Note: Descriptions are shown in the official language in which they were submitted.
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~.
.
~, . . . . .
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~
- . . . .
... ~ .... .. . . ...
- 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
