Population Of Fibrils Research Articles

Thermally induced irreversible conformational changes in collagen probed by optical second harmonic generation and laser-induced fluorescence.

Irreversible thermal conformational changes induced to collagen have been studied by optical methods. More specifically, second harmonic generation (SHG) from incident nanosecond Ng:YAG 1064 nm radiation and laser-induced fluorescence by 337 nm, pulsed nanosecond nitrogen laser excitation, at 405, 410 and 415 nm emission wavelengths were registered at eight temperatures (40 degrees, 50 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees and 80 degrees C) and normalised with respect to the corresponding values at the ambient temperature of 30 degrees C. The heating protocol used in this work, was selected to monitor only permanent changes reflecting in the optical properties of the samples under investigation. In this context, the SHG, directly related to the collagen fibril population in triple helix conformation, indicated on irreversible phase transition around 64 degrees C. On the other hand fluorescence related to the destruction of cross-linked chromophores in collagen, some of which are related to the triple helix tertiary structure, also indicated a permanent phase transition around 63 degrees C. These results are in agreement with previous results from studies with differential scanning calorimetry. However SHG and fluorescence, being non-invasive optical methods are expected to have a significant impact in the fields of laser ablative surgery and laser tissue welding.

A novel technique of taking tendon biopsies in the horse

SummaryTo evaluate a novel tendon biopsy technique for use in the horse and to assess the effect of different exercise levels on the healing process of minimally damaged superficial digital flexor tendons (SDFT), 24 Dutch Warmblood foals were randomly divided into 3 equally sized groups one week after birth. One group was box rested, one group got box rest with additional enforced exercise, and one group got free pasture exercise. Biopsies from the peripheral region of the SDFT were taken at age 2 months using a new micro-biopsy technique. At age 5 months the foals were euthanized and samples from the biopsy site and a control site were harvested. Collagen fibrillar index (CFI, the total amount of collagen as a percentage of the measured area), mass-average diameter (MAvD, mean of the diameter versus area distribution) and collagen fibrillar diameter frequency (the distribution of collagen fibril sizes expressed as a percentage of the number of collagen fibrils) were calculated. The CFI, MAvD values were significantly lower in the wound samples. It is concluded that the biopsy technique may be a useful tool for research applications. The absence of exercise-induced effects on the collagen fibril population in the wound samples suggests that the first phase of the process of tendon healing in immature equine SDFT’s sustaining a minor surgically-induced wound, is not exercise-dependent. However, findings may be different in mature horses sustaining accidental trauma.A novel technique to take biopsies from immature equine flexor tendons was developed and used to evaluate the effect of various levels of exercise on the development of the collagen fibril diameter distribution in the resulting wounds. The technique appeared to be minimally invasive, but complication rate was deemed too high for clinical use. There was no effect of exercise on mass average diameter of collagen fibrils, suggesting that the repair process, at least in its initial stage, is exercise-independent.

Decorin antisense gene therapy improves functional healing of early rabbit ligament scar with enhanced collagen fibrillogenesis in vivo

Injured ligaments heal with scar tissue, which has mechanical properties inferior to those of normal ligament, potentially resulting in re-injury, joint instability, and subsequent degenerative arthritis. In ligament scars, normal large-diameter collagen fibrils have been shown to be replaced by a homogenous population of small collagen fibrils. Because collagen is a major tensile load-bearing matrix element and because the proteoglycan decorin is known to inhibit collagen fibrillogenesis, we hypothesized that the restoration of larger collagen fibrils in a rabbit ligament scar, by down-regulating the proteoglycan decorin, would improve the mechanical properties of scar. In contrast to sense and injection-treated controls, in vivo treatment of injured ligament by antisense decorin oligodeoxynucleotides led to an increased development of larger collagen fibrils in early scar and a significant improvement in both scar failure strength (83-85% improvement at 6 weeks; p < 0.01) and scar creep elongation (33-48% less irrecoverable creep; p < 0.03) under loading. This is the first report that in vivo manipulation of collagen fibrillogenesis improves tissue function during repair processes with gene therapy. These findings not only suggest the potential use of this type of approach to improve the healing of various soft tissues (skin, ligament, tendon, and so on) but also support the use of such methods to better understand specific structure-function relationships in scars.

Ultrastructural localisation and size distribution of collagen fibrils in Glisson's sheath of rat liver: implications for mechanical environment and possible producing cells.

