The mgΔlpn mouse model for Marfan syndrome recapitulates the ocular phenotypes of the disease.

PURPOSE: Fibrillin-1 and -2 are major components of tissue microfibrils that compose the ciliary zonule and cornea. While mutations in human fibrillin-1 lead to ectopia lentis, a major manifestation of Marfan syndrome (MFS), in mice fibrillin-2 can compensate for reduced/lack of fibrillin-1 and maintain the integrity of ocular structures. Here we examine the consequences of a heterozygous dominant-negative mutation in the Fbn1 gene in the ocular system of the mgΔlpn mouse model for MFS. METHODS: Eyes from mgΔlpn and wild-type mice at 3 and 6 months of age were analyzed by histology. The ciliary zonule was analyzed by scanning electron microscopy (SEM) and immunofluorescence. RESULTS: Mutant mice presented a significantly larger distance of the ciliary body to the lens at 3 and 6 months of age when compared to wild-type, and ectopia lentis. Immunofluorescence and SEM corroborated those findings in MFS mice, revealing a disorganized mesh of microfibrils on the floor of the ciliary body. Moreover, mutant mice also had a larger volume of the anterior chamber, possibly due to excess aqueous humor. Finally, losartan treatment had limited efficacy in improving ocular phenotypes. CONCLUSIONS: In contrast with null or hypomorphic mutations, expression of a dominant-negative form of fibrillin-1 leads to disruption of microfibrils in the zonule of mice. This in turn causes lens dislocation and enlargement of the anterior chamber. Therefore, heterozygous mgΔlpn mice recapitulate the major ocular phenotypes of MFS and can be instrumental in understanding the development of the disease.

Sacroiliac Joint Ligaments and Sacroiliac Pain: A Case-Control Study on Micro- and Ultrastructural Findings on Morphologic Alterations.

BACKGROUND: The sacroiliac joint (SIJ) is a common source of low back pain. SIJ pain has shown to have negative impact on patients' quality of life. Although clinically there is an increasing interest to treat SIJ-related pain both conservatively and surgically, the underlying mechanisms related to pathology in that region are poorly understood. One hypothesis is that the SIJ ligaments are structurally altered in SIJ pain. OBJECTIVES: The given study investigated patient cases with different pain durations undergoing posterior distraction arthrodesis of the SIJ, with subsequent micro- and ultrastructural assessment of the interosseous and posterior SIJ ligaments compared with autopsy controls without known history of low back pain. STUDY DESIGN: Case-control study. Morphologic-pathological analysis of tissue samples obtained during surgery with controls from legal medicine. SETTING: Rural hospital setting in Halberstadt, Germany. METHODS: SIJ ligaments were removed from 6 patients undergoing SIJ arthrodesis for histological and ultrastructural assessment in scanning and transmission electron microscopy, and compared with 6 controls without known history of SIJ pathology. RESULTS: A number of alterations were observed in the ligaments of patients with SIJ, namely ligament disruption, collagen loosening and coiling, vascularization, and hemorrhage. In some areas, these changes were observed in proximity to healthy (structurally unaltered and intact tissues), indicative of a potential disease progression. Comparison to controls yielded a significant correlation between SIJ pathology and the level of collagen degeneration (Phi >/= 0.82; P < 0.001). LIMITATIONS: Small sample size, method of tissue removal from patients may have influenced tissue integrity. CONCLUSIONS: The combined clinical, histological, and ultrastructural analysis provided, to our knowledge, first-time evidence of morphologic SIJ ligament alteration of a nontraumatic and noninflammatory cause. Further research is necessary to elucidate these structural changes and to substantiate pain duration and patient-history-dependent changes at the ligaments of the posterior pelvis. KEY WORDS: Collagen, electron microscopy, histological and ultrastructural assessment, interosseous sacroiliac ligament, low back pain, sacroiliac joint.

A unique methionine-rich protein-aragonite crystal complex: Structure and mechanical functions of the Pinctada fucata bivalve hinge ligament.

The bivalve hinge ligament holds the two shells together. The ligament functions as a spring to open the shells after they were closed by the adductor muscle. The ligament is a mineralized tissue that bears no resemblance to any other known tissue. About half the ligament is composed of a protein-rich matrix, and half of long and extremely thin segmented aragonite crystals. Here we study the hinge ligament of the pearl oyster Pinctada fucata. FIB SEM shows that the 3D organization is remarkably ordered. The full sequence of the major protein component contains a continuous segment of 30 repeats of MMMLPD. There is no known homologous protein. Knockdown of this protein prevents crystal formation, demonstrating that the integrity of the matrix is necessary for crystals to form. X-ray diffraction shows that the aragonite crystals are more aligned in the compressed ligament, indicating that the crystals may be actively contributing to the elastic properties. The fusion interphase that joins the ligament to the shell nacre is composed of a prismatic mineralized tissue with a thin organic-rich layer at its center. Nanoindentation of the dry interphase shows that the elastic modulus of the nacre adjacent to the interphase gradually decreases until it approximates that of the interphase. The interphase modulus slightly increases until it matches the ligament. All these observations demonstrate that the ligament shell complex is a remarkable biological tissue that has evolved unique properties that enable bivalves to open their shell effectively innumerable times during the lifetime of the animal. STATEMENT OF SIGNIFICANCE: The hinge ligament shell complex is a unique functioning structural tissue whose elastic properties enable the shell to open without expending energy. Methionine-rich proteins are not known elsewhere raising fundamental questions about secondary and tertiary structures contributing to its elastic properties. The segmented and extremely thin aragonite crystals embedded in this matrix may also have unexpected elastic materials properties as they flex during compression. The structure of the interphase comprises a fascinating biological joint that connects two very different materials. The interphase materials, including the nacre, are graded with respect to elastic modulus so as to approximately match the connecting components. The interphase incorporates a thin organic rich layer that presumably functions as a gasket. This study raises many fundamental questions relevant to the diverse fields of protein chemistry, biomineralization and biological materials.

