonium salt in an aprotic solvent such as DMF or DMSO. Hyaluronic esters of ethyl, propyl, benzyl and dodecyl alcohols have been described as medical and pharmaceutical materials as medical sutures, films, microspheres, pellets, membranes, corneal shields and implants [113,114].

6-amino-6-deoxyhyaluronic acid: The amino-deoxy derivatives of carbohydrates are of interest because they are components of biological materials such as glycoproteins and bacterial polysaccharides. They are usually synthesized by catalytic reduction of the corresponding azido derivatives, which in turn can be prepared from the corresponding halodeoxy compounds [115]. A direct, high yielding, synthesis of 6-amino-6-deoxyhyaluronic acid has been achieved by selective amination of the C6-chlorinated HA in aqueous media under NH3 pressure [116].

Hyaluronic acid and methotrexate conjugate: Small molecule drugs such as antitumor compounds have been conjugated to synthetic and natural polymers. The advantages envisaged in this strategy are reduced toxicity, increased solubility and stability, localisation and controlled release of the drug. Methotrexate is used in the treatment of diseases such as inflammatory pathologies, autoimmune or neoplastic diseases. However, its therapeutic use is limited because of its high systemic toxicity. With a view to reduce the toxicity and enhance the efficacy of the drug, regioselective 6-O-methotrexyl-hyaluronic acid has been prepared by treatment of 6-chloro-6-deoxy HA with methotrexate in DMF [111].

OO O

OO HO

HO

OHN

H3C

HO

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O

Na

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1'

2'

3'

4'5'

6'

1

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4

5

6


Hyaluronic acid and taxol conjugate: Taxol (paclitaxel),

an antileukemic and antitumor agent, was first isolated

from the bark of the Pacific yew tree, Taxus bravifolia;

has a limited solubility in water. Hyaluronic acid is over

expressed at sites of tumor and provides a matrix to

facilitate invasion. Hence, to overcome the solubility

problem and to target the tumor cells, a first attempt to

synthesize an hyaluronic acid conjugate of taxol was

performed by Luo et al. by linking the taxol 2‘-OH by

way of succinate ester to adipic dihydrazide-modified HA

(HA-ADH) [117].

Recently, Lee et al conjugated paclitaxel and HA utilizing

a novel solubilization method in a single organic phase.

Hydrophilic HA was completely dissolved in anhydrous

DMSO with addition of poly(ethylene glycol) (PEG) by

forming nano-complexes. Paclitaxel was then chemically

conjugated to HA in the DMSO phase via an ester linkage

without modifying extremely hydrophilic HA [118]. The

conjugate exhibited selective toxicity toward the human

cancer cell lines (breast, colon and ovarian) that are

known to express hyaluronic acid receptors; no toxicity

was noted against a mouse fibroblast cell line at the same

concentrations.

Hyaluronic acid and mitomycin C: Mitomycin C was

linked to the HA molecule by way of an amide

bond between the drug and the carboxyl group of the

D-glucuronic acid moiety. An amide linkage formation is a

dehydration reaction and requires anhydrous systems.

However, as hyaluronic acid is difficult to dissolve in an

anhydrous organic solvent, reaction conditions were

developed to use a water based system [119]. Other

strategies envisage the use of a succinylated HA as active

intermediate for the conjugation with the drug [120].

Hyaluronic acid and camptothecin conjugate: A novel

methodology for making drug conjugates using hyaluronan

as a carrier was recently developed. This strategy involves

a completely regioselective two-step synthesis of 6-amino-

6-deoxyhyaluronan, which is then easily functionalized

making use of a simple succinate linker [116].

Hyaluronic acid and other drugs: A number of

antitumor drugs, such as doxorubicin [121] and

daunorubicin, have been linked at the amino function of

the drug to the carboxylic group of an amino acid or a

peptide spacer arm forming an amide linkage, and then

through the terminal amino group of the amino acid or the

peptide to the carboxyl group of the HA.

Hyaluronic acid and methylprednisolone: Steroid-type

of anti-inflammatory drugs such as methylprednisolone

has proved to be efficacious in reducing symptoms

associated with osteoarthritis of the knee [122]. Alkyl and

aryl esters of HA have been used in the preparations

containing methylprednisolone, both as polymer

matrices in the form of microspheres wherein the drug is

physically incorporated, and as substrates on which

methylprednisiolone is chemically linked [123,124].

Hyaluronic acid and antioxidants: Reactive oxygen

species (ROS) such as superoxide radicals generated by

metabolic processes can depolymerise hyaluronic acid

[125]. It has been demonstrated that many anti-

inflammatory drugs and free radical scavengers are

efficacious in protecting hyaluronic acid depolymerisation

by free radicals [126-128].

Hyaluronic acid and propofol conjugate: 2,6-

Diisopropylphenol (propofol) is an anaesthetic for

intravenous administration whose efficacy as a free radical

scavenger is due to its ability to form stable radicals. Its

effectiveness as a scavenger for hydroxyl radicals

generated by xanthine oxidase has been demonstrated by

assessing in vitro the depolymerisation of hyaluronic acid

in artificial synovial fluid [129]. Low-molecular-mass

hyaluronic acid oligosaccharides have major biological

effects: notably, they appear to play a role in the

recruitment and activation of inflammatory macrophages,

which may result in an exacerbation of the inflammatory

process and tissue damage [130,131]. For this reason,

propofol was proposed as a shield for hyaluronic acid

[124].

