CHAPTER 1: THE RELEVANCE OF CHITIN 1.1 CHITIN: FROM THEN TO NOW Biopolymer is a term commonly used to refer to polymers biologically synthesized by nature. Polysaccharides are one such class of biopolymers, comprising of simple monosaccharide (sugar) molecules connected by ether type linkages to give high molecular weight polymers. Compared to their renowned cousins, the nucleic acids and proteins, the polysaccharides have traditionally been considered of less significance and regarded primarily as structural materials and metabolic energy sources. Only in the latter half of the 20 th Century has interest in the chemistry of the polysaccharides witnessed a renaissance brought about by the development of new methods of isolation, extraction, separation, chemical and enzymatic modification coupled with the advent of sensitive and powerful instrumental analysis techniques. This has led to the "re-discovery", identification and functional elucidation of traditional and newly discovered polysaccharides, illuminating their inherent and essential roles in biological function. I Among the polysaccharides, cellulose and chitin are the two most abundant biopolymers in the biosphere. Despite being scientifically discovered earlier than cellulose, chitin initially received limited attention. By contrast, extensive research and development has centered on cellulose since the late 19 h Century. 2 The first explicit account of chitin was in 1811, attributed to the Frenchman, Braconnot, Professor of Natural History, Director of the Botanical Garden and member of the Academy in Sciences of Nancy, France. 3 Braconnot described and named the alkali-resistant fraction from isolates of some higher fungi "Fungine". In 1823, Odier found a material with the same general properties as fungine in the cuticle of beetles and designated it "chitin" after the Greek word "chiton" that denotes "coat of mail" in reference to the cuticle. Subsequently, the chemical character of chitin began to be elucidated. The presence of nitrogen in chitin is attributed to Payen in 1843 while Ledderhose identified glucosamine and acetic acid as the hydrolysis products of chitin in 1876. Tiemann proposed that the amino sugar was based on either glucose or mannose and Fischer and Leuchs confirmed the attachment of the amino group at the C-2 position of the monosaccharide unit. Karrer and co-workers and Zechmeister and co-workers separately performed experiments to determine that N-acetylglucosamine was the primary constituent of chitin. Last, the (1-4) linkage and its 13-D conformation was confirmed by X-ray diffraction, enzymatic and deamination reactions by Meyer and others in the first half of the 20 th Century. The discovery of chitosan is ascribed to Rouget in 1859 when he found that boiling chitin in potassium hydroxide rendered chitin soluble in organic acids. In 1894 Hoppe- Seyler named this material chitosan. Only in 1950 was the structure of chitosan finally resolved. It has taken more than a hundred years to arrive at the chemical identity of chitin and the revelation of its polymeric properties. This is not surprising considering that the term polymer was not even acceptable until the 1930's onwards! Nevertheless, the necessary foundation was laid for the eventual emergence of chitin as an important biomedical material.
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C H A P T E R 1: T H E R E L E V A N C E O F C H IT I N
1.1 C H I T I N : F R O M T H E N T O N O W
Biopolymer i s a t e rm commonly used to re fe r to po lymers b io log ica l ly syn thes ized by
na ture. Po lysacchar ides a re one such class o f b iopo lymers , compr i s ing o f s imple
monosacchar ide ( sugar ) molecu les connec ted by e ther type l inkages to g ive h igh
mo lecu la r weigh t po lymers . Com pared to the i r r enowned cous ins , the nuc le ic ac ids
and proteins , the polysaccharides have t radi t ional ly been considered of less
s ignif icance and regarded pr imari ly as s t ructural mater ia ls and metabol ic energy
sources . On ly in the la t ter ha lf of the 2 0 th Century has interest in the chemistry of the
po lysacchar ides wi tnessed a rena i ssance b rought abou t by the deve lopment o f new
methods o f i so la t ion , ex t rac t ion , separa t ion , chemica l and enzymat ic modi f ica t ion
coupled wi th the adven t o f sens i t ive and power fu l ins t rumenta l ana lys i s t echn iques .
