• Journal of Chest Surgery
• /
• v.42 no.4
• /
• pp.409-417
• /
• 2009

Background: Glutaraldehyde-fixed heterografts are prone to calcification after long-term implantation in human, and this is one of the limiting factors for the longevity of the heterografts used in cardiovascular surgery. The aim of the study was to evaluate the anticalcification effect of an ethanol and amino acids treatment on glutaraldehyde-fixed bovine pericardium. Material and Method: Bovine pericardial tissues were divided into 5 groups. Group 1 consisted of tissues fixed with glutaraldehyde, group 2 consisted of commercially available bovine pericardial valve tissues (Carpentier-Edwards PERIMOUNT), group 3 consisted of glutaraldehyde-fixed tissues treated with ethanol, group 4 consisted of glutaraldehyde-fixed tissues treated with ethanol and L-glutamic acid, and group 5 consisted of glutaraldehyde-fixed tissues treated with ethanol and homocysteic acid. The tissue microstructure was examined by light and electron microscopy. Tissue samples of each group were implanted into rat subcutaneous tissue for 3 $\sim$ 4 months and the calcium contents were measured after harvest. Result: The collagen fibers appeared to be well preserved in all the groups. The calcium contents of groups 2, 3, 4 and 5 (13.46$\pm$11.74, 0.33$\pm$0.02, 0.39$\pm$0.08 and 0.42$\pm$0.06 $\mu$g/mg, respectively) were all significantly lower than that of group 1 (149.97$\pm$28.25 $\mu$g/mg) (p<0.05). The calcium contents of groups 3, 4 and 5 were all significantly lower than that of group 2 (p<0.05). Conclusion: Treatment with ethanol alone or in combination with amino acids (L-glutamic acid or homocysteic acid) strongly prevented the calcification of glutaraldehyde-fixed bovine pericardium.

• Journal of Chest Surgery
• /
• v.31 no.10
• /
• pp.919-923
• /
• 1998

Background: Calcific degeneration is unavoidable in either homo- or heterografts implanted in the human body. We have developed a calcification-resistant cardiovascular tissue patch using a novel technique of anticalcification. Materials and methods: Fresh bovine pericardium was harvested at the slaughter house and transfered to the laboratory in Hank's solution. After trimming and fixing the pericardium, it was embedded in 4$^{\circ}C$ 0.65% glutaraldehyde for a week and then washed by phosphate-buffered saline(PBS) of pH 7.4. This prepared pericardium was then stored in 2.5% sulphonated polyethyleneoxide(PEO-SO3) solution for 2 days at room temperature and reversed by 4$^{\circ}C$ NaBH4 solution for 16 hours. To evaluate the calcification-resistance of surface modified bovine pericardium with PEO-SO3, either glutaraldehyde- treated(GA group, n=4) or PEO-SO3-treated pericardial patch(PEO-SO3 group, n=4) was implanted into adult mongrel dog to reconstruct the main pulmonary artery and the descending aorta using a partial clamp technique. After 1 month follow-up, the implanted patches were retrieved to evaluate the pathologic findings and the content of calcium and phosphorous. Results: The PEO-SO3 group showed substantially less retraction and significantly less calcium deposition than the GA group in both aortic(7.10$\pm$1.05 vs. 13.81$\pm$2.33 mg/g of dried tissue) and pulmonary positions(1.55$\pm$0.29 vs. 6.72$\pm$0.70 mg/g)(p<0.01). Phosphorous contents were also less in the PEO-SO3 group than the GA group significantly, 8.11$\pm$1.07 mg/g vs. 19.33$\pm$4.31 mg/g in the aortic and 2.58$\pm$0.40 vs. 12.60$\pm$3.40 mg/g in thepulmonary position(p<0.01). Conclusions: These findings suggest that PEO-SO3 modified bovine pericardium is highly calcification-resistant but further study is needed to evaluate the long-term biological safety and compatibility of the prosthesis.