Cross-linked Hyaluronic Acid-based materials: Hyaluronic acid is rapidly metabolised in vivo by enzymes

such as hyaluronidase [132] and by free radical oxidations

[131], which limit its use in native form as a biomaterial.

In, addition hyaluronic acid is highly soluble in water. In

order to overcome these limitations, the physico-chemical

properties of hyaluronic acid have been modified using a

variety of cross-linking reagents. These chemical

modifications have led to a number of hyaluronic acid

derivatives with a variety of physical formats such as

liquids of various viscosities, fibers, weaves, sponges and

fleeces. Cross-linked hyaluronic acids with special

properties and structure, such as different degradation rate,

different surface characteristics, different porosities, have

applications as tissue engineering scaffolds for the delivery

of cells, gene transfer, wound healing, post-surgical

adhesion prevention, and implantation of bioactive

compounds in vivo repair sites.

Ossipov et al. developed a new methodology exploiting a

protective group strategy based on initial mild cleavage of

a disulfide bond followed by elimination of the generated

2-thioethoxycarbonyl moiety ultimately affording free

amine-type functionality. The strategy therefore

encompasses a new approach for mild and highly

controlled functionalization of HA with both nucleophilic

and electrophilic chemoselective functionalities with the

emphasis for the subsequent conjugation and in situ

crosslinking [133].

Li et al. synthesized a polymeric nanogel, with tumor

targeting properties and a controllable phototoxicity,

utilizing a low molecular weight-hyaluronic acid

photosensitizer conjugate, resulting in the formulation of

self-organizing nanogels in aqueous solutions [134] while


Kim et al synthesized hyaluronic acid/quantum dots

(QDots) conjugates. The reaction was performed through

amide bond between carboxyl groups of QDots and amine

groups of adipic acid dihydrazide modified HA (HA-

ADH) assessing the possibility of HA derivatives as

target-specific drug delivery carriers for the treatment of

liver diseases [135]. In the field of delivery systems

alkylamino hydrazide hyaluronic acid (HA) derivatives

were prepared to design new biocompatible films able to

release hydrophobic drugs. These films were found to be

more resistant to degradation by hyaluronidase compared

to solutions of unmodified HA at the same concentration

[136]. Bencherif et al developed a nanostructured

hydrogel made of thiolated HA and reacted with

biodegradable POEO300MA-co-PHEMA nanogels

derivatized with acryloyl groups. The resulting scaffold

was obtained through selective atom transfer radical

polymerization (ATRP) and Michael-type addition

reactions, affording a biocompatible matrix for cell and

protein encapsulation in both tissue engineering and drug

delivery applications [137]. Skoza et al exploited the

specificity of hyaluronate lyase to biodegrade HA and

introduce a double bond in the glucuronic acid moiety

followed by the generation of free aldehyde group via

ozonolysis and the subsequent reduction of the generated

ozonide. This gives rise to a new reactive HA substrate to

be derivatized with several molecules (such as biotin,

polymers, or proteins) with application in tissue

engineering and biomaterials production [138].

A strong commercial interest drives the extensive use of

HA hydrogels in cosmetics and tissue engineering. Due to

the frequent use of HA derivatives in aesthetic surgery,

novel, biocompatible, and non-toxic dermal fillers

hyaluronic acid (HA) hydrogels were studied. Yeom et al

successfully developed new hydrogel for tissue

augmentation applications. Instead of using highly

reactive cross-linkers such as divinylsulfone (DVS) [139]

for Hylaform, 1,4-butanediol diglycidyl ether (BDDE) for

Restylane, and 1,2,7,8-diepoxyoctane (DEO) for Puragen,

HA hydrogels were prepared by direct amide bond

formation between the carboxyl groups of HA and

hexamethylenediamine (HMDA) with an optimized

carboxyl group modification for effective tissue

augmentation [140]. Crosslinked hyaluronic acid

hydrogels have been specially designed and synthesized

to promote tissue repair. A range of glycidyl

methacrylate-hyaluronic acid (GMHA) conjugates have

been synthesized and photopolymerized to lead to a

number of crosslinked GMHA hydrogels with different

physical properties such as swelling, mesh size. As

expected, the amount of crosslinking and the degree of

degradation was dependent on the amount of the

conjugated methacrylate; the more crosslinking the less

degradation rate [141,142]. Su et al. developed an

hydrogel formed by oxidated hyaluronic acid (oxi-HA)

cross-linked with adipic acid dihydrazide (ADH). HA was

oxidized by sodium periodate to create aldehyde

functional groups, which could be cross-linked by ADH.

