A comparative study of the epiligament of the medial collateral and the anterior cruciate ligament in the human knee. Immunohistochemical analysis of collagen type I and V and procollagen type III.

BACKGROUND: Recent reports in rat models have shown that fibroblasts in the epiligament, an enveloping tissue of the ligament, are not static cells and play an important role during the early ligament healing of isolated grade III injury of the collateral ligaments of the knee. Fibroblasts produce collagen types I, III and V and infiltrate within the ligament body via the endoligament. In addition, similarities have been reported between the structure of the epiligament of the medial collateral ligament and anterior cruciate ligament of the knee in rat and in human. In line with the ascribed role of the epiligament tissue and the synthesis of these collagens and their role in ligament healing, the aim of this study was to determine their presence in the normal epiligament of the aforementioned ligaments in humans, to compare their differential expression and to present a novel hypothesis about the failure of healing of the anterior cruciate ligament in contrast to the medial collateral ligament. MATERIALS AND METHODS: We used samples from the mid-substance of the medial collateral and the anterior cruciate ligament of the knee joint, acquired from 12 fresh knee joints. Routine histological analysis was performed through hematoxylin and eosin stain, Mallory's trichrome stain and Van Gieson's stain. The immunohistochemical analysis was conducted using monoclonal antibodies against collagen type I and V and procollagen type III. The number of cells in the epiligament, endoligament and the ligament tissue was assessed quantitatively through a computerized system for image analysis NIS-Elements Advanced Research and Statistica software. RESULTS: Our observations revealed certain differences in the morphology of the epiligament, as well as variations in the expression of the investigated molecules. Expression of collagen type I was mostly low-positive (1+) in the epiligament and positive (2+) in the ligament tissue of both ligaments. Expression of procollagen type III was mostly positive (2+) in the epiligament and ligament tissue of the medial collateral ligament, low-positive (1+) in the epiligament and negative (0) in ligament tissue of the anterior cruciate ligament. Expression of collagen type V was predominantly low-positive (1+) in the epiligament and negative (0) in the ligament tissue of both ligaments. The immunoreactivity for all three molecules was always higher in the epiligament of the medial collateral ligament than that of the anterior cruciate ligament. CONCLUSIONS: The results of our study illustrate for the first time that fibroblasts in the human epiligament are indeed responsible for the synthesis of the main types of collagen participating in the early ligament healing, thus corresponding to previous data of the medial collateral ligament healing in animal models. The differences between the epiligament of the investigated ligaments could add a novel explanation for the failed anterior cruciate ligament healing.

Characterization of the anchoring morphology and mineral content of the anterior cruciate and medial collateral ligaments of the knee.

The manner in which ligament connects to bone remains an area of interest for researchers, bioengineers, and clinicians. Stable fixation of an anterior cruciate ligament (ACL) graft has been shown to be paramount to preventing excess anterior tibial translation and to restoring the normal kinematics of the knee joint. In this study, the surface area of attachment and the mineral characteristics of the ACL and medial collateral ligament (MCL) attachment sites were characterized to determine the factors that contributed to ligament attachment strength. Findings from this study indicated that the area of attachment of the ACL's insertion was significantly greater than the ligament's origin (95.8 mm(2) ± 21.5 vs. 73.2 mm(2) ± 16.2, P = 0.009). Additionally, the ACL was measured to have a greater surface area of attachment when compared with the MCL (84.5 mm(2) ± 18.8 vs. 58.2 mm(2) ± 23.8, P = 0.005); although, the MCL was observed to have a greater region of calcified fibrocartilage (CFC) than the ACL (533.0 µm ± 116.9 vs. 195.5 µm ± 36.6, P = 0.0003). No significant correlation was observed between the ligament's area of attachment and the thickness of the CFC region. Measurements of ash percent suggested that the boundary region, between the CFC and host bone, possessed the least mineral content for the three regions of interest. These data suggest that ligament attachment strength can be attributed to several factors, including the ligament's area of attachment, regional thickness, and mineral content of the CFC.

Effect of sulfated glycosaminoglycan digestion on the transverse permeability of medial collateral ligament.