The ultrastructure and size distributions of collagen fibrils in Glisson's sheath were investigated in the rat liver to analyse the mechanical environment around the fibrils and their possible cells of origin. Glisson's sheath was found to contain 2 populations of collagen fibrils with different diameters and distinct localisations, namely fibroblast-associated and bile epithelium-associated. Fibroblast-associated collagen was composed of fibrils arranged in bundles and constituted the majority of the collagen in Glisson's sheath. Bile epithelium-associated collagen was represented by small dispersed groups of fibrils just beneath the basement membrane of the bile duct. The basement membrane of the bile duct was frequently reduplicated into a few or as many as 10 layers of laminae densae, with scattered collagen fibrils between these laminae. The diameters of the fibrils of both groups of collagen increased in relation to the calibre of the bile duct, whereas at any given place in Glisson's sheath bile epithelium-associated collagen fibrils had a smaller diameter compared with those of the fibroblast-associated fibrils. The increment in fibril diameter along the bile duct is considered to be correlated with the increase in mechanical stress acting on Glisson's sheath. The difference in diameter between the 2 populations as well as the incorporation of fibrils between the laminae densae of the basement membrane of the bile duct supports the view that the bile epithelium-associated collagen is produced by the epithelial cells of the bile duct, thus having a different origin from that of fibroblast-associated collagen. These findings provide the first evidence that the epithelial cells of the interlobular bile duct produce fibril-forming collagen. Furthermore, it is suggested that cholestasis stimulates the epithelial cells of interlobular bile duct to increased synthesis of fibril-forming collagen that is also produced by these cells under physiological conditions.

Cartilage fibrils of mammals are biochemically heterogeneous: differential distribution of decorin and collagen IX.

Cartilage fibrils contain collagen II as the major constituent, but the presence of additional components, minor collagens, and noncollagenous glycoproteins is thought to be crucial for modulating several fibril properties. We have examined the distribution of two fibril constituents-decorin and collagen IX-in samples of fibril fragments obtained after bovine cartilage homogenization. Decorin was preferentially associated with a population of thicker fibril fragments from adult articular cartilage, but was not present on the thinnest fibrils. The binding was specific for the gap regions of the fibrils, and depended on the decorin core protein. Collagen IX, by contrast, predominated in the population with the thinnest fibrils, and was scarce on wider fibrils. Double-labeling experiments demonstrated the coexistence of decorin and collagen IX in some fibrils of intermediate diameter, although most fibril fragments from adult cartilage were strongly positive for one component and lacked the other. Fibril fragments from fetal epiphyseal cartilage showed a different pattern, with decorin and collagen IX frequently colocalized on fragments of intermediate and large diameters. Hence, the presence of collagen IX was not exclusive for fibrils of small diameter. These results establish that articular cartilage fibrils are biochemically heterogeneous. Different populations of fibrils share collagen II, but have distinct compositions with respect to macromolecules defining their surface properties.

Effects of training on collagen fibril populations in the suspensory ligament and deep digital flexor tendon of young Thoroughbreds

SUMMARY Objective To determine the effect of a specific galloping exercise regimen on collagen fibril mass-average diameters (MAD) in the deep digital flexor tendon (DDFT) and suspensory ligament (SL) of young Thoroughbreds. Animals 12 Thoroughbred fillies, 21 ± 1 (mean ± SD) months old. Procedure 6 horses underwent a specific 18-month treadmill training program involving galloping exercise. The remaining 6 horses served as controls, undertaking low-volume walking exercise over the same period. Sections were excised from the midpoint of the DDFT and SL, and small strips were dissected from central and peripheral locations for each structure. Fibril diameters were measured from micrographs of transverse ultrathin sections, using a computerized image analysis program. An MAD value was calculated for the central and peripheral regions of the DDFT and SL for each horse. Values for both regions were compared between exercised and control horses. Results The MAD did not change significantly with exercise for either the DDFT or the SL. Conclusion Loading of the DDFT as a result of this exercise regimen was not sufficient to stimulate collagen fibril hypertrophy, in keeping with current data that indicate this tendon, compared with the SL and superficial digital flexor tendon (SDFT), is subjected to low loads. Microtrauma, in terms of reduction in fibril MAD, may have occurred in the SL at a site different from that sampled. Another possibility is that, between the trot and the gallop, loading of the SL does not increase to the same extent as that of the SDFT. (Am J Vet Res 1998;59:64–68)

