Radial Collagen Fibers Research Articles

The Mechanical Role of the Radial Fiber Network Within the Annulus Fibrosus of the Lumbar Intervertebral Disc: A Finite Elements Study.

The annulus fibrosus (AF) of the intervertebral disc (IVD) consists of a set of concentric layers composed of a primary circumferential collagen fibers arranged in an alternating oblique orientation. Moreover, there exists an additional secondary set of radial translamellar collagen fibers which connects the concentric layers, creating an interconnected fiber network. The aim of this study was to investigate the mechanical role of the radial fiber network. Toward that goal, a three-dimensional (3D) finite element model of the L3-L4 spinal segment was generated and calibrated to axial compression and pure moment loading. The AF model explicitly recognizes the two heterogeneous networks of fibers. The presence of radial fibers demonstrated a pronounced effect on the local disc responses under lateral bending, flexion, and extension modes. In these modes, the radial fibers were in a tensile state in the disc region that subjected to compression. In addition, the circumferential fibers, on the opposite side of the IVD, were also under tension. The local stress in the matrix was decreased in up to 9% in the radial fibers presence. This implies an active fiber network acting collectively to reduce the stresses and strains in the AF lamellae. Moreover, a reduction of 26.6% in the matrix sideways expansion was seen in the presence of the radial fibers near the neutral bending axis of the disc. The proposed biomechanical model provided a new insight into the mechanical role of the radial collagen fibers in the AF structure. This model can assist in the design of future IVD substitutes.

New Insights on the Composition and the Structure of the Acellular Extrinsic Fiber Cementum by Raman Analysis.

Acellular extrinsic fiber cementum is a mineralized tissue that covers the cervical half of the tooth root surface. It contains mainly extrinsic or Sharpey’s fibers that run perpendicular to the root surface to anchor the tooth via the periodontal ligament. Acellular cementum is continuously and slowly produced throughout life and exhibits an alternating bright and dark pattern under light microscopy. However, although a better understanding of the structural background of acellular cementum is relevant to many fields, such as cementochronology, periodontology and tissue engineering, acellular cementum remains rarely studied and poorly understood. In this work, we studied the acellular cementum at the incremental line scale of five human mandibular canines using polarized Raman spectroscopy. We provided Raman imaging analysis and polarized acquisitions as a function of the angular orientation of the sample. The results showed that mineral crystals were always parallel to collagen fibrils, and at a larger scale, we proposed an organizational model in which we found radial collagen fibers, “orthogonal” to the cementum surface, and “non-orthogonal” fibers, which consist of branching and bending radial fibers. Concerning the alternating pattern, we observed that the dark lines corresponded to smaller, more mineralized and probably more organized bands, which is consistent with the zoological assumption that incremental lines are produced during a winter rest period of acellular cementum growth.

Physiologically Distributed Loading Patterns Drive the Formation of Zonally Organized Collagen Structures in Tissue-Engineered Meniscus.

The meniscus is a dense fibrocartilage tissue that withstands the complex loads of the knee via a unique organization of collagen fibers. Attempts to condition engineered menisci with compression or tensile loading alone have failed to reproduce complex structure on the microscale or anatomic scale. Here we show that axial loading of anatomically shaped tissue-engineered meniscus constructs produced spatial distributions of local strain similar to those seen in the meniscus when the knee is loaded at full extension. Such loading drove formation of tissue with large organized collagen fibers, levels of mechanical anisotropy, and compressive moduli that match native tissue. Loading accelerated the development of native-sized and aligned circumferential and radial collagen fibers. These loading patterns contained both tensile and compressive components that enhanced the major biochemical and functional properties of the meniscus, with loading significantly improved glycosaminoglycan (GAG) accumulation 200-250%, collagen accumulation 40-55%, equilibrium modulus 1000-1800%, and tensile moduli 500-1200% (radial and circumferential). Furthermore, this study demonstrates local changes in mechanical environment drive heterogeneous tissue development and organization within individual constructs, highlighting the importance of recapitulating native loading environments. Loaded menisci developed cartilage-like tissue with rounded cells, a dense collagen matrix, and increased GAG accumulation in the more compressively loaded horns, and fibrous collagen-rich tissue in the more tensile loaded outer 2/3, similar to native menisci. Loaded constructs reached a level of organization not seen in any previous engineered menisci and demonstrate great promise as meniscal replacements.

