Contribution of glycosaminoglycans to the microstructural integrity of fibrillar and fiber crimps in tendons and ligaments.

The biomechanical roles of both tendons and ligaments are fulfilled by the extracellular matrix of these tissues. In particular, tension is mainly transmitted and resisted by protein (collagen, elastin) fibers, whereas compression is opposed by water-soluble glycosaminoglycans (GAGs). GAGs spanning the interfibrillar spaces and interacting with fibrils through the interfibrillar proteoglycans also seem to play a part in transmitting and resisting tensile stresses. Both tendons and ligaments showing similar composition, but different functional roles and collagen array, exhibit periodic undulations of collagen fibers or crimps. Each crimp is composed of many knots of each single fibril or fibrillar crimps. Fibrillar and fiber crimps play a mechanical role in absorbing the initial loading during elongation of both tendons and ligaments, and in recoiling fibrils and fibers when tissues have to return to their original length. This study investigated whether GAGs covalently attached to proteoglycan core proteins directly affect the 3D microstructural integrity of fibrillar crimp regions and fiber crimps in both tendons and ligaments. Achilles tendons and medial collateral ligaments of the knee from eight female Sprague-Dawley rats (90 days old) incubated in a chondroitinase ABC solution to remove GAGs were observed under a scanning electron microscope (SEM). In addition, isolated fibrils of these tissues obtained by mechanical disruption were analyzed by a transmission electron microscope (TEM). Both Achilles tendons and medial collateral ligaments of the rats after chemical or mechanical removal of GAGs still showed crimps and fibrillar crimps comparable to tissues with a normal GAG content. All fibrils in the fibrillar crimp region always twisted leftwards, thus changing their running plane, and then sharply bent, changing their course on a new plane. These data suggest that GAGs do not affect structural integrity or fibrillar crimp functions that seem mainly related to the local fibril leftward twisting and the alternating handedness of collagen from a molecular to a supramolecular level.

Morphological, biochemical and mechanical features of the cranial cruciate and lateral collateral ligaments in dogs.

The cruciate ligament and the collateral ligament play key roles in stabilization of the knee joint. Cases of serious knee joint problems presented at the, the Veterinary Teaching Hospital of Rakuno Gakuen University, Japan mostly involved rupture of the cranial cruciate ligament (CCL). Disorders in structural and biochemical components of the CCL were thought to be the causes of the knee problems. Morphological, biochemical and biomechanical features of the CCL and the lateral collateral ligament (LCL) were therefore analyzed. In the CCL, fibroblasts with ovoid and enlarged nuclei were observed mainly at the periphery of collagen bundles. The array of collagen fibrils in the LCL was slightly disoriented, but that of the CCL was tight and regular. In the LCL, the major groups of collagen fibrils were those with diameters of 70-80 and 120-130 nm. Most collagen fibrils in the CCL had diameters of 70-80 nm. The mean collagen diameters were 90 nm in the CCL and 105 nm in the LCL. The ratios of the noncollagen area to the area occupied by collagen fibrils were 43% in the CCL and 55% in the LCL. There was no difference between the amounts of HA or between the amounts of DS in two ligaments. However, the amount of CS in the CCL was about 17-times greater than that in the LCL. The expansion of and the resistance to tension exerted onto the CCL were less than those of the LCL. A high concentration of CS and low tensile strength due to small-sized collagen fibrils cause the CCL to rupture easily, especially when overextension of the knee joint occurs.

Proteoglycans and catabolic products of proteoglycans present in ligament.

The aim of the present study was to characterize the proteoglycans and catabolic products of proteoglycans present in the tensile region of ligament and explant cultures of this tissue, and to compare these with those observed in the tensile region of tendon. Approx. 90% of the total proteoglycans in fresh ligament was decorin, as estimated by N-terminal amino acid sequence analysis. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V(0) and/or V1 and/or V2) and aggrecan. Approx. 23% of decorin detected in the matrix was degraded. Intact decorin and decorin fragments similar to those observed in the matrix that retained the N-terminus were also observed in the medium of ligament cultures. Intact biglycan core protein was detected in the matrix and medium of ligament cultures, and two fragments originating from the N-terminal region of biglycan were observed in the matrix of cultured ligament. Versican and versican fragments that retained the N-terminus of versican core protein were detected in fresh matrix and medium of tendon cultures. Approx. 42% of versican present in the fresh ligament was degraded. Aggrecan catabolites appearing in the culture medium were derived from aggrecanase cleavage of the core protein. An intact link protein and a degradation product from the N-terminal region of type XII collagen were also detected in the medium of the ligament explant.

Autonomic innervation of tendons, ligaments and joint capsules. A morphologic and quantitative study in the rat.

We analyzed the neuronal occurrence of autonomic transmitters; noradrenaline (NA), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), in the Achilles tendon, medial and lateral collateral ligaments and knee joint capsule in the rat--by immunohistochemistry (IHC). In addition, the tissue concentrations of the sympathetic neuropeptide, NPY, and the parasympathetic peptide, VIP, were determined by radioimmunoassay (RIA). IHC demonstrated nerve fibers containing sympathetic vasoconstrictors--NA and NPY--and the parasympathetic vasodilator, VIP, in all tissues. NPY- and NA-positive nerve fibers were predominantly observed in larger blood vessels, whereas, nerve fibers immunoreactive to VIP were found in smaller vessels. In many nerve fibers a co-localization of the transmitters was seen. RIA showed that the concentration of NPY compared to VIP was 15-times higher in ligaments and twice as high in tendons and capsules. The differences noted may reflect a difference in vulnerability to degenerative conditions. In pathological conditions, dysregulation of autonomic transmitters in hypovascularized tissues subjected to repetitive mechanical load may contribute to tissue hypoxia leading to degeneration and rupture of tendons and ligaments.