This has led to the "re-discovery", ident i f icat ion and funct ional e lucidat ion oft rad i t iona l and newly d i scovered po lysacchar ides , i l lumina t ing the i r inheren t and
essent ia l roles in biological funct ion. I
Among the po lysacchar ides , ce l lu lose and ch i t in a re the two mos t abundan t
biopo lym ers in the biosphere. Desp i te being scient i fical ly discovered ear l ier than
cel lulose, chi t in ini tia l ly received l im ited at tent ion. By contrast , extensive research and
development has centered on cel lulose s ince the la te 19 h Century. 2
The f i rs t expl ic i t account of chi t in was in 1811, a t t r ibuted to the Frenchman,
Braconnot , P rofessor o f Natura l His to ry , Di rec to r o f the Botan ica l Garden and m em ber
of the A cadem y in Sc iences o f Nancy , France . 3 Braconnot descr ibed and na med the
alkal i - resis tant fract ion from isolates of som e higher fungi "Fung ine". In 1823, Od ier
found a mater ia l with the same general propert ies as fungine in the cut ic le of beet les
and de signated i t "chi t in" af ter the Greek w ord "chi ton" that denotes "coa t of mail" in
reference to the cutic le . Subseq uent ly , the chem ical character of chi t in began to be
elucidated. The presence o f ni t rogen in chi t in is a t t r ibuted to Payen in 1 84 3 wh ile
Ledderho se iden t if i ed g lucosam ine and ace t ic ac id as the hydro lys i s p roduc ts o f ch it in
in 1876. T iem ann proposed tha t the amino sugar was based on e i the r g lucose o r
man nose and F ischer and Leuchs conf i rmed the a t t achment o f the amino group a t the
C-2 pos i t ion o f the monosacchar ide un i t . Kar re r and co-workers and Zechm eis te r and
co-workers separa te ly per fo rmed exper iments to de te rmine tha t N-ace ty lg lucosaminewas the pr im ary con st i tuent o f chi t in . Last , the (1-4) l inkage and i ts 13-D confo rma tion
was conf i rmed by X-ray d i f f rac t ion , enzymat ic and deamina t ion reac t ions by Meyer
and others in the f i rs t half of the 2 0 th Century .
The discovery of chi tosan is ascr ibed to Rouget in 1859 when he found that boi l ing
chi t in in potassiu m hy droxide rendered chi t in soluble in organic acids . In 1894 Ho ppe-
Seyler nam ed this mater ia l chitosan. Only in 1950 was the s t ructure of chi tosan f inal ly
resolved.
I t has taken more than a hundred years to arr ive at the chemical ident i ty of chi t in and
the revelat ion o f its polym eric propert ies. This is not surpr is ing consid er ing that the
term poly me r was not even acceptable unt il the 1930 's onwards! Nev ertheless , the
necessary foundat ion was la id for the eventual emergence of chi t in as an important
Before p roceed ing fu r ther , i t i s neces sary to def ine what ch i t in and i t s p r inc ipa l
der iva t ive , c h i tosan , i s . The ear l i e r l i te r a tu re i s l i tt e r ed wi th exam ples o f the casua l
i n t e r c h a n g e b e t w e e n t h e s e t w o t e r m s a n d t h e r e h a v e b e e n i n s t a n c e s w h e n c h i t i n i s
r e fe r r ed , w here i t i s c l ear f rom a r ead ing o f the con ten t , tha t ch i tosan wa s be ing
di scus sed . On ly in the pas t t en year s , has there been m ore cons i s t en cy in the usage of
the t e rm s ch i tin and ch i tosan . The def in i t ion and descr ip t ion tha t fo l lows incorp ora tes
w h a t i s w i d e l y a c c e p t e d b y t h e c h i t i n / c h i t o s a n c o m m u n i t y t o d a y .
