38
A New Vascular Prosthesis of Bovine Origin /\1. I1hlt er. and H. Erasmi Department of Surgery. University Hospital. Cologne. Germany
Sum ma r y
x e ue bovin e Kcllagen -Geraup rothesen
An experimental study on a new biologic vasc ular graft for reconstruction of small diamet er arteries was carried out in 50 sheep. A follow- up period of two yea rs let us concl ude that the new vascular prosthesis may be "re -utilized" by the hosr. tntection and aneurys matlc degeneration could not be obse rved. An ea rly occlusion of the graft occ urred in only onc case. due to
In einer tierex perimentellen Studie wurde cine nach einem neuen Pra parat ionsverfahren hergestellte GefaBprothese zurn
Key words
Vascular graft- Xenogenous vascular prothesis - Collagen I
Befunde erlauben den SchluB. daBdurch das Prap araticn sverfahren die lIerstellung einer GefaBprothese gelungen lst. die vern Empfanger reutilisiert we rde n kann und nicht nur die Funktion einer Leitschiene er ftillt. urn sparer vom Empfange rorganismus vollsta ndig ersetzt zu \.. 'crden. Die bei xenogenen Grafts beschriebenen degenerativen vcranderungen mit Aneurysmabildung konnten nicht beobachtet we rden . Protheseninfekt ic nen traten nicht a uf Bel 50 implantiertcn bovinen Kollagenprothesen kam es nur in ein em Fall zu einem technisch bcdingten FrUhversagcn des Gcfa ficrsutze s.
In trod ucti on
Prep aration procedure
Recent advances in vasc ular surgery cannot be considered without suitable prosthetic materials. Reconstruction of arteria l blood vessels has frequ ently bee n und ert aken since the int ro duction of synthetic grafts. The first one of clinical interes t was the so-called Vinyon- N-Prosthests. created by Voorhees in 1952 (20). Since th en a lot of different vascular prost heses. pr epared with the use of synt hetics or of'btologica l origin. hav e been created. Surgical techniques have been improved and brought to wo rld -wide standards . Thus. a large number of patients suffering pain orthre ate ned by the loss of their extre mities hav e been su ccess fully tr eated . On pr inciple. synthetic and biological vas cular gralis can be differentiat ed . As far as alloplastic mat eria ls are conce rne d. Dacron- and PTFE-protheses a re su ccessful and now well established in vascular surgery. Their main use is in th e reconstruction oflarge-diameter blood vess els . As soca lled biological grafts. the autogenous sapheno us vein . and in a rema rka bly smaller num ber Dardick's umb ilical vein graft s. and ovine prost heses are of clinical inter est (J - 3. 5). The autogenous sa phenous ve in. not a vascular prosthesis in the rea l sense, ca nnot be used in 25 %- 30 % of patients due to small diameter. varicosis or after Babcock's proce du re (4. 19). Therefor e furthe r investigations on biological vascular grafts are necessary.
Any preparation procedure for xenoge nous vascular grafts known till now has been bas ed on the one descri bed by Rosenberg in 1956 (8- 10). The only common feature of the procedure we use and Rosenberg's is the use of ficin as a proteolytic enzyme . All other steps ha ve been modified . or invented by ourse lves (11-15). The materials used in this study a re femoral arte ries of bovine or igin. harvested immed iate ly afte r slaug htering cattle. They ca n be sto red in ice-water for a maximum of 24 hours until further pre paration . Proteolytic treatment is carried out to remove all cellular, muscle, an d elasti c tissu e as we ll as collagen fibers type III. This is don e by imm ersion of th e arteries in a solution containing 10 g offi cin and 3 g of'l-cyste ine per liter. The enzymatical stripping has to be ca rried out at a temperatu re of 37 °C, standard phosphate-citrat e buffer being employed to keep pH at 6.0 for 6-24 hou rs . They are then washed in ru nning wa ter for 24 hours. The absorbed enzyme is inactivated by imme rsion of the grafts in a 1 % aq uaous solution of sodium-chlorite (NaCIO,) for 24 hours. afte r which th ey are again was hed in running water, Stabilisation of the grafts requires a trea tment in a 3 % dialdehyde-starch solution for 7 days at room te mperatu re . They are once more wash ed in running water for 24 hours .
