CA1158807A - Process for the preparation of new organ transplants - Google Patents

Abstract

Abstract The organ transplants are intended to be used as a replacement (prostheses) for organs or parts of organs, for examples arteries or veins, which have undergone pathological change or are functionally impaired, The organs, which are taken from a fish, bird or higher mammal, are subjected to crosslinking of the amino groups and of the alcoholic hydroxyl groups of the peptide chains of the intercellular matix by means of a di-, tri- or poly-carboxylic acid It is advantageous subsequently to treat the crosslinked product with a dialdehyde, in order to bind amino groups which have not reacted, or to free the crosslinked product from material which poten-tially may act as an antigen, by hydrolysis with ficin, papain or a protease having a similar action, After the hydrolysis, a comprehensive crosslinking is appro-priately ensured using a dialdehyde or a di-, tri- or poly-carboxylic acid, The resulting prostheses are distinguished by chemical stability, biophysical and biochemical properties similar to those of the natural material and the absence of rejection reactions.

Description

1 1~8807 Proce;s fOL' ihe orer~arati-n O:r l1ew or~ n trcllr~ s , ~
The pros-l;het:ic mat.erial avai.:!able in reco-fl.s-trllc~
-tive surgery for organs or parts of orr~ans ~hich ha~re ~clergone a pathologi cal change or are fu~c-tionally impaiIed covers several p~s;ibilities (F. Largiar~r9 "Organ Tr~nsplantation'!, 2nd ed~-tion, Georg Thieme Verlag, Stuttgar-t 1970). Specifically9 the original organ or par-t of an organ can be replaced~
by e~dogenous ma-terial 9 for example au-tologous veins [G E Mavor e-t al., J. Cardiovasc. SurgO 16 (1975)9 1~0]~
by implan-ts made of plastic [M~E. De Bakey e-t aL, "Fif`teen Years' ~xperience with Dacron Vascular PI`OS--theses", Brochure, Baylor Coll. Med. 19r~1 (Amer. Coll.
Surg. Exhibit. 1971)~ or by approprla-t;ely prepared transplants from diverse species of animals (P. Walter and H. Schmi~tz, "Der he-terologe Gef~-~s~ersatz" ("Heterologous Vascular ~eplàce-ment"), Editio Cantor, Aulendorf 1976).
The latter originate :in the main ~rom anirnal organs or parts thereof (for example the carotid arteries of calves or cardiac valves from pigs), which after removal are freed, by means of an enzyr~a-tic hydroly-sis (for example with ficin~, from proteins which poten--tially act as antigens. The intercellular matri~
remaining after this -trea-tmen-t consists in the main of collagenous fibres of -type I. In order to implJ-t to this collagen skeleto11 ob-tained a:~-ter proteolytic hydro--lysis the density and stability necessary for a pros--thesis, a so-called "tanning" is carried out. Al~e-hydes, such as dialdehyde starch, glu-taraldehyde and paraformaldehyde, as well as g]yoxa] and polyacrolein and also aceta]dehyde and cro-t;onaldehyde, are availab]e for this tanr1ing - or crosslinking. The common characteristic of these subs-tances used for crosslinking is tha-t -they react ~rith the ~-amino groups of -the lysine residucs in the collagen peptide chain wi-th the formatio of Schif~ 7 s ~ases.
Howev~r, wlth organs pre~treated in this way, such as, for example, vascular pros-theses (U.S. Paten-t ~ j~

3509~9439 or ~,wjss Pat(l~-t 5959105) i-t h~-~s been :to~Jld tha-t the blophysical characterlstici o,:Lg:inally preisent~
such as -the elasticity :in the axial and radial direc-tions cmd also -t;he extremely smoo~h nature of the inner surface of the vessels 9 are complete]y lost as a resu]t of the enzyma-tic degradation by rneans of ficin. As a consequence of this, it has to date not been possible to meet the demands for an ideal vascular prosthesis of biological ma-terial. The same also applies in -the case of the replacement of o-ther organs or parts of organs. If 9 on the other hand, the ficin degradation is omitted, the danger of an an-tigen effect canno-t be excluded9 such as, for exc~nple,in theprocess fortnepre~
paration of na-tural tissue for implantation in accord-ance with German Offenlegungsschrift 2,519,107 A fur-ther modification of the collagen skele-ton cornprises binding aliphatic carboxylic acids covalen-tly to -the side chain of aminoacids by acylation, wlth the formation of more highly negatively charged derivatives (asis known an excessposi-tive chargepromo-tes thrombosis).
However, the procedure does not result in crosslinking in the collagen skeleton (P.N. Sawyer et al., "Vascular Grafts", Appleton Century Crofts, New York 1977, page 282 et seq.).
Despi-te the efforts described above, clinical experience teaches that the -transplants already proposecl can, in genera:L, no-t be regaIdcd as com;)]etely S,ltiS~
factory, a-t least in -the long term (H. Haimovici, "Vascular Surgery, Principles and Techniques", Mc~raw-Hill, New York 1976, page 304).
It has now been found, surprisingly, that by means of novel intermolecular and/or in-tramolecular cross]inking of the macromolecules of -the intercel]ular matrix of organs or par-ts of organs, prior to or even without the proteolytic degradation by means of ficin, the biophysical characteristics originally presen-t 9 such as, for e~,ample, in t;he case of vascular pros-thec;es, -the axial and radial elastici-ty and the smooth nature of tlle imler surface of the vessels~ are substtan-tially retained~

, - 1158~07 . ~ ~

This crosslinking dif~ers from the kno~m proce dures in that it is effected not by the formation of Schiff~s bases bu-t by chemica]ly considerably more stable acid amide bonds between the amino groups, or by ester bonds between the alcoholic hydroxyl groups~ of the peptide chains of the intercellular matrix and the carboxyl groups of a di-, tri- or poly-carboxylic acid, which is employed as an agent ~or crosslinking.
The process according to the invention comprises subjecting organs or parts of organs of fishes, birds or mammals, preferably from higher mammals, to cross-linking of the macromolecules of the intercellular matrix by the formation of amide bonds, or ester bonds, between the amino groups, or the alcoholic hydroxyl groups, of the peptide chains and the carboxyl groups of di-, tri- or poly-carboxylic acids of the alipha-tic, cycloaliphatic, aromatic or heterocyclic series.
Although it is not essential, a further treat-ment, in accordance with the following guidelines, of the resulting intercellular matrix crosslinked by amide bonds can bring certain advantages.
Initially, the matrix can be treated in a first additional process stage with a dialdehyde. The treat-ment with the dialdehyde serves in the main to bind any amino groups which have not been bound during cross-linking. As a result of this, the excess negative charge is increased, which, as experience has shown, lowers the danger of thrombosis, for example of vascular transplants (P.N. Sawyer et al., ibidem).
In place of the abovementioned treatment with a dialdehyde, the matrix can, in another (alternative) additional process stage, be freed from material which potentially acts as an antigen, by hydrolytic degrad-ation with ficin, papain or another protease of the same or similar substrate specificity. By means of the additional hydrolysis or proteolysis, organ transplants are obtained with which the possibility of an antigen effect, that is to say the triggering of an immuno-logical defence reaction with rejection of the transplant .

