Role of corneal collagen fibrils in corneal disorders and related pathological conditions

The cornea is a soft tissue located at the front of the eye with the principal function of transmitting and refracting light rays to precisely sense visual information. Corneal shape, refraction, and stromal stiffness are to a large part determined by corneal fibrils, the arrangements of which define the corneal cells and their functional behaviour. However, the modality and alignment of native corneal collagen lamellae are altered in various corneal pathological states such as infection, injury, keratoconus, corneal scar formation, and keratoprosthesis. Furthermore, corneal recuperation after corneal pathological change is dependent on the balance of corneal collagen degradation and contraction. A thorough understanding of the characteristics of corneal collagen is thus necessary to develop viable therapies using the outcome of strategies using engineered corneas. In this review, we discuss the composition and distribution of corneal collagens as well as their degradation and contraction, and address the current status of corneal tissue engineering and the progress of corneal cross-linking.

Different architectures of collagen fibrils enforce different fibrillogenesis mechanisms

According to current knowledge on collagen fibril-logenesis, collagen fibrils are formed by a cooperative process involving lateral fusion of small protofibrils. Almost all the experimental research, however, was carried out on tendon collagen, whose fibrils are characterized by approximately straight subfibrils. By contrast, in most tissues the collagen fibril sub-units follow a helical course in which geometrical constraints prevent lateral fusions, thereby implying a different mechanism where collagen fibrils grow by addition of individual microfibrils rather than by lateral fusion of pre-assembled subfibrils. The proc-ess at the origin of these fibrils may provide a simple, automatic explanation for the remarkable uniformity in fibrils size observed in most tissues without re-quiring the intervention of unknown mechanisms of diameter control. Other mechanisms of growth con-trol remain indispensable to terminate the fibril-logenesis process in tendons and ligaments.

Infrared nanoimaging of nanoscale sliding dislocation of collagen fibrils

Collagen,one of the major components in the mammalian connective tissues,plays an essential role in many vital physiological processes.Many common diseases,such as fibrosis,overuse injuries,and bone fracture,are associated with collagen arrangement defects.However,the underlying mechanism of collagen arrangement defects remains elusive.In this study,we applied infrared scattering-type scanning near-field optical microscopy to study collagen fibrils’structural properties.Experimentally,we observed two types of collagen fibrils’arrangement with different periodic characteristics.A crystal sliding model was employed to explain this observation qualitatively.Our results suggest that the collagen dislocation propagates in collagen fibrils,which may shed light on many collagen diseases’pathogenesis.These findings help to understand the regulation mechanism of hierarchical biological structure.

Endometrial Collagen Fibril Hyperplasia is Associated with Implantation Failure in Women Undergoing IVF-ET

Objective To analyse the effects of controlled ovarian hyperstimulation （ COH） on the endometrial expression of collagen fibril （CF） during the peri-implantation period in patients undergoing IVF, and its relation to endometrial receptivity （ER） in repeated implantation failure（RIF）. Methods Peripheral blood and endometrial biopsies were obtained from 45 infertile women on days 5, 7 or 9 after oocytes retrieval or ovulation in a stimulated cycle （SC） and natural cycle （NC） respectively. CF was assayed by transmission electron microscope and quantified by modified Masson dyeing. The outcome of subsequent embryo transfer（ET） was observed. Results Levels of both E2 and progesterone were higher in the peripheral blood in SC than in NC. Also the expression of CF in the stroma in each secretory phase was increased significantly in SC （P 〈O.05）. After embryo transferring, expression levels of CF in the pregnancy group dropped between the mid- and late-secretory phase, but no change in the non-pregnancy group. In the same term, all patients undergone endometrial curettage had higher pregnancy rate than those without. Conclusion Imbalance of production and degradation of endometrial CF in the secretory phase resulting from COH may be the cause of defective ER and implantation failure in some RIF patients. Endometrial curettage may improve implantation rate by inducing appropriate CF hyperplasia and degradation.

