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。     

环氧改性与戊二醛处理的牦牛心包的扫描电镜形态比较

作者将环氧改性与戊二醇固定的牦牛心包缝制成血管，然后缝合在成年家犬颈动脉上．４个月后，取出材料，作扫描电镜观察，比较两者的形态学变化，并将未被植人的牦牛心包作同步电镜观察．结果表明，环氧改性的牦牛心包更为完整，其浆膜层脱落程度小，更为光滑，胶原纤维的走向更富弹性和抗张强度。植入体内后，环氧改性的牦牛心包撕裂程度明显较低．说明环氧改性的牦牛心包的力学性能明显优于戊二醛固定的牦牛心包，材料的力学性能是由胶原纤维的走向决定的。     

不同交联改性牛心包材料的细胞毒性研究

本研究采用培养的人胚肺纤维细胞分析比较戊二醛、环氧１铬交联处理后４％甲醛保存的牛心包材料的细胞毒性。在浸泡前与浸泡１０、２０、３０天的周期中，不同交联处理的牛心包材料与浸出液的细胞增殖抑制指数显示：９１）三种交联剂处理的心包材料均有明显的细胞毒性。（２）随浸泡时期的延长，心包材料与浸出液的细胞毒性逐渐降低。（３）铬交联处理的牛心包材料与浸出液的细胞毒性显著高于其他交联材料。     

一种新的生物瓣材料——Ⅶ.醛固定剂对牦牛心包流变性能的影响

本文用软组织生物材料试验机,在37℃生理盐水环境下研究醛固定剂对牦牛心包材料应力—应变和应力松弛等性能的影响。实验结果表明:(1)用戊二醛固定的心包试样,其应力松弛明显地比新鲜试样小。固定后戊二醛使胶原纤维在低应变下的应力增加,并有超高形变出现。(2)甲醛的交联作用在PH7.5附近产生。甲醛在形成交联的同时还引起心包中基质的固化,致使试样变硬,在受拉时常呈脆性断裂,断裂伸长率减小。用0.625％甲醛处理的试样交联度低,而用1.2％和5％甲醛处理的则有较高的交联度。(3)固定后的材料,在抗张强度上均有不同程度的降低。     

环氧化合物制备丝素凝胶材料的细胞毒性研究

利用环氧化合物与丝素蛋白在冰点以下或较高温度下作用可以获得两种不同结构和性能的凝胶材料。为研究两种材料在生物医用材料方面应用的可行性,本实验测定了两种凝胶材料的浸提液对小鼠成纤维细胞(L-929)细胞增殖率的影响,初步探索了两种凝胶材料的细胞毒性,发现两种凝胶材料均呈现出较高的细胞相对增殖率,细胞毒性均为1级,都在可以医用的合格范围内;另外实验还对环氧化合物和戊二醛两种交联剂的细胞毒性进行了对比,发现环氧化合物的细胞毒性明显低于戊二醛,认为环氧化合物可以作为丝素蛋白材料改性的更为安全的交联剂。     

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     

不同化学方法改性的牛心包体外降解规律的研究

牛心包材料是一种以胶原蛋白为主要成分的天然生物材料。为了研究牛心包作为组织工程材料的可能，本实验通过采用失重法、蛋白质检测法及氨基酸测定法定量地研究和对比了几种不同改性方法处理过的牛心包的体外降解规律，力求找到一种改性方法使材料既有可控的降解性，又有较小的抗原性，以使牛心包成为一种良好的可降解性GTR材料。研究结果显示，用乙醇封端处理的心包相对于戊二醛及环氧交联处理的心包，在降解性方面更易满足可降解GTR材料的要求。蛋白质检测法及氨基酸测定法也为以后建立控制心包降解速率的数学模型奠定了良好基础。     