Thorae. cardiovasc. Surgeon 40 (1992 ) 38- 4 1 © GeorgThieme VerlagStuttgart · New York
Received for Publication : August 1, 1991
Downloaded by: Universite de Sherbrooke. Copyrighted material.
technical reasons.
Ersatz kleinkalibriger Arterien untersucht. Die tiber ei ne n Nach unte rs uchu ngszeitraum von his zu 2 Jahren gewo nnene n
Treatm ent with sodium borohydrid e (NaBH4 ) turns the collagen tub es to an insoluble sta te in acetous and basi c medium. Becoming more flexible the collagenous tissue keeps its tensile stre ngth. The br eaking strength of the fibers corres ponds to stee l wires of the same diameter (21- 23). As a side reaction, the material becomes pallid, losing its yellow tint cause d by tanning. Redu ction is carried out in an aquaous solution contai ning 1 g sodium -borohydride/ liter for 24 hours. To remove the sodium-borohydride, it is agai n was hed in run ning water . Byshrinking the grafts in heat on glass -rods ofa certai n diam eter , the wa ll of the tubes becomes imperm eable, the pr osth esis is non-por ous. Furtherm ore, a certa in elasticity, corres ponding to arterial blood-vessels, is obtaine d. Using conical glas s-rods even grafts with a conical sha pe can be pr epared "to measur e". Sterilisation is performed chemically usin g a solution whi ch conta ins 50 % ethanol and 1% propylene oxide/liter . As pr oven in pr evious investigations this pr ocedure is quite suita ble for biological mat erials (18,2 4,25 ).
Thorac. cardiouasc. S urgeon 40 (1992)
Fig.1 Prosthesis30 days afterimplantationincarotida l position. Prox. anastomotic region, H. E., 600x
Materials an d methods In animal experiments we used 50 shee p of both sexes , aged between 2 an d 4 years and of a max. weight of 75 kg. The carotid artery in 30 and the abdo mina l aorta in 20 sheep was resected and replaced by one of our newly prepa red grafts. The carotidal grafts varied from 4-5 mm in diameter, and 6-8 em in length , whereas in aortic grafts the diameter varied from 6-8 mm, and the length 8-10 ern. The grafts have been studied for a maxi mum of 2 years. Dur ing this period experimental an imals were sacrificed at given times, i. e. 3, 6, 9, 12, and 24 months postoperatively. Sonographic and X-ray investigations were perform ed before harvesting the implant ed prosth eses. After sac rificing the experimental ani mals, light and electronm icrosco pic studies were carried out. Res ults An ea rly occlusion, due to technical reasons, occurred in only one case. All othe r implanted grafts wer e functioning at the time of harvesting. Thirty days postoper atively the thickness of the implant ed graft s' walls and of the neo-intima and the width of mesh in the collagen I struct ure were ana lyzed using light-mi croscopic techniques. The thickness of the prosthetic wall varied between 300 an d 500 /L m . The neoendot helium measured 100 - 250/Lm , the width of mesh ranged from 2.5-20.5 /Lm and 30 .1-130 /Lm. Both the proximal and dista l terminations of the arterial blood vesse ls showe d a regu lar structure. The media adjoining the grafts showe d a pau city in muscle cells an d an infiltration with fibers of collagen I. Blood-vessels and pr osthesis were linked firmly by connecti ng tissue (Fig. 1). Near the suture mater ial, small amo unts of coagulate d blood and fibrin , hem osiderin and fibroblasts could be identified. In the region of anasto mosis, the inte rnal layer see med to be slightly thicker than within the grafts . The prostheses were completely coated by a newly formed intern al layer . The sub endoth elial tissu e, conta ining fibroblasts, fibrocytes, and a sma ll amount of newly built collagen, seemed to be fixed st rongly to the "ne o-intima" and the gra ft. Other cells
39
Fig. 2 Prosthesis 30days afterimplantation incarotidal position. Internal layer, prox. anastomotic region, H.E., 600x