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or implan t s i s eY~cludecl~ everL in the long terrn.
If the said prvteolysis with fi.cin~ papain or the llke is carried ou-t 9 individual arrlino groups are liberated in the int~rcelllllar llla-trix. In orcler to bind these groups and the amino groups which may not have been re.lcted during the preceding crosslinking, it is advisable, in a second additiona'L process s-tage, either to treat -the matrix wi-th a dialdehyde - as has already been described above - or to subject t;he matrix to crosslinking again, by forrr,ing amide bondc with the aid of one of the di-, tri- and po].y-carboxylic acids mentioned.
The invention is described in detail in the tex-t which follows.
The starting ma-terial used is in par-ticular arteries, veins, cardiac valves and also the peri-cardium and the like from birds and higher mammals of a suitable size. Suitable donors are both man himself (autologous transplan-ts) and also calves, cattle, horses, sheep, pigs, geese, turkeys, pheasants and other animals (heterologous transplants) Organs and parts of organs preferred for this purpose are those from anima'ls, because of their more general availability, but in par-ti-cular those from young animals, because of their excel lent elastici-ty It is self-evident that the orgc~ns ancl par-ts of organs are freed fro~n the surrounding tissuc immedia-tcly after they have been removed and that, in the case of arteries or veins, the coIlaterals are tied off by ligature The organs or parts of organs are then subjected to the process irnrnedia-tely or are stored in water, physiologica] saline solu~tion or another physio-logical aqueous solution, for exarnple T~leen 80 (poly-oxyethylene deriva-tives of the sorbitan oleates), or in a non-aqueous liquid, for example dirnethylsulphoxide, optionally wi-th -the addition of a litt;le sodium azide~
at O to -15C un-ti'l the process is ernployed The one stage which always forms par-t of the process according to the invention comprises crosslink-1 1588~)7 r~
.in~ of th~ p(.'r)-tid.C` Ch~l ins O:~ CC)n,ri ~i. t,llen.-~ 'S OI th~ -i;e~
ce:llu:Lal~rnat;rix~ lt i~ baseci on the bi.l1di.ng of two 9 three or more amino grol.aps (in -tlle main E arllinO groups of the lysine radicals) or alcoholic: hydroxyl groups of th~ peptide chains by means o: aliphatic 9 cycloaliphatic aroma-tic or heterocyclic di~ 7 tri- or poly-carboxylic acids 9 in -the form of acid amide bonds or es-ter bonds.
Amongst the carboxylic acids mentioned, suitahle acids are in particular those which con-tain neither oxo groups (aldehyde and ke-to groups) nor amirlo groups.
Di-, tri- and po].y carboxylic aci.ds which c~n be used are in particular those which do not carry any func-t;ional groups except for the carboxyl groups and in some cases hydroxyl groupsO
Preferred aliphatic dicarboxylic acids and tri.-carboxylic acids are those having not more -than 12 car bon atoms, tha-t is to say in particular oxalic acid, malonic acid, succinic acid, rnalic acid, glutaric acid, adi.pic acid, pimelic acid, suberic acid, sebacic acid and dodecanedioic acid, and also tartaric acid and mucic acid, and pre~erred -tricarboxylic acids are tricarbally-lic acid and citric acid.
Amongs-t the cycloaliphatic di-, tri- and poly-carboxylic acids, suitable acids are, inter alia, the cyclopen-tanedicarboxylic acids and the cyclohexarle-dicarboxyli.c acids, ~or examplc cyclohexane~ di.(ar--boxylic acid Aromatic dicarboxylic acids which may be men-tioned here in particular are phthalic acid, isoph-thalic acid and -terephthalic acid, and aroma-tic tricarboxy:Lic acids which may be men-tioned are trirnesic acid and -trimellitic ac:id.
Suitable he-terocyclic di-, tri- and poly-carboxy-lic acids are, inter alia, ~uran--2,5-dicarboxylic acid, tetrahydroluran-2,5-dicarboxylic acid, oxi.dised s-tar~h and carboxymethylcellulose.
The exis ting space be-tween two E--amino gro~ps acces~ib].e lor crosslinking and the solvbili-ty, in -the solven-ts, appropriate for carrying ou-t the process, ~ ~ 5 ~ 7 make a relatively narrow lower and upper limit appear advisable in the case of the preferred groups mentioned. Thus, particularly pre-ferred acids are, on the one hand, aliphatic dicarboxylic acids having 3 to 12 carbon atoms, i.e. acids ranging from malonic acid to dodecanedioic acid. On the other hand, however, higher molecular polycarboxylic acids, such as, for example, oxidised starch, have also proved particularly suitable.
Crosslinking can be effected on the basis of all of the methods customary in chemistry for forming an amide bond between an lQ amino group and a carboxyl group; in this context see, inter alia, H.D. Law, "The Organic Chemistry oE Peptides" (John Wiley & Sons Ltd., London-New York 1970). In particular - without any claim to completeness - the acid chlorides, acid azides or acid anhydrides of the carboxylic acids mentioned can be reacted with the free amino groups of the intercellular matrix. The free di-, tri- and poly-carboxylic acids can also be bonded to the free amino groups of the intercellular matrix with the aid of a suitable coupling reagent, such as the carbodiimides, for example dicyclohexylcarbodiimide.
As a rule, the reaction is carried out in an anhydrous organic solvent, such as tetrahydro~uran, dioxane, pyridine, di-methylformamide, dimethylacetamide, dimethylsulphoxide and hexa-methylphosphoric acid triamide, or in a mixture of two or more of these solvents. If a water-soluble coupling reagent, for example N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride, is employed, water is also suitable as the solvent.
The organs and parts of organs stabilised in this way can now be subjected, in an (optional) second stage of the process, to tanning or crosslinking by means of formaldehyde, or a dialdehyde, .~ - 6 -l) 7 the amino groups in the collagen-peptide chain which are possibly still free being reacted, with the formation of Schiff's bases.
This treatment is also advantageous inasmuch as, in particular, the prostheses obtained with glutaraldehyde are already ~irtually sterile.

