Effect of Immobilization on Insoluble Collagen Concentration and Type I and Type III Collagen Isoforms of Rat Soleus Muscle

Immobilization is often associated with decreased muscle elasticity. This condition is known as muscle contracture; however, the mechanism remains unclear. The purpose of this study was to clarify the mechanism governing muscle contracture in rat soleus muscle by identifying changes in ankle joint mobility, insoluble collagen concentration and type I and type III collagen isoforms following 1- and 3-week immobilizations. Following a 1-week immobilization, range of motion (ROM) of dorsiflexion declined to 90% of the control value; additionally, ROM dropped to 67.5% of the control value after a 3-week immobilization. This finding suggested that ankle joint mobility decreases in conjunction with extended periods of immobilization. Insoluble collagen concentration in soleus muscles, which was unchanged after 1 week of immobilization, increased 3 weeks after immobilization. These results may be indicative of collagen fibers with strong intermolecular cross-links contained in the muscle was made increased relatively by 3 weeks of immobilization. Therefore, the change in intermolecular cross-links may be significant in terms of progress of muscle contracture with longer periods of immobilization. On the other hand, the ratio of type III to type I collagen isoforms in muscular tissue increased following a 1-week immobilization; moreover, this ratio remained constant after 3 weeks of immobilization. These data suggested that muscle immobilization may induce type III collagen isoform expression. The increase in the ratio of type III to type I collagen isoforms do not change in parallel with the increase in the limitation in ROM; however, this phenomenon probably is not closely related to the progress of muscle contracture. The change of collagen isoform in immobilized muscle may be involved in the mechanism governing the progression of muscle fibrosis.     

Opto-thermal radiometry and condenser-chamber method for stratum corneum water concentration measurements

Opto-thermal radiometry and the condenser-chamber TEWL (trans-epidermal water loss) method are two novel technologies for in-vivo skin studies. In this paper, for the first time, we combine the measurement results of the two technologies, and study the stratum corneum water concentration distribution, water status in stratum corneum, stratum corneum thickness as well as its swelling effects, and stratum corneum water-holding capabilities. We present both the theoretical background and illustrative measurement results. PACS 87.61.Ff; 87.80.-y; 87.50.Hj     

Atomic force microscopy investigation of chemically stabilized pericardium tissue

Native and chemically stabilized porcine pericardium tissue was imaged by the contact mode atomic force microscopy (AFM), in air. Chemically stabilized pericardium is used as a tissue-derived biomaterial in various fields of the reconstructive and replacement surgery. Collagen type I is the main component of the fibrous layer of the pericardium tissue. In this study, the surface topography of collagen fibrils in their native state in tissue and after chemical stabilization with different cross-linking reagents: glutaraldehyde (GA), dimethyl suberimidate (DMS) and tannic acid (TA) was investigated. It has been found that chemical stabilization causes considerable changes in the surface topography of collagen fibrils as well as in the spatial organization of the fibrils within the tissue. The observed changes in the D-spacing pattern of the collagen fibril correspond to the formation of intrafibrilar cross-links, whereas formation of interfibrilar cross-links is mainly responsible for the observed tangled spatial arrangement of fibrils and crimp structure of the tissue surface. The crimp structure was distinctly seen for the GA cross-linked tissue. Surface heterogeneity of the cross-linking process was observed for the DMS-stabilized tissue. SDS-PAGE electrophoresis was performed in order to evaluate the stabilization effect of the tissues treated with the cross-linking reagents. It has been found that stabilization with DMS, GA or TA enhances significantly the tissue resistance to SDS/NaCl extraction. The relation between the tissue stability and changes in the topography of the tissue surface was interpreted in terms of different nature of cross-links formed by DMS, GA and TA with collagen.     