Dermatan and chondroitin sulfate glycosaminoglycans (GAGs) comprise over 90% of the GAG content in ligament. Studies of their mechanical contribution to soft tissues have reported conflicting results. Measuring the transient compressive response and biphasic material parameters of the tissue may elucidate the contributions of GAGs to the viscoelastic response to deformation. The hypotheses of the current study were that digestion of sulfated GAGs would decrease compressive stress and aggregate modulus while increasing the permeability of porcine medial collateral ligament (MCL). Confined compression stress relaxation experiments were carried out on porcine MCL and tissue treated with chondroitinase ABC (ChABC). Results were fit to a biphasic constitutive model to derive permeability and aggregate modulus. Bovine articular cartilage was used as a benchmark tissue to verify that the apparatus provided reliable results. GAG digestion removed up to 88% of sulfated GAGs from the ligament. Removal of sulfated GAGs increased the permeability of porcine MCL nearly 6-fold versus control tissues. Peak stress decreased significantly. Bovine articular cartilage exhibited the typical reduction of GAG content and resultant decreases in stress and modulus and increases in permeability with ChABC digestion. Given the relatively small amount of GAG in ligament (<1% of tissue dry weight) and the significant change in peak stress and permeability upon removal of GAGs, sulfated GAGs may play a significant role in maintaining the apposition of collagen fibrils in the transverse direction, thus supporting dynamic compressive loads experienced by the ligament during complex joint motion.

Transversely isotropic distribution of sulfated glycosaminoglycans in human medial collateral ligament: a quantitative analysis.

Decorin and its associated glycosaminoglycan (GAG) side chain, dermatan sulfate (DS), play diverse roles in soft tissue formation and potentially aid in the mechanical integrity of the tissue. Deeper understanding of the distribution and orientation of the GAGs on a microscopic level may help elucidate the structure/function relationship of these important molecules. The hypothesis of the present study was that sulfated GAGs are aligned with transversely isotropic material symmetry in human medial collateral ligament (MCL) with the collagen acting as the axis of symmetry. To test the hypothesis, sulfated GAGs were visualized using transmission electron microscopy (TEM). Three orthogonal anatomical planes were examined to evaluate GAG distributions against symmetry criteria. GAG populations were differentiated using targeted enzyme digestion. Results suggest that sulfated GAGs including DS, chondroitin sulfates A and C, as well as other sub-populations assume transversely isotropic distributions in human MCL. Sulfated GAGs in the plane normal to the collagen axis were found to be isotropic with no preferred orientation. GAGs in the two planes along the collagen axis did not statistically differ and exhibited apparent bimodal distributions, favoring orthogonal distributions with over half at other angles with respect to collagen. A previously developed model, GAGSim3D, was used to interpret potential TEM artifacts. The data collected herein provide refined inputs to micro-scale models of the structure/function relationship of sulfated GAGs in soft tissues.

Treatment with bioscaffold enhances the the fibril morphology and the collagen composition of healing medial collateral ligament in rabbits.

Porcine small intestinal submucosa (SIS) was shown to be an effective bioscaffold in enhancing the mechanical properties of healing medial collateral ligaments (MCL). The purpose of this study was to investigate whether there are corresponding improvements in morphology and tissue compositions. Fourteen rabbits were equally divided into two groups. In the SIS-treated group, a 6 mm gap was surgically created in the right MCL and a layer of SIS was sutured covering the gap. For the nontreated group, the gap-injured MCLs remained untreated. All the left MCLs were sham operated and used as controls. At 12 weeks, the status of collagen types I and V was evaluated with immunofluorescent staining. The collagen type V/I ratios were obtained using SDS-PAGE. Collagen fibril diameters were calculated from the transmission electron micrographs. The results revealed that in the SIS-treated group, the collagen fibers were more regularly aligned as were the cell nuclei. The collagen fibril diameters were 22.2% larger and the ratio of collagen type V/I was 28.4% lower than those for the nontreated group (p < 0.05). These improvements in the morphological characteristics and biochemical constituents of healing MCLs following SIS treatment are the likely reasons for improved mechanical properties.

