Interferometric evaluation of collagen concentration in tendon fibers

The dry mass concentration of collagen in native rat tail tendon fibers was studied by interferometric techniques. It could be shown that the collagen concentration of rat tail tendon fibers is not constant nor does it vary uniformly with the fiber diameter. Evidence is presented that the collagen concentration shows significant oscillations as a function of diameter. In order to explain the experimental findings, a layered model is proposed for the fiber structure; age dependent changes were also studied. It seems that the fiber structure is unaltered by ageing at the supramolecular level but its functional capacities are affected and swelling is inhibited.     

Healing parameters in a rabbit partial tendon defect following tenocyte/biomaterial implantation

Although rabbits are commonly used as tendon repair model, interpretative tools are divergent and comprehensive scoring systems are lacking. Hence, the aim was to develop a multifaceted scoring system to characterize healing in a partial Achilles tendon defect model. A 3 mm diameter defect was created in the midsubstance of the medial M. gastrocnemius tendon, which remained untreated or was filled with a polyglycolic-acid (PGA) scaffold + fibrin and either left cell-free or seeded with Achilles tenocytes. After 6 and 12 weeks, tendon repair was assessed macroscopically and histologically using self-constructed scores. Macroscopical scoring revealed superior results in the tenocyte seeded PGA + fibrin group compared with the controls at both time points. Histology of all operated tendons after 6 weeks proved extracellular matrix (ECM) disorganization, hypercellularity and occurrence of irregular running elastic fibres with no significance between the groups. Some inflammation was associated with PGA implantation and increased sulphated proteoglycan deposition predominantly with the empty defects. After 12 weeks defect areas became hard to recognize and differences between groups, except for the increased sulphated proteoglycans content in the empty defects, were almost nullified. We describe a partial Achilles tendon defect model and versatile scoring tools applicable for characterizing biomaterial-supported tendon healing.     

Growth of mineral crystals in turkey tendon collagen fibers

Bone and several other vertebrate mineralized tissues are formed by the organized growth of crystals of carbonated apatite within a matrix of type 1 collage fibers. The development of this process in isolated fibrils of young turkey leg tendons has been studied by transmission electron microscopy. Collagen banding, presumably due to ion concentration, precedes the appearance of any crystals. The smallest crystals observed are short needles in bands near the surface of the fibrils. Longer needles, up to the length of the collagen gap regions, were also seen, and, evidently at a later stage, single crystal belts extending partly or wholly through the fibrils. Finally, in mature tendon crystal platelets, seemingly derived from the cracking of belts, extend partly into the collagen overlap zone. In the least mineralized tendon, extrafibrillar mineral-containing vesicles have occasionally been observed adjacent to regions of radiating needle crystal growth in the fibrils, and, more commonly, smaller particles adjacent to bands of very small needles.     

Regeneration and repair of tendon and ligament tissue using collagen fibre biomaterials

Collagen fibres are ubiquitous macromolecular assemblies in nature, providing the structures that support tensile mechanical loads within the human body. Aligned type I collagen fibres are the primary structural motif for tendon and ligament, and therefore biomaterials based on these structures are considered promising candidates for mediating regeneration of these tissues. However, despite considerable investigation, there remains no collagen-fibre-based biomaterial that has undergone clinical evaluation for this application. Recent research in this area has significantly enhanced our understanding of these complex and challenging biomaterials, and is reinvigorating interest in the development of such structures to recapitulate mechanical function. In this review we describe the progress to date towards a ligament or tendon regeneration template based on collagen fibre scaffolds. We highlight reports of particular relevance to the development of the underlying biomaterials science in this area. In addition, the potential for tailoring and manipulating the interactions between collagen fibres and biological systems, as hybrid biomaterial-biological ensembles, is discussed in the context of developing novel tissue engineering strategies for tendon and ligament.     

Growth of mineral crystals in turkey tendon collagen fibers.

Bone and several other vertebrate mineralized tissues are formed by the organized growth of crystals of carbonated apatite within a matrix of type 1 collagen fibers. The development of this process in isolated fibrils of young turkey leg tendons has been studied by transmission electron microscopy. Collagen banding, presumably due to ion concentration, precedes the appearance of any crystals. The smallest crystals observed are short needles in bands near the surface of the fibrils. Longer needles, up to the length of the collagen gap regions, were also seen, and, evidently at a later stage, single crystal belts extending partly or wholly through the fibrils. Finally, in mature tendon crystal platelets, seemingly derived from the cracking of belts, extend partly into the collagen overlap zone. In the least mineralized tendon, extrafibrillar mineral-containing vesicles have occasionally been observed adjacent to regions of radiating needle crystal growth in the fibrils, and, more commonly, smaller particles adjacent to bands of very small needles.     