Collagen Fibril Diameters in the Rabbit Medial Collateral Ligament Scar

Previous transmission electron microscopic investigations of collagen fibril diameters in rabbit medial collateral ligament (MCL) scars have indicated a homogeneous population of small fibrils for the first 40 weeks of healing. In this study, four 8 mm MCL gap scars were studied at 78 weeks of healing and another three at 104 weeks. Results showed increased heterogeneity in the distribution of fibril diameters in all scars, with the appearance of progressively slightly larger fibrils in 78 and 104 week specimens. All longer term scars still contained roughly 90% small fibrils plus some “patches” of larger fibrils, but there was considerable variation between animals in these proportions. No scar contained the fibril populations typical of uninjured adult rabbit MCLs. These results suggest slow but on-going collagen fibril turnover and remodeling in this gap healing rabbit MCL model via currently unidentified mechanisms.

Type II collagen is a major component of bovine retinal microvessel extracellular matrix.

To determine which interstitial collagen types may be present in bovine retinal microvessel extracellular matrix (ECM). Dissociated bovine microvessels were treated with detergents and the resultant purified ECM monitored by transmission electron microscopy (TEM). Pepsin-extracted soluble ECM collagens were identified by Western blots. Collagens were further purified by neutral salt precipitation and carboxymethyl cellulose (CMC) chromatography before cyanogen bromide (CNBr) peptide mapping and two-dimensional peptide mapping of CMC-generated fractions. Interstitial collagens were localized by immunofluorescence on frozen sections. Transmission electron microscopy of detergent-purified microvessel ECM demonstrated numerous 10-50-nm collagen fibrils associated with basal laminae regardless of vessel diameter. Western blots showed that soluble ECM collagens were strongly positive for type II, moderate for type III, and weak for type I. CNBr peptide maps and two-dimensional maps of neutral salt and CMC-purified fractions confirmed the presence of type II collagen. Immunofluorescence localized type II collagen in large and small vessels of the retina. Type II collagen is an unexpected major component of bovine microvessel ECM, whereas types I and III are present in minor amounts. Type V collagen is also a substantial ECM component. Accordingly, all four types may contribute to a heterogeneous population of collagenous fibrils identified by TEM in intact isolated retinal microvessel ECM.

Collagen fibril populations in human anterior cruciate ligament allografts. Electron microscopic analysis.

We studied human anterior cruciate ligament allograft specimens by quantitative electron microscopy to analyze their collagen fibril populations. The specimens were procured at the time of second-look arthroscopy from the superficial region of the midzone of the anterior cruciate ligament grafts after synovial clearage. The grafts used for the anterior cruciate ligament reconstructions were from fresh-frozen allogenic Achilles, tibialis anterior or posterior, or peroneus longus or brevis tendons and had been implanted 3 to 96 months previously. By 12 months after surgery, the anterior cruciate ligament allografts consisted predominantly of small-diameter collagen fibrils (30 to 80 nm), which resulted in a unimodal pattern in the collagen fibril profile. The number of large-diameter fibrils (90 to 140 nm) within the allogenic tendon grafts had decreased. This predominance of small-diameter collagen fibrils persisted in almost all specimens older than 12 months. The anterior cruciate ligament allografts had collagen fibril profiles that did not resemble normal tendon grafts or normal anterior cruciate ligaments, even several years after surgery.

Stereologic studies on collagen in bovine articular cartilage.

In an ultrastructural stereologic study on bovine articular cartilage we found that collagen volume density increased with increasing distance from the joint surface and from the chondrocyte. These results not only corroborate previous biomechanical data of a vertical stiffness gradient, but they also suggest that the mechanical forces are unevenly distributed horizontally. On the other hand, although mean collagen fibril diameter showed large differences between the interterritorial compartments of the three zones, there was a population of slender fibrils in all zones and compartments. Since the coarser fibrils provide the high tensile strength (Nimni 1988), the role of the slender fibrils may be to enhance the deformability of the tissue. Moreover, in spite of substantial differences in mean fibril diameter, collagen surface densities were in the same order of magnitude in the territorial and interterritorial compartments, and only slightly lower in the pericellular compartments. The surface parameter may be important for specific molecular interactions. The collagen fibrils have different polarity, i.e. the direction of the fibrils appears to be parallel and antiparallel, about 50% running in each direction. This, together with the very high length/diameter ratio (Clark 1985), may indicate that each fibril is assembled by the concerted action of many cells. The characteristic properties of articular cartilage depend on interactions between its macromolecular components, and the present quantitative data form a basis for discussions on the specificity and regulation of such interactions.

