CA2386586A1 - 3d matrix for producing cell transplants - Google Patents
3d matrix for producing cell transplants Download PDFInfo
- Publication number
- CA2386586A1 CA2386586A1 CA002386586A CA2386586A CA2386586A1 CA 2386586 A1 CA2386586 A1 CA 2386586A1 CA 002386586 A CA002386586 A CA 002386586A CA 2386586 A CA2386586 A CA 2386586A CA 2386586 A1 CA2386586 A1 CA 2386586A1
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- CA
- Canada
- Prior art keywords
- matrix
- dimensional matrix
- acid
- aqueous solution
- chitosan
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3852—Cartilage, e.g. meniscus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/70—Polysaccharides
- C12N2533/72—Chitin, chitosan
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Dermatology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Transplantation (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Rheumatology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Vascular Medicine (AREA)
- Urology & Nephrology (AREA)
- Materials For Medical Uses (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a method for producing a biologically compatible matrix, to a matrix that can be obtained using the method, and to the use thereof. The invention also relates to an implant. The inventive matrix can be produced by dissolving, in water, chitosan in acid, by freezing the solution and by subsequently lyophilizing the same. Excess acid can be removed by neutralizing with a base before or after the lyophilization. The inventive matrix is dimensionally stable and elastic. When implanted, it induces only a minor immunoreaction.
Description
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02386586 2002-10-02 ~ ~~-~RFF.~~1 T E:F .<~ (l i1 y n _ ,$
'_'ize object is achieved according tc~ a first embodiments cif- t~:f- inTt-enticn witY~ a mE~~l-:c.,~1 try.- prO:'iuczry a x~i occ>mp~~tb1P three-dimens~oual rnatrax,. where an ;a:.m~<->z~:~ solL:tsc;r is brepared from cl:;tosan ax'lu art c-;;:ce~~;s cv.f an ar.lca, t. lie aque:~:>l1S sGll~t~C~:~ '~~; nellt~."al~~ t--i~, t:'ic~ r:e:ra:-~~7.i~ed ac~.teous soluti.<:n is frc,~:E~n, and rt::e.
a v~-~,'- ._. ~. ~ ~~ relwc~~ ~d by subl itr,at i ~~~ un~,c~r r ~ c~L~~vc:d nrer~SU:'~-~ .
iC' A t:-:r~=t~ c:=.-_.~ension~n~ rr~.xt-~;i:: .<,-i'-ri c.-~rt:_~:ara~l.e pxor~crt:i~::~
:~l~:a~r,E~ ~;,cccrdirc~, tc~ a secorit~ c~mh~~;;inverlv o~ t1-:<
.~,_'~~:~::t.or: ~~~Tith a r;ethod for prcducilig a tnioCOmpatil~le Llur.=E'-d?.ICI<.'WSIC~Ilc'i-1 Tt~truX, Wrl'la~~ art dC'('v:,~Wu:> SO.lutl0n ~.3 F~rA~arE~cl zrom crlitocan acrd an excess of an acid whzch l:: ;_:<-::Le;rr: e.3 From the croup termed by aI~=yi axed aryl r;~i_~2:':'.?:'~ t.-"~rl:'C7:'y ~ 1~~ c..c.LQ:a, '~l8 ~i.~l:~GLi:a SOlL:t_lUrl 1;
::.2-~::'' F'.'"' , ci:lu '..I-iC V.'~ t.P_i 1 S 2-t.~r'tC?VE'_~~ ~~ j' SLIYi i. :: ITicj t J ~ ~ C1 LiIlL_:E:~u - ;~~. J +~t ys i3 L~Fein;O r~~movecl befe rc.--...
r ~ c m := t> ~_;, rm ' a r a , 'm i. r a x C c a ~:
i]L .~:x.tF'h ~Y:2 2'C:CLOVa~ FJy Sur7111TLSt7.c:)t'1.
_.-. t~~~ rr~.e~~."t_c.~:i ~,f thF inver:Li~~r~. first-L';~ ar'1 a~ue;:~-L:;
>:_:~.~'~-L,-,m ..:~ ~uL°e~a~:d rzorn a r~a~c:::a":-1y c3~,a::wt'ylatt.~
_:? ' 2 ~_7'.c;Ll ,-irrd e~r1 c:Cl'~ ':J~'11 Cf1 ZS _~I e=SE-I':t 7 :~i e~
CE?s_S' . , ,'y,~r-rS 1~~ r.?E'c?:_t lrl tIZIS CWYIrIE.'':~W' i=il~~ L:l'L' ~iF: Cf t:a ;~(.~;iE'Oi.i~ S~W.lit lG'it ~S ~ri t!1 t. 2C7 d7 W 1'c:-:GE?, yr-~~~.'.,"G~7i 'y 'u 1~:~1;~'>z:- 'r.:H . B~ .his means thF: fr~3e ar:'w~nc> crrt~up~ _i x: "-t e~
~1-L:. ~:,,,s,:. ~:rw <~tt 'Least. part:i~~ 1',~ ~>ro'onated, whic?~1 .':'.'... ,..:'C'::a r~']r'~ ~.O~ll~."Ji l i t;j~ 1:1 Lit3t.E~''r . ~1'~'3f-~
al?lCiuIlt W czC' L ~''~
~.:~ L:O~ C1~. ~:.C~:1.1 . rt IilLi,~ t; mcC~C3 ! 's' L'7 E: C'.:105E.:r1 :a0 t'.~'l~~t; f..,_1F:..
_t..i ~, C:O;c~ri dl.~,~U-~'1~5. F~XCeSS~~.'f.' ~dd.lt:i.C7r1 UI øC1C.~ .., .W'~'~.1~-1~:?~l G;IiFtF' pO:~SI~~E rJEi.:c~llSE? e?XC::E;:iS ~t~:_Cj Llllly :'~E:
e~~'r,v~ed a-sU'_r:, an~< tiuus 'he k~c.,-h i.l~:~ uh .s rr~a~ It .:_i:__~~u!a .~r :.rye an-~oun~~ of auia ar~r ].a2-!~E:. :->.m!%ur~ts wf c:~~~..1 ~~::W~:~F': C';~L;iC i:1 c~ 0.':J~~ !.u ~ Iv, ~>rel:tYtdk~l,y ti. i r.c'.>
_v, in particular 0.1 tc~ '1.3 R, solut-.o:l at-t:~
- a-.'>'-~b?. r > .
.. L,ar:: .c;,::ax.l.'.' °.~u,;t~ablC=~ ac:i.C3 i~ lactic: ,-z..';::1.
T::a>
'~'~.i'i.;~',~'Li~'.~:,~ I';wr:L:1.X t. r:F'tl CO1-lta?.:1''~ ldL~.c3t',.i~
lOIiS i~l~i s i: y p_' ttER 1:? : d-1 F:1U :511 :, <;° 9I6 ~ cA 02386586-2002-~l0-02 '. H.~RfE~'f l E.k ~f,'1 (si),l c:t~~~~nte2:i~:mS of ti-!e cP:it.o_-;an. ~'he :I(~tri.x i:; p~ntic~zl~:r.y,~
SOf t ~zl~ci ~la'~tzC in t~-_is Cast:e .
'1'h-E: ciI'OC'ant- t~-t i'hi tc>saW iS r.Y'ei eral~~ y CrtG:~~I7 t:0 reS'.i'.
iI: a ;~onc.entratir~n of from 0.01 to '~'~ by ~aeig2zt, t rvfr,~rrb7y ~:n. 5 to 1'~ b;.~ we~.grlt . Inflt~e:~ce can t~
_YC=rtt:d ;HIV trne~ structure at t'~e matrix, in particZ;:l~,r : !.:~,oz~e Sive; tzrcu;gh t he corlcr-nt=~~xr:.io.n of t~?rf.-s ~:_rI_ :o:,ar: w~': i :at-~>>rl. It: iS t:~c;:;siko!.a in :.h. ~~ ~ra~~ t,- .
v., ~: t;
t~~~ ~.::e I-;;-~L._.~ ~.'._,f, e.,f :l":c-. at-ri>, tm the x;art..i,::2:1~x-t~.'~a ~,y a:e:7.;~~itt-I ~f;?~:ar_h tre ma.t.Lix ~.s inrer_~caed to ~.:>f: calani~e<~t_ GS~o<:u~~' c:Zii:cs.~>a:-I i;; prc.~ducea f=~orr, r_at.ura.l so'~:xCes, : t ~1=9:: Y:C) i,:i'..7_ft-'T-1T,'~. lTtp.~E'Ct_1~3r WE-l~yi:t;. 1'f'LP 1?:p.~E'i-lll-W t- WelO]iIr t- i';lctu. V-~n~ 2ue=:_4:E'C11 ~~:« ;iT-lc~ :~C: Pl~.?1 -E.-'' t_rlcil'1 ~
U~~U l!::~d, GL~-~7l3nlll_11~_T
I1 i~aiG :i~JilT'Cf' d:l~~ yYCyc1"d>=1C):'1 Tr''..'IOUI. ':Iif= C'~112:OSCIl ~~7.' a .'L.'.Icv.-iiCl Y.~1E3 t'11L°PE-C'..iITlE.'TlSlWlic'11 IT'~c':tZ:~n 1.
TlOt Sl.I~~E:C:i_ ar_,,,~ rep-.txiction:~ in mlat.ion try its rtolscul:~r ~~,_~iQht.
' d by s u:b w i rna t i :-~ n .-~.i:~E'f t_~lE: Lr'~e'ZI:1~, u_a1B 4adi_CR 1S rPITlO'.:TE
'.:I_QF'?- =~E~711(_t'C~ ~?rE'..''>S:JT'F:. .c.lli t.dl'JZc' Iirt''SSllF'~
Z~3i1C1eS r~J.N
Tr:ITi i.;~~~~ t:) 3 rl~d. rr~2lr~ E'X~3<_t COS'lrllt:lOnS c~Y-E' .'~-iJ..2J.;~..°:ta:~Cl 1"~\: r,I=~E' t=W >I~Il~C~.'al~is.''I: G~
L.rIE~, aCIIE'C':iS :'(7~lltiQ:'I.
. ._ '.-::il::'! ~-- ~a ,C:; .c'i" 7 1.E ~C:L~~ M;~=~ ~>~E~'ri LLSLO'1, i-t lT:cz_l ~.W -' dt: lc:.e(:
LtmrA~l~; ,~~..'e~.~.pc:rated during the lyolara:ili~atic>n.
tr.F:r~re tree focusing it is preferre~ci 'o remove excess ::,ci,.': ~.ty adding ~ base t-o adult _r:.<? IWi of the acTUeo~:s 3G :<u_'~.;t:i;~.'I ':n f.~-:'~m ':..'_r '_~.~ 7.5, preferably ~.5 t:o :'.0, Ta::Y ~l~~Llla: r _'J t=~~ . .'..'. t~: lT~c'itl=''_'.i iV:.:..I ,.TE:~,_, C~C~~~C.
j::2:C'j:iiil.''-.:W.'~: lil Z'Glet:IUIl to 2tS u.,2me_'lSlWi'1~-~. ;-itd~a.l:.'.~' . _ ,...:<;~iulr::. '1'rIe lrJat:T:.ix ;.ic~c.:s :lGt :~ry.;Ii;-; ~=,r~ r:-t~t;vlrdratlon ar::a ~.~c.:~:- r-:<:~t-. ~ilC;w- an-r~ :~;:i uctu;~a i c:'rarig~:°:> ~,::
trlFr . T:li..
-, _)') rL~3I7~c' :): 1_i'_ ~f:>'. __... ~c'~~-C.1CL;1<.-'~I .~' .Tl~~-'1~:E;_~~
'.'J~lt'Il t:rlv ti!'I~l~OI=,:
a . ~' _ ,1; _1'~ ~':>t: C1t'zS :3 yH Ui ixOIT1 t; . ~ tc_, . V bE'~:?1."E t}_E-' ~','~'!t='::iI:~:l. 'i'rIW ''~lltv'a-'tj.I'1 Std1't:',a' t0 j'.~~-c:Cipi'~.~lt_E' :i1 t~i7;:
r~;::a~:r . it. s ~:ssun7ec~ ~.rlat. Llncfer r_eut r: a1 cc~nCii tiolis r:
r,,:,l nr' ~-t ~ ,7.:W SUSF7~:1S.7.On : S fC:).::T:cC~. GL'~'ilCt'1 1'1afi ,., 1 n : iW ~ iW ~!FR 1 : v.l -I r=il 5 J I :5'=t' 9.1 Ei 5 cA 02386586 2002-10-02 ~ ~H.-IRfE\'I I ER ~~i n 1 i~
be::~:«lciul eFfe'rt ora tt=a st:ruat.u:~c of t=-le lycphili.z~d matri:~. T}ie nemtrali.: ed aqurou~> cflitosan solutiGn tr~us ~:' ... _;u:~~~e~_mwon. The c~euu~~ suluti<:,r~ ~i'.a~- r~lw:e.refor.~, C:c~.P1'lCtl.:lu t: r1 t~:° Ct'lJ.,trOSan C'C>:lCE~llt~«tlCW, iJC' i:
~ , ;
CC TiL COOL.".
'7 , : :1 1: ~ L: i. .'. c 1 -~ L r3 t. :~ C7 Zl .
f ri a met hc~cr 1 _~ke that ?r c~pos~~d b~y 2~adihal ~ ~Y~ et a.~ . , t t:r.
:r,~:'~.~. is e?:-.~t:~ri r.~nc,es a l.~;x~qe t-.flan~c~ i:u ;-r:e :,'truc-ture <-:, -,Fv'u.'-IW . :::'_..xt,-ii>l. 'nt E'7i(:E;:-'.~.' c:lCt;:tlC.' t3Clu t.>~' ~CTI~l1 1.~'?Z'r~t-1~.~:'1 1i.> ,.t::: ~:mtoas r3aUH. ~:ddit-ion of talc uu-l:~:r>u5 soluti"n F;ue:-ulr~..~iJ~.y' l~~aus tc: loc°al di:~so~ution of t_he chitosa.~:.
v.~E:it::?n i_~: : ep~ eci~Wt«ted b;,~ the PlaC~. The pore structure OJ: ':r~= T~::ztr:L.~: i S '.tll::S a~' terE~_~
~:~ .~s r'~:~sa_.~;i.; i ty leas now been fo'Lrrd, with the rued!od fol.:nea ta~s- t-t:e im-entor:s, ~or stab.~_Z iairlg the rr~;~t=ri:.c so that:
.-it:: , c>r cc'r.iderabl~.~ sma, i ler, str'u~'t:ura L cha~_-zges ocr,.m ''u'1 lla'I-i.:'lt:L yi-OCCSSI.nCf t_~f t=~1~ ITlai:.I_7.:K a~tC-"1 rE'IrlOUTal ~?:.
..:if' ~.',-'3''E-T' ,~j' ~! ~~O'~)~'11 ~_12.c;f_1OI1.
im ,t,'~~ fiis' e_'r~oc?imF.rit of thc~ :nethod of t_~Ie iliwritior_, v:he c:iit.osan :,olut~.ori has a neutral pJ3 even before the . ~ .---:G_ ~~:lg an.~i lyo;ahii:irc~tvor.. By ._=utrai ~~1'3" , s meamL
~.. tni:-~ ..<-~nr_E=ect--.on a poi in Brie r-ancJe =tic9~.:,atec~ abov.~_ ii° ~ ,'i '1":,~'_°ri!'~i-E;' l:fl ? Gr~C~F~?Y :iCC'l~,~~~'~z1 j% f:7 u' 1_1!E' CI'e.tT''L.'.C 'cWi i~f=: =~~xui:!:.i~r~:t~c:: ~.~~i'~i bare afZ~~r t~~~= iyc~plui2 :.,pat ion.
Tt:F.
-.~:~E_°W_~~:a poixW is ti-lat. tr.e ci-A~r_c~san :olur.:.cn i.s ,.:.m-Lta~.r.'.v- ne~._;tral.iee~ aad tree cr::itosan nrcc-=p~r.~xte.-~> ~=l:a .':?. ,.IAF_' mewt_.r=xl.i~.at_ic_,rt is alt~~f~ 'Tried o~.it.
_~f~l C:ctIL=.:' ~'y', ~'i~E= Ie.~;l.~i_lrlC~ 111ti'."2'~-~_x T_E't11:>~~,',iV~t.'~S ''?J:
1'E'}'i'"Wj='w:tl.Ci::. 1I'1 tr.G ~lt~:t_ C-'III~iCIC~IIT1L:I-It CW
t_rlfITWti1(~C( ~'i"
r-r,r r,e,.~.Ilt -_~,I:, the acwea is rm~t ~ r. th~~ first instanew~
~~.p.-.-ie~W to any Par':icul.ai' restrictions.
z'a ?r: is possible to use fc>r p_E~parina the aqueol.s ~~:-u.'c~:a:n ;~c~~.u~ic:n an ac-.d wh~r_ri is ::electe.=~ from tt~e cJ.-cAUU _:':>zcn-ed :>~,r i_ric%:-galni~~ ac::rds a:m:: «rg:~l:lic uc.i..:l~:
, ~:'rcW-.v-;i~=>_-,- ~7~~~3-- and arylca~~bo~yli.C acids, iIl "-zr-t ic'u i~.r h~yd.-c~f:~; c~:irboxyl ~.c ac~.c's . L<-~ctic acid ::
Ill iy. <y' )t>~.R 1:,:.~5 F':~1 X1.1 3sW 9-tu:> cA o2sas5ss 2oo2-io-o2 c:H.~iR>'EVTIER ,~I1~11 _ ;
;.
n~~~-1- i=l.i:."~.1 _::xr ~~' ~iI_ E?feT_~2_~-'C.'~ _ xa-:c.z:;l =;~ of inorganic ar_i~~;~ are hydr~;c~hloric acid, ~~lic;:->~'ht>:r i~~ ~a<.i~? and acvicxic r~~:oN~,h~.t~ sait~:. ~'x~:m~les .. c~.rct,':Ii'-' a:_id>:, are alKyl.carY~c',;~y? ~c acids him%ir:c_r 1 ::_~!-:->o:u moms, it ~e~.rig possb.lt for tht> ,-.~1k,~1 chain t.o :u a :a;~:~.:.~~:311t.. c~ldin cI l.~rancriecl. i-:xamplc-», a>_'t Lorm.wr _,.. i~:-~, ,~~efi-.:., ac.c~, _ wop-i~~r,_ic acv~d, k_~ucx~r=_c aCi i~, ._,e~.= ic_ :zc iu, l:ex~rloic flcid, h~:pt-..anc'ic ac-iG ar:d hi y7r:I
:=tr~:ig'~r:--a~l_air~ carLao:~ylic aca~~. ~lsu sui.tn:r,~le nxa C,7=iC=:i?'-);7i)~;i,~L1C aClC~S ::~V1S7C~ ~ t.0 rG' Ca'_'.''~')iaXl e:tC)1'IW, ~'1..1,C11 ex:u :7::caliG ~c_j.~~, ~;LICClnie c3C.'.Cj Or ~di~l('_ ~C'Zt~. jp1 ~ui~:.aL~le ~r~~ ~:roma>:ic carbc::y'iic acids such as berzc.~,it~
<x:~it-i ox- napjir~hoic acid Furtt:e:~-more, arni:m~> ac~i:~s, such c:c;.LLtcin.C acid UY aCpc~rtic aC~:~, c~L~ d7::C~ sul.t=~~x'r~e.
:~t'u'.h'~=",'r."'. ~~d:L'C LCi:~.c'3I'1~ :pGC~ I't-SLlit-:~; c1!E~ ~-~~W~ll:trC~ W1 L1:
. .'T,0::.-.,% Cci:~:J<~Xj%1iC 'cic:idS . T:G.; C;C1:,? ~r~y .'~.?1=L.el;;>1G
'-y-_az ~,~',r c_rm~:~o:-;yl-~c ~:cids ha~'_r:c; ~ to 1'? cwxborl a,'ann~ .
~...:::I:.i",'I:.:_ ~l: ..i":C:' ::T :l'.i~ZP 1'ljlC,rC,':;yl Cjl~Gilp S dI7;1 OWES ~.:?
;itW.'W- C. d Z I-'C: X'J i CI2-OLlj7S T~rc.PIiu, i lWt-:P_ ITtt'W, EC'u i E:
. ;X2~- tMTJ~ i:~
.:il-t: C:i','CG'; :: C izC 1C1, LdGt 1-C dc :~iu, alE~1 ~C ciC'~-.,~., .'ar~~r i C' lcie_~:);:: c~ t-r.i.c aC,~d. 'ft. is ~L7.so x:o~s~bae ~o u~;Er ;a.-t.-"y:t=Lt: r':',''a3:O::V C:drbUXaJIIC dC.'1GS, ~Or Pis-4'I1'.;~.lf.' T.fi.~.:1~E11.C.' .__ ;'ii~.l.i~
Base~> ~.~h ich can be used are cc'nT~ en" i o::a"i ba;~es such a.; , :-~,.r ~-1:<:Yrj_~1~-v alkali I:let.al hv;~dro~czdes such :~.s r~aOH. ~~t:e j':%y;' -~W; ::~rlc~l:cad i'~e Cr':O.'-'~t:='1 Jo WicilL d nE?ut~T_W 1 pij Cdh ~_~..
i;: r~ i '"i r f: a ~.. i t. h ~J~..I t. ~~ ~ ~ ~ w. C'.1.: .l t...1' .
.. a~_:'.v_rlf'I' i'~~~7.'-'.'~:1~).'-.,:.~.t.~' fOL' l~t)I.;ilr:111CT i1 Pi~?tYiX 1,':'Lt.jl ~'L~i C- ,''i~ ';t' _rl1<; ","t:Y E~' 1 . ~t7r t..lN =rld L. y .i'.~_ :;.J
~:e:' :~L~iji1.11 zE~:.~
~;-~;:~_''.. . ~i-i '.rIC? ~..:.':iClS ~,15~"'...1~ i0I C~._:~~aol',7~":7<~
~I7~~ ~a'Z3.~0:;>ctl7 ' Cl :;ester .
y _._ :r:i;;~ enne_~~ged, 5u.rprisir:g7.y, r:hat~ tine pr~.:p~x.-t=es ;:~
t.:~i~: _..rt.osan :r~a'ri.~. cap );;)e irlf?~aer~eu 'ny the choice of ,.-:E ~~C~U.::LCr'i:.~11. ~fulS 1. Llt~illZ~C1 111 i:.rlE' SUCOnEI
~n , ;~;; . t~,~, 51't:h 1 °. t.-'! 1-.~.1 ' 1 -! .'~:r ,' 9-t f~ ', ~
02386586 2oo2-io-o2 ~:: H_~.hY~.~..l.l Fh:
_ 8 ez:~!odiment of t:he method of the i.x:v~enti<):~. ':kie acids LiSF:W : "~.I1 ?l~'11.'adrE~ I-1~'Cl~:-Oxy Cdr"~~0'LyZ.ll..' r'3S'c'' u-~C:.'ad~~. l.lE:'ry c:c'~ci ro:roioert i.as arc-: obt.ain~=,-i in partic;zlar cn use c 'Lartir. ,~~~id, w_r.h whici~i the iac.tate arlicn forms ti'm coi.:;~terwon to t_he protonate<3 amino ctroup of Chitosam.