Indirect injury stimulates scar formation-adaptation or pathology?

In several animal models of osteoarthritis induced by cruciate ligament transection, a dense, scar-like tissue mass forms rapidly on the medial side of the knee joint. This mass mimics clinical fibrosis that sometimes occurs after joint surgery. It is unknown exactly why this medial tissue mass forms and what cells are involved in its formation. This study characterizes this medial mass by histology, biochemistry, and the expression of types I and III collagen mRNA. The medial mass is compared with the medial collateral ligament (MCL) and the MCL epiligament in anterior cruciate-transected and unoperated joints, and to normal skin and skin scar. The morphology of the medial mass resembled the epiligament and skin scar more than the MCL. The concentration of DNA and RNA and the RNA-DNA ratio were elevated dramatically in the medial mass compared with all other tissues including skin scar. However, the mRNA copy number and ratio of collagen types I and III mRNAs did not differ significantly among the medial mass, MCL, epiligament, and skin in either the control or the operated joints. The response of the medial mass, MCL, and MCL epiligament to cruciate transaction involves both hyperplasia and hypertrophy, but without a dramatic shift in cell phenotype. The medial mass may be a useful mimic or model of intraarticular adhesions, hypertrophic scars, ligament sprains, and arthrofibrosis.

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.

A role for calcitonin gene-related peptide in rabbit knee joint ligament healing.

Knee joint ligament healing has been shown to be improved when the torn ligament ends remain in contact, however, the rationale for these effects is unknown. The sensory neuropeptide calcitonin gene related peptide (CGRP) has potent trophic and vasodilatatory properties and as such is thought to be advantageous in wound repair. In ascertaining a role for CGRP in rabbit medial collateral ligament healing, the present study examined changes in CGRP-like immunoreactivity (CGRP-LI) and CGRP-mediated vasomotor responses in gap injured (non-contact), Z-plasty apposed (contact), and sham operated control medial collateral ligaments. At 6 weeks post-trauma, CGRP-LI decreased in the healing zone of gap injured and Z-plasty apposed medial collateral ligaments compared with controls, and non-contact ligament nerve fibres exhibited an abnormal morphology. Topical administration of CGRP (10(-13) to 10(-9) mol) caused a dose-dependent increase in ligament perfusion in each experimental group of knees. The CGRP-mediated vasodilatation associated with gap injured ligaments was not significantly different from controls (P = 0.06), whereas apposed medial collateral ligaments showed an augmented response to the peptide (P < 0.0005). These findings indicate that the beneficial effects of ligament interposition post-trauma may be related to an enhanced responsiveness to CGRP in conjunction with a more typical re-innervation profile. Conversely, the aberrant characteristics of CGRP-LI nerves occurring in gap injured tissue is suggestive of impaired CGRP release which may explain the poor functional recovery associated with these ligaments.

In-vitro cyclic tensile loading of an immobilized and mobilized ligament autograft selectively inhibits mRNA levels for collagenase (MMP-1).

To test the hypothesis that loading conditions can be used to "engineer" ligament autograft behaviors, the effect of cyclic tension on the mRNA levels of matrix molecules and collagenase in in-vivo immobilized and mobilized 6-week rabbit medial collateral ligament (MCL) autografts was examined using an in-vitro system. Femur-[autograft MCL]-tibia complexes were subjected to a tensile stress of 4 MPa at 0.5 Hz for 1 min, followed by 14 min of rest. This 15-min testing cycle was repeated for 4 h. Semi-quantitative reverse transcrip-tase polymerase chain reaction (RT-PCR) was performed on RNA from mechanically treated MCL autografts, using rabbit-specific primer sets for types I and III collagen, biglycan, decorin, fibromodulin, lumican, versican, matrix metalloproteinase-1 (MMP-1, collagenase-1), MMP-13 (collagenase-3), and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Interestingly, 4 h of culture of normal control MCLs led to increased mRNA levels for MMP-1 (P < 0.05), but there were no significant changes in MMP-13 mRNA levels. Total RNA levels in that normal MCL tissue were, however, decreased after culture (P < 0.05). In-vitro tensile loading of in-vivo mobilized autografts resulted in a significant increase in total RNA (185% of in-vitro non-loaded autografts). On the other hand, in-vitro tensile loading of in-vivo immobilized autografts resulted in no significant changes in total RNA levels compared with levels in non-loaded control grafts. MMP-1 mRNA levels in both the in-vivo mobilized (47% of non-loaded autograft) and in-vivo immobilized (38% of non-loaded autograft) MCL autografts were significantly lower than those in non-loaded control tissue following in-vitro tensile loading, but there were no significant changes in the mRNA levels for the seven other matrix molecules assessed. These results show that it is possible to selectively inhibit MMP-1 mRNA levels in autograft ligaments by supplying mechanical stimuli in vitro. The results also demonstrate that in-vivo immobilization leads to a decrease in the effects of subsequent in-vitro mechanical loading in such autografts with respect to total RNA levels. Collectively, these results demonstrate that both in-vivo and in-vitro loading have implications in the engineering of an ideal ligament graft.