The result was a colorless, transparent and injectable

hydrogel as potential vitreous substitute [143]. Other type

of hydrogel were those made by Oldinsky et al,

characterized by both a hydrogel and a hydrophobic

polymer for biomedical applications. A series of

amphiphilic graft copolymers consisting of HA, a

glycosaminoglycan, and high-density polyethylene

(HDPE), that is, HA-co-HDPE, were fabricated.

Exploiting an esterification reaction between HA and

functionalized HDPE the resulting material was a

semicrystalline, insoluble powder applicable in saline

suspension or molded form, including orthopedic tissue

repair [144].

The extreme versatility of HA is emphazised also by its

employment in material science with carbon nanotubes.

Hybrid hyaluronic acid (HA) hydrogels with single wall

nanotubes (SWNTs) were formed by cross-linking with

divinyl sulfone, producing considerable change in the

morphology of the lyophilized hybrid hydrogels compared

to HA hydrogels itself. As a final result, HA plays a dual

role of matrix and linker for the rigid reinforcing

nanofibers [145]. A similar approach was used by Mendes

et al to produce hydrogels able to be used as potential

biomaterials for the restoration of bone defects [146].

Finally, a current cutting-edge topic is the development of

biocompatible delivery systems to be used as a carrier for

genetic material. Many attempts have been made to

produce the ―perfect‖ carrier, able to overcome all the

issues related to the crossing of the biological barriers

(enzyme degradation, endocytosis, intracellular

trafficking), once administered in the body. Mok et al

produced a Green covalently linked fluorescent protein

(GFP) antisense oligodeoxynucleotide (ODN) / (HA)

conjugate via a reducible disulfide linkage, and the HA-

ODN was complexed with protamine to increase the extent

of cellular uptake and enhance the gene inhibition

efficiency of GFP expression [147,148]. Furthermore, HA

was derivatized exploiting the conjugation with

polyethyleneimine (PEI) chains through the carboxylic

groups to obtain a favorable positively charged

environment for siRNA complexation [149-151]. This

strategy could have potential applications as safe and

effective nonviral carriers for ODN and siRNA nucleic

acid therapeutics

Global markets and manufacturers

The use of hyaluronic acid (HA) has been extensively

investigated in blends with other biopolymer and growth

factors to form matrices to mimic the extracellular matrix

surrounding the living cells. In this section commercially

valuable application of hyaluronic acid as biomaterial will

be described.

The HA market is led by several manufacturers who

compete in different market segments related to their

specific application. Seikagaku Corporation,

manufacturing Artz HA viscosupplementant, is the largest


company in the global HA market with its dominance in

the Japanese market and its presence in Europe and US.

Among the competitors, Seikagaku is followed by

Genzyme Biosurgery. Q-Med dominates in the field of

dermal filler while the largest competitor in the ophtalmic

viscoelastics market is Advanced Medical Optics (AMO).

Table 1 summarizes the composition of the main

competitors in the field of HA-based biomaterials:

Table 1: Leading competitors in the HA-based treatment market (by

segment Global, 2005) [152].

Company Market share

Genzyme

Biosurgery 16.3%

Q-Med 12.3%

Fidia Farmaceutici 9.0%

AMO 7.9%

Alcon 7.3%

Chugai

Pharmaceuticals 5.3%

Santen 1.9%

Bausch&Lomb 1.9%

Inamed 1.8%

Corneal 1.5%

Bioniche Life

Science 1.3%

Others 11.1%

An emerging company in the HA manufacturer‘s scenario

is Novozymes, which has developed a patented process for

production of HA form an bioengineered strain of Bacillus

Subtilis. Also Anika Therapeutics recently strengthened its

position market acquiring part of Fidia Advanced

Biopolymers, producing hyaluronic acid-based products in

several therapeutic areas including the regeneration of

connective and structural tissues damaged by injuries,

aging or degenerative diseases.

It is worth mentioning that in 2010 the global HA

treatment market is foreseen to generate over $2 billion in

revenues, demonstrating an overall compound annual

growth rate (CAGR) in excess of 8%. The main sectors of

extreme commercial interest are those related to:

- Dermal fillers

- Ophtalmic viscoelastics

- Viscosupplementation

In addition to these sectors other areas are being deeply

investigated for their usefulness in commercial

applications such as:

- Tissue engineering

- Adhesion barriers

- Wound healing/wound dressing

Dermal fillers: HA is one of the most used materials for

dermal filler products. In fact, according to recent

statistics, more than 85% of all dermal filler procedures

occurred with a hyaluronic acid derivative [153,154].