SMAD3 deficiency promotes vessel wall remodeling, collagen fiber reorganization and leukocyte infiltration in an inflammatory abdominal aortic aneurysm mouse model

TGF-β signaling plays critical roles in the pathogenesis of aneurysms; however, it is still unclear whether its role is protective or destructive. In this study, we investigate the role of SMAD3 in the pathogenesis of calcium chloride (CaCl2)-induced abdominal aortic aneurysms (AAA) in Smad3−/−, Smad3+/− and Smad3+/+ mice. We find that loss of SMAD3 drastically increases wall thickening of the abdominal aorta. Histological analyses show significant vessel wall remodeling with elastic fiber fragmentation. Remarkably, under polarized light, collagen fibers in the hyperplastic adventitia of Smad3−/− mice show extensive reorganization accompanied by loosely packed thin and radial collagen fibers. The expressions of matrix metalloproteinases including MMP2, MMP9, and MMP12 and infiltration of macrophage/T cells are drastically enhanced in the vascular wall of Smad3−/− mice. We also observe marked increase of NF-κB and ERK1/2 signaling as well as the expression of nuclear Smad2, Smad4 and TGF-β1 in the vessel wall of Smad3−/− mice. In addition, we find that SMAD3 expression is reduced in the dedifferentiated medial smooth muscle-like cells of human AAA patients. These findings provide direct in vivo evidence to support the essential roles of SMAD3 in protecting vessel wall integrity and suppressing inflammation in the pathogenesis of AAAs.

Tectorial Membrane Material Properties in Tecta Y1870 C/+ Heterozygous Mice

The solid component of the tectorial membrane (TM) is a porous matrix made up of the radial collagen fibers and the striated sheet matrix. The striated sheet matrix is believed to contribute to shear impedance in both the radial and longitudinal directions, but the molecular mechanisms involved have not been determined. A missense mutation in Tecta, a gene that encodes for the α-tectorin protein in the striated sheet matrix, causes a 60-dB threshold shift in mice with relatively little reduction in outer hair cell amplification. Here, we show that this threshold shift is coupled to changes in shear impedance, response to osmotic pressure, and concentration of fixed charge of the TM. In Tecta Y 1870 C/+ mice, the tectorin content of the TM was reduced, as was the content of glycoconjugates reacting with the lectin wheat germ agglutinin. Charge measurements showed a decrease in fixed charge concentration from − 6.4 ± 1.4 mmol/L in wild-types to − 2.1 ± 0.7 mmol/L in Tecta Y 1870 C/+ TMs. TMs from Tecta Y 1870 C/+ mice showed little volume change in response to osmotic pressure compared to those of wild-type mice. The magnitude of both radial and longitudinal TM shear impedance was reduced by 10 ± 1.6 dB in Tecta Y 1870 C/+ mice. However, the phase of shear impedance was unchanged. These changes are consistent with an increase in the porosity of the TM and a corresponding decrease of the solid fraction. Mechanisms by which these changes can affect the coupling between outer and inner hair cells are discussed.

Virtual biopsy of rat tympanic membrane using higher harmonic generation microscopy

Multiharmonic optical microscopy has been widely applied in biomedical research due to its unique capability to perform noninvasive studies of biomaterials. In this study, virtual biopsy based on back-propagating multiple optical harmonics, combining second and third harmonics, is applied in unfixed rat tympanic membrane. We show that third harmonic generation can provide morphologic information on the epithelial layers of rat tympanic membrane as well as radial collagen fibers in middle fibrous layers, and that second harmonic generation can provide information on both radial and circular collagen fibers in middle fibrous layers. Through third harmonic generation, the capillary and red blood cells in the middle fibrous layer are also noted. Additionally, the 3-D relationship to adjacent bony structures and spatial variations in thickness and curvature are obtained. Our study demonstrates the feasibility of using a noninvasive optical imaging system for comprehensive evaluation of the tympanic membrane.

Col11a2 Deletion Reveals the Molecular Basis for Tectorial Membrane Mechanical Anisotropy

The tectorial membrane (TM) has a significantly larger stiffness in the radial direction than other directions, a prominent mechanical anisotropy that is believed to be critical for the proper functioning of the cochlea. To determine the molecular basis of this anisotropy, we measured material properties of TMs from mice with a targeted deletion of Col11a2, which encodes for collagen XI. In light micrographs, the density of TM radial collagen fibers was lower in Col11a2 –/– mice than wild-types. Tone-evoked distortion product otoacoustic emission and auditory brainstem response measurements in Col11a2 –/– mice were reduced by 30–50 dB independent of frequency as compared with wild-types, showing that the sensitivity loss is cochlear in origin. Stress-strain measurements made using osmotic pressure revealed no significant dependence of TM bulk compressibility on the presence of collagen XI. Charge measurements made by placing the TM as an electrical conduit between two baths revealed no change in the density of charge affixed to the TM matrix in Col11a2 –/– mice. Measurements of mechanical shear impedance revealed a 5.5 ± 0.8 dB decrease in radial shear impedance and a 3.3 ± 0.3 dB decrease in longitudinal shear impedance resulting from the Col11a2 deletion. The ratio of radial to longitudinal shear impedance fell from 1.8 ± 0.7 for TMs from wild-type mice to 1.0 ± 0.1 for those from Col11a2 –/– mice. These results show that the organization of collagen into radial fibrils is responsible for the mechanical anisotropy of the TM. This anisotropy can be attributed to increased mechanical coupling provided by the collagen fibrils. Mechanisms by which changes in TM material properties may contribute to the threshold elevation in Col11a2 –/– mice are discussed.