Effects of fibril- or fixed-collagen on matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1 production in the human hepatocyte cell line HLE

AIM: Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) are central to the spontaneous resolution of liver fibrosis. The mechanisms involved have been investigated in hepatic steilate cells (ISC), but not in hepatocytes. We investigated the effects of fibril- and fixed-collagen on MMP-1 and TIMP-1 production in hepatocytes, using the HLE cell line.METHODS: Fibril type T and Ⅳ collagen were prepared by HCl digestion of type T and Ⅳ collagen, respectively.For fixed-collagen, culture dishes were coated with fibril type Ⅰ or Ⅳ collagen and fixed by ultraviolet. Type Ⅰcollagenase activity was measured using fluorescein isothiocyanate-labeled type Ⅰ collagen. MMP-1 and TIMP-1 in HLE cells were measured by a one-step sandwich enzyme immunoassay.RESULTS: Both fibril type Ⅰand Ⅳ collagen significantly increased type Ⅰ coilagenase activity about two-fold compared with no fibril collagen. The effects of the fibril collagen were not affected by the coating condition. There was no significant difference in the effects on collagenase activity between cells cultured in medium containing fibril type Ⅰ collagen and those cultured in the presence of type Ⅳ collagen. Both types of fibril collagen significantly increased MMP-1 production, and showed more than 10-fold higher levels of MMP-1 than the control. The enhanced MMP-1 production by fibril collagens was unaffected by the coating condition. By contrast, TIMP-1 production was not changed by the addition of fibril type Ⅰ or Ⅳ collagen,and neither was it affected by the coating conditions.Coating with type Ⅰ collagen significantly suppressed MMP-1production by almost one-tenth compared with no coating.By contrast, lIMP-1 production was not affected by either the absence of a collagen coat or by increasing the concentration of the coating collagen.CONCLUSION: These results indicated that, in HLE cells,fibril- and fixed-collagen have opposite effects on MMP-1production without affecting TIMP production. Fibril collagen induced collagenase activity by up-regulation of MMP-1 production without affecting TIMP-1 production.By contrast, fixed collagen reduced MMP-1 production.Our results suggest that hepatocytes might also play an important role in the regulation of the hepatic fibrosis alongside HSC.

Platelet-Derived Growth Factor-A Overexpression Correlates with Atrial Fibrosis in the Patients with Atrial Fibrillation Secondary to Rheumatic Valvular Disease

Objective: To investigate the relationship between platelet-derived growth factor-A (PDGF-A) and atrial fibrosis in patients who have developed atrial fibrillation (AF) secondary to rheumatic valvular disease. Methods: 84 selected patients participated in the current study who have developed rheumatic heart disease and were going to have a cardiac surgical operation. In the current study, whole subjects were divided into two group, they were atrial fibrillation (AF) group (the quantity is thirty-nine) and sinus rhythm (SR) group (the quantity is forty-five). Before the operation, complete clinical data was available for the whole patients. During the operation, the right atrial tissue (0.3 - 0.5 mm3) was disserted from every patient. Right atrial fibrosis was observed by Masson staining and the distribution of PDGF-A in right atrium specimen was observed by immunohistochemistry. RT-PCR techniques were applied to admeasure the mRNA expressions of PDGF-A in patients’ atrial tissue. At the same time, western-Blot techniques were employed to admeasure the protein expressions of PDGF-A. Results: In baseline clinical characteristics, in both AF group and SR group, there was no apparently difference between them (P > 0.05);compared with SR group, the diameters of left atrium and right atrium in AF group were apparently increased (P Conclusion: Atrial remodeling plays an important role in patients with valvular atrial fibrillation;PDGF-A in patients with AF was highly expressed in the right atrial, and was closely related with atrial fibrosis.

羧基化的聚酰胺—胺/无定形磷酸钙诱导Ⅰ型胶原纤维矿化的研究

目的:利用羧基化的聚酰胺—胺稳定矿化物前驱相无定形磷酸钙,合成并表征同时具有有机和无机材料特性的仿生矿化复合物(ACP/CPAMAM),并观察其诱导胶原纤维仿生矿化的效果。方法:制备ACP/CPAMAM纳米复合物,并通过透射电子显微镜、选区电子衍射、粒径分析、电位分析等对其表征和验证。最终体外实验应用牛跟腱胶原纤维,借助透射电镜—能谱分析观察胶原纤维内部及周围晶体形成情况。结果:ACP/CPAMAM纳米复合物成功合成并且为无定形相,平均粒径为25.13 nm。透射电子显微镜及选区电子衍射显示,ACP的表面出现10 nm厚的低衬度包裹层,证实了ACP/CPAMAM的成功合成且其结构为非晶相。透射电镜—能谱分析显示,应用ACP/CPAMAM矿化肌腱Ⅰ型胶原纤维后可见高密度晶体,胶原纤维周围可见致密、连续的晶体形成,证实该纳米复合物可连续诱导胶原纤维矿化。结论:ACP/CPAMAM作为一种新型的纳米复合生物材料具有良好的生物活性和仿生矿化特性,能够诱导胶原纤维形成矿化产物,提示ACP/CPAMAM可能成为一种新型诱导胶原纤维仿生再矿化的纳米材料。