` - 6A -~ ~8~07 In principle~ every dialdehyde is suitable as the dialde-hyde; dialdehyde starch and in particular glutaraldehyde are preferred. The reaction with the dialdehyde is advantageously carried out in aqueous solution f for example in accordance with the method of Swiss Patent 595,105 or United States Patent 3,093,439.
Alternatively, the organs or parts of organs stabilised by crosslinking can, if desired, be subjected to the proteolytic effect of a suitable enzyme, without suffering substantial losses in their original biophysical and biochemical properties. This behavior is to be ascribed to the fact that the peptide chains of the collagen are fixed by the crosslinking, before the stabilising elements of the intercellular matrix, such as elastin, proteoglycans and also structural glycoproteins, are partially removed by the hydrolysis.
The preferred second stage of the process thus comprises an incubation of the organ material in a solution, appropriately an aqueous solution, of ficin, papain or another protease of the same or similar substrate specificity. The hydrolysis or proteolysis is preferably effected with ficin, advantageously in the pEI range from 4.0 to 5.5; it can, inter alia, be carried out in accordance with Example 3 of Swiss Patent 595,105.
The repeat crosslinking which advantageously follows the hydrolysis can be carried out with a dialdehyde o.r one of th~
abovementioned di-, tri- and polycarboxylic acids; the methods described above are suitable for this purpose.
The resulting organ transplants are then washed thoroughly with water, sterilised and stored in heat-sealed plastic bags until they are used. Sterilisation of the products is effected ~ ^ "

exclusively by chemical means, with the aid of propene-1,2-oxide (see under K-I solution in the experimental section), propiolactone (~-hydroxy-propionic acid lactone) or ethylene oxide.
To summarise, by means of the process according to the invention prostheses are obtained which are ?

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, ` L158807 par-ticularly suitable as repIacelnerlts i`or defective organ~ or parts -thereof, because thei.r interm~lecu]ar and intramolecular transverse bonds (acid amide bonds and ester bonds respectively) are dis-tlnguished by par-ticular chemical stability, their biophysical proper-ties (elasticity and rigidity) and also their biochemical proper-ties (reduced thrombo-genicity) are extremely similar to those of the natural material, as a result of the type of crosslink-ing mentioned, prior to or evelwithout proteolytic degrad-ation, and they nevertheless cause no rejection reactions, that is to say cannot have an antigen effect The advantages of the characteristic features mentioned for surgical use andfor the therapeutic results are obvious~and they have in fact already been confirmed in animal experiments.
Mongrel dogs with a body wei.ght of 20 to 25 kg~
and the artery transplants prepared according to Example 9~were used for the studies. The prostheses were implanted (a) at the two femoral arteries of each dog, immediately distal o~ the inguinal ligament, by the method of P. Walter et al. [Helv. chir. Acta 46 (1979), 81 et seq.], (b) at the two carotid arteries, (c) at the abdominal aorta~ in the caudal direction from the exits from the renal arteries CP. Walter and H. Schmitz, Der heterologe Gef~ssersatz (Heterologous Vascular Replacement), page 30~ Editio Cantor, Aulendorf 1976], (d) at the infrarenal inferior vena cava by the method of S. Horsch et al. [Langenbecks Arch. Chir. 344 (1978), 225 et seq ]. For comparison, parallel experi-ments were carried out with prostheses made of Teflon (R) and prostheses made of Dacron (R) and also with umbili-cal veins which had only been tanned with glutaraldehyde.
The occlusion rate of the implants after six weeks was measured.
It should be emphasised here that, in ~a~ticular~
implantation point a (see above) on the hip joint of the dog is subjected to high mechanical stress (flexion), 1 1 5 ~

hich frol1l ti,me tc,-time lI1cr~ac,es -to a pelmanent s-trc:is~
l other WO?'d~ GhliS -tes-t arranC~el~cnt corre~pc!nds to extreli~e:ly unfa~ourab]e physiological cond:itions.
The resul-ts ~ere -then also -th- more informa~tivec ~he implants of Teflon (P~) and -the umbi,lic,al ve:in gave an occlusion rate of 100~o ~ld the implants o:E Dacron (P~) gavc an occlusion rate of 77~09 whilst the implarlts according -to Example 9 showed an occlusion rate of 50~0O
The values ob-tained from the comparison s-tudies are s-ta-tlstically significant.
In studies under physiologically more favourable conditi,ons, specifically a-t the Ar-teria carotis, carried out in -the medium term, i.e. over a period of 6 months5 the occlusion rate of -the implants according to Exc~mple 9 fell to 20~'; the value determined is sta-tistically significant. In s-tudies in -the infrarenal region no thrombosis was observe~ in the medium term (6 months).
Comparison trials on humans are now in progress in a universit-y hospital.
Exclrnple 1 Carotid arteries from calves are freed from surrounding connective tissue and -the collaterals are tied off. After mechanical preparation, they are washed ~lith deionised water, dried off with absorben-t paper, drawn up on a glass rod with a diameter of, for example,3 mm and placerl in a rneaC;uring cy~ i,er ~illed with te-trahydrofuran, for dehydration. The con-teIlt~
of the measuring cylinder are shaken round a-t 1 -to 2 hour intervals. After 5 hours the liquid is replaced by fresh -tetrahydrofuran. After a further 2 to hours the glass rods are removed~ ~
The fol~o~ring day the ar-teries are placed in a

2% (weight/volume) solution of adipic acid chloride in tetrahydrofuran and left in this solu-tion for 24 hours.
The solution is occasionally shaken round, or circulated by rneans of a pump. The arteries are then placed i pure -te-trc~hydrofurc-an~ then in tetr~hydroIuran/water (1:1 volume/volurne) and finally in phGspha-te-buffered saline solu-tion ancl are left in each solution for 30 to t 158807 ~ 10 -60 minutes~ The arteries are now ready for ster-,lis-ation or for hydrolysis with ficin~ papain or the like.
a) Steri]isation with ~ ne-1~2-cxide The arteries are placed in a 1% (weight/volume~
solution of propene-1,2-oxide in e-thanol/water (1:1) (volume/volume) ~or 16 to ~4 hours and possibly e~en longer. They are then placed under sterile conditions in sterile PBS solutionand together with this solution are sealed into sterile plastic bags by heat-sealing.
PBS solution 320 g of sodium chloride, 8 g of potassium chloride, 51.2 g of disodium hydrogen phosphate dihydrate~
8.0g ~ potassium dihydrogen phosphate, 5.2 g of calcium chloride dihydrate and 4.0 g of magnesium chloxide hexa hydrate are dissolved in 40 litres of water.
I~ it is desired to store the arteries in an ethanol/water mixture, it suffices to place them in the abovementioned alcoholic-a~ueous propene oxide solution and to seal them, together with the liquid, in plas~ic bags by heat-sealing.
b) Sterilisation wit~ Qpiolacton ~ droxy-pr pionic acid lactone) .. . _ The following salts are dissolved in about 900 ml of deionised water: 0.236 g of sodium chloride, 0.248 g of potassium chloride, 0.363 g of calcium chloride dihydrate, 0.190 g of magn~sium chloride hexahydrate and 0.172 g o~ potassium dihydrogen phosphate. The pH
value of the solution is raised to 7.4 with a little 0.1N aqueous sodium hydroxide solution. 11.782 g of sodium bicarbonate are then dissolved in the solution and the volume is made up to 1,000 ml by adding water.
Immediately before sterilising the arteries, 8.86 ml (10.08 g) of propiolactone are added to 1 litre of this solution, the arteries are placed in the mixture and are sealed together with the liquid in a plastic bag by heat-sealing.
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The ar-teries placed in PBS solution or physio_ logical saline solution are sealed together with the ..~.