原花青素交联牛心包材料的研究

本研究针对天然交联剂原花青素处理牛心包材料的性能进行研究.采用原花青素交联牛心包组织,制备人工生物心脏瓣膜材料,并对其交联特性、力学特性、抗酶降解性能、亲疏水性能、细胞毒性试验以及溶血试验等进行分析.结果显示:原花青素或戊二醛处理的牛心包组织变性温度、力学特性、表面亲水性能和抗酶降解能力都明显提高;与传统交联剂戊二醛相比组织稳定性、亲水性能和抗酶降解能力未见显著性差异,但是最大断裂强度显著提高(原花青素交联组织最大断裂强度(13.863 0 MPa)明显高于戊二醛交联组织(10.784 2 MPa);溶血试验结果表明原花青素处理固定组织的溶血率(2.61%)远低于戊二醛处理的组织(12.54%);细胞毒性试验结果表明原花青素交联组织在1、3、5 d的细胞增殖率分别为76.19%、88.96%、100.12%,而戊二醛在相同的时间点的细胞增殖率分别为63.15%、57.28%、48.74%.原花青素交联的瓣膜材料组织结构稳定、亲水性好、毒性小且能维持较好的力学性能,作为人工生物瓣膜材料具有很好的研究前景.     

牦牛心包的组织结构

本文以资源丰富的牦牛心包为对象,对其组织结构进行了探讨,并与商品牛心包瓣材料进行对比,结果发现: (1)牦牛心包主要由胶原纤维、弹性纤维和多种结缔组织成分构成;整个厚度的心包组织可分为三层:浆膜层、纤维层和心包外结缔组织层。 (2)与美国Edwards公司和Shiley公司牛心包材料相比,牦牛心包除弹性纤维数量较少外,在织组结构上没有重大差异。 (3)从组织学角度看,牦牛心包很有希望用作生物心瓣材料。     

两种交联剂制备软骨细胞移植支架的比较

目的：试用两种交联剂对小牛真皮基质来源的支架材料进行交联,比较支架的细胞毒性、结构、生物相容性和细胞贴附的差异,为在体动物试验提供实验依据。方法将脱细胞真皮基质分为两组,分别浸入0.05%戊二醛溶液和0.2%水溶性交联剂进行交联,MTT 法检测细胞毒性和溶血率。将交联后的支架分别植入大鼠皮下,评价生物相容性。以排液法粗测孔隙率,并在电镜下观察支架的结构和孔隙大小。培养间充质干细胞并贴附于两种方法处理的材料表面,电镜下观察贴附情况。结果以戊二醛溶液和水溶性交联剂两种方法处理的支架细胞毒性检测均合格,溶血率分别为4.61%与2.97%,均符合国家标准。经戊二醛交联的支架生物相容性差,炎症反应始终存在,水溶性交联剂处理的真皮基质组织相容性较好,仅有轻微的炎症反应。水溶性交联剂制备的支架材料孔隙率为84.3%±5.0%,戊二醛制备的支架材料孔隙率为79.7%±10.8%,差异不具有统计学意义（P >0.05）。戊二醛交联的支架细胞贴附差,而水溶性交联剂制备的支架细胞贴附良好。结论应用水溶性交联剂处理的支架细胞毒性和溶血率检测均合格,具有良好的生物相容性、孔隙率和细胞贴附性,该法可以作为后期制备软骨细胞移植支架的交联方法。     

Effect of pretreatment with epoxy compounds on the mechanical properties of bovine pericardial bioprosthetic materials.

Early failures of bovine pericardial heart valves are due to leaflet perforation, tearing and calcification. Since glutaraldehyde fixation has been shown to produce marked changes in leaflet mechanics and has been linked to development of calcification, bovine pericardium fixed with the four hydrophilic epoxy formulations and their mechanical properties are studied in this paper. We measured the thicknesses, shrinkage temperatures, stress relaxations and stress-strain curves of bovine pericardiums after different treatments with (1) non-treatment (fresh), (2) glutaraldehyde (GA), (3) epoxy compounds followed by the posttreatment with GA (EP 1#, EP 2#), and (4) epoxy compounds (EP 3# and EP 4#). Results of this study showed that the hydrophilic epoxy compounds are good crosslinking agents. There are no significant differences of shrinkage temperature and ultimate tensile stress among all tissue samples pretreated with GA, EP 1# and EP 2#. However, the stress relaxations of tissue-samples pretreated with epoxy compounds followed by the posttreatment with GA (EP 1# and EP 2#) are significantly slower than that pretreated with GA, and the strains at fracture of EP 1# and EP 2# are also significantly larger than that of GA or epoxy compounds. These facts show that the bovine pericardium pretreated with the epoxy compound followed by the posttreatment with GA (EP 1# and EP 2#) possesses greater tenacity and potential durability in dynamic stress.     

