The effect of different treatment modalities on the calcification potential and cross-linking stability of bovine pericardium.

Porcine heart valves and bovine pericardium exhibit suitable properties for use as substitutes in cardiothoracic surgery, but must meet several requirements to be safe and efficient. Treatment with glutaraldehyde (GA) render some of these requirements, but calcification and degradation post-implant remain a problem. This study aimed to identify additional biochemical treatments that will minimize calcification potential without compromising the physical properties of pericardium. Pericardium treated with GA calcified severely after 8 weeks in the subcutaneous rat model, compared to tissue treated with higher concentrations of glycosaminoglycans (GAG) and commercial Glycar patches. GA, lower concentrations GAG and Glycar pericardium had high denaturation temperatures due to enhanced cross-linking. Tensile strength of GA tissue was significantly lower than GAG-treated or Glycar tissues, due to lower water content with resultant lower flexibility and suppleness. Pericardium treated with 0.01 M GAG gave acceptable denaturation temperatures, tensile strength and reduced calcification potential. All tissue treatments evoked comparable host immune responses, and no significant difference in resistance to enzymatic degradation. Ineffective stabilization and fixation of cross-links following GAG treatment, as well as limited penetration into the pericardium, resulted in GAG leaching out into the surrounding host tissue or storage medium, and prohibits safe clinical use of such tissue.

A multi-step approach in anti-calcification of glutaraldehyde-preserved bovine pericardium.

AIM: Bioprosthetic cardiovascular substitutes, manufactured from glutaraldehyde-preserved bovine or porcine tissues, are prone to calcification after implantation. The aim of the study was to evaluate the ultrastructure, material stability and calcification behaviour of glutaraldehyde-preserved bovine pericardium, treated with a multi-step anti-calcification process which addresses each of the major causes of calcification and tissue degeneration. METHODS: Bovine pericardium samples were divided into 2 groups. Group I (control) consisted of tissue fixed with 0.625% glutaraldehyde and Group II (study group) consisted of tissue fixed with 0.625% glutaraldehyde and exposed to a multi-step anti-calcification process. Ultrastructure was examined by scanning electron microscopy and material stability was assessed by mechanical testing, shrinkage temperature and enzymatic degradation. Calcification was assessed by histology (Von Kossa stain) and by atomic absorption spectrophotometry in the subcutaneous rat model. RESULTS: Bovine pericardium in the study group revealed less visible changes in the ultrastructure of the collagen matrix, improved material stability (P<0.05) and significantly (P<0.001) reduced calcification compared to control tissues (4.5+/-1.2 versus 136.03+/-11.39 ug/mg tissue). CONCLUSIONS: In conclusion, results demonstrate that the multi-step anticalcification process improved the material stability and reduced the calcification potential of bovine pericardial tissue. These improvements in the quality of the bovine pericardium should enhance the long-term durability of the tissue as a bioprosthetic substitute for cardiovascular application.

Glutaraldehyde-fixed kangaroo aortic wall tissue: histology, crosslink stability and calcification potential.

Stentless aortic heart valve substitutes, manufactured from biological tissues, are fixed with glutaraldehyde to cross-link collagen, reduce antigenicity, and sterilize the tissue. Despite improved cross linking, reduced antigenicity, and various anticalcification measures, the aortic wall tissue present in these prostheses tends to calcify. The aim of this study was to assess the morphology, collagen cross-link stability, and calcification potential of glutaraldehyde-preserved kangaroo aortic wall tissue as opposed to porcine aortic wall tissue. Porcine and kangaroo aortic wall tissues were fixed in 0.625% buffered glutaraldehyde. Histology and cross-link stability were examined. Calcification potential was determined in the subcutaneous rat model. Kangaroo aortic wall tissue was significantly (p < 0.01) less calcified than porcine aortic wall tissue (26.67 +/- 6.53 versus 41.959 +/- 2.75 microg/mg tissue) at 8 weeks. In conclusion, the histological differences between kangaroo and porcine aortic wall tissue correlate well with the reduced calcification potential of kangaroo aortic wall tissue. The reduced calcification potential could result in improved long-term durability of stentless kangaroo heart valves as bioprostheses.