长波紫外辐射对Ⅰ型胶原损伤的拉曼光谱研究

应用拉曼光谱技术研究了长波紫外(Ultraviolet-A,UV-A)辐射对Ⅰ型胶原的损伤,检测了Ⅰ型胶原及其经过90min的UV-A辐射后的拉曼光谱,得到了一个较完整的Ⅰ型胶原紫外损伤机制。实验结果表明:90min的UV-A辐射导致Ⅰ型胶原分子内氢键断裂、氢键体系发生变化,肽链的螺旋度减少,逐渐解螺旋,无规卷曲等无序构象增加。此外,UV-A辐射使Ⅰ型胶原分子内脯氨酸的羟基化程度降低。这些变化必然会引起Ⅰ型胶原三螺旋结构的损伤,并导致皮肤光老化过程中组织内胶原纤维的破坏。     

The role of telomeres in skin aging/photoaging

Recent work has substantially elucidated the mechanisms of skin aging and photoaging. In particular, a central role for telomere-based signaling can be inferred. Intrinsic aging is largely controlled by progressive telomere shortening, compounded by low grade oxidative damage to telomeres and other cellular constituents, the consequence of aerobic cellular metabolism. In sun exposed skin, UV irradiation also damages DNA and accelerates telomere shortening. Aging and photodamage appear to share a common final pathway that involves signaling through p53 following disruption of the telomere. These telomere-initiated responses, in combination with UV-induced damage to critical regulatory genes, lead to the familiar picture of "photoaging." These and other insights into the molecular basis for skin aging/photoaging may lead to enhanced management options.     

Histochemical and ultrastructural study of collagen fibers in mouse pubic symphysis during late pregnancy

Reference is usually made to the parallel orientation towards the main line of exerted tension at the pubic joint in mice, for supporting forces applied to the joint. Despite the wealth of morphological information about the extracellular matrix in this joint, little is known regarding the involvement of the crimp of collagen fibers in the dramatic transformations occurring in this region during the last 3 days of pregnancy. Examination of the collagenous architecture suggests that the biomechanical properties are directly related to fibril diameters, composition of ground substance and changes in the bundle morphology, particularly in the crimp structure. The purpose of this study was to further describe the transformation of the collagen fibers of the pubic symphysis during late mouse pregnancy. We examined the architecture of collagen fibers in the symphysis and pubic ligament through the Picrosirius-polarization method and also through scanning electron microscopy to directly visualize and measure the crimping from pregnant and virgin mice. The crimp angle and the length of five consecutive crimps were measured according to Patterson-Kane et al. [Connect. Tissue Res. 36 (1997) 253]. It could be demonstrated that the angles progressively decreased and the crimp length increased, denoting that the fibers have untwisted during the relaxation process. Our findings suggest that a disruption of the helical arrangement of the collagen containing fibers may contribute to explaining the rapid remodeling that occurs at the end of pregnancy and that is responsible for an increase in pliancy and length of the pubic ligament in mice.     

Structure and composition of arytenoid cartilage of the bullfrog (Lithobates catesbeianus) during maturation and aging

The aging process induces progressive and irreversible changes in the structural and functional organization of animals. The objective of this study was to evaluate the effects of aging on the structure and composition of the extracellular matrix of the arytenoid cartilage found in the larynx of male bullfrogs (Lithobates catesbeianus) kept in captivity for commercial purposes. Animals at 7, 180 and 1080 days post-metamorphosis (n = 10/age) were euthanized and the cartilage was removed and processed for structural and biochemical analysis. For the structural analyses, cartilage sections were stained with picrosirius, toluidine blue, Weigert’s resorcin-fuchsin and Von Kossa stain. The sections were also submitted to immunohistochemistry for detection of collagen types I and II. Other samples were processed for the ultrastructural and cytochemical analysis of proteoglycans. Histological sections were used to chondrocyte count. The number of positive stainings for proteoglycans was quantified by ultrastructural analysis. For quantification and analysis of glycosaminoglycans were used the dimethyl methylene blue and agarose gel electrophoresis methods. The chloramine T method was used for hydroxyproline quantification. At 7 days, basophilia was observed in the pericellular and territorial matrix, which decreased in the latter over the period studied. Collagen fibers were arranged perpendicular to the major axis of the cartilaginous plate and were thicker in older animals. Few calcification areas were observed at the periphery of the cartilage specimens in 1080-day-old animals. Type II collagen was present throughout the stroma at the different ages. Elastic fibers were found in the stroma and perichondrium and increased with age in the two regions. Proteoglycan staining significantly increased from 7 to 180 days and reduced at 1080 days. The amount of total glycosaminoglycans was higher in 180-day-old animals compared to the other ages, with marked presence of chondroitin- and dermatan-sulfate especially in this age. The content of hydroxyproline, which infers the total collagen concentration, was higher in 1080-day-old animals compared to the other ages. The results demonstrated the elastic nature of the arytenoid cartilage of L. catesbeianus and the occurrence of age-related changes in the structural organization and composition of the extracellular matrix. These changes may contribute to alter the function of the larynx in the animal during aging.     