• Journal of Chest Surgery
• /
• v.42 no.2
• /
• pp.157-164
• /
• 2009

Background: Various studies and experimental trials have been done to develop bioprosthetic devices to treat complex congenital heart disease due to the limited usage of homograft tissue. The purpose of the present study was to evaluate the effect of diamine bridges with using L-lysine, as compared with using ethanol. Material and Method: Porcine pericardium was fixed at 0.625% GA (commercial fixation). An interim step of ethanol (80%; 1 day at room temperature) or L-lysine (0.1 M; 2 days at $37^{\circ}C$) was followed by completion of the GA fixation (2 days at $4^{\circ}C$ and 7 days at room temperature). The tensile strength and thickness of the porcine percardium were measured, respectively. The treated pericardiums were implanted subcutaneously into three-week old Long-Evans rats for 8 weeks. The calcium content of the implants was assessed by atomic absorption spectroscopy and the histology. Result: Ethanol pretreatment ($13.6{\pm}10.0ug/mg$, p=0.008), L-lysine pretreatment ($15.3{\pm}1.0 ug/mg$, p=0.002), and both treatment ($16.1{\pm}11.1ug/mg$, p=0.012) significantly inhibited calcification, as compared with the controls $(51.2{\pm}8.5ug/mg)$. L-lysine pretreatment ($0.18{\pm}0.02mm,\;1.20{\pm}0.30kg$ f/5 mm) significantly increased the thickness and tensile strength, as compared with ethanol pretreatment ($0.13{\pm}0.03mm,\;0.85{\pm}0.36$ 1.0 kg f/5 mm) (p<0.01, p=0.035). Conclusion: The diamine bridges using L-lysine seemed to decrease the calcification of porcine pericardium fixed with glutaraldehyde, and this was comparable with Ethanol. Additionally, it seemed to enhance the thickness and tensile strength.

• Journal of Chest Surgery
• /
• v.42 no.6
• /
• pp.685-695
• /
• 2009

Background: Calcification is the most frequent cause of clinical failure of bioprosthetic tissues that are fabricated from Glutaraldehyde (GA)-fixed porcine valve or bovine pericardium. We recently used a multi-factorial approach of employing different mechanisms to investigate how to reduce the calcification of bioprosthetic tissues. The purpose of the present study was to evaluate the synchronized synergism using ethanol, L-lysine and $NaBH_4$ in glutaraldehyde treated porcine pericardium from the standpoint of calcification and tissue elasticity. Material and Method: Porcine pericardium was fixed with 0.625% GA (commercial fixation). An interim step of ethanol (80%; 1 day at room temperature) or L-lysine (0.1 M; 2 days at $37^{\circ}C$) or $NaBH_4$ (0.1 M; 2 days at room temperature) was followed by completion of the GA fixation (2 days at $4^{\circ}C$ and 7 days at room temperature). The tensile strength and thickness of the samples were measured. The treated pericardiums were implanted subcutaneously into three-week old Sprague-Dawley rats for 8 weeks. The calcium content was assessed by atomic absorption spectroscopy and the histology of the samples. Result: The amount of calcium in the pericardium pretreated with ethanol (13.6${\pm}$10.0 ug/mg, p=0.008), L-lysine (15.3${\pm}$1.0 ug/mg, p=0.002) and both (16.1${\pm}$11.1 ug/mg, p=0.012) was significantly reduced compared with the control (51.2${\pm}$8.5 ug/mg). However, $NaBH_4$ pretreatment (65.7${\pm}$61.8 ug/mg, p=0.653) and combined pretreatment that including ethanol, L-lysine and $NaBH_4$ (92.9${\pm}$58.3 ug/mg, p=0.288) were not significantly different from the controls(51.2${\pm}$8.5 ug/mg). Both the combined pretreatment using ethanol and L-lysine (7.60${\pm}$1.55, p=0.76) and the combined pretreatment that included ethanol, L-lysine and $NaBH_4$ (7.47${\pm}$1.85, p=0.33) increased the tensile strength/thickness ratio compared with that of the controls (4.75${\pm}$1.88). Conclusion: The combined pretreatment using ethanol and L-lysine seemed to decrease the calcification of porcine pericardium fixed with glutaraldehyde, as compared to single pretreatment, and it increase the tissue elasticity, but to the degree that showed synchronized synergism. $NaBH_4$ pretreatment seemed to increase the calcification of porcine pericardium, irrespective of whether single or combined pretreatment was used.

