CA2195867A1

CA2195867A1 - Membrane for regenerating body tissues and use of said membrane as a wound covering and substitute skin

Info

Publication number: CA2195867A1
Application number: CA 2195867
Authority: CA
Inventors: Gunter Bertholdt
Original assignee: Individual
Current assignee: WL Gore and Associates GmbH
Priority date: 1994-07-25
Filing date: 1995-07-20
Publication date: 1996-02-08
Also published as: EP0772425A1; WO1996003094A1; DE4426315C1; AU3162895A

Abstract

The invention concerns a membrane for regenerating body tissues which consists of a biocompatible material and is characterized by the combination of the following features: the membrane thickness ranges from 0.5 to 500 µm; it is permeable to gases and macromolecules but impermeable to cells; and it has a microstructure in the form of peaks (16) and/or troughs (14).

Description

22 JA?l.I. '9'7 15:25 KLJI~IKER SCHl'llITT-NILSON HIRSC~ E #:377g 5.3Z44 (~ , PC)LYGLOT I.~TERNATIC)NAL f~ ~ q ~ P~ 6 7 Glo~ g _~r~: 0 ~40 Br~Lnn~n St~c~, F~ ;laor S~n Fr~nci~co, ~A 9410~ ~ USA
~el ~41 5~ 5 12.880 P~X ~4 1 5~ 5 IZ-899 TRA~SLAl'IO~ FROM CE~MA~

WO ~6!03094 PCTJEP95/02g6g ~embra~e for ~egenerati~n of ~3ody Tissues ~d ~se of the ~embrane as ~ Wound Coverin~ and Skin Replacement rne inventior~ concems a rnembran~ for r~e:leration of bcdy tissues, ~ method for it~
production, .ts use f~r co-cultivatian of cells, its use as a~ifi~i~ sk~n and its use 3s p~rt ~I a wound plas~er.
The c~lter ~ellular layer o~ human skir, ~epi~erm~s~ is separated ~om the Lnder!ying cellul r layer (~ermis) by a ~asal rnembrane. ~i5 ba~al mcmbranc, which .s ~harz~tcnzed by a special lln~ atin~ stmcture (re~e crests), consists of so-cailed ECM prote~ns (extracc!lular matrix), some cf wh~ch are synthesize~ by epidem~s cells ~;eratinoc~tes) and some by derTnis ce!ls (fibroblasts). Th~ b~sal membrar.e, on the one hand, rc~ese.~ts a cellul~ b~rr~er, but, on thc other h~, pcrmits ga~ and material excha~e between the two cell layers.
~ he bas~ cells produce the cell3 of the overlying 3ayers b~ cell ~ivision and it is ~ssumed ~at ~he ~tructure of the sicin sur~ace is also det~ in~d by the ~tructure ot the rete crest~.
I~unr~ e-a~ea i~junes to the s~; in which ~.e bR~~l mcmbrane is destroyct in ~ddidon to the epidermls ~his stn~chlre can rlo longer be reforrned wi~ut ài~ficulty. In ~act, . .

22 ~JAN. ' 97 15-26 I~LIJNKER SCHMITT--~ILSON HIR~Ch ~E tt37~9 S. 4~
21 95~67 kera~inocytes can repcpulate the sWn s~ace starting from the wour~t cdges and the so-s~lled skin ~ppendzges (for ex~mple, h~irs sweat glands). However~ rhe rete crests, in particu~ar, for the rnost p~ are no longer fo~Tned sr only inadequately so. The newly fon~ed tissue is morphoiogically and f~GtiunaLly distinct for tlorm~l s~in and i~ referred to as scar tiss~e.
If deep~r $kin l~yers ~ro al~o dQm~ed, ~r ~xampE~ hird-c!egree b~ the kerarinocy~es of t~ke ski~ ~ppendages are des~oyed wi~ the~n, e~ cn orderl~ sczr formation is no longer possi~le. This means tha~ wcLmd healing is h~..pered ~nd is mostly connected w~th defomlations, !ike one-sited wntr~ctions and proliferation.
Previous tr~2t~ents havo essentially pursued the goal ~f prote~ting the wourld from i.~ection &nd fi~he~ darna~o by ~ech~cal ~ndlor ~:hrmit~ for examplel sal~es, gels~
covenrlg and pos~biy creatin~ a favorable climat for ~ow~d hea!in~ er~ are ~ifferent ~pirL~on~ conccm~ng the best ~n~itio~ for wound he211ng, one schooi ~a~oring dry wound h~aiirlg, anothcr ~noi~t wour~d heal~ng~ :30~h schools shar~ the vIew that na~ral wound healin~ should be allowed ta occur as undistur~ed as possible.
Transplarltation Of autologous skin now offers the best res~ll~ for tre~ nt ~f deep~
lar~e-~ea wounds. For thi~ purpose thc ski~ suppliod for su~.ace enl~r~ement by a specL~I
pracess ~y mea;ls of a number ~f sm~ll cuts in ~ me~h-lik~ eonfiguration (m~sh ~ra~t).
In partieula~ly severe c~cei ~,vhen 8Q 20 Y0% of the body sllrface is dar.na~ed (for example, in ce~tain ~ suf~lcient ~da~na~ed tissue i~ not availa~le for autolo~ous tr~nsplarltation. ~n these cases it is possible b~Ced on the pior.eering work of Green a~d Rheint~vald (Celr, ~, 3~1-334, 197'~) to reproduce in vitro ke~aIi~ocytes re~ e~ed ~om a b~opsy, culnL~e them ih t~.e ~orm of thin coll sheets and findlly tr3~iplant them in the patient.
To i~npr~vc m~rhpnic~l s~bility and mar.ageability of the cell layers (sheets) ~hese wcrc culnlred ~rl sponge~ e m~ices. Il~e m~t~ices generally coll~ist c~colla~en arld~or hyaluron~c actd Or polyl~ctide andfor polyelycolido. R~sorba~le and non-resorbable rneshes, as well ~ felts made of polyiac~ld~ an~or polvglycoli~e h~ve also been used. Although ~hese sponge like m~trices e~joy the adva,~tage of hig~er mecha~ical stability relative to simp~c sheets, ~he .~gheF content of Ioreign matenal, as well as the incr~ased ~ime demand neces~ fi4r c~ltunn~ ~nth cel~s, ~rc dr~wb~ck~.

