Biochemical and anisotropical properties of tendons

Tendons are formed by dense connective tissue composed of an abundant extracellular matrix (ECM) that is constituted mainly of collagen molecules, which are organized into fibrils, fibers, fiber bundles and fascicles helicoidally arranged along the largest axis of the tendon. The biomechanical properties of tendons are directly related to the organization of the collagen molecules that aggregate to become a super-twisted cord. In addition to collagen, the ECM of tendons is composed of non-fibrillar components, such as proteoglycans and non-collagenous glycoproteins. The capacity of tendons to resist mechanical stress is directly related to the structural organization of the ECM. Collagen is a biopolymer and presents optical anisotropies, such as birefringence and linear dichroism, that are important optical properties in the characterization of the supramolecular organization of the fibers. The objective of this study was to present a review of the composition and organization of the ECM of tendons and to highlight the importance of the anisotropic optical properties in the study of alterations in the ECM.     

Comparative ultrastructural analysis of different regions of two digital flexor tendons of pigs

Tendons are parallel arrays of collagenous fibers which are specialized in resisting and transmitting tensile forces. In this work we examined the structure of the superficial digital flexor tendon (SDFT) and the deep digital flexor tendon (DDFT) of pigs, which are considered "wrap around" tendons and so receive compression and tension forces. In both tendons, fibrocartilaginous areas were observed in the regions subjected to compression plus frictional loading. Histological and ultrastructural analyses of the tensional region showed an extracellular matrix (ECM) rich in collagen bundles, that were all arranged in the same direction. Fibroblasts were seen closely associated with the collagen bundles. Chondrocyte-like cells and high levels of glycosaminoglycans (GAGs) were observed in the compressional regions. The collagen bundles in the compressional region were arranged in several directions and were associated with proteoglycans (PGs). The crimp pattern detected in the tensional region showed that the collagen fibrils were ordered aggregates which formed helical superstructures.     

关节软骨内胶原纤维各向异性的红外光谱学显微成像研究

福尔马林溶液对于固化关节软骨组织、防止在长时间测量过程中组织的分解退化起到很好的作用,但对福尔马林溶液浸泡后软骨组织的结构变化(固化)过程及其胶原纤维各向异性的改变却鲜有研究。采用傅里叶变换红外光谱显微成像技术与偏振技术相结合的方法,通过关节软骨内胶原纤维(蛋白)的红外光谱特征吸收峰(Amide Ⅰ,Amide Ⅱ带)的吸光度随福尔马林溶液浸泡时间及偏光角度的变化来研究福尔马林溶液对软骨组织结构即胶原纤维各向异性的影响,并利用与各向异性方程拟合得到的决定系数(R2)对胶原蛋白纤维各向异性程度进行表征。研究发现,关节软骨Amide Ⅰ和Amide Ⅱ带的各向异性随着福尔马林浸泡时间的增长而愈加明显(Amide Ⅰ带变化尤为明显),说明福尔马林溶液中甲醛分子诱发了胶原蛋白分子新的交联,最终获得较好的固化效果,有利于关节软骨的各向异性分析。本研究将为今后关节软骨样本的制备、储存及各向异性研究提供参考。     

利用红外光谱评价文物微环境中有机酸对蚕丝纤维化学结构影响

研究文物微环境污染因素对文物材料影响是分析文物老化原因和妥善保存文物的重要基础。利用红外光谱(FTIR/ATR)剖析了文物微环境中甲酸、乙酸气体对蚕丝纤维结构影响。结果表明:低浓度甲酸气体能减弱纤维分子内氢键,使酰胺Ⅰ(1 617cm-1)谱峰减弱、酰胺Ⅱ谱峰(1 515cm-1)变窄、无规线团构象的酰胺Ⅲ谱峰(1 230cm-1)增强、纤维结晶度下降;当浓度高于8.1mg.m-3时,呈β-折叠构象的肽链段(GlyAla)n特征谱峰(1 000,975cm-1)增强、纤维结晶度提高。分析认为呈无规线团构象的短肽链发生β-折叠构象转变。乙酸气体对酰胺Ⅰ和酰胺Ⅱ谱峰影响不明显,但能引起无规线团构象增加和纤维结晶度降低,其作用弱于甲酸气体。本研究为进一步分析丝织品保存环境污染物的危害作用提供基础数据。     