A comparison of in vivo gene delivery methods for antisense therapy in ligament healing.

To determine the most efficient in vivo delivery method of oligonucleotides for antisense therapy in ligament healing, fluorescence-labelled phosphorothioate oligodeoxynuleotides (ODN) were introduced into 12 rabbit ligament scars 2 weeks after injury using haemagglutinating virus of Japan (Sendai virus: HVJ)-conjugated liposomes. We compared the efficiency of cellular uptake of fluorescence as a percentage of all cells in each scar using three delivery procedures: (1) direct free-hand injection into the ligament scar using a conventional syringe; (2) systematic direct sca injection using a repeating 10 microliters dispenser and a square mesh grid system; and (3) injection into the feeding (femoral) artery. Results showed that there was a significant difference in fluorescence uptake by scar cells on day 1 after injection between the three delivery methods: (1) direct free-hand, 9.7 +/- 7.6% (average +/- s.d.); (2) systematic direct, 58.4 +/- 15.9%; and (3) intra-arterial, 0.2 +/- 0.1%. Systematic direct injection was most efficient and it resulted in 25.9 +/- 13.0% of scar cells being labeled at 7 days after transfection. We then introduced antisense ODN for the rabbit proteoglycan, decorin, into ligament scars with this delivery method and confirmed a significant inhibition of decorin mRNA expression in antisense-treated scar tissues in vivo both at 2 days (42.3 +/- 14.7% of sense control +/- s.d.; P < 0.0025) and 3 weeks (60.5 +/- 28.2% of sense control +/- s.d.; P < 0.024) after treatment, compared with sense ODN-treated scars. Decorin was significantly suppressed also at protein level in antisense-treated scars at 4 weeks (66.6 +/- 35.7% of sense control +/- s.d.; P < 0.045) after treatment. These results demonstrate that in vivo transfection efficiency in ligament scars is 'delivery system dependent' and that introduction of antisense ODN for the small proteoglycan, decorin, with this delivery method can lead to significant suppression of its expression over 3 weeks both at mRNA and protein levels. Thus, an effective model for the potential manipulation of scar composition and quality in ligament healing has been established.

Decrease in fibronectin occurs coincident with the increased expression of its integrin receptor alpha5beta1 in stress-deprived ligaments.

Stress deprivation secondary to immobilization leads to atrophic changes in periarticular soft tissues. The changes in ligaments include a disorganization of collagen and cellular ultrastructure with varied biochemical alterations resulting in a functionally weaker tissue. This study tests the hypothesis that alterations in fibronectin (Fn) and the expression of its integrin receptor alpha5beta1 in ligament fibroblasts accompany the extracellular matrix remodeling which occurs in stress-deprived knee ligaments. The left knees of eighteen New Zealand white rabbits were surgically immobilized in acute flexion. Fibroblasts within three nine week and three twelve week stress-deprived anterior cruciate ligaments (ACLs) and medial collateral ligaments (MCLs) demonstrated markedly increased immunostaining for the beta1 and alpha5 integrin subunits, as compared to fibroblasts in the contralateral unoperated control ligaments. The effects of stress deprivation on the concentration of Fn was measured by competitive ELISA on the remaining twelve rabbits. Decreases in Fn of 54.0 percent and 63.7 percent occurred in the ACL after nine and twelve weeks of stress deprivation when compared to contralateral controls. The MCL had less of a decrease, losing 37.7 percent and 41.7 percent at nine and twelve weeks, respectively. These results suggest an important role for the Fn-specific integrin receptor alpha5beta1 in remodeling stress-deprived periarticular ligamentous tissue, and the importance of maintaining normal stresses on periarticular ligaments to prevent the degradation of extracellular matrix components such as Fn.