;li,;~:, r"a~tzix :,h:~:,~r~i:igh temperature re:~is.ta:Zr_e and m c.wn, ?~oz. ~=xart;pi e, k~e autoclavec~l v'rithotit diffzcul ty.
IJc ca_~.~~e'_z:ati -errs other sicrr:s of cieco"rp~~:i ti:~r:
or of ; >:e nlar.ri:; <ypsar '~ur:n r ti-lis. ~~'he matrix als~~ sho;:s a high e!asti.ct~~ arld mechanical stren~~th. Tiuus, a sheet-like tT~;x'.: r .i:: c-:i,!ta~c'uz ~mc~ the ~qu~~ous c-hi CCSan i:md ~~;~ i pact ..
a~i,rr.l,~t:i~:,r~ int.ca r-s=tri a3ih and then area=,:ir:g .i~:i ~, ;:aul~:i:ii~._r:u, b<> > olled ur:~ '..~rii~hout _i:i::f ~~~c-il. .z,-;~lty .rx;d v:~_~_.rnut 'r:a=:c::r~.ri-vc3the matri;t.
i ;;
i;:1 r.:.:c:: ~~~rcarrc.l eni~oci:im~=nt cf t-he Iner:iod of t he ut'.'~~.'t'-'1C~~.i, ._i1G StabJ.Ilt:lT:C_j. t?~f~=:Ct of :hE
1VC~,2:0~:~'c3~iS'_'i-C''2- ciI"il.~ Cr..C::i:W;}'~ 1C c3CldS c:,~.'FJE'ar:~ eS.rH'r: .' f t:i'1-G' 0111 ~-~.0~ _~I'1 ~.~lut..ou. i~~ zoot neutra.iized _before tile ~r~e:.irlg. ~?ftex~
%!? ;.ue '-c~F~hii iu,af.io:"~, residues of e:~ress acic.3 which i"ire _,.. a 'i1 p_'esc.~nt are rer;zowed by eciui?i.i~ration with base, eaaz.~~ ~_ ~f~.0'.-' is Na;~H. 'a'hE= matt:i.x is t.h~:;1 waared ~~~~ti"1 ';~~:~ Fir , ~:.~h ci~ m~z- c~ntai r: a phosr:hata ;.~t.f f c_>r (':' . 't N, r)H
at_;<_!._.. . , . I'iic structural cimr;,gc::s _:z the: matr:.x axe :~.r~
. . ~_.'-.. . ::,7ac-:~ cw'r:r i~zer.:~bly ~ c=s<~ 'rzar~ v~;tt: 4hee ~rrat: i :1 ~-ies:,::r~i.i~ed iai~ r~lGdi hally. i 4. is particu7.a~~iy adva.:-:tag~:c,m i .~ r.h~~ ~w~c~ond em~bodimer~t of the metrioc9 ;~)f t :'ze i rven t i ~~~n '. C' .. 'L I-l f: : :r ., :, i,. ~ > : > d I': S O 1. L1 t 1 C: Xl t O ~) a :
i E~ is t.:C a i 1. ~. c' C~ ~ V F~' n u'? a 'L C~' :" F.' :.i':e f:~e~~<:_ryF~cruilibx~:t-.~.~_~I uz~dc~r ,::lkaii:m: c;m:ditic:~n~::
_ . _._ t::rlrv=1 ziC, ~r:v::l~ie:'~WleCE=-~sar]'. ':lie :?1c.'<r_. C'<ah ,.~"'..~:~uzi_i,~'r y';";~,~.=c.',~y~~ di?'CCt1~; dnil, f_Ot example, c'ol0i'1~.::E~d 1_~'y :~ C' t; f1 P ITt~~' ~:~ G ~. :Ti < ~ W. S O f t h f_~ m ar" i_ h G Q C! .~ t 1'1 <! 1 r:'J B n t' _ C> n t a ~: r.! 1 L
35 in a. mat-rix w:i th a uniform por= st.rur_.ture, which s<~f~-: ~ri~~ ~:>li.able. I:f, for ex~lmple, the m.~trir. :s c~or-tnr~~4.~:ed i;~~ F;=ensure from the fingers, it- xe:~umes i ~:.s t_J.' 1_~~.Ii~ L ~~;<:~~rc'lCId1-I': E3lcl~:tr~..Ca.i ~ ~i eiit°r t:iE
~?rE'S~illrl_ ;:i _ _=::lo'.wd. 'I'hr~ rnatr::~: can be molaed wxtho~.it breaking car i 1c~ ~t~ o:: BIER 1 ..lfs F_~~ =31-1 .'.t<t~ <.~lh:; cA o2sas5as 2oo2-io-o2 <'HaRPEWiEk ~~';<t1a _.
~cra:r;;bl..ir_:r a:':c~ C~iri, for e~;amp:'e, he zol~ed up_ ~io L:.>iz~~ rol~:erent asserr~b? ae~es of ce~~s it is necessary yor t-nr=a r:,at-T: ix to '.zav~; suffice ~~nt< strength c'.urinc~ t.t:
°, ~w.~? i:rl i ~ar_ion rr:l' cell s and rc~ be nroken do~~~.n on2y oT~'er~ a ~.w~r',,.r pro': o:~ce.d Fex-~ o~:.. 'i'l:e ra'.'. V cf hreuk~.ioV.>1-1 can ~~e ct-~:-ir.ro l ~r~_~ -.J:i a t~-ie caegree of deacetyJ.atio:l of c~hitosaT< .
IBIS ~: SE ;7f w uhltOSO?1 'vs':':~'jl at:l.ll ~Idp d VE"_''l' j2?t~~l C~2Ci2~::','l?
~ s.:t.,=av4i~~::z it :s p;~~s:i~u.e nor breaks:;~wvrr-1 ~.i.mus ~_~f 1;! a~-~: <v ,~,.;-i,':.3 one lw,~nt:; ;,o be ;~cti~ev~'c=d, ':'iaE-. ln~=.r.r.ix .s observed t; c' b~? part.i~~.1->'v~x~l~' h~aiz ~'1 ~~:~..~r;'~"~ri~':i.E:' t~:'~-7e=I-1 2<.1'1E W~ll -t:»dIi ~'SCiS vi C~E:~::~r E'e !' ~~Eai:G-'t.l't.~i'pl~ ~7r t~n~. 'T'~':~=' ~PCfx'E,'P .'_7'~ i-Ic':>ctCE't'l-lc~3t-1 L~: :.~:1._ze_:: t.: tle PJ-dcct:yl gre:ups of chito:w:n. i?y degr'=t~
~J~' t:~CciC~'t.4'ic"~:.lJIU '1S LI1Gd11t: t-he rat.lC CJf WllciCGt'~'ldi.E=C.1 -; : ~~.- '''~~ 'a1 of azn:ino Jr-e,ups presE:~l~t ~ni.r o grov,_lt ~ '0 4.. ~.. to,~
(tc~;,=: ':-~z :. r.ee amino c~zroups and ar<.t~~lar.?d amine . :'.i.;L.r~ . _It is aSS~;nc~d t:'lat at a d~_c xGe vL
. : l ~ O' ", c3 C:''~ t ';' 1 d t_ . 0I i C-~ t. ~ g l~ ~ c'~IT.O I ~.):: 01:
S S t ~ l_I C- L 1; z r:: :.: c~ I''=,' i O Z ITt~;' ('.~
lli VlCl~:~ c._ Ec~S Ot tYte YTlctLriX. i~~1ESE: ?IIIOr~7hQ::~ u.''.~~clS
W.w."i r~?: E ;v_;lTicl~;i~' I~~r~ c~C.~c-iC!CeC~ )uE'ttr4_x~ 1~?1' e.IlZyCIlES . 1L
1S 1~~':la;~.
'~:~'a''a''xZ~l.e t''J ~.:1_ _L,1C::':C:E'? r.iE? :-~:lt E,~ a: l= W~'1? Crl ~'~'=E.' ~';:?': i i.X ..,.
,..'.'.o..i.._~!.1 ._f. :":.'. ~~lO:~-yr ~:I.l~ ;::'u]~,~SLE:i:1 _-r:..~ rlc,=
~~ia,'..~t'u.Cill.;~
_. . ~:t_'~:':~':.
.:::- ~:m:,t:e _ ize c,f t:'~E rr;atri.s c~dzi LurtiiN~r bra inf~luencf~ci ius- t.:lc~ Yat.c ,.~ ~;~hic~l-, the ~~~~a<>o:Is c:r:~tosan sol~:t:ion i ~r~~r.c=1. ~r~ yin ~, connection, rwps.c3 ?vree~irc, i~~x <<~:am~~! ~~
~(~ ..i ..iq~jic~ :-lit~.ticxc:=za, z°esi~l~.s in ~rr.a:l7 h,~-<~ diary-,<~ter;v, ',~:~"E':C-E~~S :a~.:.::>'.~: fre~Z~.?1C~F, iC7r F'J~.ci:TIL.7lt~' 1.n t:he !-dn(QC fl:Qnt :~ 1 ~ "~= , -e u~u '.ts. ir. largo cores . The telr.zaerature :a:,r tr~:c~ir:g the '~izitosan solution is pre~:e_ably chosen _-.. '-'i:= Yes,.:i_~~ t=rOm --GOC)°C: t.0 -4~-'~', 1G'=r=eid~..v~;~
G~°C ~;:~
35 -i~~i'~.. I;-;_ t icuiarl.y preferably ~0°C ;:« -1~~~~~~_ ~':c- ~v:at"ri~ o~~','~iz:able usincr ''1e m~t.h~x? c~e~~~c:'ibE-.~t~ abc:v<.e ::~. t:C.'('v? ~(.'r:!' ~%rC?iJert~E:S ~.~'y' ::t~IiliJdr'15.'7I2 Y~'Li_:i :T:r::t:T'lvE:~I
;:=:~;a'.~:~ i:? ri:~? L'~2::10i r3Zt<. 'I';lE'.~ 11'LVE'21~3.OY1 4E1F~I_E~fCra-? ~.I,..S'CJ
i~ iJ~-cv~ )TE=.R 1'::-(t3 t'.-1T '1-! Z~7 9-IIiS CA 02386586 2002-10-02 E-~H.~RYEV..I,IER nic~lt - 1p _ re.:ares to a matrix as ~~~.~ta=rted usiro~ t2ue metr:od c:~~~=~cri.bc-.~c1 a~~o-,re_ The matrix;. is dimensienaliy sable, .'>~!'r <irt~ f .,~ !:"C~~7.Le, c-'-.1'Si~ Catl t:~l~_1 ~ EdSl 1y ~.i2 X:Z'OCPS:_;~'i,~
w;uCl, fi;~Y. E:~:~~:T:j~lu, ht~ C'llt t,C'~ t.~'.w C~PS;I'PC~ :;.i2l~E' :>a:vpi_v.
It cm: bE_: at.:toclawed and thud> st.erWized ~~imply with~,ut:
ric:c~~mj~os.: t i or: rF'actzons occurr ing . No furtt:r-~r ~.~;r~staricE"s sIc.i-I as, collagen or alginate axe necessary fo= :=.tab: 1iz=n« the matrix. The aegree of acetyl.atc-,rt ,~:nc~: trm:;~, f:;x. P~:amp:lt~, tht: ~;t.vorbai~i'i.tf ~~ '~.he rnatxw::
'L.~.an be i~h~~~er_~ Wit~~to:~t- iestric'~i..-~r,~ end pan whms t»~
~~ir~-,'st~ c.~ t _~ t:~e particular: requirt:r;~erats . The ~.t=ructt.lw :~f tr:EJ :r~atr~x, iri _~artiC~a:J.ar the pore si~t~, cart k>E~
z.r.~=_ur~nrnd t hrough .rle <_-.oraoent,rat::c~n c>r the aquecn..m;
r_f~:i t~s"n sc~l.ut~.an and t:he f'ree~ rrg conditions.
~s "'P1~;~' C.~mE':lSiJrlcxL~~jl su3~71-E ci.i'1Cl ~le~ii~J-'_E': rrW;trl?C 1:
~:~.:L._IJ.I:tf~1 1~.r:""1: v:lc Iria'r-1.~: ~lc~:_: d ct'.'t:-_e' i::nic~ri r1';
~._ (.., l i t i - i_; I ~ -L. :1. 1 .
,il~ y.tle,: r'lcll:x-J.~~ C:c_I: ~~ rn:::,xl~lCW 35 C~~'Slreu tilr~7.1~r1 tl'tE ~.a~"~~F' r:un.:.~~=- of a:r,:r~o groups . 1n a preferred err~odi;<:ent t:tc -:~:ree-di mensio:.al matrix, l igands are ~c>va:lently ;
ro::_°o~,Ta7.~,IZtly btwand to t~r_e chitosan rr~atri:~, Freferab::y tc; t hi, 'r r=a amino groups of chitrasar~ _ L.go:ods which c.r3n :~~ ~s ed a_ e, tan examp-! e, iii owtn promaters, protcir>, i~~r.-ionE~~-, ~mparin, heparari suiratE"r~, ch.ndroit2lu sn_llfr °r:~s, uFxtt°an :~ulfrates or a mixture of thecae :~Lli.:~yrc:lC~~.S. 'j'7< .~lG7dIic'~S ~?E.'~c~.'_dl:i~.~' Si~rV~ tii e0:_~t~Gl eliW~
ir-y~>xa~.~a cr>;.;. ~:~x~~l.ifE:ratior;.
:Eei.l c~r-c~~~a;:h ~~.I the :nat~ix -~s wurt~:ex: i:npr~cwed i.f t.'aEr.
;a;t.r:;: i.~ ~::~~:tE~d ~.~~.th au::~riocacvus :.-ii.~zir:_ r y ;1,.,~ tnr~e.-di~nt.t~.si.~,mi:~ matrix ~~~- t:a ~r~~,>Errtxo:: can m aso.l.i~i phase im a c,l:~ture .~e;~cto(ce.:i i-,-<.'': i:~I _;' ) . ~i~hr ITtdtr.X :a:lUWS ~~ ~J~r"~l Il:l~~l StrtrICTth :'r1 ivl:ia.'.lt'E'~ TCIE'G~~.Url. It f~~~ W lI>V ~rrlE'rC~ECl ttidt_ t~lr' md:x~.X
;~T-C~aI:~L.iJS C"E:rl.1 <-~T ~aG:Ct1 n iv y(t ~' v(ER ) ., ' -I si F'.1v 51.1 .'t n'; n I t> ~ 02386586 20(72-l0-02 <.'H.~FPF~'I' I EIZ e~ f % 15 - :L 2 -acne LT-~.atri.~r: .t.imul~;tes ce.i1 <.Iro~wth. Lt car. therefore imk:lan' ~~ci diz'ectl~;.~, i . a. wi rl~out. ~~,>'evio»:. coloni zati oLZ
1.~'s' ~'G ~ .i:v: . T:f t:fir trlE. LT?-e~i~ a:~ti~C.~,i_~rl, E"1C3W~C7,~I'iC.71.15 Ct''11S ~I ,'_',L~r 3- 11 , .~.y. t: : 7, t il i T.Ilc'.l t i- l ~i (~. td G I: <3 .i. y ,~.' C:1 S S 0 1 ~J 1 I1 Q .
c ;'1~ matr~.. is aLsc~ suitax~le fcr ~.~e as c~~11 i:rplant, ir:
1;:3~.r:,,_~1:.7.,'it~ ~O? C~'il: ti ~.c=t~~ t=r'yp17.:1~ ~"'e11~. Zrl t:'..., r-t=!T:I:E_''C:t 1 __'.r( ' t '.S ~rPiG': rA~ ?:O t.. t0 'LlSI°_ ciTl'y' GJr'I7E'Y 1.C~ 1.,:,.
:, ~'l !'; W l ~.'-e.~. ~~. c: 1 ~: ~. . '1'~i u' t: (,.-' '.~ 1 :.i u.. . ~
c.~.i rC: f~ !_: r~ ~ :? 1 'y' C. d )! f: Tl x~ ~:' oIl s ~ i . i_ :l~.~i pn~:~er.'t ~~y b:. opsy and t:w~tuxed cm the cell rY~atxzx, a:~d ..
:':i~~' C~~.:1 ~ :-'.'CL~!~'.~_il=t _i C ~IE??1 : LT'.C~i~-~ntE.'C~ :iI!~
t:.lF." ~it3t 1'G':at_ .
1 2' ~'::>to .ctl:' . E.' l ~'Ct;1.0:1 T"taclL r l O7-:S ~rE' ~:a.iri.;
.d:l.'.2d : : V
~-,1-~-~'--1',:~a'~:,~ G:'vva_-.ir' 'C( r-~?f? C:iJ..~.OI:a cit (OT( ()f t.I~l~' t~'lI'(-'W_ nl..T~E:ii.il~'..~Y'ii:i1 lT~c~t=r1X V~)lt.~l. ~I1WOC3E?"lOLis 5t''4':il CC11.S (~~i-'Ilc, 1 G=, m_W :;t i tierce i 4~!-.ic-~~ - stimulated by the resp.=cti ve arowt:l ar; t.c~rc 0i' ,:'rP :.aur-roLlL=~~3i=i~ t. i ssue -- dif ~>'~r ent fate or:
1y _. .. .'_r,F a ::_e of the t. ranrpiant , dnc3 «ws r>c~ r c>
F .
<._,:: ~ :~::-i.= azr-icrl with c.ar:..ilaae cells Lwr reTl~.'wed io~nati.o;l v'~ j ~'. ~.' ~ 1 1. :Z (_'-'. C', c'~ I- t 1 ~. ~1 C7 G . 1 ~: a. ~.i 1. i d (~ ~ ~v c( t. i~ d yr c3, n t i.~. Ca t:_ .~t°_!~omp~~r4u ~~sit:~ piez-ious mat~-ic~.s Lnadt: ct various a:. t ~ ;-:> :~1: ~..r ~-ami.cs . ''~lze ~f : Tr,~t er ~.,:.a r~~rri~in 1:z ;:.i-:;-.
r_ ~', :~ ..n::.na~~fT c~.jer. a~ ter _ t~=L,e yzar:~ be. re~og~li zed ,..
'_;.~_-e'i;.7zi a:lG '.~:t~ re_ie;:t.e~j. ~:~:r-:~ ;turec~._dimcr.vional r.-~atz.i:.c <~~ar: h:.:: ~_ _,=c~rii ~:e~i k>oriu 1?y luu,mam ar.c~ ::.y arl.illi;~.l c:e.i ~ws i ~ ;
:vs <v : ,-y.7 ~:- E:. f i' ~ 1'Tt I10 .t S a ~ Ci r~ g C7 : ~.' h c, L- ~L I
. ~ h ~:c Y ~~: :~ E_ l l s c: N i3 ~»r-r_icul err'' ~,T sui t_ab? a because tricky inciure a negligib i a :r~.rnun~~.cc7ical respar_~.~e in the ~.ecipiexlt. siaark cell:::
oz:"s~ ~:cii.~'i:c~~7'J 1.1;:~E.~C'1 uS ~;~:~Gdn Yi=k I. c=;C:~~rt'W1'1t, I''tW° ~~:c3TCl~:I~' f;.i_ 1 .,.- -. ,..
a ,.
..rl!-~' i...'~G .? '~.J t C. i C:u .
i ~'~
iz;a:l~_r:t ~~x tt-:P :i.n~renti~r: which st.im;.l:'~lte~:~ only a :,'1 : ": i : : iO 1 I I ;.T L: _'! t' L' E3 S ~-~ O r( ~~ E' 1 ~ <l ;~ V e7 :''_ t ci ~~ E' C:) 1.1 'u 1 'V C t'Wly.~ S f:' ;~
''l e' tl~F c?...~3ltnE'11S1C~I7.c11 TriatrlX ciE'NG:Il.~.iFe[~ ,;i~0~'E'_.
WrSl::tz ,~. ';vlonlred key ervdo_1er»us ce.! 1:~. i-t:us, ~:vi-P:~~.ihlE-;:i ~, a tnixl sheEt-like z:~atrix which ~-.a~ be:-:1 ..._....~.iSe~i tvit~l keratocytes under s~,=er.ile cc,ndition~-~ ~x~
~:'1 StiJ.'~cW1 E t_(( 1. t-:Ll?'2 ITicW1121T1; Cc~I7 ~er-1'E.' ci5 Sk~.L"1 trcln ~p~ dIIY_ _ :''r~<: coioT:ized matrix caz~ be remcw-ec3 Lri~ru the cultur to nm~~.fit-_:e~. mn:_ltr sterile Can<litioms in t.~m~ operati:ig t 1 0 : W~ tW ~I(:h 1 . : -1 ~ ll.~l p 1 -l .'.i~ ; <,~-ici:, cA o2sas5as 2oo2-io-o2 f H.aRYE~T 1 ER ~ tt 1 t~
l~ _ t; ~i?ei i: t'I- aI:c~ bF= ~ldCpC~ On ~1':~ C.L~ct1'1~~ WO~J:1C1 ~11Y. ~c?CE.' . 1. t ._. thexi ;ac c. s~k~?e for end~pf~no,.zs rFll:~ t~~ rnic~rate i.r.to ~:t'..-; n:~~t_~~ix '.c> ,'.oi~u tissue.
:I'1 a rCL: ) C~~ C: '~ :1-f P i : t ~ °, ~:. X t l ~ ], C J_ 2. ~ ~ l V a ~' :1 5 G ~. S O 1-i C7 S 2~ 1 ~'J ~. E~ _ i' r": c :.z.~.:..ri:c in tty~ ca.E? is cclc:nized .~~itr: li:~e:~ ; ells ir, a ~,~ tnbl P Cll? lure medium. 'The? a:sei;ualac~~~ ~:i.E Cell , t~~n . c':h :;u~~ ::r~.: -_-~l.so 1:7c stak_ailizeci hm the rt-~a~r~x, is r:~~:;=rz .::r.~::a.itc>~in ;:1e ~atie.:;_.~s body arvd tr:c:ft? can, l e;itclillti_~G-, d_~~'~St tkie LL:I-aCtlW~r= Ot ~ Clc~iTd~E_-'C:1 iiVE_-'Y.
