The Red Kangaroo pericardium as a material source for the manufacture of percutaneous heart valves.

BACKGROUND: The kangaroo pericardium might be considered to be a good candidate material for use in the manufacture of the leaflets of percutaneous heart valves based upon the unique lifestyle. The diet consists of herbs, forbs and strubs. The kangaroo pericardium holds an undulated structure of collagen. MATERIAL AND METHOD: A Red Kangaroo was obtained after a traffic fatality and the pericardium was dissected. Four compasses were cut from four different sites: auricular (AUR), atrial (ATR), sternoperitoneal (SPL) and phrenopericardial (PPL). They were investigated by means of scanning electron microscopy, light microscopy and transmission electron microscopy. RESULTS: All the samples showed dense and wavy collagen bundles without vascularisation from both the epicardium and the parietal pericardium. The AUR and the ATR were 150±25µm thick whereas the SPL and the PPL were thinner at 120±20µm. The surface of the epicardium was smooth and glistening. The filaments of collagen were well individualized without any aggregation, but the banding was poorly defined and somewhat blurry. CONCLUSION: This detailed morphological analysis of the kangaroo pericardium illustrated a surface resistant to thrombosis and physical characteristics resistant to fatigue. The morphological characteristics of the kangaroo pericardium indicate that it represents an outstanding alternative to the current sources e.g., bovine and porcine. However, procurement of tissues from the wild raises supply and sanitary issues. Health concerns based upon sanitary uncertainty and reliability of supply of wild animals remain real problems.

Microstructural alterations owing to handling of bovine pericardium to manufacture bioprosthetic heart valves: A potential risk for cusp dehiscence.

INTRODUCTION: Cross-linking and anti-calcification of prosthetic heart valves have been continuously improved to prevent degeneration and calcification. However, non-calcific structural deteriorations such as cuspal dehiscences along the stent still require further analysis. MATERIAL AND METHOD: Based upon the previous analysis of an explanted valve after 7 years, a fresh commercial aortic valve was embedded in poly(methyl methacrylate) (PMMA) and cut into slices to ensure the detailed observation of the assembly and material structures. A pericardial patch embossed to provide the adequate shape of the cusps was investigated after paraffin embedding and appropriate staining. The microstructural damages that occurred during manufacturing process were identified and evaluated by light microscopy, polarized microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: The wavy collagen bundles, the key structure of the pericardium patch, were damaged to a great extent at suture sites along the stent and in the compressed areas around the stent post. The fixation of the embossed pericardium patch along the plots of the stent aggravated the microstructural modifications. The damages mainly appeared as the elimination of collagen bundle waviness and delamination between the bundles. CONCLUSION: Considering the modes of failure of the explant, the damages to the collagen bundles may identify the vulnerable sites that play an important role in the cusp dehiscence of heart valve implants. Such information is important to the manufacturers. Recommendations to prevent in vivo cusp dehiscence can therefore be formulated.

Correlation between structural changes and acute thrombogenicity in transcatheter pericardium valves after crimping and balloon deployment.

INTRODUCTION: Transcathether heart valve replacement has gained considerable acceptance during the last decades. It is now part of the armamentarium for aortic valve replacement. The procedure proved to be highly efficient. However the issues of the blood compatibility and tissue durability were not raised and the adverse events were probably under-reported, according to observations of thrombosis after deployment. MATERIAL AND METHOD: Bovine pericardium leaflets were sewn inside a 26mm diameter stainless steel stent to manufacture these valves (one control and two experimental). The correlation between the trauma and the acute thombogenicity of bovine pericardium leaflets, after crimping and ballooning, was investigated via an in vitro blood flow with labeled platelets. These leaflets were processed for histology: scanning electron microscopy, light microscopy, and transmission electron microscopy. RESULTS: The control specimens showed a regular pericardium structure with some blood cells deposited on the collagen fibrous surface (inflow) and scarce blood cells deposited on the serous surface (outflow). After crimping and ballooning, the structure of the pericardium was severely injured, eventually with delaminations and ruptures. The blood cell uptake was considerably increased compared to the control. CONCLUSION: It would therefore be appropriate to pay more attention to the design of the valves. Specifically, the incorporation of a buffer tissue or fabric between the pericardium and the metallic stent is suggested. The issue of ballooning deserves detailed and in depth investigation regarding the lifetime of the device.

Transcatheter heart valve crimping and the protecting effects of a polyester cuff.