Characterization of Fibers in an Existing Network for High Speed System (10Gb/s or Greater) Compatibility

With OC-192 communications systems being commercially available and higher bit rate systems in development, prudent telecommunications network administrators are testing their installed fibers to determine if they can be successfully used at 10 Gb/s and higher. Together with New Brunswick Telephone (NBTel), the Fiber Optics Group at UNB have tested various installed fibers and cables in the NBTel network for their losses at wavelengths of 1244, 1310, 1550, and 1625 nm, as well as for strain and polarization mode dispersion (PMD). Weather conditions, age, place of installation, and cable types have also been considered. Aging does not seem to affect the performance of the fibers. Although most fibers are high-speed system compatible when looking at attenuation measurements, about 40% of the fibers tested would not meet the 10 Gb/s OC-192 system manufacturer's design guidelines concerning PMD.     

Characterization of Fibers in an Existing Network for High Speed System (10Gb/s or Greater) Compatibility

With OC-192 communications systems being commercially available and higher bit rate systems in development, prudent telecommunications network administrators are testing their installed fibers to determine if they can be successfully used at 10 Gb/s and higher. Together with New Brunswick Telephone (NBTel), the Fiber Optics Group at UNB have tested various installed fibers and cables in the NBTel network for their losses at wavelengths of 1244, 1310, 1550, and 1625 nm, as well as for strain and polarization mode dispersion (PMD). Weather conditions, age, place of installation, and cable types have also been considered. Aging does not seem to affect the performance of the fibers. Although most fibers are high-speed system compatible when looking at attenuation measurements, about 40% of the fibers tested would not meet the 10 Gb/s OC-192 system manufacturer's design guidelines concerning PMD.     

Atomic force microscopy on human trabecular bone from an old woman with osteoporotic fractures

AFM images were taken of the exterior surface of a single trabecula, extracted from a human femoral head removed during surgery for a hip fracture in an old women with former fractures. The images showed a dense structure of bundled collagen fibrils banded with 67 nm periodicity. Bundles were seen to run in parallel in layers confirming the collagen structure seen by other techniques. Single collagen fibrils were seen to cross the bundles, thus forming cross-links between neighboring bundles of collagen fibrils. Some of these crossing fibrils did not have the 67 nm band pattern and their dimensions were about half compared to the neighboring collagen fibrils. Very little mineral was found on the surface of the trabecula. An AFM image of a fracture plane was also displayed. The trabecula was extracted from a region close to the hip fracture. However, there were in this case no obvious features in the images that could be linked directly to osteoporosis, but altered collagen banding and collagen protrusions may alter mechanical competence. A path to extensive studies of the nanometer scale structure of bone was demonstrated.     

