Development of an ovine collagen-based composite biosynthetic vascular prosthesis

Clinical

Maleriais 9 (1992) 211-223

of an Ovine Collag Biosynthetic Vascular Glenn _A.

wards

Department of Veterinary Clinic and Hospital, University of Melbourne, Princes Highway, Werribee, Victoria 3030, Australia

Bio Nova International

Pty Ltd, 36 Muster

Terrace, North Melbourne, Victoria 3051, Australia

Abstract: The search for an ideal vascular prosthesis to bypass peripheral vascular obstructive lesions is necessary where autologous tissues are either unavailable or unsuitable. This paper will outline the development and use of vascular conduits, principally of biological origin. The clinical benefits and limitations of these materials are discussed. The development of a composite biosynthetic prosthesis (Omniflowa) is described, together with the testing methods used to determine and predict its suitability for use as an arterial substitute. The ovine biosynthetic prosthesis has significantly improved surface and mural properties over previous attempts at producing prostheses for vascular reconstruction. Immunohistological studies on samples recovered from dogs after 4 years show that the original ovine collagen is still present after 4 years, and it is further augmented by the deposition of new, host-derived connective tissue.

We will discuss the clinical benefits and limitations of these materials and describe the development of a composite biosynthetic prosthesis (Omniflow@), together with the testing methods used to determine and predict its suitability for use as an arterial substitute. Special mention will be ma material’s composite structure may overcome some of the limitations of the purely synthetic or biological materials.

TRODUCTION Atherosclerosis and its sequelae are known to be a major cause of death in the Western world. An important manifestation of atherosclerosis is peripheral vascular disease, a progressive obliterative disease of the arterial tree particularly of the lower limbs, resulting in severe claudication, rest pain or ischaemic changes. Therapeutic measures used in cases of peripheral vascular disease are aimed at reducing the progression of the isease and the reduction of its manifestations. In mild cases, conservative methods such as dietary changes, drug therapy and lumbar sympathectomy may be adequate. Surgical methods of management have included endarterectomy procedures, balloon catheter angioplasty and laser thermal angioplasty, and for severe and extensive obliterative lesions, bypass procedures using vascular conduits of various origins. This paper will outline the development and use uits principally of biological origin.

DEVELOPMENT

L CRAFTS

The so-called ‘classical period ’ of vascular surgery probably began with the pivotal wor Guthrie. Between 1901 and 1905 explored the basic patterns of healing of vascular tissues and described techniques for successful arterial reconstruction and tram The ‘modern era ’ of vascular surgery, however, only began following the development of the radiological visualization of the arterial tree, safe 211

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blood transfusion, heparin anticoagulation anaesthetic tecllniques.3

Autogenous

and safe

tissue

A major milestone in the surgical treatment of peripheral obstructive disease occurred with the use of reversed autologous saphenous veins as femoropopliteal bypass conduits, by Kunlin in 1949.4 To date, the autogenous saphenous vein continues to be the material of choice for small diameter arterial bypass procedures, and has been described as the ‘Gold Standard’ against which all potential alternatives must be compared. The success of the autogenous saphenous vein is principally due to the fact that it is a living, natural vessel with a healthy thromboresistent endothelial lining, is flexible, is of adequate size and availability, and is nonantigenic.5 In common with all veins, the saphenous vein has a number of valves along its length which must be rendered ineffective for the vein to be used as an arterial substitute. This is commonly achieved by harvesting the vein from its tissue bed and reversing it, or less commonly by breaking the valves, by various methods, the vein may be left in situ.’ Long-term clinical results for the reversed autogenous saphenous vein (for femoropopliteal reconstruction) report cumulative patency rates of between 50 and 44 % following 10 years implantation Following initial inconsistent, and generally poor, results, the in-situ vein technique has recently been resurrected5 with clinical results reported to be equal or superior to those of the reversed saphenous veins, especially to the distal arteries of narrow diameter. The autogenous saphenous vein is, however, not available to all patients requiring lower limb revascularization. The vein may be unsuitable or absent in a number of patients due to prior removal, varicosities, duplication, sclerosis or inadequate diameter. Between 10 and 15 % of patients in need of a bypass conduit are reported to require an alternative conduit.‘j The saphenous vein is also itself not the perfect arterial conduit. Evidence of significant deterioration of vein grafts has been reported with over 30% of autogenous saphenous veins developing serious defects threatening the long-term function of the graft.7 Failures result from stenotic lesions’, ’ in the medium term and aneurysmal changes or progressive atherosclerotic lesions following 3 to 6 years of implantation.7

The development and use of reliable alternatives to the autologous saphenous vein for lower extremity revascularization continue, therefore, to be a major goal of those involved in the production or use of vascular prostheses. Several biological and synthetic alternative grafts and prostheses are currently available, but none have fully matched the performance of the autologous saphenous vein. Fresh arterial autografts would theoretically appear to be the ideal arterial substitute. The procurement of adequate lengths for peripheral revascularization, however, remains a problem and the use of arterial autografts is usually limited to short segments including renal and carotid artery reconstruction. The use of autologous arm veins has been reported by a small number of authors when the saphenous vein is unavailable.