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2~ JAN. ' g7 1!~:2~ ~LUIYKFI~ SCHMI-rT-hlI.~O~ HIRSCH DE p'~770 5, 5je~4 ~t q5~i7 ~ 1 impl~ntatio~1 system havin~ ~s essenti~l co~nponen~ a PTFE ~nembrane ur,th sp~ified pore di~t~r alld specifie~ thicknes3 in whirl~. the cel]s c~pable of fo~ning cer~
~iological p~oducts ~e c ~ rçd i5 knqw~. from WCJ ~3~1~700. The syste.rn is n~t us~d fa~
rc~çne~tion of skin.
A m.ern~r~ t ~a~ bc ~L~ed ~ ~ r~pl~ccment i5 alrcady knowr. frar~ ~P o 46 4~5. ~he me~r.bra~e cons s~s of a ~ioeump~lible maten~l an~ is perfora~ed. I~e iloles a2e ~e~u~r'y a~Tar.~ed ~nd haYe a diarneter be~veen 10 and 1000 ,um. Hale pe~ra~io~ is suppo~e~ to suppolt gr~wth of t~ membrane ~.d skin re~eneration ~Iioro~olor~es of im~l~~te~ autolo~ou~ or ~.e~erologous '~esatin~c~ftcs ~re formed ~ the h~le region~. The membrane is stiil ~r.der clirLical testin~.
r;derlylng ~ask of thE in ~ntion is tc devis~ ~ mem~r~.e s~t supports rccOnstruCtinn of li~ ing bio~gical tis~ue and especislly regeneration of ~ody tissu~ ~nd an skir., ac w~ll as ~o of~r 1 process for prodsletion oi: the ~nem~rsne.
This task is sol~red accor~ing t~ the invcntion by a metnbrane having ~.e fea~re~ of the eh~sacteri~ng p3rt of Cla~rn 1 and producible ~sccording to the p~oce~s of C'aim 27.
The ,n~ention is e~sent~ally based on the firldin~ shat the basal membrane w~th wel fa~ed rete crese~ f d~cssive ~ t~ ce for fi~cti~nslity of the ~kin. It ~ proco~d~
from ~.e fact th~t ~,o~.al cell ~n~ EClvl ~trueturcs form wL.en ~ppropnate st~ting c~n~itio~s z~e ere~;ed fur this ~hese ~on~itio~s include ~ot onl~l- the fac~ L~iat epidermal ~11 laye~s corne in con~a~ h de~21 iayers, b~ll also L~iat a Ce~a~n thr~c-dime~stonal structure must addition~lly be pr~e~t that ~reates re~ions ~ith di~çrent condit~ans, fo~ e~ample, ir~ te~ms of o~yEen sll~ply ar ~oc~l eoncen~a~icn of cer.~;n signa) mol~cuies. Only b-~ these condi~ions is a cascade ~f e~ents leading to com~l~te tissue re~eneraticn ~i~ge~ed.
In its ~onn a~ld fill~clion ~ membr~e ~cc~rding ~o the inve~.tiorl represent~ anequlYalent of ~Se b~s~ m~msbrarle of human skin. ~f it corLsists of resorb~bie mate~.al, il ~an f~.ction ~s "pl~ce~ho1~r ' of the basal membrane of hu~ ski~ and ~s~rr.e its r~t~on ~ntil the ~oll have consauct~d their o~ ~nctional structures.