INTRODUCTION: Prior to deployment, the percutaneous heart valves must be crimped and loaded into sheaths of diameters that can be as low as 6mm for a 23mm diameter valve. However, as the valve leaflets are fragile, any damage caused during this crimping process may contribute to reducing its long-term durability in vivo. MATERIAL AND METHOD: Bovine pericardium percutaneous valves were manufactured as follows. The leaflets were sutured on a nitinol frame. A polyester cuff fabric served as a buffer between the pericardium and the stent. Two valves were crimped and one valve was used as control. The valves were examined in gross observation and micro-CT scan and then the leaflets were processed for histology and analyzed in scanning electron microscopy, light microscopy and transmission electron microscopy. RESULT: Crimping of the valves resulted in the increase thickness of the leaflets and there was no evidence of additional delamination. The heavy prints of the stents were irregularly distributed on the outflow surface in the crimped devices and were shallow and did not penetrate throughout the thickness of the leaflets. However, the wavy microscopy of collagen fiber bundles was well preserved. They were found to remain individualized without any agglutination as shown by the regular banding appearance. CONCLUSION: Crimping of self-deployable valves per se caused only minor damages to the leaflets. However, the procedure could be refined in order to minimize areas of high pressure and swelling of the tissue that can be accompanied with flow surface disruption and increase of the hydraulic conductance. The incorporation of a polyester buffer serves to prevent the deleterious effects that may be caused if the pericardium tissue were in direct contact with the nitinol stent.

Ultrasonic atomization and subsequent desolvation for monoclonal antibody (mAb) to the glycoprotein (GP) IIIa receptor into drug eluting stent.

An eluting-stent system with mAb dispersed in the PLLA (poly (L-lactic acid)) was validated in vitro. Specifically designed spray equipment based on the principle of ultrasonic atomization was used to produce a thin continuous PLLA (poly (L-lactic acid)) polymer coating incorporating monoclonal antibody (mAb). This PLLA coating was observed in light microscopy (LM) and scanning electron microscopy (SEM). The concentration of the monoclonal antibody (mAb) to the platelet glycoprotein (GP) IIIa receptor and the eluting rate were then measured by a radioisotope technique with (125)I-labelled GP IIIa mAb. An in vitro perfusion circuit was designed to evaluate the release rates at different velocities (10 or 20 ml min(-1)). The PLLA coating was thin and transparent, uniformly distributed on the surface of the stent. Three factors influenced its thickness: PLLA concentration, duration and gas pressure. The concentration of mAb was influenced by the duration of absorption and the concentration of the mAb solution; the maximum was 1662.23 + or - 38.83 ng. The eluting rate was fast for the first 2 h, then decreased slowly and attained 80% after 2 weeks. This ultrasonic atomization spray equipment and technological process to prepare protein eluting-stents were proved to be effective and reliable.

Posttransplant antibodies and fresh venous allograft failure in dogs.

Small diameter arterial reconstruction is usually achieved by use of the autologous long saphenous vein. As an alternative to this blood conduit, the venous allograft has been used with some success in the past, but is likely to be the target of an immune rejection reaction from the host. This study was designed to characterize humoral immune reactions possibly involved in the outcome of venous allografts. Ten mongrel dogs received a histoincompatible femoral vein allograft and an autograft as interposition grafts to both femoral arteries. They were investigated for donor-specific antibody development using donor splenocytes and cultured vascular endothelial cells (EC). Serum samples were collected at surgery, at 2 weeks, and every month until graft occlusion occurred. All autografts were patent at retrieval except one, and all allografts underwent thrombosis. In all dogs, donor-specific IgG development was observed that appeared specifically at 4 weeks and lasted until graft occlusion was detected. All reactive sera were cytotoxic to donor EC except one, and this reactivity was completely lost after serum absorption on donor splenocytes. This latter absorption resulted in the total loss of flow cytometric reactivity against donor EC in 3 dogs, whereas a low reactivity was still present in 4 dogs. Immunoblotting analysis showed a posttransplant reactivity against various protein bands on donor EC. Absorption of the reactive serum on donor splenocytes resulted in the loss of reactivity to proteins of approximately 40, 30, and 22 kDa in most experiments. Moreover, as demonstrated by immunofluorescence on cryostat sections of explanted grafts, IgG deposition was seen mainly in the media and the adventitia of the allografts but not in autografts. These results suggest that a donor-specific antibody response directed mainly against MHC antigens might play a role in the thrombosis of histoincompatible venous allografts, thus decreasing the patency rate.

Fresh venous allografts in peripheral arterial reconstruction in dogs. Effects of histocompatibility and of short-term immunosuppression with cyclosporine A and mycophenolate mofetil.