• Journal of Chest Surgery
• /
• v.31 no.11
• /
• pp.1023-1030
• /
• 1998

Background: Calcific degeneration limits durabilities of the bioprosthetic tissues implanted in the human body. The direct coupling sulphonated polyethyleneoxide(PEO-SO3) to the bioprosthetic tissues after glutaraldehyde(GA) fixation and the removal of residual aldehyde groups from the tissues can augment the effect of calcification-resistance. Materials and methods: To study the anti-calcification effect by PEO-SO3 modification and the removal of the residual aldehyde groups of tissues, surface modified bovine pericardia(BP-PEO-SO3) were preserved in aseptic saline to wash out GA(saline group) and 0.65% GA solution(GA group). And then above two groups and PERIGUARD (Bio-vascular. Co.) (product group) were evaluated with respects to calcium contents and microscopic findings using in vivo implantation models at carotid and femoral artery and peritoneum of 8 adult dogs. Results: In the tissues retrieved from carotid artery, calcium content was significantly decreased in saline group than in other two groups(saline; 2.89±0.31 vs. GA; 6.14±1.08 vs. product; 22.82±5.00 mg/g of dried tissue; p<0.05). In the tissues retrieved from femoral artery and peritoneum, calcium amount was also decreased in saline group than in other two groups, but not reached the significant difference between groups. On the other hand, the pathologic findings of pericardial tissues showed marked destructuction in GA group compared to the other two groups. Conclusions: In this study, covalently PEO-SO3 bound to bovine pericardium decreased calcifications and the anti-calcification effect of BP-PEO-SO3 could be augmented by the washing out the residual aldehyde groups using saline after GA fixation. Conclusively, the PEO-SO3 modified bovine pericardium is highly resistant to calcification and can be useful for the development of calcification-resistant cardiovascular patches and valves.

• Journal of Chest Surgery
• /
• v.43 no.3
• /
• pp.235-245
• /
• 2010

Background: Our goal was to evaluate anti-calcification effects of decellularization and diverse fixing methods including preincubation of the bovine pericardium with ethanol. We also assessed changes in mechanical properties. Material and Method: Harvested bovine pericardium was decellularized with 0.25% sodim dodecysulfate and then treated with 5 methods of fixation: (1) 0.5% glutaraldehyde (GA) for 14 days, (2) 0.5% GA for 5 days, 2% GA for 2 days and 0.25% GA for 7 days, (3) 0.5% GA for 5 days, 2% GA for 2 days, 0.25% GA for 7 days, and then 70% ethanol for 2 days, (4) 0.5% GA for 5 days, a mixture of 2% GA and 70% ethanol for 2 days, and 0.25% GA for 7 days, (5) 0.5% GA for 5 days, a mixture of 2% GA, 65% ethanol, and 5% octanediol for 2 days and then 0.25% GA for 7 days. All treated bovine pericardia were tested for histological variables, lipid content, and mechanical properties including tensile strength and thermal stability. A total 10 kinds of differently treated bovine pericardia were implanted into rat subdermis and harvested 8 weeks later. Harvested pericardia were evaluated for calcium content. Result: No protein denaturation was observed microscopically after decellularization. There was a 32% mean decrease in tensile strength index after decellularization in the bovine pericardium group fixed. Octanediol preincubation attenuated the decrease in tensile strength and maintained thermal stability. TG and cholesterol were not affected by decellularization but were decreased by organic solvent. Calcium content was decreased after decellularization, and organic solvent preincubation decreased calcification in the non-decellularized bovine pericardium group. Conclusion: Decellularization and organic solvent preincubation have anti-calcification effects but decellularization may cause mechanical instability. A method of decellularization and fixation that does not cause damage to matrices will be needed for evaluation of the next step in using tissue-engineering for replacement of cardiac valves.