Homologous

tissues

The first successful clinical report of the use of an arterial homograft was made by Gross et al. in 1949.* The use of the arterial homograft subsequently increased rapidly between 1950 and 1955, but they were later found to suffer a high incidence of degenerative changes including aneurysm formations and their use was subsequently abandoned. Despite this, a small number of groups continue to study and clinically utilize either fresh or preserved homologous saphenous veins. It was suggested that vein allografts recovered from cadavers were less susceptible than arteries to ischaemic changes, as they are not only weakly antigenic, but may be less dependent on the vasa vasorum for nutriti0n.l’ With only isolated exceptions, however, the clinical results achieved with venous homografts have been disappointing. lo Considerable evidence also indicates that, despite earlier beliefs, homograft antigenicity does play a major role in its mode of failure.ll Another homograft, the modified human umbilical vein (MHUV) was first used clinically in 1974.12After initial unsuccessful attempts to use the unmodified umbilical vein, Dardik et al. subsequently selected glutaraldehyde treated umbilical veins with an external polyester (Dacron) mesh for initial clinical trials for limb salvage.12 While early reports ofprostheses recovered from patients showed a general retention of graft architecture, a desmoplastic response around the polyester mesh and patency rates similar to those of autogenous saphenous vein, 12,l3 the long-term results were not as

Ovine collagen-based composite biosynthetic vascular prosthesis

promising. Increasingly, reports of flow surface wrinkling, intimal breakdown, infection and aneurysmal degeneration began to appear. In 1986, Hasson et a1.l4 reported on their review of 60 MHUV implants of greater than 2 years duration. Only 15 of these prostheses had remained patent. Eight prostheses (57 %) were found to have aneurysms and three (21 O/o) showed preaneurysmal changes of diffuse dilatation and mural ulceration. Only one prothesis appeared to be completely normal. The authors considered that the mural degeneration may be caused by a chronic immunological reaction, the likely mechanism being insufficient collagen cross-linking or a time-dependent reversal of glutaraldehyde cross-links. Dardik et on their 10 year al. I5 more recently reported experience with 907 prostheses in 715 patients. In those implants of greater than 5 years duration 36 % had aneurysms and a further 21% showed dilatory changes. With the advent of Duplex scanning techniques for the evaluation of prosthetic function in situ, an aneurysm incidence of 40 to 47 % has now been reported for MHUV prostheses after 3 years implantation.16 Heterologous

tissues

Studies into the preparation and use of heterologous tissues began in 1954 with work on the modified bovine arterial heterograft by Rosenberg et all7 These studies developed from the idea that vessels of heterologous origin might be made nonantigenic by the removal of immunoreactive parenchymatous tissue, mainly smooth muscle, enzymatically to leave a nonreactive insoluble collagenous framework which may then be chemically cross-linked to provide strength and durability. Bovine carotid arteries were placed in the proteolytic enzyme ficin (1%) for 3 h and then tanned with a 1.3 % solution of dialdehyde starch. The modified bovine arterial graft produced in this way was first implanted into the femoropopliteal position in man in 1962.18Further clinical experience with the modified bovine heterograft appeared to be promising and its use in arterial reconstruction procedures increased rapidly. Longterm results, however, demonstrated a high incidence of aneurysms and a decreased resistance to infection.19 Sawyer and coworkerszO have more recently reported on the development of a negatively charged glutaraldehyde tanned (NCGT) bovine heterograft for which improved properties are 18

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claimed. Improved clinical performance has also CGT bovine been reported,lgx ‘O although the heterograft has yet to gain wide clinical acceptance. Synthetic materials

Of the various synthetic prosthese polyester fabric (Dacron) and expan fluoroethylene (PTFE) continue to b to any large extent. It has been well established that for their a porous structure is advantageous performance since it allows host tissue ingrowth to provide a solid anchorage for the neointima.” The porosity of Dacron prosthe s, most of which are based on a woven or knitt textile construction, however, necessitates that most must be preclotted prior to use to fill their interstices with bloo coagulum and prevent catastrophic blood loss following implantation into t Textile prostheses have prov be excellent for such a.5 for thoracic or larger diameter use, abdominal aortic replacement, where the blood flow rate is sufficiently high that surface thrombogenicity is less important than lQ~g”terrn mechanical strength and ease of fabrication. oor clinical results were, however, reported in narrow diameter prostheses (< 8 mm) especially for below-k~.ee reconstruction.” These poor results stimulated further changes, including the development of a noucrimped knitted prosthesis invested with an adherent, spirally wound, external plastic coil to render the wall resistant to kinking and compression and to improve the flow surface characteristics. Improved patency rates have een reported to be associated with these grafts.‘” The coating of fabric prostheses with fibrin glue,2” albuminz4 and collagenz5 has also been described in an attempt to decrease the problems associated with surface thrombogenicity and to decrease the requirement for preclotting of the prosthesis prior to implantation. PTFE, an expanded type of Te on, was created by William Gore in 1970 and was quickly recognized for its extreme inert properties. It was introduced into vascular surgery in 1972.‘” Following initial clinical use for arterial reconstructions, however, a high incidence of aneurysm formation soon became apparemZi and the grafts were subsequently reinforced with a thin outer wrap of PTFE orientated at right angles to the inner layer. As with other prostheses, clinical results were initially very promising, but it soon became apECM 9