Ultrastructural examination of dentin using focused ion-beam cross-sectioning and transmission electron microscopy

Focused ion-beam (FIB) milling is a commonly used technique for transmission electron microscopy (TEM) sample preparation of inorganic materials. In this study, we seek to evaluate the FIB as a TEM preparation tool for human dentin. Two particular problems involving dentin, a structural analog of bone that makes up the bulk of the human tooth, are examined. Firstly, the process of aging is studied through an investigation of the mineralization in ‘transparent’ dentin, which is formed naturally due to the filling up of dentinal tubules with large mineral crystals. Next, the process of fracture is examined to evaluate incipient events that occur at the collagen fiber level. For both these cases, FIB-milling was able to generate high-quality specimens that could be used for subsequent TEM examination. The changes in the mineralization suggested a simple mechanism of mineral ‘dissolution and reprecipitation’, while examination of the collagen revealed incipient damage in the form of voids within the collagen fibers. These studies help shed light on the process of aging and fracture of mineralized tissues and are useful steps in developing a framework for understanding such processes.     

Electron microscopic determination of silver incorporation in collagen fibers as a model of organic-metal chiral supramolecular structure with optical anisotropic properties

The high positive linear dichroism (LD) exhibited by silver-impregnated collagen fibers are thought to be caused by an ordered binding of silver nanoparticles to the collagen fibers. A correlation between LD, established by image analysis, and the size of silver particles as measured by electron microscopy was investigated in the same sections of silver-impregnated tendon collagen fibers. Silver particles were found to be periodically ordered along the collagen fibers, with some preference for the 67 nm collagen period and collagen a bands. The silver particle diameter for a sample of n=279 was 18.51 nm on average, with 95% of the values ranging between 17.90 and 19.14 nm. The finding that interference colors are correlated to abnormal dispersion of birefringence characteristics supports the concept that collagen fibers induce an ordered binding of silver distributed along the fibers.     

Investigation of the cosmetic ingredient distribution in the stratum corneum using NanoSIMS imaging

In order to understand the mechanisms of action of cosmetic ingredients, it is important to establish the distribution of the component agents within the epidermis of the skin. To date, time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used to detect cosmetic ingredients in the skin. However, it is technically difficult to investigate the distribution of the agents in the stratum corneum using TOF-SIMS. Therefore, an analytical method with higher spatial resolution is required. In this study, we investigated an imaging analysis technique based on NanoSIMS to detect cosmetic ingredients in the skin. Pig skin was used as a model for human skin. The sample was treated with a cosmetic formulation containing ¹⁵N-labelled pseudo-ceramide (SLE). The sample was frozen with liquid nitrogen and cross-sections were cut using a cryomicrotome. As a result, the fine layer structure of the corneocytes was clearly observed by using NanoSIMS. Our studies indicate that SLE penetrates into the stratum corneum via an intercellular route. We conclude that application of NanoSIMS analysis can contribute to a better understanding of the function of cosmetic ingredients in the skin.     

Picrosirius-polarization staining method as an efficient histopathological tool for collagenolysis detection in vesical prolapse lesions

The Picrosirius-polarization method has been indicated as a selective histochemical stain for collagen detection in tissue sections. This method can also be of value for studying collagen degradation given that, under polarized light, collagen displays birefringence due to its molecular order. The aim of this study is to highlight this staining method as an additional instrument for a rapid and excellent confirmatory diagnosis of the presence of collagenolysis in connective tissue in the vaginal wall with vesical prolapse lesion, in tissue sections. Dramatic changes in collagen morphology were found in vaginal mucosa in vesical prolapse disorder: they were weakly stained by Sirius red and under polarized light appeared as thin, pale (weakly birefringent), greenish, and with fibers more scattered, while the histoarchitecture of the organ showed a disrupted appearance. Thus, in the present study, we showed in vaginal mucosa in the vesicle prolapse that corroded collagenous framework appears as fragmentary and irregularly separated collagenous structures, that are weakly birefringent, corresponding to a molecular disorganization of these fibers caused by collagenolysis.     