Goad results have been achieved w~3er1 thc~ thrGe_..
cii?:ve_nwor.a_i matrix i: cclcini~ed bv,~ auto ~.ogo-as r-:hr-:r_drc~cY,-t_es . Zhe matrix c:ar. then by transp7 anted t:c~
1'= tlle== ~z1_Jp' aprlatr~ ~Jlu''ces on the car',~ila:~e wi:ere nc-w mc~; ti'~,<~r~ .--_:_ib5tanc-e call tt-ien develop. Tfii~ provi; f:>~
c4 raw t, a:~-,~a_n Cages compared a,>i th the methacz currenr_'! y i n use, of tza:'apJ.a.r~~ation c,~f cartilage ~velis wi.t_h<.nTr :-,_y-;~oz;:. r:az* : .i.x. ''_'he hi<:h robal''.~ J.it~- G;;visting iri t~::
S
tr:~:.r_,':oca ~:~ut the periosteuz~: u;~11 be darr;aGed arid b«z:c~
i,,~~'7.Ls wily r~._~.~ groV. irito the new ~art;.J_cx~~N 1<~yc~x', w. t;lu .>,a'r,:~t~~y._,ent <~irc~c:mr~it:ion c1 t_t.E~ r:ew uarti lage, <:=.-f; :..c-icW ?~v~ tlTf= i:r,~lar.t o+- tx:e in~~enti c,r:.
... _ ..___<:':ii'~.~~.=r',s Clt, Ut:.;lf-'r i=E_i ~.'.' Vf1-llC~n iy'!'~f~
,'ivZti~.~.,.~.L~ i .:~~?1.i_iIl.: Zlny 2. I'1E=.' ltlGt.I 1.?: 'crf~-' Owtf-?0::~;te::~~, r',C:LQt....lIlUC~~u.:r:.. , r1E'~~~tt_C>C_'y'tt:S, ~UW (? tildrl.~OW St2IIl Cf~ll ~ nr rl~Lliorla~.
Cel~~;..
iu.m~:r:' ion -~s explained in morE_ detai Z below ~~,r.. th Ol! I-rvE~r t;IlC~E? t~: , ~.'JC:Zc~~ tJ.CtlWE'.S dnu ~yT 1T;~'..-'d:J.~'.~ O~
t~XdrCl~]it".,-,'..
fv~gnc'cs _::o-~.' s~e~Wvic:al:iy:
Fig. 1. a:i i.maae GT a II-tatrix t~.~iCt. iawtate f-:~:;:._-: tE=r ~ :'~r~ , r.:rs:; ac;~ueous c:hi t_f:~~:an s~,-.~ 1,z'' i~;,rl l~na~.ri.nc~ b<'r~:-:
3J rTtm.:;.r.al.~~~c;d rvriore the fxe~zin~ ir: -he~ ~:~rr~du~~tion a., t.l~a Inat.Tx;
F.ic~~ 2 a. irlldc~e of a matrix, the Irtatrix haz'z:zg bf:=fin t=:m.lili.bl-ated a.tex- l,~~ophil~.r.ation w:th _'~iaGH (5'~ .~.~y !
to ,l~ ;l~ ;rF,4: 1":.~- F':~,Z. :;i.~ ;s<t- q.~E;:; cA o2sas5as 2oo2-io-o2 n'H.~1RF'E~'IiER ~i~ll.
_ a~y ..
~,=eig~~t y ~ n t.'ze prodmct:~pn of the ritatri::; t~h:e c:ou=-lteriola is for:rc°d in (aby lar_tate and in (b) ~y phosphate;
Fig. 3 a rr,i.rrograph cf the matr -x oo~.Urli:,ed 'p,.~
chondY°rlcyteC .
~.~camp~e 1 : Ge:~eral :rethocZ for pr Uduc. _ r: c3 a three-dim~en ~ior.a:i =rutr-ix t 1st a:-nbodiment ) 1.G c:t-:ito~=an a.s c3:~sso~.vred b;~ stirrirm~ in ci~lute aaue~:~us Gc=ld ,=~t ~l pH betwEPn .. and 6. The desix'ec~i x~H is tll~~n ~Ca~,l~.l~>2:EW1 'tJl2'.,:-1 C1l.~.a2:f'C~ I'~c~~~'13. ~~rlE; SlWutlC:;'i..1 ~S tY~i3n5fax~"C'C~
lilYtW1 1'ESS't.': G~ ci 71.11t.a1~.iE.' Sflci~~ ciT'iC~ iI'a.Wn dt -~C ti;~
~:'rI a ~~'<: s s a l u:~ f t h t h a f r ~. z ~=n s o ~. a t f on f .r f _~ 1_ T ~:-:~:I :,I T_'r3C1 1 T:ti? F~ ZyiJ~~l= 1.12:E;:I a:ZQ 2,i F: wu i:~?x- ~.~ t3inU'~:'r_-.;,1 ~:~p' ~',1~% ~ ::'fl,~r 1f:1'1 ;_lI_C~Ur XE~:~,lWf~r; ~~r~ 551:: E:? f i7 _ l_ .':> 'r:~;~~ . The ::p~;:~cxf re:':idme ran "n_e c~my:.Ic:1'~d witheul~
f:ar 'he::w ~wrvii~-ut.icn for col.:ni~~zt.a.~r: :w;wf_3.1.s.
?~u ExamplA 2: ~:~e.zera~. rnethad ~ox- r%r oduc; r_c~ a t:or~u-a : m~:~ ~ .f c~ri~~l rr.c~ t~ i.x. t ~:ld ertll~Pdimar.t 1 l~h~t:os:~r-I f;; u.:~solved by :~t.izrir~g i.:z <~i-flute «qtLec~us ri.~..~yry~:V Cet~'~0::'y'~1.C t~C-id cz~ d ~JH beC:VJCt~rl f altd ~. i't'it~
C~l.~?%r SC:l.l~t;.?.Gtl 15 t_rUZf_.'n 1n <~ VPSSP1. C'~f G'.lltd~ue S~'Lc:
.:--~r ~1 t-erry:f«a~-wre of from -_5G no -5'~C. Trlc vESSel wsr.h yrw.;.F~a. ~~vlu4ion is transferred into <;: :.3~o~.~hili2Ex.', t.~:F_~ ',~.~at~=~r is removed by st.i:'vlzma:t~.on under reduc.-F=,a Nz ~-a:=.!. ~. a~' f'? . ~ '_ te~ ~ . f iuP& ) . 'I'21~ ~;~.~c~nay res is ~_«:~
3'.l C: E:.'I"tci Li: LI7C; ca f' i:C'r J: E'IrIOV~.1 O~ I<r1E Cvcit~E-T_-~7~i S;Ll~7~ lITt~t10:1 ;~.~.:z.~if~ci Yl ~.c_~d?um i:-:~d~c~xir-:~ :~,~lllr;on ~'aw 2:~i' w~igr;; f hc~,:rc . ''1'~e spongy Inat:r iy i:~ thesrZ .~:a~.hed wi t.~.
~:ic:;;;>z:.::Ee;3 ,,~at.c~r until ti:e pH :is t=~:~iezvab<<. L~l~ c<~Ils.
Example 3: i=xc~ducti-~~n of a pc~rouC c:~tc~sa:1 matx:~x f <~~~;~zntex.:i >>r1 : 1 actate) f; . ? <x c>i= c:r-I:~ t~:~san and i~ . 11-i.3 g of lacr_i.r. ,--;icicl (5t7~
~.n ',;;<at;w, pl:.~-crla~ect 3:~'om ?nerck K:.a:, Dazms~;~cir_.l were 1~! n~ ~~y ~IF'R 1'.:.~5 F'~,1 :'.7-! :3n' ~.tF35 cA 0238658s 2002-10-02 ~'H.1RPE~TIFFi ~~ia1"i dissi:'i~.red by stirring in 9.?9 g of deio~lized water, and tL~t, t~l_~ Boas ad~;usted tc-> ~ Gl~t.h 1 M sodium lZydrox_de c': L L1 t. 1 i? I'. . Z't l E: C: 1 G' a r ~ O 1 ll t. 1- a n '.~: ez S ~. I
t :L t: 1 az ~ 1 y t r U 2 ~ 1"A c3 ~
::~'~C arid rhel t:ie sc~~venc caas remo~.>ed by> a;~b1_~mar-_on ~l. ' O . l75 lFzi .
:;3m: ~ t.~ressure o.
=, s~'i~ir~, f .1_e-perm foam with a :,'~JOn~ Blast ic:ity ',ryas ~~,~:.::«i:lec? _ '''?~e : oa~c~ was used ~,~it.hout: iuy~thrr - , :e..):i.... ~~ i_c=t1C>?1 : ;?I.' (:'i7l.G'1.!.'~:clt' C~Il ~y' ~.~=' ~L.1S .
~~t-lE: _'Ilcit.2'.? X 1S
'.E-'~ill:.E?(: iI: . ?.C;. ~. Tu'. :lctS <1 l.lr:li.OrlT;, flI:t7l~,' :'i.rl7Ct.l_:~~'i7 ::-~a -dare. TJo uist:c~rt i.C?ns are dE~tectable, and t~he matrix ~;1,~.,~:.a; a nra..f~.w-n f r.t~-p~~re struwt,.~r~: thrayhout .
Example 4 : Pr~W.z~.~~:ion 0L a pcro~as c.:hi tosan mat r i. x i~_~ruxit<_=:r:_c~r~: ~~~~~tate! i 1st cmbcc.(irner~tl ;z :vf chitc~san ,rmi e.1 g of acetic acid ('.~00~) wNr~~
,-.-s~~l:~.~:F»~ x:y s:-.irring in 9."9 ~ of daiorl~.z:~d water, a:m~
t~tuc, t>H v:~ras adj as Beer to ~i ',»i t2n ;L M socii um l:ydroY i _ic=
~-c~'.ut.ec,m ~';i~- ~lt..~;~r solr.lticn was, ti~'stl;T :-xo~en ~r __ '= '~C'. r.rW them the solvE~nt was ~~emoved lay sublimat :.c>:
i~:n;?i- r a Fr'c~s~ u~ a 'w:~ O . U'~ hFa .
.->~ =:rr5a.s~e , f:irm_.pore fow was obtained.
ExamQle 5: ~roduCtiorl of a pc;rc~us chitcosan rnat:: ~x r.~,.~..mter ~ c.n : priwcp~rnte >
~:i . . cn at cli t ~~ pan and CI . G37 rnl of ortho~phosphoric a'~ i d :e 't~ iJ.:l1":W >-' tj5~ (.~ ) '.~JErE' Ql=rsCil :jE.~-'C~ b~! S G.1T'L'1 T1C~ iii .;,i' ~r~?ior:i.~:ac.? ~f;at_E=::~ , ~~nd thF~ pH w~~s a~:~yu stud t:0 7 w ;.
trl ~c~~ _;ociit;sr; ry~~r_c~x-i.cie soluti,-.vn. ~'.'ie ciN;~r soliirien was c :-;,..: ::rozer: ~.;t ;~4:;~. ~z;ld then tam so'.~vent: v:Us ..~~rl;~',,w;u: ~7~; ~:ubl~,matiOn tender n plE:~~s~rE: :2: ~..~.0'p h:='a.
.; c.
a '.vwi tP, fine-pore foam with a spongy e~i.ast~.city was ~:}i->t ~~inec~. 'T tie foam was used without furt.t~~-r :.;uri.ticw~:i.~~m '-<~r c:oioriization by cells.
':Ot.-tn' 0~ ~IF;R 1E': lei F_1; 51~ .'3f1;( 9.E(i:~ ~ 02386586 2002-10-02E
CH~1RPEITiEF ,a;o)i Z c.
'F:zr~~.e 6: e'rc~dt:(_tioru ~?f :~ yorc:ws ci'_ztr~<~;an :r:~t~ 1., ~~':'~~z:m::~'r-im~ : c~Iut~.rnat~ ) . ~ p .-ot ru...~:u~:~i.n q ~f r~~.:.t.ai:l:racid (~t5-t~;:!aa arll: c_'~,'i '.-~ ''<.(;~.)-r;i~rt:~ c3zasvlved ~t.irrirv,,a iC1.00 cT
by _~_' ;y 'aF.v;~oaa:.~-c..;;~ ar, -i::cr---tm-wand d:~~juter.;.
p;-i '~,~ , ~~;ith iM
.::;~c.-_,:rrl.'i_,r~:r~:vz.ide soitrt:i<:r:r.~::m ~vE~a solution wa . -:-< ~t--r: pit ?=~~~ -, ar~~d ~.'se:r vr~.~ sv ~
vcnr '.~:a:~
_ _ ~:~'u .. ~-~~'y., ~.....-:;.1 d ~.. ~ C ~, J . iJ r, l-!:=w,< L::Ir L..1 i7i t i.:tiC~e2.:1.:.:'t: ,f'Z,~~<a <:> i~ .
.. m~~'112-E', i_ . yprC: ~C7dIT1 ~'lt~V1=rC~ ~'a ~p;;:~lCT'y' r-'12.~'.St;.'!.C1'V N~i1 i::' s: ~.i .l. : :' f=~ ( ~ .
E:~i ~TCiy ~ t3 'I
. - ~; : t ~ ~~:i. ~~<,s.~:n arn:l 0 . 1 1:13 ~ :~j La<-:t.i c ,-:ICid ~war:
'?:i: E-,1:<=;i 1-w.T stzrring ~-1~ ~~.-7~a g r:f dE :-7n:.;r=c~ v..~::rtF=x, ,::l~:
',I-tF- :''r-::i' Sv.lli:l0ii y'd~ j'.it.Ni~~.'~~ t[-VZ~Tl ,:t-: ~-1'''(-:.
..'iiE
~- cj"~,; it::-i ~"~ ':~,'v s t i~il rF':T10 ~ h'C~ ~>_v' =~ LlrJ 1. llllc~ t 1 O7T'. lili'~E'r' ;:t cJ
_W E->~~ .laa-~ U i_ (J . t:. f~l't~rs . ~I'llEv .'_~~70:1c~'v' :.~ E~ = ;
~~A[' W? r ~ic':;~=i t. 1. E=a fl , 'v:~l.: lt;T~. ~1~'C1C0%WLC~E~ :=.O~11t1'_7I1 ~ tO-~'~',-, .C'1' WE::1.CJ}lt: ) ~'.>TY .::-,!
a :", .,.. .., '11::~ ,; h'ce7~ Cvc,S~-:!.-'C~ s~: 1 t;h ~- W 1 C~I~ 1. 'E~s;t '::d t r::'T: tli2:.
.t?1.i =<-r~ . _J: y::t'~~ ?'e'~;~ 1~:yG .LZ1 ail Clcrt~t-:u t':l;lve f'0, Tn.
~.;.r... :4 :<m>i.v!:c~ci .r1 :':ica. ....
.. C.'~.~rrrq~.t3xa~.ive r~xampls 1:
:j s:?'~~ C_:-I ~:. L,..7Lidii c~ll.C~ v. . l~7=~i ifl C'r ,.. i-t-.~-:IL~~~!i:_)S~ri~.':~Y-.1.C: c:l: :r~
:,;~-::r . ~ ii s~.'~ i ;E, c3 h,' ;t. it r -i:rg ir: 7 . C;tJ ~r c~f <-L~ion~rF~~u _,-~~~, ~:;~ ,..t_:c~ cl~a~ ~~7~,lt::ior. ~.~:c,s ~:i.r_~,r.ly Wc:=en =!
T~Ir~olvrert ~r~us t:rlcan r~~r:~o~:FC? ir,: sui,i.rma+.':;u ,...., ..c=; t~1'a _ .:t~:;C E: Ofi: ~~ . 'a~ il'cl . 'I'f1E ~~Ori~:i~' .C'c?:
1 :'~ut? ~r;rt:.
_.~ :. s- L!-''~-'. l.ri .'iGC7.i.Lil'.l ,VC~L"GXlf~f-'_ St:?''_Llt ~ OI"1 'f~;?"' _~ ~ ~iC?i,.lL"S a::;~.1;_~
. :,r' , ':u;~ :!':i-C~ ',~li..t1 ~..LrlC7:Wa:e:C~ YJ<~te.':" ~.li'7;:11. t.
"r?.':rcil . ~~Wy.t;a rr':_;t.,C.,.:~'~ :2l1 a :alrli:>:lE:: 'r,>fl.LCe ~W~TT1. !'I:E lrivL3:lr: _, '..:°c( _... ~ .2.~1.
i t; - r-~ ~L;~ '.t[ r? 1 - : 1.'s : -~ 1 ~i a~ 3 t1 i n.1 r3,'-? CA 02386586 2002-10-02 -~ r'H.~hF'FFvT I I:K r~ n;_ r~
F'.~~~rmp~e 8 : ?r ociuct i:;rl of a t~ort:~us cF-l:i t.o~;<:n rnat:. i.x ..
t;1 ,, _.'r-1 ~ 1f E:~ ~ ' t E. I '~. :-~; t i C_' ': ~ 3. Llllll O ~ i i '1 -:~
c'~ t. 1. O i 1 e: ) ~ A~ - C~ Z 11 t~ c'. I ('~. ~.
-: C' 1 ':i i n:~ . '1a c~f chi t c:~:~ar: '.~~e-ra pLs~ i.n i ~! ml of v.'t;e.r and the i>ii v..<~~. ~:.,~;u~tNci t:v . .,ith 1 N! hlydc:och:Lo:ric aciu, and r!z,v ,~.~
n.i:r:;-nrE~ ~~r~ shirred _~t~~ 2~ 1'.ours. l.~e r~F-i was ad-jjust:~:c:
. . . ~,:,: ;,-i~:li-_-tg ~ F~a 'slaOH arc= atom, ~.l'.i if= :~tmrr:z:c~, t ~~ ~.x'~ t:. t.a:.rLi r ;.~c. i c3 :nd ':~? . 0 ~ :~ r~~ '1 -~~t-hy_'~
~. ~~ ."~ ~.t-.~.,'1~_. ,_.top~r~~pyYl ) Carbo<-W i.uir'.e L F:DF.i ) t::.~e aci;iwc~
;:r; . <_ii.~~"c:=~v~-d. ::..E_~T ll~-~ ing bee-.~. st.~ r:-E~;j a~: rw;-:."n, - <a,_:o-., t~ r E.: f;::>x a 'L= _ hE.r ;s ?loL r~> , tJm ~rf'.va~ ad ;
Li~;t, ;=~:;i.
..... . .,".t''1 1. :T T'fc'.~u~, .-~il':C~ t.:t'.r~
~ir~~'~'1~71t..~2::~'.W~'.~ ~_L1'-;~~_. ..., ~_taTij ::d_;_riE~,l iii::ii .~ Z pt jP~.::I-il'<c.'Cl ~n'c3t:~Y. TIZE
in'd;:sl'ee~:7 j:.: ~~ E=' C' .1 rJ Z ~ ci t-'? :'.~ < : ~ E;.', y ~' ~; L a 1 V E=C~ W J t.
I1 i~ . ~_ ~l ~ -~ C) l: f 1 cz L' r 1 C c1 C ~ _ 1.
~.~;; _" _, ~t:f7 , :w~~ra:-r -~-:a=r.3 ~rorn Ni.~x~~k ~"~a~:, i>armr;~~ac~t) ._ . , e::. ~ r: ?.~i;-r g G.: svcarE'I-. '~~Y." j~H ';vc3~. ud~llS~E'_~ t. C' . it:rL .i:~ ~~od; um i~~-dx°oxide solutic~rl, ana t_ILe "o-Luvi::>,'L
v~.'.~is:: l: t:C>'<'i'i: i-tt: <~~ "l_.'. 'I'~7E' 473CF~r 'fJciS Crue'T1 I'[:r'CLOV~CZ t:y' ~;u.;:: i .;rmt:i ~:~n :L::;E=r a pressur« -of t~ . G'-; ~;pu .
.- ':,t: i.t:.::'. '."F>:>!:~~' ?~E~~-~:L ~liCV:t~~ E.)}7i c:; ,t.:~~~ . 1'rlE
S~:UilEaE-~_' C-'.3,x1 '..'?t~
. _ ~ '... ,=tlOLit: '_lI~ l~t-lE-x C.iLi~ L.'.1C<xv.~G:A.
?'. ~xr2tttpl~ 9: Ps°~::clt~t'tloZ2 of ~ ~.Y:~ec--dimel:~io;tal matr..
r'1. . _'1 ='L'T.7i:: ~ ~ -. .E=-~ ~:E.' 1 j c~C'.!'1E='~:1 brl f:~C'r ~.~:>
..m_ ~ZC]-1_.Wil~J- v E~ c E:~"'11 i.C~~<?i'1 '~'JE:I:F- ~L;t 1:1 ~.!I ITl'~. Ua'. W::~~~r i:Ili~
t:riC-_' .t -L ..., ' ;~ ~ y ,:: ~.
?i' ~.'.rs; ._. -iu::t.~cf ,_~. 3 t~~itn 7. r~: h';.'ts:>c _~ _- acLd.
__ : , . !.,!~J ~_'-t a°. '._~Jc~T'."v ~ -STI~.)i.-rtl t :1 C E ~~: W C
.<. ~ ~10i._ '_': _ , '. .'7E-? ~~..i. E:.; Y
_ ..~._ i;.._,. .~:a:> -.,a~~.l~t: ~°t-.c~ ~~i~ . . ~ w~ r.~z .. T~
'~lriCj~I ~:n~:~ t.rl~::~:.
_ G _u L -'.1"~- i_--s~.l'Tr- 1.-Fi,S~I c3I-t_t . . V~~' ~( Wf ~.i -E~t~l'y'~
_. ;.~1,,~ ~ ~.;- .~ ~::t i_r:~~L~: :~F~s-1 ) c:a;r bor ~i in-,i,:ie --I~-:.r~
) we :-E
..s'..1 "~:....f. : :.~ (r V' clC:~~lEi'~ ,3T'1~ C~1S~.1J1'.'tW .1 X''y' .~'~
l i. T :LIlc:X _ r~t~'~'~~_ '~ (:CT 1)::=:f~f1 ~>u. ~'1.I-T E:C~ at'. .i'~ 01I1 tE~lrLyE=I~~,L:LI~ E' cir c~llZ-2.r7~_'"
.. , ., ._ , C. r:E:S ~)f'~ v:~-'~.'. dC3,j17CL'.E?o t_~> ~~ v.'Lr_.rt 1 iJ
E.SeaiJi~. ~:'flo_~
. . t ~-;t.~-::<.~'.; 1i : ;.:L:red of r ~rLd ~,a-h~d N>ir ri J. ~,f .,,'clt e-.'T . :'_1~ '.J'rf~~':=~.>l.c?ii~ Uld.<-r' t=C1E~T~ C~1.~'SO~Y.TeC~
'rJlr~: ,1. Z..L1-~ CI
af3 t _, I.',', ',IE'It l" .[~'l j-yl.S. .I-~ :''~1'I ~.~Ei:; ~ 02386586 2002-10-02 F: ' H(.~NF'E~I'iEv;ft ,'lt:r;,';
.... y_'j _.
