酒精对戊二醛固定牛心包影响的实验研究

目的探讨酒精处理对戊二醛固定牛心包体内钙化和力学性能的影响。方法牛心包经戊二醛固定后,再用酒精处理,未经酒精处理组作为对照组。将两组牛心包埋植于大鼠皮下,分别于3周和2个月后取出,然后通过原子吸收光谱法和VonKossa钙染色评估两组牛心包钙含量变化。进行力学性能测试和热皱缩温度测定评估两组牛心包的物理性能。结果组织学检查显示酒精再处理对牛心包胶原纤维结构无明显破坏;大鼠皮下埋植实验显示酒精再处理能显著抑制戊二醛固定牛心包的钙化,3周时酒精再处理的戊二醛固定牛心包组钙含量是(1.08±0.30)mg/g,酒精未处理组钙含量是(24.55±7.16)mg/g;2个月时酒精再处理戊二醛固定牛心包组钙含量是(1.20±0.43)mg/g,酒精未处理组钙含量是(120.22±37.36)mg/g,差异有统计学意义;酒精再处理后牛心包的断裂伸长率增大,抗拉负荷增大,但抗拉强度改变无统计学意义;酒精再处理后牛心包的热皱缩温度下降。结论酒精处理戊二醛固定的牛心包动物体内实验显示有显著的抗钙化作用,并且具有良好的力学性能。     

牛心包脱细胞处理后免疫原性的研究

牛心包用0.5%胰蛋白酶处理3 h,探讨其作为组织工程心脏瓣膜支架材料的可行性。大鼠被分成新鲜牛心包组、脱细胞处理牛心包组、空白对照组三组,分别测定其血清中产生的特异性抗牛心包抗体IgG的OD值,结果发现IgG含量在术后7 d三组间比较无统计学意义(P>0.05),其余各时点间比较差异有显著性(P<0.05),在相应时点新鲜牛心包产生的IgG值比脱细胞处理的牛心包高,且新鲜牛心包产生的IgG值在术后2月达峰值,而脱细胞处理牛心包产生的IgG在术后1月达峰值。组织学观察发现脱细胞处理后牛心包完全清除了细胞成分,纤维网架结构轻度松散,但基本保持完整。结果表明,这种脱细胞方法能完全清除牛心包上的细胞成分,降低免疫原性。     

生物交联剂京尼平交联牛心包生物支架材料的性能

脱细胞牛心包膜因其具有优良特性而成为组织工程生物瓣膜中理想的支架材料.采用冻融+表面活性剂法对牛心包组织进行脱细胞处理后,用戊二醛或京尼平对其进行表面修饰固定.通过HE染色及扫描电镜观察脱细胞效果,并对交联组织进行厚度检测、含水量和接触角测试、力学检测、交联指数和差示扫描量热(DSC)测定、体外降解、溶血试验以及细胞毒性试验,判断两种交联方法对脱细胞牛心包基质的影响,从而选择出交联脱细胞组织的最好方法.结果显示,冻融+表面活性剂法脱除细胞彻底,戊二醛或京尼平交联后组织厚度明显增加分别约20%,25%,亲水性良好,力学特性稳定,交联指数都高达90%,且28 d降解率分别为5%和3%,交联后组织稳定性高.但京尼平组溶血率(0.37%)远小于戊二醛组溶血率(13.77%),且细胞毒性很低.作为瓣膜组织工程支架材料,京尼平交联脱细胞牛心包膜比戊二醛交联效果具有更好生物相容性,交联效果好,是较好的交联方法.     