The three-dimensional ultrastructure of the collagen fibers, reticular fibers and elastic fibers: a review]

Fibrous components of the connective tissue are light-microscopically classified into three types: collagen fibers, reticular fibers and elastic fibers. The present paper reviews the three-dimensional ultrastructure of these fibrous components, mainly based on our studies by scanning electron microscopy. The collagen fibers are shaped like tapes or cords about 1 to 20 microns in diameter. Each fiber is a bundle of fibrils running roughly parallel to each other. These collagen fibrils vary in diameter from 30 to 300 nm depending on their locating area of the body, and show a repeating pattern of depressed and protruding segments on the surface. The reticular fibers consist of collagen fibrils about 20-40 nm in diameter, which run singly or in small bundles. They are usually interwoven elaborately to form thin lace-like sheets or sheaths attaching to basal laminae of such cells as epithelial, endothelial and muscular cells. These fibers are considered to play an important role not only in adhering the cells to the collagen fibers, but also in constituting the skeletal framework suitable for individual cells and tissues. The elastic fibers consist of two different components: elastin and fibrillin. Elastin forms unit fibrils of 0.1-0.2 micron thickness which are arranged in bundles or laminae specific to individual organs and tissues. Fibrillin, on the other hand, forms microfibrils about 10 nm in diameter running in or along elastin bundles. These microfibrils also form delicate networks separate from elastin components. For a comprehensive understanding of the fibrous components in the connective tissue, the author proposed categorizing them into two systems: the collagen fibrillar system as a supporting framework of tissues and cells, and the fibrillin-elastin fibrillar system for distributing stressing forces uniformly in tissues.     

Regeneration of liver parenchyma and sinusoidal cells following partial hepatectomy

Partial hepatectomy (30 percent of the liver weight) was performed in 225 female Wistar rats, ages 6 weeks, 6 and 9 months. The animals were sacrificed in intervals between 4 hours and 21 d after the operation. The automated microscopic image analysis after Simon et al. (1984) and histoautoradiography were utilized for evaluation of liver cell regeneration. Despite measurement of more than 1,000 cells per animal we were not able to confirm enlargement of hepatocytes as the first reaction to partial hepatectomy The number of liver cells per unit area increased whereas sinusoidal size remained rather constant, corresponding to a decrease in average cell size and in the standard deviation of cell area. The importance of binucleated hepatocytes in the process is emphasized. Measurement of the nucleoli and of the mean extinction values of liver cell nuclei as a measure of the DNA content did not provide any essentially new perspectives. The investigation did however emphasize the importance of the extent of the hepatic resection and the age of the experimental animals on the intensity and subsequent course of liver cell regeneration. Proliferation of the sinusoidal cells themselves after partial hepatectomy could not definitively be demonstrated to be age-dependent.     

Mechanical properties of native and reconstituted rat tail tendon collagen upon maturation in vitro

Native and reconstituted rat tail tendon collagen were tested mechanically after in vitro maturation by incubation. The mechanical strength of the native tendons increased upon incubation and attained maximum strength values similar to those of tendons matured and aged in vivo. This finding indicates that the same stabilizing process occurs both in vivo and in vitro. However, the mechanical strength values similar to those of tendons matured and aged in vivo. This finding indicates that the same stabilizing process occurs both in vivo and in vitro. However, the mechanical strength increased at an initial higher rate in vitro than in vivo. The mechanical strength of fibrils reconstituted from purified tail tendon collagen increased during incubation in air as previously reported for fibrils prepared from skin collagen. Fibrils prepared from tail tendon and skin collagen shared common mechanical and thermal stability characteristics upon the incubation. However, distinct qualitative mechanical characteristics for fibrils of the two collagens were found. These characteristics showed a resemblance to those of the respective source tissues. The results indicate that the same process is responsible for the gain in mechanical strength of native tissues and reconstituted collagen fibrils. Thus, reconstituted collagen fibrils seem a useful model for studying mechanical stability changes during maturation of collagen.     

Comparative study of the angle-resolved backscattering properties of collagen fibers in bovine tendon and cartilage

In a biological tissue, light scattering is based on the size and type of scatterers seen as refractive index variations that describe the optical properties shown. In this paper, we have implemented the variable incidence angle technique of multiple angle of illumination experiment on tendon and cartilage samples whose dominant constituents are genetically different types of collagen fibers, type I and type II, respectively. It is found that tendon displays a much greater angular anisotropy in its optical backscattering coefficient than the healthy cartilage. We propose that this is due to a more uniform distribution of fine fibrils than is found in tendon. Rayleigh-Gans approximation is used to give qualitative support to this idea.     

Role of storage on changes in the mechanical properties of tendon and self-assembled collagen fibers

Fibrous collagen networks are the major elements that provide mechanical integrity to tissues; they are composed of fiber forming collagens in combination with proteoglycans (PGs). Using uniaxial tensile tests we have studied the viscoelastic mechanical properties of rat tail tendon (RTT) fibers and self-assembled collagen fibers that were stored at 22 degrees C and 1 atm of pressure. Our results indicate that storage of RTT and self-assembled type I collagen fibers results in increased elastic and viscous components of the stress-strain behavior consistent with the hypothesis that storage causes the introduction of crosslinks. Analysis of the elastic and viscous mechanical data suggests that the elastic constant of the collagen molecule in RTT is about 7.7 GPa. Measurement of the viscous component of the stress-strain curves for RTTs and self-assembled collagen fibers suggests that PGs may increase the viscous component and effectively increase the collagen fibril length.     