could not be found. Elastic fiber s and muscle cells wer e missing as well as so-called vasa vaso rum. Ninety days postop eratively the inner layer in the vascular prosth esis was att ached smoot hly. In the anas tomotic regions small vessels were in evidence. The inner layer had been differentiated to a smooth endothelial layer (Fig. 2). In the central regions of the grafts, a lot of polygonal cells besides adjusted cells form ed like spindles could be found. These cells could be identified as smoot h-muscle cells by their form and structure as well as by stai ning . This could be proved by electron-microsco pic techniques (Fig. 3). Results after 6 months showed a rectilineal evolvement of the findings 90 days after implantation. The endothelial layer was now well differentiated, showing no differ ence to normal blood vessels. One year after implantation, the endothelial layer had become even smoot her. Even the anastomotic regions showed a uniform distrib ution of cells an d connecting tissue. A lar ge amount of cells ha d invaded the grafts by now. Smooth-muscle cells had increase d distinctly in numb er . The walls of the grafts still seemed to be unchan ged 720 days postoperatively. Thickness correlated to the form er implant ed vascular pr osth esis. The endothelial layer was completely differ entiated by now to a small one-cell layer , a "neo-endothelium" (Fig. 4). Gra fts had been accept ed and incorp orated completely by the host.
Downloaded by: Universite de Sherbrooke. Copyrighted material.
A New Vascular Prosth esis of Bonin e Origin
Thorae. eardiovase. Surgeon 40 (1992)
M. Wa lter, and JI. Erasmi Fig.3 Smoothmuscle cells inthe graft.Electron-microscopic study
Fig.4 ' Neo-enoothelium"inthemiddle ofthegraft. 720days afterimplantationin abdominalaorticposition, H. E., 600x
reached by shrinking the grafts in heat. Furthermore, by this means the graft becomes non -porous, its inner surface is sealed. Thus prepared, collagen implants are not only used as a "live rail" by the host , but are invaded by cells, which finally lead to incorporation and "revitalisation " of the implanted pros thesis (21, 23). Aneurysmal dilatation, a well-known prob lem in biological vascular grafts (1, 2, 6, 7, 16-1 8), could not be demonstrated . The biological gr aft present ed in this paper underwent an increasing invas ion by fibrobla sts, rising from the surrounding tissue. It was not discernible whether cellular structure s aro se from circulating blood. Alread y 90 days after implantation the inner surface of the graft is covered completely by a thin layer, which can be interpreted as a "neo-endothelium". The increasing invasion by cells is quite remarkable. During a period of 2 years cells ar e gradually differ entiat ed into elastic and mu scular elements. At last, the layers of the graft walls correspond to regular arteries. This process had not come to a final stand-still after 2 yea rs. Destruction of the grafts' walls was not evident. The gra ft is, ra ther, incorporat ed by the host. The xenogenous grafts known till now have been quite susceptib le to infection (17). Besides th e degeneration ofthe graft , this is considered to be the most sever e complication in biological vascular prostheses and has led to resign ation finally. Seege and Amgwerd (18) reported a 4-6 % rate of infection using xenografts in arterial recon struction . Schlosser even had an infection rate ofl2 % (17). We did not observe infections in our experimental study. Thus , no comments can be mad e on the behavior of the new collagen graft in case of infection. Local treatment of infection in experimental studies is quite exceptional and mentioned quite infrequ ently in the liter ature. This data should therefore be judged with caution. Hyperp lasia of the intimal layer, one of the main problems in small ves sel replacement. was not in evidence. The patency rate was invariable in both ca rotida! and aortic positions. Our new graft seemed to be similar in handling and consistency to autogenous veins. We draw the following conclusions from our experiment al study. The new biological vascular graft seems to be quite suitabl e for sma ll-vessel replacement du e to its stabi!ity, elasticity, the lack of thrombogenicity, and absence of antigenic reactions. Her eby must be borne in mind that res ults obta ine d over a period of 2 years have to be considered critically. Nervertheless, our results are encouraging enough to carry out furth er investigations on this probl em.