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parent that PTFE prostheses do not work well when extended below the knee and in low flow situations. ‘a Failures were reported to result from the development of neointimal hyperplastic stenoses, especially at the distal anastomosis,2g and the progression of distal arterial disease.“g These changes were suggested to result from stresses across the anastomoses created by the low radial elasticity (compliance) of PTFE relative to that of the host artery.

Mandrel-grown fibrocollagenous composite biosynthetic materials Biosynthetic vascular prostheses have been defined by Baier3’ as the combination of tissue and synthetic materials, usually stabilized with some cross-linking reagent, that are meant to function from the start of implantation as thromboresistant conduits inciting minimal reactions in their blood flow surface while fostering integration with surrounding host tissue. Hufnage131 described the use of a porous Dacron mesh implanted over a stent, in this case Teflon, to produce a fibrocollagenous tube containing the supporting mesh within its wall. In 1969, Sparks3’ subsequently experimented with a device called a ‘tissue die ‘, which consisted of an outer tubular shell of stainless steel punctured by many small holes, an inner highly polished tubular mandrel of stainless steel and a middle anchoring skeletal tube of Dacron. The tissue die was implanted subcutaneously for 5 to 12 weeks, after which it was disassembled, leaving an autogenous tissue tube containing Dacron buried within its walls. The use of autogenous grafts produced by this method for use in a femoropopliteal bypass was reported. In 1973, Sparks33 further reported on various mandrel preparations, using silicone rods covered with Dacron mesh, without the use of rigid metallic stents. Clinically, femoropopliteal artery grafts were produced in situ by the implantation of the silicone/Dacron matrix of the desired diameter through a stab wound over the common femoral artery running distally to the level of the popliteal artery. Six to eight weeks later, the ends of the graft were mobilized, the silicone rod was withdrawn and the graft was anastomosed proximally and distally to the obstructed arterial segment. Although early evaluation by Sparks revealed 90 % patency in 10 femoral popliteal bypasses at 1 to 9 months,33 a review of 48 of these patients at 7 years was quite discouraging. 34 This report was then followed by

further reports which described low patency rates associated with a high incidence of complications, including early thrombosis, aneurysmal degeneration and late lumenal narrowing.35 Structural weakening and aneurysm formation resulted from a lack of cross-links between the collagen fibres and poor maturation of the collagenous tube during the short implant period. Guidoin et aL3’ further commented that tissue repair varies from species to species, between individuals and from site to site within an individual. Strict equivalence of mandrel performance in diseased humans, relative to healthy animals, cannot, therefore, be expected. These prostheses have now been generally abandoned for arterial reconstruction in humans.

AN OVINE COLLAGEN-BASED COMPOSITE BIOSYNTHETIC PROSTHESIS The combined concepts of the ‘Sparks mandrel ’ and the possibility of fixation of tissues with glutaraldehyde led Ketharanathan and Christie3’ to construct a mandrel-grown ovine collagen prosthesis, which may be transplanted between species. This ovine biosynthetic prosthesis is formed when silicone mandrels covered with a polyester mesh become encapsulated with ovine collagen following implantation for 12 to 13 weeks beneath the cutaneous trunci muscles of adult sheep, a process which ensures that the polyester mesh forms an integral part of the prosthetic structure. 11,36-38(Fig. l(a)). The tubes are excised and trimmed of the excess fat and connective tissue, then fixed with 2 % glutaraldehyde, processed and stored in 50 % ethyl alcohol. A major advantage of this technology has been the possibility of producing prostheses of predetermined dimensions, fashioned by the judicious selection of the appropriate mesh/mandrel combination for diameter size and physical characteristics. Following the initial reports of the successful use of this prosthesis (Omniflow-Bio Nova International, Melbourne, Australia) in both animal models and man, 11,36*38the prosthesis has been in-vivo evaluaand subjected to in-vitro tion 30,37,39-44 In selecting the testing methods used, it was recognized that a vascular conduit needs to maintain its structural integrity in a biologically dynamic and challenging environment for the lifetime of the patient, and that some predictive information on its

Ovine collagen-based composite biosynthetic vascular prosthesis

215

Fig. 1. (a) The ovine biosynthetic

prosthesis (6 mm i.d.). (b) Histology, using H&E staining, of an explant showing the fully integrated mesh fibres within a compact collagen matrix. (Bar = 50 pm.)

potential performance is important in assessing the material. The physical and chemical characterization of the prosthesis is an important initial step in establishing the potential usefulness of the material. Physical and chemical characterization forms the basis of the vascular prosthesis standards adopted as an American National Standard.4’ Such information is, however, difficult to interpret for biological-based materials because of their inherent variability and anisotropy. A novel set of test criteria may need to be developed for each material type. A number of investigators have proposed a definition of the properties or performance criteria of an ideal prosthesis.30,46 In general, an ideal prosthesis is expected to have the following characteristics : has biocompatibility, allowing for healing with some tissue incorporation. ciently elastic and easy to handle and suture.