温度对鱼鳞胶原蛋白二级结构的影响

从草鱼鱼鳞中提取酶溶性胶原蛋白(PSC),通过SDS-PAGE电泳分析为典型Ⅰ型胶原蛋白且达到电泳纯。在此基础上利用傅里叶变换红外光谱(FTIR)、拉曼光谱(Raman)和圆二色谱(CD)研究了温度对鱼鳞胶原蛋白二级结构的影响。FTIR分析表明：鱼鳞胶原蛋白具有典型的胶原蛋白特征吸收带,酰胺Ⅰ、酰胺Ⅱ和酰胺Ⅲ带的特征吸收频率分别出现在1658,1552和1238cm-1处。随温度升高,酰胺A和酰胺B峰位向低波数移动,1658cm-1处吸收峰裂解成多个吸收峰;1552cm-1处的吸收峰在35℃微略红移,随后发生明显蓝移;1238cm-1处吸收峰随温度升高向低波数移动。在拉曼光谱中,胶原蛋白的酰胺Ⅰ、酰胺Ⅱ和酰胺Ⅲ带的特征吸收频率分别出现在1669,1557和1245cm-1处,都较红外光谱的波数高;此外,921和855cm-1处脯氨酸的特征谱峰在拉曼光谱中体现出来。圆二色谱分析表明,胶原蛋白溶液在221.6和204.4nm分别有一正、负峰,具有典型胶原蛋白三螺旋结构的特征圆二色谱峰型。胶原蛋白冻干品的FTIR光谱和Raman谱线大都在35～60℃时发生波数和强度改变,而胶原蛋白乙酸溶液的CD谱线在20～35℃之间发生剧烈改变。由此可以判断胶原蛋白在固态和溶液状态下,变性温度存在一定差异,胶原蛋白冻干品比其乙酸溶液更稳定。     

航天诱变育种甜椒品系的红外光谱分析

首次采用傅里叶变换红外光谱法(FTIR)对航天诱变育种甜椒品系种子和一般大田生产的甜椒品种种子进行了对比研究,比较了它们红外光谱的异同。甜椒种子的红外光谱主要由蛋白质的吸收带和碳水化合物的吸收带组成。航天育种的两个甜椒品系种子(SP11和SP18)红外光谱的主要吸收峰的峰位、峰形与普通甜椒品系种子相同,表明经航天诱变的甜椒其主要化学成分和基本结构并未发生变化。太空甜椒种子,在2 854,1 652, 1 542以及1 160～1 062 cm-1范围内的吸收都较普通甜椒种子增加。2 855 cm-1峰是CH₂—伸缩振动,1 652 cm-1处的吸收峰为酰胺Ⅰ带,是CO的伸缩振动峰,1 542 cm-1的吸收峰是酰胺Ⅱ带,是N—H的弯曲振动和C—N的伸缩振动,1 160 cm-1处的峰可能为碳水化合物的C—O的伸缩振动引起。表明航天诱变使甜椒种子的蛋白质和碳水化合物含量增加。     

胃炎和胃溃疡组织细胞的显微拉曼光谱研究

利用共焦显微拉曼光谱研究了胃炎组织和胃溃疡组织的粘膜细胞。结果表明,与胃炎组织细胞比较,溃疡组织细胞中胞嘧啶的振动峰781 cm-1相对较弱,而腺嘌呤和胸腺嘧啶793,823cm-1处的峰强有所增加; 对应于α螺旋结构蛋白质酰胺Ⅰ和酰胺Ⅲ带的1654和1230～1270cm-1的强度均相对减小,色氨酸特征峰1332cm-1和苯丙氨酸特征峰1003cm-1强度有所降低,同时与色氨酸残基微环境变化密切相关的1554频移到1556cm-1,且强度有所增加;脂类的特征谱线1073 cm-1向高波数频移到了1078cm-1, 对应于CH₂扭转振动的1303cm-1与来自于CH面内变形振动的1268cm-1强度比值减小。上述变化表明两种疾病组织细胞中的核酸,蛋白质和脂类在成分、结构和构型上都存在明显差异;研究发现利用C—H弯曲振动的谱线1449cm-1和蛋白质酰胺Ⅲ带的谱线1660cm-1的强度比值A₁₄₄₉/A₁₆₆₀区分两种疾病具有较好的准确性。     