Evaluation of kangaroo pericardium as an alternative substitute for reconstructive cardiac surgery.

BACKGROUND: Bioprosthetic materials (human, bovine and porcine) are used in various cardio-thoracic repair and replacement procedures because of excellent performance and low thrombogenicity. These bioprosthetic substitutes fail due to degeneration and calcification. This study examines the morphology, tensile properties and calcification potential of kangaroo pericardium in vitro and in vivo. METHODS: Bovine (control tissue) and kangaroo pericardium, fixed in 0.625% buffered glutaraldehyde, were examined by light and scanning electron microscopy. A standard method was used for biaxial testing. Pericardial strips (10 x 5 mm) were implanted subcutaneously into male Wistar rats and retrieved after 4, 6 and 8 weeks and examined by Von Kossa's stain technique and atomic absorption spectrophotometry. RESULTS: Histology revealed serosa and fibrosa cell layers in both tissues. Electron microscopy showed a densely arranged collagen matrix in kangaroo pericardium. Kangaroo pericardium calcified significantly less than bovine pericardium at 4 weeks (0.80+/-0.28 versus 21.60+/-4.80 microg/mg) at 6 weeks (0.48+/-0.08 versus 32.80+/-14.4 microg/mg) and at 8 weeks (2.40+/-1.20 versus 30.40+/-17.20 microg/mg), respectively. CONCLUSIONS: Kangaroo pericardium has a densely arranged collagen matrix with a higher extensibility and significantly lower calcification potential. Therefore, kangaroo pericardium could be used as an alternative substitute in cardiac surgery because of its low calcification potential.

Evaluation of kangaroo aortic valved conduits in a juvenile sheep model: preliminary findings.

BACKGROUND: Biological heart valve substitutes, manufactured from either porcine or bovine tissue, have been in use for more than 30 years. Despite low thrombogenicity and excellent performance, bioprosthetic heart valves tend to degenerate and calcify early in young patients because of patient and valve related factors. The aim of this study was to examine the calcification behavior of glutaraldehyde-preserved kangaroo heart valves in a juvenile sheep model. METHODS: Porcine (n = 10) and kangaroo (n = 10) valved conduits were implanted in the descending aortic position of juvenile sheep and retrieved after 6, 8, and 12 months. Retrieved valved conduits were examined for morphological changes and calcification of the valve tissue, using Von Kossa's stain technique and atomic absorption spectrophotometry. RESULTS: Structural valve deterioration, characterized by increased stiffness and severe calcification, occurred in 100% of the porcine conduits within 4 months. Kangaroo valve leaflets were significantly (p < 0.001) less calcified at 6 months (3.39+/-1.80 microg/mg), 8 months (5.86+/-4.57 microg/mg), and at 12 months (14.38+/-6.72 microg/mg), compared to porcine valves at 3 months (176.45+/-42.88 microg/mg ) and at 4 months (154.67+/-52.67 microg/mg ). Porcine aortic wall tissue was more calcified (118.24+/-42.86 microg/mg) than kangaroo aortic wall tissue (79.55+/-26.40 microg/mg). CONCLUSIONS: Kangaroo heart valves calcify less than porcine heart valves. These findings suggest that a different donor valve tissue has a lower calcification potential probably due to a difference in the morphological ultrastructure. This could result in improved long-term durability of kangaroo heart valves.

Kangaroo versus porcine aortic valve tissue--valve geometry morphology, tensile strength and calcification potential.

OBJECTIVE: Valve related factors and patient related factors are responsible for calcification of valvular bioprostheses. Recent studies showed different donor and recipient species have different influences on the total calcification rate of bioprostheses. This study was performed to evaluate and compare Kangaroo aortic valve leaflets with porcine aortic valve leaflets. Experimental design. Prospective study. Setting. Cardio-thoracic experimental research of a university department. MATERIALS AND METHODS: Glutaraldehyde-fixed Kangaroo and porcine valve leaflets were evaluated in vitro according to valve geometry (internal diameter and leaflet thickness), morphology (light and electron microscopy) and tensile strength. In vivo evaluation consisted of implantation in a rat model for 8 weeks, Von Kossa stain for calcium and atomic absorption spectrophotometry for total extractable calcium content. RESULTS: Kangaroo valves indicated a smaller internal valve diameter as well as a thinner valve leaflet (p<0.01, ANOVA) at corresponding body weight, less proteoglycan spicules in the fibrosa, increased elasticity (p<0.05) and low calcification potential (p<0.01, confidence interval 95%). CONCLUSIONS: Kangaroo aortic valve leaflets have different valvular qualities compared to porcine valve tissue. Kangaroo valve leaflets are significantly superior to porcine valve leaflets as far as calcification is concerned. These results are encouraging and suggest further in vivo evaluation in a larger animal model before clinical application can be considered.