1. It

3. It is haemocompatible and ~o~immu~ogeni~. 4. It has strength and durability, both physically and biologically. 5. It is reproducible. Methods used to define the physical and chemical characteristics of the ovine biosynthetic prosthesis are designed to provide quantitative information related to these performance criteria.“‘, 4* The possible relationship between such criteria and the series of tests used within our laboratories is shown in Table 1. A number of these tests are also used routinely during the production process to ensure that quality assurance standards are met.42

Structural integrity and Morphology

The structure of the ovine biosynthetic vascular both prosthesis has been describe , using lightll.38,41 and scanning electron microscopy.3oz41 IX-2

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G. A. Edwards, G. Roberts

Table 1. Relationship criteria

of laboratory

tests

to performance

Table 2. Physical and chemical characteristics of the Omniflow prosthesis (Average values and standard deviations, n = 99)

Structural integrity and biocompatibility potential of tissue incorporation Morphology Collagen content Shrink temperature Porosity

Collagen (% dry wt) Shrink temperature (“C) Porosity” (ml cm-2 min-l x 10m3) Compliance (% inner in radius x 10m7) Critical surface tension (dyne cmds)

Elasticity, ease of handling Compliance

a As a control, the modified human umbilical average porosity of 5 x 10e3 ml cm-2 min-l.

Surface haemocompatibility Infrared spectrum Critical surface tension In-vivo performance Strength, Tensile Fatigue In-vivo

durability and burst strength strength durability

Both techniques show that the mesh becomes fully encased with collagen during its implantation in sheep (Fig. l(b)). Collagen deposited between the mesh and silicone mandrel produces the smooth compact intimal layer of the resulting prosthesis. The thickness of this layer exhibits some variation, being least at positions where the knots in the mesh are found and greatest where no knot is present. The inner surface of the intimal layer where it has been in contact with the silicone mandrel is composed of closely packed elongated fibroblasts. Collagen is found in a circumferentially orientated array parallel to the surface of the prosthesis. Where it surrounds the mesh the collagen fibres generally show a circular arrangement with growth between the individual filaments of the mesh fibres (Fig. l(b)). Fibroblasts aligned with individual synthetic and collagen fibres are found throughout the tissue matrix. Foreign body giant cells and macrophages are found adjacent to the mesh fibres.38.41 Small vessels are only occasionally observed within the wall of the prosthesis and specific stains indicate that the tissue matrix contains little elastin.41 Where elastin is present, it is associated with the small vessels or in the outermost layers. Collagen content

The measurement of the total collagen content of the ovine biosynthetic prosthesis has been reported by Roberts et aL40 and Wadelton and Roberts4’ and is used as a quality control measure of the structural integrity of the prosthesis. Collagen analysis is performed by calorimetrically measuring the hydroxyproline liberated in acid hydrolysis, essentially

44.7 * 10.9 81.3kO.88 <3 3.08 * 1.3 23-26 veins had an

according to the method described by Stegemann47 The percentage dry weight of collagen in the ovine biosynthetic prosthesis is 44.69 2 10.89 % (n = 198)40,42 (Table 2). Immunohistological studies by Ramshaw et aL41 of the collagens within the wall of the prosthesis show that type III collagen is distributed throughout the prosthesis, but is particularly prominent in the innermost layer of the graft and around individual polymer fibre filaments where fibroblast proliferation is evident. The ratio of type III to type I collagen by electrophoretic analysis indicates the presence of a high content of type III collagen (40-500/)o w h en compared to normal dermis. The high content of type III collagen is consistent with that found in foreign body granuloma tissue surrounding sutures in liver tissue and provides useful physical properties, as normal blood vessels are characterized by a high content of this collagen type. Shrink temperature

The shrink temperature (T,) or thermal denaturation temperature of a protein is the temperature at which the collagen molecules within the tissue matrix become denatured.40 The T, is used as an index of the extent of cross-links induced by the glutaraldehyde processing, between and within the collagen fibrils. The T, curve for fully tanned collagen based materials plateaus at just over 80 “C as all available cross-linking sites are occupied. The T, for the processed ovine biosynthetic prosthesis is measured to be 81.3 & 0.9 “C (Table 2). Collagen materials fully cross-linked with glutaraldehyde generally resist resorption in situ, and their structural integrity is maintained.20 Incomplete tannage, on the other hand, may result in the biodegradation of the material, and is reflected in a low T,. Porosity