A Quantitative Ultrastructural Study of Collagen Fibril Formation in the Healing Extensor Digitorum Longus Tendon of the Rat

A study was made of collagen fibril populations in healing tendon using a window lesion in rat extensor tendon. Two environments were thus created. One (the lesion area) where stress levels were reduced; the other (the non-lesion area) where stress levels were increased. In both areas a population of small diameter (&lt; 50 nm) collagen fibrils were synthesized. Lesion area fibrils increased their diameter only slowly. Non-lesion area fibrils added to a population which increased its diameter distribution comparatively rapidly. It is suggested that in the lesion area fibrils were synthesized in response to tissue damage and low levels of stress. These may be of type III collagen. In the non-lesion area fibrils were synthesized in response to raised levels of stress. These may be of type I collagen. Implications of these events, especially as they might relate to gap formation in flexor tendon surgery are discussed.

Ultrastructural morphometry of matrical changes induced by exercise and food restriction in the rat calcaneal tendon

The ultrastructural morphometry of collagen fibril populations in 24 calcaneal tendons obtained from 12 Fischer 344 rats were studied to elucidate matrical changes induced by food restriction and/or endurance exercise. Rats were randomly assigned to four equal groups: ad libitum control (AC), ad libitum exercise (AE), restricted diet control (RC) and restricted diet exercise (RE) groups. Beginning from 6 weeks of age, animals in the two food restriction groups were fed 60% of the mean food consumption of ad libitum fed rats. Then, starting from 6–7 months of age, the rats in the two exercise groups performed 40–50 min of treadmill running at 1.2–1.6 miles h −1 every day for a total of 10 weeks. Endurance training did not significantly alter body weight, but food restriction with or without exercise resulted in a significant loss of body weight. In ad libitum fed controls, food restriction alone did not significantly alter the mean collagen fibril CSA, but predisposed a preponderance of small-sized collagen fibrils. Endurance training per se induced a significant (32%) increase in mean fibril CSA (P < 0.05), but this adaptive response to exercise was prevented by food restriction, as indicated by a 33% decline in fibril CSA (P < 0.05). These findings demonstrate that dietary restriction modifies the adaptation of tendon collagen morphometry in response to endurance training, and that weight loss is better achieved with food restriction than endurance exercise.

Synchrotron x-ray diffraction studies of the cornea, with implications for stromal hydration

The intermolecular and interfibrillar spacings of collagen in bovine corneal stroma have been measured as a function of tissue hydration. Data were recorded from low- and high-angle x-ray diffraction patterns obtained using a high intensity synchrotron source. The most frequently occurring interfibrillar spacing varied from 34 nm in dry corneas to 76 nm at H = 5 (the hydration, H, is defined as the ratio of the weight of water to the dry weight). The most frequently occurring intermolecular Bragg spacing increased from 1.15 nm (dry) to approximately 1.60 nm at normal hydration (H approximately 3.2) and continued to increase only slowly above normal hydration. Most of the increase in the intermolecular spacing occurred between H = O and H = 1. Over this hydration range the interfibrillar and intermolecular spacings moved in tandem, which suggests that the initial water goes equally within and between the fibrils. Above H = 1 water goes preferentially between the fibrils. The results suggest that, even at normal hydration, water does not fill the interfibrillar space uniformly, and a proportion is located in another space or compartment. In dried-then-rehydrated corneas, a larger proportion of the water goes into this other compartment. In both cases, it is possible to postulate a second set or population of fibrils that are more widely and irregularly separated and therefore do not contribute significantly to the diffraction pattern.

Rotary shadowing of collagen monomers, oligomers, and fibrils during tendon fibrillogenesis.

Collagen monomers, oligomers, and fibrillar structures were isolated from chick tendons at various stages of development and studied by rotary shadowing. Monomers of Type I collagen, solubilized in 0.15 M NaCl solutions, were mostly present as collagen, pN-collagen, and pC-collagen with few procollagen molecules. They did not form polymers, nor were they associated with a carrier. Dimers of fibrillar collagen molecules were arranged in a 4-D stagger, suggesting that this was the preferred molecular interaction for the initiation of collagen fibrillogenesis. Type XII collagen molecules were mostly free, but some were attached by their central globular domain to one end of free fibrillar collagen molecules. Tenascin and Type VI collagen were also identified. The fibril populations consisted of collagen and beaded structures. These fibrils consisted of beads (globular domains) about 23 nm in diameter, separated by a period about 27 nm in length. Beads were linked by filamentous structures. These beaded fibrils probably represent the microfibrils of elastin.