降低医用牛心包致热原性研究

目的比较不同浓度酒精溶液洗脱医用牛心包中残留戊二醛的效果,通过酒精溶液洗脱作用降低其致热反应。方法牛心包经戊二醛固定后,再用酒精溶液处理,未经酒精溶液处理组作为对照组。分别采用50％、60％、70％、80％、90％酒精溶液对医用牛心包进行浸泡洗脱处理,应用气相色谱法测定洗脱液中戊二醛的浓度,比较不同浓度酒精溶液洗脱戊二醛效果;并采用2005年版《中华人民共和国药典》附录热原检查的方法,检测经60％、70％、90％不同浓度酒精溶液洗脱后牛心包致家兔体温升高的差别。结果洗脱实验中,60％和70％酒精溶液对医用牛心包冲洗后,洗脱液中戊二醛含量分别达18．21‰±0．14％。和11．91‰±0．09‰,优于其余浓度组（n=3,P〈0．01）,60％酒精溶液组优于70％酒精溶液组（n=3,P〈0．05）;热原实验,60％、70％、90％酒精溶液处理组、戊二醛对照组家兔体温升高值分别为0．5℃、0．6℃、0．9℃、0．8℃,其中以60％的酒精溶液效果最优,与对照组相比差异有统计学意义（n=3,P〈0．05）,其余两组与对照组比较差异无统计学意义。结论60％酒精溶液处理可以有效洗脱医用牛心包中残留戊二醛,并且可明显降低其致热原性。     

染料介导光氧化固定牛带瓣颈静脉的生物特性评价

为了解染料介导光氧化固定牛颈静脉的生物稳定性,抗钙化性和免疫原性,新鲜的牛颈带瓣静脉经染料介导光氧化法固定,分别进行化学消化和胃蛋白酶消化,上清液进行梯度聚丙烯酰胺电泳来了解其生物稳定性;将氧化固定的材料埋植于SD大鼠皮下,3周后取出,测钙含量、进行钙染色以及组织学检查了解其抗钙化性和免疫原性;采用戊二醛固定以及新鲜的牛带瓣颈静脉作为对照。染料介导光氧化固定的牛带瓣颈静脉经化学和酶消化之后的上清液电泳之后出现极淡的蛋白条带,明显淡于新鲜的未处理的材料,戊二醛固定的材料几乎不出现蛋白条带。皮下埋植3周后,钙含量测定,染料介导光氧化组显著低于戊二醛组,统计学上差异有显著性意义;钙染色,戊二醛组可见大量的钙沉积,新鲜组也可见钙沉积,但比戊二醛组少,染料介导光氧化组只见少量钙沉积;组织学检查提示光氧化组细胞浸润明显少于其他两组。体外动物实验提示,染料介导光氧化固定的牛带瓣颈静脉具有生物稳定性,低的钙化性和低免疫原性。     

不同方法处理牛心包的体外力学和抗钙化性能评价

比较传统GA法（A组）、三氯化铝与乙醇联合处理法（B组）、多聚环氧化物处理法（C组）和光氧化法（D组）四种不同方法处理牛心包后力学与抗钙化性能。对新鲜牛心包随机分为四组,用四种方法处理后,测定组织的极限抗拉强度、断裂伸长率、热皱缩温度,比较其力学性能;建立大鼠皮下植入模型,包埋2月后取出标本测定组织钙含量,并做HE染色及von Kossa染色。极限抗拉强度：B组〉A组=C组〉D组;断裂伸长率：C组=D组〉B组〉A组;热皱缩温度：A组=B组〉C组〉D组;钙含量测定：A组〉〉C组〉B组〉D组（P〈0.05）;HE染色和von Kossa染色结果符合以上测定值。三氯化铝与乙醇联合处理组的组织交联程度与力学强度最佳,光氧化处理组与多聚环氧化物（简称PC）处理组的组织柔韧性更好。在抗钙化性能方面,光氧化处理组最好,三氯化铝与乙醇联合处理组与多聚环氧化物处理组次之,GA处理组最差。     