酯化前后酵母菌的红外光谱比较

对啤酒酵母酯化前后的红外光谱进行了分析。啤酒酵母的红外光谱图主要由蛋白质的吸收带、碳水化合物的吸收带组成。1 652 cm-1处的吸收峰为酰胺Ⅰ带,是CO的伸缩振动,1 542 cm-1的吸收峰是酰胺Ⅱ带,振sup>振动和N—H的弯曲振动引起的。1 454 cm-1处的吸收峰为CH₃和CH₂的弯曲振动峰;1 160 cm-1处出现的峰可能为细胞壁的主要成分——碳水化合物中C—O的伸缩振动峰;1 080 cm-1处的吸收峰是由啤酒酵母中的RNA,DNA或细胞壁中存在的碳水化合物或醇中的C—O伸缩振动引起的。用甲醇酯化后在1 744和1 454 cm-1处的吸收峰强度增加,说明酵母菌细胞表面的羧基发生了酯化反应。酯化后细胞的主要成分和结构保持完整。     

傅里叶变换红外光谱法研究PLGA微球中葡激酶突变体(K35R)的二级结构

用复乳溶剂挥发法制备了葡激酶突变体(K35R,DGR)的PLGA微球,并用傅里叶变换红外光谱法(FTIR)定量研究了PLGA微球内DGR的二级结构。 将可增强分辨率的傅里叶去卷积技术与高斯曲线拟合技术共同用于对微球内DGR酰胺Ⅰ带的定量分析,发现DGR酰胺Ⅰ带共包含9个红外吸收峰,并对各组份进行了归属。 此外,还对微球内DGR结构稳定性相关的1 623和1 650 cm-1吸收峰进行了讨论。     

Response of hydrogen bonding to low-intensity 50 Hz electromagnetic field in typical proteins in bi-distilled water solution

Samples of hemoglobin and bovine serum albumin in different bi-distilled water solutions were exposed to a 50?Hz electromagnetic field at the intensity of 1?mT to investigate the response of hydrogen bonding to the applied field after exposure of 3?h by means of Fourier Transform Infrared spectroscopy. Spectral analysis evidenced a significant decrease in the absorbance signal of the Amide I vibration in exposed samples of hemoglobin and bovine serum albumin water solutions. In addition, Fourier self-deconvolution analysis and min-max normalization applied in the mid-infrared region to exposed and unexposed hemoglobin samples revealed a significant increase in the absorbance signal of the Amide II band and an up-shift toward the high energies of 1.5?cm^?1 after exposure. Similar findings were observed after exposure of bovine serum albumin. These results can be easily explained assuming that hydrogen bonding in the secondary structure of these proteins in bi-distilled water solutions was enhanced after exposure to 50?Hz electromagnetic field.     

生态滤池出水悬浮物的元素分析和红外光谱分析

对生态滤池出水悬浮物的碳氢氮三元素和红外光谱进行了分析比较。元素分析结果表明,生态滤池出水悬浮物的无机成分比普通滤池稍高,用中颗粒惰性填料时复合床生态滤床的出水悬浮物无机物含量高。出水悬浮物的红外吸收光谱图主要由蛋白质的吸收带、碳水化合物的吸收带组成。1 655 cm-1处的吸收峰为酰胺Ⅰ带,是CO的伸缩振动,1 542 cm-1的吸收峰是酰胺Ⅱ带,是N—H的弯曲振动和C—N的伸缩振动,1 235 cm-1附近是酰胺Ⅲ带,是C—N的伸缩振动和N—H的弯曲振动引起的。单层普通滤池和单层生态滤池出水SS(suspend substance)红外光谱图差别很小;采用中颗粒填料的生态滤池,出水SS的红外光谱图中,1 042 cm-1处的吸收峰相对强度比粗颗粒填料的高,而2 924和2 852 cm-1的甲基伸缩振动峰和1 720～1 200 cm-1区域的相应吸收峰强度,比用粗颗粒填料的低。     

