Different crimp patterns in collagen fibrils relate to the subfibrillar arrangement.

Collagen fibril ultrastructure and course were examined in different connective tissues by PLM, SEM, TEM, and AFM. In tendons, collagen fibrils were large and heterogeneous with a straight subfibrillar arrangement. They ran densely packed, parallel, and straight changing their direction only in periodic crimps where fibrils showed a local deformation (fibrillar crimps). Other tissues such as aponeurosis, fascia communis, skin, aortic wall, and tendon and nerve sheaths showed thinner uniform fibrils with a helical subfibrillar arrangement. These fibrils appeared in parallel or helical arrangement following a wavy, undulating course. Ligaments showed large fibrils as in tendon, with fibrillar crimps but less packed. Thinner uniform-sized fibrils also were observed. Fibrillar crimps seem to be related to the subfibrillar arrangement being present only in large fibrils with a straight subfibrillar arrangement. These stiffer fibrils respond mainly to unidirectional tensional forces, whereas the flexible thinner fibrils with helical subfibrils can accommodate extreme curvatures without harm, thus responding to multidirectional loadings.

Periarticular ligament changes following ACL/MCL transection in an ovine stifle joint model of osteoarthritis.

Anterior cruciate ligament (ACL) injuries often lead to significant functional impairment, and are associated with increased risk for induction of degenerative joint disease. However, few studies have described the effect of ligament transection on the remaining intact knee ligaments. This study sought to determine specifically what impact combined ACL/medial collateral ligament (MCL) transection had on the remaining intact knee ligaments, particularly from the histological, biochemical, and molecular perspectives. Twenty weeks post-ACL/MCL transection, the cut ends of sheep MCLs were bridged by scar, while the posterior cruciate ligaments (PCLs) and lateral collateral ligaments (LCLs) seemed gross morphologically normal. Water content and cell density increased significantly in the MCL scars and the intact PCLs but were unchanged in the LCLs. Collagen fibril diameter distribution was significantly altered in both MCL scar tissue and uninjured PCLs from transected joints. MMP-13 mRNA levels in MCL scars and PCLs from ligament transected joints were increased, while TIMP-1 mRNA levels were significantly decreased in the PCLs only. This study has shown that some intact ligaments in injured joints are impacted by the injury. The joint appears to behave like an integrated organ system, with injury to one component affecting the other components as the "organ" attempts to adapt to the loss of integrity.

Effect of GDF-5 on ligament healing.

The effects of growth and differentiation factor-5 (GDF-5) on ligament healing were studied using a gap injury model of the medial collateral ligament in rat knee joints. The administration of GDF-5 once at the time of surgery significantly improved the mechanical properties of the femur-ligament-tibia complex. At 3 weeks after surgery, 30 microg of GDF-5 improved the ultimate tensile strength of the complex by 41%, and the stiffness by 60%, compared with the vehicle control (p < 0.05 for both; Fisher's PLSD test). The observation with a transmission electron microscopy revealed that GDF-5 increased the diameter of collagen fibrils in the repair tissue, which was considered to be a possible mechanism for the positive result in the biomechanical testing. Quantitative PCR and in situ hybridization revealed enhanced type I procollagen expression by GDF-5, and the PCR analysis also revealed that the GDF-5 treatment reduced the expression of type III procollagen relative to type I procollagen. The PCR analysis further showed that the expression of decorin and fibromodulin was relatively reduced against type I procollagen by the growth factor, which was considered to be responsible for the increase of collagen fibril diameter in the repair tissue. No adverse effects were observed, and the use of GDF-5 was considered a promising approach to facilitate ligament healing.

Ultrastructural morphometry of anterior cruciate and medial collateral ligaments: an experimental study in rabbits.

This study presents morphometric analyses of collagen subfascicle area fraction and collagen fibril diameter distributions for the anterior cruciate (ACL) and medial collateral (MCL) knee ligaments from transmission electron micrographs of ligament cross sections of five mature, female New Zealand White rabbits. Statistically significant differences in subfascicular area fractions were found between the ACL and MCL (0.89 +/- 0.02, 0.97 +/- 0.01, respectively; p less than 0.001). Mean fibril diameters for the ACL and MCL were also significantly different (0.059 +/- 0.005, 0.085 +/- 0.011 microns, respectively; p less than 0.025). Fibril eccentricity (a measure of parallel alignment of collagen fibrils within the ligaments, defined as the ratio of minor to major axes of elliptical fibril outlines) was 0.89 +/- 0.03 and 0.85 +/- 0.08, respectively, for the ACL and MCL; these data were not significantly different (p greater than 0.1). The relative amount of variation in the pooled fibril diameter data due to variation between animals, ligaments, locations within ligaments, and among fibrils at individual locations are reported. The variation of fibril diameter distributions between the ACL and MCL was substantially greater than the variation between different locations within each ligament cross section as well as between different animals. The structural differences reported may help explain known differences in the biomechanical properties of the ACL and MCL.

