嵌合型组织工程牛心包片在大鼠皮下包埋效果的初步研究

目的:探索"体外去细胞-明胶再基质化-碳化二亚胺/N-羟基琥珀酰亚胺(以下均简称EDC/NHS)交联-体内自体细胞植入"的"嵌合型组织重构理论"在牛心包片处理过程中的科学性和可行性,以寻找组织工程生物瓣膜理想的支架材料。方法:采用"明胶再基质化-EDC/NHS交联"对脱细胞牛心包组织进行嵌合处理后植入大鼠皮下,通过HE染色对比新鲜组、脱细胞组、戊二醛组、嵌合组植入前后组织形态学改变,并对各组牛心包片进行厚度检测、含水量和力学检测、钙含量测定试验,判断该嵌合方法对牛心包基质的影响。结果:基线水平抗张强度测定结果显示,嵌合组牛心包抗张强度与戊二醛组(P=0.09)及脱细胞组(P=0.56)相比较并无显著差异远低于新鲜组(P=0.001);钙含量远低于新鲜组(P0.001)及戊二醛组(P0.001)。包埋2月后,嵌合组牛心包钙含量远低于新鲜组(P0.001)及戊二醛组(P0.001)。大鼠机体对明胶嵌合组处理牛心包的浸润程度最轻,然后是戊二醛组、脱细胞组,而新鲜组的浸润程度最重。在大鼠皮下降解率明显低于脱细胞组,炎症细胞浸润程度显著低于新鲜组,与戊醛组相当。结论:作为瓣膜组织工程支架材料,嵌合型牛心包膜比戊二醛交联效果具有更好生物相容性,钙化降解低,是较好的交联方法。     

组织工程化肺动脉带瓣管道纤维支架制作方法初步探讨

目的:观察采用酶加去污剂的方法脱去猪肺动脉带瓣管道的细胞和基质的效果,并通过物理和化学等方法对其进行测定,评价其作为组织工程支架材料的可行性。方法:采用0.25%胰蛋白酶和1%的Triton依次对新鲜猪肺动脉带瓣管道进行脱细胞脱基质处理。通过厚度、含水量、抗拉强度及热皱缩温度测定其物理性质与新鲜猪肺动脉对比;通过测定其蛋白多糖含量间接反应基质脱除的大小;通过新西兰幼鼠皮下埋植试验观察异种移植后炎症反应的大小。结果:采用0.25%胰蛋白酶和去污剂1%Triton能够有效脱除肺动脉壁和瓣膜中的细胞和基质成分,产生多孔隙性、低免疫原性的支架材料,初步具备良好组织工程支架材料的基本特点。     

小口径全生物化异种移植血管制备方法的实验研究

目的 探索和评价酶-去污剂法联合肝素结合处理犬颈动脉,以构建小口径全生物化移植血管.方法 将犬颈动脉分为两组,均采用酶-去污剂法脱细胞处理,其中一组再行肝素结合处理;对两组样本进行组织学和扫描电镜观察及机械性能测试以评价脱细胞效果;甲苯胺蓝染色观察肝素结合情况;凝血时间实验观察肝素发挥作用的持续时间.结果犬颈动脉经脱细胞后,细胞成分去除完全,细胞外基质保持完好,机械性能无明显改变;肝素结合于管壁全层;经肝素处理后的血管标本凝血时间明显延长.结论 酶-去污剂法联合肝素结合处理可以作为制备小口径异种移植血管的新方法 .     