Discussion
The replacem ent of small-diameter vessels ha s not been solved satisfactorily so far. Even autogenous venous grafts, with which the best results are obtained nowada ys, ar e not a suitable material in every case (3, 6, 19). Further investigations have to be carried out to improve prosthetic materials for the reconstruction of arterial blood vesse ls damaged by occlusion or aneurysm. The new xenogen vascular graft presented in this pap er undergoes enzymatic debridement by an efficient proteolytic enzyme ficin, derived from the sap of the fig tr ee (23). Finally a tub e resu lts formed only offibers of collagen type I. By redu ction , for the first time in history, the collagen fibers ar e rendered into an insoluble state (22, 24). Elasticity is
References 1
1
3
4
Anders. A., R.lIiiring, undE. Renk : Langzeiterfahrunge n mit formalinbehandelten allogenen Saphena - und Nabelvenenim plantaten in der Shun tchirurgie. ln: Keese, K. E. Ied.l Gefal3 und Patient., BadenBade n, Witzstroc k Verlag 1980 . 209 Baier , R. E., C. K. Akers, and S. Perlmutter: Processed Human Umbilical Cord Veins for Vascular Reconstructive Surgery. Trans . Am. Soc. Artif Intern. Organs 22 (19 76) 5 14 B6hmig, H. 1.. G. Zeidler, F. Hamoncourt, und B. Fischer: Orthograder freier Vene nbypass in der Behan dlung Iemoropo plitealer arterie ller Versch liisse : 10 Jahres Ergebnis. Angio 10 (1988 ) 85 Cranley. 1. 1.. and C. D . Haf ner: New Prosthetic Materialscompa red with Autogenous Saphenous Vein for Occlus ive Arterial Disease of the Lower Extremity . Sugery 89 (1981) 2
Downloaded by: Universite de Sherbrooke. Copyrighted material.
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Thom e. ca rdiova sc. Surgeon 40 (1992)
A New Vascula r Pros th es is ofBovine Orig in Dardtk. If.. J. M. Ibrah im. M. Kahn. and 1. J. Dardik : Long-term Expe rience with the Glutaraldeh yde-stabilized Human Cord Vein in 634 Lower Extre mity Hevasculartz ation Bypass Procedures. In: Mercier . C . R. Shy (eds .) Gluta ralde hyde-stabilized Umbilical Cord Vein lDardik-Biografil in Vascular Reconstruction. Res. and Clin.
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Gerou la nos . S t.: Bioprothesen . Huber Verlag. Bern 1985 Rosenb erg. N: Dialdehyde Sta rch-ta nned Bovine Heter ografts. In: Stan ley. J . C.. W. E. Burkel, S. M. Lindena uer . R. H. Bartl ett . J. G. Turcotte Ieds .) Biologic and Synthe tic Vascular Protheses.• New York. Grune & Stra tton 1982.4 23 Ros enberg. N.. J. Henderson. and 1. F. Douglas: The Use of Arteria l Implants Prep ar ed by Enzymatic Modification of Arteriai ll eterografts. II. The Physical Properties of Elstica and Collagen Componen ts of the Arte rial Wall. AMAArch . Surg . 74 (1957)89 Rosenberg. N . 1. Henderson. and G. H. Lord: Collagen Arter ial Prosth esis of Heterologous Vas cular Origin: Physical Propertie s and Behaviour as an Arterial Graft. In: Sawyer . P. N. (ed.l Biophysical Mechanism s in Vascular Hemostasis and Intravascular Throm bosis. New York. Appleton. Cent ury. Crofts 1965 . 316 Ros enberg. N.. E. R. Kaugh eran. J. Henderson. G. /I. Lord. and J. F. Doug/as: The Use of Segmental Arterial Implants Prepared by Enzymat ic Modlflcatlon of Heter ologous Blood Vessels. Surg. Foru m 6