Heparinized bovine pericardium as a novel cardiovascular bioprosthesis

A novel chemical modification of biological tissues was developed by the direct coupling heparin to bovine pericardium (BP). The heparinization involves pretreatment of BP using GA and followed by grafting heparin to BP by the reaction of residual aldehyde and amine group of heparin. BP was modified by direct coupling of heparin and the effect of heparin coupling on calcification was evaluated in vitro and in vivo. Heparinized BP was characterized by measuring shrinkage temperature, mechanical properties, digestion resistance to collagenase enzyme, in vitro cytotoxicity, and in vivo calcification. Thermal and mechanical properties showed that the durability of heparin-treated tissue increased as compared with fresh tissue and GA-treated tissue. Resistance to collagenase digestion revealed that heparin-treated tissue has greater resistance to enzyme digestion than did fresh tissue and GA-treated tissue. Heparinized tissue had shown to be non-cytotoxic, however, relatively high cytotoxicity was observed in the GA-treated tissues due to the release of GA. In vivo calcification study demonstrated much less calcium deposition on heparin-treated BP than GA-treated one. Obtained results attest to the usefulness of heparinized BP for cardiovascular bioprostheses.     

Evaluation of the degree of cross-linking in UV irradiated porcine valves.

A porcine heart valve was irradiated by Ultraviolet (UV) rays (10 W, 254 nm) for 2, 4, 8 and 24 hours at 4 degrees C to cross-link the structural collagen matrix. The degree of cross-linking was evaluated by assaying the released amount of hydroxyproline (Hyp) from the matrix, and comparing it with the positive controls of valves treated by glutaraldehyde (GA) solution (0.625 wt%) and the negative controls of non-treated fresh valves. The undigested weight ratio of the specimens increased by increasing the UV irradiation time. The undigested weight of the leaflets, tunica interna and tunica externa of the fresh, GA-treated and UV-irradiated specimens after collagenase digestion was compared. As UV irradiation increased, the amount of released hydroxyproline was gradually decreased until 8 hours of irradiation, after which the released hydroxyproline-reduction occurred slightly until 24 hours of irradiation time in this system. A total 47.68% of the hydroxyproline in the valve was cross-linked by UV irradiation after 24 hours, while 73.74% of the hydroxyproline in the positive control was crossed-linked. Light microscopic observation revealed that the typical crimp pattern of collagen fibers decreased and was rearranged into a dense flattened pattern as the UV irradiation induced interfibrilar cross-linking. GA-treated valves demonstrated a denser matrix pattern than the UV-irradiated specimens. Cross-linked collagenous tissue prepared by UV irradiation would be useful for improving durability and reducing the disadvantages related to using a chemical cross-linking agent.     

Glatiramer acetate-specific antibody titres in patients with relapsing / remitting multiple sclerosis and in experimental autoimmune encephalomyelitis

Glatiramer acetate (GA) is an immunomodulatory drug approved for the treatment of clinically isolated syndrome (CIS) and relapsing/remitting multiple sclerosis (RRMS). As an antigen-based therapy, GA induces GA-specific antibodies in treated patients and animals. GA-specific antibodies do not neutralize therapeutic effects on relapses and disability. Rather, it has been suggested that GA-specific antibodies may be associated with improved clinical outcomes. We evaluated antibody responses in eight patients with RRMS treated with GA for 15 months and antibody responses in GA-treated C57BL/6 mice before and after induction of experimental autoimmune encephalomyelitis (EAE). There were no significant differences from pretreatment levels of total IgE or GA-specific IgE in patients with RRMS. Total IgG1, IgG3 and GA-specific IgG4 were significantly increased at 15 months of GA treatment. Antibody type and titre were not associated with clinical outcomes, i.e. expanded disability status scale (EDSS) score, disease burden on magnetic resonance images (MRI) or clinical relapses. In contrast, mice with EAE showed a marked increase in GA-specific IgE and GA-specific IgG1 antibody responses. GA-treated mice demonstrated improved clinical symptoms and lower mortality than untreated controls. Our results suggest that antibody responses to GA are heterogeneous among patients with RRMS, with no apparent association between antibody response and clinical outcomes. Clinical improvements in EAE-induced GA-treated mice suggest that GA-specific IgE and IgG1 may contribute to GA treatment effects in EAE.     