Microscopical investigation of canine anterior cruciate ligament and patellar tendon: collagen fascicle morphology and architecture.

The collagen structure of the canine anterior cruciate ligament (ACL) and patellar tendon (PT) was examined by using light and scanning electron microscopy. The collagen waviness known as a crimping was found to occur in ACL and PT fascicles. This waviness, seen at the periphery of fascicles, is very smooth, and its amplitude seems to decrease from the periphery toward the fascicular center. It appears as a periodic collapse of the fascicle in two dimensions. Two models of the architectural patterns of the ACL and PT wavy fascicles are presented. The constituent collagen fibrils are either parallel or twisted relative to the fascicle axis, giving rise to planar and helical wave patterns, respectively. There is a distinct difference between the ACL and PT collagen structure. The helical wave pattern occurs in both PT and ACL while the planar waveform is found only in the centrally located ACL fascicles. In addition, there is less variability in fascicular size and density over the PT cross-section than in ACL.

A quantitative analysis of matrix alignment in ligament scars: a comparison of movement versus immobilization in an immature rabbit model.

This investigation quantified the alignment of fibrillar matrix in normal rabbit medial collateral ligaments (MCLs) and in healing MCLs from animals treated with or without knee immobilization. Twenty-four immature female rabbits were given complete midsubstance injuries to their right MCLs. Fifteen of them had that knee pin immobilized in flexion, while the remaining nine were allowed unrestricted cage activity. Animals were sacrificed in groups of three at intervals of 3, 6, or 14 weeks after injury, and both healing MCLs and unoperated contralateral controls were fixed in situ for subsequent removal, freeze-fracture, and preparation for scanning electron microscopy (SEM). A random sampling of SEM photographs followed by automated, statistically validated image processing was used to quantify alignment of matrix in all samples. Results showed that nonimmobilized MCL scars in this model do remodel over 14 weeks of healing, returning to normal alignment values in that time. Surprisingly, MCL scars in immobilized knees were even better, with mean matrix alignments falling statistically within normal MCL limits at all healing intervals studied. If not due to an unknown sampling or fixation artifact, these results suggest that gross knee flexion and extension is not a prerequisite for scar matrix alignment in this immature model of ligament healing.

Rigid immobilization alters matrix organization in the injured rat medial collateral ligament.

The effects of mobilization on matrix reorganization and density after ligament injury were studied in rat medial collateral ligaments using scanning electron microscopy (SEM). Both medial collateral ligaments of 14 Sprague-Dawley rats were sharply incised transversely at their midpoint. A 1.14-mm threaded Kirschner wire was driven through the tibia and into the femur of the right leg (through the knee) to immobilize that knee at 90 degrees of flexion. Four additional rats were used as controls. The right medial collateral ligament of the control rats was exposed in the same manner as the experimental rats and the wound closed without damaging the ligament. Rats were sacrificed on the 7th and 14th days postinjury and the ligaments evaluated by SEM. The electron micrographs from this study demonstrated that early on, the tissue at the injury site is disorganized on a gross scale with large bundles of poorly organized matrix. Large "defects" were present between bundles in the substance of the ligament and appeared as holes in the ligament around the injury site. As healing progressed, the matrix in the mobilized specimens appeared to bridge the injury site more rapidly and completely with fewer "defects" and thus higher density than the immobilized specimens.

The interrelation of fiber bundles in the anterior cruciate ligament.

The anterior cruciate ligaments (ACL) of dogs, humans, and rabbits were studied by light and scanning electron microscopy after fixation in situ. In all species, the ACL was composed of multiple 20 microns-wide collagen fiber bundles separated by columns of cells in fibrous capsules. These bundles were in turn grouped into fascicles of varied size. The fascicles were surrounded by thin membranous sheets that ran through the ligament forming single or multiple layers between fascicles. Splaying of the ACL at insertion was created by increased volume in the cellular intervals. Bending of the fiber bundles occurred in this region--which corresponds to the fibrocartilaginous zone. We propose that the cell layers accommodate compressive forces and the membranes allow slipping among fascicles without compromising blood supply.