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Rosenberg. N.• an d G. Lord : Prestenotic Turbulence in Arteries as a Cause for Mural Thrombosis and Embolism. J. Card iovasc. Surg. 6
Sc hafe r. K.. F. Utz. und If. Stiegler: Erfahru ngen mit boviner Carotis als cenuersae . In: Muller-Wiefel. H. Ied .) Gera8ersa tz.. Baden Baden . Witzstr ock Verlag 1980. 82 Schlosser. V.. B. Vrbany, und N. Thieme: Spater gebnisse nac h hete rologem Gefa8ersa tz in der reko nst ru ktiven Arter ienchi ru rgie. Chi-
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Sege. 5.. und D. A mqu-erd : Der bovine Graft als GeraBersatz bel arter iellen Verschltissen der un teren Extre mitiit. ln: Muller -Wlefel. H. Ied.l Gefafler satz. Baden -Baden . Witzst rock Verlag. 1980. 70 Seqesser, L. liO". und B. Faidutti: Ocr infra popliteale Bypass beim [ungeren En.....achsenen - Lan gzeitresultate im Vergleich. Anglo 7 11985)257 Voorhee s. A. B.. A. Jaret zki, and A. II. Blakemore: Use of Tubes
Const ructed from Vinyon MW Cloth in Bridging Arter ial Defects, Prelimin ary Report . Ann. Surg. 135 (1952 ) 332 Walt er. M.. U. Brenner. W. Hotz miitler. and 1. M, Milller: Experi men ts with Biological Material for Closure of Incisiona l Hernias. Laborato ry Animals 21 (1987) 195 Wall er. Mo . V. Brenner. IV. Holz mutler. und 1. .\1. uuner. Bovines Per icard zum VerschluB von Ba uchwanddefekten . Z. expo Chir. Tran spl. Kunstl. Organ e 20/6 (1987) 328 Walt er. At.. V. Bren ner. H. Kell er. und J. M. Miiller: Experime ntelle Unte rsuchungen mit einem biologischen Material zum VerschluB von Bauchd eckend efekten. Langenbecks Arch. Chir. 363 09851 253 Walter. AI.. J. .\1. .\filller. H. Kelle r. un d U. Brenner: Ossifikation am Kollagenin terponat. Akt. Tra uma . 15/ 6 (1985) 254 Walt er. M.• und P. Walt er: Dermodress zur Versorgung grob flachiger Haut defekte. Chir. Praxis 32 (1984)1 2i
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Ros enberg. N.. G. If. Lord. and J. Henderson: Collagenous Arteri al Graft of Bovine Origin: Seven- Year Observ ations in the Dog. Surgery
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Rosenberg, N.. A Martinez . and P. N. Sawyer: Tanned Collagen Arter ial Prosthesis of Bovine Carotid Origin in Man. Ann . Surg. 164 119661247
Prio.Doz. Dr. M. Walter
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Rosenberg, N.. 1. E. Thompson . and 1. M. Kes hishia n: The Modified Bovine Arterial Graft. Arch. Surg. 111 (1976) 222 Soqi.A : P. Walter. M. /I . Walter. S. Mahler. Y. Be" Yakar. and M. At Feuchturanqe r. Der modres s: A New Tem porary Skin Substitute for Exten sive Dee p Burn Covera ge. P!ast. Reconstr. Surg. II(1985) 223
Chir. Univ-Klini k Kdln-Lindenthal Chir. Kreislauflabor Jose ph-Stelzma nn-Str . 9
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6711970195 1
IS
D-5000 Koln 41
Germa ny
Downloaded by: Universite de Sherbrooke. Copyrighted material.