To date, no arterial substitute has been shown to be as effective as the autologous saphenous vein in peripheral revascularization procedures. In the present study, the venous allograft was evaluated as a vascular substitute in terms of patency and induction of host immune reactivity, whether used in major histocompatibility complex-incompatible, major histocompatibility complex-incompatible dogs. The immunosuppressive drug therapies were given for a period of 31 days, beginning 1 day before transplantation, and consisted of the use of cyclosporine A, mycophenolate mofetil, or a combination of both. All histoincompatible allografts were thrombosed at 4 or 8 weeks after transplantation with antibody development and cell-mediated cytotoxicity in the graft, whereas histocompatible allografts showed late stenosis without immunologic reactions directed toward donor cells. Given alone, neither cyclosporine A nor mycophenolate mofetil improved the overall patency of venous allografts; thrombosis occurred shortly after cessation of immunosuppression. Still, the cyclosporine A-mycophenolate mofetil combination therapy led to a 100% patency rate at 20 weeks after implantation and immune reactions were markedly reduced. This study shows that the fresh vein allograft is still an attractive and functional alternative to the autologous saphenous vein if the host immunologic reactions are controlled by cyclosporine A-mycophenolate mofetil immunosuppression.

Disposable filters for microaggregate removal from extracorporeal circulation.

A comparative study of three commercially available blood filters for use in extracorporeal circuits was made in dogs. All filters were efficient in removing infused microclots from the circulation, but all caused mild thrombocytopenia and two produced minimal hemolysis. In dogs infused with microclots, only those animals without blood filters in the infusing circuit showed evidence of pulmonary microembolism at autopsy. It was concluded that while the filters have minimal disadvantages, their potential in reducing microembolism in extracorporeal circuits far outweighs these disadvantages.

Implantable artificial lung. Preliminary report.

The ultimate treatment of chronic respiratory insufficiency is pulmonary replacement by an artificial organ, homologous lung transplantation, or chronic paracorporeal respiratory supplementation. The woven capillary membrane oxygenator appears to be a major development toward implantable artificial organs. The four units tested are made up of screens 3.5 by 4.0 cm. of capillary tubing 0.3 mm. I.D. by 0.64 mm. O.D. assembled into rectangular blocks. Units made up by five, ten, twenty, and forty screens have been assembled and tested according to the protocol suggested by Galletti. The maximum oxygen transfer rate with blood was 48 ml. per minute per square meter. Water carbon dioxide transfer rate was 23.1 ml. per minute per square meter. The pressure drops in the liquid phase were 8.5, 15.3, 13.8, 17.6 mm. Hg at 1 L. per minute flow. These results indicate that the woven capillary membrane lung is an acceptably efficient oxygenator. The characteristics of design and performance suggest that this oxygenator can be made to be implanted into the chest or used as a paracorporeal respiratory assistance device.

Preclotting of knitted Dacron prosthesis. A scanning electron microscope study.

A scanning electron microscope study of preclotting on knitted Dacron prosthesis is reported. Five steps of the interaction are well identified: (1) before any blood contact (virgin Dacron), (2) during the first 3 minutes (fibrin and platelet aggregates), (3) fifth minute of contact (clotting), (4) 15 minutes of contact with heparinized blood (thin fibrin network), and (5) the following minutes (invasion of fibrin, which enmeshes blood cells).

Nature of deposits in a tubular membrane oxygenator after prolonged extracorporeal circulation: a scanning electron microscope study.

Although silicone fibers are among the most compatible with tissue and blood, numerous deposits are observed after their prolonged usage in a capillary membrane oxygenator, even when the blood has been properly heparinized. The scanning electron microscope (SEM) study shows that the morphology of these deposits varies greatly, depending upon the part of the unit from which the sample is taken. The area close to the inlet is the most severely affected. The outlet zone is affected to a lesser degree, and the areas in between are only slightly affected.

An X-ray photoelectron spectroscopy study of the external surface of explanted microporous polyurethane vascular prostheses.

XPS has been used to examine the external surfaces of microporous polyurethane vascular prostheses implanted for up to 6 months in dogs. The phenomena of bilirubin absorption and physical degradation were investigated, using three different chemical washes to clean the prostheses. Very little evidence for chemical change was found, indicating a predominant role for the mechanical or biochemical ablation of degraded material.

Hydrophobic and fibrillar microporous polyetherurethane urea prosthesis: an ESCA study on the internal and external surfaces of explanted grafts.