It has long been recognized that the porosity of an

Ovine collagen-based composite biosynthetic vascular prosthesis

implanted synthetic material is a critical determinant in the subsequent host tissue reaction to that implant.” The significance of the porosity of a biological material on the host response however, remains controversial. It has been recognized that ‘host tissue incorporation into the ovine biosynthetic prosthesis does occur.3g An important application of porosity measurement of biological prostheses is that damage to the prosthetic wall during collection and processing will be reflected by sharp increases in porosity.“O, 42 The porosity of the ovine biosynthetic prosthesis (Table 2) is measured by connecting the sample prosthesis to a burette filled with 50 % ethyl alcohol and pressurized to 25 kPa with air. The amount of water flowing through the wall over a 5 min period is measured, and the porosity is recorded as the volume of fluid lost (in ml cm-’ mine1 x 10-3).40

Elasticity : compli

Compliance is a useful measure of the radial elasticity of a vascular prosthesis.40,48 It may be defined as the percentage change in radius per unit pressure increase between 50 and 100 mm Hg40 and is measured using a strain gauge similar to that and host artery should have closely related compliance values.30*4g%50 A compliance mismatch has been suggested to cause alterations in blood flow patterns and shear stresses, resulting in intimal damage and thrombus formation.“’ The development of anastomotic neointimal hyperplasia has also been reported to be related to a compliance mismatch and the subsequent transanastomotic stresses between the prosthesis and the host artery.‘l Intimal hyperplasia is the abnormal, continued proliferation and overgrowth of smooth muscle cells in response to endothelial injury at anastomotic sites. Generally, biologically derived grafts have a more desirable compliant wall relative to the stiffer synthetic materials. Recent work has also described an increased progression of atherosclerosis distal to low compliant PTFE grafts compared to more compliant (autogenous saphenous vein) ASV in femoropopliteal reconstructions.” The compliance of the ovine biosynthetic prosthesis measured in our laboratories was 3.08 + 1.3 (Table 2). Walden et ~1.~’in comparing the compliance of human femoral arteries and veins with MHUV, eterografts, knitted Dacron and PTFE compliance values of 5.9, 4.4, 3.7, 26, 1.9

217

and 1.6, respectively. A number of studies with these materials have reported results suggesting that increased patency correlates with a decreased compliance mismatch.4g~‘o Hamilton et al.,‘” on the other hand, found a strong correlation between initial compliance of biological vascular prostheses and an increased compliance with in-vitro collagenase treatment. They suggest that prostheses with high initial compliance may thus be more susceptible to enzymatic degradation in situ, with subsequent mural degeneration mation such as that seen in the heterograft. The compliance of the ovine biosynthetic prosthesis being intermediate to that of the biological and synthetic prostheses may, therefore, combine atencies associated the advantages of improved with the biological alternative with the durability of the synthetic prostheses. Surface haemocompatibility In-vitro

methods of surface characterization and invivo performance are both used as important indicators of the haemocompatibility of the prosthetic surfaces. Infrared spectroscopy

The infrared spectrum (IRS) of a material surface is a very useful ‘fingerprint’ of the surface structure, and finds additional application in identifying surface contamination by substances such as lipids.30,40 The IRS is measured in our laboratories with a Perkin-Elmer model 298 spectrophometer, using a KRS-5 internal reflectance crystal. The I of the ovine biosynthetic prosthesis exhibits lume wall compositions dominated by proteinaceous components and diminished lipid contents over prior-art specimens. 3o The similarity in spectral patterns of tracing between ovine biosynthetic prosthesis and host arterial (dog) flow surfaces suggests that both have a similar surface chemistry. Critical surface tension

Critical surface tension (CST) is a measure of the free surface energy of the blood contact surface of the prosthesis. It is determined by plot of the cosine of the contact angle (8) ma by several purified liquids of various surface tensions placed on the surface against the surface tension of that liquid. 3oThe CST is the surface tension below which complete spreading is observed. Thus, the straight

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G. A. Edwards, G. Roberts

Fig. 2. Histology, using Sirius Red staining of an explant, showing an anastomosis between the aorta (A) and an ovine biosynthetic prosthesis (V) with a polyester mesh bundle (M). A thin layer of pannus (P) is seen extending over the anastomosis. (Bar = 100 pm.) (From Ref. 39, with permission.)

line intercept corresponding to zero contact angle (i.e. cos 19= 1) is the critical surface tension. A CST in the range 22-28 dyne cme2 has been suggested to be favourable for optimal blood compatibility,30 characteristic of endothelialized surfaces, and has been strongly correlated with longterm vascular prosthetic patency.30 The CST for the ovine biosynthetic prosthesis, measured at the Arvin-Claspan Center in New York3’ as well as our own laboratories, indicate that the prosthesis has an average CST of 23 to 26 dyne cmm2 (Table 2).