Immune response in patients receiving a bioprosthetic heart valve: lack of response with decellularized valves

Conventional biological heart valves treated with glutaraldehyde (GA) reveal a limited lifespan due to calcification. This is assumed to be an immune response initiated process, which is not seen with decellularized valves. However, their immunological potential is still a matter of debate. Therefore, serum samples from patients undergoing heart valve surgery were obtained before (Pre), after (Post), and 9-12 months after operation (Follow Up). Immunoglobulin G (IgG) and M (IgM) antibodies against porcine collagen I and α-Gal (Gal-alpha1,3-Gal-beta1,4-GlcNac-R) were determined for decellularized and GA treated valves. Antibody titers for collagen type I revealed no significant alteration for both types of valves. However, a considerable anti-α-Gal antibody response was observed in patients with GA-treated porcine valves. In detail, IgM antibodies were increased during follow up (p<0.05), whereas decellularized valves revealed a minor decrease in the IgM response (p<0.001). IgG antibodies were considerably increased with GA-treated porcine (p<0.05) and bovine (p<0.01) xenografts, whereas there was lack of response with decellularized valves. This indicates that GA treatment is not sufficient to eliminate immune response to the α-Gal epitope completely. Future investigations will have to verify whether immune response to α-Gal can be linked to the limited durability of conventional valves.     

Immunogenicity and effector functions of glutaraldehyde-treated rabbit and mouse immunoglobulin G

Rabbit and mouse IgG treated with glutaraldehyde (GA) were immunogenic in homologous species. Glutaraldehyde treatment induced in the IgG molecule two types of antigenic determinants. One of them was found on the monomeric fraction of GA-treated rabbit IgG (haptenic determinant) and the other on the polymeric fraction (structural determinant). The haptenic determinants were found also on monoaldehyde-treated rabbit IgG and GA-treated Fab and Fc fragments. It was demonstrated that rabbit and mouse antibodies are specific for GA-treated IgG and have species specificity. While GA treatment did not alter the antigen binding capacity of rabbit IgG antibody, its effector functions (except protein A binding) were much affected. Thus it was found that GA treatment enhances IgG ability to react with rheumatoid factor, reduces drastically its capacity to activate the complement system, abolishes the cytophilic properties of IgG and accelerates its catabolic rate. The possible blocking effect of GA on the amino acid residues (mainly Lys) situated in or very close to the effector sites of the IgG molecule is suggested.     

Cells, rather than extracellular matrix, nucleate apatite in glutaraldehyde-treated vascular tissue.

Glutaraldehyde (GA) causes a large increase in [Ca(2+)](i) and [P(i)](i) and calcification of porcine aortic valve fibroblasts. Calcification in GA-treated vascular tissue is likely to begin intracellularly, but the potential role of extracellular matrix has not been taken into account in earlier studies. To compare the role of cells and matrix in calcification, intestinal pouches made of a lipid-extracted rat small intestine were prepared. Lipid-extracted porcine aortic valves, or cells cultured from those same valves, were placed in intestinal pouches, sealed, fixed with GA, and grafted in rat subcutis. Cells in the pouches calcified in 3 weeks whereas the valvular matrix did not calcify for 9 weeks. Cellular calcification spread to the wall of the intestinal pouches and grew heavier after 9 weeks. Similarly, smooth muscle cells calcified exclusively in GA-treated rat aorta grafted in rat subcutis for 3 weeks. Calcification of extracellular matrix was seen after 9 weeks. Cells initiate calcification and extracellular matrix serves as a substrate for the subsequent growth of apatite in GA-treated vascular tissue.     