原花青素处理去细胞牛心包细胞的相容性研究

目的考察原花青素处理去细胞牛心包的细胞相容性。方法采用L929细胞经原花青素处理去细胞牛心包浸提液培养后,噻唑蓝试验（MTT）法测定其相对增值率;将L929细胞与原花青素处理去细胞牛心包直接接触培养,逐日观察细胞生长状态;将L929细胞种植于原花青素处理的去细胞牛心包表面,扫描电镜观察其生长情况。结果原花青素处理去细胞牛心包性质稳定,细胞毒性程度为0～1级。L929细胞与心包材料直接接触培养生长良好,形态学无明显改变。结论原花青素处理去细胞牛心包细胞相容性好。     

组织工程纳米心包片的基础实验研究

目的:运用组织工程学方法和纳米技术对牛心包进行处理,研究其免疫原性,以期制备免低疫原性的组织工程补片。方法:采用明胶再基质化、碳化二亚胺/N-羟基琥珀酰亚胺(简称EDC/NHS)交联和高速搅拌复乳溶剂挥发法对牛心包进行相关处理,之后植入大鼠皮下进行观察,通过HE染色对比纳米组、交联组、脱细胞组和新鲜组四组植入前后的组织形态学改变,并对各组心包片进行机械性能、厚度、含水量和钙含量检测;免疫组织化学法检测心包片表面CD4的沉积。结果:肉眼观察到大鼠皮下降解率纳米组明显低于脱细胞组,与交联组相当,脱细胞组降解较快;抗张强度测定结果显示,纳米组抗张强度与交联组(P=0.76)及脱细胞组(P=0.61)相比较差异无统计学意义,但远低于新鲜组(P<0.01);钙含量纳米组远低于新鲜组(P<0.001),与交联组相当;免疫组化结果显示各组心包片表面均有明显的CD4沉积,纳米组的排斥反应远低于新鲜组,与交联组相当。结论:组织工程学方法和纳米技术结合制备的纳米心包片具有低免疫原性,低降解率和抗钙化的优良特性。与交联组相比,纳米微球的嵌合并未明显影响心包片的机械性能和免疫原性,有望成为构建组织工程补片缓释模型的理想载体。     

心包替代材料的动物实验研究

本实验将戊二醛处理的牛心包、猪肠衣和合成材料分别移植于山羊心包上,半年后取材进行大体肉眼评估、组织病理学检查及钙原子吸收光谱测定。结果显示:生物材料较合成材料为优,生物材料中的猪肠衣较牛心包为优。     

CHAPS联合十二烷基硫酸钠的除垢剂动脉脱细胞支架材料制备

目的采用3-[(3-胆酰胺基丙基)二甲基铵基]-1-丙磺酸盐(CHAPS)和十二烷基硫酸钠(sodium do-decyl sulfate,SDS)的联合除垢剂方案对脐动脉进行脱细胞,评价脱细胞的有效性和支架材料的力学性能改变情况。方法分离新鲜脐带获取人脐动脉,通过对脐动脉进行CHAPS联合SDS脱细胞处理,获得脱细胞血管支架,对其进行苏木素-伊红、Masson、Verhoeff′s Van Gieson(EVG)等组织染色和扫描电镜分析,并进行DNA及胶原定量分析;于支架上接种成纤维细胞,通过细胞相容性的程度评估除垢剂残留情况;对血管支架材料进行爆破压力及应力应变曲线分析,评价支架材料力学性能改变。结果组织染色结果显示脱细胞支架材料血管壁细胞消失,与未脱细胞的脐动脉相比较,其胶原纤维和弹力纤维结构无明显差别,电镜下可见血管细胞消失,支架材料成多孔隙结构;DNA定量显示DNA浓度极低,胶原蛋白浓度无明显改变;人成纤维细胞可以很好附着于支架材料上并生长;脱细胞支架材料抗爆破压力及应力应变曲线与脐动脉比较无明显变化。结论 CHAPS联合SDS的脱细胞方法能彻底去除脐动脉血管壁细胞成分,且较为完整地保留了血管的组织结构。脱细胞后的血管支架材料具有良好的生物相容性和力学性能。     

牛心包组织的钙化与蛋白聚糖的关系

用Triton X-100等四种表面活性剂处理牛心包组织,对处理液作蛋白聚糖定量分析,对处理后的组织作蛋白聚糖超微形态观察,并将处理后的材料植入动物皮下21天,通过材料钙含量分析判断钙化程度。结果表明,牛心包组织的钙化与其蛋白聚糖有关。     