In-vivo performance

Extensive in-vivo testing of the ovine biosynthetic prosthesis has indicated that the material is basically haemocompatible with the lumen becoming covered with a thin, smooth neointima of compacted fibrin. ‘I, 36-3g, 43,44 Complete endothelialization of 35 mm segments of the prosthesis implanted into the infra-renal aortae of dogs occurred within 9 months. Endothelialization, however, remained incomplete in dogs receiving aorto-iliac bypass prostheses up to 4 years.3g,43,44 In an extensive series of 63 ovine biosynthetic implants, used as aorto-iliac bypasses in dogs for up to 4 years duration3’ the thrombus-free surface area varied from 80 to 100% in all but three prostheses. Subintimal haemorrhagic staining of the flow surface within the hood of the end-to-side aortic anastomosis was evident in 80% of the prostheses, but this did not appear to compromise

the intimal surface. A pannus ingrowth composed of fibroblasts and smooth muscle cells, extending from the tunica intima and media of the host arteries over the surface of the prosthesis (Fig. 2), increased in length up to 20 mm with implant duration, but failed to cause significant narrowing of the anastomoses. Immunohistological examination of samples recovered from the midsection of prostheses, using specific monoclonal antibodies (MAbs) to different collagen types, has been used to study the neo- or pseudo-intimal lining which had formed on the prosthesis. 44 These studies showed that, at 4 years, type III host-derived collagen was a predominant component of the new flow surface, with type I collagen being less abundant. Type V collagen was also found to be present, apparently associated with the endothelial cell layer, and evident in early explants, for example, at 6 months, rather than older, 4 year explants.

Strength and durability

Collagen is the major component of a large number of biomaterials.53 In certain applications, such as for burn or wound management, a material with rapid collagen turnover is used to provide a template for new tissue deposition. In vascular prostheses, however, it is necessary to minimize the turnover of the collagen so that structural integrity of the device is maintained. Control of collagen

Ovine collagen-based composite biosynthetic vusculav prosthesis

turnover can be achieved by the chemical crosslinking of the collagen during the manufacturing process. Glutaraldehyde has been the most commonly used agent to induce these cross-links, thereby increasing the material’s biocompatibility and durability.2Q. 53 While chemically cross-linked biological materials have been used for arterial substitutes in the absence of suitable autogenous saphenous vein for over 3.5 yearsI many of them have undergone biodegradation with subsequent aneurysm formation after 2 or more years.14-16’lg Despite the expectation from extensive animal implantation and in-vitro studies of a more favourable result, problems with these prostheses have come to light only after prolonged clinical use. The concept of strength and durability testing of a vascular prosthesis to predict its likely clinical performance is thus a critical and controversial issue. Classic techniques used in the mechanical evaluation of textile grafts45 are not always applicable to prostheses of biological origin. Each device may require a novel set of evaluation criteria, which will be a function of the intrinsic properties of the device. The durability of an implantable vascular device is a function of both the material strength and the biological response to the material. In the case of biological materials, the durability is, therefore, dependent on their primary connective tissue structure together with the degree and stability of the cross-links induced by the chemical modification. We have adopted an approach for the durability testing of the ovine biosynthetic prosthesis similar to that described by Hayashi.j4 This is a three-way approach involving in-vitro mechanical testing, invitro fatigue testing and in-vivo biocompatibility and endurance testing. In-vitro mechanical

testing

The mechanical strength of the prosthesis is determined most easily by both tensile and burststrength testing methods. Temile testing, as described by several investigators, j4 is one of the more difficult properties to interpret due to the natural variations in wall thickness and lack of homogenicity in most biological tissues. Ovine biosynthetic prostheses, like most natural biological tissues, exhibit a typical exponential stress-strain curve with a large initial deformation at low loads. The prostheses are also anisotropic and exhibit twice as much compliance in the circumferential direction than longitudinally,

219

but are four times stronger lo~~it~di~aliy, ties which are probably related to the orient the collagen fibrils. The test does show, however; that the forces required to achieve a primary fracture in the collagen structure are far greater than hose encountered in vivo. Although uniaxial tensile testing is convenient for routine assessment of material elasticity and strength, these tests cannot fully predict the multiaxial behaviom of the intact prosthesis under pressure, since a pressurized tube is under a complex state of stress. Burst strength testing, using uncut tubed prostheses, has been described’” and is a more appropriate test of the ultimate strength of a material, which depends on the integral structure of its wall. Because of the low porosity of the ovine biosynthetic prosthesis, direct pressurization is an effective method by which to measure the burst strength. The sample is connected between a reservoir containing 50% ethanol and a peristaltic pump with a pressure monitoring gauge. The flow rate is increased against a control valve in increments of 15 kPa until 250 kPa (168X mm Hg) is reached.40 All Omnitlow grafts are able to support this pressure. MHUV has been reported to sustain a pressure of greater than 1000 mm. HgS5 In general, commercial biologically based prostheses do not fail as a result of low initial strength. Stress testing beyond physiological limits may, in fact, produce failure modes not encountered in the implant environment. Mechanical strength testing, however, is a useful measure of the structural integrity of a commercial prosthesis. All Omniflow grafts are pressurized to 35 kPa prior to selection and packaging.40, 42 Fatigue testing

