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。     

染料介导光氧化固定牛颈静脉血管片体外内皮化

目的研究染料介导光氧化固定牛颈静脉的细胞毒性．初步探讨此种材料体外内皮化的可能性,方法实验组为染料介导光氧化固定组。对照组为戊二醛固定组。采用羊的颈内静脉内皮细胞作为细胞源,细胞种植于管腔一侧,时间为5d。动态观察细胞生长情况,扫描电子显微镜检查种植细胞前后的材料和免疫组织化学因子Ⅷ染色情况。结果光氧化固定组．采用扫描电子显微镜观察,在种植细胞前,在材料表面无细胞,而在种植细胞后,在材料表面密布一层细胞。戊二醛固定组．采用扫描电子显微镜观察,在种植细胞前后无差别,在材料表面见戊二醛固定时残留的少许细胞。光氧化同定组．材料表面种植的细胞因子Ⅷ染色为阳性。结论染料介导光氧化固定牛颈静脉血管片无明显细胞毒性．在体外可以很好地内皮化。     

生物瓣材料人工种植内皮细胞的研究

牲瓣材料表面人工形成单层内皮细胞可望成为生物瓣防钙化并延长其使命寿命的最有前途的方法之一，解除戊二醛的毒性作用是种植细胞在生物瓣表面存活的前提。用Ｌ－谷氨酸处理戊二醛固定保存６个月以上的牛心包材料，成功地接种了培养的血管内皮细胞，使生物瓣内皮细胞种值的进一步研究成为可能。     

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.     

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.     

新型抗钙化牛颈静脉管道材料的体外细胞毒性评价

背景：Triton X-100、环氧氯丙烷联合改性处理戊二醛固定的牛颈静脉管道是一种新型抗钙化右心管道材料,其生物相容性方面的研究较少。 目的：评价新型抗钙化牛颈静脉管道的体外细胞毒性。 方法：通过CCK-8法检测新型抗钙化牛颈静脉管道(实验组)及单纯戊二醛处理牛颈静脉管道材料浸提液(对照组)对L-929小鼠成纤维细胞的毒性作用,以第2,4天为检测时间点,计算细胞相对增殖率、对材料毒性进行分级。 结果与结论：CCK-8法细胞毒性试验显示新型抗钙化牛颈静脉管道材料浸提液第2,4天L-929细胞增殖率均在85%以上,毒性分级为1级,无细胞毒性,且显著优于对照组(P < 0.05)。提示经戊二醛、Triton X-100、环氧氯丙烷联合处理制备的新型抗钙化牛颈静脉管道材料无细胞毒性。     

改造天然生物组织为血管支架材料的预处理方法

目前 ,临床对血管替代物的需求量越来越大 ,传统的来源已不能满足需要。组织工程化血管的出现使得这一问题有望得到解决。构建组织工程化血管就必然涉及血管支架的预制。生物支架材料是血管支架中的一大类 ,它在细胞黏附及促细胞生长等方面优于人工合成的支架材料。由于生物性材料自活体取出后即开始降解 ,同时不同材料间也存在着种群差异 ,不宜保存和直接应用 ,故需采用一些预处理方法来解决这些问题 ,预处理目的就是在移植生物性材料前 ,降低其抗原性、提高其抗酶降解能力 ,并较长时间地保持其良好的力学性能和组织结构。这些处理方法包括运用戊二醛、多聚环氧化合物、碳化二亚胺、京尼平及原花色素等化学试剂进行交联的化学方法和应用光氧化等进行交联的物理方法。本文详细地叙述了各种预处理方法的机理及相应各种材料处理前后免疫原性、生物稳定性、力学性能、细胞毒性、抗钙化能力等特性的变化 ,并对各种方法的优缺点做一简要的评述。总之 ,生物性材料预处理的发展趋势是继续深入研究和开发细胞毒性小的天然交联剂 ,完善并拓宽光氧化交联的应用     

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.     