1-::=C'(_:LC(~1~0 1I'1 4:c1tt~2:, purchase:G~
,:3L1<.~ fI:Cll', MW2C.~
P;~:~aA. Parratadt:'tby stirring in 9 . "~ ~ water, the ~H
ez ., ;_:~_:ja:W:~~i~rit1 1 N sC)cliL:rn hydrG:~,:i~~t? .~:i, !_c, so!tlti <:arlc7t-..m~ soiut:ion Fir~~ily, ,.i~t::
vans ~.rr~<.i:n <~t; --2~''C.
~:cit~~.r= ',.ras ed by SLlh:i_iinat.lOn a ~~r~ssure~~f T?P1O-.~ umtPr Ci .. h;_'~
~:.~
~"-: ~,'hi~.ct ;,~onc~z.,~ r~siCIue v,~~,tc; ,).~t:ni:ie~:! . e'l'm spc.ncTe canl k7e "-:°'i r:i.'.'i.~.~mt ~t:_'_rie.r ,~~;!ri tio,~.t:_ i-;''Ii.
~,,af,axn~~le' 10: GenE,ral io; ~::~ec:~L.lr_~= yr)I° ~~eer-:.nr <_.I::vi ~icur~~~t:.,:ig ;.m j M ;~~r-1 tl-~.-_~_ t..r,rtE,~ci~mc:ns;.onal matr~i~:
'.t:~ec~ _,jlrner-tvic!ma L ma t: r_ iX csbi:aaned ~s in E:~can-y:~les 1 I'_:. ~:, s:- au~-or~wed in PAS alt 12i°'~ ~:or ~u r~iru~_es. ~:'1-:c' i:~~<~"'.'.-~L.'~:. ~._ T:t-LE',I: at:<?r;..:~ i~llU ['OIYtIJ-LE:?tE-'1'y LVE~Y~EC~ wl;,.I:
,! :. ~ :l 1 a Ol . ~. t? ~ 1. .~', 'AVl a. C~ ('1 ~1 c-3 V f? Ci~ ~ ~ I-2 C
L.1 1 t t1 I' f' C1 1 I~! ~ S ~a .!. t 'c, r~ '1 r~
r7z' :.;z~a_ lrcdi~.Jna arm then pu~ ~ir~:c~t-:i-r~ ~~r.t:o a :nar_~ i:~
's~7al.:.i_:J tlwa'.: ~.'?E?c'':i ~.;Lltr dp~.7X(?t:)'Y:.edt_.Et .:OZI'E'<jLll.Y~?TiEl'1'CS cx:~~.':
._ . 1 ? : ~ ~ : ~.: %.: ~:: :'t:i w1 r t'_ r..'=- := Ll 1 ~ __.,J ~.L t', t=
':='_Tl~) C': Y'7a W 1 h 4 . :.' l-: F= Iflcl'-. 7- 1 :';. t:7 7 ':' C].
;;;,w..=t': .:~-m i)~~ --erlct~red wn~.~er s'er~le condo r.:_:ans anr~, r ~:...
tt,'~.c_!r.! ai f:e, :-:rt~el c;f".t ~u .
~'.~c::.sa~~e 11: t::ultL.J-: z.r~<f c;f 'vlz~~::d~~~'~,.'',~.~ ,)1a cf.~ th:r t-~>--i..: r;:..:::~ z corm J;:a t :: .i r .; i ;
,:'.,v _~":p:J~x- l,~i;.~~ndrocyt:.e;= cbt~:ineU :.~y ~~io:~sy :~.c a°e:' ;.i~l:e=l,"j ~.rf: <.:~rl~;7IZt;';.C-;.iC'}r.t:,C ip~,j'~J~:;1~1 f;'l~Ol.'~_;]T', !C'E.~.~1 ..~
:~.F-1J, ~ i.l~r~:»C~r-:i .~.I~C: . , r_~;o-_ i =: ':-1.r-E?C?--C~7YrlOIiSiG:lC1 I:lBti1?: X:~IOCiLIC-4~C~ c~:3 2I1 ~'.?:cxll:T~.~t' .~r ~"'i _i:~i:~.-.u.; i~' c~.'.;tC)CZdVt3C:~ fir. 1..~~~:i~~ =Or l~l :i~l:'alf~E'S.
'~:JE:
c."'.>f:i ~_':y;.E~.~srJPt:.t.t?C: IlldrY_1?: W..l:j ..Lit.
cw~7TJr'C>~:~;"ieit0'.:.~' .lri.<~ ',.
~s'LieT -.~ E~ C'OI1C1? L.1C)I-:S ct17C1 ~Llt J Il e~ C:,lll t=L11 ~: VE'-~~>~~ . mfli.:
t~ mn'. r,J~F-~,~z crlonc:irocytes witYl the uult.,.~r~~ m~.~r~ium wEJ,rce rrA~-pro ~:>ut uIJ ::':e ~t:~eE~ dimension<<1 Irlat=-i:k_ and incubat~c? x r..
'r' _ ~.>.r '_ t-o a1 da~rs . J~uming tiW s permd, tt:e ce1 1 ~;
~.:'!j'u:, LI:i'i7 t~t~::' ~:,tlrE~t~' _C ~:lIE.'Xl;.i? cf.cil t;ieli.:'1:~~.
:Il':Ir:rxt.I-i.X 's:~".il :arc~~~::-,~1 was YC.-'moved urFcW:Y' :~terilE? c:ondit.iJra and c~.ar:
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<:~ft.~Rf'E.WIER ;j)(~~y - .L~
i.. _]E.".<1 :gyp l.ril~l~ C~IZt.F:~ .
3 tl;w:s:; the co.or.ized c.hzto~;=.n matrix ,, ~:.i,_ro,w«~ica.,1~,' I:-Eagnlfi.E:d imak~fe. 'F'r.Ea ~.,=~;~:er regi'__.._, ;Il~~.:.C,.".t~''Ci Jr?';~ ci.l c::1'~I~iiNi eIiC~ !~C?:l~C7St?(~ '~!f ~~~jrl~x'1'."'.-~1.
_ i t;.' E;: ' :1 t '::' .~ i_.1- E. ~i Cz ~ .r F_. ;=7 ~ i, C C C, 'F C E.' ~L L S W } 11 (T: r_ }~ d '..' E'. C~ P_ \,' f' 1 Cj ~; , t '~~
,':!n t:'.'IE' lT:Fatr:i.X c~t:I~i.l:C1 CiitLi.~..~=Ilcl. '~hF3 c:e~ ~.~
O,rC~W irit0 ;.t:E~
;JV:,_ E.r:; ~;t ~,L;t' !",1~; .; 1 :~:. <~:lO OI"1T1 '.'1T ~:E' C..1CIE_a.lSlr'.~_,~i.~.
1 ..' v ~~~~~yy ~)rt:)F'.>~='-t:lt:.S O~ t.,f":EC~'112:.03rtI1 Tllc~t:Y1'::. lrP
Sy.Y'ClI'1~[~i.V
ir.l _uea:Ce~cl r~~ t:hF= I~roduwt:ic>ri Inetl:ocl aTmi b5' t: k~~
;ve:'v.rlt:E;.ric~=1 ~.~ed. Tr:e effect of the counte.rion as cir~zr 1 'T:-c;':t1 s:C::'l',;'u<a r' LSCil'1 C):_ ~~lc~ . ~d (~iCWICI~7l-Ee % j dIlCj _'t7 ir::'_::ur.),:~r-mr.z-~~ :.~x~-srnplE-~ 'L.) . ~orl~ ::Wt.ri.w.E:.s :~,~t~.ri:
prodtn~eE~ 1':-~r ::.s ::U~.~.._;i~ .-:: t:c~a:u arid t.ne apprc~~il-iate~ ~.E>;_~:In ~.~a~.:E ~
, r--,t:j ;:~1':F' :aC:i i.llt:li)II w;'lu hf'.iTVpv'1.TIC~ i:k~C?
:.C'~.~~f'.lt~
..._.:.!..! a71~~'' 1:'I'1 'I fl r:- ~Oc~Ill .~'.'.=lk:l= t:i?S.l.CxLlE.'~
:~.'EW,:E'. t:j:~_-i;
C=C~t_:-w1.1..)clt.E-eu.1: ~:~!W~_L1S r~~clOH. ~Jri tI;>H- :' i.11"tGir=.C <-u~
_... ,.I.t a'-- i'.:;1 , -~~ c,~ . a:as , ~he ::t ~-I:1~.~t.i~ral v'.lanaF. i r-~
~,ai;.. '.?: E: I: e;_rui ~:Lk:!~:dtlo:~ t~:it:.h I3~U11 is Eii ~;tl:u;.~t:l.v,~ :i ~':::::i :~~-_ t.'s~= v:v phe'~;~1-!czt:t~ t?-ic3_ a?b. 'rh~ ruYfrA!~e O'~
i'c, ~~le'.::~ 7.~.:~ ..~~ _°~IfIC:Y(_ifi.ruC'W c.IIZC~ ~~1:>DVS Ll-'S,~'.r' '~:i1=Or1Q11IlE:E'u ...".i;..i;l;?a'.1.:.7. ~T~:1C'-: ilft;.:~:.X !)~:lu.cLIIlt?a ~I'1 1=:?'~c~l'!~':~1.~ I ,.., '"t'~_'.:t:i.?1C't_L" I!'~EJI'G c.lsx:-',:_;.C i?:lc~ St='c:fi,:.~ tr:1%x?1 ;
tar: YTU:~~l': ~.'~, iia~
~;,:'"~ ~':i7: ~~t: ~ iii ~?>ci,'Ty~' .... .. rr C.'c;~:7 ~E' !'.OrilC'Z'"C~'.~~E'C~ ?IiCj, ,~~:
t'l-le;~ :vrE--~;,~='m;i: is remcvecl, elast.-..lcwll'y' rFStmnE=~ :t,e :~z_cri.ria~ !:~im agailz. ~° can likev:i~:E= bE? bE-eltt :- ~ :sr::i ~_r~ i1-,~ ;~~ a.rrout, break.~rl;; .
,;:
'vr,-, a~2ect ~,i ~.!~le pr~~:lu~Vr;lcrn mE3rhcW vs dear ;-r '~'i.::::i~i::l'. 1I'1~7 L'~J.'_; i ci:lE~ :'d . 1-:'l C jI<.J C_ci-',C'. C.' i_ f :1;7 , ..
t i..:.::IUi.: i ~_' > ; , 'klf? 1 ~y;,"t . (: z C'1 ~: i._ IPI?l1t'.T:.~,11 :e'-~Cl ;;ac?t ~%,I: E:' l :iE:
..~.! ~ _: w:i. i '.'~='., 'v:ii-~rt?x.1:7 ':.'J. 31 L_?'lE :'CEClt_tJ.?'.
I'C:u''1'~'''1'~ . a::.x .. , "-1~,,.~,i~y,:vi t:' tai':1<.1 4.'cil: f'!-'CiliJvt-'C: c3it:r-'.L' t:.i~E_ ~.._,,..,~.>l:il~e.w..l~liJTa :....Z.
t:~,:'_::~~'Mc:,at:.:ior1 . ~t.~l ~~czuc3CU~s t~J~O~:. ~lhe unifbr'tri struct.t.txE~
~;i: '-.yv~.~ .~-,-ar~ac~~ c,f ~xre Ittar.r'ix i~c1_t:arly ~~Twicien'.. io _''.s. ;. ~! , ~JC=-lllC~ \.':l Y u.Lli~11;1' i rfrC O'.'. ITI~ ~ OZ ~~. s. N
. ,':lE~~ lTlcit::-' _~. :K.
~~E'~ili::'.Ev':' l.l~i l'1.G. .. ShGWS ~:;-2I- ~')et:r~'Z ;:iOft.rleSS ~:ilC~
jii (.!'.'. fa;,' '11;R .1~>':7(,I F:,.I =>11 :i;li' ~l.Llj:> CA 02386586 2002-10-02 r. (..'IL.aRf'1-;~'1 fflv ~'Jfl',:':3 ~Jl~~t~y.7.t:,~' r.~'1~r1 ;:~1E lTlat.2:~lX ~,X'OI?L .~1C,~. at-i.
'L'h~..~ _r_: ofl~rt.iF:s of L1'1e mal~1':ic~s ar-f~ stl~~unari.~~.d in a ~yi-~l.ut.~zt~-.'r ~nznpa~-ison in 'fable 1 .
.fl:i;', t1. ~;l':. ::. :iti f.="~, :>1i a~li <,l~fj:iCA023865862002-10-02[F, tH:~flf'[.\'ll~~ a1;!:.'-1 _ ~ ry Table 1 ~.~Ulilx)cij i.:ji.)Ci Oi t:rlE? F~2:-O~Jc=L-tl E?:-'i O~ iJGi'Oll~
~~'1I'F:t~_ i_77_IT.:=-I7:-,1 C~IicZ1 CtW t-ClScill :Cvcit.YlC~
....._ .... _ __ . __. ..._ _ _ . ____.. __. _._. _____. ~._ ~.._ ~_ .~ _.._..
'ytl~?'_E"-.'a.0I1 Pr~C~llCt:tt)'1 l~~tl<-)r Of t Ic:r i I ITnE'r~l~[i'~ ! :T'u~C:t -::'C
_._ __ _ .__.__~ ".
_ _ _., ____.
m )>~~. ; ':.~ctat.e : I ... _--,~.hit.F
_ _._~ __-_ = _ _ t .:~ r'lic i 1 a~_;:t~tate -, ___. __ .~r~it.F
_ _ fi. ___ _ __ _. _ _ ___ ____ _ _ i l ILa.~'..:._~.~.'~ - ~ f:.. ~ . a, f,,~t.,d t-~,, .._ ~ .. ~_ _ - vJhi t_ t~
_ _ __.. _ _ ___.~ _..~_..-.~__ _.
j -.. .; l ~~. I E- 1 y.~.lt.hl'll ~ C: e'.a~ ~-...~ ~: _ i "_ .__.._...~ .___ _... _ _ ....__.._ __._. . . ''l"~11 .' f ~ _ , - ~ .- .. ~ i ~'~ '' ,' ' ° __ ____.I L;~ _t a t' = I ~ 'N~z z. t. ~~
~ ry,~ ~, ,_~,r;;vt~ Lrnosph~~~,~-_._._.__~.__________ _..~....~ whir.
f. _' '.'_._ ~- '' -_ t~
I_. _. ..___... _ ___-__~_.__ _ _ .. . ..._ r...~ ~ ."ttr _. _ F a ik~ility ~eha~.ror i l:izati.ri cW :an ~
1 the. rna Crir_ ~d~.~itio=~~ 2'' of l ~ mlli d r_ wa t~C-~ ~ ~ [1 ~ i_ ) ._~-:~:r~:>?f.__... -. -h-,t++'rabl~ y-+' ..~,rlir_e,-__-_ 3 y:~,:r~--_____. -____ __.
i Jf-LE~x:.hly ._-arcely :~o chan9e~
~~ I
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nri'~.:. ,~n:~ ~-~
~~ iarf i f lE:-rib.':e, ~ .:~~rt:i~7.e:ai~rowr_ L'-~.'cl~Jr1 .uvlilVt I :_C-~:JS~:I...C',1 ~.N:1 V
~_ 1 ._z 4i L. t t. 1'v~f_! )~ir' t .l i ,':, E~..-F?.~ f v'::~C.) c? E?.:~:
_ t ~____ ___' _1. _ __ _ _ _. __...__ __ _ _ ____ i _ _ .
_.....~
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n ~v:l_s_.4~ __ ~ar.ga~
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t C
i C' ~~., a 1:. L
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I
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~ i.i.G~~-S s)IrVI'1 __. _, - 1 _..._.._L__ .
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_ D.l..a~<.>-!_~irng _.r>.r_c~s,~r. ic:
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t-~F, ars.u_ou::~
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l :l j ~~-'It?!-?~!c,u eZ Il ~_)f- ~"~],' SNd~ Sll~ial~3t_1C>
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i i t~ I' ;~ Ii ~ ?;. , t -'. :t; F =t '_ " : -! :S ~i ,' ~ ~ f s ~ 02386586 2002-10-02 : (. H.IR f'f\'~' f I:F ~'] (! :' ~ t'C.'ilc>'vr31 t=y WdtC~.L~ ~~y :~Ll~>.~.'.tLlcl~..i_OIi ~ Fqui'yil.:~~~tioz: wit~i T~IaGH
1'I:iv; ~!'7'J~1'l~.i~:;I-1 TF_>~at~5 to :A 11'WZ;I~1GC~ iCtx ~I-Oa:'~LlCIIiCl c1 k~~::c':mrpati't~le three-~iimens-~r~Ial matrix, to r~ mat~ i.x '-,r -- _ t. ~ a a th t he method oz t~~e ir_~vent.ic'n, a:~u to ~..l-,t~
,~.__~_.,..:ab_ V:._ EI.>_ Cfle1'"r~~~ _ t:::..~;;;i:_~=_w~'~~~e ~.sccE<sscs '_:3;rc heen achie--Jeci in reerrvt .~ ,-t: ttm~ ~3r4~:~ u' IneG-c~:..i. tran:..-plarls.:-;. h3owev~°u, 'i :' _-~ ~ ~i:~~ e-~rn:~.r~ ~~e t.'.lro~:ah r1-,~ ~:n-.,:~~.:i amo'.l:.ts ~~
1 dor=,--x~
i :7; rs c~v.~.i lub':E , whi oh lea:; l ed t ~-~ '-~>rlcit~w.-abLEe ;r.-ai I':c ti ;; ~.~i.~_v_c=r:t_, ~N,q,li: i:.c1 t':rar:rF~laI;Lv,: . :~~ zl..lc',~t-~e:~
l:rc~ir : ~':v:- ., -1 ,-:r yat_h:~~gelm :.-m;,' >n~>>_ rrar~srni.t t.~:t.~ wi t..h aon;~,...
o~U~:nS, a=~ ~",-as the case at, lE:zs:_ occa~i~~naily with .__. ~,E7'Ti'='.°> ,-~'St~FfC'Yallt~ T:Ci.~IT': F~_DS. lrl c~:~C~z tlOn. Y:YdTISpZdIlt:-F'i ur <7~:Y7:: ,rI E-' : E.'~~"~?CTr]?. ~.eE~ aS fG2'-2tin ''')~ i_tl~~ x ~C1~%lE=Eli. ~ ,'i _rmn:er_a ~:v_,r.elr.. :~f~_.er tht~ t.: G= sp~~r_t~tion rne patient _., f:~':f:j t=iC?ri:' CjEr'~E.'i2CaaWIlt OT: ~I;~Tl~.Il~-SLa-i1?IE<:a~lV$
iTt'c:eilC:ciiTlE:'I1L:S
~>1_~:L'Z~~:pri 1 E=;~ E<Ct "! vrl YE'clCt? u'711~'. . raft=EIii~t=> .~it~Vfr rEnCE?I1t-.-i"_f _... i~~~~~rI ma~;e j.:c:~ Gultu~'F antifici~:1 arg~~~I:: from cel i.
..v.:.!.t.:::~E:':=,, t~Y::~ .-onsider'ak:~le su~.-ce~'se~ have i:>E~~IU
~ci'1iE=vc.'~~
. . . _ :". , ~.:_ E c;'.: . < : 1 ~ e_: i. 2 7 ~ :'-, ,_ t' . .. _. i.~E' 1 T:!:~ ~ '..'i a E':a Cill ...
t; ,.- ilt':-..,.t'rl,~i.. ,'u.lt,..1:~: "!.'17.i~::~': i.~c~ll :JL
~11:3~,ie':!~
'y''r~I l_''=~-I~E' :C; I' uhE' :2Ei_~' .°_YEr:~IFnI:;:>, 'Gx' E~:;;~Ttl~Jle 1I'1 t.",y.
~~ aF'r= C~ ~ crl W tr . Mh l 3 c~ ~ t l ~ l t'. 1:~ ~ 02COc1tZ QX- ~.:,0"~W
~.7dZ't Cci_1 x ~,:i~?u: i.)e-% r~-dI-l,v!c3!'I~t=C~. dtli~ ~~. °~.-':iu)at=:11011:W:t.W.~ S vYE3 ll$P<'~
'.h~~' ':rt? r:t:t. =rCC'Cnl.~~ nS LO::EL~:I bV trlt~ ie.~?p1E~C'S
=('~I:i~:t:_1~St'~-t.E_111, t~~'ldt' 1.~~. ~O S3V o]n:: rf~~~Ct10I2 redC'~.10i1 l;:ui:
y:.."i" :" .
=!.j;:~;''i;j~0~.' Of h:i:OWI1 ITic3'-~Y'ldl.S . CJ;~ : ''1E<. ITli~t Z'1~ c3=
E' C:.Ci-11c'l~t::1 ,~ ~ 1. l,7 :i . -: ~. t E; C.'l i ;S: E:? C~ . .:. T:. :1 C "~ i t Ci : 3 I :
. :~ (.Vt' E:' V t' Y : '~' O 7 ~ r a C3 E<' I1 _ t i w;: ~;-m: i~., ~ :"trd cr;ly m:lu.~C~.rit1'' f.)Y' l;L.oc3tlcl::c~
1~'. ~_l. ~ .:il I:~;.Ilic::1 ~WtlE':1t.,5 x:'E.'C:C',~.t~ .L: 1:>
i;ir~~.?l~liCt"1 :I~t:~;:I1 _::-. i k)0:.~ :.t:H c'e~1 I-;=.~ c.'.UE'. dT-~c~ 1. t:~rlLl~ aSSC>C'i 3t;E.'C~ v~_.
L; I1 t;,ylE.
': f ..,:- F. .
- 1: i. ._ ..,.. ~:,.:.~.- c': ~t.,xc':v:::.E:~il:.r'.rE:dS;I~m:~
lnt~fr2~E«,'C ~~'.-: t,I.C.iIii~ ~;1-I-u;:~
I:u.,3': t-: ,. rI<3'.:::?~a.:i i . l~.Ii. i.:-C?_ic3l'1 i S 3 f?~~1't loll 1_1- "xt.dCWi: ~ ~..d'=P:'~
L O . ( .n. D ~ )f ER 1 ~ ' -f .'S F.~T 51 ~ ~ ~- ~ -f t~ CA 02386586 2002-l0-02 ~.~H:~RPE~T 1 FR ':al 0 fi ti 1i fL-~ I1 carif'1 1 ~s c~~OY c'? ir:e~C~ f r'C:n tY?f: eXJ:S!W lt~i=7riS <) t z i ;::'~z <>i..o;~:; . ' t: i s ~~m c~minopo-.'~ysaccliar' i df~ tpoly-~ i -,~ ._ ~::1'm:~~,~~~::~zi=:f?i wYi:icli a.~ ,z~~ecin r_r,~:~ ~ E~;-ia'ra:l ;:e'~~t.or ,:.:r :=xainz~i:_ u= _~:tu:re rnat-Ei ial :w :'or e:_Cap~ i~iati.r~g dx~lg~~ .