AngⅡ诱导大鼠BMSCs联合脱细胞牛心包体外构建工程化心肌组织的实验研究

目的：探讨血管紧张素Ⅱ（AngⅡ）诱导骨髓间充质干细胞（BMSCs）分化为心肌细胞的能力,及诱导后BMSCs体外联合脱细胞牛心包构建工程化组织心肌的可行性。方法分离培养大鼠BMSCs,用含0.1μmol/L AngⅡ的完全培养基诱导培养第3代BMSCs 24 h,然后换用完全培养基连续培养;对照组采用完全培养基连续培养。采用免疫荧光法检测诱导后的BMSCs表达心肌特异蛋白cTnT、β-MHC情况;透射电镜观察诱导后的细胞超微结构。用去污剂-酶消化法对新鲜牛心包行脱细胞处理,然后将诱导后4周的BMSCs接种于脱细胞牛心包生物支架上培养3 d后,对其进行观察检测。结果 AngⅡ诱导后的BMSCs向心肌细胞分化,可表达cTnT和β-MHC,对照组则不表达cTnT和β-MHC。电镜结果显示诱导后的细胞间可见类肌节组织及明显的桥粒连接;新鲜的牛心包经去污剂-酶联合四步法脱细胞处理及京尼平交联后,HE染色观察脱细胞的效率接近100%。扫描电镜观察发现脱细胞处理后的牛心包表面无细胞残留且表面排列杂乱,放大后可见有2μm小孔。观察体外构建的工程化心肌显示诱导后的BMSCs可良好地黏附于脱细胞牛心包生物支架表面并生长增殖,且少量可渗透到支架内部。结论 AngⅡ诱导的大鼠BMSCs可向心肌细胞方向分化,表达心肌特异蛋白cTnT和β-MHC。将其种植于脱细胞牛心包生物支架上,表面黏附良好,且可渗透到支架内部及血管周围,有望用于构建具有血管网络化的组织工程化心肌。     

灭菌处理牛心包制备脱细胞支架的内皮化

文题释义：组织工程：主要研究种子细胞、生物材料、构建组织和器官4个方向,其核心是建立由细胞和生物材料构成的三维空间复合体,对病损组织进行形态、结构和功能的重建并达到永久性替代,具有良好的发展前景和广阔的应用市场。牛心包脱细胞支架：经化学和物理方法去除牛心包中的细胞,形成无免疫原性或低免疫原性材料,构建组织工程支架。 背景：组织工程学研究常用的动物组织不可避免地存在各种微生物附着,而无菌是组织工程材料临床应用的一项基本要求。 目的：观察体积分数75%乙醇灭菌对牛心包性能及生物相容性的影响。方法：将牛心包组织分别用无菌PBS(对照组)、含1%抗生素(青霉素/链霉素/两性霉素B溶液)的PBS、氯己定及体积分数75%乙醇进行灭菌处理。采用LB固体培养基评价4种方法的杀菌效果;采用VB染色评估4组处理对牛心包组织结构的影响;通过CCK-8实验测定4种处理抽提液的细胞毒性。将体积分数75%乙醇灭菌处理的牛心包制作为脱细胞支架,与人脐静脉内皮细胞共培养,观察细胞的黏附与内皮化效果。结果与结论：①体积分数75%乙醇和氯己定处理24 h的牛心包满足完全灭菌的要求,1%抗生素处理组和对照组可见明显菌落形成;②VB染色显示,体积分数75%乙醇、氯己定和1%抗生素处理的牛心包胶原纤维呈波浪状排列整齐,结构紧凑,弹性纤维含量较少但结构清晰;③体积分数75%乙醇灭菌处理的牛心包不影响L929细胞的增殖活性,培养1-3 d内的细胞存活率均在100%以上;氯己定灭菌处理的牛心包有很强的细胞毒性,导致细胞死亡;④人脐静脉内皮细胞可在脱细胞支架表面正常生长与黏附;在20 d的种植期内,第8-12天脱细胞支架表面黏附的细胞最多;⑤结果说明,体积分数75%乙醇能够有效消灭附着在牛心包上的所有微生物,不会影响牛心包的组织学完整性与生物相容性。ORCID: 0000-0002-3448-8230(刘飞) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

环氧交联牛心包材料内皮化的抗钙化作用研究

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 ,提高其耐久性打下基础。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确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效果 ,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     