S
41
A New Vascular Prosthesis of Bovine Origin /\1. I1hlt er. and H. Erasmi Department of Surgery. University Hospital. Cologne. Germany
Sum ma r y
x e ue bovin e Kcllagen -Geraup rothesen
An experimental study on a new biologic vasc ular graft for reconstruction of small diamet er arteries was carried out in 50 sheep. A follow- up period of two yea rs let us concl ude that the new vascular prosthesis may be "re -utilized" by the hosr. tntection and aneurys matlc degeneration could not be obse rved. An ea rly occlusion of the graft occ urred in only onc case. due to
In einer tierex perimentellen Studie wurde cine nach einem neuen Pra parat ionsverfahren hergestellte GefaBprothese zurn
Key words
Vascular graft- Xenogenous vascular prothesis - Collagen I
Befunde erlauben den SchluB. daBdurch das Prap araticn sverfahren die lIerstellung einer GefaBprothese gelungen lst. die vern Empfanger reutilisiert we rde n kann und nicht nur die Funktion einer Leitschiene er ftillt. urn sparer vom Empfange rorganismus vollsta ndig ersetzt zu \.. 'crden. Die bei xenogenen Grafts beschriebenen degenerativen vcranderungen mit Aneurysmabildung konnten nicht beobachtet we rden . Protheseninfekt ic nen traten nicht a uf Bel 50 implantiertcn bovinen Kollagenprothesen kam es nur in ein em Fall zu einem technisch bcdingten FrUhversagcn des Gcfa ficrsutze s.
In trod ucti on
Prep aration procedure
Recent advances in vasc ular surgery cannot be considered without suitable prosthetic materials. Reconstruction of arteria l blood vessels has frequ ently bee n und ert aken since the int ro duction of synthetic grafts. The first one of clinical interes t was the so-called Vinyon- N-Prosthests. created by Voorhees in 1952 (20). Since th en a lot of different vascular prost heses. pr epared with the use of synt hetics or of'btologica l origin. hav e been created. Surgical techniques have been improved and brought to wo rld -wide standards . Thus. a large number of patients suffering pain orthre ate ned by the loss of their extre mities hav e been su ccess fully tr eated . On pr inciple. synthetic and biological vas cular gralis can be differentiat ed . As far as alloplastic mat eria ls are conce rne d. Dacron- and PTFE-protheses a re su ccessful and now well established in vascular surgery. Their main use is in th e reconstruction oflarge-diameter blood vess els . As soca lled biological grafts. the autogenous sapheno us vein . and in a rema rka bly smaller num ber Dardick's umb ilical vein graft s. and ovine prost heses are of clinical inter est (J - 3. 5). The autogenous sa phenous ve in. not a vascular prosthesis in the rea l sense, ca nnot be used in 25 %- 30 % of patients due to small diameter. varicosis or after Babcock's proce du re (4. 19). Therefor e furthe r investigations on biological vascular grafts are necessary.
Any preparation procedure for xenoge nous vascular grafts known till now has been bas ed on the one descri bed by Rosenberg in 1956 (8- 10). The only common feature of the procedure we use and Rosenberg's is the use of ficin as a proteolytic enzyme . All other steps ha ve been modified . or invented by ourse lves (11-15). The materials used in this study a re femoral arte ries of bovine or igin. harvested immed iate ly afte r slaug htering cattle. They ca n be sto red in ice-water for a maximum of 24 hours until further pre paration . Proteolytic treatment is carried out to remove all cellular, muscle, an d elasti c tissu e as we ll as collagen fibers type III. This is don e by imm ersion of th e arteries in a solution containing 10 g offi cin and 3 g of'l-cyste ine per liter. The enzymatical stripping has to be ca rried out at a temperatu re of 37 °C, standard phosphate-citrat e buffer being employed to keep pH at 6.0 for 6-24 hou rs . They are then washed in ru nning wa ter for 24 hours. The absorbed enzyme is inactivated by imme rsion of the grafts in a 1 % aq uaous solution of sodium-chlorite (NaCIO,) for 24 hours. afte r which th ey are again was hed in running water, Stabilisation of the grafts requires a trea tment in a 3 % dialdehyde-starch solution for 7 days at room te mperatu re . They are once more wash ed in running water for 24 hours .
Thorae. cardiovasc. Surgeon 40 (1992 ) 38- 4 1 © GeorgThieme VerlagStuttgart · New York
Received for Publication : August 1, 1991
Downloaded by: Universite de Sherbrooke. Copyrighted material.
technical reasons.