龙血竭对肺纤维化大鼠肺组织TGF-β/Smads信号通路分子mRNA表达的影响

目的观察龙血竭对肺纤维化大鼠肺组织TGF-β信号通路分子TGFβR及Smad4 mRNA表达的影响,探讨其防治肺纤维化的作用及机制。方法将30只SD大鼠随机分为3组:肺纤维化模型组,龙血竭治疗组和正常对照组,每组10只,雌雄各半。模型组和治疗组大鼠均以博来霉素(5 mg/kg)气管内滴入诱导肺纤维化;对照组以等量生理盐水气管内滴入。从第2 d起,治疗组大鼠给予龙血竭(180 mg/kg,溶于2 mL生理盐水中)灌胃,对照组和模型组大鼠则以等量生理盐水灌胃。所有大鼠于第29 d早晨处死。HE染色检测肺组织病理改变;原位杂交检测肺组织TGFβR及Smad4 mRNA表达;免疫组化法(S-P法)检测型胶原纤维。结果模型组肺组织内炎性细胞计数为22243.60±5011.55,治疗组为12913.78±5640.12,两组比较差异有统计学意义(P<0.01)。经龙血竭治疗后大鼠肺内TGFβR mRNA表达较模型组降低(P<0.01)。治疗组Smad4 mRNA表达与模型组比较差异无统计学意义。治疗组和模型组比较,型胶原纤维表达减弱(P<0.01)。结论龙血竭能有效地减轻肺纤维化大鼠的纤维化程度,其机制可能与抑制肺内TGFβR mRNA的表达,从而阻止型胶原过度沉积有关。

牙本质表面状态对粘结界面影响的激光扫描共聚焦显微镜研究

目的 :研究不同表面状态下形成的牙本质湿粘结界面的异同。方法 :以RhodamineB为荧光剂 ,用激光扫描共聚焦显微镜 (LSCM)分别观察干燥或湿润粘结时形成的牙本质粘结界面的异同。结果 :湿粘结时 5种粘结系统在牙本质粘结界面都有良好的渗透。以丙酮为溶剂的粘结系统干燥粘结时形成的混合层的厚度有明显的降低 ,牙本质小管中树脂突较细 ,中断现象增多 ,长度缩短。结论 :干燥粘结时 5种粘结剂在牙本质表面的渗透性减弱。

牛跟腱Ⅰ型胶原纤维的微观结构与理化性能分析

采用酸酶结合法成功制备了牛跟腱Ⅰ型胶原纤维,其二级结构与Ⅰ型胶原的结构特征相符;AFM结果发现,胶原纤维粗细均匀,具有D周期横纹结构,直径为(135±0.20)nm,在溶液中分散均匀且呈现多孔的微观网络结构;其抗张强度与拉断伸长率分别达到1.293 MPa、93.77%;其疏水性能较纯Ⅰ型胶原有所提高,接触角为68.9°,吸水倍率、保水率与透水汽率分别为37.13%,19.44%和2749.77 g/(m2/d);其孔隙率为89.54%,平均累积吸附与累积解吸附的比表面积分别为8.4251和12.8046 m2/g,表现出孔体积大、比表面积小和孔尺寸大的特点。牛跟腱Ⅰ型胶原纤维的微观结构与理化性能分析对天然胶原纤维的深度加工、成型及应用提供了新的、可靠的理论依据。