~2 JAI~ 7 15: :27 KLI.JNI~E~ ;~CHh~ITT--NILSOI~ HIRS~!-I ~E ~7~9 S. 6~44 21 95~67 lhe speci~ adva~tag~ of the inYention is that the decisivc s~rting conditions for natura~ ,egc~c,~ti~n of a complex tissue can be ~rcated in a cell cultu~e or in the body with a mi~ nount of foreign matenal and time.
Ille mcmbrane pesm ts s~ucrured adhesion of macromolecules, ~hich promot~
~dhesion or ncn~d~esion of ~pecifi~ cell ty~es o~ triggcr o~he~ cell reactsons. 1~ is 2~s~
possible to isol~e specific cellq by cell sorting ~nd disL~ ute them teiiberately in th~
recesses oF the ~embrane in a specified ratio. Since t~c ba~ cm~rane is present ;~ many other ~ody tissues, the membra~e according to ~e inver.tion is also suitable ~r re~enerAti~n Gf thi~ tissu~.
~ he rnemhr~nP according t~ the invention rc~ t~ an llppro~ matc m~rphological image in the b~al men~.brane of h~an skin. The in~rinsiGally sf.a~lc mierost~ucn~re is preferentially ch~ucL~ e~ by pro~ cio~;s and recesses ~istributed in a rEgula~ pattem ~hat can be descnbed, for cxam~le, by the superposition of hYo orL~.ogonal si~e wavcs. One coul~
then i~ken the micros~c~e of ~hc membrane in principle to t~e sh~pc of ~n e~g c~ton, which also poCce~ses r0si~ance to shear forces rhe transiuons be~wee~ thc p¢o~usionE 3nd recesses ~re p~eferably s~;ooth and eontinuous ar d formet w~out ed~cs. ~Iowevcr, in many appli~tiorls it can ~8 su~cient if oruy ~rotru~ions ~r ~nly revesses are prescribed. A
co~tin~lous LransitiorL ~e~een the protrusiQns 3nd recesses is not ~soLutel~ essen~ial either~
but advantageous.
FiL~n~ w~-~ pro~sions for medical applicstio~ are kn~ from r TS Patent 5.18~ 4.
This ~ cture is supposed to con~ibute to th~ fact t~ut the film does not S~tp 50 readily on ~he skin b~ist~rs cov~red with them7 ~he freely accessible surf~cc being ~ub~tant~ly r~duced by the prc~usiolls ~,d friction on the ~ee side decidcdly reduced on this aecount However, t~cse f~lms are not lntanded fo~ regeneration of bcdy ~iss~es, nor ~rculd thcy be suita~le fior ~his if only be~ause ~f their relatiYeiy high dlicknl~ss of 0.~ ~o; O mm.
~ e l~r~ t;n~ ~urfac~ stn~ct~e sf the rnembrane accor~ng to ~he ~nvention perrr~its membranes ~o l~e stacke~ o~ e~ch other so that ca~lties a~e foImed~ which, Gn t~e arle hand, abso~b ~iquid~ ~y c~pill;~s~ action orl cont~ct wit~ thcm and In which, on the other hand, ce~ls carl grow uithout be~g ~ehAn~cal!~ d~ma6cd. ~lch &ITangements ha~e proven themselves 22 JAN. ~ 97 15~ LUhJKER SCH~lITT--NTLSON HIRSCI-i CE ~t3'779 S. 7~44 - 21 95~61 in con~unction wi~h wo~nd pl~sters, which might al50 ~e at~ributable to tll~ fact ~ha~ surface enlargement of ~bout 4Q% .s achie-~ed in comp~Mson ~ith a fla~ mem~rane and irnproved ~xygen supply to the sells is the~eby gu~rar~teed.
Thc r.~embrLne has ~ thichless of O.S ~o 500 ~rn, e~pecially about 2 to 20 llrn. k ger~eFally applies that, depe~iny on ~e mcterial, thc membranc shoul~ b~ de~igne~ as t}~n as possible, but rnust still remairl mana~eable.
The pore si~e of the me;nbrane pr~ferably lies below 3 ,~m~ The pores should be creatcd so ~ha~ they ~e pe;Tlleable t~ gases like oxygen and ~o ~ac~omolecules, but tL~r ea~le to cells.
The spacing of the ~ idual p~otrusion~ or rPcesses ~ro~ each other, i~l o~her word~, the ~ id pa~tern, is ~bout 50 to 503 ~um, pre~Fer~bly about 200 ,um. The magnLtude of hei~ht .d depth, i.c., the arn~ii~de of the protrusion plus re,,ess ~ies at 50 to 1000 ~m, esp~eia~ly ~etwecn 100 and 500 ~m, preferahly at ~bout l50 to 300 ~m.
~ he ~naterial of which the membr~ne consists is preferably a mater~al th~t can be ~ro3cen down by ~iYiI1~5 cells and whose degrad~tian prO~ucts have no adverse ef~ec~ on the skir. regcner~tiorl pracess~ Polyglycolide, polylactiae, copolymers of the~e ~ubsta~.ees or thei~ ~n~atiYes, polycaprol~ctoae~ polydiox~or~es, pol~pho~ph~ G~ polysulf~nes and polyuretha~es are preferred. Polyhydruxybu~yr.~ acld, collager. crossllr~ed ~vith z cr~linking age~t or hy2llh-on~c acid are ~'so suita~9e. Addstion~i ex~mples of appropriat~
~aterials a~e mentioned i~ EP 0 4~ 42~, to ~rhose ~is los~-e rc~rence is expressly made.
Jn any e~ent, ~h~ m~tenal m~st be stable until suf~cien~ ~utologous st~uc~ures are built up, i.e., preferably ab~ut 5 to l0 days.
Pro~ction of the intn~sie~ly s~ble microstructure occ~s by ordinary metho~s, An embossin~ pr~cess is preferre~ ln which e~her en~rav~ng rolls ~re used as embossing die, Qc is known, for ex~mp~e, from US Patent 3,484,835, or cG~-sj,ol~dine en~r~ving pi~tes. T~e microporoslty ca~ also be produced by kndW51 methods. A fclt-like 0! ~rous e~tmdsd mater,al ean also be uset ~ s~in~ mQterial for the membr~e. Thc Inech~nical ~nt c.hemica~ pr~pe~ies ~f ~he mem~rane car~ be preciscly adjusted by sele~ting appropnate mhnrln~ers~