Isolation of intact collagen fibrils from healing ligament.

The inability to isolate intact collagen fibrils has limited the study of their growth and structure. Although intact fibrils have been isolated from echinoderms and from embryonic chick tissues, no method has previously succeeded in isolating intact collagen fibrils from a postfoetal vertebrate tissue. Having previously observed that gentamicin weakens rat tail tendon, we hypothesized that gentamicin may weaken interfibrillar bonds and that intact collagen fibrils might be isolated from tissue treated with gentamicin. In this study medial collateral knee ligaments of Sprague Dawley rats were transected and then harvested 24, 48 or 96 h postoperatively. These specimens were placed in gentamicin or phosphate-buffered saline for 72 h, vortexed for 1 h, incubated in gentamicin for an additional 24 h, and vortexed again for 1 h. Negatively stained specimens were examined with a transmission electron microscope. The phosphate-buffered saline specimens yielded only broken fibrils. The gentamicin specimens yielded both broken and intact fibrils. The latter and tapering ends and consisted of molecules orientated such that their amino termini pointed toward the tip and their carboxy termini pointed toward a short central region where the molecular polarity reversed.

An immunohistochemical study of enthesis development in the medial collateral ligament of the rat knee joint.

The changing distributions of collagens and glycosaminoglycans have been studied at the attachments of the medial collateral ligament during postnatal development. The ligament is of particular interest because it has a fibrocartilaginous attachment to the femoral epiphysis, but a fibrous one to the tibial metaphysis. Ligaments were examined in rats killed at birth and at 2, 4, 6, 8, 10, 20, 30, 45, 60, 90 and 120 days after birth. Cryosections were immunolabelled with monoclonal and polyclonal antibodies against types I and II collagen, chondroitin 4 and 6 sulfate, dermatan and keratan sulfate. Although the ligament is attached at both ends to bones that develop from cartilage, there was a striking difference in collagen labelling. Type II collagen was only found in spicules of calcified cartilage in bone beneath the tibial enthesis after ossification had commenced, but there was a continuous band of labelling at all stages of development at the femoral enthesis. Initially, the cartilage at the femoral attachment lacked type I collagen, but by 45 days labelling was continuous from ligament to bone. Continuity of labelling was seen much earlier at the tibial enthesis, as soon as bone had formed. There were also marked changes in glycosaminoglycan distribution. Keratan sulfate was present at both entheses up to 45 days, but only at the femoral enthesis thereafter. Both attachments labelled throughout life for dermatan sulfate, but chondroitin 4 and 6 sulfate were only found at the femoral end. The results suggest that enthesial cartilage at the femoral attachment was initially derived from the cartilaginous bone rudiment but was quickly eroded on its deep surface by endochondral ossification as bone formed at the attachment site. It was replaced by fibrocartilage developing in the ligament. This mechanism allows enthesis cartilage/fibrocartilage to contribute to the growth of a bone at a secondary centre of ossification in addition to dissipating stress at the ligament-bone junction.

Medial collateral ligament healing one year after a concurrent medial collateral ligament and anterior cruciate ligament injury: an interdisciplinary study in rabbits.