胶原纤维溶胀液的流变特性研究

采用酸酶法制备牛肌腱胶原纤维,采用旋转粘度仪研究了不同浓度胶原纤维溶胀液的流变性能,并采用幂律方程对流变性能数据进行非线性回归拟合,得到胶原纤维在不同温度下的流变模型。研究结果表明,在实验范围内,浓度分别为0.5%,1.0%和1.5%的胶原纤维溶胀液的剪切应力均随着剪切速率与胶原纤维含量的增加而增大;随着温度的升高,胶原纤维溶胀液的表观粘度呈下降趋势,当体系温度达到36℃时,胶原纤维溶胀液的表观粘度下降急剧,胶原纤维发生热变性;胶原纤维溶胀液流体出现"剪切稀化"现象,表现出假塑性流体的特征,流变模型的拟合相关系数r2≥0.991,说明幂律模型能有效地表征胶原纤维溶胀液的流变性能。胶原纤维溶胀液的流变学特性研究为天然胶原纤维的进一步开发及利用提供了理论和实验依据。

积雪草苷对兔耳增生性瘢痕组织中胶原纤维及TGF-β1表达的影响

目的:探讨积雪草苷用于兔耳增生性瘢痕后对胶原纤维组织结构形态的改变和TGF-β1表达水平的影响。方法:健康成年日本大耳白兔20只,随机分为实验组(高中低浓度积雪草苷治疗)和对照组(生理盐水、模型),共5组,且均通过手术制成兔耳瘢痕模型。术后21d形成成熟的兔耳瘢痕模型,实验组和生理盐水组分别早晚各一次局部注射高浓度积雪草苷1 000mg/ml、中浓度500mg/ml、低浓度250mg/ml和0.9%NaCl 0.2ml,空白对照组不做任何处理。用药后28d切取标本,采用测微尺、瘢痕硬度精密测定仪测量瘢痕和正常兔耳皮肤厚度和硬度,光镜下观察苏木精-伊红染色、Masson三色法胶原染色的组织结构变化情况;采用免疫组织化学法检测瘢痕和正常皮肤组织中TGF-β1表达的变化。结果:与模型组相比,积雪草苷组瘢痕组织增生层明显比模型组薄,且未见丰富的血管和炎细胞,胶原纤维减少,排列稀疏,TGF-β1表达下降,且表现出一定的浓度依赖性。结论:积雪草苷可明显抑制增生性瘢痕,其抗瘢痕的作用可能通过降低TGF-β1的表达有关。

人发角蛋白人工腱诱导自体腱形成中胶原原纤维的形态学变化

目的观察人发角蛋白(HHK)人工腱诱导自体腱形成过程中胶原原纤维形成的形态学变化。方法制备肌腱损伤动物模型,植入HHK人工腱,于术后3、6、9、12、16周取出,进行光镜及电镜观察。结果自体腱形成过程中,先后有一级、二级和三级胶原原纤维形成。结论HHK人工腱诱导自体腱形成过程中,人工腱植入区肌腱断端和腱膜下的腱细胞去分化,分泌大量的胶原蛋白,并在细胞外生成一级胶原原纤维,再逐级融合形成二级和三级胶原原纤维,最终多数腱细胞胶原化而消失。

关节软骨胶原纤维增强特性

目的在COMSOL关节软骨计算中引入胶原纤维增强特性,并分析引入前后模型结果的差异。方法以软骨中胶原纤维的走向为基础,对胶原纤维的应力单独建模,将胶原纤维的应力写入到原来软骨多孔弹性模型中。为避免应变2次项的出现采用了函数调用方式,同时提高求解器的迭代次数以更好收敛。结果胶原纤维增强模型表面的Y方向初始位移仅为15μm,是非增强模型的17.6%。增强模型的X方向正应变仅为非增强模型的10%,而近表面的X方向正应力超过非增强模型的10倍。结论在COMSOL关节软骨计算中实现了胶原纤维增强特性的引入,为软骨胶原纤维损伤提供了计算模型和理论分析。胶原纤维通过横向增强约束了竖直方向的应变,提高了软骨承载能力,改善了软骨的力学性能。

原子力显微镜对胶原各级结构和聚集形态的观测

使用原子力显微镜（AFM）观测了胶原各级结构及典型体外聚集形态，对用AFM研究胶原的适用范围、优点和不足进行了系统的概括，并对制样方法和检测条件进行了探讨。AFM能观测到胶原分子的形态，也能用于检测和表征胶原的超分子聚集体——胶原原纤维，其周期横纹清晰可见，还能检测到由原纤维组装而成的直径为1-2肌m的胶原纤维。AFM在用于研究胶原其它体外聚集形态时，也得到了很好的结果。虽然AFM的应用存在一些局限性，如对样品的表面平整度要求较高、扫描范围的缩放不够灵活，但其具有制样手段方便快捷、分辨率高等优势，使其有望在胶原及其它生物大分子的研究中发挥重要的作用。