-is ac-~ arma3e is. ~rlat: i t e.an 1>e :.~oit:ple~f~7-y absorbed trm~ k:~c~ciy. <.i:itcsdrl can bra disso.Lved ii water und~:z .-:I.gnr.1-y ~c~.~?ic colidit~;;~:m> !pI-I ~ 6.> t:hrc:~uc:~h pr:~t.ot~at i~-»i f t_T-iF tree ainz,:o arol.ps . ; t- i s reprecipi taf.~ted f roTrl r h.=_ ~z~:z,E;~us solalticln ~uride:r alkaline emiditi~~~::s (pH > '- i .
ecinani_-m m ans rI-z ~t r:'s:itosan c~a ,~,Ee I', aepe:,d n ~ r; a ~ n t»:ru'iFd and znr~acessed :under mild c-ondi tior.s.
-.w.' ' , s'~ ~. , ~~;~:.-1-.~ro;~c:~es a v:~ri~le .~or t u~~nsplantatio:l 1_1;.U c1 pi-ti ltil. Wii:.C~l cOilS3.':~ts Of c3 I'ldtLX lIlL.C) W~lJ.Cr2 <: JI?s ha~;E=- ;:~ro~:r.. ~c;r this purpose, f_:irr=t z; cii:itos~n a~:; i=t-_~'. ,~_i contai.r:inu ~! i.ving cells ~s r_c:duc~ed. ~'r:is ~'.US.I 1t"=.'::'11 , .. t..IZF'1~1 F':1~':1 :'):uE'_'u :::'1 e1 SErTTILIf.''lT.lt';:ik!ZE~ LTterP.brv.'if-' y- f-, I'? : ::; .1 E' . ~T'~"1 ~'Er' C r:. l t C7 :~ d r1 i ~i p I' ':-! C i jJ l L ct ~: E l'~ a : l L~.
c~~x'rns :--=n ~rmcrossli:u~ed matrix zm ~,~=hvch -,she dells arf, _.. i'_~t~rlb-ated.
Il,ac:izn~l~v <~~ a:1., ',F~iomateri;Al.s Z~9a: .0:.:), y.p. 113', :' ) c:~.-. ~cw ik_E~~- a matri x for .-i ssuc-. reqe:~erati:~:i.
.'::.' t~_;c:,, '~:lil Cil ~~. ~~~ ~~~:'-~~ tE'GCa<:t'.~'~u:.E.~d ~~ f-C7!~ t L-;. :-~
a_"- u~_r: ~:~«:'se ,i i ssc:~ vwd In '~! . U M acetic acid to :r~suLt ~n >c'l::r~ic>r_~. ~:;zt~ll ~ Chit_~NaI: Cvnt:Fnt. Gf trc~':~i J. c0 3~ ia~,~
',.:~f-icrht. ~':~e s.~iut.ion is frc~en and t_he :water' and tt_<:>
f~xCC':;a '~"et:~c.'_ r9~~lC~ ci.r'~~ rerhCVErd k7y lYGrJI'111-izitaGl'1. 'T'~-!~_~
..: F J c: ;i.'=: i i ; : ~, f: c'~ .:.)':: ~ - G :~ V,'~ 11 C ! i ': Q r': t;
C c 1 r1 ~. X1 ~: : 1 1 ~: G ci :;a ~ ~.'> f _'4.~ l W .'. f'~2c~I'l~-C'''.sl I',1' t:-~iE' L,'O~W'tl.l.li:.,:.iv.lOr'1 C,'.W'.'1~'~~-t::lC':IS. 'ha'-' et'~: -E.'rc~~~f'' Cf ld:Tlt'til- Caf lr:P ~7p2:-fJS 1n i.llEN rL..il l -11:C_J. rCt3~l~;., 1 '; 1.~: ~.1':~.'? L..;~yf_' s~ -..L~-;f~ f.LITm 'r~it' i t-':_ih.L\ 1 V~_,_7r111.'.~.rE'i~
T,:'".. ~:.:, i~ 7c,:v~C'.rir~'<-.u ;~:'a .~ltf ~~:7!~ .::Plu~'W: _... l~n i . i;7.: :x t.: i~;: i l-i W riiY:.;~..lc~ I n~l~-.'nU.,' I'1f-W~llrM, .'.t:!? ":1,'_0v:;:.,..
p_'i _...... _., rc=~i::y; and '~.i.ziW-ly .3-_s:c.l~.:r:s. Rerie4rf~a ~:f~~;:,.",.,:.vl ~I~y' (:1f tf?E,-' CE'l:lr.OSc~n .-'.t~2lCti;~E'. Can bW
C~YC?Vt?I-itEei,~
t~y ~_-dui liLvrat. i~~r: im di? ute rSaOH or by way-;.hi n~3 wirh a:1 t'~llc'-i:"1('ll :>(.~I:I~".-~, ~O? E'X~IT':~~r.' 1~~~, ~r~!J, ',?~', '70.
'l'~'.,~' ,,.;Y,W ri..'.a::f-r~~ ::m~zw_. ws ~Cr C~'iiS pwry:.o~C ey,-~li.br~,ated ii~
! ('~ n;: iW rlF;a 1:: . d v t _1T 5 L i aa? ~.t6 cA o2sas5as 2oo~2-ioY-o2 ~
H.~RPE~~i i ,t:R ~%; on M Nn0)3 for aL»ut 10 mil:zltes and v.=a~ll~d with wat:t~r ar.c~ F~BS (phosphate--bufiexed physia~.oc~ical sal~,.ne ~:r~~~ i.:tion; . t_;r. eql.lilibrat=~.on -r; dil.uts IJaO:-~, howevter , the rm:tr;x shrinks snd ~t~GtaS Chane~es in i's struct~Lr(e vfhch are on l y paYt l;~' revers i b.! e~ 0:'1 redvlc.vin~ t he pi-I t-: o ir: ..-jeidirion, t?'~w mritr;x show: numerous ~ r _;:clusior:s. t>ett:er resLl:Lts are '~btazned ~~>ri E'C~l~l ~ ~.~'Jrc~t.l0r'1 V.%2t~1 ~Tl EthaIiO~. SG~''Y1F'c . 'j't~_ Z-rITIOV~
e: ~~' r;cvi u:,~ors, ir:i tzally ~ vaca:um ~s ~-~rief .._ ar~plied WJ v;3'.:tl-~'.1 V.':~'il d~~SOZZ.iiC i,'-..iC"~ '101.. .. 1'',1r a :tE'r' r:adVc?nCu~=;F' rr.er.r_ionf.~(~ is that the rnai=r.i:~. _.. st=E~ri.~z~~rl duxvz'~g t:..i:E
~q'a~.libr.utio.r: -'zaolsgh the t.:~at_nent ;r.~ith alcoh._u .
,~~~L~_~r-t:o<.vf~ss, t,ae rj;er_hod is comaarat-ivel~, cornplicate~:.
iv;~,: Ee:1 i i tit<r membrarve i s 5 ta;b7 1 i :~ ed i. -;; an ethan; ~.l ~~~.wie~ , ~. ;:2-.ange i.r. ; he pore s ; ~-,~cvt~.wr, ~~,c:cu:rs bccau:~,~:
:'riii.;i~Si.'i :Tl-c,;' :iE.' 10Gc1~1'y l~lc>('JW 'E:i~ tlrr t~'1B L'yd~eL-drW~
l.il,a;; L'1'1C~ purC' :~t'I:ulCt'.~;I-E' 1S y.?'t~1'~J '::;--:c.,~'~0~%f?d.
't~'r:W
f,'vd::ated InaC:~:i;.; is :lescribed as sit L- ~r~d f exible, b1_;~
..;rlo~.r.=s c~r~:l.~- Lo~n~' stxLnt~ti-i. 'This r_or_siciera'ol~.~ impedes .('~ =)r~~.i:E~r~;'~:y-? CO,~ ti1(' fCf~3tr;.li,:. Zr uilF- IT,.:~.t:C.a.,'.~:
:S Cllt= tCJ tt:ip r~;nil~~,l;OX~I i.~::'~e ~rlci~>E ~OY ~XaInp.l E-' LOI' C'lllr.~T lilC~
Cartl.~dC~E!
~:mr-1 :..> cc~ i;~m'm car's i.lage, :-t~ 1s -~ a>ry __~ s~.l;l brokml .
_~:LiC(::'r:.t~e, Ct~'tllYlrig CW thA cE:~W .S .1S IT'ttiC~A di~LlCU~t brol : r rJC::I:%_iliSi: ~. f ;1:~W 1r. i S t <'3Si.i~' vC;I: _-1tW c. ~~.1.(r c911 .y ~d~7I~1~r:1.;.~ JJV :'.""~ri1~111c~t:.lCUI=:a, ~(:W E'_::~r.'!x'Wc~ ~?r. t-,'~".c::l~fC-!rr L::'-~
ir;.~ rrc:_h (.':,Wture ~nedimrr. Ti~i.s :ne~ns t::~=it .mp''alrt:
~.~.-7. W : va (1':~ ' 7 nt:C.u SY r L1G t;l.irC C girl ~E ~?1 OC~L7:'E_-C.: W
L W'1 ~:I10L~I~!
i~E :.t~J'aci:l 1'.tc'~trl~'~eS' Ol~'a~~i' 's'J::t:~'1 C~Yt~c7.L
C1~I"tlCLlltY. TI-1 -;:~.j-'; Y i <y:;j , tS,~C:; s?=: ~ ~ z' Pr cl~ _ C~~SI.~:-1~'E: u)'_G ,'.~t VduCt.l;i»
..,_ Ii.:. 1-.'~ t.f:;.i' 1: ' fa""p . : ~->I- rti ;i TW.1~-xlv:.iSG', i~l.~.-iF:I- :?-u~;,~.'. i']~ t-~:Ili t .iSct"1 , c-.1 l: t. ! C'I: c.~2.'~ f'Y<:)at:'r-: cY I T eCC ! ~y', f-.',~i E-~x~r.~ ~~W: im l~.~,u .ci riv~w.~~en, or, "~~~:l:c~re tre f reezi.zlr.~, . . , a-i:"E' ~?2c.'C1~~1 tcit_t?d ~3.~'~ C:JF1 lalit~E'1' cl~)~.c:~ ~.:'1E~ <=.j:-1G1t7.G:l: Snl~.i_i'2 ii.a~.~Ir. iW rClEi SY,T-;1C'tlll~E'S 2u'!''v? I~1('>t Ca E'S
C:i°:.~.1E'Cl.
W
'T'hi_: 1r?~.~EI1t:10r: 18 b~~:~~d Oxl C:'lr: OA7~et~t OI: ~.rC>V~dina ;i;E?'.: _7'>;~ uC.'.Cl:JI:~~:W'~'l~,.lC"? rl~I c;. ~J1(:ICOIPF7:~l2:.li~~..~, t=i':r~-'F:-(~.L.:Cnf.'rl_:lQTlu :Tlcil~:r~« 'J:f'1::C:ii Zc;C~LllrlJ_a ~F':rS P°:~Owt ~Y~ Zf~dd5 tQ
St~~)~iE?
.:~~er tn~xtn~~r~=s l fi 1 ~ '' ' if '_' CIE K l i -~ :~ !'.~-', > 1 1 :S r~ ' $I -; fj v CA
02386586 2002-10-02 ~ ~~-~RFF.~~1 T E:F .<~ (l i1 y n _ ,$
'_'ize object is achieved according tc~ a first embodiments cif- t~:f- inTt-enticn witY~ a mE~~l-:c.,~1 try.- prO:'iuczry a x~i occ>mp~~tb1P three-dimens~oual rnatrax,. where an ;a:.m~<->z~:~ solL:tsc;r is brepared from cl:;tosan ax'lu art c-;;:ce~~;s cv.f an ar.lca, t. lie aque:~:>l1S sGll~t~C~:~ '~~; nellt~."al~~ t--i~, t:'ic~ r:e:ra:-~~7.i~ed ac~.teous soluti.<:n is frc,~:E~n, and rt::e.
a v~-~,'- ._. ~. ~ ~~ relwc~~ ~d by subl itr,at i ~~~ un~,c~r r ~ c~L~~vc:d nrer~SU:'~-~ .
iC' A t:-:r~=t~ c:=.-_.~ension~n~ rr~.xt-~;i:: .<,-i'-ri c.-~rt:_~:ara~l.e pxor~crt:i~::~
:~l~:a~r,E~ ~;,cccrdirc~, tc~ a secorit~ c~mh~~;;inverlv o~ t1-:<
.~,_'~~:~::t.or: ~~~Tith a r;ethod for prcducilig a tnioCOmpatil~le Llur.=E'-d?.ICI<.'WSIC~Ilc'i-1 Tt~truX, Wrl'la~~ art dC'('v:,~Wu:> SO.lutl0n ~.3 F~rA~arE~cl zrom crlitocan acrd an excess of an acid whzch l:: ;_:<-::Le;rr: e.3 From the croup termed by aI~=yi axed aryl r;~i_~2:':'.?:'~ t.-"~rl:'C7:'y ~ 1~~ c..c.LQ:a, '~l8 ~i.~l:~GLi:a SOlL:t_lUrl 1;
::.2-~::'' F'.'"' , ci:lu '..I-iC V.'~ t.P_i 1 S 2-t.~r'tC?VE'_~~ ~~ j' SLIYi i. :: ITicj t J ~ ~ C1 LiIlL_:E:~u - ;~~. J +~t ys i3 L~Fein;O r~~movecl befe rc.--...
r ~ c m := t> ~_;, rm ' a r a , 'm i. r a x C c a ~:
i]L .~:x.tF'h ~Y:2 2'C:CLOVa~ FJy Sur7111TLSt7.c:)t'1.
_.-. t~~~ rr~.e~~."t_c.~:i ~,f thF inver:Li~~r~. first-L';~ ar'1 a~ue;:~-L:;
>:_:~.~'~-L,-,m ..:~ ~uL°e~a~:d rzorn a r~a~c:::a":-1y c3~,a::wt'ylatt.~
_:? ' 2 ~_7'.c;Ll ,-irrd e~r1 c:Cl'~ ':J~'11 Cf1 ZS _~I e=SE-I':t 7 :~i e~
CE?s_S' . , ,'y,~r-rS 1~~ r.?E'c?:_t lrl tIZIS CWYIrIE.'':~W' i=il~~ L:l'L' ~iF: Cf t:a ;~(.~;iE'Oi.i~ S~W.lit lG'it ~S ~ri t!1 t. 2C7 d7 W 1'c:-:GE?, yr-~~~.'.,"G~7i 'y 'u 1~:~1;~'>z:- 'r.:H . B~ .his means thF: fr~3e ar:'w~nc> crrt~up~ _i x: "-t e~
~1-L:. ~:,,,s,:. ~:rw <~tt 'Least. part:i~~ 1',~ ~>ro'onated, whic?~1 .':'.'... ,..:'C'::a r~']r'~ ~.O~ll~."Ji l i t;j~ 1:1 Lit3t.E~''r . ~1'~'3f-~
al?lCiuIlt W czC' L ~''~
~.:~ L:O~ C1~. ~:.C~:1.1 . rt IilLi,~ t; mcC~C3 ! 's' L'7 E: C'.:105E.:r1 :a0 t'.~'l~~t; f..,_1F:..
_t..i ~, C:O;c~ri dl.~,~U-~'1~5. F~XCeSS~~.'f.' ~dd.lt:i.C7r1 UI øC1C.~ .., .W'~'~.1~-1~:?~l G;IiFtF' pO:~SI~~E rJEi.:c~llSE? e?XC::E;:iS ~t~:_Cj Llllly :'~E:
e~~'r,v~ed a-sU'_r:, an~< tiuus 'he k~c.,-h i.l~:~ uh .s rr~a~ It .:_i:__~~u!a .~r :.rye an-~oun~~ of auia ar~r ].a2-!~E:. :->.m!%ur~ts wf c:~~~..1 ~~::W~:~F': C';~L;iC i:1 c~ 0.':J~~ !.u ~ Iv, ~>rel:tYtdk~l,y ti. i r.c'.>
_v, in particular 0.1 tc~ '1.3 R, solut-.o:l at-t:~
- a-.'>'-~b?. r > .
.. L,ar:: .c;,::ax.l.'.' °.~u,;t~ablC=~ ac:i.C3 i~ lactic: ,-z..';::1.
T::a>
'~'~.i'i.;~',~'Li~'.~:,~ I';wr:L:1.X t. r:F'tl CO1-lta?.:1''~ ldL~.c3t',.i~
lOIiS i~l~i s i: y p_' ttER 1:? : d-1 F:1U :511 :, <;° 9I6 ~ cA 02386586-2002-~l0-02 '. H.~RfE~'f l E.k ~f,'1 (si),l c:t~~~~nte2:i~:mS of ti-!e cP:it.o_-;an. ~'he :I(~tri.x i:; p~ntic~zl~:r.y,~
SOf t ~zl~ci ~la'~tzC in t~-_is Cast:e .
'1'h-E: ciI'OC'ant- t~-t i'hi tc>saW iS r.Y'ei eral~~ y CrtG:~~I7 t:0 reS'.i'.
iI: a ;~onc.entratir~n of from 0.01 to '~'~ by ~aeig2zt, t rvfr,~rrb7y ~:n. 5 to 1'~ b;.~ we~.grlt . Inflt~e:~ce can t~
_YC=rtt:d ;HIV trne~ structure at t'~e matrix, in particZ;:l~,r : !.:~,oz~e Sive; tzrcu;gh t he corlcr-nt=~~xr:.io.n of t~?rf.-s ~:_rI_ :o:,ar: w~': i :at-~>>rl. It: iS t:~c;:;siko!.a in :.h. ~~ ~ra~~ t,- .
v., ~: t;
t~~~ ~.::e I-;;-~L._.~ ~.'._,f, e.,f :l":c-. at-ri>, tm the x;art..i,::2:1~x-t~.'~a ~,y a:e:7.;~~itt-I ~f;?~:ar_h tre ma.t.Lix ~.s inrer_~caed to ~.:>f: calani~e<~t_ GS~o<:u~~' c:Zii:cs.~>a:-I i;; prc.~ducea f=~orr, r_at.ura.l so'~:xCes, : t ~1=9:: Y:C) i,:i'..7_ft-'T-1T,'~. lTtp.~E'Ct_1~3r WE-l~yi:t;. 1'f'LP 1?:p.~E'i-lll-W t- WelO]iIr t- i';lctu. V-~n~ 2ue=:_4:E'C11 ~~:« ;iT-lc~ :~C: Pl~.?1 -E.-'' t_rlcil'1 ~
U~~U l!::~d, GL~-~7l3nlll_11~_T
I1 i~aiG :i~JilT'Cf' d:l~~ yYCyc1"d>=1C):'1 Tr''..'IOUI. ':Iif= C'~112:OSCIl ~~7.' a .'L.'.Icv.-iiCl Y.~1E3 t'11L°PE-C'..iITlE.'TlSlWlic'11 IT'~c':tZ:~n 1.
TlOt Sl.I~~E:C:i_ ar_,,,~ rep-.txiction:~ in mlat.ion try its rtolscul:~r ~~,_~iQht.
' d by s u:b w i rna t i :-~ n .-~.i:~E'f t_~lE: Lr'~e'ZI:1~, u_a1B 4adi_CR 1S rPITlO'.:TE
'.:I_QF'?- =~E~711(_t'C~ ~?rE'..''>S:JT'F:. .c.lli t.dl'JZc' Iirt''SSllF'~
Z~3i1C1eS r~J.N
Tr:ITi i.;~~~~ t:) 3 rl~d. rr~2lr~ E'X~3<_t COS'lrllt:lOnS c~Y-E' .'~-iJ..2J.;~..°:ta:~Cl 1"~\: r,I=~E' t=W >I~Il~C~.'al~is.''I: G~
L.rIE~, aCIIE'C':iS :'(7~lltiQ:'I.
. ._ '.-::il::'! ~-- ~a ,C:; .c'i" 7 1.E ~C:L~~ M;~=~ ~>~E~'ri LLSLO'1, i-t lT:cz_l ~.W -' dt: lc:.e(:
LtmrA~l~; ,~~..'e~.~.pc:rated during the lyolara:ili~atic>n.
tr.F:r~re tree focusing it is preferre~ci 'o remove excess ::,ci,.': ~.ty adding ~ base t-o adult _r:.<? IWi of the acTUeo~:s 3G :<u_'~.;t:i;~.'I ':n f.~-:'~m ':..'_r '_~.~ 7.5, preferably ~.5 t:o :'.0, Ta::Y ~l~~Llla: r _'J t=~~ . .'..'. t~: lT~c'itl=''_'.i iV:.:..I ,.TE:~,_, C~C~~~C.
j::2:C'j:iiil.''-.:W.'~: lil Z'Glet:IUIl to 2tS u.,2me_'lSlWi'1~-~. ;-itd~a.l:.'.~' . _ ,...:<;~iulr::. '1'rIe lrJat:T:.ix ;.ic~c.:s :lGt :~ry.;Ii;-; ~=,r~ r:-t~t;vlrdratlon ar::a ~.~c.:~:- r-:<:~t-. ~ilC;w- an-r~ :~;:i uctu;~a i c:'rarig~:°:> ~,::
trlFr . T:li..
-, _)') rL~3I7~c' :): 1_i'_ ~f:>'. __... ~c'~~-C.1CL;1<.-'~I .~' .Tl~~-'1~:E;_~~
'.'J~lt'Il t:rlv ti!'I~l~OI=,:
a . ~' _ ,1; _1'~ ~':>t: C1t'zS :3 yH Ui ixOIT1 t; . ~ tc_, . V bE'~:?1."E t}_E-' ~','~'!t='::iI:~:l. 'i'rIW ''~lltv'a-'tj.I'1 Std1't:',a' t0 j'.~~-c:Cipi'~.~lt_E' :i1 t~i7;:
r~;::a~:r . it. s ~:ssun7ec~ ~.rlat. Llncfer r_eut r: a1 cc~nCii tiolis r:
r,,:,l nr' ~-t ~ ,7.:W SUSF7~:1S.7.On : S fC:).::T:cC~. GL'~'ilCt'1 1'1afi ,., 1 n : iW ~ iW ~!FR 1 : v.l -I r=il 5 J I :5'=t' 9.1 Ei 5 cA 02386586 2002-10-02 ~ ~H.-IRfE\'I I ER ~~i n 1 i~
be::~:«lciul eFfe'rt ora tt=a st:ruat.u:~c of t=-le lycphili.z~d matri:~. T}ie nemtrali.: ed aqurou~> cflitosan solutiGn tr~us ~:' ... _;u:~~~e~_mwon. The c~euu~~ suluti<:,r~ ~i'.a~- r~lw:e.refor.~, C:c~.P1'lCtl.:lu t: r1 t~:° Ct'lJ.,trOSan C'C>:lCE~llt~«tlCW, iJC' i:
~ , ;
CC TiL COOL.".
