枝化状聚乙二醇交联改善猪去细胞主动脉瓣的力学性能比较

目的 采用不同相对分子质量枝化状聚乙二醇(PEG)交联巯基化的猪去细胞瓣,观察其对去细胞瓣膜力学性能的影响.方法 将25例猪去细胞主动脉瓣随机分为5组:PEG3400组、PEG8000组、PEG12000组、PEG20000组和对照组,常温反应时间4 h.测定残余巯基计算交联效率;静态拉伸实验测定力学性能.结果 4 h交联率依次为92.40%、89.88%、87.87%和87.46%,相对分子质量越小,交联效率越高,PEG3400组与其他各组比较,差异有统计学意义(P＜0.05);力学测试显示,相对分子质量越大,力学强度越高,呈一定的正相关,PEG12000和PEG20000组抗拉强度分别为(3.22±0.41)MPa和(3.19±0.15)MPa,与对照组比较,差异有统计学意义(P＜0.05).结论 相对分子质量12000 Da和20000 Da枝化状聚乙二醇交联可有效改善猪去细胞瓣的力学性能,有望用于构建新型复合的组织工程瓣膜支架材料的研究应用.

Abstract：

Objective Branched polyethylene glycols (PEG) with different molecular weight were cross-linked to the porcine decellularized aortic valve (DAV) and the effects of PEG cross-linking on the mechanical properties were investigated. Methods A total of 25 porcine DAVs were randomly assigned into 5 groups: PEG3400, PEG8000, PEG12000, PEG20000 and control. The reactive time was 4 h at room temperature. The efficiency of crosslinking was calculated by measuring the residual thiol group. The mechanical properties were obtained by static tensile test. Results The efficiency of crosslinking was 92. 40% , 89. 88% , 87. 87% and 87. 46% in PEG3400, PEG8000, PEG12000, PEG20000 groups, respectively. As compared with other groups, the PEG3400 group had significantly greater increase in the crosslinking degree (P ＜ 0. 05). Tensile test showed the tensile strength of PEG12000 and PEG20000 groups was (3. 22 ±0.41) and (3. 19 ±0. 15) MPa, respectively, and significantly different from that in control group (P ＜ 0. 05). The tensile strength and young' s modulus had a positive correlation with the molecular weight of PEG. Conclusion Branched PEG with the molecular weight of 12 000 Da and 20 000 Dacan effectively crosslink to the porcine decellularized aortic valves and improve their mechanical behaviors, and has the potential to be used as the material of fabricating new hybrid scaffold of tissue engineering heart valves.     

枝化状聚乙二醇交联改善猪去细胞主动脉瓣的力学性能比较

Objective Branched polyethylene glycols (PEG) with different molecular weight were cross-linked to the porcine decellularized aortic valve (DAV) and the effects of PEG cross-linking on the mechanical properties were investigated. Methods A total of 25 porcine DAVs were randomly assigned into 5 groups: PEG3400, PEG8000, PEG12000, PEG20000 and control. The reactive time was 4 h at room temperature. The efficiency of crosslinking was calculated by measuring the residual thiol group. The mechanical properties were obtained by static tensile test. Results The efficiency of crosslinking was 92. 40% , 89. 88% , 87. 87% and 87. 46% in PEG3400, PEG8000, PEG12000, PEG20000 groups, respectively. As compared with other groups, the PEG3400 group had significantly greater increase in the crosslinking degree (P ＜ 0. 05). Tensile test showed the tensile strength of PEG12000 and PEG20000 groups was (3. 22 ±0.41) and (3. 19 ±0. 15) MPa, respectively, and significantly different from that in control group (P ＜ 0. 05). The tensile strength and young' s modulus had a positive correlation with the molecular weight of PEG. Conclusion Branched PEG with the molecular weight of 12 000 Da and 20 000 Dacan effectively crosslink to the porcine decellularized aortic valves and improve their mechanical behaviors, and has the potential to be used as the material of fabricating new hybrid scaffold of tissue engineering heart valves.     

应用四枝化状聚乙二醇-乙烯砜基交联去细胞带瓣管道的实验研究

目的利用四枝化状聚乙二醇-乙烯砜基（polyethylene glycol-VS,PEG-VS）交联去细胞主动脉带瓣管道,制备新型组织工程复合支架,研究其力学和生物学性能。方法采用胰酶法制备兔主动脉去细胞带瓣管道,利用四枝化状PEG-VS与兔去细胞带瓣管道交联构建复合支架,体外静态条件应用力学测试仪检测去细胞管道和复合支架力学性能。将纯种新西兰大白兔30只随机分为3组,每组10只,对照组：正常兔主动脉带瓣管道;去细胞组：去细胞带瓣管道;复合支架组：PEG复合支架。构建兔颈总动脉瓣膜移植模型,术后28 d分别用超声心动图检测3组移植管道通畅率,用HE染色、扫描电子显微镜观察微观形态和炎症细胞浸润,免疫荧光染色检测复合支架体内内皮化程度。结果体外力学测试结果示：PEG复合支架弹性模量及最大负荷力均较去细胞管道有明显提高（P弹性模量=3.1×10-9,P最大负荷力=1.1×10-6）。术后血管彩色多普勒超声心动图提示：复合支架组管腔通畅率高于对照组（P=0.054）和去细胞组（P=0.019）,腔内血栓形成率、管腔形变率明显降低;HE染色、扫描电子显微镜检测显示：复合支架组体内内皮化程度增高,内皮细胞于支架上均匀附着;免疫荧光检测显示：支架表面内皮细胞标记物CD34阳性率高于对照组和去细胞组。结论利用四枝化状功能化PEG-VS改性去细胞主动脉瓣带瓣管道,可明显改善组织工程支架生物力学性能和组织相容性。     

枝化状聚乙二醇交联去细胞瓣复合材料抗钙化性能研究

目的 探讨枝化状聚乙二醇(PEG)交联去细胞瓣复合材料的皮下抗钙化性能.方法 N-丁二酸-S-乙酰基巯基乙二醇酯(SATA)巯基化修饰去细胞瓣,以相对分子质量为20×103的枝化状丙烯酰化PEG交联获得复合材料(PEG组).以戊二醛固定天然瓣(GA-NAV组)和戊二醛固定去细胞瓣(GA-DAV组)为对照.取3组瓣膜样本各18枚,植入新西兰白兔背部皮下,分别于2、4、8周取出行Von Kossa染色观察钙沉积,原子吸收光谱法测定其钙含量.结果 GA-NAV组钙化发生快且进展迅速,2、4、8周的钙含量依次为(22.39±1.79)、(93.05±6.08)、(174.97±8.34) μg/mg,GA-DAV组钙化程度较轻,钙含量分别为(5.57±2.11)、(31.15 ±5.16)、(72.54±6.50) μg/mg,而PEG组未见明显钙化发生,对应钙含量仅为(0.46±0.07)、(1.68±0.10)、(2.92±0.25) μg/mg,相同时间点组间差异有统计学意义(P＜0.05).结论 相对分子质量为20×103的枝化状丙烯酰化PEG交联去细胞瓣复合材料具有良好的抗钙化性能.     

枝化状聚乙二醇对牛跟腱Ⅰ型胶原结构与构象稳定性的影响

目的 观察枝化状聚乙二醇对牛跟腱Ⅰ型胶原结构与构象稳定性的影响.方法 以牛跟腱Ⅰ型胶原为原料,以枝化状聚乙二醇为交联剂,在有效交联的前提下,通过测定羟脯氨酸含量观察聚乙二醇交联对胶原酶解稳定性的影响,采用示差扫描量热法(DSC)观察胶原热变性温度,采用圆二色光谱法(CD)观察聚乙二醇对胶原三螺旋构象稳定性的影响.结果 枝化状聚乙二醇交联组24 h降解率为26.79%,而对照组则达97.39%,两者比较差异有统计学意义(P＜0.05).交联组热变性温度为(79.1±1.2)℃,与对照组(68.6±1.9)℃比较,差异有统计学意义(P＜0.05);CD谱测定特征性吸收峰差异无统计学意义(P＞0.05).结论 枝化状聚乙二醇可显著提高其热变形温度和抗酶性分解能力,而对其三螺旋结构与构象无明显不利影响,是较理想的生物高分子交联剂,可有望用于胶原分子的交联改性.

Abstract：

Objective The triple helix of collagen is the basis of its biological function, such as cell adhesion and tissue remodeling. Crosslinking of collagen with chemical agent will improve the biomechanical properties. However, the effects of differernt crosslinking agents on the structure and conformation stability of type Ⅰ collagen are rarely investigated. Methods The branched polyethylene glycol ( PEG)derivative ( MW 12 kD) was used as crosslinking agent, and allowed to react with bovine achilles' s tendon type Ⅰ collagen modified by succinimidylacetylthioacetate (SATA). The ability of resistance of crosslinked collagen to enzymatic degradation was investigated by measuring the release of hydroxyproline, and differential scanning calorimeter ( DSC ) was taken to determine the thermal denaturation temperature. The effect of PEG on the riple helix of collagen was studied by circular dichroism ( CD) . Results The resistance ability of PEG crosslinked collagen was strongly enhanced when compared with that of control group ( P ＜0. 05 ) , and the thermal denaturation temperature was also significantly rised. CD demonstrated that PEG crosslinking did not result in the destruction of the triple helix conformation of type Ⅰ collagen. Conclusion Branched PEG derivative used in this study is a promising polymer crosslinking agent that may be utilized in the modification of type Ⅰ collagen.     

去细胞和戊二醛处理对猪主动脉瓣内源性逆转录病毒影响的比较

我们应用聚合酶链反应(PCR)及逆转录．聚合酶链反应(RT-PCR)方法对去细胞猪瓣、猪外周血、犬腹主动脉内移植去细胞猪瓣1个月后外周血，并同时对经戊二醛处理猪主动脉瓣加以对照研究．探讨这两种方法对猪内源性逆转录病毒(PERV)的影响。     

去细胞猪主动脉瓣叶的获取和内皮细胞的种植

目的　探讨猪主动脉瓣叶去细胞后作为组织工程心脏瓣膜支架的可行性。　方法　经胰酶 - EDTA、表面活性剂和核酸酶处理 ,去除猪主动脉瓣叶的细胞成分 ,测定瓣叶去细胞前、后的生物力学特性 ,并在其表面种植新生牛主动脉内皮细胞 (BAECs) ;分别行大鼠皮下包埋实验。　结果　猪主动脉瓣叶中的细胞成分能完全去除 ,获得完整无细胞的纤维网状支架 ,断裂强度和断裂伸长率无明显变化 ;种植的 BAECs在去细胞瓣叶表面可形成一层连续的细胞层 ,其分泌前列环素 (PGI2 )的能力同直接种植在 2 4孔板中的比较 ,差异无统计学意义 (P>0 .0 5 )。　结论　猪主动脉瓣去细胞后获得的纤维支架可以用来构建组织工程瓣膜 ,适宜于血管内皮细胞的生长。     

NO／cGMP通路调节降低由去细胞和聚乙二醇化血红蛋白导致的血管收缩

NO／cGMP通路的活化调节平滑肌细胞松弛，因此，尽管结合5-kDa的聚酰亚胺（乙烯）一乙二醇（聚乙二醇，PEG）在体内能降低血管收缩作用，但在作为血液替代品的去细胞血红蛋白（Hb）的应用中，血管收缩仍然是一主要障碍。为研究一项最近开发的去氧聚乙二醇化血红蛋白（Deoxy-PEGHb）和操纵NO／cGMP通路如何调节离体小鼠心脏的血管收缩。     

高交联玉米淀粉的非晶化特性

研究了以三氯氧磷为交联剂的高交联玉米淀粉的制备方法,报道了高交联玉米淀粉颗粒随反应取代度增加而逐渐非晶化的现象.采用偏光显微镜和广角X-射线衍射对其由多晶态向非晶态的渐变过程进行了研究,提出高交联玉米淀粉中存在不同于原淀粉多晶颗粒态的只含无定形结构的非晶颗粒态。对非晶颗粒态高交联玉米淀粉颗粒的粒度分布的进一步研究结果还表明,此时的淀粉颗粒发生了轻度的膨胀。     

碳化二亚胺交联去细胞猪肺动脉带瓣管道物理性能的研究

目的研究碳化二亚胺（carbodiimide,EDC）交联去细胞猪肺动脉带瓣管道体外的物理性能。方法在相对无菌条件下取得20个猪带瓣肺动脉,沿肺动脉瓣膜交界处纵向剪开分成3份（每份样本都由肺动脉管壁及其瓣膜组织组成）。将3份样本用抽签法随机分为3组：A组为对照组,为新鲜猪肺动脉带瓣管道样本,不再进一步处理;B组为去细胞组织样本,采用胰蛋白酶＋去污剂Triton X-100对猪带瓣肺动脉样本进行去细胞处理;C组为EDC交联去细胞组织样本,即在B组处理的基础上进一步行EDC交联处理。测试肺动脉组织含水量、厚度、抗张强度和热皱缩温度,评价所有样本的物理性能。结果胰蛋白酶＋去污剂能完全去除猪肺动脉带瓣管道组织的内膜层内皮细胞和管壁及瓣膜组织的细胞和部分基质成分,保留比较完整的纤维支架成份。与A组相比,B组肺动脉壁样本含水量增加（P=0.000）,肺动脉瓣膜含水量变化不明显,肺动脉壁（P=0.000）和瓣膜（P=0.000）的厚度减少、抗张强度下降（P〈0.01）、热皱缩温度无明显变化。与B组比较,C组肺动脉管壁样本含水量减少（P=0.000）,肺动脉瓣膜样本含水量变化不明显。肺动脉管壁和瓣膜样本的厚度变化不明显、抗张强度增加（P〈0.01）、热皱缩温度增加（P=0.000,0.000）。与A组相比,C组肺动脉壁和瓣膜含水量变化不明显,肺动脉壁和瓣膜的厚度减小（P=0.000,0.000）,抗张强度无明显差别,热皱缩温度增加（P=0.000,0.000）。结论 EDC交联去细胞方法能够加强去细胞肺动脉带瓣管道的抗张强度、降低去细胞肺动脉管壁的含水量。     

Failure properties of ascending thoracic aortic aneurysms with dysfunctional tricuspid aortic valves

 Open in a separate windowOBJECTIVESAscending thoracic aortic aneurysms (ATAAs) often coexist with dysfunctional tricuspid aortic valves (TAVs). How valvular pathology relates to the aortic wall mechanical properties requires detailed examination.METHODSIntact-wall and layer-specific mechanical properties from 40 and 21 patients with TAV-ATAAs, respectively, were studied using uniaxial tensile testing, longitudinally and circumferentially. Failure stress (tensile strength), failure stretch (extensibility) and peak elastic modulus (stiffness) measurements, along with histological assays of thickness and elastin/collagen contents, were compared among patients with no valvular pathology (NVP), aortic stenosis (AS) or aortic insufficiency (AI).RESULTSIntact-wall stiffness longitudinally and medial strength and stiffness, in either direction, were significantly lower in AI patients than in AS and NVP patients. Intact-wall/medial thickness and extensibility in either direction were significantly lower in AS patients than in AI and NVP patients. In contrast, intact-wall/medial stiffness circumferentially was significantly higher in AS patients than in NVP patients, consistent with the significantly increased medial collagen in AS patients. Failure properties and medial thickness and elastin/collagen contents were significantly lower (more impaired) in females. The left lateral was the thickest quadrant in NVP patients, but the 4 quadrants were equally thick in AS and AI patients. There were significant differences in strength and stiffness among quadrants, which varied however in the 3 patient groups.CONCLUSIONSThe aortic wall load-bearing capacity was impaired in patients with ATAA in the presence of TAV stenosis or insufficiency. These findings lend biomechanical support to the current guidelines suggesting lower thresholds for elective ascending aorta replacement in cases of aortic valve surgery.     