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:l.iqii~ a plas-tic b,ag by hcat~c;ee~ir)g,, Ste:rilis-ation ls e:`f~cted by e~pocilnO; the sea~le.l plas-tic bags l,o etl.~lc~ne o~ le ~;a~ 'or 4 hours at 25 to 30C :in a steril--isation apparatus9 in accordance ~J~ -th a ~,no~n method.
Exc~ 2 ~ our mechan:ically prep.lred cal~ arterieC, are individually drawn U.p on to 4 mm -thick glass rods arld placedina 500rlll rneasuring cylinder fillecl wi.th pyridiI~e.
One hour later -the ar-teries are so stiff that -the glass, rods can be removed. The liquid is decanted and replaced by fresh pyridine. Af-ter one hour -the pyri-dine is renewed agaln. Af-ter-a further one hour, the arteri.es are placed in a mixture which has been prepared in the following way^ 2 ml (2.5 g) of adipic acid chloride are injected into a mixture of 98 ml of pyridine and 2 ml o:E di.methyl~ormamide, with s-tirri.ng~
a fine precipitate of adipylpyridinium chloride forms.
~hen it has se-tt,led, it ls covered with a cribriform plate made of porcelaln. The ar-teries are -then placed on the cribriform plate, so that they are entirely covered by the liquicl bu-t do no-t come into ContaG~; with the precipita-te. 18 hours later -the ar-teries are rernoved from the liquid and immersed in K-I solution (see Example 9 below) for 30 minutes. They are then washed four times with 0.05N sterile aqueous ace-tic acid and twice with s-teri.le phospha-te ~u.~fer (1115 molar) of pH 8. Finally, they are p:l.aced in s-ter1.:le 1'13S
solu-tion and sealed together Wi th this lic1uid into a p~caci-tic bag by heat-sealing.
Exa~le 3 10 calf ar-teries are prepared mechanically and placed ver-tically for -thrce hours in a 500 ml measuring cylinder filled wi-th -te-trahydrofura1l. They c~re then placed indiviclually in glass -tubcs al~out 2 cm w:ide and are each covered wi-th a layer o~ 40 ml of a 0.06% (weight/
volume) solu-tion o~ adiplc acid in -te-trc~hydrofuran/water (4.1~ (vollm1e/voll.lme). ~î-l;er or1e hour llo ml o~` a 2%
(weigh-t/volume) solution o['~icyclo~!exy:Lc~rbo~iiriiide:in tetra-hydrofuran areadded to each t,ube and l~ixed wl-th -the . 12 ~
sc~ t~ c.~ )r~s(~rl L~ .J`.:ir3g c~ d ~/i.o :i~ J~ .r1O
Afte-~ ~ hollrs tll{ arteries are pLaced in 1~-I soLut~orJ
(see E~ p`]e 9 bel.ow)~ ~he foLlo\Jjng d~-ly thoy are each washed witLl ~lO ml of s-t rile et'lanol/water (1.1) (VO~ 'o]~ n(l I`rit') ~0 li~] 0~ r.il(~ p~lo~)]~at;~b~frcr (1~15 r~ c!-', of p~-I 8 and are placed in s-~erile P~f'~ solution~ Fin-ally 9 they are sealed together wit.h -l.he PBS solu~tion in plastic bags by hea-t~seali.ngO
_~,.ramE~e 4 After mechanical prepara-ti.on 10 calf a~-teries are pl.aced in a 500 ml measuri--lg~ cylînder filled ~rith dime-thylformamide. A:E-ter ~ hou.rs -the arter:ies are placed individually in glass -tubes ~i-th an internal diameter of abou-t 2 cm and 40 ml of a 0.06/o (wei.ght;
volume) solution of adipic acid in dimet}lylforfnamide/
wa-ter (~:1) are poured over each ar-tery~ After one hour, 40 Ml ol a 2~o (vol.un~e/volume) solution of dicyclo hexylcarbodiimidt-~ in dimethy.l1`ormamicle ar2 adcLed to each -tube and mi~ed wi-th the adipic acid solu-tion poured in earlier, by shaking and swi.nging round. 3 hours lat:er -the liquid is poured off frorn each tube and replaced `by 80 ml of K-I solution in each case. I~le follo~lng day the arteries are each washed with l~0 ml of sterile ethanol/~later (1:1) (volume/voL.~une)c~d~ith llO ml of sterile ph(-sp}lcl-r buffer (1~L5 molar) of pH 8. Finally, they are placed in sterile PBS sol.u-tion and sealed -toge-th~r w:i.-th thls solution in a plastic bag by hec-l-t-seal.:i.ng.