'7 , : :1 1: ~ L: i. .'. c 1 -~ L r3 t. :~ C7 Zl .
f ri a met hc~cr 1 _~ke that ?r c~pos~~d b~y 2~adihal ~ ~Y~ et a.~ . , t t:r.
:r,~:'~.~. is e?:-.~t:~ri r.~nc,es a l.~;x~qe t-.flan~c~ i:u ;-r:e :,'truc-ture <-:, -,Fv'u.'-IW . :::'_..xt,-ii>l. 'nt E'7i(:E;:-'.~.' c:lCt;:tlC.' t3Clu t.>~' ~CTI~l1 1.~'?Z'r~t-1~.~:'1 1i.> ,.t::: ~:mtoas r3aUH. ~:ddit-ion of talc uu-l:~:r>u5 soluti"n F;ue:-ulr~..~iJ~.y' l~~aus tc: loc°al di:~so~ution of t_he chitosa.~:.
v.~E:it::?n i_~: : ep~ eci~Wt«ted b;,~ the PlaC~. The pore structure OJ: ':r~= T~::ztr:L.~: i S '.tll::S a~' terE~_~
~:~ .~s r'~:~sa_.~;i.; i ty leas now been fo'Lrrd, with the rued!od fol.:nea ta~s- t-t:e im-entor:s, ~or stab.~_Z iairlg the rr~;~t=ri:.c so that:
.-it:: , c>r cc'r.iderabl~.~ sma, i ler, str'u~'t:ura L cha~_-zges ocr,.m ''u'1 lla'I-i.:'lt:L yi-OCCSSI.nCf t_~f t=~1~ ITlai:.I_7.:K a~tC-"1 rE'IrlOUTal ~?:.
..:if' ~.',-'3''E-T' ,~j' ~! ~~O'~)~'11 ~_12.c;f_1OI1.
im ,t,'~~ fiis' e_'r~oc?imF.rit of thc~ :nethod of t_~Ie iliwritior_, v:he c:iit.osan :,olut~.ori has a neutral pJ3 even before the . ~ .---:G_ ~~:lg an.~i lyo;ahii:irc~tvor.. By ._=utrai ~~1'3" , s meamL
~.. tni:-~ ..<-~nr_E=ect--.on a poi in Brie r-ancJe =tic9~.:,atec~ abov.~_ ii° ~ ,'i '1":,~'_°ri!'~i-E;' l:fl ? Gr~C~F~?Y :iCC'l~,~~~'~z1 j% f:7 u' 1_1!E' CI'e.tT''L.'.C 'cWi i~f=: =~~xui:!:.i~r~:t~c:: ~.~~i'~i bare afZ~~r t~~~= iyc~plui2 :.,pat ion.
Tt:F.
-.~:~E_°W_~~:a poixW is ti-lat. tr.e ci-A~r_c~san :olur.:.cn i.s ,.:.m-Lta~.r.'.v- ne~._;tral.iee~ aad tree cr::itosan nrcc-=p~r.~xte.-~> ~=l:a .':?. ,.IAF_' mewt_.r=xl.i~.at_ic_,rt is alt~~f~ 'Tried o~.it.
_~f~l C:ctIL=.:' ~'y', ~'i~E= Ie.~;l.~i_lrlC~ 111ti'."2'~-~_x T_E't11:>~~,',iV~t.'~S ''?J:
1'E'}'i'"Wj='w:tl.Ci::. 1I'1 tr.G ~lt~:t_ C-'III~iCIC~IIT1L:I-It CW
t_rlfITWti1(~C( ~'i"
r-r,r r,e,.~.Ilt -_~,I:, the acwea is rm~t ~ r. th~~ first instanew~
~~.p.-.-ie~W to any Par':icul.ai' restrictions.
z'a ?r: is possible to use fc>r p_E~parina the aqueol.s ~~:-u.'c~:a:n ;~c~~.u~ic:n an ac-.d wh~r_ri is ::electe.=~ from tt~e cJ.-cAUU _:':>zcn-ed :>~,r i_ric%:-galni~~ ac::rds a:m:: «rg:~l:lic uc.i..:l~:
, ~:'rcW-.v-;i~=>_-,- ~7~~~3-- and arylca~~bo~yli.C acids, iIl "-zr-t ic'u i~.r h~yd.-c~f:~; c~:irboxyl ~.c ac~.c's . L<-~ctic acid ::
Ill iy. <y' )t>~.R 1:,:.~5 F':~1 X1.1 3sW 9-tu:> cA o2sas5ss 2oo2-io-o2 c:H.~iR>'EVTIER ,~I1~11 _ ;
;.
n~~~-1- i=l.i:."~.1 _::xr ~~' ~iI_ E?feT_~2_~-'C.'~ _ xa-:c.z:;l =;~ of inorganic ar_i~~;~ are hydr~;c~hloric acid, ~~lic;:->~'ht>:r i~~ ~a<.i~? and acvicxic r~~:oN~,h~.t~ sait~:. ~'x~:m~les .. c~.rct,':Ii'-' a:_id>:, are alKyl.carY~c',;~y? ~c acids him%ir:c_r 1 ::_~!-:->o:u moms, it ~e~.rig possb.lt for tht> ,-.~1k,~1 chain t.o :u a :a;~:~.:.~~:311t.. c~ldin cI l.~rancriecl. i-:xamplc-», a>_'t Lorm.wr _,.. i~:-~, ,~~efi-.:., ac.c~, _ wop-i~~r,_ic acv~d, k_~ucx~r=_c aCi i~, ._,e~.= ic_ :zc iu, l:ex~rloic flcid, h~:pt-..anc'ic ac-iG ar:d hi y7r:I
:=tr~:ig'~r:--a~l_air~ carLao:~ylic aca~~. ~lsu sui.tn:r,~le nxa C,7=iC=:i?'-);7i)~;i,~L1C aClC~S ::~V1S7C~ ~ t.0 rG' Ca'_'.''~')iaXl e:tC)1'IW, ~'1..1,C11 ex:u :7::caliG ~c_j.~~, ~;LICClnie c3C.'.Cj Or ~di~l('_ ~C'Zt~. jp1 ~ui~:.aL~le ~r~~ ~:roma>:ic carbc::y'iic acids such as berzc.~,it~
<x:~it-i ox- napjir~hoic acid Furtt:e:~-more, arni:m~> ac~i:~s, such c:c;.LLtcin.C acid UY aCpc~rtic aC~:~, c~L~ d7::C~ sul.t=~~x'r~e.
:~t'u'.h'~=",'r."'. ~~d:L'C LCi:~.c'3I'1~ :pGC~ I't-SLlit-:~; c1!E~ ~-~~W~ll:trC~ W1 L1:
. .'T,0::.-.,% Cci:~:J<~Xj%1iC 'cic:idS . T:G.; C;C1:,? ~r~y .'~.?1=L.el;;>1G
'-y-_az ~,~',r c_rm~:~o:-;yl-~c ~:cids ha~'_r:c; ~ to 1'? cwxborl a,'ann~ .
~...:::I:.i",'I:.:_ ~l: ..i":C:' ::T :l'.i~ZP 1'ljlC,rC,':;yl Cjl~Gilp S dI7;1 OWES ~.:?
;itW.'W- C. d Z I-'C: X'J i CI2-OLlj7S T~rc.PIiu, i lWt-:P_ ITtt'W, EC'u i E:
. ;X2~- tMTJ~ i:~
.:il-t: C:i','CG'; :: C izC 1C1, LdGt 1-C dc :~iu, alE~1 ~C ciC'~-.,~., .'ar~~r i C' lcie_~:);:: c~ t-r.i.c aC,~d. 'ft. is ~L7.so x:o~s~bae ~o u~;Er ;a.-t.-"y:t=Lt: r':',''a3:O::V C:drbUXaJIIC dC.'1GS, ~Or Pis-4'I1'.;~.lf.' T.fi.~.:1~E11.C.' .__ ;'ii~.l.i~
Base~> ~.~h ich can be used are cc'nT~ en" i o::a"i ba;~es such a.; , :-~,.r ~-1:<:Yrj_~1~-v alkali I:let.al hv;~dro~czdes such :~.s r~aOH. ~~t:e j':%y;' -~W; ::~rlc~l:cad i'~e Cr':O.'-'~t:='1 Jo WicilL d nE?ut~T_W 1 pij Cdh ~_~..
i;: r~ i '"i r f: a ~.. i t. h ~J~..I t. ~~ ~ ~ ~ w. C'.1.: .l t...1' .
.. a~_:'.v_rlf'I' i'~~~7.'-'.'~:1~).'-.,:.~.t.~' fOL' l~t)I.;ilr:111CT i1 Pi~?tYiX 1,':'Lt.jl ~'L~i C- ,''i~ ';t' _rl1<; ","t:Y E~' 1 . ~t7r t..lN =rld L. y .i'.~_ :;.J
~:e:' :~L~iji1.11 zE~:.~
~;-~;:~_''.. . ~i-i '.rIC? ~..:.':iClS ~,15~"'...1~ i0I C~._:~~aol',7~":7<~
~I7~~ ~a'Z3.~0:;>ctl7 ' Cl :;ester .
y _._ :r:i;;~ enne_~~ged, 5u.rprisir:g7.y, r:hat~ tine pr~.:p~x.-t=es ;:~
t.:~i~: _..rt.osan :r~a'ri.~. cap );;)e irlf?~aer~eu 'ny the choice of ,.-:E ~~C~U.::LCr'i:.~11. ~fulS 1. Llt~illZ~C1 111 i:.rlE' SUCOnEI
~n , ;~;; . t~,~, 51't:h 1 °. t.-'! 1-.~.1 ' 1 -! .'~:r ,' 9-t f~ ', ~
02386586 2oo2-io-o2 ~:: H_~.hY~.~..l.l Fh:
_ 8 ez:~!odiment of t:he method of the i.x:v~enti<):~. ':kie acids LiSF:W : "~.I1 ?l~'11.'adrE~ I-1~'Cl~:-Oxy Cdr"~~0'LyZ.ll..' r'3S'c'' u-~C:.'ad~~. l.lE:'ry c:c'~ci ro:roioert i.as arc-: obt.ain~=,-i in partic;zlar cn use c 'Lartir. ,~~~id, w_r.h whici~i the iac.tate arlicn forms ti'm coi.:;~terwon to t_he protonate<3 amino ctroup of Chitosam.
;li,;~:, r"a~tzix :,h:~:,~r~i:igh temperature re:~is.ta:Zr_e and m c.wn, ?~oz. ~=xart;pi e, k~e autoclavec~l v'rithotit diffzcul ty.
IJc ca_~.~~e'_z:ati -errs other sicrr:s of cieco"rp~~:i ti:~r:
or of ; >:e nlar.ri:; <ypsar '~ur:n r ti-lis. ~~'he matrix als~~ sho;:s a high e!asti.ct~~ arld mechanical stren~~th. Tiuus, a sheet-like tT~;x'.: r .i:: c-:i,!ta~c'uz ~mc~ the ~qu~~ous c-hi CCSan i:md ~~;~ i pact ..
a~i,rr.l,~t:i~:,r~ int.ca r-s=tri a3ih and then area=,:ir:g .i~:i ~, ;:aul~:i:ii~._r:u, b<> > olled ur:~ '..~rii~hout _i:i::f ~~~c-il. .z,-;~lty .rx;d v:~_~_.rnut 'r:a=:c::r~.ri-vc3the matri;t.
i ;;
i;:1 r.:.:c:: ~~~rcarrc.l eni~oci:im~=nt cf t-he Iner:iod of t he ut'.'~~.'t'-'1C~~.i, ._i1G StabJ.Ilt:lT:C_j. t?~f~=:Ct of :hE
1VC~,2:0~:~'c3~iS'_'i-C''2- ciI"il.~ Cr..C::i:W;}'~ 1C c3CldS c:,~.'FJE'ar:~ eS.rH'r: .' f t:i'1-G' 0111 ~-~.0~ _~I'1 ~.~lut..ou. i~~ zoot neutra.iized _before tile ~r~e:.irlg. ~?ftex~
%!? ;.ue '-c~F~hii iu,af.io:"~, residues of e:~ress acic.3 which i"ire _,.. a 'i1 p_'esc.~nt are rer;zowed by eciui?i.i~ration with base, eaaz.~~ ~_ ~f~.0'.-' is Na;~H. 'a'hE= matt:i.x is t.h~:;1 waared ~~~~ti"1 ';~~:~ Fir , ~:.~h ci~ m~z- c~ntai r: a phosr:hata ;.~t.f f c_>r (':' . 't N, r)H
at_;<_!._.. . , . I'iic structural cimr;,gc::s _:z the: matr:.x axe :~.r~
. . ~_.'-.. . ::,7ac-:~ cw'r:r i~zer.:~bly ~ c=s<~ 'rzar~ v~;tt: 4hee ~rrat: i :1 ~-ies:,::r~i.i~ed iai~ r~lGdi hally. i 4. is particu7.a~~iy adva.:-:tag~:c,m i .~ r.h~~ ~w~c~ond em~bodimer~t of the metrioc9 ;~)f t :'ze i rven t i ~~~n '. C' .. 'L I-l f: : :r ., :, i,. ~ > : > d I': S O 1. L1 t 1 C: Xl t O ~) a :
i E~ is t.:C a i 1. ~. c' C~ ~ V F~' n u'? a 'L C~' :" F.' :.i':e f:~e~~<:_ryF~cruilibx~:t-.~.~_~I uz~dc~r ,::lkaii:m: c;m:ditic:~n~::
_ . _._ t::rlrv=1 ziC, ~r:v::l~ie:'~WleCE=-~sar]'. ':lie :?1c.'<r_. C'<ah ,.~"'..~:~uzi_i,~'r y';";~,~.=c.',~y~~ di?'CCt1~; dnil, f_Ot example, c'ol0i'1~.::E~d 1_~'y :~ C' t; f1 P ITt~~' ~:~ G ~. :Ti < ~ W. S O f t h f_~ m ar" i_ h G Q C! .~ t 1'1 <! 1 r:'J B n t' _ C> n t a ~: r.! 1 L
35 in a. mat-rix w:i th a uniform por= st.rur_.ture, which s<~f~-: ~ri~~ ~:>li.able. I:f, for ex~lmple, the m.~trir. :s c~or-tnr~~4.~:ed i;~~ F;=ensure from the fingers, it- xe:~umes i ~:.s t_J.' 1_~~.Ii~ L ~~;<:~~rc'lCId1-I': E3lcl~:tr~..Ca.i ~ ~i eiit°r t:iE
~?rE'S~illrl_ ;:i _ _=::lo'.wd. 'I'hr~ rnatr::~: can be molaed wxtho~.it breaking car i 1c~ ~t~ o:: BIER 1 ..lfs F_~~ =31-1 .'.t<t~ <.~lh:; cA o2sas5as 2oo2-io-o2 <'HaRPEWiEk ~~';<t1a _.
~cra:r;;bl..ir_:r a:':c~ C~iri, for e~;amp:'e, he zol~ed up_ ~io L:.>iz~~ rol~:erent asserr~b? ae~es of ce~~s it is necessary yor t-nr=a r:,at-T: ix to '.zav~; suffice ~~nt< strength c'.urinc~ t.t:
°, ~w.~? i:rl i ~ar_ion rr:l' cell s and rc~ be nroken do~~~.n on2y oT~'er~ a ~.w~r',,.r pro': o:~ce.d Fex-~ o~:.. 'i'l:e ra'.'. V cf hreuk~.ioV.>1-1 can ~~e ct-~:-ir.ro l ~r~_~ -.J:i a t~-ie caegree of deacetyJ.atio:l of c~hitosaT< .
IBIS ~: SE ;7f w uhltOSO?1 'vs':':~'jl at:l.ll ~Idp d VE"_''l' j2?t~~l C~2Ci2~::','l?
~ s.:t.,=av4i~~::z it :s p;~~s:i~u.e nor breaks:;~wvrr-1 ~.i.mus ~_~f 1;! a~-~: <v ,~,.;-i,':.3 one lw,~nt:; ;,o be ;~cti~ev~'c=d, ':'iaE-. ln~=.r.r.ix .s observed t; c' b~? part.i~~.1->'v~x~l~' h~aiz ~'1 ~~:~..~r;'~"~ri~':i.E:' t~:'~-7e=I-1 2<.1'1E W~ll -t:»dIi ~'SCiS vi C~E:~::~r E'e !' ~~Eai:G-'t.l't.~i'pl~ ~7r t~n~. 'T'~':~=' ~PCfx'E,'P .'_7'~ i-Ic':>ctCE't'l-lc~3t-1 L~: :.~:1._ze_:: t.: tle PJ-dcct:yl gre:ups of chito:w:n. i?y degr'=t~
~J~' t:~CciC~'t.4'ic"~:.lJIU '1S LI1Gd11t: t-he rat.lC CJf WllciCGt'~'ldi.E=C.1 -; : ~~.- '''~~ 'a1 of azn:ino Jr-e,ups presE:~l~t ~ni.r o grov,_lt ~ '0 4.. ~.. to,~
(tc~;,=: ':-~z :. r.ee amino c~zroups and ar<.t~~lar.?d amine . :'.i.;L.r~ . _It is aSS~;nc~d t:'lat at a d~_c xGe vL
. : l ~ O' ", c3 C:''~ t ';' 1 d t_ . 0I i C-~ t. ~ g l~ ~ c'~IT.O I ~.):: 01:
S S t ~ l_I C- L 1; z r:: :.: c~ I''=,' i O Z ITt~;' ('.~
lli VlCl~:~ c._ Ec~S Ot tYte YTlctLriX. i~~1ESE: ?IIIOr~7hQ::~ u.''.~~clS
W.w."i r~?: E ;v_;lTicl~;i~' I~~r~ c~C.~c-iC!CeC~ )uE'ttr4_x~ 1~?1' e.IlZyCIlES . 1L
1S 1~~':la;~.
'~:~'a''a''xZ~l.e t''J ~.:1_ _L,1C::':C:E'? r.iE? :-~:lt E,~ a: l= W~'1? Crl ~'~'=E.' ~';:?': i i.X ..,.
,..'.'.o..i.._~!.1 ._f. :":.'. ~~lO:~-yr ~:I.l~ ;::'u]~,~SLE:i:1 _-r:..~ rlc,=
~~ia,'..~t'u.Cill.;~
_. . ~:t_'~:':~':.
.:::- ~:m:,t:e _ ize c,f t:'~E rr;atri.s c~dzi LurtiiN~r bra inf~luencf~ci ius- t.:lc~ Yat.c ,.~ ~;~hic~l-, the ~~~~a<>o:Is c:r:~tosan sol~:t:ion i ~r~~r.c=1. ~r~ yin ~, connection, rwps.c3 ?vree~irc, i~~x <<~:am~~! ~~
~(~ ..i ..iq~jic~ :-lit~.ticxc:=za, z°esi~l~.s in ~rr.a:l7 h,~-<~ diary-,<~ter;v, ',~:~"E':C-E~~S :a~.:.::>'.~: fre~Z~.?1C~F, iC7r F'J~.ci:TIL.7lt~' 1.n t:he !-dn(QC fl:Qnt :~ 1 ~ "~= , -e u~u '.ts. ir. largo cores . The telr.zaerature :a:,r tr~:c~ir:g the '~izitosan solution is pre~:e_ably chosen _-.. '-'i:= Yes,.:i_~~ t=rOm --GOC)°C: t.0 -4~-'~', 1G'=r=eid~..v~;~
G~°C ~;:~
35 -i~~i'~.. I;-;_ t icuiarl.y preferably ~0°C ;:« -1~~~~~~_ ~':c- ~v:at"ri~ o~~','~iz:able usincr ''1e m~t.h~x? c~e~~~c:'ibE-.~t~ abc:v<.e ::~. t:C.'('v? ~(.'r:!' ~%rC?iJert~E:S ~.~'y' ::t~IiliJdr'15.'7I2 Y~'Li_:i :T:r::t:T'lvE:~I
;:=:~;a'.~:~ i:? ri:~? L'~2::10i r3Zt<. 'I';lE'.~ 11'LVE'21~3.OY1 4E1F~I_E~fCra-? ~.I,..S'CJ
i~ iJ~-cv~ )TE=.R 1'::-(t3 t'.-1T '1-! Z~7 9-IIiS CA 02386586 2002-10-02 E-~H.~RYEV..I,IER nic~lt - 1p _ re.:ares to a matrix as ~~~.~ta=rted usiro~ t2ue metr:od c:~~~=~cri.bc-.~c1 a~~o-,re_ The matrix;. is dimensienaliy sable, .'>~!'r <irt~ f .,~ !:"C~~7.Le, c-'-.1'Si~ Catl t:~l~_1 ~ EdSl 1y ~.i2 X:Z'OCPS:_;~'i,~
w;uCl, fi;~Y. E:~:~~:T:j~lu, ht~ C'llt t,C'~ t.~'.w C~PS;I'PC~ :;.i2l~E' :>a:vpi_v.
It cm: bE_: at.:toclawed and thud> st.erWized ~~imply with~,ut:
ric:c~~mj~os.: t i or: rF'actzons occurr ing . No furtt:r-~r ~.~;r~staricE"s sIc.i-I as, collagen or alginate axe necessary fo= :=.tab: 1iz=n« the matrix. The aegree of acetyl.atc-,rt ,~:nc~: trm:;~, f:;x. P~:amp:lt~, tht: ~;t.vorbai~i'i.tf ~~ '~.he rnatxw::
'L.~.an be i~h~~~er_~ Wit~~to:~t- iestric'~i..-~r,~ end pan whms t»~
~~ir~-,'st~ c.~ t _~ t:~e particular: requirt:r;~erats . The ~.t=ructt.lw :~f tr:EJ :r~atr~x, iri _~artiC~a:J.ar the pore si~t~, cart k>E~
z.r.~=_ur~nrnd t hrough .rle <_-.oraoent,rat::c~n c>r the aquecn..m;
r_f~:i t~s"n sc~l.ut~.an and t:he f'ree~ rrg conditions.
~s "'P1~;~' C.~mE':lSiJrlcxL~~jl su3~71-E ci.i'1Cl ~le~ii~J-'_E': rrW;trl?C 1:
~:~.:L._IJ.I:tf~1 1~.r:""1: v:lc Iria'r-1.~: ~lc~:_: d ct'.'t:-_e' i::nic~ri r1';
~._ (.., l i t i - i_; I ~ -L. :1. 1 .