不同试剂脱除猪胸主动脉壁细胞的对比研究

目的:用不同方法去除猪胸主动脉壁细胞,并比较去细胞效果,为组织工程瓣膜或带瓣管道提供良好的实验材料。方法:采用十二烷基硫酸钠、胆脂酸钠、曲拉通（TritonX100）制备猪胸主动脉脱细胞基质。将实验分为十二烷基硫酸钠组、胆脂酸钠、曲拉通组和空白对照组。标本进行大体、光镜、电镜观察,力学性能的检测并做比较。结果:胆脂酸钠无法完全脱除主动脉壁内细胞,十二烷基硫酸钠和曲拉通都能完全脱除猪胸主动脉细胞,曲拉通较好地保持了胶原纤维和弹性纤维的原有排列和分布,并有较好的力学性能。后两者脱细胞时间无显著差别。结论:曲拉通可以成功用于制备大血管脱细胞基质,为组织工程瓣膜或带瓣管道的研究提供材料。     

人脱细胞羊膜负载大鼠骨髓间充质干细胞构建组织工程膀胱的研究

目的 探讨人脱细胞羊膜(Human acellular amniotic membrane ,HAAM)负载大鼠骨髓间充质干细胞(MSCs)进行膀胱替代修复的可能性.方法 用去污剂洗涤和酶消化方法 制备HAAM.将大鼠MSCs在体外培养、扩增、传代.取第4代干细胞用1%二甲亚砜(DMSO)预诱导8 h,然后应用膀胱匀浆上清液诱导7 d.将诱导分化后的MSCs接种于HAAM表面进行短暂体外培养,获得组织工程膀胱.将雄性大鼠60只随机分为3组,行半膀胱切除术,然后分别采用负载干细胞后的HAAM(A组)、HAAM(B组)以及单纯缝合(C组)的方法 进行修补,每组20只.分别于术后2、4、8 w测定膀胱容量、进行膀胱造影并取材观察组织再生情况.结果 去污剂洗涤联合酶消化法能有效去除人羊膜中的细胞成分,光镜及电镜观察无细胞成分残留,细胞相容性好.大鼠膀胱重建术后,A、B两组与C组膀胱最大容量(Volmax)比较有极显著性差异(P＜0.01),而A、B两组之间膀胱最大容量比较无显著性差异(P＞0.05).组织学观察,2 w时HAAM即已吸收降解,膀胱移行细胞和平滑肌细胞开始形成,A组吻合修补区较厚,平滑肌细胞规则且丰富.结论 HAAM作为膀胱移植物进行膀胱修补吸收降解迅速,负载MSCs后构建的组织工程膀胱是理想的替代修补材料.     

猪主动脉瓣去细胞基质的制备

目的完全去除猪主动脉瓣细胞,取得良好的去细胞支架材料。方法用5 g/L胰酶对标本中的某些基质和细胞加以消化后,再应用不同去垢剂（十二烷基硫酸钠、脱氧胆脂酸钠、Triton X-100）加以洗脱,结合溶液渗透压改变等去除猪主动脉瓣细胞;标本进行大体、光镜、电镜观察和热皱缩温度的检测。结果胆脂酸钠无法完全脱除猪主动脉瓣细胞,十二烷基硫酸钠和曲拉通均可完全去除猪主动脉瓣细胞,Triton X-100组的主动脉瓣胶原纤维和弹性纤维得以较好保持。结论Triton X-100可以成功脱除猪主动脉瓣细胞,为组织工程瓣膜的研究提供材料。     

Complete dynamic repopulation of decellularized heart valves by application of defined physical signals-an in vitro study