牦牛心包的化学改性及其防钙化能力的研究

本文用川西牦牛心包为原料,初步分析了牦牛心包组织的主要成分。通过对牦牛心包组织中胶原的化学改性,用大白鼠为钙化实验动物,研究了经戊二醛固定后牦牛心包组织中胶原的羧基和氨基与组织钙化的关系。实验结果表明,增加组织中胶原的羧基含量,可加速组织在动物体内的钙化;减少羧基含量,则可降低组织的钙化,而氨基对组织钙化是乎没有影响。TEM研究结果,说明胶原是导致牦牛心包组织钙化的主要成分之一。X-衍射分析钙化的牦牛心包组织,说明组织在大白鼠体内的钙化与生物瓣在人体内钙化的形式相同,均是以Ca_(10)(PO_4)_6(OH)_2的形式沉着于组织中。此外还用EDAX,组织学,SEM等方法研究了牦牛心包组织在动物体内的钙化情况。     

用六次甲基二异氰酸酯(HDI)作为牦牛心包材料交联剂的探索实验

为寻求一种优于戊二醛(GA)的新型生物瓣膜材料交联剂,作者从可行性探索的角度,用气态六次甲基二异氰酸酯(HDI)处理川西牦牛心包材料。通过对心包材料的收缩温度(T_s)和氮基酸组成的测定,对气态HDI与心包胶原组织的反应作了初步分析。此外还评价了材料的生物相容性和缓钙化性能,测定了材料的力学性能。实验结果表明:HDI可以作为心包材料的交联剂,但必须防止HDI与心包作用的同时,也与心包中含有的水份反应。经HDI处理的牦牛心包材料具有良好的生物相容性和力学性能。与戊二醛处理的牦牛心包材料相比,其缓钙化能力有所提高,证明HDI有可能成为一种生物瓣膜材料的交联剂。     

Acellular vascular tissues: natural biomaterials for tissue repair and tissue engineering

Various research groups around the world are actively investigating cardiovascular prostheses of biological origin. This review article discusses the need for such bioprosthetics and the potential role for natural tissues in cardiovascular applications such as cardiac valves and vascular grafts. Upon implantation, unmodified natural materials are subject to chemical and enzymatic degradation, seriously decreasing the life of the prosthesis. Therefore, methods such as glutaraldehyde and polyepoxide crosslinking treatments and dye-mediated photooxidation have been developed to stabilize the tissue while attempting to maintain its natural mechanical properties. Also, residual cellular components in a bioprosthetic material have been associated with undesired effects, such as calcification and immunological recognition, and thus have been the motivation for various decellularization processes. The effects of these stabilization and decellularization treatments on mechanical, biological and chemical properties of treated tissues have been investigated, specifically with regard to calcification, immunogenicity, and cytotoxicity concerns. Despite significant advances in the area of cardiovascular prostheses, there has yet to be developed a completely biocompatible, long-lasting implant. However, with the recent advent of tissue engineering, the possibility of applying selective cell seeding to naturally derived bioprosthetics moves us closer to a living tissue replacement.     

Crosslink formation in porcine valves stabilized by dye-mediated photooxidation

Bovine pericardial and porcine valve materials stabilized by dye-mediated photooxidation have shown potential for bioprosthetic valve use. Previously, in vitro and in vivo stability of these materials was demonstrated through enzymatic, chemical, extraction, rat subcutaneous, and functional challenges. Here, we examine the stability of photooxidized porcine aortic valves through amino acid, crosslink, and hydrothermal isometric tension analysis. Photooxidation reduced intact histidine residues from 17.0 to 0 residues per 1000, indicating the photooxidative alteration of this amino acid. Diphenyl borinic acid-derivitized hydrolyzates of proteins were separated by high-performance liquid chromatography, which identified several amino acid crosslinks that appeared with photooxidation that were absent in untreated controls. Thermal relaxation analysis indicated a significantly higher (p < 0.0002) thermal stability for photooxidized porcine cusps than that of untreated controls, with mean relaxation times for untreated cusps of 14,000 +/- 4650 versus 22,900 +/- 2480 s for photooxidized cusps. In summary, porcine aortic valve tissue treated by dye-mediated photooxidation contains new chemical species and exhibits properties consistent with intermolecular crosslink formation, which explain the increased biostability of this material and its potential for use in bioprosthetic devices.     