3D打印淫羊藿苷/脱钙骨基质修复兔股骨髁骨缺损

文题释义：脱钙骨基质：主要由93%胶原、5%可溶性蛋白及2%残余矿化基质组成的商业化生物材料，包含不同浓度的骨形态发生蛋白、生长因子及转化生长因子，具备良好的骨传导与骨诱导能力，可以单独或联合其他材料广泛用于骨折、骨不连、骨肿瘤、骨融合、股骨头坏死等治疗中。结构模型指数：是描述骨小梁组成结构中板层结构和杆状结构比例的参数。如果结构中骨小梁主要为板层结构，那么结构模型指数接近于0；如果结构中骨小梁主要为杆状结构，那么结构模型指数接近于3，该值越小骨小梁越成熟。背景：淫羊藿苷可促进骨髓间充质干细胞的增殖与成骨分化，具有良好的促成骨性能。脱钙骨基质具备良好的骨传导与骨诱导能力，可以单独或联合其他材料广泛用于骨修复中。 目的：观察3D打印淫羊藿苷/脱钙骨基质材料的缓释性能、细胞相容性与体内成骨性能。方法：利用3D打印技术制备淫羊藿苷/脱钙骨基质材料与脱钙骨基质材料，检测淫羊藿苷/脱钙骨基质材料的体外缓释性能。将骨髓间充质干细胞分别接种于两种材料表面，以单独培养的细胞为对照，于设定的时间点进行活/死染色、MTT、碱性磷酸酶活性与骨钙素含量检测。取新西兰大白兔30只，建立股骨髁骨缺损模型后分3组处理：对照组不植入任何材料，一组植入脱钙骨基质与同种异体兔骨髓间充质干细胞复合物，另一组植入淫羊藿苷/脱钙骨基质材料与同种异体兔骨髓间充质干细胞复合物，术后4，12周进行Micro-CT、组织学与力学性能检测。结果与结论：①3D打印淫羊藿苷/脱钙骨基质材料具有缓释性能，在28 d时淫羊藿苷释放量达总量的(54.9±7.9)%；②活/死染色显示，接种1 d后两组材料表面的细胞数量较少，随着培养时间的延长，细胞数量明显增多，至7 d时细胞形态良好，并且淫羊藿苷/脱钙骨基质材料表面的细胞更加均匀、数量更多；③MTT检测显示，淫羊藿苷/脱钙骨基质组培养7，10，14 d的细胞增殖快于其余两组(P < 0.05)；④淫羊藿苷/脱钙骨基质组培养7，10，14 d的碱性磷酸酶活性与骨钙素含量均高于其余两组(P < 0.05)；⑤术后12周Micro-CT显示，植入材料两组均可见大量的骨小梁，其中淫羊藿苷/脱钙骨基质组骨小梁数量更多、骨小梁厚度增加、骨小梁分离度更小；⑥术后12周组织学显示，植入材料两组可见大量骨组织形成，其中淫羊藿苷/脱钙骨基质组新生骨量最多；⑦淫羊藿苷/脱钙骨基质组抗压强度高于其余两组(P < 0.05)；⑧结果表明，3D打印淫羊藿苷/脱钙骨基质具有良好的缓释性能、细胞相容性与骨诱导性能。ORCID: 0000-0002-5272-812X(张虎雄) 中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程     

In vivo calcification of glutaraldehyde-fixed cardiac valve and pericardium of Phoca groenlandica