Dermal fillers for face rejuvenation were proposed on the

market many years ago and received a large interest with

the introduction of injectable bovine collagen. As the

procedures for wrinkle correction with dermal fillers

became more popular, the search began for safer, longer

lasting fillers [155-157]. The next generation of filler after

those made of bovine collagen was crosslinked hyaluronic

acid derivatives, which was an improvement over bovine

collagen in that it did not require a skin test for

hypersensitivity and appeared to be longer lasting. The

search for safer and more effective dermal fillers has

continued because of the patients requirements to have

fillers which predictably last longer than 6 months. In spite

of a number of new temporary fillers composed of

crosslinked hyaluronic acid that are available in Europe

and are undergoing clinical testing in the United States, it

is not fully understood what parameters control the

performance of these fillers [158]. Nevertheless, the

customer demand for such a products is very high and

demonstrated by the list below, summarizing the most used

crosslinked HA-based materials as dermal fillers (Table 2):

Several studies have been carried out to explain the

properties [159] and the mechanism of action of dermal

fillers. Wang et al. proposed that the injection of

crosslinked hyaluronic acid stimulates collagen synthesis,

partially restoring dermal matrix components damaged by

environmental conditions (air, UV radiation). They

hypothesize that this stimulatory effect may be induced by

mechanical stretching of the dermis, which could activate

the dermal fibroblasts. These findings suggest that cross-

linked hyaluronic acid may be useful for stimulating

collagen production therapeutically, particularly in

atrophic skin conditions [160]. Table 2: Producers, products and functions of the most used dermal

fillers [152].

To improve the efficacy of the injections and the whole

final esthetic result, Kenner proposed the simultaneous

administration of HA and botulinum neurotoxin to

strengthen and improve the rejuvenation effect of the

treatment, showing for the first time that hyaluronic acid

and botulinum neurotoxin can be delivered in combination

in the same syringe and at the same time for the treatment

of the upper face [161,162]. Hasson et al evaluated

Esthélis, a new formulation sold as injectable non-animal

crosslinked hyaluronic acid. It is characterized by a

polydense cohesive matrix (CPM®), which produces a gel

of uniform consistency with better biointegration to the

Company Market share Product

Q-Med 71,2% Restylene

Inamed 10,6%

Hylaform

Captique Juvederm

Corneal 8,5% Juvederm

Surgiderm

Merz Pharmaceuticals 1,9% Hyal-System

Boletero

Fidia 1,3% Hyal-System

L.E.A. Derm 1,1% Juwederm

Mentor 0,9% Puragen

Others 4,4% -


tissues and a longer duration in the treatment of atrophic

scars [163]. Monheit et al. studied a new type of dermal

filler based on a Dermal gel extra (DGE) crosslinked HA-

based material containing lidocaine, engineered to resist

deformation and degradation. The results obtained on

patients indicate that DGE could provide a comfortable

and cost-effective dermal filler option for clinicians and

patients [164].

Table 3: Producers, products and relative market share of the mainly used

dermal fillers [152].

Brand Name Manufacturer Function

Hylaform Inamed Moderate to severe wrinkles

Hylaform Plus Inamed Moderate to severe wrinkles

Restylene Touch Q-Med AB Superficial lines

Restylene Q-Med AB Moderate to severe wrinkles

Restylene

Perlane Q-Med AB Deep dermis and lips

Restylene SubQ Q-Med AB Add volume

Restylene Lipp Q-Med AB Lips

Juvederm 24 LEA Derm Medium to deep wrinkles

Juvederm 24HV LEA Derm Mid dermis and lips

Juvederm 30 LEA Derm Mid dermis and lips

Juvederm 30HV LEA Derm Medium to deep wrinkles

and lips

Esthelis Anteis Mid or deep dermis and lips

Puragen Mentor Mid dermis and lips

Yeom et al. proposed a new generation of dermal fillers

based on HA for tissue augmentation. Instead of using

highly reactive crosslinkers such as divinyl sulfone (DVS)

for Hylaform, 1,4-butanediol diglycidyl ether (BDDE) for

Restylane, and 1,2,7,8-diepoxyoctane (DEO) for Puragen,

HA hydrogels were prepared by direct amide bond

formation between the carboxyl groups of HA and

hexamethylenediamine (HMDA) with an optimized

carboxyl group modification for effective tissue

augmentation [140]. The research mentioned above

indicates products which are under the spotlight of the

cosmetic and pharmaceutic industries; the market segment

of this type of products is mainly subdivided among the

companies listed in Table 3:

Ophthalmic viscoelastics: Hyaluronic acid is widely use

in ophthalmology due to its viscosupplementant properties

and its presence in the cornea, the sclera and the vitreous

humor [165]. Ophthalmic viscoelastics are HA-based

products used to maintain the structure of the eye‘s

capsule, mainly in the phacoemulsification (removal of

the eye‘s vitreous humor) step of cataract surgery [166],

but also in other surgical procedures such as vitreoretinal

surgery, anterior segment surgery, glaucoma surgery, and

corneal transplantation [167]. HA-based ophthalmic

viscoelastics are used in every cataract procedure and

intraocular lens implantation. Maintaining the eye‘s

structural integrity without the fluid vitreous humor,

viscoelastics also provide lubrification and protection to

sensitive corneal tissue. Some studies have been carried

out to produce a biocompatible materials for the vitreous

humor replacement after vitrectomy using a new

crosslinked HA through adipic acid dihydrazide bridges

[143] and for cell sheet delivery applications [168]. By

bonding to the fragments of the cloudy lens, viscoelastics

act to facilitate natural lens removal toward the end of the

phacoemulsification process. In this sector the main

competitors are listed in Table 4:

Table 4: Producers, products and relative market share of the manly used

ophthalmic viscoelastics [152].