The optimal treatment for concurrent injuries to the medial collateral and anterior cruciate ligaments has not been determined, despite numerous clinical and laboratory studies. The objective of this study was to examine the effect of surgical repair of the medial collateral ligament on its biomechanical and biochemical properties 52 weeks after such injuries. In the left knee of 12 skeletally mature New Zealand White rabbits, the medial collateral ligament was torn and the anterior cruciate ligament was transected and then reconstructed. This is an experimental model previously developed in our laboratory. In six rabbits, the torn ends of the medial collateral ligament were repaired, and in the remaining six rabbits, the ligament was not repaired. Fifty-two weeks after injury, we examined varus-valgus and anterior-posterior knee stability; structural properties of the femur-medial collateral ligament-tibia complex; and mechanical properties, collagen content, and mature collagen crosslinking of the medial collateral ligament. We could not detect significant differences between repair and nonrepair groups for any biomechanical or biochemical property. Our data support clinical findings that when the medial collateral and anterior cruciate ligaments are injured concurrently and the anterior cruciate ligament is reconstructed, conservative treatment of the ruptured medial collateral ligament can result in successful healing.

Myofibroblasts in the healing lapine medial collateral ligament: possible mechanisms of contraction.

The specific objective of this study was to determine the chronology of the appearance of the myofibroblast in the healing ligament. The overall goal of our work is to elucidate the cellular mechanism of contraction in this tissue. The myofibroblast has been found to be responsible for wound contraction in many tissues and to be the cause of the contracture in several pathological conditions. This cell type contains the actin isoform previously thought to be unique to smooth muscle cells and displays certain characteristic features at the ultrastructural level. In 26 New Zealand White male rabbits, the right medial collateral ligament was transected, whereas the left medial collateral ligament received a sham operation. The central third of the ligament (ligament scar tissue) was evaluated at 2, 3, 6, 8, 10, and 12 weeks postoperatively by immunohistochemical techniques, transmission electron microscopy, and Western blot analyses. Three other rabbits served as anatomic controls. During the early reparative phase (2 and 3 weeks after transection), there was an increase in the number of cells containing alpha-smooth muscle actin as well as augmentation of the alpha-smooth muscle actin content within each cell--a finding attributed to smooth muscle cells and pericytes associated with neovascularity. No myofibroblasts were detected at this stage, immediately postoperatively, or in the sham-operation controls. Ligaments in the remodeling phase of healing (6, 8, 10, and 12 weeks) exhibited alpha-smooth muscle actin in fibroblasts (myofibroblasts) as well as in vascular pericytes and smooth muscle cells. During this stage of healing, transmission electron microscopy demonstrated an increase in the number of cells displaying myofibroblastic features. It was estimated that at 12 weeks of healing 10% of the cells at the site of injury were myofibroblasts. This is the first definitive finding of myofibroblasts in the injury site of the healing ligament, to our knowledge. The appearance of myofibroblasts in the 6-12 week healing period, the interval during which the ligament has been shown to contract in studies by other investigators, is a rationale for a hypothesis that a cellular contractile apparatus comprising alpha-smooth muscle actin (i.e., the myofibroblast) may contribute to the recovery of original ligament length (and normal in situ strain).

Collagens in an adult bovine medial collateral ligament: immunofluorescence localization by confocal microscopy reveals that type XIV collagen predominates at the ligament-bone junction.

To understand the structure and function of medial collateral ligament, collagens present in an adult bovine ligament were determined. The mid-section of the ligament was powdered and extracted with 4M guanidinium hydrochloride, and the residue was digested with pepsin to solubilize the collagens. Type I collagen was the major fibril collagen recovered in the pepsin solubilized fraction, with types III and V each representing about 5% and 2%, respectively. Type VI collagen was the major collagen present in the guanidinium hydrochloride extract, and it accounted for about 40% of the proteins in the extract or 4% of the tissue dry weight. Type XII and XIV collagens were also detected in the guanadinium hydrochloride extract as minor components. Immunofluorescence localization using confocal microscopy showed that type XII and XIV collagens are associated with the ligament fibrillar network and that type XIV collagen was prominent at the ligament-bone junction. These data reinforce the notion that these collagens are associated with the type I collagen fibrillar network in connective tissues. In view of high mechanical stresses that exist at the ligament-bone interface, presence of type XIV collagen in high concentration at this junction may contribute to the modulation of the biomechanical properties of this tissue.