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- 2~ JAh~. ' 9~ 15: ~ K.LUNKER SCHh1ITT-NI~ N ~1IRSCH DE ~3779 ~. 8f44 En~raving tcchr.ology is ordinar~ly spplied for pr~duction of ~e embossing rolls o.
pia~e5 requ~red for the ~mbossing procees. Two method~ ~re av~lablo in en~ra~
~echnolagy~ One method is res~cted prirnar,ly to me~h~uc~l ~nethods in which 3 s~uc~e i en~bossed ~3 a ~urfacc by me~s of an apprDpriately designed tip and th~s stmchL-~ serves a~
master ~or die shapir~g when it h9!i harden~ o~her method i3 lascr cngrav~ng, wh~ch mostly ffnds ~ppli~ation on rotatin~, c~ramic-co3ted ro~ls, duriI2g which a he!ic~lly scar nir g l~ser beam is mod~ ted by s~rlin~ a gr~y ton~ pa~ten~. A co~espor,dingly ~odulated ~rzvin~ tool is ~ls~ applic.qble here as mee~niçPl analog.
The f,rst n~ed rnethod producing engra~ing pl~tes or rolls that have a sFecifi~
a~angeme~t of gec~net;i~ recesse~ in the F~rm of cuboids, spheres, tru~c~e~ cone~, e~o., betw~e:l which p~ resid~l sur.~*ces co~tinue to e cist, which ~ e f~ther round~d bY
etc~g to~h~iq-le~. TG pr~t~e ~ mbrFne ac~ordine ro th~ ir,~en~ion. by me~ns of t~s engravin~ ~chnolcgy ~ su~face ~f 20 x 20 mm. of ur~ rd~r~ed steel w~s engrav~d u~h ~
graYing t~OI in the folm~ of a py~amid w~th s~uaro ~ase ~t a ~ifvr~n ed~e leng~h of ~. ' n~rn (ed~es and pe ~s rounde~ o~f) so tna~ tlse spacin~ from py.~i pe~ to p~amid peElk wa~
Q.2 T~r~ This f~ ~ then h~.-dened and sert~ed as r~cte~ fior ernbossin~ mo'ds to be c~ ~om it.
It ;3 possibie to prodLce m~lch more co~pl{~x thrce-dimensional strucr~e3 ~-.h the ~ee~r.d-n~ne~ en~r~Y,n~ technology. !aser ~ r~ving. Laser engr~ving technclo~y is th~ re preferred in conjunction w~th t},e prcs~nt ~nven~iGn beeause ~f ~he sought corr.p!ex ;3 s~ucture. The aec~lracy of ~s metho~, howcver~ is n~nall~ sharply lirnited by the q~ali~ c,~ the gray tone ~a~er~. Gray to,~e p tterns are there~r~ disperls~d ~ith aco~rdin~ :o the ~n~ention d ,nstead ~he laser is controllcd by stipu;ation of mathesrl~tic~ fi.lnctio~ so that ~ surface i~ produced in ~hc ~izc ~nd ~rangerrlerlt o~the ~. ~aT n~emb~r.c s~ruc~es, in which the tr~nsitions between prol!rusions and recesse~ are pre~e;ably smoo~h.
For thi5 purpo~e th~ laser r~uires a ~ o~respond.ng z va!~e for each coordinate of .he x-y plar.e, which ~ives the dcp~ to be peodluced so ~t, vie~e~d over the surf~ce, a relief s~, ctl:~e appro~ ting the ba~siaI membrane is ~-oduced. A n~th~m~tical appr~x:mation of the eg~ c~on shape~ b~21 mernbrsne 8tIUCtl~ can l~e ~them~ic~l~ dcse~ibed as 22 JAN '97 -15:29 KUJNKER ~H~IITT-I'J-L.;~Ot~ HIR~;CIi [~E l:t~"79 ~;,9~44 orthogonal ir.terference of two sine 03cill,~tiolls of the same ~piitudc, ~requency ~nd phaseT
fios exzmple, in ~,e Fonn;
2 - t~x, ~) = A"~sin(2n)~x/3~0 ~ A~sin(2~)"yl300, with A ~ A" + AJ = 3~a 11m for amp'it~des ~n~ w~elcnE~u ~f 300 ~m.

Sirnplified, this c~ b~ expressed by:

z~x~ y~ - A"sin~x~ t Atsin~y) or ~(x, yl = A~ccs(x~ + A~cos~y).