OBJECTIVE: Cardiovascular tissue engineering is a novel concept to develop ideal heart valve substitutes. The objective of this study was to use decellularized porcine pulmonary valves, ovine cells and dynamic tissue culture to obtain viable and biomechanically stable constructs, resembling native aortic heart valves. METHODS: Endothelial cells and myofibroblasts were obtained from ovine carotid arteries. Porcine pulmonary valves were decellularized enzymatically, reseeded and cultured using a hydrodynamic bioreactor system over a time period of 9 or 16 days. Controls were grown over an equivalent time period under static conditions. Specimens of each valve were examined biochemically (cell proliferation, DNA, collagen, 4-hydroxyproline, elastin and glycosaminoglycans), histologically (hematoxylin-eosin, Movat-pentachrome and immunostains) and mechanically (radial and circumferential strength). RESULTS: Histology and biochemical assays demonstrated the removal of almost all cells after decellularization with preservation of the extracellular matrix. Recellularization under pulsatile conditions was significantly improved after 9 and 16 days compared to static conditions. Biochemical and mechanical analysis revealed a continuous increase of cell mass, collagen and elastin contents and strength under pulsatile culture conditions compared to significantly lower values in the static controls. CONCLUSION: This study demonstrated the superiority of the hydrodynamic approach of cellular reseeding to replace decellularized porcine heart valves with ovine cells with almost complete preservation of extracellular matrix integrity.     

Temporal regulation of extracellular matrix components in transition from compensatory hypertrophy to decompensatory heart failure

OBJECTIVE: Extracellular matrix, particularly type I fibrillar collagen, provides tensile strength that allows cardiac muscle to perform systolic and diastolic functions. Collagen is induced during the transition from compensatory hypertrophy to heart failure. We hypothesized that cardiac stiffness during decompensatory hypertrophy is partly due to a decreased elastin:collagen ratio. MATERIALS AND METHODS: We prepared left ventricular tissue homogenates from spontaneously hypertensive rats (SHR) aged 30-36 weeks, which had compensatory hypertrophy with no heart failure, and from SHR aged 70-92 weeks, which had decompensatory hypertrophy with heart failure. Age- and sex-matched Wistar-Kyoto (WKY) rats were used as normotensive controls. In both SHR groups, increased levels of collagen were detected by immuno-blot analysis using type I collagen antibody. Elastin and collagen were quantitated by measuring desmosine/isodesmosine and hydroxyproline spectrophometrically, respectively. To determine whether the decrease in elastin content was due to increased elastinolytic activity of matrix metalloproteinase-2, we performed gelatin and elastin zymography on left ventricular tissue homogenates from control rats, SHR with compensatory hypertrophy and SHR with heart failure. RESULTS: The elastin:collagen ratio was 0.242 +/- 0.008 in hearts from WKY rats. In SHR without heart failure, the ratio was decreased to 0.073 +/- 0.003 and in decompensatory hypertrophy with heart failure, the ratio decreased to 0.012 +/- 0.005. Matrix metalloproteinase-2 activity was increased significantly in SHR with heart failure compared with controls (P < 0.001). The level of tissue inhibitor of metalloproteinase-4 was increased in compensatory hypertrophy and markedly reduced in heart failure. Decorin was strongly reduced in decompensatory heart failure compared with control hearts. CONCLUSIONS: Since collagen was induced in SHR with heart failure, decorin and elastin were decreased and the ratios of gelatinase A and elastase to tissue inhibitor of metalloproteinase-4 were increased, we conclude that heart failure is associated with adverse extracellular matrix remodeling.     

Optimal cell source for cardiovascular tissue engineering: venous vs. aortic human myofibroblasts

Arterial vascular cells have been successfully utilized for tissue engineering in human cardiovascular structures, such as heart valves. The present study evaluates saphenous vein-derived myofibroblasts as an alternative, easy-to-access cell source for human cardiovascular tissue engineering. Biodegradable polyurethane scaffolds were seeded with human vascular myofibroblasts. Group A consisted of scaffolds seeded with cells from ascending aortic tissue; in group B, saphenous vein-derived cells were used. Analysis included histology, electron microscopy, mechanical testing, and biochemical assays for cell proliferation (DNA) and extracellular matrix (collagen). DNA content was comparable in both groups. Collagen and stress at maximum load was significantly higher in group B. Morphology showed viable, layered cellular tissue in all samples, with collagen fibrils most pronounced in group B. In conclusion, saphenous vein myofibroblasts cultured on biodegradable scaffolds showed excellent in vitro tissue generation. Collagen formation and mechanical properties were superior to aortic tissue derived constructs. Therefore, the easy-to-access vein cells represent a promising alternative cell source for cardiovascular tissue engineering.     