The ESCA study gives a good qualitative and quantitative elemental analysis of internal and external surfaces of foreign materials. Microporous hydrophobic Mitrathane (a polyetherurethane urea) grafts were implanted as blood conduits in dogs for up to 6 months. Surface analysis of explanted grafts demonstrated the presence of different contaminants: sodium, chlorine, silicon, in patent grafts, i.e. those implanted for 1 month and less. The sulphur probably comes from the presence of proteins on the surface of the polymer and the high level of nitrogen is also protein-related. At 6 month implantation, the grafts were occluded and a decrease of proteins on the surface was observed. The values of N/C and O/C ratios are also reported. For the virgin material, these ratios correspond to the quantity of hard and soft segments; but, for the explanted grafts, these parameters are also influenced by the presence of proteins due to the Versaclean washing which did not wash away all the proteins on the surface of the polymer. The SEM photographs showed a certain degradation of polyurethane after 6 month of implantation. However, by ESCA study, it is difficult to compare the surface of virgin and explanted grafts because it is masked by the presence of proteins.

Lipid uptake in synthetic vascular prostheses explanted from humans.

Previous in vivo studies in humans and dogs have revealed an atherosclerosis-like phenomenon in which lipid penetration within arterial prosthesis wall was observed. The primary goal of the present study was therefore to investigate the occurrence of this lipid retention in ePTFE prostheses implanted in humans and therefore identify potential risk factors related to this phenomenon. Lipid uptake in 367 ePTFE microporous vascular prostheses explanted from humans was studied using Fourier transform infrared spectroscopy. The assignment of the infrared absorption features clearly revealed the presence of strongly bonded unsaturated fatty acids to the microporous structure of the prostheses. A one-way ANOVA statistical analysis showed that the lipid uptake in the synthetic vascular prostheses depended on the duration of implantation of the prosthesis and on the sex of the patient. A two-way ANOVA showed that a relationship existed between the estimated lipid uptake and the internal diameter of the prosthesis. These results confirm that the lipid uptake phenomenon depends on some clinical factors related either to the patients or to the prostheses' morphological parameters.

In vivo performance of the polyesterurethane Vascugraft prosthesis implanted as a thoraco-abdominal bypass in dogs: an exploratory study.

Among the various prototype vascular prostheses that have been developed over recent years as small vessel substitutes, the Vascugraft polyurethane device produced by Braun-Melsungen AG has a number of attractive features. As well as having high mechanical compliance similar to that of the arterial tree, it has been manufactured from a specially synthesized poly(ester urethane) with improved biostability and its microfibrous structure provides a highly porous wall with open communicating pores. With a view to evaluating the in vivo biofunctionality and biostability of this prosthesis in the dog, 10 mm diameter grafts were implanted as thoraco-abdominal bypasses for prescheduled periods of 1 months and 12 months, and their performance monitored in terms of gross morphology, histology and the measurement of the chemical and physical properties of the explanted and cleaned specimens. Both grafts were patent at retrieval. Each had a smooth and glistening flow surface without organized mural thrombi and showed the development of a thin collagenous internal capsule with the presence of endothelial-like cells. Both grafts were well encapsulated externally and revealed a small distal bend or kink which is frequently observed by any thoraco-abdominal bypass in dogs. The fresh explanted prostheses were cleaned by a new enzyme treatment which provided specimens for microscopic, mechanical and thermal analyses, as well as studies of the surface and bulk chemistry. By comparing the results from the explanted and cleaned material with those of the virgin prosthesis, we have observed some deterioration in the integrity of the microfibrous structure, some loss in mechanical performance, marginal changes in molecular weight, and an apparent microphase separation of the hard and soft segment domains at a depth of a few microns. While the biofunctionality of a 10 mm calibre device has been demonstrated, additional in vivo studies are recommended to assess the biofunctionality at different diameters and the biostability over longer periods of implantation.

Cellular reaction to the Vascugraft polyesterurethane vascular prosthesis: in vivo studies in rats.

The biocompatibility of Vascugraft, a polyesterurethane vascular prosthesis manufactured by Braun-Melsungen AG, was assessed by immunofluorescence and histological studies. Discs, 1 cm2, of Vascugraft prosthesis were implanted into the peritoneal cavity of rats. Results were compared with Impra, GORE-TEX and Mitrathane prostheses and a control group. Animals were killed at 1, 2, 6, 9 and 12 wk. Total T cells, T helper cells, T suppressor cells and activated T lymphocytes expressing Interleukin-2 receptors were quantified by a cytofluorometric technique in the peripheral blood of rats. For each period of implantation, all vascular prostheses showed no significant change in the percentage of total T cells, T subsets and T cells expressing Interleukin-2 receptors when compared to the control group. Histological examination of the tissue reaction surrounding the Vascugraft revealed a mild inflammatory reaction, similar to the one observed with both polytetrafluoroethylene grafts. However, the rate and the degree of encapsulation were different between grafts. The Vascugraft prosthesis was well encapsulated 2 wk after implantation, whereas inhibition of fibroblastic proliferation into the graft wall and surrounding both polytetrafluoroethylene grafts was observed for each period of implantation. The Mitrathane prosthesis exhibited a moderate inflammatory response, characterized by a high level of activation on fibroblasts compared to other grafts.