l~.,e obtaine~ m~tnx of ~ values ~9 t~en ~5ed ~ ~ b2~ for pulsc-frequeney mo~ul~tion of ~he laser beam ~nd t~anslatory x-y control of ~he work tabie.
l~is can ~e transferred tG rotatory laser engr~Ying ,n wnl~ h y ~ r~ ~, w~,ere r is the radiuj cf ~he roll being engraved and ~ the anguiax position.
~ Iicrorellef production by laser en~raving i3 often applied irl conjunction wi~n rotating, mosely cer~mic-ca~te~ rolls~ but can ~Is~ ~e u~e~ to n1~ coppe. ~ hard ~ tal and ~Iso in conjunction ~l~h flat piate~. rt hag ~urrled O~lt ~.at laser m~hini~g of hard met~
i5 most ~ppropri6te for mic~orclief s~ruc~L~es of the mer~bran~ accord.ng to the ir.ventior~.
By usln~ a laser beam ~tb ~ foca~ ur.~th of 2 to 20 ~m, which was modul~ed in a~lation ou;put (~ulse n~mber, intens.ty) loy t~e aforementioned fo;mu~a and scanned ovEr the workplece with a rcquired step width (~ ast ~ , a defined co~i~uous micro~eli~f s~ucture could be produceà ~hat ~rould n~t be ar~in~ble with ~ 3ray tone pattem. ~elief sEruetures in the range from 10 x 10 ~m to IOCG X 1000 ~In werc a~hieved ~wiEh lcfer~ilce to spacing an~ hei~ht di~e~ence~ ber~eer~ the protr~sions and recesses~. ~aterial-specific di~contL~luities ~ &bla~ian charzctenstics ~an be equalized by correction factors in ~he m~thematic~l descrip~ion of ~.e s~cture.
The very precisely produced 33-~tn1c~ed dio sur~ace produced t~iLll this m~ethcdgal,~e ~c~ ~o~d z~esults not cnly }n th~ usu~l ccram~ic coating~, bu~ especially Lq ~a.rd tool 22 JP~N. ' 97 15: 30 LJN~ER SCHlqlITT~ lI 51:3N HIRSC 2 1 ~ 5 3 ' 7 metal. ~n the case of ceram~'c en~raYing, ~le erl~ravet surface sesYe~ as rrlacter in a subse~uent em~o~sin~ process in ord~r to produce a metal die roll o~ plar.e die surface. ~D
this case thc master ,s cmbosse~ in ~n unhar~ened toll surface or plane surface (so-c~lled rol~
embossing) by s~ep~se rot~tory ~r trar~S~atorS~ metho~s. Th~s ro~l w~c then hardened and a~ain ser~es as rTlaster in ~ ~ec~nd roil ~mbossing ~f prodYction ~f a producti~n roLI, in which t~is master i~ rolled out on tne produc~Ian roiI.
A fiim can then be embo~ed wi~h this produCtion roll in arl ord~na~y em.bossin~
calend~r eiiher ~ith un~on ~natin~ r~ils or a "washed~ soft mating roli. ~t i~ ~dv~ntageous in iaser cng~a~ ing directly ln a ha~d metal sur~ace that the ~r~t r~ll ernbossing and su~sequent hardes~irlg drop out.
l~he .~icroreLçf ca~. r~nally ~Iso be p~;oduced ~n a ~ird manner in addition tc mechanic~l engra~,~in~ and laser e~.~s~avin~, whi~ or~ ~ of the fact th4t ~ tissuc imFression of the der:nis Is produeed in ~hich a skin biop~v is pe~forrne~ and the der nis then extracted, dried a~d coated ;~th polye~her. ~he crosslirL~ed polye~er layer is ~hen copied frorr, the d~rmis {negati~e) ~nd serves ~s m~cter fo~ a hart3 gypSllm impression (positive). A steel mold i~ finahy fortned from the hard gypsum n~o-~. The ~icsorclief so obtained rep~esents .,ai ap~roxir~lation of the ~truc~use of the b~a~ membr~ne A skjn biopsy was obt~ined, fcr exa~nple, by means o~ a derrnatome ~;CastrovieJodc tome, Stor~ In~ rnerlt) ~ith 3 depth set~ln~ of O.S to I mm, The samp~e was cu~ on a surface of ~0 x ~.0 mm ~d ~ncu~a.~d for t 5 to 48 hours in ~.2% trypsin at 4~C. As soon as a fir~t epidetmis loose~r~g wa~, v~sible on u~e comers, ~c piece wa~, t~ sLl~d to a ~m~l~ petr~
dish 3~ mm in ~li~m~te~ . 2 mL of 10% fetal cal~semm ~FCS). The cpiderrnis w~, then sep~r~ted thcre from the derm~s ~ e3ns o~ two ~ent ~rceps. llle desmis ~as trsnsfierred to a dry p~i di~,h ~d there spre~d flat v~ h the epiderm.~l sidc up. 'rhe tissue W~5 frccd cf a~hcr,ng ~nedium by su~ion and coatcd with th~ i~p~essicn ~ s ~mpregum (ESPE, Se~feld, polyether, two-component system), in which a s!ighl pn.5;~c wa~, exerted on the impre~ n m~,s. A~er 1~ m~llutes she pc~ly~ther layer w~s puPed off and stre~hed in arl appropr~ate [Tran~latc~sncce sic;;iter~l~sllr;ot ot~'cin~ 3c~ ,cn~;"cl..~ .l.s~"c""~wased]ml~hth~ve bcer~ int~rlded.l ~, ~2 JA~ 7 15-3~ iNKER 50~1hlITT-hILSON HIf~SCH ~ 2 1 7 ' '3 ~ 44 Fr~me. A positive impc~siGn w/l~ pt~d~od from thi5 ~e~tive impres~i~n a~er about 24 hG~s ~h supcr hard gyp~um, as is common, for example, in den~l IRbcratcry tech~iqu~.
Thi~ h~rd xyp9u5n posi~ve impression servei as ~a~ter for a metal impr~s~i~n A~er a ~laste~ is produced in one QJ the three meti.o~s iust descnted, the actual embo~s.ng di:s ~e produced in thc form af cmboss~ng rolls or embossing plates b,y castin~
L~'C ma~tcr ~ & silicane .~ol~ o~ a5 a ~teel m~ld. A second mo~d is produccd by in pressicn frotn th~ m~ld 50 pr~duced s~ ~hat a po~itive and negativc mold are obtaine~.
A speci~ steel sn.old was produccd acc~rdirlg to the cu51~0~ry den~ l~boratory teehniq~:e *cn. ~e h~rd g~pswn m~ld, which sho~ he tissue i~npressinn.
Shape-mated po~.itiei~e a~d n~gat.ve impressinns were pro~ed from sillcon~ f~e~ c rnicror~lief 7.}ui,t had been produc~d by en~-/in~, be it ~ith geo~ tric st~npin~ or ~er beam-macrune~ micr~reiief rolls. Thls was carr~d ou~ in a vacuu~ c~se;ing uni~ ~teYpe C~Q-~;Ç~ om ~c ~C Co. 11~.~ two-come~nent sys~em 3ilicone and cunn~ ~gent of ~leH~K Ca. was use~ here, in which silicone ~ith ~ Shorc h3.rdnes~ of ~ wa~ ~ed. A negative impression ~4-as fi~st pr3~uce~, ~cm whieh an a~dl~ionQl limpres~ion ~thc positieve improssion) was producede~ after the c~oled ne~a~i~e unpressicn ha~i 'oeen &oate~ h spra~ p~i~ ag~t (H~K~. T ~o channels were Tn~de in tne mold wkich pem~lt meten~l to ~low betwe~r. the two ~cmplemonta;y moid F~
Both mlembr~les ~n.~ a hy3iuro~e acid s~bili~ed ~y e~tenfica~ion (~iya~f ~rom ~idia Advar.ccd Biopolyrr~er~) and fro~ opol~mer con~istlng of poiy~lycoli~ acid (~CiA) ~nd trimethyle:l.e ear~or.ate ~TMC~ (r~tio ~5:3~) wcre p~odt;ce~ h the embossing die~, so produced.
g membrane FroduG~ r. ~vm ~ya~, films o f hyal~on~c acid 11 OOu~ esterif~e~
~ith berlzyl alcohol ~yz~f ~ 1 of Fidi~ Ad~ ar.ced Poiyn~.ers~ w~th a ~h~ckrless of O.Q$ mm were cur Into pie~cs me3~ E 20 x 20 ~ e pi~ceS were we~.te~ O~ bo~h sides ~r.th about 50 IlL dimetky~o~ de ~iF ~om Merck~, placed in the sii;cone mold and presse~.
~nder vacuum for 30 s~cor~ds, llle em~osse~ membranes ~-cre c~refiLlly rrmoved fi~m t.he mold with for~ps and ~ fetred to pure e~yl ~Icohol. Aflte. 5 to 60 minutos rhe ~r.cmbranes wcre w~shed Lr~ physiological sal~rle ~0.~% NaCI). The m~e~l~rane3 were y , . ..