The idea l s t ruc ture o f ch i t in is a l inear po lysacch ar ide o f [3 - (1-4) -2-ace tamido-2-de oxy-
D - g l u c o p y r a n o s e w h e r e a l l r e s id u e s a r e c o m p r i s e d e n t i r e ly o f N - a c e t y l - g l u c o s a m i n e
res idues o r in ch i t in j a rgo n , fu l ly ace ty la ted . L ike wise , the idea l s t ruc ture o f ch i tosan ,the p r inc ipa l der iva t ive o f ch i tin i s a l inear po ly m er o f 13- ( l -4 ) -2-amino-2-de oxy- D -
g l u c o p y r a n o s e w h e r e a ll re s i d u e s a r e c o m p r i s e d e n t i re l y o f N - g l u c o s a m i n e r e s i d u e s o r
fu l ly deac e ty la ted . The shor t - fo rm n o ta t ions fo r idea l ch i t in and ch i tosan a re dep ic ted
in F igure 1 .1 and employed th roughout th i s book .
C H 2 O H
N H A c
\
C H 2 O H
O
O H O
N H 2 n
C h i t in C h i t o s a n
A c - -
OII
- - C - - C H 3
Figure 1 .1 : Idea l i zed r epresen ta t ion o f ch i t in and ch i tosan
T r a d i t i o n a l s o u r c e s o f t h e b i o p o l y m e r , h o w e v e r , d o n o t r e s u l t in 1 0 0 % a c e t y l a t e d c h i ti no r 1 0 0 % d e a c e t y l a t e d c h i t o s a n a n d i n r e a l i t y , t h e b i o p o l y m e r e x i s t s a s a c o - p o l y m e r a s
represen ted in F igure 1 .2 , where the numbers in square b racke t s on the ex t r eme le f t
r ing as s igns the s ix carbon s in the g lucopy rano se r ing f rom C-1 to C-6 . Spec i f i ca l ly ,
t h e s u b s t i tu t i o n a t t h e C - 2 c a r b o n o f t h e g l u c o p y r a n o s e r in g c a n b e e i t h e r w i t h t h e
a c e t a m i d o g r o u p o r a n a m i n o g r o u p . T h e d i f f e r e n ti a t io n b e t w e e n c h i t i n a n d c h i t o s a n i s
to consider their respective acetyl content . W hen the num ber of acetam ido group s is
more than 50% (m ore comm only 70-90% ) the b iopolym er i s te rmed chi tin . In chi tin
terminology, the num ber of ace tamido groups i s te rmed the degree of ace ty la t ion (DA).On the o ther hand, when the degree of deacety la t ion (DD) or the amino group i s
predom inant , the b iopolym er i s te rmed chi tosan .
[6]C H 2 O H
[4 ] / ~ O [ ' , , , , ] F C H 2 O H q
I I I ~ 4, ~ , V n / l ~ _ ) - - - 0 O H
_ IR
OR = - - C / /
" C H 3
R = - - H
a n d x > 5 0 % - - - > c h i t in
a n d y > 5 0 % - - > c h i to s a n
Figure 1.2: Chem ical structural representat ion of chit in and chitosan
depic t ing the co -polym er charac ter o f the b iopolym ers
Instant differentiat ion between chit in and chitosan can be at tained from their solubil i ty
and n itrogen content . 4 Chit in is soluble in 5% Lithium chloride/N , N-
Dimethylacetamide solvent [LiCI /DMAc] and insoluble in aqueous ace t ic ac id whi le
the conv erse is t rue of chitosan. The nitrogen content in purif ied samp les is less than
7% for chit in and mo re than 7% for chitosan .
In this book, the term chit in wil l be used general ly to refer to chit in and i ts derivatives,
including chitosan. W hen direct references are ma de to chitosan or any chit in
deriv ative, the spe cific nam es will be used. Ch itin also has three crysta lline form s, c~, 13
and T. Unless oth erwise specif ied, chit in denotes a-chit in.
Despi te i t s ear ly discovery, and being the second most abundant polysacchar ide af ter
cellulose with an est im ated annual production of at least 10 l~ tons per yea r in the
biosphere, chit in has remained an almost unused biomass resource late into the 20 th
Century. 5 W hy did ch it in lang uish in obscurity for so long?