Sensory neuropeptidergic pattern in tendon, ligament and joint capsule. A study in the rat.

The normal occurrence of sensory neuropeptides in tendons, ligaments and joint capsules in the rat was analyzed by immunohistochemistry and radioimmunoassay (RIA). Nerve fibres immunoreactive to substance P (SP), calcitonin gene-related peptide (CGRP), neurokinin A, galanin and somatostatin were identified in the Achilles tendon as well as the collateral ligaments and joint capsule of the knee. The neuropeptidergic fibres were predominantly found in the epiligament and paratenon. However, SP- and CGRP-positive fibres were also seen in the proper ligament and tendon tissues. RIA showed higher concentrations of SP and CGRP in tendons than in ligaments and capsules. The morphological and quantitative data obtained on sensory neuropeptides in normal tendons, ligaments and joint capsules may be used as a reference for tissue analysis in painful and inflammatory conditions of the locomotor apparatus.

Characterization of a large chondroitin sulfate proteoglycan present in bovine collateral ligament.

Bovine collateral ligament synthesized a 35S-labeled large proteoglycan species which eluted with a Kav of approximately 0.27 on Sepharose CL-2B and contained only chondroitin sulfate chains with a molecular mass of approximately 32 kDa. Fluorography of the 35S-labeled core proteins derived from the large ligament proteoglycan revealed a broad range of molecular masses above approximately 200 kDa, which was of comparable size to the four major endogenous core protein bands derived from this proteoglycan detected with 5/6/3-B-3, an antibody directed against terminal unsaturated chondroitin-6-sulfate disaccharides. The core proteins derived from the large ligament proteoglycan exhibited immunoreactivity of 12/21/1-C-6, an antibody specific for a peptide epitope common to both the G1 and G2 domains of aggrecan. Four major core protein bands with molecular masses greater than approximately 200 kDa derived from the large ligament proteoglycan, were detected using the antibodies raised against versican from bovine aorta or human fibroblasts. Compared with aggrecan, the 35S-labeled large ligament proteoglycan was distributed over a broader range of buoyant densities in an associative caesium chloride density gradient. This polydispersity may be indicative of differences in the degree of glycosylation as well as heterogeneity in the size of the large ligament proteoglycan core proteins. The 35S-labeled large ligament proteoglycan also demonstrated the ability to form complexes with an aggrecan aggregate preparation, the majority of which could not be dissociated by the presence of HA10-50. These findings indicate that the large chondrotin sulfate proteoglycan synthesized by bovine collateral ligament may be a versican-like proteoglycan which exhibited the potential to form like protein-stabilized complexes.

Biochemical analysis of collagens at the ligament-bone interface reveals presence of cartilage-specific collagens.

The collagen fibrils of some ligaments attach to bone by passing through a zone that consists of nonmineralized and mineralized fibrocartilages. Very little, however, is known about the cells, the biochemical composition, the extracellular matrix organization, and function of these fibrocartilages. In this study, the collagens present in the fibrocartilages of the bovine medical collateral and anterior cruciate ligaments femoral attachments to bone were isolated, characterized, and their distribution at these sites was assessed by laser confocal microscopy. Types II, IX, and XI collagens were identified after pepsin digestion of the tissues in addition to the types I and V collagens. Immunoblotting using specific polyclonal antibodies confirmed the presence of types II and IX collagens at these sites. Immunofluorescence using confocal microscopy showed that type II collagen was prominent in the nonmineralized area and to a lesser extent in the mineralized zone of the insertion. Type IX collagen showed similar distribution as type II collagen. Type II collagen isolated from the ligament-bone interface contained half hydroxypyridinium cross-linking residues when compared to type II collagen isolated from articular cartilage of the same animals. These data indicate that the fibrocartilaginous zones at the ligament-bone interface are cartilaginous in nature. The cartilage collagens may play a role of anchoring the ligament to bone or the cartilage-like tissue may participate in the modulation of the mechanical stresses which are known to exist at the soft tissue-hard tissue interface.

Ultrastructural immunolocalization of type-VI collagen and chondroitin sulphate in ligament.

Immunological methods were used to determine the identity of the major components comprising a network of electron-dense seams (described by the authors in a previous work) within the extracellular matrix of medial collateral ligament (MCL) from humans and rabbits. Tissue obtained from MCL midsubstance was subjected to pre-embedding labelling with colloidal gold at the electron microscopic level with monoclonal antibodies (MAbs) against type-VI collagen and chondroitin sulphate (CS), before and after digestion with chondroitinase ABC and testicular hyaluronidase. Tissue labelled with anti-type-VI MAbs showed gold conjugates attached to the microfilamentous component of the seams both before and after enzyme digestion, which confirmed the identity of the beaded microfilaments as type-VI collagen. Treatment of the tissue with anti-CS MAbs resulted in labelling of undigested tissue only. In these treatments, gold particles were found attached to granules that were interspersed throughout the network of type-VI microfilaments. Both the granules and gold labels were absent from the network following enzyme digestion. Thin nonbeaded microfilaments that did not label with anti-type-VI MAbs also were present within the seams. The loss of these nonbeaded microfilaments following enzyme digestion suggested that they might represent strands of hyaluronan. The codistribution and sequestering of type-VI collagen and CS within discrete seams or channels suggests that these regions of the MCL midsubstance may contain higher concentrations of water than the surrounding dense fibrillar matrix.