22 CA~. ~ ~?7 15 ~?1 KLUNKER SCHMITT-~ILSON HIRSCH C?E #3~5 ~ 12t4~

21 ~5~Q,~!7 inc~.??~?vr~c~ in zpprop~iate ~1?~ for stYnli7~ion and isra,iiated at 25 kGy The Hya~f film is prcsen. in gel fonn, ~?hosc perrneabi'~it~? depends on ~hc degree of croc~iinkin~ of tho gel~ ~he macromolecules diffi;se throu~h the ~?el, wti. h is ~quipped with.
"u??~er spa~ es!' . Depending on the d~grec of CrOSSIin?l??? in g, ~yFe cf ~na~romolecule and rl~nber of hydrophilic g:oups, ~he gel tl~s a h~gh~r or ~owcr ~eg~e?~ of pç~bi~ i~r. A
pe~neabilit~?? of the membrane produced frorn Hyaff film ~om mo~ecules berw?een, 0~000 a~?d 50,0C~ d~!ton is a~tagcous.
In ~he ca~e of rnembrane production frotn PGP~-T~C these copolymers wtre extm~edwith the sm~es~ p??ossiblc spi~e~ets ~fiber d~ameter abo~t 30 llm) ~nd procec~ed to a thir~ fel~
~thic ~i~ss B00 ti_? I 000 ?,lm~. ~,i I felt Wi~S? wets_~ ~th ac~tonc ~nd prcssed b~ctw~ en ~?0 shape mated s?teel ~olds at abiv?Ut 19_? C ~.th a prcssure 0?. about 4.137 bar (S?i.~? p? i) fO? Zbi4?$1t 8 seco~ds. The reslllt wa3 a nlicroporous mcmbrane with a Wci~s of a~out 40 ~m.
Th~ in~e~tiori wil1 be ~escnbe~ below w~th referencP to ~e acccmpany~ng dr~wing.In the traw~ng:

Figu~e 1 sho~s a per~pective ~iew of the rne~r,bra~e with ~e ~nicros~ct~e and a h~lf unfol~e~ cuter membra~c, Fi~,ure ~ shows a schern~tic cross section thr~ugh a ~o~d plaste.;
Fi~re 3 shows a ~chcinatic Yiew of stac~ed membranes scparated by a gr~d.

For wound coYerirlg ~f large third degrec bums ~hc Ir.embrane ~.,cor~u~.~ to ~e invention wlth the microst~ucture ~efcrence r.o~ I O ~n Figu~ 1) is placed dire~tly cn ~ie wound. Kerat~nocytes are isolated from a biopsy of the pa;ient and reprudueed for 2 to 3 ~eeks in a c~l1 culture. These ker~in~cytes a~e 5uspe~ded ~d flxc~ to the men~rane with microsL-ucture using fibrin a~hesive. An o.~ter m~brane differ~t firom the membr~ne ~ceording to the u~Yentiorl ~reference no. i~ in Fi~e 1) i3 plaeed above it~ Thekera~ ocyt~ coliect in th~ reces~es 14 ~nd ~here rorTn a 'tcntic~l" m~ss f~r optimal gro~th cond~ ns~ since a stren~h-incre~ing di~fererltiation of celis necd not be waited for d~nn%
use of sus~ensions, as wnuld be thc ca~e ;.n the p~d~ction of sheets, the patier~t c~ be cared 1~