,il~ y.tle,: r'lcll:x-J.~~ C:c_I: ~~ rn:::,xl~lCW 35 C~~'Slreu tilr~7.1~r1 tl'tE ~.a~"~~F' r:un.:.~~=- of a:r,:r~o groups . 1n a preferred err~odi;<:ent t:tc -:~:ree-di mensio:.al matrix, l igands are ~c>va:lently ;
ro::_°o~,Ta7.~,IZtly btwand to t~r_e chitosan rr~atri:~, Freferab::y tc; t hi, 'r r=a amino groups of chitrasar~ _ L.go:ods which c.r3n :~~ ~s ed a_ e, tan examp-! e, iii owtn promaters, protcir>, i~~r.-ionE~~-, ~mparin, heparari suiratE"r~, ch.ndroit2lu sn_llfr °r:~s, uFxtt°an :~ulfrates or a mixture of thecae :~Lli.:~yrc:lC~~.S. 'j'7< .~lG7dIic'~S ~?E.'~c~.'_dl:i~.~' Si~rV~ tii e0:_~t~Gl eliW~
ir-y~>xa~.~a cr>;.;. ~:~x~~l.ifE:ratior;.
:Eei.l c~r-c~~~a;:h ~~.I the :nat~ix -~s wurt~:ex: i:npr~cwed i.f t.'aEr.
;a;t.r:;: i.~ ~::~~:tE~d ~.~~.th au::~riocacvus :.-ii.~zir:_ r y ;1,.,~ tnr~e.-di~nt.t~.si.~,mi:~ matrix ~~~- t:a ~r~~,>Errtxo:: can m aso.l.i~i phase im a c,l:~ture .~e;~cto(ce.:i i-,-<.'': i:~I _;' ) . ~i~hr ITtdtr.X :a:lUWS ~~ ~J~r"~l Il:l~~l StrtrICTth :'r1 ivl:ia.'.lt'E'~ TCIE'G~~.Url. It f~~~ W lI>V ~rrlE'rC~ECl ttidt_ t~lr' md:x~.X
;~T-C~aI:~L.iJS C"E:rl.1 <-~T ~aG:Ct1 n iv y(t ~' v(ER ) ., ' -I si F'.1v 51.1 .'t n'; n I t> ~ 02386586 20(72-l0-02 <.'H.~FPF~'I' I EIZ e~ f % 15 - :L 2 -acne LT-~.atri.~r: .t.imul~;tes ce.i1 <.Iro~wth. Lt car. therefore imk:lan' ~~ci diz'ectl~;.~, i . a. wi rl~out. ~~,>'evio»:. coloni zati oLZ
1.~'s' ~'G ~ .i:v: . T:f t:fir trlE. LT?-e~i~ a:~ti~C.~,i_~rl, E"1C3W~C7,~I'iC.71.15 Ct''11S ~I ,'_',L~r 3- 11 , .~.y. t: : 7, t il i T.Ilc'.l t i- l ~i (~. td G I: <3 .i. y ,~.' C:1 S S 0 1 ~J 1 I1 Q .
c ;'1~ matr~.. is aLsc~ suitax~le fcr ~.~e as c~~11 i:rplant, ir:
1;:3~.r:,,_~1:.7.,'it~ ~O? C~'il: ti ~.c=t~~ t=r'yp17.:1~ ~"'e11~. Zrl t:'..., r-t=!T:I:E_''C:t 1 __'.r( ' t '.S ~rPiG': rA~ ?:O t.. t0 'LlSI°_ ciTl'y' GJr'I7E'Y 1.C~ 1.,:,.
:, ~'l !'; W l ~.'-e.~. ~~. c: 1 ~: ~. . '1'~i u' t: (,.-' '.~ 1 :.i u.. . ~
c.~.i rC: f~ !_: r~ ~ :? 1 'y' C. d )! f: Tl x~ ~:' oIl s ~ i . i_ :l~.~i pn~:~er.'t ~~y b:. opsy and t:w~tuxed cm the cell rY~atxzx, a:~d ..
:':i~~' C~~.:1 ~ :-'.'CL~!~'.~_il=t _i C ~IE??1 : LT'.C~i~-~ntE.'C~ :iI!~
t:.lF." ~it3t 1'G':at_ .
1 2' ~'::>to .ctl:' . E.' l ~'Ct;1.0:1 T"taclL r l O7-:S ~rE' ~:a.iri.;
.d:l.'.2d : : V
~-,1-~-~'--1',:~a'~:,~ G:'vva_-.ir' 'C( r-~?f? C:iJ..~.OI:a cit (OT( ()f t.I~l~' t~'lI'(-'W_ nl..T~E:ii.il~'..~Y'ii:i1 lT~c~t=r1X V~)lt.~l. ~I1WOC3E?"lOLis 5t''4':il CC11.S (~~i-'Ilc, 1 G=, m_W :;t i tierce i 4~!-.ic-~~ - stimulated by the resp.=cti ve arowt:l ar; t.c~rc 0i' ,:'rP :.aur-roLlL=~~3i=i~ t. i ssue -- dif ~>'~r ent fate or:
1y _. .. .'_r,F a ::_e of the t. ranrpiant , dnc3 «ws r>c~ r c>
F .
<._,:: ~ :~::-i.= azr-icrl with c.ar:..ilaae cells Lwr reTl~.'wed io~nati.o;l v'~ j ~'. ~.' ~ 1 1. :Z (_'-'. C', c'~ I- t 1 ~. ~1 C7 G . 1 ~: a. ~.i 1. i d (~ ~ ~v c( t. i~ d yr c3, n t i.~. Ca t:_ .~t°_!~omp~~r4u ~~sit:~ piez-ious mat~-ic~.s Lnadt: ct various a:. t ~ ;-:> :~1: ~..r ~-ami.cs . ''~lze ~f : Tr,~t er ~.,:.a r~~rri~in 1:z ;:.i-:;-.
r_ ~', :~ ..n::.na~~fT c~.jer. a~ ter _ t~=L,e yzar:~ be. re~og~li zed ,..
'_;.~_-e'i;.7zi a:lG '.~:t~ re_ie;:t.e~j. ~:~:r-:~ ;turec~._dimcr.vional r.-~atz.i:.c <~~ar: h:.:: ~_ _,=c~rii ~:e~i k>oriu 1?y luu,mam ar.c~ ::.y arl.illi;~.l c:e.i ~ws i ~ ;
:vs <v : ,-y.7 ~:- E:. f i' ~ 1'Tt I10 .t S a ~ Ci r~ g C7 : ~.' h c, L- ~L I
. ~ h ~:c Y ~~: :~ E_ l l s c: N i3 ~»r-r_icul err'' ~,T sui t_ab? a because tricky inciure a negligib i a :r~.rnun~~.cc7ical respar_~.~e in the ~.ecipiexlt. siaark cell:::
oz:"s~ ~:cii.~'i:c~~7'J 1.1;:~E.~C'1 uS ~;~:~Gdn Yi=k I. c=;C:~~rt'W1'1t, I''tW° ~~:c3TCl~:I~' f;.i_ 1 .,.- -. ,..
a ,.
..rl!-~' i...'~G .? '~.J t C. i C:u .
i ~'~
iz;a:l~_r:t ~~x tt-:P :i.n~renti~r: which st.im;.l:'~lte~:~ only a :,'1 : ": i : : iO 1 I I ;.T L: _'! t' L' E3 S ~-~ O r( ~~ E' 1 ~ <l ;~ V e7 :''_ t ci ~~ E' C:) 1.1 'u 1 'V C t'Wly.~ S f:' ;~
''l e' tl~F c?...~3ltnE'11S1C~I7.c11 TriatrlX ciE'NG:Il.~.iFe[~ ,;i~0~'E'_.
WrSl::tz ,~. ';vlonlred key ervdo_1er»us ce.! 1:~. i-t:us, ~:vi-P:~~.ihlE-;:i ~, a tnixl sheEt-like z:~atrix which ~-.a~ be:-:1 ..._....~.iSe~i tvit~l keratocytes under s~,=er.ile cc,ndition~-~ ~x~
~:'1 StiJ.'~cW1 E t_(( 1. t-:Ll?'2 ITicW1121T1; Cc~I7 ~er-1'E.' ci5 Sk~.L"1 trcln ~p~ dIIY_ _ :''r~<: coioT:ized matrix caz~ be remcw-ec3 Lri~ru the cultur to nm~~.fit-_:e~. mn:_ltr sterile Can<litioms in t.~m~ operati:ig t 1 0 : W~ tW ~I(:h 1 . : -1 ~ ll.~l p 1 -l .'.i~ ; <,~-ici:, cA o2sas5as 2oo2-io-o2 f H.aRYE~T 1 ER ~ tt 1 t~
l~ _ t; ~i?ei i: t'I- aI:c~ bF= ~ldCpC~ On ~1':~ C.L~ct1'1~~ WO~J:1C1 ~11Y. ~c?CE.' . 1. t ._. thexi ;ac c. s~k~?e for end~pf~no,.zs rFll:~ t~~ rnic~rate i.r.to ~:t'..-; n:~~t_~~ix '.c> ,'.oi~u tissue.
:I'1 a rCL: ) C~~ C: '~ :1-f P i : t ~ °, ~:. X t l ~ ], C J_ 2. ~ ~ l V a ~' :1 5 G ~. S O 1-i C7 S 2~ 1 ~'J ~. E~ _ i' r": c :.z.~.:..ri:c in tty~ ca.E? is cclc:nized .~~itr: li:~e:~ ; ells ir, a ~,~ tnbl P Cll? lure medium. 'The? a:sei;ualac~~~ ~:i.E Cell , t~~n . c':h :;u~~ ::r~.: -_-~l.so 1:7c stak_ailizeci hm the rt-~a~r~x, is r:~~:;=rz .::r.~::a.itc>~in ;:1e ~atie.:;_.~s body arvd tr:c:ft? can, l e;itclillti_~G-, d_~~'~St tkie LL:I-aCtlW~r= Ot ~ Clc~iTd~E_-'C:1 iiVE_-'Y.
Goad results have been achieved w~3er1 thc~ thrGe_..
cii?:ve_nwor.a_i matrix i: cclcini~ed bv,~ auto ~.ogo-as r-:hr-:r_drc~cY,-t_es . Zhe matrix c:ar. then by transp7 anted t:c~
1'= tlle== ~z1_Jp' aprlatr~ ~Jlu''ces on the car',~ila:~e wi:ere nc-w mc~; ti'~,<~r~ .--_:_ib5tanc-e call tt-ien develop. Tfii~ provi; f:>~
c4 raw t, a:~-,~a_n Cages compared a,>i th the methacz currenr_'! y i n use, of tza:'apJ.a.r~~ation c,~f cartilage ~velis wi.t_h<.nTr :-,_y-;~oz;:. r:az* : .i.x. ''_'he hi<:h robal''.~ J.it~- G;;visting iri t~::
S
tr:~:.r_,':oca ~:~ut the periosteuz~: u;~11 be darr;aGed arid b«z:c~
i,,~~'7.Ls wily r~._~.~ groV. irito the new ~art;.J_cx~~N 1<~yc~x', w. t;lu .>,a'r,:~t~~y._,ent <~irc~c:mr~it:ion c1 t_t.E~ r:ew uarti lage, <:=.-f; :..c-icW ?~v~ tlTf= i:r,~lar.t o+- tx:e in~~enti c,r:.
... _ ..___<:':ii'~.~~.=r',s Clt, Ut:.;lf-'r i=E_i ~.'.' Vf1-llC~n iy'!'~f~
,'ivZti~.~.,.~.L~ i .:~~?1.i_iIl.: Zlny 2. I'1E=.' ltlGt.I 1.?: 'crf~-' Owtf-?0::~;te::~~, r',C:LQt....lIlUC~~u.:r:.. , r1E'~~~tt_C>C_'y'tt:S, ~UW (? tildrl.~OW St2IIl Cf~ll ~ nr rl~Lliorla~.
Cel~~;..
iu.m~:r:' ion -~s explained in morE_ detai Z below ~~,r.. th Ol! I-rvE~r t;IlC~E? t~: , ~.'JC:Zc~~ tJ.CtlWE'.S dnu ~yT 1T;~'..-'d:J.~'.~ O~
t~XdrCl~]it".,-,'..
fv~gnc'cs _::o-~.' s~e~Wvic:al:iy:
Fig. 1. a:i i.maae GT a II-tatrix t~.~iCt. iawtate f-:~:;:._-: tE=r ~ :'~r~ , r.:rs:; ac;~ueous c:hi t_f:~~:an s~,-.~ 1,z'' i~;,rl l~na~.ri.nc~ b<'r~:-:
3J rTtm.:;.r.al.~~~c;d rvriore the fxe~zin~ ir: -he~ ~:~rr~du~~tion a., t.l~a Inat.Tx;
F.ic~~ 2 a. irlldc~e of a matrix, the Irtatrix haz'z:zg bf:=fin t=:m.lili.bl-ated a.tex- l,~~ophil~.r.ation w:th _'~iaGH (5'~ .~.~y !
to ,l~ ;l~ ;rF,4: 1":.~- F':~,Z. :;i.~ ;s<t- q.~E;:; cA o2sas5as 2oo2-io-o2 n'H.~1RF'E~'IiER ~i~ll.
_ a~y ..
~,=eig~~t y ~ n t.'ze prodmct:~pn of the ritatri::; t~h:e c:ou=-lteriola is for:rc°d in (aby lar_tate and in (b) ~y phosphate;
Fig. 3 a rr,i.rrograph cf the matr -x oo~.Urli:,ed 'p,.~
chondY°rlcyteC .
~.~camp~e 1 : Ge:~eral :rethocZ for pr Uduc. _ r: c3 a three-dim~en ~ior.a:i =rutr-ix t 1st a:-nbodiment ) 1.G c:t-:ito~=an a.s c3:~sso~.vred b;~ stirrirm~ in ci~lute aaue~:~us Gc=ld ,=~t ~l pH betwEPn .. and 6. The desix'ec~i x~H is tll~~n ~Ca~,l~.l~>2:EW1 'tJl2'.,:-1 C1l.~.a2:f'C~ I'~c~~~'13. ~~rlE; SlWutlC:;'i..1 ~S tY~i3n5fax~"C'C~
lilYtW1 1'ESS't.': G~ ci 71.11t.a1~.iE.' Sflci~~ ciT'iC~ iI'a.Wn dt -~C ti;~
~:'rI a ~~'<: s s a l u:~ f t h t h a f r ~. z ~=n s o ~. a t f on f .r f _~ 1_ T ~:-:~:I :,I T_'r3C1 1 T:ti? F~ ZyiJ~~l= 1.12:E;:I a:ZQ 2,i F: wu i:~?x- ~.~ t3inU'~:'r_-.;,1 ~:~p' ~',1~% ~ ::'fl,~r 1f:1'1 ;_lI_C~Ur XE~:~,lWf~r; ~~r~ 551:: E:? f i7 _ l_ .':> 'r:~;~~ . The ::p~;:~cxf re:':idme ran "n_e c~my:.Ic:1'~d witheul~
f:ar 'he::w ~wrvii~-ut.icn for col.:ni~~zt.a.~r: :w;wf_3.1.s.
?~u ExamplA 2: ~:~e.zera~. rnethad ~ox- r%r oduc; r_c~ a t:or~u-a : m~:~ ~ .f c~ri~~l rr.c~ t~ i.x. t ~:ld ertll~Pdimar.t 1 l~h~t:os:~r-I f;; u.:~solved by :~t.izrir~g i.:z <~i-flute «qtLec~us ri.~..~yry~:V Cet~'~0::'y'~1.C t~C-id cz~ d ~JH beC:VJCt~rl f altd ~. i't'it~
C~l.~?%r SC:l.l~t;.?.Gtl 15 t_rUZf_.'n 1n <~ VPSSP1. C'~f G'.lltd~ue S~'Lc:
.:--~r ~1 t-erry:f«a~-wre of from -_5G no -5'~C. Trlc vESSel wsr.h yrw.;.F~a. ~~vlu4ion is transferred into <;: :.3~o~.~hili2Ex.', t.~:F_~ ',~.~at~=~r is removed by st.i:'vlzma:t~.on under reduc.-F=,a Nz ~-a:=.!. ~. a~' f'? . ~ '_ te~ ~ . f iuP& ) . 'I'21~ ~;~.~c~nay res is ~_«:~
3'.l C: E:.'I"tci Li: LI7C; ca f' i:C'r J: E'IrIOV~.1 O~ I<r1E Cvcit~E-T_-~7~i S;Ll~7~ lITt~t10:1 ;~.~.:z.~if~ci Yl ~.c_~d?um i:-:~d~c~xir-:~ :~,~lllr;on ~'aw 2:~i' w~igr;; f hc~,:rc . ''1'~e spongy Inat:r iy i:~ thesrZ .~:a~.hed wi t.~.
~:ic:;;;>z:.::Ee;3 ,,~at.c~r until ti:e pH :is t=~:~iezvab<<. L~l~ c<~Ils.
Example 3: i=xc~ducti-~~n of a pc~rouC c:~tc~sa:1 matx:~x f <~~~;~zntex.:i >>r1 : 1 actate) f; . ? <x c>i= c:r-I:~ t~:~san and i~ . 11-i.3 g of lacr_i.r. ,--;icicl (5t7~
~.n ',;;<at;w, pl:.~-crla~ect 3:~'om ?nerck K:.a:, Dazms~;~cir_.l were 1~! n~ ~~y ~IF'R 1'.:.~5 F'~,1 :'.7-! :3n' ~.tF35 cA 0238658s 2002-10-02 ~'H.1RPE~TIFFi ~~ia1"i dissi:'i~.red by stirring in 9.?9 g of deio~lized water, and tL~t, t~l_~ Boas ad~;usted tc-> ~ Gl~t.h 1 M sodium lZydrox_de c': L L1 t. 1 i? I'. . Z't l E: C: 1 G' a r ~ O 1 ll t. 1- a n '.~: ez S ~. I
t :L t: 1 az ~ 1 y t r U 2 ~ 1"A c3 ~
::~'~C arid rhel t:ie sc~~venc caas remo~.>ed by> a;~b1_~mar-_on ~l. ' O . l75 lFzi .
:;3m: ~ t.~ressure o.
=, s~'i~ir~, f .1_e-perm foam with a :,'~JOn~ Blast ic:ity ',ryas ~~,~:.::«i:lec? _ '''?~e : oa~c~ was used ~,~it.hout: iuy~thrr - , :e..):i.... ~~ i_c=t1C>?1 : ;?I.' (:'i7l.G'1.!.'~:clt' C~Il ~y' ~.~=' ~L.1S .
~~t-lE: _'Ilcit.2'.? X 1S
'.E-'~ill:.E?(: iI: . ?.C;. ~. Tu'. :lctS <1 l.lr:li.OrlT;, flI:t7l~,' :'i.rl7Ct.l_:~~'i7 ::-~a -dare. TJo uist:c~rt i.C?ns are dE~tectable, and t~he matrix ~;1,~.,~:.a; a nra..f~.w-n f r.t~-p~~re struwt,.~r~: thrayhout .
Example 4 : Pr~W.z~.~~:ion 0L a pcro~as c.:hi tosan mat r i. x i~_~ruxit<_=:r:_c~r~: ~~~~~tate! i 1st cmbcc.(irner~tl ;z :vf chitc~san ,rmi e.1 g of acetic acid ('.~00~) wNr~~
,-.-s~~l:~.~:F»~ x:y s:-.irring in 9."9 ~ of daiorl~.z:~d water, a:m~
t~tuc, t>H v:~ras adj as Beer to ~i ',»i t2n ;L M socii um l:ydroY i _ic=
~-c~'.ut.ec,m ~';i~- ~lt..~;~r solr.lticn was, ti~'stl;T :-xo~en ~r __ '= '~C'. r.rW them the solvE~nt was ~~emoved lay sublimat :.c>:
i~:n;?i- r a Fr'c~s~ u~ a 'w:~ O . U'~ hFa .
.->~ =:rr5a.s~e , f:irm_.pore fow was obtained.
ExamQle 5: ~roduCtiorl of a pc;rc~us chitcosan rnat:: ~x r.~,.~..mter ~ c.n : priwcp~rnte >
~:i . . cn at cli t ~~ pan and CI . G37 rnl of ortho~phosphoric a'~ i d :e 't~ iJ.:l1":W >-' tj5~ (.~ ) '.~JErE' Ql=rsCil :jE.~-'C~ b~! S G.1T'L'1 T1C~ iii .;,i' ~r~?ior:i.~:ac.? ~f;at_E=::~ , ~~nd thF~ pH w~~s a~:~yu stud t:0 7 w ;.
trl ~c~~ _;ociit;sr; ry~~r_c~x-i.cie soluti,-.vn. ~'.'ie ciN;~r soliirien was c :-;,..: ::rozer: ~.;t ;~4:;~. ~z;ld then tam so'.~vent: v:Us ..~~rl;~',,w;u: ~7~; ~:ubl~,matiOn tender n plE:~~s~rE: :2: ~..~.0'p h:='a.
.; c.
a '.vwi tP, fine-pore foam with a spongy e~i.ast~.city was ~:}i->t ~~inec~. 'T tie foam was used without furt.t~~-r :.;uri.ticw~:i.~~m '-<~r c:oioriization by cells.
':Ot.-tn' 0~ ~IF;R 1E': lei F_1; 51~ .'3f1;( 9.E(i:~ ~ 02386586 2002-10-02E
CH~1RPEITiEF ,a;o)i Z c.
'F:zr~~.e 6: e'rc~dt:(_tioru ~?f :~ yorc:ws ci'_ztr~<~;an :r:~t~ 1., ~~':'~~z:m::~'r-im~ : c~Iut~.rnat~ ) . ~ p .-ot ru...~:u~:~i.n q ~f r~~.:.t.ai:l:racid (~t5-t~;:!aa arll: c_'~,'i '.-~ ''<.(;~.)-r;i~rt:~ c3zasvlved ~t.irrirv,,a iC1.00 cT
by _~_' ;y 'aF.v;~oaa:.~-c..;;~ ar, -i::cr---tm-wand d:~~juter.;.
p;-i '~,~ , ~~;ith iM
.::;~c.-_,:rrl.'i_,r~:r~:vz.ide soitrt:i<:r:r.~::m ~vE~a solution wa . -:-< ~t--r: pit ?=~~~ -, ar~~d ~.'se:r vr~.~ sv ~
vcnr '.~:a:~
_ _ ~:~'u .. ~-~~'y., ~.....-:;.1 d ~.. ~ C ~, J . iJ r, l-!:=w,< L::Ir L..1 i7i t i.:tiC~e2.:1.:.:'t: ,f'Z,~~<a <:> i~ .