Calcification remains the main reason for failure of bioprosthetic valves. The aim of this study was to evaluate the in vivo calcification response of a new bioprosthetic valve, derived from cardiac tissue of Phoca groenlandica. Aortic and pulmonary leaflets, bovine, and Phoca groenlandica pericardia were fixed in buffered glutaraldehyde solution. Tissues were divided into four groups: group 1, bovine pericardium (BP); group 2, pulmonary leaflets; group 3, seal pericardium; and group 4, aortic leaflets. All samples were implanted subdermally into four sets of eight female 12-day-old Wistar rats for 21 days. The tissues were divided into two parts for calcium measurement, and histology with hematoxylin-eosin, von Kossa, and Weigert Van Gieson staining. All groups experienced significant calcification. Group 1 with 1.39 mg/g (0.34) before and 125.78 mg/g (21.48) after implantation (p < 0.001), group 2 with 1.50 mg/g (0.43) before and 151.85 mg/g (19.1) after (p < 0.001), group 3 with 3.15 mg/g (0.62) before and 116.38 mg/g (33.74) after (p < 0.001), and group 4 with 1.84 mg/g (0.52) before and 126.95 mg/g (13.37) after (p < 0.001). Explant samples showed foreign body response, disorganized collagen, and obvious calcification. The cardiac valve and pericardium of Phoca groenlandica calcify to the same extent as the BP.     

Calcification of chemically treated bovine pericardium.

One of the most important problems arising in cardiac bioprostheses made with bovine pericardium and, more generally, with biologically-derived tissues is tissue calcification. The present study assessed four chemical treatments on patches of bovine pericardium, intended to avoid or minimize calcification. Pericardium specimens were treated with: A) 0.5% glutaraldehyde; B) 0.5% glutaraldehyde + 4% formaldehyde; C) same as A, but with a further neutralization treatment; D) acylation of fresh bovine pericardium. Circular samples of 1 cm diameter were subcutaneously implanted in the abdominal region of three groups of six rats. The explants were retrieved after 2, 4 and 8 weeks. The calcium content and the histological results showed better behaviour for C and D samples than with the commonly used fixation methods (A and B). The lowest calcification was observed with treatment D, even though its morphological structures were somewhat modified with homogenation of collagen bundles. Among the glutaraldehyde-based treatments, treatment C appears to be the most promising because the pericardium shows slower calcium accumulation with a diffusive pattern.     

Study on the Preparation of Decellularized Scaffold of Bovine Pericardium

Objective: To search for the best procedure on preparation of acellular bovine pericardium,so to provide scaffolds for constructing tissue-engineering Methods:The bovine pericardiums were treated with 5 methods,which were divided into 6 groups.Group A:Fresh bovinepericardium;GroupB:Trypsin-detergentgroup;GroupC:Freeze-thaw-detergent24 h group;Group D:.Freeze-thaw-detergent 48 h group;Group E:Freeze-thaw-nuclease group;Group F:Detergent-nuclease group.Then,by HE staining and scanning electron microscope to observe the effects of decellularization and fibrous changes among the 6 groups;by water content testingmechanical testing to observe the changes in physical properties of the matrix;by detecting the DNA content of each group to determine the effect of decellularization qualitatively;by cytotoxicity test to detect the biocompatibility of bovine pericardium in each group.Results:The 5 methods can all remove the cellular components effectively,compared with the fresh bovine pericardium,the water content of each decellularized group were increased (P＜0.05),while the DNA content decreased (P＜0.05),with statistically significant differences.Of group E,the fibers were a little disorder,with the largest tension and the elastic modulus increased,while the rupture tensile rate decreased.Compared with fresh bovine pericardium,the largest tension of the other decellularization groups were all decreased (P＜0.05).The fibers of group B,group D were irregularly arranged and also with ruptures,both the elastic modulus and the rupture tensile rate decreased(P＜0.05).In group C and F,the fibers were dense and their direction was normal,the elastic modulus and the rupture tensile rate were similar to the fresh bovine pericardium (P＞0.05).Cytotoxicity results showed that the cell toxicity of group B,group C,group D,group E and group F were respectively 0.9,0.6,1.0,1.0 and 0.5,each group were qualified toxicity test,in which group C and group F were with the lowest cytotoxicity.Conclusion:Group C and group F can remove the cell components of bovine pericardium successfully,while maintaining the major structural components and the histological and biological properties of bovine pericardium,and with low cytotoxicity.However,group C is more economical than group F,and easier to operate.So the method on freeze-thaw-detergent 24 h can be the best choice to produce a decellularized bovine pericardium.     

Fixation of vascular grafts with increased glutaraldehyde concentration enhances mechanical properties without increasing calcification

Our objective was to study the effect of glutaraldehyde (GLU) concentration, heat, and photooxidation on mechanical properties and calcification of bovine pericardium grafts in an in vivo model. Fresh pericardia were treated as follows: 0.625% GLU for 7 days (standard); 0.625%, 1%, and 3% GLU at 4 degrees C for 20 days and 50 degrees C for additional 20 days; irradiation in cross-linking medium with metilene blue at 0 degrees C for 8 hours. Tissues were subjected to tensile mechanical tests (n = 76). Fixed patches were subcutaneously implanted in mice for 50 days (n = 16 per treatment). Calcification was assessed by atomic absorption spectrophotometry (n = 55) and von Kossa staining (n = 28). Analysis of variance and Tukey's test were used for statistical analysis. The 3% GLU and 3% GLU + heat treatments showed an enhancement of the mechanical properties above standard treatment. No significant difference was found in calcification between treatments. The 3% GLU treatment enhances the mechanical properties of the tissue above standard treatment without increasing calcification and without applying heat; therefore it is recommended for high-strength applications. Supplementary treatments to decrease calcification could be combined with this methodology to obtain a high-strength-low-calcification biomaterial for manufacturing of long-term cardiovascular grafts.     

Bone tissue engineering using human adipose-derived stem cells and honeycomb collagen scaffold

Human adipose-derived stem cells (ASCs) have the capacity to regenerate and the potential to differentiate into multiple lineages of mesenchymal cells. The aim of this study was to investigate the possibility of using honeycomb collagen scaffold to culture ASCs in bone tissue engineering. The osteogenic capacity of ASCs in vitro, was confirmed by histology and measuring the expression of cbfa-1. After that, ASCs were cultured for up to 14 days in the honeycomb scaffold to allow a high density, three-dimensional culture. Scanning electron microscopy data showed that the scaffold was filled with the grown ASCs, and calcification, stained black with von Kossa, was confirmed. Furthermore, The ASC-loaded honeycomb collagen scaffolds cultured for 14 days were subcutaneously transplanted into nude mice, and excised after 8 weeks. Bone formation in vivo was examined using HE stain, von Kossa stain, and osteocalcin immunostain. Those histological views showed significant positive stains in the samples of osteogenic medium in the three types of stain. These results suggest that this carrier is a suitable scaffold for ASCs and will be useful as a three-dimensional bone tissue engineering scaffold in vitro and in vivo.     

Treatment of bioprosthetic heart valve tissue with long chain alcohol solution to lower calcification potential

The use of glutaraldehyde-treated biological tissue in heart valve substitutes is an important option in the treatment of heart valve disease. These devices have limited durability, in part, because of tissue calcification and subsequent tearing of the valve leaflets. Components thought to induce calcification include lipids, cell remnants, and residual glutaraldehyde. We hypothesized that treatment of glutaraldehyde-treated bioprosthetic heart valve material using a short and long chain alcohol (LCA) combination, composed of 5% 1,2-octanediol in an ethanolic buffered solution, would reduce phospholipid content and subsequently lower the propensity of these tissues to calcify in vivo. Phospholipid content of glutaraldehyde-treated porcine valve leaflets and bovine pericardium was found to be 10.1 +/- 4.3 (n = 7) and 3.9 +/- 0.48 (n = 2) microg/mg dry tissue, respectively, which was reduced to 0.041 +/- 0.06 (n = 7) and 0.21 +/- 0.05 (n = 4) microg/mg dry tissue, respectively, after LCA treatment. Calcification potential of the treated tissues was assessed using a rat subcutaneous implant model. After 60 days of implantation, calcium levels were found to be 171 +/- 32 (n = 11) and 83 +/- 70 (n = 12) mg/g dry weight for glutaraldehyde-treated porcine leaflets and bovine pericardium, respectively, whereas prior LCA treatment resulted in reduced calcium levels of 1.1 +/- 0.6 (n = 12) and 0.82 +/- 0.1 (n = 12) mg/g dry weight, respectively. These data, taken together, support the notion that treatment of glutaraldehyde-treated tissue with a short and long chain alcohol combination will reduce both extractable phospholipids and the propensity for in vivo calcification.     