First, i f we contrast chit in to cellulose, one explana tion cou ld be the source o f the raw
material . Cellulos e is readily available from trees and cotton plants; two tradit ional
sources that date back centuries, almost to the beginning o f civil ization. Coup led with
the centuries of experienc e in exploit ing and using this resource, i t is readily
unders tandable that ce l lu lose has been wel l ent renched as a b iopolymer of s igni f icant
uti li ty. Con versely, sources o f chit in comprising o f shells of crustaceans, insects andfungi, are more difficult to exploit and therefore, perceived to be of insignificant
usefulness except as food and decorative ornaments . Second, looking back a t the br ief
historical account of chit in and chitosan described above, the biopolym er belonged
prima rily in the realm o f the naturalists , who se preocc upation lay in identifying and
charac terizing chit in and chitosan in plants and animals. These included isolating and
determining the composit ion, the structure, macrostructure and physical propert ies and
establishing the ir biochemica l and biological roles. 6 A mo dicum of chit in che mistry
only began to appea r in the late 1930 's. Very l i t tle else was reported in the ensuin g
years from its discovery unti l the 1950's. A third factor is the difference in the physical
proper t ies of chi t in in compar ison to cel lu lose where the s t rong hydrogen-bondingnetw ork in the former led to a mo me ntary dead-end to i ts man ipulation and use. The
search for an effective solution to this si tuation was the beginning of a series of on-
going e fforts to defy the chemical and structural inertness o f chit in.
The turnaround from anonymity can probably be at tr ibuted to two unrelated yet vital
events. This was the advent o f the canning industry that came into being in the 1800's.
Canning enabled the preservat ion of food for extended per iods of time. Obviously , the
seafood process ing industry o f which crabmeat and other shel lf i sh p roducts were a par t
of embraced canning and grew. Coupled wi th an increas ing consumpt ion by a growing
world population and the globalization of food taste, the shellf ish waste that was
generated gradually increased, eventually leading to the need to resolve thisaccu mu lating industrial waste prob lem in the 20 h Century.
1.4 F R O M W A S T E T O N IC H E M A T E R I A L S
Commencing in the 1950's, a more concerted effort began to appear in the scientif ic
l i terature. A natural course in f inding a solution to the abundan t shellf ish waste from
seafood process ing i s the in teraction between indust ry and Academ ia. One exam ple i s
Tottori , Japan, famous for i ts snowcrabs and associated seafood processing facil i t ies
that generated such shellf ish wastes. I t is probab ly no coincide nce that Professo r
Shigehiro H irano of Tottori Universi ty (now retired) becam e an eminent pioneer in
chit in research since the late 1960's. This exam ple duplicated in similar si tuations
around the world from the 1950's onward no doubt has changed the course, or at least
fast-forw arded the chit in story outcom e. Eventually, this resulted in the increased
unde rstanding of the science and techno logy o f chit in that culminated in the first
"mi les tone" as i t were , wi th Professor Mu zzarel l i ' s landm ark book. 7
Ne xt bega n the giant leap in the s tudy and applicat ion o f chi t in and ch itosan ar is ing
from tw o fortui tous events . Firs t, industr ial interest turned into activi ty that establ ished
the regular avai labi l i ty of the biopo lym er for research and com mercial ex ploi tat ion.Today , p roducers o f ch i tin and ch i tosan span the g lobe and a re found in Nor th
Am erica, Eu rope and Jap an and increasing ly from China, Ind ia and So uth East Asia. 8
The qua l i ty o f the product i s var ied . The predom inant ch i t in and ch i tosan products a re
produced and con sumed m ain ly in Japan . Increas ingly , Korea is p roducing a mix of
products a im ed a t an expor t marke t .