牙本质不同表面状态对其粘接界面影响的扫描电镜观察

目的研究牙本质表面的干燥或湿润状态对牙本质粘结界面微观结构的影响。方法用扫描电镜观察和比较两类5种粘结系统在干燥或湿润的牙本质表面进行粘结时形成的粘结界面的微观结构。结果湿粘结时5种粘结系统在粘结界面获得良好的渗透图象;干燥粘结时,两种以丙酮为溶剂的粘结系统在干燥粘结时形成的粘结界面,与湿粘结时有明显的区别,混合层较薄,并有明显的不完全渗透的结构和混杂层的形成;以酒精和水为溶剂的粘结系统在两种不同粘结状态时粘结界面的超微结构没有明显的区别。结论使用新型的湿粘结系统时,牙本质粘结表面应该保持一定的湿润度,以保证粘结剂在粘结界面的良好渗透,获得较高的粘结强度。

应用原子力显微镜对不同种属Ⅰ型胶原纤维微观结构的研究

目的 :利用原子力显微镜 (AFM)对猪和牛的Ⅰ型胶原蛋白在溶液中的聚集行为进行微观结构的形态学研究。方法 :将Ⅰ型胶原蛋白溶液 ,滴至新鲜剥离的云母片上 ,空气中干燥后 ,用原子力显微镜观察。结果 :发现来自猪和牛的I型胶原蛋白在浓度为 3 2 μg ml时以单体形式和聚集体形式混合存在 ,在浓度为 4mg ml时两种I型胶原蛋白自发装配成胶原纤维 ,形成多孔网状结构 ,孔隙大小为 :牛 ,10 0～ 15 0nm ;猪 ,170～ 2 0 0nm。孔隙率 :牛 ,5 75个 μm2 ;猪 ,193个 μm2 。平均粗糙度为 :牛 ,3 1nm ;猪 ,8 7nm。均方根粗糙度为 :牛 ,4 0nm ;猪 ,11 4nm。平均高度 :牛 ,5 0 9nm ;猪 ,5 2 3nm。Rp v :牛 ,72 1nm ;猪 ,10 2 7nm。结论 :利用原子力显微镜能够准确直观地观察到Ⅰ型胶原蛋白形成的胶原纤维的种属差异性 。

大鼠脾损伤愈合的组织学动态观察

目的 :动态观察大鼠不同形式脾损伤愈合的组织学变化 ,评估脾破裂保守及保脾治疗的理论依据。方法 :三种脾破裂模型分为 1w、2w、3w、4w、5w动态组织学观察脾脏愈合的过程。结果 :1w为血肿期 ;2w、3w为脾脏结构再生起始期 ,红细胞淤积消散 ,创面充血减少 ,T、B淋巴细胞开始聚集 ;4w、5w为再生期 ,T、B淋巴细胞归位 ,小动脉周围淋巴鞘 (periarteriolarlymphocyticshealths,PLAS)结构逐渐清晰 ,淋巴细胞密度增大 ,出现胶原纤维瘢痕。结论 :脾脏外伤有较好的自愈能力 。

基于低温等离子体技术的胶原纤维表面改性和鞣制化学作用

就低温等离子体对胶原和皮革表面处理以及辅助皮革鞣制过程进行了剖析。通过不同等离子体气体对胶原纤维表面的改性处理后,可以增加或引入新的活性官能团,包括羧基、羟基、磺酸基、氨基等,降低胶原纤维与鞣剂分子间的反应活化能,有助于提高铬的吸收与结合率,减少铬用量,降低废液中铬的排放量;或增进了原本与胶原交链作用较弱或鞣性较差的金属离子,如Ti、Al、Fe、Si等,与胶原之间的反应活性,从而使这些无毒的金属离子能够取代铬鞣;或在聚合性气体介质中(如丙烯酸及丙烯酸酯类、偶链剂等),通过等离子体接枝聚合,直接在胶原蛋白质分子链间形成网络化的交链作用,从而实现无铬清洁鞣制技术。

原子力显微镜对天然猪皮胶原纤维精细结构的研究

简要介绍了原子力显微镜这一新型表面测试技术在猪皮胶原精细结构研究中的应用方法。初步探讨了猪皮胶原在原纤维水平上的精细结构。