4 cal:L` ar-tex:ies are :Iree~cl froln con.necti.ve tissue~
in the usual way and the collaterals are -ti.ed off~
They are -then placed in G0 ml of 0.02~o (weight/volurne) aqueous adipic acid 501u-tion for 2 hours. They are then placed in 60 nrl of an aqueous solu-tion which con--tains acllpic acid in a concerltra-ti.on of 0.2 g per :Litre and N-ethyl-N~-(3-dirnethylalnirlopropyl)-cclrbocdi.imide hydrochloricle in a concentra~tion of 25 g per li-tre.
~I-ter 20 h.ours, the ar-~teri.es are placed in Jt~ solution and 24 hours la-ter -they a:r e p Laced in s terile PBS
solu-tion. They are sea] ed -toge ther wi-th the :Latter `` 1 1588t~7 . .
~ 13 -solution iIl sterile plastic bags by heat-sealjr~gO
~ _6 Mechanica3.ly prepared arteries are placed in 60 ml of a solution of 0.1yo (weight/volume) suberic acid in wa-ter for 2 hours and are then placed in 60 ml of an aqueous solution which contains, per litre, 0.24g of suberic acid and 25 g of N-ethyl-N9-(3-dimethylamino-propyl)-carb~diimide hydrochloride. After 20 hours the arteries are placed in K-I solution and after a further 24 hours they are placed in sterile PBS solution and sealed together with this solu-tion in sterile plastic bags by heat-sealing.
Example 7 Calf carotid arteries are treated with adipic acid chloride in tetrahydrofuran using the procedure described in Example 1. The resulting arteries are then not sterilised but are hydrolysed with ficin in the following way:
1 litre of water is warmed to 37C. 10 g of ficin are added, with stirring; in some circumstances the fi~in only partly dissolves. 1 g of cysteine is then added, and dissolved, with stirring. The pH
value of the solution is raised to 6.0 with 1N aqueous trisodium citrate solution If the liquid is turbid, it is filtered through cotton fabric and freed from the turbidity in this way. Its temperature meanwhile falls to about 25 to 30C.
m e arteries are placed in a measuring cylinder and the clear ficin solution is poured over them. The measuring cylinder is placed in a waterbath at 37 to 38~, so that its contents warm to about 37C. The reaction has ended at the latest after 3 hours at 37C. The ficin solution is now poured off and discarded. The arteries are washed for a quarter of an hour with running deionised water at about 20C. The water is then poured off. The arteries are still in the measuring cylinder. 1 litre of an aqueous solution which con-tains 11.9 g o~ sodium chlorite (NaClO~) is poured over them. A~ter 18 hours at room temperature, the liquid , , ..... ~ ... . .......... ~ .. , . .. -- .
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~-~ 1158807 is decanted off and the arteries are ~as,hed for c~out a quarter of an hour with running deionised water at about 20C~ The arteries are now ready for sterilis-ation or for after-treatment ~ith a suitable aldehyde.
Exa~
Calf carotid arteries are treated with adipic acid chloride in tetrahydrofuran using the procedure described in Example 1 The resulting arteries are then not sterilised but are hydrolysed with ficin in the following way:
1 litre of citric acid/phosphate buffer solution of pH 4.5, prepared by the method of T.C. McIlvaine [J. Biol. Chem. 49, 183 (1921)], is initially intro-duced. 10 g of ficin are added, and dissolved) wi-th stirring. 1 g of cysteine is then added, and dissolved, with s-tirring The arteries are placed in a measuring cylinder and the ficin solution described above is poured over them. The subsequent treatment is in accordance with the method of Example 7.
Example 9 As described in Example 7, calf carotid ar-teries are first crosslinked with adipic acid chloride in tetrahydrofuran and then subjected to hydrolysis with fici~.
Subsequently they are agaln draw~ up on gla~s rods and placed in a 4% a~ueous glutaraldehyde solution.
After 24 hours the rods are removed and the arteries are washed for at least 90 minutes in running water.
Finally, they are placed in K-I solution and sealed in a plastic bag by heat-sealing.
K-I solution 720 ml of water and 720 ml of ethanol are mixed.
16.9 ml (14 1 g) of propene-1,2-oxide are added to this solution. This solution is al~ays freshly prepared and used for sterilisation immediately after it has been prepared.
Example 10 7 calf pericardia are freed from fat,scraped - ~ ~5~37 - ~5 -s~bs-t~n~ti.all.y s~oo-t:h ~ i,]?1:.lccc1 ill r-;oo m:l. o:L t~-t:r~ d~o :Curan i.?:` .1 .Challow CliSho rl'he ~ lli.CI iS rep:la,oed tby f~esh tetrally~.Iofllran ai~ter t da-r and a~ter 2 days~
~:E-ter a total of 4 days~ -the per;,cardla are placed irl 500 ~ll of a 2~ ~weigh-l,!volur.le) solut:io~l oi adipic acid chloride in te-t:rah.ydrofl1ran and left in this soluti.on for 1 day. The liquid is then poured off. The pericardia are placed l~or half an hour in ethanol/water (1:1, volurne/vol~ e), this opela~ic)n being (.c~rrded out three times, and are -then washed for hal~ a.n hour :in running water. They are then placed in a phosphate--buf~ered saline solu-ticn o~ pH 4.5. They are now ready for hydrolysis with f`icin, papain or another sui.t-- able enz~ne.
The pericard.ia crosslinled with adil-)ic acid chlor-ide are in~Ine-rsed in a ficin solu-tion as c~escribed in Example 7 or 8. The temperature is 37C. A~ter ,

3 hours the f~cin sol.u-tion is poured of and the peri.-cardia are washed with runni,ng deionised wa-ter for a quarter of an hour. They are then placed-in an æ.queous solution which contains 11,9 g of sodiwn cI-lori.~e per li-tre, 18 hours later the solu-tion is poured offO
The pericardia are washed with run~ling dei.onised water for a quarter of an hour. They are now ready for after-treatment wi-th a suitable aldehyd.e or for ster:i.lis-ati on, The per:icardia which have been crossl:inked w:i.th adipic acid ch:Loride and hydrolysed wi.th ficin are pl.aced ir,. a 1~ (weigh-t/voll~ne~ aqueous solu-tion of glu-tara;Lde hyde for 24 hours, ~reed from excess aldehyde by washing for t~v.hour,s in running water and s-terilised either with K-I .solution or with e-thylene oxide.
a) S-teril..i,sation with K-l solu-l-i.on The abovementioned pericardia, which finally have been tre.ated wi-th glutaraldehyde and washed, are placed in I~ I soluti,on and seale(l together with this solu-l;ion in a pl,asti.c b~g by hea-t-seal.irlg.
b) ~-te:ri:l,i, a-tion wi-th e~ .ene o~i~e Each of the abovementioned pericardia, which 1 588t~7 6 ~
finally have been treated wi-th glutaraldehyde and washed, is sealed together withO.9% saline solution in a plastic bag, by heat-sealing. The sealed bags are exposed to ethylene oxide gas for ~ hours at 25 to 30~
in a sterilisation apparatus.
Example 1 1 Calf carotid arteries are prepared mechanically, as described in Example 1, and washed with wa-ter, They are then placed in 200 ml of tetrahydrofuran and this liquid is replaced by fresh tetrahydrofuran al~ter 1$ 2 and 4 days. After a total of 7 days, the arteries are placed in a 3.2 % (weight/volume) solution of dodecanedioic acid dichloride in tetrahydrofuran and are left in this solution for 48 hours. They are then placed in tetrahydrofuran for 16 hours, in tetrahydro-furan/buf~er solution (1:1 volume/vol~ne) of pH 4.5 for 8 hours and finally in aqueous buffer solution of pH 4.5 for one day. They are now ready for sterilisation or ~or hydrolysis with ficin.
Exam~ 12 As described in Example 7 or 8, calf carotid arteries are first crosslinked with adipic acid chloride in tetrahydrofuran and then subjected to hydrolysis with ficin. After washing out the sodium chlorite solution as in Example 7, the arteries are placed in 0,5% ~ueous glutaraldehyde solution, After 48 hours the liquid is poured of~. The arteries are then wa,shed for half an hour with running wa~er. E'inally, they are placed in 0.9% sodium chloride solution and introduced together with this liquid into plastic bags and the bags are heat-sealed. The sealed plastic bags are sterilised by exposing to ethylene oxide gas, as described in Example1 under section c).
Examp~
550 g of maize starch are introduced into 3 litres of water and stirred for a quarter of an hour, A solu-tion of 705 g of sodium metaperiodate in 9 litres of water is then added dropwise in the course of 1 hour.
The suspension is stirred for a further 1~ hours. It . .