AMO 39,1% Healon

Alcon 36,4% DisCoVisc

Santen 9,6% Opegan

Bausch&Lomb 9,3% AmVisc Plus

Staar Surgical 2,6% StaarVisc

Others 3,0% -

Some efforts have been made to prepare crystalline lens

made of biodegradable hyaluronic acid and a non-

degradable polymeric gel, showing good biocompatibility

in the body [169].

The use of these materials is also extended to the eye‘s

cosmetic surgery. In fact, soft-tissue fillers have been

successfully used for volume augmentation in the orbit, the

tear trough, the upper eyelid/sub-brow region and in the

brow and temple region. Non-animal stabilized hyaluronic

acid fillers are the most commonly used in orbital and

periorbital volume augmentation. As a matter of the fact,

the role of injectable soft-tissue fillers in treating

periorbital pathologies is gaining much interest, due to the

results obtained in the treatment of paralytic

lagophthalmos, lower eyelid retraction and cicatricial

ectropion [170].

Viscosupplementation: Intra-articular viscosupplementa-

tion was approved by the Food and Drug Administration

(FDA) in 1997. HA viscosupplementation is a treatment

for osteoarthritis (OA) sufferers where injectable, artificial

produced HA supplements the body‘s own HA. The

product acts to reduce joint pain while increasing joint

mobility [171,172]. The positive effects of HA are due in

part to the fact that it influences physical properties, such

as viscoelasticity and lubrification, and HA molecules

interacts with the tissues in the joints. HA products are

delivered as a series of injections and provide varying

lengths of pain relief. It should be recognized that HA

viscosupplementation doesn‘t cure osteoarthritis but rather

serves as an intermediate treatment for patient who have

not been successfully treated with pharmaceuticals [173].

The pain relief activity of this treatments is of course, not

limited to OA itself but also to disease-related pain

persistent after arthroscopic surgery [174], such as

meniscectomy or cartilage damage in joints.

Strauss et al. highlighted as intra-articular hyaluronic acid

viscosupplementation was gaining popularity as a

treatment option in the nonoperative management of

patients with osteoarthritis. Recent clinical studies have


demonstrated that the anti-inflammatory, anabolic, and

chondroprotective actions of hyaluronic acid reduce pain

and improve patient function. With evidence mounting in

support of the efficacy of this treatment modality for

patients with osteoarthritis, its potential use in patient

populations affected by pathologies of weight-bearing joint

such as knee [175] and ankles [176] is deeply investigated.

Bencke et at. showed that the number of patients with

osteoarthritis (OA) is expected to be growing.

Viscosupplementation, in which hyaluronic acid is injected

into the knee joint, has evolved into an important part of

our current therapeutic regimen in addressing the patient

with knee pain due to OA. Although suffering from lack of

an "evidence-based" approach there is a growing body of

data demonstrating the efficacy of HA in decreasing pain

and improving function in patients with knee OA. The

extensive use in clinic of HA has led to the introduction on

the market of various forms of HA, although little data are

available to justify one over the other [177]. Further proofs

about the successful concept of HA as viscosupplementant

were given by Mathieu et al during in vitro studies on

rheological behavior of synovial fluids mixed with linear

and crosslinked HA. They showed as in vitro tests

highlighted the stability of HA over a period of 6 week

without relevant alteration of the rheologic behavior and

no pronounced degradation by catabolytic enzymes [178].

The pharmaceutical companies are very active in this field

since these are high-demand products. The leading

companies in the viscosupplementation field and the

relative HA-based products are summarized in Table 5.

Table 5: Manufacturers, products and share market of the most used

viscosupplementants [152].


Seikagaku corporation 35,80% ARTZ

Genzyme Biosurgery 26,10% Synvisc (Hylan G-F20)

Fidia Pharmaceuticals 14,30% Hyalgan

Chugai Pharmaceuticals 8,40% Suvenyl

Bioniche Life Science 2,00% Suplasyn

Others 13,40% -

Tissue engineering: The production of artificial 3D

matrices is nowadays a challenging topic [179],

considering the in vitro tissue growth for surgical

application a strong market need. Several approaches have

been proposed to promote the cell growth on 3D-matrices

made of biocompatible components. Due to a strong

market demand for those kind of artificial tissues research

in regenerative medicine is rapidly expanding to cope with

this new need. Based on the concept of tissue regeneration

triggered by HA-based materials, some products are

commercially available for the treatment of deep dermal

lesions such as non healing ulcers, deep II degree and III

degree burns [180] (HyalograftTM

3D), for autologous skin

replacement (Laserskin®), for autologous fibroblasts and

keratinocytes replacement (Tissuetech) and for wound

healing/dressing, whose use will be discussed later on in

this chapter.

In general, there is nowadays a trend towards supplying

cells with a material to speed up the tissue healing process.

Hydrogel encapsulation made of different biopolymers

provides cells with a three dimensional environment

similar to that experienced in vivo and therefore may allow

the maintenance of normal cellular function in order to

produce tissues similar to those found in the body [181].