r-Hirudin inhibits platelet-dependent thrombosis during cardiopulmonary bypass in baboons.

BACKGROUND: Systemic anticoagulation is required during cardiopulmonary bypass (CPB) to inhibit the activation of platelets, the coagulation system and ultimately thrombus formation. Unfractionated heparin is most commonly used, but it is neither entirely safe nor completely effective. The use of protamine sulphate to reverse the anticoagulant effect further complicates the use of heparin. The clinical need for a heparin substitute is therefore obvious. We evaluated the efficacy of r-Hirudin, a potent and specific inhibitor of thrombin, as anticoagulant in a baboon model of cardiopulmonary bypass. METHODS: Ten baboons, divided into two groups of five each, were used. The one group received 0.7 mg/kg r-Hirudin as a bolus before CPB was started, followed by a constant infusion of 1.4 mg/kg/hr for the 90 min of CPB. The other group received a bolus of 2.5 mg/kg heparin before the start of CPB, followed by maintenance dosages to maintain the activated clotting time (ACT) >400 sec. RESULTS: Adequate anticoagulation was obtained with both anticoagulants. Haemodilution due to priming the extracorporeal system with Ringer's lactate and appropriately anticoagulated donor blood, was equivalent in both groups. During CPB with heparin, but not with hirudin, there was a significant increase in the number of circulating platelet aggregates, thrombin-antithrombin (TAT) complexes and 111In-labelled platelet accumulation in the oxygenator. After the initial decrease in platelet count due to haemodilution, it further decreased significantly during CPB with heparin but remained relatively constant when r-Hirudin was used. CONCLUSIONS: Our results strongly suggest that r-Hirudin is superior to heparin especially with respect to its inhibitory effect on platelet dependent thrombogenesis caused by the biomembranes of the oxygenator.

Laboratory evaluation of tissue heart valves. A comparative assessment.

Tissue of commercially prepared tissue heart valves were evaluated and compared with aluminium treated, fixed porcine valve tissue in vitro (tensile strength, scanning and transmission electron microscopy) and in vivo (calcification potential after subcutaneous implantation in the rat model). Valve leaflets (n = 40) were divided into four groups according to the method of treatment: Group I (fixed in 0.652% glutaraldehyde, control), Group II (fixed and treated with aluminium), Group III (fixed and treated with Toluidine blue) and Group IV (fixed and treated with watersoluble alkyl sulphate). Tensile strength was not influenced in Group II and III (p > 0.05). Group IV indicated a significant (p < 0.05) reduction in tensile strength. Scanning electron microscopy revealed damage and loss of surface endothelium in Group III and IV respectively. Transmission electron microscopy indicated damage to underlying matricial cells in Group III and IV. Calcification potential was significantly (p < 0.001) reduced in Group II to IV. We conclude that damage ultrastructure could contribute to the reduced tensile strength in Group IV and that reduced tensile strength might have an influence on the long-term durability of tissue heart valves. Antimineralization treatment of tissue heart valves does retard calcification but is yet unable to inhibit the process completely.

Interstitial pH during myocardial preservation: assessment of five methods of myocardial preservation.