A more appropriate in-vitro measure of material durability is its resistance to fatigue. The fatigue strength of a vascular prosthesis can be determined by cyclic pressure loading.3” Our laboratories use two methods for testing the fatigue strength of vascular prostheses : a high load, low cycle test and a low load, high cycle test. A high load, low cycle test or accelerated fatigue test subjects the prosthesis to a pulsatile flow of physiological saline at 37” C, und stress for up to 96 h (135 beats min-l at a pulse ressure of 0 to 25 kPa).40 The test samples are, t efore, subjected to pulse rates and pressures greater than those encountered physiologically. The integrity of the y measuring the prosthetic wall is determined porosity of the wall at 24 h intervals.

220

G. A. Edwards,

I I

0

5

I

10 BEATS (Millions)

I

15

20

Fig. 3. Results of the low load, high cycle or mock physiological fatigue test for modified human umbilical vein (HUVG) (n = 2) and ovine biosynthetic prostheses (Omniflow) (n = 6). Structural integrity is determined by the measurement of porosity over the duration of the test. 0, omniflow; +, MHUV.

Thirty specimens were subjected to this test and all survived with only moderate increases in porosity. All specimens tested also had a final burst strength in excess of 175 kPa. Two prostheses were pumped for extended periods of 264 and 2016 h prior to failure. Allowing for biological variability, it is evident that the ovine biosynthetic prosthesis does not weaken significantly under these conditions. There was no evidence of aneurysm or sites of dilatation. Baier3’ similarly reported excellent physical integrity by subjecting the ovine biosynthetic prosthesis to similar tests for 1 million pulses in a test bath deliberately kept nonsterile in order to challenge the prosthesis with infective microorganisms during mechanical stressing. A low load, high cycle test or mock physiological in-vitro test exposes the prosthesis to cyclical loading within physiological parameters. The test prostheses are attached to a pulsatile pumping system similar to the AFT test. The pulse pressure ranges from 80 to 140 mm Hg with a pulse rate of 135 to 140 beats minl for the equivalent of 20 million beats. The integrity of the prosthetic wall is again determined by intermittent measurement of the porosity. In a comparative study, six ovine biosynthetic prostheses and two modified human umbilical vein prostheses were subjected to this test. All prostheses withstood the full duration of the test. The performance (porosity) of the MHUV prostheses remained comparable to the ovine biosynthetic prostheses for the first 12 million beats (Fig. 3). After this, a trend toward deteriorating integrity was found for the MHUV prostheses relative to the

G. Roberts

ovine biosynthetic prostheses. In the final burst strength tests after 20 million beats, all ovine biosynthetic prostheses withstood pressures in excess of 175 kPa, while both MHUV samples burst at around 120 kPa. Histologically, the wall of the post-test ovine biosynthetic prostheses remained intact. The MHUV prostheses, however, exhibited an inner compact layer of smooth muscle cells and a thicker outer layer of loosely woven connective tissue and elastic fibres which showed extensive separation of the elements. The ovine biosynthetic vascular prosthesis thus appears to have excellent physical integrity following both in-vitro mechanical and fatigue testing. This property is probably a result of the fully integrated structure of the synthetic and biological component of the prosthesis, in which the fibrils of the polyester mesh are closely invested with mature, compact cross-linked collagen fibres (Fig. 1(b)). It has been reported, however, that the loss of structural integrity of collagen-based devices could also be due to the resorption of collagen from the device in situ, possibly due to the reversal of the chemically induced cross-links5’ The biocompatibility and biodurability of the prosthesis‘ must, therefore, be ultimately determined by long-term implantation within an in-vivo model.

In-vivo durability testing

Ketharanathan and Christie3(j were the first to report the results of dog implants in 1980. Ovine biosynthetic prostheses placed into the infrarenal aortae of 7 dogs maintained a 100 % patency for up to 3 years. During the period of evaluation, there was no angiographic evidence of dilation or of progressive stenosis. Excellent host tissue incorporation was reported externally. Christie et aZ.38and Perloffll also reported on the use of these prostheses of various diameters and lengths placed into the infrarenal aortae (n = 9), common iliac arteries (n = 7) and aorto-iliac (n = 10) bypass positions in dogs and the infrarenal aortae of rats (n = 29). A 100% patency rate was maintained in the three positions in dogs for up to 3 years. Seventy-nine percent (23/29) of the prostheses implanted into rats remained patent at 6 months. Ketharanathan and Christie36 reported on a comparative study in which both ovine biosynthetic and PTFE prostheses were implanted as parallel cortiliae bypass grafts in 10 dogs. All prostheses

