Octanediol treatment of glutaraldehyde fixed bovine pericardium: evidence of anticalcification efficacy in the subcutaneous rat model

OBJECTIVE: Anticalcification strategies of glutaraldehyde-fixed xenograft tissue aim to extract lipids or to neutralize toxic aldehyde residuals. The purpose of this study was to evaluate the efficacy of octanediol compared to standard treatments of glutaraldehyde-fixed bovine pericardium in the subdermal rat model. Octanediol treatment is an ethanolic solution (40%) containing a long chain aliphatic alcohol (5% 1,2-octanediol) that removes lipids without diminishing the stability of collagen. METHODS: Octanediol and standard glutaraldehyde fixed bovine pericardium were both implanted in 24 Sprague-Dawley rats, explanted after 30-75 days (12 animals each) and submitted to X-ray (score 0-4), histology, electron microscopy and elemental analysis by spectroscopy (Ca and P content). Unimplanted octanediol and standard glutaraldehyde fixed pericardium served as control. RESULTS: At 30 days octanediol-treated pericardium showed calcium content of 0.20+/-0.1 vs 20.07+/-36.79 mg/g dry weight for standard pericardium. The difference was also evident at 75 days: calcium content of 2.36+/-7.38 mg/g dry weight for octanediol vs 165.61+/-23.35 mg/g dry weight for standard (p<0.0001). Differences were also detected at X-ray (mean score 0.7+/-0.6 octanediol vs 3.8+/-0.4 standard at 75 days). Equally, mean P content was 11.69+/-21.33 mg/g dry weight for standard vs 0.60+/-1.45 mg/g dry weight for octanediol samples at 30 days, and 90.90+/-12.61 mg/g dry weight for standard vs 1.42+/-4.34 mg/g dry weight for octanediol at 75 days (p<0.0001). At electron microscopy collagen appeared well preserved regardless of the type of treatment; in octanediol treated pericardium cell membranes almost disappeared and only few profiles of endoplasmic reticulum and rare mitochondria were visible. CONCLUSIONS: Treatment with octanediol strongly prevents calcification of glutaraldehyde fixed bovine pericardium in rat subdermal model, even in the long-term. Evidence of octanediol efficacy may entail important implications for new generation bioprosthetic valves.     

心脏瓣膜修复材料钙化和抗钙化研究进展

心脏瓣膜生物替代材料发生临床失效的一个主要原因是钙化,其机理非常复杂,涉及一系列决定因素,如含钙细胞外液与膜内磷酸脂反应,形成磷酸钙盐沉积.目前最有效的抗钙化治疗策略有：戊二醛固定组织后加入钙化抑制因子,易钙化位点的剔除或化学修饰,戊二醛固定的改良以及其他化学交联方法.就心脏瓣膜修复材料钙化和抗钙化动物实验研究和临床试验研究进展作一综述.     

混合醇对戊二醛鞣制的牛心包防钙化改性的实验研究

目的　本实验首次应用混合醇 (即 1、2 -丙二醇 ,1、3-丙二醇 ,2、3-丁二醇 ) ,对戊二醛处理后的牛心包进行化学改性 ,防止钙化。方法　将戊二醛处理后的牛心包用混合醇进行化学改性 ,植入离乳的SD大鼠背部皮下 ,6 0d后直接做组织钙定量检测、光镜检测、超微结构观察、测定热皱缩温度及组织湿度。结果　混合醇改性后的牛心包试片平均钙含量为 (1 817± 0 91) μg/mg干重 ,与对照组GA试片的平均钙含量 (176 32± 4 3 2 7) μg/mg干重相比 ,钙化明显减轻 (P <0 0 1)。光镜下显示混合醇改性试片较少炎性细胞浸润 ,胶原纤维结构保持良好 ;GA试片 ,则有大片状钙盐沉积及较多炎性细胞浸润 ,胶原纤维结构破坏严重。混合醇防钙化改性 2 5℃作用时 ,抗钙化效果明显好于 4℃作用温度 (平均钙含量分别为 (1 817± 0 91)及 (10 8 85± 5 6 99) μg/mg干重 ,(P <0 0 5 )。三组的热皱缩温度、组织湿度无显著性差异 (P >0 0 5 )。结论　⑴戊二醛鞣制的牛心包经混合醇改性后 ,可显著减轻钙化。⑵混合醇抗钙化改性的效果与鞣制温度密切相关 ,在 2 5℃条件下抗钙化效果优于 4℃作用温度。     