22 JAN. ' 97 15: Q1 ~LU~KER 5cH~ITT-~JILsoN HIR5CH D7 1 q 5 8 6 7 #3779 S 13~44 for a:~er ~ much shorter wazting time. Because of collection of kerat~noe~es in the rec~sses this can ~e dis~ibuted in p~icul~rly mil~ fashion. This type or a?plic~tio~ is to h~ prefesred over ~hin, un~fr~rm applic21t.on.
The outer ~nembrane 12 consists, for e~a2nple~ of p~l~ure~ane, microporcus polyethvlene or PT~E. It is pe~eable ~o gases and wster vqpor~ but imp~mle~ble to llquids as~d gemls.
Fig~e 2 3hows a wo~n~ plaster accordin~ to th~ .nven~ion eonsistin~ ~ an outer membrGno 12 an~ s~ stack of rcsorbable rne~branes }O with m~crost~u~l~c, which a:e separ~ted frorr~ each other ~y resorbable g~u~e 18. The gauze pre-~cnts the mem~rancs f;om lyin~ ~oo tightly on each othcr (Figure 3). ~axin~ ty ~olume is ob~ ed when the pro~usion~ 1~ of an upper membrane 10 lie above the recesses !4 of a lower membrane 10.
The ~auze can serve to ~lue the membrancs together at sites ~0, for e7~np~e, 'oy bnef thertr~
sof;enin~ of ~he ~au~e maten~l. rhe ga~o can optionally be omittcd. The resorbable membranes are p~ rated in order to allo~ blood an~ secretions to pass through. Thc outer .~em~rane 12 ~ protrude on the cdges abo~e the inner ~nembral~e ar~d be proYided w~th ~n adhesive in thi~ region ~n order t~ permi~ fi~;ation of ~e wour.d pi~s~er ~o the 5kin.
nlc c~cked membrasles folrn a cushi~n into which blood and ~erretions can pcnerrate bv capiilary Porces. 1310Gd co2gulation occ~rs on these membran~s, The keradnQcytes migrate along the protrusions occupied by fibr~n into the plaster and 6dherc ther~
preferentially. Wound he~liny ~ceurs essentially wi~out sc~r fcrma~ion. When the pia~tet is w~hdr~wn a~er ~.ealing or ~or chPnginy, ~he tn~mb~ane layers rlear the wound ren~ai~ on the weund, whose surface they protcct ~nd whose r~-wly forrned stnlcn~es are ~etained. P~in re~ep~ors rem~in cove~ed as well so that removal of the woun~ p}2~iter in patients pa~ticularly sensiti~e to pain Can ocCur ~thout problem~.
Finaliy7 thc me;nbrano C~l a~so ~e used for culti~atior~ of cc115, espe~ially fol co~
c~lltiYation of di~rently di~exntiated cell~ ~or reg~neration of tissues. Thes- can be used, for ~xample, fo~ ~an6plant~tiGn, f~r investi~ation of ~iisu~ ctio~, for syn~hesis of highly effcctivc macromoiecules or aS 2 biologic~l scnsor. The memb~ane is stretched ~n a fr~m.o and bcth sides of the frame are e7cposed ~n succcssion to a cell sL~pension of ~wo differen~
t~

:. .

22 JAN. ' 97 1~:32 KLJ~KER ;-CHMLTT-NILS',N ~IRSCh l::E ~37'79 S. 1~44 2 1 ~ J f3 ~ /

ce~l ~pes. A~ter se~ t~tion ar.d fi~ation cf oGe ccll typ~ th~ frame is tL~rned a~d t.he s~ne process repeated with a SUSFerLSiOn of the other celi type.
Fcr co-cs~lti~a~ion of human skin cells on the microstnlctur-d membrane ~lormal ~urnan fibroblasts ~e~e ~ oduced to o~e side of ~.e ~,cm~rane i~ a cor.ccntration o~SO,OûO
to 1 00,000 cellY pe~ cm in ~ M m,edi-lsn ~ibco) ~ith 10% ~é~al calf S~ in~for a we~k at 37~C. Thc lr.embr~~e w~q thçn nlrned and cov~red w~th no~nal hu;nan kera~ acyt~s ~n Rhein~a;ld and 5reen mcti~t (Cell, 1 g75) + 10% fet21 c31f serum in a corlcen~ati~n of 50,~0 to 100,000 ceils per C.~n2. This co~cuinare ~as incuba,ed at 37-C fcr 2 to 4 w~eks, ~ f

Claims

1. Membrane for regeneration of body tissues made of a biocompatible material, characterized by the combination of the following features:
- a thickness in the range from 0.5 to 500 µm, - permeable to gases and macromolecules, but impermeable to cells, - a microstructure in the form of protrusions (16) and/or recesses (14).

2. Membrane according to Claim 1, characterized by the fact that the membrane is permeable to molecules between 20,000 and 50,000 dalton.

3. Membrane according to Claim 1 or 2, characterized by the fact that the membrane is resorbable.

4. Membrane according to one of the Claims 1 to 3, characterized by the fact that the membrane has a uniform thickness.

5. Membrane according to one of the Claims 1 to 4, characterized by the fact that the protrusion (16) and recesses (14) are arranged regularly in a grid pattern.

6. Membrane according to the Claims 1 to 5, characterized by the fact that the protrusions (16) and recesses (14) are all designed the same and correspond in size.

7. Membrane according to one of the Claims 1 to 6, characterized by the fact that the microstructure is instrinsically stable.

8. Membrane according to Claims 1 to 7, characterized by the fact that the protrusions (16) and recesses (14) of the microstructure directly border each other and grade into each other.