O&e

collagen-based composite biosynthetic vascular prosthesis

remained patent for periods up to and exceeding 23 months. No prosthesis showed aneurysmal dilatation. Fibrinous tissue ingrowth through the wall of the PTFE prostheses resulted in progressive and irregular laminal narrowing. This was not seen in the tanned ovine collagen grafts. Wilson and Klement 43 later reported on a comparative in-vivo study in which ovine biosynthetic prostheses and MHUV prostheses were individually implanted as aorto-iliac arterial bypasses in dogs for up to 3 years without the use of anticoagulation or antiplatelet agents. A total of 47 ovine biosynthetic grafts were implanted, 12 of which were examined at over 1 year follow-up and four examined after 3 years. Of the 15 Dardik biografts, 5 were explanted at 1 to 2.5 years followup. Graft patency was 80.8 % (38 out of 47) for the ovine biosynthetic prostheses compared with 73.3 % (11 out of 15) for the Dardik biografts. None of the prostheses showed fusiform aneurysm formation. ardik biografts, however, were distinguished by extensive wrinkling and ridging of the flow surface in association with a more extensive coverage of mural thrombus, presumably due to turbulent blood flow induced by the irregular flow surface. These findings are essentially similar to those of early clinical changes associated with mural degenerationl” The authors commented that the presence of the reinforcing mesh incorporated near the inner surface of the ovine biosynthetic grafts may be important in preventing this problem. In contrast, the reinforcing polyester mesh in the Dardik biografts is applied external to the graft and this allows considerable focal deformations to occur at the inner aspect of the graft wall resulting in the wrinkling and ridging of the flow surface and focal out-pouching which effectively became ‘mini-aneurysms ‘_ Histopathological examination showed that foci of microcalcification and cartilaginous metaplasia were present in both prosthetic types, as were areas of haemorrhage and fibrin infiltration into the graft wall. These findings were, however, considerably more extensive in the Dardik biografts than in the ovine biosynthetic prostheses. Microcalcification has been rep in numerous glutaraldehyde-fixed connective t structures implanted in the body for long periods.53 There was no evidence in this study that this had any significant effect on prosthetic function. The extent of such calcification dex of degenerative change in the in this sense, the lesser extent of 19

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such changes in the ovine biosynthetic grafts may be a favourable indicator in regard to their longterm biological fate. Biodurability of the ovine biosy~t~~ti~ prosthesis was further tested in our laboratories by the implantation of 63, 100 to 120 mm long, ‘6 mm internal diameter prostheses into the aorto-iliac bypass position in dogs for varying periods up to 4 years.3g The explanted samples were examined for patency, structural stability and morphology, including the absence of aneurysms, and the extent of any thrombus formation. A cumulative patency of 66% at 4 years was achieved. Gross examination of patent prostheses showed a smooth neointimal surface and good host tissue incorporation on the adventitial surface. No aneurysmal or other changes likely to threaten the function of the prostheses were detected. The failures reported were largely the result of technical errors, with only one directly related to a defect in the prosthesis (exposed mesh), resulting in thrombosis. A highly species-specific MAb, which can distinguish sheep type III collagen from that of dog, was also used for immunoperoxidase staining to demonstrate the persistence of the original, glutaraldehyde-stabilized ovine type III collagen after 4 years of implantation.3g An Ab to type III collagen with a different species specificity was also used to show the presence of newly deposited, hostderived type III collagen in the prosthesis. The persistence of the original collagen after 4 years implantation, still forming an integral composite structure with the polyester mesh, and the deposition of new host-derived collagen contribute to the observed structural integrity of the prosthesis and minimize the likelihood of aneurysm formation which has been observed for other biological, vascular replacements. 14-=,lg

The first clinical implants of the ovine biosynthetic (OmniAow@‘) prosthesis for arterial substitution in the lower limbs of 14 patients for limb salvage were reported by Christie et d3” A patient was reported to have had successful left and right main coronary artery bypasses with the prosthesis. The Omniflow prosthesis has been marketed in Europe and Australia since 1984 with in excess of 1000 confirmed peripheral implants. Clinical reports of prosthetic performance are consistent with ECM 9

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G. A. Edwards, G. Roberts

the experimental data, showing no deterioration of the prosthesis up to 5 years implantation, and a cumulative patency rate of 44 % has been reported for limb salvage for that period.56

10.

11.

CONCLUSIONS The Omniflow mesh-reinforced tissue prosthesis shows significantly improved surface and mural properties over previous attempts at producing biological tubes for vascular substitution or repair. The improved prosthesis characterized here overcomes previously noted problems with the ‘Sparks mandrel concept’ by specific control of the growth period in ovine subdermal locations prior to harvesting, and by preservation with glutaraldehyde-based tanning fluids to strengthen significantly the tissue tubes, as well as to suppress immunological sites. The result is the production of tissue tubes with a fully incorporated reinforcing mesh, displaying high structural and mechanical integrity and lumenal wall properties associated with adequate haemocompatible performance. The ovine biosynthetic prostheses also possess the advantages over biological tubes of natural origin of being readily available, convenient to use and obtainable in a variety of lengths, diameters and shapes.

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