Fan et al. synthesized galactosylated hyaluronic acid

(GHA) prepared through the covalent coupling of

lactobionic acid with HA. The derivative was then used to

make highly porous three-dimensional sponges composed

by a blend of chitosan and GHA. The mixture formed a 3D

matrix stabilized by the electrostatic interaction between

the two biopolymers. The matrix was applied to

hepatocytes for liver-specific functions analysis. The

addition of GHA not only improved the wettability and

changed their mechanical properties, but also significantly

influenced the cell attachment ratio. Moreover, liver

functions of the hepatocytes entrapped in the CS/GHA

scaffolds, such as albumin secretion, urea synthesis and

ammonia elimination were improved in comparison with

those in similar scaffolds made of chitosan [182].

Kasahara et al. have developed an original three-

dimensional (3D) scaffold to meet improved

biomechanical and biological requirements. The constructs

were made of chitosan-based hyaluronic acid hybrid

polymer fibers. Previous studies have shown that these

hybrid polymer fibers have superior adhesion of

chondrocytes and the ability to maintain chondrocyte

phenotype. The study aimed at regenerating hyaline-like

cartilage with sufficient mechanical properties combining

a volume-reduced 3D scaffold and a bioreactor system.

The implantation of such a biocomposites was proposed as

a model approach for various cartilaginous lesions [183].

Other studies, such that of Lee et al. focused on the

fabrication of 3D highly porous scaffolds and their

biochemical binding affinity to realize various biomimetic

functionalizations. This was achieved by using a binding

mechanism that incorporated molecules, which strongly

bind to each other. A good example of such a binding

molecule pair was avidin and biotin. In this work, a porous

hybrid scaffolds composed of natural materials such as

collagen and HA was prepared to evaluate morphology,

mechanical strength, in vitro enzymatic degradation and

cytotoxicity of the scaffolds as well as cell growth within

the scaffolds [184]. Other attempts were proposed by Chou

et al., who prepared a glue made of autologous fibrin as a

potential scaffold with very good biocompatibility for

neocartilage formation. However, fibrin glue has been

reported not to provide enough mechanical strength, but

with many growth factors to interfere the tissue growth.

Gelatin/hyaluronic acid/chondroitin-6-sulfate (GHC6S)

tricopolymer sponge was prepared as scaffold for cartilage

tissue engineering and showed very good results in cell

seeding and cell distribution [185]. The results indicated

that the chondrocytes cultured in GHC6S-fibrin glue

would effectively promote extracellular matrix secretion,

inhibiting the degradation. The evidence could support that


GHC6S-fibrin glue would be a promising scaffold for

articular cartilage tissue engineering [186].

Hu and al reported the formation of biocompatible

hydrogels using physically cross-linked biopolymers.

Gellation of silk fibroin (from B. mori silkworm) aqueous

solution was effected by ultrasonication and used to entrap

blended, uncrosslinked, hyaluronic acid (HA) without

chemical cross-linking. This is a novel approach to HA

hydrogel systems, which otherwise require chemical cross-

linking. Further, these systems exploit the beneficial

material and biological properties of both polymers. These

novel non-chemically cross-linked blend hydrogels may be

useful for biomedical applications due to biocompatibility

and the widespread utility of hydrogel systems [187].

Adhesion barriers: Adhesions are a major cause of post-

surgical morbidity and mortality and entail a substantial

medical economic burden. It has been estimated that 90%

of patients undergoing major abdominal surgery and 55%

to 100% of women undergoing pelvic surgery develop

adhesions. These phenomena are tightly linked to two risk

factors: tissue injury and inflammation response. Clinically

significant outcomes associated with adhesions include

chronic pelvic and abdominal pain, bowel obstruction, and

infertility. Consequently, methods for preventing such

adhesions have been the focus of extensive research. Few

barrier devices based on polysaccharides such as

hyaluronic acid or oxidized regenerated cellulose are in

commercial use, since part of them are based on synthetic

polymers (polytetrafluoroethylene) [188,189]. One of the

most used adhesion barrier is Seprafilm®, a composite

made of HA and carboxymethylcellulose employed in a

wide range of applications such as in bowel obstruction in

gynecological malignancies [190], tendons repair [191]

and after myomectomy, which is a procedure to surgically

removed uterine fibroids from the uterus. This procedure

often causes adhesion formation and decreases subsequent

fertility. This was the reason to evaluate the effectiveness

of several antiadhesion barrier materials in preventing

adhesion after myomectomy. Tsuji et al. prospectively

classified 63 women undergoing myomectomy alone into

four groups according to the type of antiadhesion material

used: Hyaluronic acid-carboxymethylcellulose film

(Seprafilm®), Dextran 40 (10% Dextran 40 Low

Injection®), factor 13 with fibrinogen (Beriplast

®) and

control. Seprafilm® was highly effective and was superior

to the other antiadhesion materials tested in preventing

uterine adhesions after myomectomy [192]. Other HA-

based product available on the market is INCERT®-S

(Anika), an anti-adhesion barrier gel for use in spinal

surgeries such as discectomies with a laminectomy or

laminotomy.