Bioprosthetic tissue preservation by filling with a poly(acrylamide) hydrogel

Glutaraldehyde (GA) fixation has been used for more than 40 years as the preferred treatment to suppress immunogenicity and increase durability of bioprosthetic tissues (BPT) used in heart valve prostheses. This fixative and its reaction products have, however, been implicated in the calcific degeneration and long-term failure of these devices. The current study investigates stabilization of BPT and the mitigation/prevention of calcification by filling aortic wall samples with a synthetic poly(acrylamide) (pAAm) hydrogel, with and without pre-treatment with GA. Histological and gravimetric analysis showed full penetration of the acrylamide (AAm) into the fresh tissue, while only partial filling could be achieved with GA pre-fixed tissue. The observed decrease in amino-group content (0.157+/-0.012-0.123+/-0.021 micromol/mg, p<0.03) and corresponding increase in shrinkage temperature (67.2+/-0.8-78.1+/-1.8 degrees C, p<0.0001) when fresh tissue was filled, indicate the participation of tissue-amines in a process that leads to BPT crosslinking. These effects were much less pronounced when the tissue was pre-fixed with GA. Filling increased the tensile stiffness of fresh tissue (to levels half that of 0.2% GA fixed tissue), but decreased the stiffness of GA pre-fixed tissue. When compared to standard 0.2% GA fixed samples, fresh tissue filled with AAm showed 88% (p<0.0001) less calcification while exhibiting similar resistance toward degradation by protease. Filling did not result in significant decreases in calcification when the tissue was pre-fixed with GA.     

Quercetin-crosslinked porcine heart valve matrix: Mechanical properties,stability, anticalcification and cytocompatibility

Bioprosthetic heart valves, prepared by glutaraldehyde (GA) crosslinking, have some limitations due to poor durability, calcification and immunogenic reactions. The aim of this study was to evaluate the crosslinking effect of a natural product, quercetin, on decellularized porcine heart valve extracellular matrix (ECM). After crosslinking, the mechanical properties, stability, anticalcification and cytocompatibility were examined. The results showed that the tensile strength of quercetin-crosslinked ECM was higher than that of GA-crosslinked ECM. After crosslinking with quercetin, the thermal denaturation temperature of ECM was clearly increased. Quercetin-crosslinked ECM could be stored in D-Hanks solution for at least 30 days without any loss of ultimate tensile strength and elasticity. After soaking in D-Hanks solution for 36 days, there was only 11.55% non-crosslinked excess quercetin released and no further release thereafter. Cell culture study shows that no inhibition on proliferation of vascular endothelial cells occurred when the quercetin concentration was lower than 1 μg ml^?1. This non-cytotoxic concentration was 100 times higher than that of GA. The resistibility of quercetin-crosslinked ECM to in vitro enzymatic hydrolysis was comparable to that of GA-crosslinked ECM. An in vitro anticalcification experiment showed that quercetin crosslinking could protect ECM from deposition of minerals in simulated body fluid. The present study demonstrated that quercetin can crosslink porcine heart valve ECM effectively, which suggests that quercetin might be a new crosslinking reagent for the preparation of bioprosthetic heart valve xenografts and scaffolds for heart valve tissue engineering.     

Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering

Porous scaffolds for skin tissue engineering were fabricated by freeze-drying the mixture of collagen and chitosan solutions. Glutaraldehyde (GA) was used to treat the scaffolds to improve their biostability. Confocal laser scanning microscopy observation confirmed the even distribution of these two constituent materials in the scaffold. The GA concentrations have a slight effect on the cross-section morphology and the swelling ratios of the cross-linked scaffolds. The collagenase digestion test proved that the presence of chitosan can obviously improve the biostability of the collagen/chitosan scaffold under the GA treatment, where chitosan might function as a cross-linking bridge. A detail investigation found that a steady increase of the biostability of the collagen/chitosan scaffold was achieved when GA concentration was lower than 0.1%, then was less influenced at a still higher GA concentration up to 0.25%. In vitro culture of human dermal fibroblasts proved that the GA-treated scaffold could retain the original good cytocompatibility of collagen to effectively accelerate cell infiltration and proliferation. In vivo animal tests further revealed that the scaffold could sufficiently support and accelerate the fibroblasts infiltration from the surrounding tissue. Immunohistochemistry analysis of the scaffold embedded for 28 days indicated that the biodegradation of the 0.25% GA-treated scaffold is a long-term process. All these results suggest that collagen/chitosan scaffold cross-linked by GA is a potential candidate for dermal equivalent with enhanced biostability and good biocompatibility.