Viscoelasticity of the vessel wall: the role of collagen and elastic fibers.

The aortic wall contains collagen fibrils, smooth muscle cells, and elastic fibers as the primary load-bearing components. It is well known that the collagen fibrils bear loads in the circumferential direction, whereas elastic fibers provide longitudinal as well as circumferential support. Stiffening of the vessel wall is associated with loss of elastic tissue and increases in the collagen content: however, little is known about the mechanism of vessel wall stiffening with age. The purpose of this review is to attempt to relate structural changes that occur to the collagen and elastic fibers to changes in the viscoelastic behavior that are associated with aging. Analysis of the viscoelastic mechanical properties of collagen fibrils from tendon, skin, and aortic wall suggest that the collagen fibrils of aortic wall are different than those of other tissues. The elastic spring constant of the collacen fibrils in vessel walls is significantly less than that found in tendon, suggesting that the presence of type III collagen in aortic wall increases the flexibility of the collagen fibrils. Furthermore, it is hypothesized that changes in the interface between collagen fibrils, elastic fibers, and smooth muscle during aging and in connective tissue disorders leads to changes in the viscoelasticity of the vessel wall.     

Optical anisotropy of collagen fibers of rat calcaneal tendons: An approach to spatially resolved supramolecular organization

Optical anisotropic characteristics investigated by polarization microscopy have been valuable for the study of the oriented organization of collagen fibers in tendons. However, topographic differences in supramolecular organization of collagen fibers along extensive areas in tendons have not yet been described. Here, the statistical variability of the oriented organization of collagen fibers along extensive areas (10(5)-10(6)microm(2)) of 7-microm thick unstained sections of rat calcaneal tendons were studied by assessing their birefringence with polarization microscopy and image analysis, and the periodic frequency distribution in their birefringent images with the fast Fourier transform (FFT). Various levels of birefringence intensity were determined principally by image analysis procedures, and periodicity in the birefringent images was revealed by FFT line profile and spectrum image patterns. Present results support the idea of a helical distribution for collagen bundles along the tendon long axis, and of a statistical architecture for the rat calcaneal tendons in terms of variability in the oriented distribution of their collagen fibers.     

Combined staining of elastic and collagen fibers for histopathological and differential diagnosis in aorta pathologies

Histochemical techniques are important for diagnosis of connective tissue fibers involved in different aorta pathologies. The aim of this study was to develop a histochemical staining method that identifies both elastic and collagen fibers simultaneously in the same section of aortic wall. Fragments of aorta from deceased dogs were processed according to standard histological technique for paraffin embedding. Sections were stained with orcein with counterstained with either light green or single step Gomori trichrome, and also orcein stain and picro-sirius red. Orcein stain combined with picro-sirius red allowed visualization of both elastic fibers (brown) and collagen fibers (red) with light microscopy using bright field illumination. Moreover, under polarized light microscopy only collagen fibers were detected, appearing as reddish birefringent fibers, confirming that picro-sirius red-stained fibers were collagen fibers. The combination staining by orcein and picro-sirius solutions provided visualization of both collagen and elastic fibers in the same section, facilitating identification of these connective tissue fibers in the aortic wall.     

Isolation and partial characterization of collagen fibrils, fibers and fiber-bundles from insoluble calf dermis

Collagen fibrils, fibers and fiber bundles have been isolated from insoluble calf skin chips by disaggregation with a neutral salt buffer followed by differential centrifugation. The ratio of type I collagen/type III collagen was relatively constant, regardless of the size of the fibrous materials. On the other hand, the ratios of glycosaminoglycans/collagen and non-collagenous glycoproteins/collagen were significantly higher in the thinner fiber fractions. However, the ratio of hyaluronic acid to dermatan sulfate was unchanged. The role of the non-collanenous constituents in collagen fibrillogenesis in vivo is discussed.     

Quantification of collagen and elastic fibers using whole‐slide images of liver biopsy specimens

Histological evaluation of fibrosis after a liver biopsy is crucial for evaluating the pathology of patients with chronic liver disease. Previous studies have reported quantitative analyses of fibrosis using images of collagen‐stained sections. However, analysis of these studies requires manual selection of the region of interest. In addition, the quantification of elastic fibers is not considered. The present study was conducted in order to measure both the collagen and elastic fiber area ratios using Elastica van Gieson‐stained whole‐slide images (WSIs) of liver biopsy specimens. High‐resolution WSIs provide precise color classification, enabling accurate detection of even fine collagen and elastic fibers. To minimize the influence of pre‐existing fibrous tissue, median area ratios of the collagen and elastic fibers were independently calculated from the image tiles of the WSIs. These median area ratios were highly concordant with area ratios after the pre‐existing fibrous tissues were manually trimmed from the WSI. Further, these median area ratios were correlated with liver stiffness as measured by transient elastography (collagen: r = 0.73 [P < 0.01], elastic: r = 0.53 [P < 0.01]). Our approach to quantifying liver fibrosis will serve as an effective tool to evaluate liver diseases in routine practice.