9. Membrane according to Claim 8, characterized by the fact that the transitions between the protrusions and recesses are smooth and continuous without edges.

10. Membrane according to one of the Claims 1 to 9, characterized by the fact that the microstructures of the top and bottom of the membrane correspond.

11. Membrane according to Claim 10, characterized by the fact that the microstructure can be described by the superposition of two sine or cosine curves.

12. Membrane according to Claim 11, characterized by the fact that the structure can be described by the formula z(x, y) = A*sin(x) + A*sin(y) or z(x, y) = A*cos(x) + A*cos(y), in which A denotes amplitude.

13. Membrane according to one of the Claims 1 to 12, characterized by the fact that the thickness of the membrane is 2 to 20 µm.

14. Membrane according to one of the Claims 1 to 13, characterized by the fact that the membrane consists of a crosslinked gel.

15. Membrane according to one of the Claims 1 to 14, characterized by the fact that the membrane is microporous and the pore size lies below 3 µm.

16. Membrane according to one of the Claims 1 to 15, characterized by the fact that the spacing (a) from protrusion to protrusion (16) or from recess to recess (14) is 50 to 500 µm, preferably about 20 µm.

17. Membrane according to one of the Claims 1 to 16, characterized by the fact that the amplitude of the protrusions plus recess is about 50 to 1000 µm.

18. Membrane according to Claim 17, characterized by the fact that the amplitude is 100 to 500 µm.

19. Membrane according to Claim 18, characterized by the fact that the amplitude is 150 to 300 µm.

20. Membrane according to Claims 1 to 19, characterized by the fact that the material consists of natural ECM molecules or synthetic molecules.

21. Membrane according to Claim 20, characterized by the fact that the material for the membrane is chosen from the group comprising polylactide, polyglycolide, copolymers of these substances or their derivatives, polycaprolactone, polydioxanone, polyphosphazene, polysulfone, polyurethane, polyhydroxybutyric acid, hyaluronic acid or collagen crosslinked with a crosslinking agent.

22. Use of the membrane according to the Claims 1 to 21 for cultivation of cells.

23. Process for cultivation of cells using a membrane according to the Claims 1 to 21 in which autologous keratinocytes are fixed on the membrane.

24. Use of the membrane according to one of the Claims 1 to 21 for co-cultivation of differentiate cells.

25. Process for co-cultivation of differentiated cells using a membrane according to one of the Claims 1 to 21 in which the membrane is stretched in a frame and exposed on both sides to corresponding cell suspensions.

26. Artificial skin consisting of a membrane according to Claims 1 to 21.

27. Artificial skin consisting of a membrane with cell culture produced according to the process according to Claim 23 or 25.

28. Use of the membrane according to one of the Claims 1 to 21 for covering burns.

29. Use of the membrane produced according to the process of Claim 23 to 25 for covering of burns.

30. Wound plaster consisting of at least one membrane according to one of the Claims 1 to 21 and an outer membrane (12) that is gas-permeable, germ-tight and fluid-tight.

31. Wound plaster according to Claim 30, characterized by a stack of membranes (10) that are optionally separated from each other by gauzes (18) of restorable material.

32. Wound plaster according to Claim 30 or 31, characterized by the fact that the membranes (10) are perforated in order to allow blood and secretions to pass through.

33. Wound plaster according to one of the Claims 30 to 32, characterized by the fact that the outer membrane (12) protrudes on the edges above the inner membrane (10) and is coated in this region with adhesive (20).

34. Process for production of a membrane according to one of the Claims 1 to 21,characterized by the fact that the microstructure of membrane is produced with an embossing die having a corresponding relief structure.

35. Process according to Claim 34, characterized by the fact that the embossing die has a hard metal surface in which the relief structure is produced directly by laser engraving.

36. Process according to Claim 35, characterized by the fact that the laser device for production of relief structure is NC-controlled.

37. Process according to Claim 35 or 36, characterized by the fact that the laser device has a laser beam with a width from 2 to 20 µm.

38. Process according to Claim 35 to 37, characterized by the fact that a silicone die that serves as embossing die for the membrane is embossed with the laser engraved hard metal die.

39. Process according to Claim 34, characterized by the fact that the relief structure of the embossing die is produced by taking a tissue impression.

40. Process of Claim 39, characterized by the fact that the tissue impression is an impression of the dermis.

41. Process according to Claim 40, characterized by the fact that the tissue impression is produced in the form of a hard gypsum impression in which the dermis is recovered by skin biopsy and coated with an impression mass and the impression mass after it has sufficiently hardened is separated by the dermis and molded in hard gypsum.

42. Process according to Claim 41, characterized by the fact that the embossing die is produced by molding the hard gypsum impression.

43. Process according to Claim 34, characterized by the fact that the membrane consists of a hyaluronic acid film stabilized by esterification that pressed in a mold for 30 seconds under vacuum.

44. Process according to Claim 43, characterized by the fact that the mold is a silicone mold.

45. Process according to Claim 34, characterized by the fact that the membrane consists of a felt of copolymer fibers that is pressed in mold.

46. Process according to Claim 45, characterized by the fact that copolymer fibers consists of two-thirds polyglycolic acid (PGA) and one-third trimethylene carbonate (TMC).

47. Process according to Claim 45 or 46, characterized by the fact that copolymer fibers have a diameter of about 30 µm.

48. Process according to one of the Claims 45 to 47, characterized by the fact that pressing is carried at a temperature of about 195 °C and a pressure of about 4 bar for about 8 seconds.