CA2386586A1 - 3d matrix for producing cell transplants - Google Patents

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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'_'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.
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.~,_'~~:~::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;
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_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~ .

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'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 .

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~~'1I'F:t~_ i_77_IT.:=-I7:-,1 C~IicZ1 CtW t-ClScill :Cvcit.YlC~
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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.