Other examples of dangerous adhesions are peritoneal

adhesions. These are among the most frequent cases of

tissue connections that form within the abdominopelvic

cavity following surgery or other injuries. They can cause

major medical complications. For this reason, researchers

are combining barrier devices and pharmacological agents

to be used in the prevention of this type of adhesion

formation. It was hypothesized that an adhesion barrier,

which also delivers anti-adhesion drugs, can address both

physical and physiological causes for adhesion formation.

For this reason an in situ cross-linking hyaluronan

hydrogel (barrier device) was prepared containing the

glucocorticoid receptor agonist budesonide [193]. A

similar approach to prevent peritoneal adhesions was the

application of physical barriers, which can separate the

injured regions during peritoneal healing. The hybrid

cross-linked HA-nanoparticles system appears to be a

biocompatible and highly effective adhesion barrier. This

―hybrid system‖ is able to prevent postoperative peritoneal

adhesions and combine the biocompatibility and ease of

application of in situ cross-linkable hydrogels with the

controlled release features of polymeric nanoparticles

[194]. Furthermore, postoperative peritoneal adhesions can

have serious, potentially lethal consequences.

Pharmacotherapy and barrier devices can reduce adhesion

formation to varying degrees, but their efficacy is limited

by rapid clearance from the peritoneum and lack of

biological activity, respectively. To overcome these

limitations, Yeo et al. have delivered tissue-type

plasminogen activator (tPA), which is deficient in the first

2-3 postoperative days, using a highly cross-linked in situ

forming hyaluronan gel. They demonstrated this

formulation's anti-adhesion activity in animal model that

involved recurrent adhesions [193]. Bioresorbable

membranes composed of hyaluronic acid and

carboxymethylcellulose (HA/CMC) are the most effective

method to prevent intra-abdominal adhesions; however,

their efficacy may be limited to the site of application.

Previous studies have shown that the intraperitoneal

administration of a neurokinin-1 receptor antagonist (NK-

1RA) reduces adhesions; however, the co-administration

of HA/CMC plus an NK-1RA has not been studied. The

co-administration of HA/CMC plus NK-1RA not only

increases the efficacy of the membrane at the site of

application, but also significantly reduces adhesions

formation at distal unprotected sites. This combination

may represent an emerging concept in more effective

adhesion prevention throughout the peritoneum [195].

Zhao et al. prepared a sodium carboxymethylcellulose-

Hyaluronic acid-Carboxymethylchitosan (CMCS) blend

films. The results suggested that the material was held

together by strong molecular interaction and hinted good

compatibility in the blend films. The blend films are

expected to be used as controllable degradation

biomaterials in the field of postsurgical hemostasis and

anti-adhesion [196].

Wound healing and dressing: Successful repair of

wounds and tissues remains a major healthcare and

biomedical challenge in the 21st Century. In particular,

chronic wounds often lead to loss of functional ability,

increased pain and decreased quality of life. Advanced

healing therapies employing biological dressings, skin

substitutes, growth factor-based therapies and synthetic

acellular matrices, all of which aim to correct irregular and


dysfunctional cellular pathways present in chronic wounds,

are becoming more popular [197]. Commercially available

HA-based products (for healing and dressing) on the

market are Hyalofill-f, Hyalofill-R, Connettivina,

Connettivina Plus, Jaloskin®, Hyalomatrix®. Hyalofill

family products are absorbent, soft and conformable

fibrous fleece (F) or rope (R) composed of HYAFF®, an

ester of hyaluronic acid. Jaloskin® is a transparent film

dressing for the treatment of superficial moderately

exuding wounds. Hyalomatrix® is a bilayered, sterile,

flexible, and conformable wound dressing that acts as an

advanced wound care device.

HA uses in wound healing are strongly justified by its non-

antigenic properties and the number of forms it can be

manufactured, such as gels, creams to sheets of solid

material through lightly woven meshes. Epidermal

engraftment is superior to most of the available

biotechnologies and, as such, the material shows great

promise in both animal and clinical studies of tissue

engineering. Ongoing research works focus on the ability

of the biopolymer to enhance angiogenesis and the

conversion of chronic wounds into acute wounds [198].

Moreover, Gao et al investigated the biological roles of

hyaluronan (HA) fragments in angiogenesis acceleration.

Studies have confirmed that oligosaccharides of HA (o-

HA) are capable of stimulating neovascularization in vitro

and promoting blood flow or angiogenesis in animal

models. However, few laboratories have studied the

function of o-HA as an exogenous treatment in injured

tissue repair in vivo. It is thought that o-HA may lose its

activities when used topically in vivo due to its small size,

which may be absorbed quickly by the surrounding tissues.

A special slow-releasing gel containing a mixture of o-HA

at defined size was prepared and the healing effects, by

topical application to an acute wound model, was studied.