We investigated changes in myocardial pH during cardioplegic arrest with five methods of preservation at 15 degrees +/- 1 degree C. Twenty-five dogs were subjected to cardiopulmonary bypass for 150 minutes. Group I (control) had hypothermia only. Group II received THAM-buffered blood cardioplegia, group III a bicarbonate-buffered blood cardioplegic solution, group IV infusions of hyperkalemic blood, and group V oxygenated St. Thomas 2 solution. After 120 minutes of ischemia, interstitial pH in group I was markedly depressed (6.4 +/- 0.07; p < 0.01). The pH in groups II and IV was well maintained (7.23 +/- 0.05 and 7.27 +/- 0.07) and differed significantly (p < 0.05) from that of the remaining groups. The pH in groups III and V was less well maintained (7.14 +/- 0.02 and 7.01 +/- 0.05), with no significant difference (p > 0.05) between these two groups. Postreperfusion functional recovery after 45 minutes was 24% +/- 6% in group I, 92% +/- 3% in group II, 82% +/- 5% in group III, 84% +/- 4% in group IV, and 66% +/- 6% in group V. Creatine kinase levels were significantly (p < 0.01) increased and ultrastructural damage was more prominent in group I compared with the remaining groups. Myocardial water content significantly increased in all groups. We conclude that a strongly buffered blood-based cardioplegic solution is more effective in preventing interstitial acidosis during moderate hypothermia and that maintenance of an optimal tissue pH plays an important role in postischemic functional recovery.

Processing factors as determinants of tissue valve calcification.

The effect of different processing factors on tissue valve calcification were studied in the subdermal rat model. Factors evaluated, were the influence of tissue ischaemia (4 degrees and 25 degrees C), different blocking reagents (KH2PO4, T6, MgCl2 and AlCl3), fixation pressures (0, 10 and 20 mmHg) and the pH (3.72 and 7.40) of the fixative. Tensile strength tests performed, showed that all blocking reagents tend to weaken valve tissue. Histologically calcification originated mainly in the spongiosa of the valve leaflets. Tissue ischaemia at 4 degrees C significantly (p less than 0.05) decreased the calcification potential of the valve tissue. Ischaemia at 25 degrees C significantly (p less than 0.05) increased this process. Blocking reagents KH2PO4, T6 and MgCl2 significantly (p less than 0.05) reduced calcification of valve tissue. AlCl3 pretreatment virtually prevented it to such an extend that the calcification process was down to 92% of the control group. Different fixation pressures had no influence on the calcification potential of the tissue. AlCl3 was dependent on a low pH (3.72) to act as a blocking reagent on tissue calcification. It is concluded that certain processing factors do influence the calcification potential of valve tissue. These factors should be considered when constructing bioprostheses with glutaraldehyde-treated porcine valves.

The effect of potassium phosphate, magnesium chloride and T6 as blocking reagents on tissue valve calcification.

Potassium phosphate, Magnesium chloride and T6 as blocking agents on tissue valve calcification have been studied in vitro and in vivo. In vitro tests indicate that untreated leaflets (control group) tend to calcify significantly more than treated leaflets (p less than 0.05). Calcification in the treated leaflets tends to be varied with no significant difference (p greater than 0.05) between the different groups. In vivo findings indicate that leaflets in the control group calcify as early as 5 weeks after implantation while treated leaflets showed no significant signs of calcification at this stage. Results deduced from the extractable calcium contents as well as from histological examination, emphasise the need of a blocking reagent to decrease calcification on a valvular bioprosthesis.

[Extensive endarterectomy of the left anterior descending artery]. / Uitgebreide endarterektomie van die afdalende tak van die linkerkoronêre arterie.

Diffuse obstructive disease of the left anterior descending coronary artery presents a new challenge to the surgeon especially if the heart and septal muscle supplied by this vessel is ischaemic but viable. Venous skips were utilized in the past to overcome this problem, but recently extensive endarterectomy of this system has been advocated. The case studies of 5 patients are presented who have undergone extensive endarterectomy of the left anterior descending artery and coronary bypass grafting.

Coronary artery bypass at the University of the Orange Free State Medical School. Medium-term follow-up of the first 100 cases.

The increasing number of patients undergoing coronary artery bypass surgery has made the investigation and evaluation of the results of the procedure imperative. The first 100 patients who underwent this type of surgery at the University of the Orange Free State Medical School were followed up for a period of 3-8 years in order to obtain a prognostic pattern. The results of the study indicate that the procedure carries a low mortality rate and that the medium- to long-term results are encouraging. Eighty-one per cent of the patients are still actively pursuing their usual daily activities and 50% are completely free of symptoms.