Atomic Force Microscopy of bulk tendon samples: affect of location and fixation on tissue ultrastructure

Atomic Force Microscopy (AFM) is a surface characterisation technique which analyses topology. To date, AFM studies of tissue ultrastructure have focussed on single collagen fibrils extracted from different tissues prior to analysis. Using sample preparation techniques used in electron microscopy studies, this work uses AFM to analyse the collagen ultrastructure of bulk samples from bovine deep digital flexor tendons (DDFTs). DDFT ultrastructure in regions of the tendon which experience different loading conditions are compared. Samples are analysed post-freezing and post-aldehyde fixation with either 10% formalin or 4% glutaraldehyde in order to investigate the affect of tissue preservation on tissue ultrastructure. The results demonstrate that both fibril diameter and repeat unit of the tendon vary between different regions in the dorsoventral plane, with regions subjected to both tensile and compressive forces exhibiting smaller fibril diameter and repeat unit compared to regions subjected to tensile forces alone. These differences are detectable regardless of the tissue preservation technique used. However these measured differences do vary with preservation techniques with aldehyde-fixed samples exhibiting smaller fibril diameters and larger repeat units compared to frozen samples. These results demonstrate that AFM is a highly suitable technique for the characterisation of different ultrastructures in bulk samples but that it is important to be consistent in the choice of preservation technique.     

Monitoring the thermally induced structural transitions of collagen by use of second-harmonic generation microscopy

The thermal disruption of collagen I in rat tail tendon is investigated with second-harmonic generation (SHG) microscopy. We investigate its effects on SHG images and intensity in the temperature range 25 degrees-60 degrees C. We find that the SHG signal decreases rapidly starting at 45 degrees C. However, SHG imaging reveals that breakage of collagen fibers is not evident until 57 degrees C and worsens with increasing temperature. At 57 degrees C, structures of both molten and fibrous collagen exist, and the disruption of collagen appears to be complete at 60 degrees C. Our results suggest that, in addition to intensity measurement, SHG imaging is necessary for monitoring details of thermally induced changes in collagen structures in biomedical applications.     

二维红外相关光谱法研究温度对交联胶原结构的影响

采用傅里叶红外光谱和二维相关分析研究了改性前后胶原在升温(25～115℃)过程中结构的变化。结果显示,改性前后胶原的特征吸收峰强度降低,峰值向低波数移动,其中酰胺II带的变化最明显,降低了～10cm-1,表明维系胶原三股螺旋结构稳定的氢键被破坏,结构发生改变。在1 515cm-1处自相关峰强度最强,说明温度对酰胺II带的影响最大。与未改性胶原相比,改性胶原的相关程度更弱,表明改性胶原结构受温度影响要小,交联提高了胶原的热稳定性;改性后胶原结构变化的顺序也不一样。由此可见,二维红外相关分析法能提供由温度引起的胶原结构动态变化的微观信息,对进一步研究改性胶原结构和功能之间的关系有一定的意义。     

生物吸附剂ZL5-2对六价铬离子吸附作用的红外光谱分析

研究了Agrobacterium.sp产生的具有吸附作用的蛋白聚糖类物质ZL5-2对Cr(Ⅵ)的吸附作用,并对吸附Cr(Ⅵ)前后及解吸附后的ZL5-2进行了红外光谱分析。结果表明,ZL5-2对Cr(Ⅵ)吸附的最佳pH为0.5～1.5,随着pH增大,吸附作用减弱。60 min时吸附达到平衡,80 min后所有的Cr(Ⅵ)都被吸附。被吸附的Cr(Ⅵ)可以被解吸附,解吸率为13.6%～67.9%。通过红外光谱分析,发现吸附后ZL5-2在3 400 cm-1附近OH,NH伸缩振动峰强度减弱,并发生了约8 cm-1的位移,2 900 cm-1附近C—H伸缩振动减弱,1 600 cm-1处的酰胺Ⅰ带伸缩振动的相对强度减弱,并发生约13 cm-1的位移。解吸附后,OH,C—H伸缩振动又恢复到原来水平,而NH、酰胺Ⅰ带的伸缩振动未恢复到原来水平,推测该吸附过程存在可逆吸附和不可逆吸附,以可逆吸附为主。     