脱细胞天然支架生物力学性能变化及预处理改善组织相容性的实验研究

目的观察脱细胞猪主动脉瓣的生物力学性能变化,探讨不同预处理改善天然支架组织相容性的效果。方法新鲜猪主动脉瓣经酶加去污剂法去除细胞,力学测试仪检测其最大负荷、最大应力、最大应变和弹性模量的变化,苏木精-伊红（HE）染色、Ⅰ型胶原免疫组化染色和扫描电镜观察其病理形态学变化;将脱细胞瓣膜分别予磷酸缓冲液、多聚赖氨酸和未灭活胎牛血清包被处理,然后种植大鼠主动脉肌成纤维细胞,甲基噻唑基四唑试验检测细胞黏附率,HE染色和扫描电镜观察形态学变化。结果酶加去污剂法能完全脱去瓣膜细胞,基本维持胶原纤维的空间结构,但其最大负荷、最大应力及弹性模量下降,最大应变上升（P〈0．05）;胎牛血清预处理去细胞瓣能显著提高肌成纤维细胞的黏附率,促进细胞生长、分化和增殖,并在瓣膜表面形成连续的细胞层（F值=129．26,P=0．000）。结论酶加去污剂法可较完全去除猪主动脉瓣膜细胞并保持细胞外基质的三维结构,但其生物力学性能有所下降;胎牛血清预处理能改善脱细胞瓣天然支架的细胞黏附、生长和繁殖。     

去细胞组织工程同种心脏瓣膜的免疫学特征

目的构建去细胞的组织工程同种心脏瓣膜,探讨其免疫学特性变化。方法取液氮保存的人同种主动脉带瓣管道,采用低渗液-去污剂(1％去氧胆酸,1％DOA)-核酸酶[20mg／L核糖核酸酶(RNase)和200 mg／L去氧核糖核酸酶(DNase)]去细胞法去除同种心脏瓣膜、管壁组织及肌肉中所有的细胞成分,保留完整的细胞外基质,构建去细胞的组织工程同种心脏瓣膜;采用免疫组织化学方法测定HLA-DR抗原在同种心脏瓣膜组织中的表达;行大鼠皮下移植试验,评价组织学变化及Van Kosaa银染色法定性分析、原子吸收光度计法定量分析组织的钙化程度。结果去细胞同种心脏瓣膜的白细胞抗原-DR(HLA-DR)表达较深低温液氮保存同种心脏瓣膜(HVA)显著下降;大鼠皮下移植8周后,去细胞同种心脏瓣膜组织中的炎性细胞浸润显著减少,定性分析表明去细胞组瓣叶和管壁组织中呈黑色颗粒的钙盐沉积较深低温液氮组显著下降,尤管壁组织更为显著;定量分析亦表明去细胞瓣叶和管壁组织的钙化程度显著下降(瓣叶:去细胞组0．83±0．17 mg／ g,深低温液氮组[(1．39±0．26)mg／g,P<0．05];管壁:去细胞组(2．35±2．58)mg／g,深低温液氮组[(42．66±7．46)mg／g,P<0．05)]。结论去细胞同种心脏瓣膜的免疫原性显著下降,有望减轻移植后免疫反应,延长同种心脏瓣膜的使用寿命。     

Long-term clinical results after aortic valve replacement with mechanical heart valves and mitral valve replacement with porcine valves]

Long-term clinical results of aortic valve replacement (AVR) with mechanical heart valves and mitral valve replacement (MVR) with porcine valves were analysed. Sixty-three patients received isolated AVR and 48 received isolated MVR. Sixty-eight patients with MVR including double or triple valve replacement were also added in order to evaluate the primary tissue failure (PTF). The patients with operative deaths were excluded. Survival rate at 11 years in AVR was 68 +/- 10% and 67 +/- 15% in MVR without statistical difference. At 11 years, 76 +/- 8% of the patients in AVR were free from valve-related complications in contrast with the poor result of 34 +/- 31% in MVR (p less than 0.01). Main cause of this poor result in MVR was PTF as indicated in following event free rates; 83 +/- 9% at 7 years, 61 +/- 25% at 10 years and 49 +/- 31% at 13 years. There was no statistical difference between patients of above 50 years and below 49 years in PTF. Valve-related death event free was 93 +/- 5% in AVR and 86 +/- 11% in MVR at 11 years (not significant), however, there was statistical difference in re-operation event free rate as 94 +/- 5% in AVR and 76 +/- 11% in MVR at 11 years (p less than 0.001). These results suggest that the use of porcine valves in mitral position is confined to the selected patients.     