Ersatz kleinkalibriger Arterien untersucht. Die tiber ei ne n Nach unte rs uchu ngszeitraum von his zu 2 Jahren gewo nnene n
Treatm ent with sodium borohydrid e (NaBH4 ) turns the collagen tub es to an insoluble sta te in acetous and basi c medium. Becoming more flexible the collagenous tissue keeps its tensile stre ngth. The br eaking strength of the fibers corres ponds to stee l wires of the same diameter (21- 23). As a side reaction, the material becomes pallid, losing its yellow tint cause d by tanning. Redu ction is carried out in an aquaous solution contai ning 1 g sodium -borohydride/ liter for 24 hours. To remove the sodium-borohydride, it is agai n was hed in run ning water . Byshrinking the grafts in heat on glass -rods ofa certai n diam eter , the wa ll of the tubes becomes imperm eable, the pr osth esis is non-por ous. Furtherm ore, a certa in elasticity, corres ponding to arterial blood-vessels, is obtaine d. Using conical glas s-rods even grafts with a conical sha pe can be pr epared "to measur e". Sterilisation is performed chemically usin g a solution whi ch conta ins 50 % ethanol and 1% propylene oxide/liter . As pr oven in pr evious investigations this pr ocedure is quite suita ble for biological mat erials (18,2 4,25 ).
Thorac. cardiouasc. S urgeon 40 (1992)
Fig.1 Prosthesis30 days afterimplantationincarotida l position. Prox. anastomotic region, H. E., 600x
Materials an d methods In animal experiments we used 50 shee p of both sexes , aged between 2 an d 4 years and of a max. weight of 75 kg. The carotid artery in 30 and the abdo mina l aorta in 20 sheep was resected and replaced by one of our newly prepa red grafts. The carotidal grafts varied from 4-5 mm in diameter, and 6-8 em in length , whereas in aortic grafts the diameter varied from 6-8 mm, and the length 8-10 ern. The grafts have been studied for a maxi mum of 2 years. Dur ing this period experimental an imals were sacrificed at given times, i. e. 3, 6, 9, 12, and 24 months postoperatively. Sonographic and X-ray investigations were perform ed before harvesting the implant ed prosth eses. After sac rificing the experimental ani mals, light and electronm icrosco pic studies were carried out. Res ults An ea rly occlusion, due to technical reasons, occurred in only one case. All othe r implanted grafts wer e functioning at the time of harvesting. Thirty days postoper atively the thickness of the implant ed graft s' walls and of the neo-intima and the width of mesh in the collagen I struct ure were ana lyzed using light-mi croscopic techniques. The thickness of the prosthetic wall varied between 300 an d 500 /L m . The neoendot helium measured 100 - 250/Lm , the width of mesh ranged from 2.5-20.5 /Lm and 30 .1-130 /Lm. Both the proximal and dista l terminations of the arterial blood vesse ls showe d a regu lar structure. The media adjoining the grafts showe d a pau city in muscle cells an d an infiltration with fibers of collagen I. Blood-vessels and pr osthesis were linked firmly by connecti ng tissue (Fig. 1). Near the suture mater ial, small amo unts of coagulate d blood and fibrin , hem osiderin and fibroblasts could be identified. In the region of anasto mosis, the inte rnal layer see med to be slightly thicker than within the grafts . The prostheses were completely coated by a newly formed intern al layer . The sub endoth elial tissu e, conta ining fibroblasts, fibrocytes, and a sma ll amount of newly built collagen, seemed to be fixed st rongly to the "ne o-intima" and the gra ft. Other cells
39
Fig. 2 Prosthesis 30days afterimplantation incarotidal position. Internal layer, prox. anastomotic region, H.E., 600x
could not be found. Elastic fiber s and muscle cells wer e missing as well as so-called vasa vaso rum. Ninety days postop eratively the inner layer in the vascular prosth esis was att ached smoot hly. In the anas tomotic regions small vessels were in evidence. The inner layer had been differentiated to a smooth endothelial layer (Fig. 2). In the central regions of the grafts, a lot of polygonal cells besides adjusted cells form ed like spindles could be found. These cells could be identified as smoot h-muscle cells by their form and structure as well as by stai ning . This could be proved by electron-microsco pic techniques (Fig. 3). Results after 6 months showed a rectilineal evolvement of the findings 90 days after implantation. The endothelial layer was now well differentiated, showing no differ ence to normal blood vessels. One year after implantation, the endothelial layer had become even smoot her. Even the anastomotic regions showed a uniform distrib ution of cells an d connecting tissue. A lar ge amount of cells ha d invaded the grafts by now. Smooth-muscle cells had increase d distinctly in numb er . The walls of the grafts still seemed to be unchan ged 720 days postoperatively. Thickness correlated to the form er implant ed vascular pr osth esis. The endothelial layer was completely differ entiated by now to a small one-cell layer , a "neo-endothelium" (Fig. 4). Gra fts had been accept ed and incorp orated completely by the host.