A novel chemical modification of bioprosthetic tissues using L-arginine

A novel chemical modification of biological tissues was developed by the direct coupling of bioactive molecule, L-arginine to bovine pericardium (BP). The modification involves pretreatment of BP using GA and followed by grafting arginine to BP by the reaction of residual aldehyde and amine group of L-arginine. BP was modified by direct coupling of bioactive molecules and the effect of L-arginine coupling on calcification and biocompatibility was evaluated in vitro and in vivo.Modified BPs were characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro plasma protein adsorption and platelet adhesion, and in vivo calcification. Thermal and mechanical properties showed that the durability of arginine treated tissue increased as compared with fresh tissue and GA treated tissue. Resistance to collagenase digestion revealed that modified tissues have greater resistance to enzyme digestion than did fresh tissue and GA treated tissue. Lower protein adsorption and platelet adhesion were observed on modified tissue than non-modified tissue. In vivo calcification study demonstrated much less calcium deposition on arginine treated BP than GA treated one. Obtained results attest to the usefulness of L-arginine treated BP for cardiovascular bioprostheses.     

Stabilized collagen scaffolds for heart valve tissue engineering

Scaffolds for heart valve tissue engineering must function immediately after implantation but also need to tolerate cell infiltration and gradual remodeling. We hypothesized that moderately cross-linked collagen scaffolds would fulfill these requirements. To test our hypothesis, scaffolds prepared from decellularized porcine pericardium were treated with penta-galloyl glucose (PGG), a collagen-binding polyphenol, and tested for biodegradation, biaxial mechanical properties, and in vivo biocompatibility. For controls, we used un-cross-linked scaffolds and glutaraldehyde-treated scaffolds. Results confirmed complete pericardium decellularization and the ability of scaffolds to encourage fibroblast chemotaxis and to aid in creation of anatomically correct valve-shaped constructs. Glutaraldehyde cross-linking fully stabilized collagen but did not allow for tissue remodeling and calcified when implanted subdermally in rats. PGG-treated collagen was initially resistant to collagenase and then degraded gradually, indicating partial stabilization. Moreover, PGG-treated pericardium exhibited excellent biaxial mechanical properties, did not calcify in vivo, and supported infiltration by host fibroblasts and subsequent matrix remodeling. In conclusion, PGG-treated acellular pericardium is a promising scaffold for heart valve tissue engineering.     

Effects of double cross-linking technique on the enzymatic degradation and calcification of bovine pericardia

The strength, resorption rates, and biocompatibility of collagenous biomaterials are profoundly influenced by the method of cross-linking. The in vitro and in vivo calcification and enzymatic degradation of bovine pericardia (BP) after a series of surface modifications were studied as a function of exposure time. Collagenase degradations of modified BP were monitored by scanning electron microscopy and tensile strength measurements. Bovine pericardium was modified by a combination of different tissue fixatives such as glutaraldehyde (GA), carbodiimide (EDC), diisocyanate (HMDIC), and polyethylene glycol (PEG). GA-PEG-EDC-PEG and GA-PEG-HMDIC-PEG combination treated BP retained maximum stability in collagenase digestion compared to GATBP. In vitro calcification studies and in vivo rat subcutaneous implantations of modified pericardium have shown substantial reduction in the calcification of double cross-linked BP with PEG modification. Further, the biocompatibility aspects of pericardial tissues were established by platelet adhesion and octane contact angle. It seems that cross-links involving amino and carboxyl residues may provide new ways of controlling biodegradation and calcification.