l 1588()7 is then filtered through filter-paper. The residue on the filter is sllspended in 1.5 litres of water and -the suspension is filterecl again. This process of suspend-ig and fil-tering is repeated five times The residue on the filter, which is still moist, is then introduced into 3 litres of acetone and the rnixture is stirred vigorously for half an hour and then filtered. The filter residue is dried for 42 hours at 40C in vacuo Afterwards, it is ground to ~ fine powder in a ball mill.
13 g of the fine powder are suspended in 1 litre of deionised water. The pH value is adJusted to 8.80 by adding saturated sodium bicarbonate solution.
Arteries, which - as described in Example 7 or 8 -ha~e been crosslinked with adipic acid chloride and hydrolysed with ficin, are then placed in the suspension of aldehyde starch obtained above. The liquid is kept in continuous motion with the aid of a vibrator. After 24 hours the liquid is poured off. The arteries are washed in running water for 30 minutes. They are then placed in PBS solution, sealed together with this liquid in a plastic bag by heat-sealing and steril-ised by exposing to ethylene oxide gas.
Example 14 550 g of maize starch are added to 3 litres of water and the mixture is stirred for a quarter of an hour. A solution of 705 g oX sodium motaperiodate in 9 litres of water is then added dropwise in the course of 1 hour. The suspension is stirred for a further 18 hours. It is then filtered through filter-paper. The residue on the filter is suspended in 1.5 litres of water and the suspension is filtered.~ The process of suspending and filteri.ng off is repeated five times.
Afterwards, the residue is suspended in 3 litres of water. A solution of 695 g of potassium perman-ganate in 12 litres of water is added dropwise in the course of 1 hour, with continuous stirring. The sus-penslon is stirred for a further 18 hours. The mixture is then allowed to stand for 2 hours. During this time a large proportion of the manganese dioxide formed .
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--~ J 1 1 5880 7 settles out. The supernatant suspension is trans ferred care:Eul1y~ with the aid of a siphon, into another vessel; the sediment is discarded.
The suspension is filtered through fi.lter paper. Th~?
residue on the filter is suspended in 1.2 litres of water and the suspension is filtered. The process of suspen~
ding and filtering off is repeated three times. The filter residue is then taken up in 9 litres of 0.5N
hydrochloric acid at 0C and stirred for 1 hour. The suspension is then filtered through filter-paper. The residue on the filter is suspended in 1.2 litres of water and the suspension is filtered. The process of suspending and filtering off is repeated three times. The filter residue is then suspended in 3 litres of acetone and the suspension is stirred for half an hour and filtered. The filter residue is dried for 42 hours at 40C in vacuo and is then ground to a fine powder in a ball mi,ll.
2 g of this powder, which consists of oxidised starch containing a large number of carbo~yl groups, are suspended in 1 litre of water. Mechanically prepared arteries are placed in 150 ml of this s~pension for 2 hours.
The liquid is agit,ated gently w~lth the aid of a vibra-tor. The arteries are then placed in 200 ml of a ' suspension which contains, per litre, 0.26 g of the ab,ovementioned powder and also 25 g of N-ethyl-N~-(3-dimethylaminopropyl)-carbodiimide hydrochloride.
The suspension is subjected to continuous gentle vibra-tion, After 24 hours the liquid is poured off. The arteries are washed for half an hour under running water.
They are then placed in 150 ml of citric acid/phosphate buffer solution of pH 4.5 and ~ubjected to the ficin treatment described in Example 8.

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l 158807 Example 15 Following mechanical preparation 10 calf arteries are placed in a shallow dish and covered with S00 ml of a 1~ solution of citric acid in water(weight/volume). After two hours the arteries are placed in a 500 ml measuring cylinder and covered with a solution of 0.2 g citric acid and 10 g N-ethyl-N'-~3-dimethylamino-propyl)-carbodiimide hydrochloride in 500 ml water. After one day the arteries are washed under running water for 20 minutes and sub-sequently placed in a sterile physiological saline solution. They are sealed together with the liquid in a plastic bag by heat-seal-ing and, as described in Example 1 c), sterilized with ethylene ~ oxide.
i Example 16 Twenty mechanically prepared calf arteries are drawn up on glass rods, placed in a one litre measuring flask and covered with one litre tetrahydrofuran. After two and one half hours the glass rods are carefully removed from the arteries. The tetra-hydrofuran is renewed. After a further five and one half hours the tetrahydrofuran is renewed a second time. Sixteen hours later, i.e. the next morning, the tetrahydrofuran is replaced with a solution of 22 g terephthalic acid dichloride in one litre tetra-hydrofuran. From time to time the liquid is stirred gently with a glass rod. After 24 hours, i.e. the next morning, the solution is replaced with pure tetrahydrofuran. Six hours later the tetra-hydrofuran is replaced with a mixture made of 500 ml each tetra-hydrofuran and citric acid/phosphate buffer solution of pH 4.5.
After two hours the mixture is replaced with a citric acid/phosphate buffer solution of pl~ 4.5. After being left for 16 hours the ficin .
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~ 15880~

treatment mentioned in Example 8 is carried out.
Example 17 Ten calf arteries are mechanically prepared and placed vertically in a 500 ml measuring cylinder filled with tetrahydro-furan. After two and after four hours the liquid is replaced with fresh tetrahydrofuran. After a total of eight hours the liquid is replaced with a solution containing 2 g per litre tri-mesic acid (benzene-1,3-5-tricarboxylic acid) and 20 g per litre dicyclohexylcarbodiimide in tetrahydrofuran. The mixture is swirled a few times and ailowed to stand overnight at room tempera-ture. The next day the arteries are taken out of the solution, washed in three portions with a total of about one litre ethanol/
water (1:1) (volume/volume) and subsequently placed in a citric acid/phosphate buffer solution of pH 6. They may subsequently be treated with icin, as described in Example 7.
Instead oE trimesic acid trimesitinlc acid (=pyridine-2,

4,6-tricarboxylic acid) or a mixture of the two acids in any ratio can also be used.
Example ]8 Eight mechanically prepared arteries are put on ylass rods and placed in lhis state in a 500 ml measuring cylinder in the bottom of which a small magnetic stirrer is placed. Above the magnetic stirrer at a distance of about 1.5 cm from the bottom there is a perforated plate made of porcelain. The glass rods stand on this plate. 500 ml tetrahydrofuran-dimethylsulphoxide (1:1) (volume/volume) is poured in and the magnetic stirrer is set in motion. After two hours the arteries have become sufficient-ly stiff and the glass rods are removed. The liquid is carefully ~ - 20 -.