Second, the m om entum genera ted by the se r ies of In te rna t iona l C hi t in /Chi tosan
Co nferen ces begin ning w ith the f irs t a t M IT, US A in 1977 and w ith the 8 h recently
he ld in Yam aguchi , Japan in 2000. The European C hi tin Soc ie ty and the Japanese
Chit in and Chitosan Society also further s t imulated act ivi ty with their own meetings
and the latest , The Asia-Pacif ic Chit in/Chitosan Symposium series , begun in the mid-
1990s, adds to the on-going internat ional forum on the biopolymer.
A p p l i c a t i o n a r e a
W ater t rea tment
Agr icu l ture
Text i le and p aper
B io t echno logy
Food /H ea l t h
Supp lemen t s
Cos me t i c
Biomedica l
Specif ic u s e
Coagula t ing / f loccula ting agents for po l lu ted was te
waters
Rem oval / recovery of meta l ions f rom aqu eous was te
water
Plant el ici torAnt imicrobia l agents
Plant seed coat ing
Fert i l izer
Fibers for text i le and woven fabrics
Paper and f i lm
Chr oma tog r aphy pack ing
Enzyme immobi l iz ing mater ia l s
Natural thickeners
Food addit ives including pet food
Foo d proce ssing (e .g. in sugar ref ining)Fil t rat ion and clar if icat ion
Hypocholes te ro lemic agent ( s l imming agents )
Ingredients for hair and skin care (condit ioners)
Burns and wou nds dress ings for hum ans and
animals
Biomaterial (e .g. absorbable sutures)
Ant icoagulan t o r an t i th rombog enic m ateria ls
(as sulfated-chit in derivat ives)
Hemostat ic agents (as chi tosan)
Drug del ivery, gene del ivery
Table 1 .1 : Ap plicat ions of chi t in, ch i tosan and their derivat ives 9
for a better unders tanding of the production, science and technology and uti l ization of
chitin, sustaining the research cycle that is so necessary for the future applications and
scientific k now ledge o f chit in .
I n d u s t r y
- .Agriculture
J a p a n M a r k e t
(106 U S$/y ear) l~
Bio-fertilizer 21 -
- 11 0ood and beverages
Cosm etics and toile tr ies
i Chi topack KQ 8025
_Waste and dr inking water treatment
T o t a l
W o r l d M a r k e t
(10 6 U S$ /yea r) 11
2.3
90
Biom edical 2000 1250 ,
Imm obilized Cell and culture " - 45
3
- 14 0
2 0 2 4 1 9 1 5
Table 1.2" Potential size of the market for chitin, chitosan and their derivatives
Today the exciting promise of chit in has been delivered and more importantly, is set to
play a significa nt role in the biom edical field as a biom aterial o f the 21 st Century.
Perhaps chitin may even one-day surpass i ts older and better-known sibling, cellulose.
1.5 B I O M E D I C A L A P P L I C A T I O N S
Tow ards the close of the 20 h Century, the chitin c om m unity has becom e increasingly
enthusias tic over the biomedical opportunities for this mater ia l . W hy is this so? A
plethora of processing methods including i ts chemistry and character ization together
with a more in-depth knowledge of biomedical applications including cell / t issueinteractions , have em erged. As a result , the breadth of biomedical applications spanned
by chitin continues to expand, making chitin a mater ia l increasingly impossible to
ignore. The possibil i t ies appear endless . The surge of chit in in the biomed ical
direction is rooted f rom the po stulation that there wo uld be better acceptance o f the
mater ia l by the human biological system due to i ts natural or igins and close analogy to
body consti tuents . Together with the potentia l of limitless supply of this renewable
mater ia l , these major inf luences have perpetually thrust chit in to the forefront as a
biomater ia l . Will this "gold-mine" o f opportunity be realized?
The aim of this book is to address these biomedical applications of chitin . Each cr it ical
aspect that can shed light on these issues will be put forward. In the ensuin g chapters,
the foremost biomedical applications that have been proposed for chit in , wound
dressings, blood-interaction mater ia ls , or thopedic applications , t issue engineer ing
scaffolds and drug delivery will be surveyed. This will be followed by an assessment
of whether chit in can and will meet the diverse biomedical applications proposed by