傅里叶红外光谱研究血清白蛋白构象

用傅里叶红外光谱法研究了ＢＳＡ及其水溶液的红外光谱。通过对其酰胺Ｉ带傅里叶自转积谱分析,为其及部分水溶液中的二级结构构象进行了指认。结果表明,ＢＳＡ水溶液状态与固态时的二级结构是不同的。随着溶液浓度的降低,酰胺Ｉ带二级结构峰存在明显的位移现象,即１６０９．８６ｃｍ＾－１位移到１６０８．２４ｃｍ＾－１,１６３３．８５位移到１６３８．３６ｃｍ＾－１,１６５３．６９ｃｍ＾－１位移到１６５６．１０ｃｍ＾－     

Implications of obesity for tendon structure, ultrastructure and biochemistry: a study on Zucker rats

The extracellular matrix consists of collagen, proteoglycans and non-collagen proteins. The incidence of obesity and associated diseases is currently increasing in developed countries. Obesity is considered to be a disease of modern times, and genes predisposing to the disease have been identified in humans and animals. The objective of the present study was to compare the morphological and biochemical aspects of the deep digital flexor tendon of lean (Fa/Fa or Fa/fa) and genetically obese (fa/fa) Zucker rats. Ultrastructural analysis showed the presence of lipid droplets in both groups, whereas disorganized collagen fibril bundles were observed in obese animals. Lean animals presented a larger amount of non-collagen proteins and glycosaminoglycans than obese rats. We propose that the overweight and lesser physical activity in obese animals may have provoked the alterations in the composition and organization of extracellular matrix components but a genetic mechanism cannot be excluded. These alterations might be related to organizational and structural modifications in the collagen bundles that influence the mechanical properties of tendons and the progression to a pathological state.     

Origin of the variability of the mechanical properties of silk fibers: 4. Order/crystallinity along silkworm and spider fibers

The poor crystallinity of proteic fibers has fuelled an ongoing debate over their exact organization. We present a full‐range Raman comparison of Nephila madagascariensis spider and Bombyx mori silkworm silks that sheds some new light on that matter. On the one hand, a large variability is observed along the fibers in the −200 to 200 cm⁻¹ spectral window, which is sensitive to the long‐range order signature of polyamide chains. This questions the validity of previous literature data considering silk fiber as a homogeneous material. On the other hand, the ‘amide I’ band is almost independent of the targeted point, which sets a limit to this widely used structure probe. In‐line mapping of the fibers showed that the extension of the ordered zones ranges between 1 and 3 µm. The correlation between the macromechanical behavior (the stress–strain curves) and the nanomechanics (Raman low wavenumber signatures) under controlled tensile strain demonstrates a Prevorsek's type microstructure: the macromolecular chains belong to both ordered and amorphous ‘regions’. Copyright © 2014 John Wiley & Sons, Ltd.     

明胶与AgBr晶体颗粒相互作用的研究

为了探讨明胶在照相乳剂中的行为 ,用红外光谱法研究了明胶与AgBr晶粒间的相互作用 ,证实了明胶 AgX照相乳剂的晶粒吸附有明胶 .红外光谱分析结果表明 ,明胶与AgBr晶粒间的作用除了明胶与Ag+ 的化学亲和力外 ,明胶与Br- 之间还存在静电作用 .而且 ,明胶的疏水侧链与AgBr晶粒疏水区之间还存在另一种作用力 ,该作用力可能是范德华力 ,它对乳剂红外光谱的影响显示在 10 0 0～ 12 0 0cm- 1.由于用鱼胶和骨胶制作的乳剂在AgBr的晶体形态和乳剂的感光性能方面都显示出明显的差异 ,因此 ,还比较了这两种明胶与AgBr的作用特性