The therapy by o-HA was compared with high molecular

weight HA and the known angiogenesis stimulator, VEGF,

suggesting that o-HA therapy might be useful in acute

wound repair [200]. Ferguson et al worked on HA

fragments together to bioresponsive polymers to enhance

the circulation time and stability of biologically active

proteins and peptides, while reducing their

immunogenicity and toxicity. Recently, crosslinked

material made of HA at different molecular weights

blended with arginine and epidermal growth factor (EGF)

[201] and dextrin-EGF [202] conjugates were proposed,

the latter exploiting the so called Polymer-masked

UnMasked Protein Therapy (PUMPT) concept. The

hypothesis is that conjugation of a biodegradable polymer

to a protein would protect it and mask activity in transit,

while enabling controlled reinstatement of activity at the

target site by triggered degradation of the polymeric

component. It has been developed and shown as potential

modulators of impaired wound healing. In this contest, HA

fragments (Mw 90,000 g/mol) were conjugated to trypsin

as a model enzyme. HA-trypsin conjugates exhibited 52%

greater stability in the presence of elastase, compared to

free trypsin, demonstrating the potential use of HA

conjugates as modulators of tissue repair. Ibrahim et al.

showed that exogenous hyaluronic acid oligomers (o-HA)

stimulate functional endothelialization, though native long-

chain HA was more bioinert and possibly more

biocompatible. Additionally, the author created hydrogels

containing high molecular weight HA (1 × 106 Da) and o-

HA mixtures (HA-o: 0.75-10 kDa) crosslinked with

divinylsulfone (DVS). The greatest endothelial cell (EC)

attachment and proliferation was observed on gels with an

high amount of o-HA. The study showed that the

beneficial EC response to o-HA and biocompatibility of

HA is mostly unaltered by their chemical derivatization

and crosslinking into an hydrogel [203].

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Natural Product Communications

2011 Volume 6, Number 4

Contents

Original Paper Page

Myrtenal, a Controversial Molecule for the Proper Application of the CIP Sequence Rule for Multiple Bonds

L. Gerardo Zepeda, Eleuterio Burgueño-Tapia and Pedro Joseph-Nathan 429

A Facile and Efficient Four-Step Enantioselective Synthesis of (+)-Vernolepin from (+)-Minimolide, the

Major Germacranolide of Mikania minima

Sandra M. Bach, Fernando R. Díaz, Horacio Bach and César A. N. Catalán 433

Use of the Plant Bellardia trixago for the Enantiospecific Synthesis of Odorant Products

Alejandro F. Barrero, M. Mar Herrador, Pilar Arteaga, Alexis Castillo and Alejandro F. Arteaga 439

Botcinolide/Botcinin: Asymmetric Synthesis of the Key Fragments

Jacinto Ramírez-Fernández, José Manuel Botubol, Antonio J. Bustillo, Josefina Aleu, Isidro G. Collado and

Rosario Hernández-Galán 443

C-6 Regioselective Bromination of Methyl Indolyl-3-acetate

Oscar R. Suárez-Castillo, Myriam Meléndez-Rodríguez, Lidia Beiza-Granados, Indira C. Cano-Escudero,

Martha S. Morales-Ríos and Pedro Joseph-Nathan 451

Synthesis of Pyrrolidinoindolines from 2-(2-Oxo-3-indolyl)acetates: Scope and Limitations

Martha S. Morales-Ríos, Ernesto Rivera-Becerril, Daphne E. González-Juárez, Juan Benjamín García-Vázquez,

Joel J. Trujillo-Serrato, Angelina Hernández-Barragán, and Pedro Joseph-Nathan 457

Review/Account

Biomimetic Cyclization of Geraniol Derivatives, a Useful Tool in the Total Synthesis of Bioactive

Monocyclic Terpenoids

Valentina Merlini, Marco Luparia, Alessio Porta, Giuseppe Zanoni and Giovanni Vidari 465

Drimenol: A Versatile Synthon for Compounds with trans-Drimane Skeleton

Manuel Cortés, Virginia Delgado, Claudio Saitz and Veronica Armstrong 477

Recent Advances in the Synthesis of Sesquiterpenolides from Umbelliferae

Francisco M. Guerra, F. Javier Moreno-Dorado, Zacarías D. Jorge and Guillermo M. Massanet 491

Diterpene Lactones with Labdane, Halimane and Clerodane Frameworks

Lúcia Silva, Arlindo C. Gomes and Jesus M. L. Rodilla 497

Stereoselective Synthesis of Five Biologically Active, Naturally Occurring Medium and Large Ring Lactones

J. Alberto Marco and Miguel Carda 505

Enantioselective Synthesis of Alkaloids from Phenylglycinol-Derived Lactams

Mercedes Amat, Núria Llor, Rosa Griera, Maria Pérez and Joan Bosch 515

Aconitum lipo-alkaloids – Semisynthetic Products of the Traditional Medicine

Botond Borcsa, Dezső Csupor, Peter Forgo, Ute Widowitz, Rudolf Bauer and Judit Hohmann 527

Synthesis of Pterocarpans

Leticia Jiménez-González, Carmen Hernández-Cervantes, Míriam Álvarez-Corral, Manuel Muñoz-Dorado and

Ignacio Rodríguez-García 537

Hyaluronan: From Biomimetic to Industrial Business Strategy

Erminio Murano, Danilo Perin, Riaz Khan and Massimo Bergamin 555

Hyaluronan: from biomimetic to industrial business strategy

Documents

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