Biocompatibility of the Vascugraft: evaluation of a novel polyester urethane vascular substitute by an organotypic culture technique.

To evaluate the biocompatibility of chemically and structurally modified polyurethane elastomers for use as blood vessel replacements, small squares of vascular prostheses were cultured in direct contact with endothelium from chick embryo aorta using an organotypic culture assay. The polyurethane materials tested were: Vascugraft (fibrous, open pore structure); commercial Hydrophilic Mitrathane prosthesis (high porosity, smooth surface, non-permeable, closed pore structure); experimental hydrophobic Mitrathane (less porosity but a fibrous, open pore structure, similar to Vascugraft). The commercial expanded polytetrafluoroethylene prostheses Impra and reinforced GORETEX were included as controls on account of their extensive clinical application in the femoropopliteal position. After 5 d incubation at 37 degrees C biocompatibility was assessed in terms of average area of migrating cells on the biomaterial, total number of cells surrounding the explant and level of adhesion between the cells and the biomaterial. The Vascugraft prosthesis promoted the growth of a continuous monolayer of cells on its surface. This behaviour was equivalent to Impra and reinforced GORETEX materials in terms of cell density and area of cell migration but appeared to be superior for cell adhesion. From a second series of cell culture tests, in which the extractables leached from the biomaterials were added to the nutrient medium, it was concluded that none of the biomaterials tested released cytotoxic contaminants.

Microporous hydrophilic polyurethane vascular grafts as substitutes in the abdominal aorta of dogs.

Polyurethanes are emerging as promising biomaterials. A microporous vascular graft fabricated from Mitrathane, a new polyetherurethane urea, appeared to be particularly interesting according to in vitro evaluation and was tested in vivo as an infrarenal aortic substitute (i.d. 5 mm) in 24 dogs. After implantation for scheduled periods ranging from 4 h to 6 mnth, morphology evaluation and healing analysis of the grafts were performed. At harvesting, 18 grafts were patent and 6 were thrombosed. The thrombosed grafts had been implanted for 4 h (1 graft), 1 mnth (2 grafts) and 6 mnth (3 grafts). No macroscopic deposits other than red mural thrombi were observed. At 1 mnth complete external encapsulation was observed in 2 grafts. The capsule then contracted and became thinner; over the long term it shrank, became translucent and incomplete at 6 mnth. Yellow and brown stains appeared on many of the grafts. Characterization of the brown stains showed them to be iron (Fe3+) and the yellow stains were associated with the deposition of a bile pigment (bilirubin).

Quantitative analysis of the surface morphology and textile structure of the polyurethane Vascugraft arterial prosthesis using image and statistical analyses.

The surface morphology and textile structure of the Vascugraft polyurethane arterial prosthesis were investigated. Novel methods of image analysis and the presentation of statistical data were used to obtain quantitative results of the surface morphology of the non-woven microfibrous structure of prostheses of three different sizes. These techniques have identified apparent differences in the distributions of thickness and orientation of the microfibres between the internal and external surfaces and between the three prostheses investigated.

Radiosterilization of albuminated polyester prostheses.

The study reported here is concerned with the radio-sterilization of Dacron vascular prostheses coated with crosslinked albumin. gamma-Radiations have no effect on the mechanical properties of the polyester fibres or on their crystallinity, whether irradiated in a dry state or immersed in saline. Special attention has been paid to the release of the albumin, or protein fragments from the reticulum using 125I-labelled albumin as a radiotracer. The albumin leakage depends upon the type of Dacron fabrics considered but the values derived from radioactivity measurements are always greater than those directly measured, which indicates a radio-induced break of the bond between iodine and albumin; this has nothing to do with the break of the association between albumin and Dacron. Moreover no cytotoxicity of the irradiated immersion medium has been observed using a test based on organotypic culture in liquid medium. Thus radio-sterilization of an albuminated polyester vascular prosthesis immersed in saline appears to be a suitable procedure.