Feasibility study using a natural compound (reuterin) produced by Lactobacillus reuteri in sterilizing and crosslinking biological tissues

Bioprostheses derived from biological tissues have to be fixed and subsequently sterilized before they can be implanted in humans. Currently available crosslinking agents and sterilants used in the fixation or sterilization of biological tissues such as glutaraldehyde and formaldehyde are all highly cytotoxic, which may impair the biocompatibility of bioprostheses. Therefore, it is desirable to provide an agent suitable for use in biomedical applications that is of low cytotoxicity and may form sterile and biocompatible crosslinked products. To achieve this goal, a natural compound (reuterin), produced by Lactobacillus reuteri in the presence of glycerol, was used by our group. It is known that reuterin has antibacterial, antimycotic, and antiprotozoal activities. Additionally, as in the case with formaldehyde, reuterin may react with the free amino groups in biological tissues by using its aldehyde functional group. Therefore, it was speculated that reuterin could be used as a crosslinking agent and a sterilant for biological tissues in the same way as glutaraldehyde and formaldehyde. In the study, the production of reuterin, produced by Lactobacillus reuteri under control conditions, was reported. Preparative chromatography was used to purify reuterin. Also, the minimal inhibitory concentration and minimal bactericidal concentration of reuterin and its antimicrobial activity on a contaminated tissue were investigated. In addition, the cytotoxicity of reuterin was evaluated. Glutaraldehyde, the most commonly used sterilant in the sterilization of biological tissues, was employed as a control. Furthermore, the feasibility of using reuterin as a crosslinking agent in fixing biological tissues was studied. Fresh and the glutaraldehyde-fixed tissues were used as controls. The results obtained in the minimal inhibitory concentration and minimal bactericidal concentration studies and in the sterilization study of a contaminated tissue indicated that the antimicrobial activity of reuterin is significantly superior to its glutaraldehyde counterpart. In addition, the results obtained in the 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide assay showed that reuterin is significantly less cytotoxic than glutaraldehyde. Additionally, it was found that reuterin is an effective crosslinking agent for biological tissue fixation. The reuterin-fixed tissue had comparable free amino group content, denaturation temperature, and resistance against enzymatic degradation as the glutaraldehyde-fixed tissue. In conclusion, the results obtained in this study indicate that reuterin is an effective agent in the sterilization and fixation of biological tissues.     

Biochemical changes and cytotoxicity associated with the degradation of polymeric glutaraldehyde derived crosslinks

The reversibility of glutaraldehyde crosslinks has been suggested as a reason for failure of long-term bioprosthetic implants. The stability of such crosslinks was investigated in tendons and model compounds. Small but cytotoxic levels of glutaraldehyde were still released from crosslinked tendons even after these tendons were extensively rinsed for up to 6 months. The toxic effect was evidenced by the death of fibroblasts surrounding a midsection piece of rinsed crosslinked tendon, while the end section pieces did not show toxic effects. The formation and stability of glutaraldehyde modified [14C]-L-lysine derivatives were investigated. The polymerization of glutaraldehyde with amino compounds was initially fast but continued to proceed slowly for months. Degradation of high-molecular-weight soluble polymers was detected by gel filtration chromatography. Low-molecular-weight soluble materials were also released from insoluble products which were formed when high concentrations of glutaraldehyde and radioactive lysine were reacted. These chemical and biological studies suggest that local cytotoxicity of glutaraldehyde crosslinked bioprostheses may be due to unstable glutaraldehyde polymers that persist in the interstices of crosslinked tissues.     