Efficacy of the Chitosan Posttreatment in Calcification Prevention of the Glutaraldehyde-Treated Porcine Aortic Noncoronary Cusp Implanted in the Right Ventricular Outflow Tract in Dogs

Abstract: Calcific tissue failure results in poor performance of the bioprosthetic heart valve. Chitosan post-treatment has been shown to be effective in calcification prevention of the glutaraldehyde-treated bovine pericardium when implanted subdermally in rats for 12 weeks. The present study investigated the effectiveness of the chitosan posttreatment in prevention of calcification of the glutaraldehyde-treated porcine aortic noncoronary cusp 5 months after implantation in the right ventricular outflow tract (RVOT) in mongrel dogs. Either 0.625% glutaraldehyde-treated (Group 1, n = 6) or glutaralde-hyde-chitosan-treated (Group 2, n = 6) porcine aortic noncoronary cusp with the aortic wall was sewn to the RVOT. Gross histological observations showed moderate calcification of the glutaraldehyde-treated cusps, but no calcification was noticed in the glutaraldehyde-chitosan-treated grafts at 5 months. This was confirmed by results of quantitative analyses for calcium in half of each ex-planted cusp with aortic wall. The calcium content of the 0.625% glutaraldehyde-treated cusps (Ca, 40.6 ± 24.9 mg/g dry wt) was significantly (p < 0.01) higher than that of glutaraldehyde-chitosan-treated cusps (Ca, 1.3 ± 0.29 mg/g dry wt). These findings suggest that chitosan post-treatment is effective in complete calcium mitigation of the glutaraldehyde-treated porcine aortic noncoronary cusps implanted in the RVOT in dogs.     

用牛颈静脉制备抗栓 抗钙化性右心流出道修复材料的实验研究

目的论证用牛颈静脉制备抗栓、抗钙化右心流出道修复材料的可行性。方法用EX-313交联牛颈静脉带瓣胶原管道并用肝素处理其内壁,裁成带单瓣的补片,用其扩大7条犬的肺动脉,同时建立用戊二醛制成的补片扩大肺动脉对照模型。在6个月内观察补片的抗栓、抗钙化、内皮化及组织重构的情况。结果在抗栓、抗钙化、内皮化及组织重构方面EX-313及肝素处理的牛颈静脉明显优于戊二醛处理者。结论经亲水性交联剂EX-313及肝素处理内壁的牛颈静脉有可能成为制作重建右心流出道的基本材料,在进一步研究的基础上它也可能成为组织工程带瓣管道的支架材料。     

戊二醛结合肝素鞣制牛颈静脉带瓣管道抗钙化的实验研究

目的用动物皮下包埋的模型,用戊二醛鞣制的牛颈静脉带瓣管道(VBJVC)作为对照,来研究戊二醛结合肝素鞣制的牛颈静脉带瓣管道是否具有更好的抗钙化能力和抗衰老能力。方法把牛颈静脉带瓣管道分为两组,实验组(戊二醛结合肝素组)和对照组(戊二醛组),分别包埋在两组实验兔的皮下。90d后取出牛颈静脉带瓣管道片,进行钙的定量分析和形态学检查。形态学分析包括光镜下HE染色和Von-Kossa染色,进行钙化程度的比较。结果比较戊二醛结合肝素鞣制的VBJVC(A组)较之传统的单纯的用戊二醛鞣制的VBJVC(B组),其结果有明显的统计学意义。(1)标本用原子分光光度计测量组织钙含量,A组(13.92±5.03)mg/g,B组(34.99±12.33)mg/g,A组的组织钙化明显低于B组;(2)光镜下用Von-Kossa钙染色,按照Carpentier的分级方法,A组,0度2个,Ⅰ度3个,Ⅱ度1个。B组,0度1个,Ⅰ度1个,Ⅱ度2个,Ⅲ度2个,A组的组织钙化明显低于B组。结论经过戊二醛结合肝素鞣制的VBJVC,较之单纯用戊二醛鞣制的VBJVC,有较好的抗钙化能力。     