1S88t)7 ~,'.
sucked off with the aid of a siphon and replaced with the same volume of tetrahydrofuran/dimethylsulphoxide (l:l)(volume/volume).
After a further six hours the liquid is replaced a second time.
The next morning the liquid is replaced with a solution of 10 g - furan-2,5-dicarboxylic acid dichloride in 250 ml tetrahydrofuran, :
again with the help of a siphon, 250 ml dimethylsulphoxide is added and this mixture is allowed to react for 24 hours. The magnetic stirrer remains constantly in motion. The liquid is then replaced .'t with pure dimethylsulphoxide and one hour later with a citric acid/
phosphate buffer solution of pH 5. About two hours later the buffer ,~ solution is renewed and allowed to stand overnight. The arteries are now ready for the ficin treatment according to Example 7 or 8.
Instead of furan-2,5-dicarboxylic acid dichloride tetra-hydrofuran-2,5-dicarboxylic acid dichloride or a mixture of the two ;~ acid chlorides can be used.
Example 19 - Ten mechanically prepared calf arteries are put on glass rods and pl~ced in a 500 ml measuring cylinder in which there is a ;20 saturated solution of D,L-camphoric acid (D,L-1,2,2-trimethylcyclopentan-1~3-cis-dicarboxylic acid) :in water. After 4 hours the arteries are placed in a 500 ml measuring cylinder without the glass rods being removed and covered with a solution of 0.25 g D,L-camphoric acid and 10 g N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide-hydrochloride in 500 ml water. Four hours later the glass rods are carefully removed from the arteries. The next day the arteries are washed for half an hour under running water, placed in a sterile physiological saline solution and sealed together with the liquid in a plastic ~,~
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bag by heat-sealing. Immediately afterwards they are sterilized with ethylene oxide, as described in Example 1 c).

Example 20 .
Ten aorta from pigs, each about 15 cm long, are placed in a two litre measuring cylinder and covered with a tetrahydrofuran--~ dimethylformamide mixture (9:1) (volume/volume) until the volume ,~ comes to two litres. The liquid is renewed twice in the course of twenty-four hours. The liquid is then replaced with a mixture that contains 2 g per litre naphthalene,l,8-dicarboxylic acid and 20 g per litre dicylcohexylcarbodiimide in a tetrahydrofuran-dimethyl-formamide mixture (9:1) (volume/volume). The liquid is swung around several times at intervals of about two hours. The follow-ing day it is replaced with pure tetrahydrofuran, two hours later by a mixture of the same parts (volume/volume) tetrahydrofuran and a citric acid/phosphate buffer solution of pH 5 and a further two hours later with the same buffer solution of pH 5 alone. The aorta are now ready for the ficin treatment as described in Examples 7 ~nd 8.
Exam ~
Following mechanical preparation five trachea from rabbits are`placed in a 200 ml glass beaker, covered with 150 ml tetrahydro-furan and left for four hours. Thereafter, the liquid is replaced with fresh tetrahydrofuran, and again replaced a further four hours later with fresh tetrahydrofuran. The next day the liquid is poured off. Then 150 ml of a 3% solution (weight/volume) of 1,2,3,-4-tetrahydronaphthalene-1,4-cis-dicarboxylic acid dichloride in tetrahydrofuran is poured over the trachea and left for one day.
The liquid is then replaced with tetrahydrofuran, four hours later r ; - 22 -; !.

