Abstract
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.
배경: 이종조직의 석회화는 기존에 사용된 돼지 판막이나 소 심낭을 이용한 생체 인공조직의 임상 실패의 주된 원인으로 알려져 있다. 최근에 항석회화 효과를 높이고자 기전이 다른 여러 처치를 병합하는 시도들이 보고되고 있다. 본 연구에서는 작용기전이 다른 ethanol, L-lysine, $NaBH_4$을 병합 처리하였을 때 예상되는 상승 효과를 석회화와 조직의 신축력(elasticity)을 통해 알아보고자 하였다. 대상 및 방법: 0.625% Glutaraldehyde ($4^{\circ}C$에서 2일, 상온에서 7일간)고정한 돼지 심낭을 80% Ethanol (상온에서 1일), 혹은 0.1 M L-lysine ($37^{\circ}C$에서 2일), 혹은 0.1 M $NaBH_4$ (상온에서 2일)로 처리 한 후 각각의 두께(thickness)와 장력(tensile strength)을 측정하였다. 각각의 항석회화 처리한 돼지심낭을 생후 3주된 쥐의 피하조직에 이식하고 8주 뒤 칼슘을 정량하고 조직학적 소견을 관찰하였다. 결과: 0.625% glutaraldehyde 고정만 시행한 군($51.2{\pm}8.5$ ug/mg)과 비교하여 80% Ethanol 처리한 군($13.6{\pm}10.0$ ug/mg, p=0.008)과, L-lysine 처리한 군($15.3{\pm}1.0$ ug/mg, p=0.002), 그리고 80% Ethanol과 L-lysine 처리한 군($16.1{\pm}11.1$ ug/mg, p=0.012)은 통계적으로 의미 있게 칼슘의 침착량이 적었지만, $NaBH_4$만 처리한 군($65.7{\pm}61.8$ ug/mg, p=0.653)과 80% Ethanol, L-lysme, $NaBH_4$로 모두 처리한 군($92.9{\pm}58.3$ ug/mg, p=0.288)은 칼슘의 침착량이 더 많았다. 80% Ethanol과 L-lysine으로 처리한 군과 80% Ethanol, L-lysine, $NaBH_4$로 모두 처리한 군의 장력/두께 비율(tensile strength/thickness ratio)은 각각 $7.60{\pm}1.55$, $7.47{\pm}1.85$로 glutaraldehyde 고정만 시행한 군의 $4.75{\pm}1.88$보다 증가되어 있는 경향을 보였다(p=0.76, p=0.33). 결론: Ethanol과 L-lysine을 돼지심낭에 병합처치 하였을 때, Ethanol과 L-lysine을 단독처치 하였을 때와 비교하여, 비슷한 항석회화 효과와 조직의 신축력을 증가시키는 경향을 보였지만 병합처치에 의한 상승효과를 확인하지는 못하였다. $NaBH_4$는 단독처치 혹은 병합처치하였을 때 모두 석회화를 증가시키는 경향을 보였다.