Tympanic Membrane Collagen Fibers: A Key to High‐Frequency Sound Conduction

To investigate the significance of tympanic membrane collagen fiber layers in high frequency sound transmission. Human cadaver temporal bone study. Laser Doppler vibrometry was used to measure stapes footplate movement in response to acoustic stimulation. The tympanic membrane was altered by creating a series of slits and applying paper patches to isolate the effects of specifically oriented collagen fibers. Three groups of membrane alterations were evaluated: 1) circumferentially oriented slits involving each quadrant to primarily disrupt radial fibers, made sequentially within superior-anterior, inferior-anterior, inferior-posterior, and superior-posterior quadrants; 2) the same slits made in the reverse order; and 3) radially oriented slits from the umbo to the annulus to primarily disrupt circumferential fibers. For each group, measurements of the middle-ear cavity pressure, ear canal pressure, and stapes velocity were made each time the tympanic membrane was altered. Regardless of the order in which the circumferentially oriented slits were made, there was a consistent decrease in stapes velocity above 4 kHz for the third and fourth cuts compared to the control. The mean decrease in the range of 4 to 12.5 kHz was 11 dB for the third patched slit and 14 dB for the fourth patched slit (P < .01). Radially oriented slits appear to produce smaller effects. Radial collagen fibers in the tympanic membrane play an important role in the conduction of sound above 4 kHz.

The acute structural changes of loaded articular cartilage following meniscectomy or ACL-transection

Objective Meniscectomy and anterior cruciate ligament (ACL) rupture have been identified as precursors of osteoarthrosis (OA) in clinical reviews and animal experiments. In this study, the acute effects of these injuries on articular cartilage matrix deformation, preserved in a loaded state using a cryopreservation technique, were studied by scanning electron microscopy (SEM).Method Whole knee joints from adult White New Zealand rabbits (N=87) were loaded ex vivo, using a simulated quadriceps pull under static and cyclic loading conditions, following medial meniscectomy or transection of the ACL. Specimens were plunge-frozen while under load, or following a recovery period, and prepared for SEM by cryofixation. Using SEM and photographic images, the medial tibial plateau cartilage was assessed both qualitatively and quantitatively.Results After meniscectomy, significantly increased bending and crimping of radial collagen fibers occurred with static loading. Compared to intact knees, the area of tibial cartilage showing an indentation was increased by 80% (P< 0.05), the articular cartilage thickness was significantly more reduced when under load (for high force long duration static loading, intact joints had 53%±3 reduction in cartilage thickness compared to 39%±4 after meniscectomy, P< 0.05), and it took nearly twice as long for the cartilage thickness to recover following loading. These post-meniscectomy differences were either not present or were minimal when the joint was allowed to extend when loaded. ACL-transection slightly increased collagen deformation in the deeper zones, but only with cyclic loading.Conclusion The findings indicate that, with static loading, significantly increased deformation of articular cartilage collagen structure can occur following meniscectomy, but is minimized by joint motion. This increased deformation may be relevant to the etiology and progression of joint degeneration.

Maturation of tympanic membrane layers and collagen in the embryonic and post-hatch chick (Gallus domesticus)

The surface area of the chick (Gallus domesticus) tympanic membrane (TM) increases by as much as 400% from hatching to 70 days of age (Cohen et al. [1992] J. Morphol. 212:187-193). The present study is concerned with the processes that contribute to this remarkable size increase. Middle-ear specimens were harvested in embryos aged between E10 and E18, and in post-hatch animals between days P1 and P57. Specimens were embedded in paraffin, cut in serial sections, stained for collagen fibers, and examined with light microscopy. Four locations were examined in each specimen: the anterior and posterior perimeter of the TM, the TM, over the extra-stapedius, and the TM at the tip of the extra-columella. The thickness of the epithelial, respiratory, and lamina propria layers was measured at each location. The radial collagen fibers in the lamina propria were also counted at each location, and fiber density per square micrometer was determined at each age. Thickness of the epithelial and respiratory layers remains relatively constant throughout development in all areas of the TM, whereas the lamina propria at the extra-columella and extra-stapedius continues to thicken with increasing age. Collagen density also increases during development, and this is attributed to an increase in fiber number and a reduction in the space between fibers. The results suggest that collagen may be synthesized first in the central regions of the TM and then later in more peripheral areas of the TM.