Biocompatibility study of biological tissues fixed by a natural compound (reuterin) produced by Lactobacillus reuteri

Glutaraldehyde-fixed biological tissues have been used extensively to fabricate various bioprostheses. However, the tendency for glutaraldehyde to markedly alter tissue stiffness and promote tissue calcification is a well-recognized drawback of this crosslinking agent. To overcome the aforementioned deficiency with the glutaraldehyde-fixed bioprostheses, a fixation technique using a natural compound (reuterin) produced by Lactobacillus reuteri to crosslink biological tissues was developed by our group. It was reported that reuterin inhibits the growth of gram-positive and gram-negative bacteria as well as yeasts, fungi, and protozoa. The study was conducted to evaluate the biocompatibility of the reuterin-fixed tissues with or without ethanol sterilization implanted subcutaneously in a growing rat model. Fresh and the glutaraldehyde-fixed counterparts were used as controls. The results showed that both glutaraldehyde and reuterin are effective antimicrobial agents in the sterilization of biological tissues. The degrees in inflammatory reaction for the reuterin-fixed tissues with or without ethanol sterilization were significantly less than their glutaraldehyde-fixed counterparts throughout the entire course of the study. Additionally, the reuterin-fixed tissues have comparable tensile strengths and resistance against degradation as the glutaraldehyde-fixed tissues. The results obtained at 12-month postoperatively showed that the glutaraldehyde-fixed tissue without ethanol sterilization became significantly stiff and calcified. However, it was found that ethanol sterilization of the glutaraldehyde-fixed tissue may inhibit calcification. Additionally, reuterin fixation may inhibit tissue calcification as compared to glutaraldehyde fixation. These observations implied that the biocompatibility of the reuterin-fixed tissue is superior to the glutaraldehyde-fixed tissue.     

Monitoring the self-assembly of chitosan/glutaraldehyde/cysteamine/Au-colloid and the binding of human serum albumin with hesperidin

A new method for monitoring, in real-time, the self-assembly of chitosan/glutaraldehyde/cysteamine (CGC) on the gold surface and the immobilization of Au-colloid on CGC membrane with piezoelectric quartz crystal impedance (PQCI) are firstly proposed. Cyclic voltammogram and electrochemical impedance spectroscopy were also use to investigate the formation of Au-colloid/CGC. The viscosity-average molecular weight of chitosan used was firstly estimated as 16.8 x 10(5) with piezoelectric quartz crystal (PQC) sensor. On the basis of the analysis of the multi-dimensional information provided by PQCI, two stages existed in chitosan drying course: the frequency shift of the first stage was controlled by viscoelastic of the liquid, while the total frequency shift was due to mass change. The cross-link ratio of glutaraldehyde with chitosan was about 0.13, while for glutaraldehyde with cysteamine was about 0.217. PQCI also showed that the Au-colloid immobilization is a first-order reaction, while the HSA immobilization is a sum of two exponential functions, e.g., adsorption and re-arrangement. The association of the immobilized HSA with the purified hesperidin was monitored, and the association constant was estimated as 3.42 ml mg(-1) by Scatchard analysis.     

Biodegradable biocomposite non-woven matrices based on PDLLA- and elastin-solubilized proteins/elastin

Poly(D,L-lactide) (PDLLA) was synthesized by ring-opening polymerization of D,Llactide. Non-woven PDLLA matrices were prepared by an extrusion/winding process. The process conditions were optimized and the surfaces of these matrices were modified by glow-discharge treatment and/or glutaraldehyde incorporation for immobilization of elastin-derived proteins (ESP) to the matrix to increase the biocompatibility and also to improve the bioactivity of the matrix. Glow-discharge conditions were optimized. Ethylene diamine (EDA) and Ar were used as the active monomers in the plasma phase. When EDA was used, the glow-discharge treated PDLLA matrices were first allowed to be reacted with glutaraldehyde, although, when Ar used, the treated matrices were used directly for ESP immobilization. The higher degree of immobilization was obtained for EDA and glutaraldehyde. The ESP-incorporated PDLLA matrices were further treated with elastin by cross-reaction of the ESP molecules on the matrix surfaces with elastin. Scanning electron microscopy (SEM) studies showed that ESP were homogeneously deposited the surface of the matrix.