Cytochemical evidence for a proteoglycan-associated filamentous network in ligament extracellular matrix.

The purpose of this investigation was to examine the extracellular matrix of rabbit ligament before and after digestion with glycosaminoglycan degrading enzymes. In order to preserve and enhance the visibility of negatively charged tissue components, particularly the glycosaminoglycan-containing proteoglycans, the cationic stains ruthenium red (RR) and ruthenium hexamine trichloride (RHT) were used. Cross-sections of the midsubstance of 10-month-old (mature) rabbit medial collateral ligaments fixed using conventional procedures revealed a sparse population of stellate-shaped cells that did not appear to be interconnected. Similar tissue fixed in either RR or RHT showed an extensive network of thin, electron-dense "seams" that interconnected cells and appeared to irregularly subdivide the extracellular matrix (ECM). These seams mainly consisted of a meshwork of microfilaments throughout which small granules were dispersed. Numerous 14-nm microfibrils, as well as mature elastic fibers were also present within the seams. The size and shape of the microfilaments, together with their threadlike, beaded appearance suggested that they could be Type VI collagen. The seam granules were easily removed with chondroitinase ABC, chondroitinase AC II, and mild (0.18 M) salt treatment. Only chondroitinase ABC succeeded in removing additional granules, tentatively identified as proteodermatan sulphate molecules, that were periodically located at d band sites along the Type I collagen fibrils. These results suggest that the seam granules are not dermatan sulphate containing proteoglycans, and further, that these proteoglycans may be sequestered into specific zones within the ECM through loose association with the seam microfilaments. While the functional significance of the seams remains unknown and their specific composition clearly requires further study, it is likely that they represent important functional (e.g., viscoelastic) or biological (e.g., nutritional) subdivisions of ligament substance.

Ultrastructure of early calcification in cervical ossification of the posterior longitudinal ligament.

A case of ossification of the cervical posterior longitudinal ligament was investigated with the electron microscope. The posterior longitudinal ligament was composed of bundles of collagen fibers intermingled with occasional fibroblasts and rare blood vessels. Some ligaments contained matrix vesicles in the vicinity of degenerated cells. Hydroxyapatite crystals were frequently precipitated within the matrix vesicles. These findings are similar to the fine structure of the early stage of calcification in normal and pathological calcifying tissues described previously. In this study, the calcification process of the posterior longitudinal ligament suggests that matrix vesicles originate from degenerated cells, and acquire hydroxyapatite crystal deposits. Some eventually coalesce to form a large calcifying mass. Substantial amounts of collagen fibers comprising the ligament may serve an important role in orienting apatite crystal precipitation.

Tension studies of human knee ligaments. Yield point, ultimate failure, and disruption of the cruciate and tibial collateral ligaments.

Ultimate failure strengths of human tibial collateral and anterior and posterior cruciate ligaments were determined at two different loading rates (12.5 and fifty centimeters per minute) using an Instron Tension Analyzer. The posterior cruciate ligament was significantly stronger than the tibial collateral and anterior cruciate ligaments, which were of equal strength. At ultimate failure the ligaments were intact macroscopically but electron microscopy revealed widespread disruption of the collagen fibrils. Only after further application of stress did actual macroscopic disruption occur, suggesting that microscopic failure of the collagen fibrils in grossly intact ligaments may be a significant cause of clinical instability.

A comparative evaluation of the mechanical properties of the rabbit medial collateral and anterior cruciate ligaments.

The biomechanical properties of the medial collateral and anterior cruciate ligaments from 30 New Zealand White rabbits were measured. Because of its complex geometry, the ACL was divided into two portions (medial and lateral) to provide uniform loading. This allowed an examination of the intra-ligamentous properties. A laser micrometer system was used to measure the cross-sectional area for tensile stress and a video dimension analyzer was used to measure the strain. The mechanical properties (stress-strain curves) of the MCL and ACL were different, with the modulus (determined between 4 and 7% strain) in the MCL (1120 +/- 153 MPa) more than twice that of either portion of the ACL (516 +/- 64 and 516 +/- 69 MPa for the medial and lateral portions, respectively). This higher modulus correlated with the more uniform and dense appearance of the collagen fibrils examined with scanning electron microscopy (SEM).