.. m~~'112-E', i_ . yprC: ~C7dIT1 ~'lt~V1=rC~ ~'a ~p;;:~lCT'y' r-'12.~'.St;.'!.C1'V N~i1 i::' s: ~.i .l. : :' f=~ ( ~ .
E:~i ~TCiy ~ t3 'I
. - ~; : t ~ ~~:i. ~~<,s.~:n arn:l 0 . 1 1:13 ~ :~j La<-:t.i c ,-:ICid ~war:
'?:i: E-,1:<=;i 1-w.T stzrring ~-1~ ~~.-7~a g r:f dE :-7n:.;r=c~ v..~::rtF=x, ,::l~:
',I-tF- :''r-::i' Sv.lli:l0ii y'd~ j'.it.Ni~~.'~~ t[-VZ~Tl ,:t-: ~-1'''(-:.
..'iiE
~- cj"~,; it::-i ~"~ ':~,'v s t i~il rF':T10 ~ h'C~ ~>_v' =~ LlrJ 1. llllc~ t 1 O7T'. lili'~E'r' ;:t cJ
_W E->~~ .laa-~ U i_ (J . t:. f~l't~rs . ~I'llEv .'_~~70:1c~'v' :.~ E~ = ;
~~A[' W? r ~ic':;~=i t. 1. E=a fl , 'v:~l.: lt;T~. ~1~'C1C0%WLC~E~ :=.O~11t1'_7I1 ~ tO-~'~',-, .C'1' WE::1.CJ}lt: ) ~'.>TY .::-,!
a :", .,.. .., '11::~ ,; h'ce7~ Cvc,S~-:!.-'C~ s~: 1 t;h ~- W 1 C~I~ 1. 'E~s;t '::d t r::'T: tli2:.
.t?1.i =<-r~ . _J: y::t'~~ ?'e'~;~ 1~:yG .LZ1 ail Clcrt~t-:u t':l;lve f'0, Tn.
~.;.r... :4 :<m>i.v!:c~ci .r1 :':ica. ....
.. C.'~.~rrrq~.t3xa~.ive r~xampls 1:
:j s:?'~~ C_:-I ~:. L,..7Lidii c~ll.C~ v. . l~7=~i ifl C'r ,.. i-t-.~-:IL~~~!i:_)S~ri~.':~Y-.1.C: c:l: :r~
:,;~-::r . ~ ii s~.'~ i ;E, c3 h,' ;t. it r -i:rg ir: 7 . C;tJ ~r c~f <-L~ion~rF~~u _,-~~~, ~:;~ ,..t_:c~ cl~a~ ~~7~,lt::ior. ~.~:c,s ~:i.r_~,r.ly Wc:=en =!
T~Ir~olvrert ~r~us t:rlcan r~~r:~o~:FC? ir,: sui,i.rma+.':;u ,...., ..c=; t~1'a _ .:t~:;C E: Ofi: ~~ . 'a~ il'cl . 'I'f1E ~~Ori~:i~' .C'c?:
1 :'~ut? ~r;rt:.
_.~ :. s- L!-''~-'. l.ri .'iGC7.i.Lil'.l ,VC~L"GXlf~f-'_ St:?''_Llt ~ OI"1 'f~;?"' _~ ~ ~iC?i,.lL"S a::;~.1;_~
. :,r' , ':u;~ :!':i-C~ ',~li..t1 ~..LrlC7:Wa:e:C~ YJ<~te.':" ~.li'7;:11. t.
"r?.':rcil . ~~Wy.t;a rr':_;t.,C.,.:~'~ :2l1 a :alrli:>:lE:: 'r,>fl.LCe ~W~TT1. !'I:E lrivL3:lr: _, '..:°c( _... ~ .2.~1.
i t; - r-~ ~L;~ '.t[ r? 1 - : 1.'s : -~ 1 ~i a~ 3 t1 i n.1 r3,'-? CA 02386586 2002-10-02 -~ r'H.~hF'FFvT I I:K r~ n;_ r~
F'.~~~rmp~e 8 : ?r ociuct i:;rl of a t~ort:~us cF-l:i t.o~;<:n rnat:. i.x ..
t;1 ,, _.'r-1 ~ 1f E:~ ~ ' t E. I '~. :-~; t i C_' ': ~ 3. Llllll O ~ i i '1 -:~
c'~ t. 1. O i 1 e: ) ~ A~ - C~ Z 11 t~ c'. I ('~. ~.
-: C' 1 ':i i n:~ . '1a c~f chi t c:~:~ar: '.~~e-ra pLs~ i.n i ~! ml of v.'t;e.r and the i>ii v..<~~. ~:.,~;u~tNci t:v . .,ith 1 N! hlydc:och:Lo:ric aciu, and r!z,v ,~.~
n.i:r:;-nrE~ ~~r~ shirred _~t~~ 2~ 1'.ours. l.~e r~F-i was ad-jjust:~:c:
. . . ~,:,: ;,-i~:li-_-tg ~ F~a 'slaOH arc= atom, ~.l'.i if= :~tmrr:z:c~, t ~~ ~.x'~ t:. t.a:.rLi r ;.~c. i c3 :nd ':~? . 0 ~ :~ r~~ '1 -~~t-hy_'~
~. ~~ ."~ ~.t-.~.,'1~_. ,_.top~r~~pyYl ) Carbo<-W i.uir'.e L F:DF.i ) t::.~e aci;iwc~
;:r; . <_ii.~~"c:=~v~-d. ::..E_~T ll~-~ ing bee-.~. st.~ r:-E~;j a~: rw;-:."n, - <a,_:o-., t~ r E.: f;::>x a 'L= _ hE.r ;s ?loL r~> , tJm ~rf'.va~ ad ;
Li~;t, ;=~:;i.
..... . .,".t''1 1. :T T'fc'.~u~, .-~il':C~ t.:t'.r~
~ir~~'~'1~71t..~2::~'.W~'.~ ~_L1'-;~~_. ..., ~_taTij ::d_;_riE~,l iii::ii .~ Z pt jP~.::I-il'<c.'Cl ~n'c3t:~Y. TIZE
in'd;:sl'ee~:7 j:.: ~~ E=' C' .1 rJ Z ~ ci t-'? :'.~ < : ~ E;.', y ~' ~; L a 1 V E=C~ W J t.
I1 i~ . ~_ ~l ~ -~ C) l: f 1 cz L' r 1 C c1 C ~ _ 1.
~.~;; _" _, ~t:f7 , :w~~ra:-r -~-:a=r.3 ~rorn Ni.~x~~k ~"~a~:, i>armr;~~ac~t) ._ . , e::. ~ r: ?.~i;-r g G.: svcarE'I-. '~~Y." j~H ';vc3~. ud~llS~E'_~ t. C' . it:rL .i:~ ~~od; um i~~-dx°oxide solutic~rl, ana t_ILe "o-Luvi::>,'L
v~.'.~is:: l: t:C>'<'i'i: i-tt: <~~ "l_.'. 'I'~7E' 473CF~r 'fJciS Crue'T1 I'[:r'CLOV~CZ t:y' ~;u.;:: i .;rmt:i ~:~n :L::;E=r a pressur« -of t~ . G'-; ~;pu .
.- ':,t: i.t:.::'. '."F>:>!:~~' ?~E~~-~:L ~liCV:t~~ E.)}7i c:; ,t.:~~~ . 1'rlE
S~:UilEaE-~_' C-'.3,x1 '..'?t~
. _ ~ '... ,=tlOLit: '_lI~ l~t-lE-x C.iLi~ L.'.1C<xv.~G:A.
?'. ~xr2tttpl~ 9: Ps°~::clt~t'tloZ2 of ~ ~.Y:~ec--dimel:~io;tal matr..
r'1. . _'1 ='L'T.7i:: ~ ~ -. .E=-~ ~:E.' 1 j c~C'.!'1E='~:1 brl f:~C'r ~.~:>
..m_ ~ZC]-1_.Wil~J- v E~ c E:~"'11 i.C~~<?i'1 '~'JE:I:F- ~L;t 1:1 ~.!I ITl'~. Ua'. W::~~~r i:Ili~
t:riC-_' .t -L ..., ' ;~ ~ y ,:: ~.
?i' ~.'.rs; ._. -iu::t.~cf ,_~. 3 t~~itn 7. r~: h';.'ts:>c _~ _- acLd.
__ : , . !.,!~J ~_'-t a°. '._~Jc~T'."v ~ -STI~.)i.-rtl t :1 C E ~~: W C
.<. ~ ~10i._ '_': _ , '. .'7E-? ~~..i. E:.; Y
_ ..~._ i;.._,. .~:a:> -.,a~~.l~t: ~°t-.c~ ~~i~ . . ~ w~ r.~z .. T~
'~lriCj~I ~:n~:~ t.rl~::~:.
_ G _u L -'.1"~- i_--s~.l'Tr- 1.-Fi,S~I c3I-t_t . . V~~' ~( Wf ~.i -E~t~l'y'~
_. ;.~1,,~ ~ ~.;- .~ ~::t i_r:~~L~: :~F~s-1 ) c:a;r bor ~i in-,i,:ie --I~-:.r~
) we :-E
..s'..1 "~:....f. : :.~ (r V' clC:~~lEi'~ ,3T'1~ C~1S~.1J1'.'tW .1 X''y' .~'~
l i. T :LIlc:X _ r~t~'~'~~_ '~ (:CT 1)::=:f~f1 ~>u. ~'1.I-T E:C~ at'. .i'~ 01I1 tE~lrLyE=I~~,L:LI~ E' cir c~llZ-2.r7~_'"
.. , ., ._ , C. r:E:S ~)f'~ v:~-'~.'. dC3,j17CL'.E?o t_~> ~~ v.'Lr_.rt 1 iJ
E.SeaiJi~. ~:'flo_~
. . t ~-;t.~-::<.~'.; 1i : ;.:L:red of r ~rLd ~,a-h~d N>ir ri J. ~,f .,,'clt e-.'T . :'_1~ '.J'rf~~':=~.>l.c?ii~ Uld.<-r' t=C1E~T~ C~1.~'SO~Y.TeC~
'rJlr~: ,1. Z..L1-~ CI
af3 t _, I.',', ',IE'It l" .[~'l j-yl.S. .I-~ :''~1'I ~.~Ei:; ~ 02386586 2002-10-02 F: ' H(.~NF'E~I'iEv;ft ,'lt:r;,';
.... y_'j _.
1-::=C'(_:LC(~1~0 1I'1 4:c1tt~2:, purchase:G~
,:3L1<.~ fI:Cll', MW2C.~
P;~:~aA. Parratadt:'tby stirring in 9 . "~ ~ water, the ~H
ez ., ;_:~_:ja:W:~~i~rit1 1 N sC)cliL:rn hydrG:~,:i~~t? .~:i, !_c, so!tlti <:arlc7t-..m~ soiut:ion Fir~~ily, ,.i~t::
vans ~.rr~<.i:n <~t; --2~''C.
~:cit~~.r= ',.ras ed by SLlh:i_iinat.lOn a ~~r~ssure~~f T?P1O-.~ umtPr Ci .. h;_'~
~:.~
~"-: ~,'hi~.ct ;,~onc~z.,~ r~siCIue v,~~,tc; ,).~t:ni:ie~:! . e'l'm spc.ncTe canl k7e "-:°'i r:i.'.'i.~.~mt ~t:_'_rie.r ,~~;!ri tio,~.t:_ i-;''Ii.
~,,af,axn~~le' 10: GenE,ral io; ~::~ec:~L.lr_~= yr)I° ~~eer-:.nr <_.I::vi ~icur~~~t:.,:ig ;.m j M ;~~r-1 tl-~.-_~_ t..r,rtE,~ci~mc:ns;.onal matr~i~:
'.t:~ec~ _,jlrner-tvic!ma L ma t: r_ iX csbi:aaned ~s in E:~can-y:~les 1 I'_:. ~:, s:- au~-or~wed in PAS alt 12i°'~ ~:or ~u r~iru~_es. ~:'1-:c' i:~~<~"'.'.-~L.'~:. ~._ T:t-LE',I: at:<?r;..:~ i~llU ['OIYtIJ-LE:?tE-'1'y LVE~Y~EC~ wl;,.I:
,! :. ~ :l 1 a Ol . ~. t? ~ 1. .~', 'AVl a. C~ ('1 ~1 c-3 V f? Ci~ ~ ~ I-2 C
L.1 1 t t1 I' f' C1 1 I~! ~ S ~a .!. t 'c, r~ '1 r~
r7z' :.;z~a_ lrcdi~.Jna arm then pu~ ~ir~:c~t-:i-r~ ~~r.t:o a :nar_~ i:~
's~7al.:.i_:J tlwa'.: ~.'?E?c'':i ~.;Lltr dp~.7X(?t:)'Y:.edt_.Et .:OZI'E'<jLll.Y~?TiEl'1'CS cx:~~.':
._ . 1 ? : ~ ~ : ~.: %.: ~:: :'t:i w1 r t'_ r..'=- := Ll 1 ~ __.,J ~.L t', t=
':='_Tl~) C': Y'7a W 1 h 4 . :.' l-: F= Iflcl'-. 7- 1 :';. t:7 7 ':' C].
;;;,w..=t': .:~-m i)~~ --erlct~red wn~.~er s'er~le condo r.:_:ans anr~, r ~:...
tt,'~.c_!r.! ai f:e, :-:rt~el c;f".t ~u .
~'.~c::.sa~~e 11: t::ultL.J-: z.r~<f c;f 'vlz~~::d~~~'~,.'',~.~ ,)1a cf.~ th:r t-~>--i..: r;:..:::~ z corm J;:a t :: .i r .; i ;
,:'.,v _~":p:J~x- l,~i;.~~ndrocyt:.e;= cbt~:ineU :.~y ~~io:~sy :~.c a°e:' ;.i~l:e=l,"j ~.rf: <.:~rl~;7IZt;';.C-;.iC'}r.t:,C ip~,j'~J~:;1~1 f;'l~Ol.'~_;]T', !C'E.~.~1 ..~
:~.F-1J, ~ i.l~r~:»C~r-:i .~.I~C: . , r_~;o-_ i =: ':-1.r-E?C?--C~7YrlOIiSiG:lC1 I:lBti1?: X:~IOCiLIC-4~C~ c~:3 2I1 ~'.?:cxll:T~.~t' .~r ~"'i _i:~i:~.-.u.; i~' c~.'.;tC)CZdVt3C:~ fir. 1..~~~:i~~ =Or l~l :i~l:'alf~E'S.
'~:JE:
c."'.>f:i ~_':y;.E~.~srJPt:.t.t?C: IlldrY_1?: W..l:j ..Lit.
cw~7TJr'C>~:~;"ieit0'.:.~' .lri.<~ ',.
~s'LieT -.~ E~ C'OI1C1? L.1C)I-:S ct17C1 ~Llt J Il e~ C:,lll t=L11 ~: VE'-~~>~~ . mfli.:
t~ mn'. r,J~F-~,~z crlonc:irocytes witYl the uult.,.~r~~ m~.~r~ium wEJ,rce rrA~-pro ~:>ut uIJ ::':e ~t:~eE~ dimension<<1 Irlat=-i:k_ and incubat~c? x r..
'r' _ ~.>.r '_ t-o a1 da~rs . J~uming tiW s permd, tt:e ce1 1 ~;
~.:'!j'u:, LI:i'i7 t~t~::' ~:,tlrE~t~' _C ~:lIE.'Xl;.i? cf.cil t;ieli.:'1:~~.
:Il':Ir:rxt.I-i.X 's:~".il :arc~~~::-,~1 was YC.-'moved urFcW:Y' :~terilE? c:ondit.iJra and c~.ar:
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Claims (9)
1. A method for producing a biocompatible three-dimensional matrix, where an aqueous solution is prepared from chitosan and an excess of an acid, the aqueous solution is neutralized, the neutralized aqueous solution is frozen, and the water is removed by sublimation under reduced pressure.
2. A method for producing a biocompatible three-dimensional matrix, where an aqueous solution is prepared from chitosan and an excess of an acid which is selected from the group formed by alkyl and aryl hydroxy carboxylic acids, the aqueous solution is frozen and the water is removed by sublimation under reduced pressure, with excess acid being removed before or after the removal by sublimation.
3. A method as claimed in claim 1 or 2, where the acid is lactic acid.
4. A method as claimed in any of the preceding claims, where the acid is removed or neutralized by adjusting the pH of the aqueous solution by adding a base to from 5.0 to 7.5, preferably 5.5 to 7.0, in particular 6.0 to 7Ø
5. A three-dimensional matrix obtainable by a method as claimed in any of claims 1 to 4.
6. A three-dimensional matrix as claimed in claim 5, where adhesion factors, hormones and/or growth factors are bound to the chitosan framework.
7. A three-dimensional matrix as claimed in claim ~, where the matrix is coated with autologous fibrin.
8. the use of the three-dimensional matrix as claimed in any of claims 5 to 7 as solid phase in a culture reactor.
9. The use of the three-dimensional matrix as claimed in any of claims 5 to 7 as matrix for implants.
An implant comprising a three-dimensional matrix as claimed in any of claims 5 to 7.
An implant comprising a three-dimensional matrix as claimed in any of claims 5 to 7.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19948120A DE19948120C2 (en) | 1999-10-06 | 1999-10-06 | 3D matrix for the production of cell transplants |
| DE19948120.2 | 1999-10-06 | ||
| PCT/EP2000/009809 WO2001025321A1 (en) | 1999-10-06 | 2000-10-06 | 3d matrix for producing cell transplants |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2386586A1 true CA2386586A1 (en) | 2001-04-12 |
Family
ID=7924675
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002386586A Abandoned CA2386586A1 (en) | 1999-10-06 | 2000-10-06 | 3d matrix for producing cell transplants |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP1232203B1 (en) |
| JP (1) | JP3471781B2 (en) |
| AT (1) | ATE271579T1 (en) |
| AU (1) | AU759066B2 (en) |
| CA (1) | CA2386586A1 (en) |
| DE (2) | DE19948120C2 (en) |
| IL (1) | IL148853A0 (en) |
| WO (1) | WO2001025321A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100400112C (en) * | 2002-08-02 | 2008-07-09 | 久保木芳德 | Medical material made of titanium fiber |
| WO2015123779A1 (en) * | 2014-02-20 | 2015-08-27 | Polyvalor, Limited Partnership | Lyophilized polymer scaffold compositions, processes for preparation and use in anabolic wound repair |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10117234A1 (en) * | 2001-04-06 | 2002-10-10 | Alvito Biotechnologie Gmbh | Porous and non-porous matrices based on chitosan and hydroxycarboxylic acids |
| JP4859315B2 (en) * | 2001-09-26 | 2012-01-25 | 京セラ株式会社 | Articular cartilage defect repair material and joint defect repair member using the same |
| CA2504625A1 (en) | 2001-10-30 | 2003-05-08 | Alvito Biotechnologie Gmbh | Use of chitosan materials |
| DE20215659U1 (en) | 2002-10-11 | 2004-02-19 | Alvito Biotechnologie Gmbh | Reagent kit for culturing cells |
| DE10317578A1 (en) * | 2003-04-16 | 2004-10-28 | Alvito Biotechnologie Gmbh | Using material based on chitosan and an acid in orthopedics, particularly as replacement for periosteum, also as matrix for growing or encapsulating cells |
| DE102004047115B9 (en) * | 2004-09-27 | 2007-10-31 | Universität Hamburg | Method for producing a wound dressing |
| US8993540B2 (en) | 2009-03-16 | 2015-03-31 | University Of Memphis Research Foundation | Compositions and methods for delivering an agent to a wound |
| FR2994185B1 (en) * | 2012-08-02 | 2015-07-31 | Sofradim Production | PROCESS FOR THE PREPARATION OF A POROUS CHITOSAN LAYER |
| US9662400B2 (en) | 2013-03-14 | 2017-05-30 | The University Of Memphis Research Foundation | Methods for producing a biodegradable chitosan composition and uses thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02207785A (en) * | 1989-02-08 | 1990-08-17 | Asahi Chem Ind Co Ltd | Porous carrier for cell culture |
| JPH02261838A (en) * | 1989-04-03 | 1990-10-24 | Sumitomo Cement Co Ltd | Preparation of porous chitosan material |
| JP2571887B2 (en) * | 1991-11-27 | 1997-01-16 | リグナイト株式会社 | Method for producing chitosan porous body |
| JP4472804B2 (en) * | 1999-05-18 | 2010-06-02 | 学校法人松本歯科大学 | Bone filler and method for producing the same |
-
1999
- 1999-10-06 DE DE19948120A patent/DE19948120C2/en not_active Expired - Fee Related
-
2000
- 2000-10-06 IL IL14885300A patent/IL148853A0/en unknown
- 2000-10-06 AU AU77861/00A patent/AU759066B2/en not_active Ceased
- 2000-10-06 JP JP2001528483A patent/JP3471781B2/en not_active Expired - Fee Related
- 2000-10-06 EP EP00967842A patent/EP1232203B1/en not_active Expired - Lifetime
- 2000-10-06 WO PCT/EP2000/009809 patent/WO2001025321A1/en active IP Right Grant
- 2000-10-06 AT AT00967842T patent/ATE271579T1/en not_active IP Right Cessation
- 2000-10-06 DE DE50007153T patent/DE50007153D1/en not_active Expired - Fee Related
- 2000-10-06 CA CA002386586A patent/CA2386586A1/en not_active Abandoned
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100400112C (en) * | 2002-08-02 | 2008-07-09 | 久保木芳德 | Medical material made of titanium fiber |
| WO2015123779A1 (en) * | 2014-02-20 | 2015-08-27 | Polyvalor, Limited Partnership | Lyophilized polymer scaffold compositions, processes for preparation and use in anabolic wound repair |
| US11844874B2 (en) | 2014-02-20 | 2023-12-19 | Ortho Regenerative Technologies Inc. | Lyophilized polymer scaffold compositions, processes for preparation and use in anabolic wound repair |
Also Published As
| Publication number | Publication date |
|---|---|
| IL148853A0 (en) | 2002-09-12 |
| DE50007153D1 (en) | 2004-08-26 |
| WO2001025321A1 (en) | 2001-04-12 |
| JP3471781B2 (en) | 2003-12-02 |
| DE19948120C2 (en) | 2001-11-08 |
| EP1232203A1 (en) | 2002-08-21 |
| EP1232203B1 (en) | 2004-07-21 |
| JP2003511120A (en) | 2003-03-25 |
| ATE271579T1 (en) | 2004-08-15 |
| AU7786100A (en) | 2001-05-10 |
| AU759066B2 (en) | 2003-04-03 |
| DE19948120A1 (en) | 2001-04-19 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |