Advantages of decellularized bovine pericardial scaffolds compared to glutaraldehyde fixed bovine pericardial patches demonstrated in a 180-day implant ovine study.

Glutaraldehyde (GA)-fixed bovine pericardial patches remain the cardiovascular industry standard despite reports of degradation, thickening, inflammation, calcification and lack of tissue remodelling. Decellularization provides the opportunity to attenuate some of these immune-mediated processes. This study compared the mechanical and morphological integrity of bovine pericardium that is GA-fixated (Glycar® patches) or decellularized (BPS), using a proprietary protocol, following implantation in an ovine model. The impact of the processing methods on tissue strength and morphology was assessed prior to implantation. Pericardial patches were then implanted in the descending aorta and main pulmonary artery of juvenile sheep (n = 6 per group) for 180 days, and clinically evaluated using echocardiography. At explanation, patches were evaluated for strength, calcification and biological interaction. Histology demonstrated a wave-like appearance of well-separated collagen fibers for BPS scaffolds that provided pore sizes adequate to promote fibroblast infiltration. The collagen of the Glycar® patches showed loss of collagen fiber integrity, making the collagen densely compacted, contributing to insignificant recipient cell infiltration. The clinical performance of both groups was excellent, and echocardiography confirmed the absence of aneurysm formation, calcification and degeneration. Explanted Glycar® patches demonstrated cells in abundance within the fibrous encapsulation that separated the implant from the host tissue. More importantly, the fibrous encapsulation also contributed to patch thickening of both the explanted aorta and pulmonary patches. The decellularized pericardial scaffolds demonstrated recellularization, resistance to calcification, re-endothelialization and adequate strength after 180-day implantation. The proprietary decellularization protocol produced pericardial scaffolds that could be considered as an alternative to GA-fixed pericardial patches.

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.

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.

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.