Is 10 minutes the optimum immersion time in 0.6% glutaraldehyde for human pericardium?

BACKGROUND AND AIM OF THE STUDY: Pericardial fixation with 0.6% glutaraldehyde is usually assessed by measuring the shrinkage temperature of the tissue: the higher the shrinkage temperature, the greater the degree of cross-linking induced between collagen molecules. Animal pericardium studies have shown maximum response to be obtained after brief immersion (10 min). Our aim was to evaluate the effect of glutaraldehyde immersion time on shrinkage temperature of human pericardium which, to our knowledge, has not yet been studied. METHODS: Pericardial strips were harvested from 40 patients undergoing cardiac surgery. Time of immersion in glutaraldehyde ranged from 3 min to 6 months. Fresh untreated human pericardium samples were used as controls. The relationship between shrinkage temperature and time of treatment with glutaraldehyde was studied using a regression analysis. RESULTS: Glutaraldehyde treatment of pericardial tissues caused an increase in shrinkage temperature that was related biphasically to the time of immersion in glutaraldehyde. Mathematical expression of this curve permitted glutaraldehyde immersion time to be evaluated in relation to the degree of optimal shrinkage temperature. The time required for optimal fixation with glutaraldehyde, as measured by shrinkage temperature, was 100+/-0.77 min. CONCLUSION: Our results suggested that a 10-min exposure to glutaraldehyde was insufficient for 'correct' fixation of human pericardium. Inadequate glutaraldehyde exposure of human pericardium may explain mid and long-term failures reported with this tissue in cardiac surgery.     

Mechanisms underlying hypertrophic remodeling and increased stiffness of mesenteric resistance arteries from aged rats

The mechanisms associated with structural and mechanical alterations of mesenteric resistance arteries from aged rats were investigated by using pressure myography, confocal microscopy, immunofluorescence, and picrosirius red staining. Arteries from old rats showed: (i) increased wall and media thickness, greater number of smooth muscle cell (SMC) layers but decreased density of SMC; (ii) increased number of adventitial cells; (iii) hypertrophy of nuclei of SMC and endothelial cells; (iv) increased stiffness associated with increased total collagen content and collagen I/III deposition in the media; and (v) similar content but changes in elastin structure in the internal elastic lamina. Hypertrophic outward remodeling in aged rat resistance arteries involve adventitial cells hyperplasia, reorganization of the same number of hypertrophied SMC in more SMC layers leading to thickened media and endothelial cell hypertrophy. Fibrosis associated with collagen deposition and changes in elastin structure might be responsible for the increased stiffness of resistance arteries from aged rats.     

Biomechanical characterization of decellularized and cross-linked bovine pericardium