Downloaded by: Universite de Sherbrooke. Copyrighted material.
A New Vascular Prosth esis of Bonin e Origin
Thorae. eardiovase. Surgeon 40 (1992)
M. Wa lter, and JI. Erasmi Fig.3 Smoothmuscle cells inthe graft.Electron-microscopic study
Fig.4 ' Neo-enoothelium"inthemiddle ofthegraft. 720days afterimplantationin abdominalaorticposition, H. E., 600x
reached by shrinking the grafts in heat. Furthermore, by this means the graft becomes non -porous, its inner surface is sealed. Thus prepared, collagen implants are not only used as a "live rail" by the host , but are invaded by cells, which finally lead to incorporation and "revitalisation " of the implanted pros thesis (21, 23). Aneurysmal dilatation, a well-known prob lem in biological vascular grafts (1, 2, 6, 7, 16-1 8), could not be demonstrated . The biological gr aft present ed in this paper underwent an increasing invas ion by fibrobla sts, rising from the surrounding tissue. It was not discernible whether cellular structure s aro se from circulating blood. Alread y 90 days after implantation the inner surface of the graft is covered completely by a thin layer, which can be interpreted as a "neo-endothelium". The increasing invasion by cells is quite remarkable. During a period of 2 years cells ar e gradually differ entiat ed into elastic and mu scular elements. At last, the layers of the graft walls correspond to regular arteries. This process had not come to a final stand-still after 2 yea rs. Destruction of the grafts' walls was not evident. The gra ft is, ra ther, incorporat ed by the host. The xenogenous grafts known till now have been quite susceptib le to infection (17). Besides th e degeneration ofthe graft , this is considered to be the most sever e complication in biological vascular prostheses and has led to resign ation finally. Seege and Amgwerd (18) reported a 4-6 % rate of infection using xenografts in arterial recon struction . Schlosser even had an infection rate ofl2 % (17). We did not observe infections in our experimental study. Thus , no comments can be mad e on the behavior of the new collagen graft in case of infection. Local treatment of infection in experimental studies is quite exceptional and mentioned quite infrequ ently in the liter ature. This data should therefore be judged with caution. Hyperp lasia of the intimal layer, one of the main problems in small ves sel replacement. was not in evidence. The patency rate was invariable in both ca rotida! and aortic positions. Our new graft seemed to be similar in handling and consistency to autogenous veins. We draw the following conclusions from our experiment al study. The new biological vascular graft seems to be quite suitabl e for sma ll-vessel replacement du e to its stabi!ity, elasticity, the lack of thrombogenicity, and absence of antigenic reactions. Her eby must be borne in mind that res ults obta ine d over a period of 2 years have to be considered critically. Nervertheless, our results are encouraging enough to carry out furth er investigations on this probl em.
Discussion
The replacem ent of small-diameter vessels ha s not been solved satisfactorily so far. Even autogenous venous grafts, with which the best results are obtained nowada ys, ar e not a suitable material in every case (3, 6, 19). Further investigations have to be carried out to improve prosthetic materials for the reconstruction of arterial blood vesse ls damaged by occlusion or aneurysm. The new xenogen vascular graft presented in this pap er undergoes enzymatic debridement by an efficient proteolytic enzyme ficin, derived from the sap of the fig tr ee (23). Finally a tub e resu lts formed only offibers of collagen type I. By redu ction , for the first time in history, the collagen fibers ar e rendered into an insoluble state (22, 24). Elasticity is
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