Skin layer detection of optical coherence tomography images

In this paper, we present an image processing algorithm to automatically and more precisely detect the boundary between the main skin layers: stratum corneum, epidermis, and dermis. The aim of the proposed skin layer detection algorithm is to assist the dermatologists to measure the epidermal thickness (ET) for skin diseases diagnosis and also to assist pharmacologists so that they can make a better decision for prescribing according to the advancement of the skin disorders characterized with ET change.     

Porosity estimation of aged mortar using a micromechanical model

Degradation of concrete structures located in high humidity atmospheres or under flowing water is a very important problem. In this study, a method for ultrasonic non-destructive characterization in aged mortar is presented. The proposed method makes a prediction of the behaviour of aged mortar accomplished with a three phase micromechanical model using ultrasonic measurements. Aging mortar was accelerated by immersing the probes in ammonium nitrate solution. Both destructive and non-destructive characterization of mortar was performed. Destructive tests of porosity were performed using a vacuum saturation method and non-destructive characterization was carried out using ultrasonic velocities. Aging experiments show that mortar degradation not only involves a porosity increase, but also microstructural changes in the cement matrix. Experimental results show that the estimated porosity using the proposed non-destructive methodology had a comparable performance to classical destructive techniques.     

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.     

AFM study of the extracellular connective tissue matrix in patients with pelvic organ prolapse

The method of atomic force microscopy (AFM) is used for the first time for morphological investigation of pathological changes in the extracellular matrix of skin connective tissue upon the prolapse of pelvic organs (common disorder among women). Skin samples of patients with clinically proven pelvic-organ prolapse and of patients that do not have any connective tissue related disease (control group) are investigated via AFM. The AFM study reveals that the extracellular matrix of the skin connective tissues from patients with pelvic-organ prolapse diverges from the normal in various organization levels including both micro- and nanotexture (packing of collagen fibers and fibrils, respectively). The results of AFM study of the normal and pathologically changed skin connective tissues are in good agreement with the data of clinical morphological analysis, which indicates the potential of AFM as an independent diagnostic tool.     

Effects of aging and maternal protein restriction on the muscle fibers morphology and neuromuscular junctions of rats after nutritional recovery

Changes in the nutritional status of mothers may predispose their offspring to neuromuscular disorders in the long term. This study evaluated the effects of maternal protein restriction during pregnancy and lactation on the muscle fibers and neuromuscular junctions (NMJs) of the soleus muscle in the offspring of rats at 365 days of age that had undergone nutritional recovery. Wistar rats were divided into two groups: control (CG) – the offspring of mothers fed a normal protein diet (17%) and restricted (RG) – offspring of mothers fed a low protein diet (6%). After lactation, the male pups received standard chow ad libitum. At 365 days, samples of soleus muscle were collected for muscle fiber analysis (HE staining, NADH-TR reaction and ultrastructure), intramuscular collagen quantification (picrosirius red staining) and NMJs analysis (non-specific esterase technique). The cross-sectional area of type I fibers was reduced by 20% and type IIa fibers by 5% while type IIb fibers increased by 5% in the RG compared to the CG. The percentage of intramuscular collagen was 19% lower in the RG. Disorganization of the myofibrils and Z line was observed, with the presence of clusters of mitochondria in both groups. Regarding the NMJs, in the RG there was a reduction of 10% in the area and 17% in the small diameter and an increase of 7% in the large diameter. The results indicate that the effects of maternal protein restriction on muscle fibers and NMJs seem to be long-lasting and irreversible.     

The use of the diffusion approximation for simulating radiation and thermal processes in the skin

Radiation and thermal processes in skin exposed to solar radiation are simulated based on the diffusion model of radiative-conductive heat exchange. Using the model proposed for the parameters corresponding to radiation with a wavelength of 800 nm, the contributions of thermal radiation induced by the skin and the reflection and refraction effects are estimated, and the photoprotective properties of titanium dioxide nanoparticles (TiO₂) when introduced into the stratum corneum are studied.