Extensor tendon repair: An animal model which allows immediate post-operative mobilisation

In adult rabbits, an experimentally transected digital extensor tendon was repaired. The technique employed a tongue, created from the proximal tendon, folded over to bridge the transection and reattached distally. Animals were allowed immediate post-operative mobilisation. Tendon material, 14–120 days post-operatively, was examined ultrastructurally in control and experimental animals from four different zones within the repaired tendon. The operation was well tolerated and adhesion free. Associated with the repair, two types of collagen fibril populations were observed. Firstly, in areas where tendon tissue had been removed, there was a population of fibrils with a narrow range of diameters. Secondly, in areas where tendon tissue dia subjected to an increased level of stress per unit cross-sectional area, a population of fibrils with a range of diameters similar to that of controls but with a marked increase in the percentage of small diameter fibrils. The relevance of these observations to human tendon repair is discussed.

Extensor Tendon Repair: An Animal Model Which Allows Immediate Post-Operative Mobilisation

In adult rabbits, an experimentally transected digital extensor tendon was repaired. The technique employed a tongue, created from the proximal tendon, folded over to bridge the transection and reattached distally. Animals were allowed immediate post-operative mobilisation. Tendon material, 14-120 days post-operatively, was examined ultrastructurally in control and experimental animals from four different zones within the repaired tendon. The operation was well tolerated and adhesion free. Associated with the repair, two types of collagen fibril populations were observed. Firstly, in areas where tendon tissue had been removed, there was a population of fibrils with a narrow range of diameters. Secondly, in areas where tendon tissue was subjected to an increased level of stress per unit cross-sectional area, a population of fibrils with a range of diameters similar to that of controls but with a marked increase in the percentage of small diameter fibrils. The relevance of these observations to human tendon repair is discussed.

The regulation of size and form in the assembly of collagen fibrils in vivo.

A possible mechanism for regulating the lateral growth of collagen fibrils in vivo is considered. A growth inhibitor associated with a particular part of the long semiflexible collagen molecule restricts that part of the molecule to the surface of the growing assembly. Lateral accretion ceases when these inhibitors form a complete circumferential layer around the fibril surface. Cell-mediated removal of the inhibitors allows lateral growth to proceed to a second limiting layer, and so on to subsequent limiting layers. In this way, cycles of inhibitor removal and limited lateral accretion permit growth to be synchronized over large populations of fibrils. Observed diameter distributions in bundles of embryonic and neonatal fibrils are those expected from a mechanism of this kind. The mechanism depends on the existence of axial order (D-periodicity) in fibrils, but not on any specific lateral packing of molecules. Rather, contacts between newly assembled molecules are presumed to be partly fluid-like in lateral directions (except where covalent cross-links have formed). Some initial fluidity in lateral packing prior to cross-linking does not preclude the subsequent emergence of quasi-crystalline packing as cross-links form. The cylindrical shape of fibrils in vivo may also be attributable in part to fluidity of intermolecular contacts at the growing surface.

D-periodic distribution of collagen type IX along cartilage fibrils.

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.

Do collagen fibrils of the periodontal ligament shrink with age?

Do collagen fibrils of the periodontal ligament shrink with age? Certainly not! Based on the quantitative estimation of over 25000 periodontal ligament collagen fibrils at 3 periodontal levels of 28 freshly extracted human teeth, using 10‐ to 75year‐old subjects and high resolution electron micrographs evaluated planimetrically, the present study demonstrates with data obtained from 2 sampling procedures that the diameter of the average collagen fibril, i.e., 54 to 59 nm, is rather constant throughout the human life span. This is particularly true for the vast majority of comparatively large fibrils (i.e., 57 to 64 nm), while a numerically limited population of small fibrils (i.e., 40 to 48 nm) in fact increased in diameter with age. These results are discussed in view of analogous data for other connective tissues and of the functional and metabolic status of such tissues.

Biomechanics of collagen fibril populations in regenerating tendons

Biomechanics of collagen fibril populations in regenerating tendons