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with a mixture of tetrahydrofuran and a citric acid/phosphate buffer solution of pH 5 (1:1) (volume/volume), and a further four hours later with a pure citric acid/phosphate buffer solution of pH S. One day later the ficin treatment (see Example 8) is carried out.
Example 22 Six pieces of trachea from calves, each about 12 to 15 ; cm long, are placed vertically in a two litre glass beaker and enough tetrahydrofuran is poured in that all the trachea pieces L0 are covered by the liquid. After three hours the liquid is poured off and replaced with fresh tetrahydrofuran. The liquid is swirled every two hours. After a total of eight hours the liquid is renew-ed a second time. After a total of twenty-four hours the liquid ;.
is replaced with a solution of 2% (weight/volume) adipic acid chlor-'' ~ ide in tetrahydrofuran. The liquid is swirled about every three to four hours. The following day the liquid is poured off and fresh tetrahydrofuran is poured over the trachea pieces. About two hours and again four hours thereafter the liquid is swirled. After a further two hours- it is replaced with a mixture of tetrahydro~uran and a citric acid/phos-phate buffer solution of pH 4.5 (1:1) (volume/volume) and one day later with a pure aqueous citric acid/phosphate buffer solution.
After standing for one day the ficin treatment is carried out as described in Example 8.
Example 23 ;~ ~ Following mechanical preparation a human umbilical vein is placed on an approximately 5 mm thick glass rod and together with it placed in a glass tube that is closed at one end and is approximately 2.5 cm wide and approximately 40 cm long. Enough "
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. , : , - -t 1~8~07 tetrahydrofuran is added that the vein is covered. After two hours the glass rod is carefully pulled out of the vein and the tetra-; hydrofuran is renewed twice in the course of the next twenty-four hours. The liquid is then poured off and replaced with a 2.5%
solution (weight/volume) of naphthalene-1,2-dicarboxylic acid dichloride in tetrahydrofuran. One day later the liquid is replaced with pure tetrahydrofuran, two hours later with a mixture of the ; s~me parts (volume/volume) tetrahydrofuran and a citric acid/phos-phate buffer solution of pH 4.5, and a further four hours later with a pure citric acid~phosphate buffer solution of pH 4.5. The ficin treatment, as described in Example 8, is carried out the following day.
Example 24 One hundred collaterals of calf and cattle carotid art-eries, each about 2 to 4 cm long, are placed in a 200 ml glass beaker and covered with 200 ml tetrahydrofuran. The liquid is re-newed twice in the course of twenty-four hours. Approximately every two hours the content of the glass beaker is carefully swung around. The following day the liquid is replaced with a tetrahydro-; 20 furan solution which contains 20 ml per litre adipic acid chloride ; and 38 ml per litre triethylamine. This liquid is also swirled from time to time. The following day the liquid is poured off and 200 ml tetrahydrofuran is poured over the collaterals. Two '~ hours later the liquid is replaced with a mixture of tetrahydro-; furan and a citric acid/phosphate buffer solution of pH 4.5. (1:1) (volume/volume), and a further two hours later with a pure citric acid/phosphate buffer solution of pH 4.5. One day later the collaterals are washed for a quarter of an hour under running ;
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8~V~7 water r placed in a sterile physiological saline solution, sealed together with the liquid in a plastic bag by heat-sealing and sterilized with ethylene oxide as described in Example 1 c).
Example 25 Ten calf jugular veins, each about 20 to 25 cm long, are put individually on glass rods approximately 10 mm in diameter and together with it placed in glass tubes that are hermetically closed at one end and have an inner width of 2.5 cm and a length of approx-imately 40 cm. Each glass tube is filled with enough tetrahydro-furan that the blood vessels are completely covered by the liquid.
After four hours the glass rods are removed and the li~uid is re-newed. After a fur-ther four hours the liquid is renewed a second time. The following day the liquid is replaced with a 2% solution (weight/volume) of succinic acid dichloride in tetrahydrofuran.
From time to time, about every two to four hours, the tubes are carefully swirled. The next day the tubes are emptied, the veins are placed together in a 500 ml measuring cylinder and covered with approximately 500 ml tetrahydrofuran. Four hours later the liquid is replaced with a mixture of the same parts tetrahydroEuran and a citric acid/phosphate buffer solution oE pll ~.5. ~ further four hours later it is replaced with a pure citric acid/phosphate buffer solution oE pH 4.5. One day later the veins are subjected to the ficin treatment according to Example 8.
Example 26 -Ten calf carotid arteries, each approximately 20 cm long and having an inner width of about 6 to 7 mm, are carefully put on to 8 mm thick glass rods that are rounded off at both ends, are placed in a 500 ml measuring cylinder and covered with enough ' tetrahydrofuran that all the carotid arteries are covered by the liquid. After two hours the glass rods are carefully removed.
The carotid arteries are now quite stiff; they keep the inner diam-eter of 8 mm and afterwards prove to be even more elastic. They are placed in fresh tetrahydrofuran which is renewed after four hours. The following day the liquid is replaced with a 2~ (weight/
volume) solution of glutaric acid dichloride in tetrahydrofuran and left standing for one day. The liquid is then replaced with pure tetrah~drofuran and four hours later with a mixture of equal parts (volume/volume) tetrahydrofuran and a citric acid/phosphate buffer solution of pH 4.5. A further four hours later the liquid is replaced with a citric acid/phosphate buffer solution of pH
4.5. The following day the carotid arteries may be subjected to the ficin treatment described in Example 8.
Example_27 Ten round pieces 3 cm in diameter are cut from a calf pericardium and placed in water for half an hour. Each piece is individually placed on a spur-shaped former which itself is about 8 mm in diameter and which projects about 2 cm from a plate. The pericardium pieces are pressed on all sides from the centre out on to the former with a smooth modelling stick and gradually modelled on to it. They thereby obtain the shape of a sack approximately 8 mm in diameter and 12 mm high. If the former together with the pericardium pieces put over it are placed under a 100 watt spot lamp for 20 minutes, the pericardium pieces dry on it and can be stripped off the block without losing their shape. They are now all placed together in a glass beaker in which there is 150 ml tetrahydrofuran and are carefully pressed under the surface of the liquid. The tetrahydrofuran is renewed after three and after six hours. The following day the liquid is replaced with a 3.2%
solution (weight/volume) of dodecanedioic acid dichloride in tetrahyd-rofuran and the pericardium sacks are left in it for twenty-four hours. The liquid is then replaced with pure tetrahydrofuran, four hours later with a mixture of tetrahydrofuran and a citric acid/
phosphate buffer solution of pH 4.5 (1:1) (volume/volume) and a further four hours later with a pure citric acid/phosphate buffer solution of pH 4.5. The next day the pericardium sacks are placed in a sterile physiological saline solution, sealed together with the liquid in plastic bags and sterilized immediately afterwards with ethylene oxide. They may be used as a tympanic membrane sub-stitute.
Example 28 Five calf arteries, each about 22 cm long and approximate-ly 5 mm wide, are each carefully drawn up on a 3 mm thick, U-shaped glass rod, in which the two legs of the U-shaped glass rod are about 3.5 cm apart. The arteries together with the glass rods are placed in a round ~lass dish approximately 25 cm in diameter and covered with enough tetrahydrofuran that the surface o the liql~id is about 2 cm above the arteries. The glass dish is cover-ed with aluminum foil so that the tetrahydrofuran does not evap-orate. After two hours the glass rods are carefully removed and the tetrahydrofuran is renewed. The arteries now maintain the U-shape. After a furtherfive hours the tetrahydrofuran is renewed a second time. After a total of twenty-four hours the liquid is replaced with a 2.9~ (weight/volume) solution of pyridine-2,3,6-tri-carboxylic acid trichloride in tetrahydrofuran and left to stand ~ `.
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1158~07 for twenty-four hours, covered as before with aluminum foil. The solution is then po~red off and pure tetrahydrofuran is poured over the arteries. Two hours later the liquid is replaced with a mix-ture of equal parts (volume/volume) tetrahydrofuran and a citric acid/phosphate buffer solution of pH 4.5, and a further two hours later with a pure citric a?id,'phosph2te buffer solution of pH 4.5.
The following day the arteries are subjected to the ficin treatment as described in Example 8. 'rhe U-shaped arteries are placed in -the ~easuring cylinder in such a way that the curvatur~ projects downwards and the legs ^f the U project upwards. Following treat-ment with the ficin and sodium chlorite the arteries are placed in a large, approximately 8 cm deep glass dish and washed with runn-ing deionised water Lor a quarter of an hour. A~terwards the water is poured off ~nd the arteries are plac~d in a 0.1% (weight/~olume) aqueous solution of slutaraldchyde. Three days later the liquid is poured off. The arteries are then washed for half an hour under running water. They are then placed in 0.9% sodium chlorite solu-tion, filled together with the solution in plastic bags which are sealed and then sterilized with ethylene oxide as described in Example 1, section c).

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Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of new organ transplants from organs and parts of organs of fish, birds and mammals, characterised in that the said organs or parts of organs are subjected to crosslinking of the macromolecules of the inter-cellular matrix by the formation of amide bonds between the amino groups of the peptide chains, and of ester bonds between the hydroxyl groups of the peptide chains, and the carboxyl groups of dicarboxylic acids, tricarboxylic acids or polycarboxylic acids of the aliphatic, cycloaliphatic, aromatic or heterocyclic series.

2. A process according to claim 1, characterised in that di-, tri- or polycarboxylic acids which contain neither aldehyde or keto groups nor amino groups and in particular those which carry only carboxyl groups and in some cases hydroxyl groups are used.

3. A process according to claim 2, characterised in that the aliphatic carboxylic acids used are those having 3 to 12 carbon atoms, the cycloaliphatic carboxylic acids used are the cyclopen-tane- and cyclohexane-dicarboxylic acids, the aromatic carboxylic acids used are phthalic acid, isophthalic acid and terephthalic acid, trimesic and trimellitic acid and the heterocyclic carboxylic acids used are furan- and tetrahydrofuran-2,5-dicarboxylic acid, oxidised starch and carboxymethylcellulose.

4. A process according to claim 1 characterised in that the resulting intercellular matrix crosslinked by amide bonds is additionally treated with formaldehyde or a dialdehyde.

5. A process according to claim 4, characterised in that the dialdehyde used is dialdehyde starch or glutaraldehyde, preferably the latter.

6. A process according to claim 1, characterised in that, in the resulting intercellular matrix crosslinked by amide bonds, material which potentially can act as an antigen is additionally degraded by hydrolysis with ficin, papain or another protease of the same or similar substrate specificity.

7. A process according to claim 6, characterised in that the hydrolysis is carried out with ficin.

8. A process according to claim 6, characterised in that the resulting matrix, which is crosslinked by amide bonds and is antigen-free, is additionally treated with a dialdehyde.

9. A process according to claim 6, characterised in that the resulting matrix, which is crosslinked by amide bonds is antigen-free, is additionally subjected to further crosslinking by the formation of amide bonds between free amino groups and the carboxyl groups of dicarboxylic acids, tricarboxylic acids or polycarboxy-lic acids of the aliphatic, cycloaliphatic, aromatic or hetercyclic series.

10. Organ transplants obtained by the process according to one of claims 1 and 6.