Maturation of tympanic membrane layers and collagen in the embryonic and post‐hatch chick (Gallus domesticus)

The surface area of the chick (Gallus domesticus) tympanic membrane (TM) increases by as much as 400% from hatching to 70 days of age (Cohen et al. [1992] J. Morphol. 212:187–193). The present study is concerned with the processes that contribute to this remarkable size increase. Middle-ear specimens were harvested in embryos aged between E10 and E18, and in post-hatch animals between days P1 and P57. Specimens were embedded in paraffin, cut in serial sections, stained for collagen fibers, and examined with light microscopy. Four locations were examined in each specimen: the anterior and posterior perimeter of the TM, the TM over the extra-stapedius, and the TM at the tip of the extra-columella. The thickness of the epithelial, respiratory, and lamina propria layers was measured at each location. The radial collagen fibers in the lamina propria were also counted at each location, and fiber density per square micrometer was determined at each age. Thickness of the epithelial and respiratory layers remains relatively constant throughout development in all areas of the TM, whereas the lamina propria at the extra-columella and extra-stapedius continues to thicken with increasing age. Collagen density also increases during development, and this is attributed to an increase in fiber number and a reduction in the space between fibers. The results suggest that collagen may be synthesized first in the central regions of the TM and then later in more peripheral areas of the TM. J. Morphol. 233:257–266, 1997. © 1997 Wiley-Liss, Inc.

Scanning electron microscopy of "fibrillated" and "malacic" human articular cartilage: technical considerations.

Specimens of articular cartilage from human knees with gross evidence of malacia (dull appearance and/or softness) or fibrillation (exposed fibrous strands and/or staining with India ink) were prepared for scanning electron microscopy (SEM) and compared to cartilage from apparently intact regions. Vertical cryofractures were made through the center of each specimen, so the matrix collagen structure and its relationship to surface features could be examined. Soft, dull, malacic cartilage was characterized by the presence of numerous clefts among the collagen fibers within the most superficial region of the cartilage. In one form of this condition, these clefts did not extend through the articular surface. In a second form, usually observed where the tangential zone was normally thin or absent, the free ends of radial collagen fibers were exposed, but the deeper layers were intact. Two forms of fibrillation were also identified. The first is created by separation of the superficial lamellae which curl up from the tangential layer and form frondlike projections above the normal plane of the joint surface. In the second, deep radial fibers are exposed by vertical fissures. This second form is associated with advanced damage to the joint. The early stages of cartilage failure are characterized by debonding among the major collagen fiber tracts. This process may initiate in the deep tangential zone where the radial fibers cross into the surface. The patterns of the degenerative changes are dictated by the original architecture of the collagen matrix. The microscopic findings do not correlate adequately with conventional gross grading. SEM provides useful information about injured articular cartilage.

Deformation of loaded articular cartilage prepared for scanning electron microscopy with rapid freezing and freeze-substitution fixation.

To investigate the effect of joint loading on collagen fibers in articular cartilage, 45 knees of adult rabbits were examined by scanning electron microscopy. The knees were loaded at the patella with a simulated "quadriceps force" of 0.5-4 times body weight for 0.5 or 25 minutes, plunge-frozen, and fixed by freeze-substitution with aldehydes. Six knees were loaded for 3 hours and then fixed conventionally. Fixed tibial plateaus were examined and then freeze-fractured through the area of tibiofemoral contact, dried, coated, and examined by scanning electron microscopy to assess the overall deformation of the tibial articular surface and matrix collagen fibers. With tissue prepared by conventional fixation used as a standard, the quality of fixation was graded by light and transmission electron microscopy of patellar cartilage taken from half of the freeze-fixed knees. In loaded specimens, an indentation was present where the femur contacted the tibial plateau. The diameter and apparent depth of the dent were proportional to the magnitude and duration of the load; no dent was seen in the controls. The thickness of the cartilage at the center of the indentation was reduced 15-80%. Meniscectomy always produced larger deformations in otherwise equivalent conditions. Icecrystal damage to cells was evident by transmission electron microscopy and scanning electron microscopy, but at magnifications as high as x 30,000 the collagen fibrils prepared by freeze-substitution and conventional aqueous methods were identical. In loaded regions, the collagen matrix of the tibial cartilage was deformed in two ways: (a) radial collagen fibers exhibited a periodic crimp, and (b) in regions where an indentation was created by the femoral condyle, the radial fibers were bent, in effect creating a tangential zone where none had existed before. The radial fibers apparently are loaded axially and buckle under normal loads.