The ultrastructure of sensory nerve endings in human anterior cruciate ligament.

The sensory innervation of the anterior cruciate ligament (ligamentum cruciatum anterius) of the human knee joint was studied by light- and electron microscopy. The connective tissue between the synovial membrane and the cruciate ligament contains small Ruffini corpuscles and lamellar corpuscles with several inner cores. The connective tissue septa between the individual fascicles of the cruciate ligament contain Ruffini corpuscles and free nerve endings. The free nerve endings are innervated by C-fibres and myelinated A-delta fibres. The afferent axons of Ruffini corpuscles are myelinated and measure 4-6 microns in diameter, those of the lamellar corpuscles with several inner cores measure about 6 microns in diameter. It is discussed, whether these receptors of the anterior cruciate ligament may influence the muscle tone via polysynaptic reflexes.

Lesions of the alar ligaments. In vivo and in vitro studies with magnetic resonance imaging.

STUDY DESIGN: This study analyzed anatomic characteristics of the alar ligaments and the possibility of imaging them with magnetic resonance imaging. Also determined was whether artificial ruptures of the alar ligament can be recognized experimentally. OBJECTIVE: To determine the ability of magnetic resonance imaging to visualize normal, torn, resected alar ligaments. SUMMARY OF BACKGROUND DATA: There are no studies about computed tomography or magnetic resonance imaging findings of alar ligaments and after anatomic sections. Direct visualization of the complete ligament is not possible for computed tomography. No precise diagnostic method for showing a ruptured alar ligaments has been described. Magnetic resonance imaging seems to be the method of choice for distinguishing between normal and pathologic soft tissue. METHODS: Fifteen specimens from accident victims underwent anatomic dissection. In addition, ligaments from three groups were examined: 1) eight volunteers, 2) seven patients, and 3) 17 fresh cadaveric specimen before anatomic exploratory dissection. In seven of these specimens, one ligament was cut to simulate an artificial disruption and magnetic resonance imaging was repeated. RESULTS: Lesions of the alar ligaments were found in four of 15 prepared specimens. Using magnetic resonance imaging, the alar ligaments could be identified in all volunteers, patients, and specimen except one. No ruptures were found in the 17 specimens. Of the seven resected specimens, all cuts could be demonstrated by magnetic resonance imaging. CONCLUSION: Magnetic resonance imaging is useful for showing lesions of the alar ligaments because of a high soft tissue contrast, plane independence imaging, possibility of functional scans, and secondary reconstruction from three-dimensional data sets.

The biomechanics of lateral knee bracing. Part I: Response of the valgus restraints to loading.

To better understand the role of preventive knee braces in injury prevention, a biomechanical study using fresh frozen cadaveric knees (N = 18) was conducted. Ligament tensions and joint displacements were measured at static, nondestructive valgus forces as well as low-rate destructive forces. After quantifying and establishing individual ligament contributions to valgus restraining function, knees were then braced with two different laterally applied preventive braces, the McDavid Knee Guard and the Omni Anderson Knee Stabler. The effects of lateral bracing were analyzed according to valgus force, joint line opening, and ligament tensions. Valgus applied forces, with or without braces, consistently produced medial collateral ligament (MCL) disruptions at ligament tensions surprisingly higher than the anterior cruciate ligament (ACL) and higher than or equal to the posterior cruciate ligament (PCL). Although large joint displacements were necessary for complete ligament failure, bundle disruption in the MCL, ACL, and PCL was noted at much smaller joint openings. In Part I of this study, no significant protection could be documented with the two preventive braces used. Also, four potentially adverse effects were noted: MCL preload, center axis shift, premature joint line contact, and brace slippage.

The human posterior cruciate ligament complex: an interdisciplinary study. Ligament morphology and biomechanical evaluation.