BACKGROUND AND AIM OF THE STUDY: Although bovine pericardium has been used extensively in cardiothoracic surgery, its degeneration and calcification are important limiting factors in the continued use of this material. The study aims were to decellularize bovine pericardium and to compare the biomechanical properties of fresh and decellularized bovine pericardia to those treated with different concentrations of glutaraldehyde (GA). METHODS: An established protocol for decellularization using sodium dodecyl sulfate was used, and histological analysis performed to validate the adequacy of decellularization. Contact cytotoxicity was used to study the in-vitro biocompatibility of variously treated pericardia. Mechanical testing involved uniaxial testing to failure. Mechanical properties of the fresh and decellularized pericardia (untreated and treated with 0.5% and 0.05% GA) were compared. RESULTS: Histological analysis of decellularized bovine pericardium did not show any remaining cells or cell fragments. The histoarchitecture of the collagen-elastin matrix appeared well preserved. Untreated decellularized pericardium was biocompatible in contact cytotoxicity tests with smooth muscle and fibroblast cells. The GA-treated tissue was cytotoxic. There were no significant differences in the mechanical properties of fresh and decellularized pericardia, but there was an overall tendency for GA-treated pericardia to be stiffer than their untreated counterparts. CONCLUSION: An acellular matrix, cross-linked with a reduced concentration of GA, can be produced using bovine pericardium. This biomaterial has excellent biomechanical properties and, potentially, may be used in the manufacture of heart valves and pericardial patches for clinical application.     

The pericardial bioprosthesis: altered tissue shear properties following glutaraldehyde fixation

BACKGROUND AND AIM OF THE STUDY: Glutaraldehyde-fixed bovine pericardial tissue used in the construction of valvular bioprostheses undergoes repeated bending stress during the cardiac cycle. To bend smoothly, internal tissue shearing is required. The effect of glutaraldehyde fixation on internal shear properties of this material was examined. METHODS: Pericardium from each of 12 bovine hearts was cut into two pieces; one piece was retained as fresh tissue, the other was glutaraldehyde-fixed. Circular samples were then mounted and installed in a shear testing apparatus. For each sample, the shear stress versus shear strain characteristics were measured in circumferential and radial directions at strain rates of 1.0, 0.1 and 0.02 s(-1) while immersed in a 20 degrees C bath; similar measurements were made on six fresh and six fixed samples at 37 degrees C. In addition, the stress relaxation properties were measured by holding the tissue at maximum shear for 100 s after each of the three shear deformations, and recording force generated with time. RESULTS: The shear stress-strain test on fresh tissue (n = 12) showed non-linear behavior at the three shear rates. The shear modulus for fresh tissue increased from <1.0 kPa to 5 kPa at a shear strain approaching 1.0, and results were identical in radial or circumferential directions. For glutaraldehyde-fixed pericardium (n = 12), shear modulus increased promptly to 15-20 kPa at a strain of 0.2, and did not vary with strain rate. Shear relaxation was similar in fresh and fixed tissue. CONCLUSION: Fresh pericardium sheared easily at low shear stresses, with minimal resistance developing until the shear strain exceeded 0.5, while glutaraldehyde-fixed tissue displayed a marked resistance to shearing, with an immediate rise in shear stress at low strain. No differences were detected in shear properties between radial and circumferential directions. Such marked tissue stiffening may be a factor in collagen fiber disruption, leading to bioprosthetic heart valve failure.     

Evaluation of Biaxial Mechanical Properties of Aortic Media Based on the Lamellar Microstructure

Evaluation of the mechanical properties of arterial wall components is necessary for establishing a precise mechanical model applicable in various physiological and pathological conditions, such as remodeling. In this contribution, a new approach for the evaluation of the mechanical properties of aortic media accounting for the lamellar structure is proposed. We assumed aortic media to be composed of two sets of concentric layers, namely sheets of elastin (Layer I) and interstitial layers composed of mostly collagen bundles, fine elastic fibers and smooth muscle cells (Layer II). Biaxial mechanical tests were carried out on human thoracic aortic samples, and histological staining was performed to distinguish wall lamellae for determining the dimensions of the layers. A neo-Hookean strain energy function (SEF) for Layer I and a four-parameter exponential SEF for Layer II were allocated. Nonlinear regression was used to find the material parameters of the proposed microstructural model based on experimental data. The non-linear behavior of media layers confirmed the higher contribution of elastic tissue in lower strains and the gradual engagement of collagen fibers. The resulting model determines the nonlinear anisotropic behavior of aortic media through the lamellar microstructure and can be assistive in the study of wall remodeling due to alterations in lamellar structure during pathological conditions and aging.