牛颈静脉带瓣管道的抗钙化研究进展

应用带瓣管道重建右心室流出道是治疗复杂先天性心脏病的一项重要手段。近年来,戊二醛处理的牛颈静脉带瓣管道被广泛应用于临床,但术后管道钙化、衰败问题一直未得到解决。目前抗钙化处理方法有去细胞、染料介导光氧化、环氧化合物、组织工程等,但单一方法往往不能取得令人满意的效果,多种方法联合处理将是组织工程技术发展的方向。     

Improved postfixation treatment of glutaraldehyde fixed porcine aortic valves by monosodium glutamate

The use of bioprosthetic valves remains limited due to poor long-term durability primarily because of tissue calcification-associated degeneration. Release of locally cytotoxic residual aldehyde after glutaraldehyde fixation is one of the major causes of this degeneration. In this study, monosodium glutamate was used as postfixation treatment to bind residual aldehyde in order to block its toxic effects. Thirty-six pieces of fresh porcine aortic valves were fixed by 0.625% glutaraldehyde for 14 days, and then 18 of them were treated with 1% monosodium glutamate for another 3 days before they were implanted subcutaneously into the backs of two groups of rats (n = 9 in each group) for 45 and 90 days, respectively. Retrieved specimens were examined grossly, and calcium analysis and measurements of tissue collagen and water content were carried out. The results showed that, compared with glutaraldehyde fixed specimens, monosodium glutamate postfixation treated specimens had less calcification (calcium 104.93 + 50.94 versus 141.58 +/- 58.10 at 45 days and 103.07 +/- 76.48 versus 199.33 +/- 53.44 at 90 days, micrograms/mg dry weight, p < 0.01), higher collagen content (hydroxyproline 5.50 +/- 1.29 versus 3.58 +/- 1.48 at 45 days and 5.64 +/- 0.87 versus 4.25 +/- 0.65 at 90 days, micrograms/mg wet weight, p < 0.01), and higher water content (68.00 +/- 6.95% versus 61.33 +/- 8.83% at 90 days, p < 0.05) (mean +/- SD, paired t test). We conclude that monosodium glutamate couples with residual aldehyde, which significantly reduces calcification of glutaraldehyde fixed porcine aortic valves while preserving a higher tissue collagen and water content after implantation. The preserved tissue collagen and water content of the implants is closer to that of unimplanted native valves.     

醇对戊二醛鞣制的牛心包防钙化改性的实验研究

目的本实验采用几种醇改性戊二醛处理后的牛心包,希望寻找一种理想的生物瓣处理方法。方法分为五组,即新鲜对照组及戊二醛、2,3-丁三醇、1,3-丙二醇、1,2-丙二醇组。分组将牛心包试片植入SD大鼠背部皮下60d后,行组织钙定量测定及组织学检测。结果2,3-丁二醇改性后的牛心包试片钙平均含量为3.99±0.96ug/mg干重,与GA组、1,2-丙二醇,1,3-丙二醇试片的钙平均含量(241.89±7.79、244.26±6.39、167.32±12.29ug/mg干重)相比,钙化,钙化明显减轻(均P＜0.01),但与对照组(4.11±2.23ug/mg干重)相比无显著性差异(P＞0.05)。病理组织学检查显示2,3-丁二醇组和1,3-丙二醇组试片仅有轻度形态学改变。而GA组和1,2-丙二醇组试片有严重的胶原变性,大片钙化。结论戊二醛鞣制的生物材料经2,3-丁二醇改性后,可显著减轻钙化。     

内皮化环氧交联牛心包材料动物体内抗钙化的研究

目的研究生物材料内皮化延缓钙化的效果，从材料学上改进生物瓣性能，以提高其耐久性。方法将环氧交联的牛心包材料体外内皮化后同时进行犬股动脉间位移植和腹部皮下包埋，与单纯戊二醛处理者和单纯环氧处理者进行钙化程度对比。结果钙含量测试结果以内皮化环氧交联材料皮下包埋最低，未内皮化材料中以环氧处理者钙含量较低。结论环氧交联牛心包材料可实现体外快速内皮化，生物材料的内皮化“活化”有赖于表面全部被自体存活内皮细胞覆盖才能实现，完全的内皮化确有抗钙化作用，用两种动物模型研究生物材料抗钙化和内皮化时各有优缺点