Tricuspidisation of the aortic valve with creation of a crown-like annulus is able to restore a normal valve function in bicuspid aortic valves

Objective: To evaluate the early results of a new method to repair malfunctioning bicuspid aortic valves by creating a tricuspid valve with a crown-like (i.e. anatomic) annulus. Material and methods: Twelve patients (ages from 10 to 27 years) with chronic regurgitation (and flow-dependent stenosis) of a bicuspid aortic valve underwent repair with the principle of creating a tricuspid valve and a crown-like annulus. The fused leaflets were trimmed and reinserted underneath the existing aortic annulus to create one new native cusp. The third leaflet was fashioned out of a xenopericard patch and was inserted underneath the existing annulus as well to restore the crown-like anatomy of a normal aortic annulus. A tricuspid aortic valve with a morphologically normal annulus was thus created, which resulted in improved coaptation of the leaflets. The repair was immediately assessed by transesophageal echocardiography (TEE) with the heart loaded at 50%. In two patients, a second run helped fine-tune the repair. Median cross-clamping time was 82 min. Follow-up ranged from 3 to 46 months (median 13 months). Results: No significant complication occurred. The function of the aortic valve was excellent with trivial or mild regurgitation in 11 patients and moderate regurgitation in 1 patient. There was no stenosis across the valve. The repair remained stable over time. Remodelling of the left ventricle occurred as expected. Conclusions: Aortic valve repair is feasible in some dysfunctioning bicuspid aortic valves. Tricuspidisation of the valve can result in excellent systolic and diastolic functions. The creation of a crown-like annulus results in improved coaptation of the cusps and could lead to more reliable outcome. Although long-term results are needed, this anatomic correction seems to be a good alternative to valvular replacement in certain sub-groups of patients.     

人正常主动脉瓣、风湿性主动脉瓣间质细胞的生物学行为比较

目的　比较人正常主动脉瓣、风湿性主动脉瓣瓣膜间质细胞体外培养的生物学特性。方法　从人正常主动脉瓣和风湿性病变主动脉瓣组织分离培养瓣膜间质细胞 ,在细胞形态学和生长动力学方面进行比较研究。结果　体外培养的正常主动脉瓣瓣膜间质细胞与风湿性主动脉瓣瓣膜间质细胞在形态学方面差异无显著性 ,但风湿性病变瓣膜间质细胞排列更加紊乱 ,极性消失 ,有交叉重叠现象 ,细胞浆内有较多残余小体。在接种相同的细胞密度和相同的培养条件下 ,两者细胞生长率为 (2 .0 5± 0 .1 0 ) %、(2 .1 3± 0 .1 2 ) %、分裂指数为 (2 1 .32± 1 .46)‰、(2 4 .2 5± 1 .98)‰、克隆形成率为 (45 .2 0± 2 .50 ) %、(48.60± 2 .82 ) %差异无显著性 (P >0 .0 5)。结论　正常与风湿性病变主动脉瓣瓣膜间质细胞在体外培养状态下 ,其形态学和生长动力学 ,具有相似的生物学特性     

Bovine pericardium versus porcine aortic valve: comparison of tissue biological properties as prosthetic valves

The choice of a bioprosthetic valve substitute remains controversial with the major concern being primary tissue failure after implantation. We compared biological properties of the two most frequently used bioprosthetic valve materials, bovine pericardium and porcine aortic valve, before and 90 days after subcutaneous implantation in rats. Before implantation, tissue collagen and water content were measured in nine pieces of bovine pericardium and porcine valves, each fixed in 0.625% glutaraldehyde; calcium, tissue collagen, and water content were measured in another nine pieces of the same tissues after 90 days' implantation. Bovine pericardium had higher collagen content than that of porcine valve (hydroxyproline, 7.98 +/- 0.05* vs. 4.56 +/- 0.02 micrograms/mg, dry weight) but lesser water content (72.16 +/- 3.22%* vs. 87.36 +/- 1.62%) before implantation (*p < 0.001, mean +/- SD, t test); after implantation, bovine pericardium still maintained higher collagen content (hydroxyproline, 4.89 +/- 0.04* vs. 2.61 +/- 0.06 micrograms/mg, dry weight) but contained the same amount of water (60.24 +/- 5.08% vs. 61.43 +/- 9.00%) and calcium (214.43 +/- 34.34 vs. 199.33 +/- 53.44 micrograms/mg, dry weight) (*p < 0.001, mean +/- SD, t test). We conclude that bovine pericardium has superior intrinsic biological properties for prosthetic valve manufacture. With proper integration of properties and design it will in some applications be superior to the porcine aortic valve.