To study the structural and functional properties of the human posterior cruciate ligament complex, we measured the cross-sectional shape and area of the anterior cruciate, posterior cruciate, and meniscofemoral ligaments in eight cadaveric knees. The posterior cruciate ligament increased in cross-sectional area from tibia to femur, and the anterior cruciate ligament area decreased from tibia to femur. The meniscofemoral ligaments did not change shape in their course from the lateral meniscus to their femoral insertions. The posterior cruciate ligament cross-sectional area was approximately 50% and 20% greater than that of the anterior cruciate ligament at the femur and tibia, respectively. The meniscofemoral ligaments averaged approximately 22% of the entire cross-sectional area of the posterior cruciate ligament. The insertion sites of the anterior and posterior cruciate ligaments were evaluated. The insertion sites of the anterior and posterior cruciate ligaments were 300% to 500% larger than the cross-section of their respective midsubstances. We determined, through transmission electron microscopy, fibril size within the anterior and posterior cruciate ligament complex from the femur to the tibia. The posterior cruciate ligament becomes increasingly larger from the tibial to the femoral insertions, and the anterior cruciate ligament becomes smaller toward the femoral insertion. We evaluated the biomechanical properties of the femur-posterior cruciate ligament-tibia complex using 14 additional human cadaveric knees. The posterior cruciate ligament was divided into two functional components: the anterolateral, which is taut in knee flexion, and the posteromedial, which is taut in knee extension. The anterolateral component had a significantly greater linear stiffness and ultimate load than both the posteromedial component and meniscofemoral ligaments. The anterolateral component and the meniscofemoral ligaments displayed similar elastic moduli, which were both significantly greater than that of the posteromedial component.

Histological response to carbon fibre.

The soft tissue response to carbon fibre was studied histologically one and a half years after being used to reconstruct the lateral collateral ligament of the human knee. A remarkably consistent pattern was seen in the induced ligament. The basic pattern was a "composite unit", consisting of a core of carbon fibre enveloped in a concentric manner by coherent layers of fibroblasts and collagen fibres. This new structure seemed to have been induced by continuous irritation caused by the physical structure of the carbon fibres; it is unlikely ever to acquire the structure of a natural ligament. However, it is biologically compatible and is biomechanically sufficient as long as the entire tow of carbon fibres is preserved.

Physical characteristics of the axial interosseous ligament of the human sacroiliac joint.

BACKGROUND CONTEXT: Although the sacroiliac joint has occupied a place in medical literature since at least the eighteenth century, its role in normal function and dysfunction of the back and hip remains controversial. The controversy persists, because there is still no suitable method to study the role of stability and mobility at the sacroiliac joints in vivo. One cost-effective approach to understanding such complex, deeply placed structures is biomechanical modeling. Unfortunately, very few data on the mechanical properties of tissues in this region are currently available to modelers. PURPOSE: The objective of this preliminary project was to determine some mechanical properties of the axial interosseous ligament (AIL), and to investigate the histology of the ligament. STUDY DESIGN/SETTING: A modified split plot design was used in conjunction with descriptive statistics to identify AIL characteristics. PATIENT SAMPLE: This was a cadaveric study. OUTCOME MEASURES: The study used descriptive statistics to categorize the ultimate failure strength of the AIL, and to describe the macro-constituents of the ligament. METHODS: Eighteen sacroiliac joints were harvested from nine fresh female cadavers (age range, 54 to 92). Data from 10 joints submitted to mechanical testing are reported. The eight remaining joints were used for histologic analysis of the AIL by light microscopy. RESULTS: The AIL proved to be relatively weak, with a mean failure load of 381 N (SD 43.7N) and a peak stress of 1.73 MPa (SD 0.99 MPa). Histologically, the AIL contained significantly less collagen than most other "typical" ligaments. CONCLUSIONS: The AIL failure loads are just slightly higher than those for the ligamentum flavum in the spine, a tissue composed mainly of elastic fibers. In contrast, the AIL has negligible elastin content. Because the AIL represents about 14% of the total area of interosseous sacroiliac ligaments, its mechanical properties should be useful to modelers of the joint. In addition, it appears that injury to the AIL would do little to compromise the mechanical integrity of the sacroiliac joint. Further study of this ligament seems warranted.

The annular ligament attachment to the normal human stapes footplate. A scanning electron microscopic study.

Following disarticulation of the stapes from the oval window, the free edge of the adult human stapes was examined with the scanning electron microscope. Regions of the articulating surface of the rim were examined and found to vary considerably. Anterior, posterior, and lateral ligaments are described, each with tympanic, intermediate, and lateral components.

Thin histological sections prepared from large thick sections: a new technique.

A method is described for obtaining thin (1 microm) sections for light microscopy from large area thick (100 microm) sections of low viscosity nitrocellulose embedded specimens of human spinal osteoligamentous material.