The natural involution of the sheep proximal sesamoidean ligament is due to depletion of satellite cells and simultaneous proliferation of fibroblasts: Ultrastructural evidence.

The rapid involution that happens in some muscles of ungulate fetlock joints has never been investigated at an ultrastructural level. In this study, the proximal sesamoidean ligament (PSL) of sheep was chosen as a model to investigate, at the cellular level, the transition from muscle to connective structures that occurs during early development. In particular, we were interested in observing the presence of satellite cells and fibroblasts, detecting fluctuations in their numbers in the postnatal developing PSL, and evaluating putative apoptotic mechanisms. Interestingly, some features were shared by both PSL involution and muscle ageing; the most relevant being the significant and rapid decrease in the number of satellite cells together with a quick proliferation of fibroblasts in the muscle-connective transitional area (MCT-TA). Electron microscopy and immunohistochemical analyses revealed putative cellular mechanisms that led to a progressive involution of the muscle portion of the PSL during postnatal growth. Our findings showed a fast transition from muscle to connective tissue due to the depletion of satellite cells, apoptosis of some muscle fibres, and simultaneous proliferation of fibroblasts originating from mesenchymal progenitors or from differentiation of satellite cells typically located at the border between muscle and connective tissue of the PSL.

A lambda-shaped retractor lentis muscle in the yellowfin goby Acanthogobius flavimanus.

We identified a morphologically uncommon piscine retractor lentis muscle in the yellowfin goby Acanthogobius flavimanus. This lentis muscle has a shape similar to the Greek small letter lambda (λ). The two legs of the muscle are attached to the retinal periphery at the ventral eyecup, while the tip is connected to the lens surface by a ligament. Scanning electron microscopy showed that the fibers of the lentis muscle run along the length of both the anterior and posterior legs. Immunolabeling with antiacetylated tubulin antibody and neuronal tracing with DiI of the whole lentis muscle revealed that the anterior leg is innervated by one or more nerves. The topographic distribution of ganglion cells in the retina was investigated to identify the visual axis. Three high cell density areas were observed in the dorso-temporal, ventro-nasals and ventro-temporal retina. These findings suggest that the λ-shaped lentis muscle may enable accommodatory movement of the lens toward the temporal as well as the nasal and/or ventral retina.

The Red Kangaroo pericardium as a material source for the manufacture of percutaneous heart valves.

BACKGROUND: The kangaroo pericardium might be considered to be a good candidate material for use in the manufacture of the leaflets of percutaneous heart valves based upon the unique lifestyle. The diet consists of herbs, forbs and strubs. The kangaroo pericardium holds an undulated structure of collagen. MATERIAL AND METHOD: A Red Kangaroo was obtained after a traffic fatality and the pericardium was dissected. Four compasses were cut from four different sites: auricular (AUR), atrial (ATR), sternoperitoneal (SPL) and phrenopericardial (PPL). They were investigated by means of scanning electron microscopy, light microscopy and transmission electron microscopy. RESULTS: All the samples showed dense and wavy collagen bundles without vascularisation from both the epicardium and the parietal pericardium. The AUR and the ATR were 150±25µm thick whereas the SPL and the PPL were thinner at 120±20µm. The surface of the epicardium was smooth and glistening. The filaments of collagen were well individualized without any aggregation, but the banding was poorly defined and somewhat blurry. CONCLUSION: This detailed morphological analysis of the kangaroo pericardium illustrated a surface resistant to thrombosis and physical characteristics resistant to fatigue. The morphological characteristics of the kangaroo pericardium indicate that it represents an outstanding alternative to the current sources e.g., bovine and porcine. However, procurement of tissues from the wild raises supply and sanitary issues. Health concerns based upon sanitary uncertainty and reliability of supply of wild animals remain real problems.

Type VII Collagen in the Human Accommodation System: Expression in Ciliary Body, Zonules, and Lens Capsule.

Purpose: To investigate intraocular expression of COL7A1 and its protein product type VII collagen, particularly at the accommodation system. Methods: Eyes from 26 human adult donors were used. COL7A1 expression was analyzed in ex vivo ciliary epithelium by microarray. Type VII collagen distribution was examined by Western blot analysis, immunohistochemistry. and immuno-electron microscopy. Results: COL7A1 is expressed by pigmented and nonpigmented ciliary epithelia. Type VII collagen is distributed particularly at the strained parts of the accommodation system. Type VII collagen was associated with various basement membranes and with ciliary zonules. Anchoring fibrils were not visualized. Conclusions: Type VII collagen distribution at strained areas suggests a supporting role in tissue integrity.

Decellularized Iliotibial Band Recolonized with Allogenic Homotopic Fibroblasts or Bone Marrow-Derived Mesenchymal Stromal Cells.

Decellularized scaffolds present promising biomimetic approaches in various fields of tissue engineering. Different tissues have been selected for decellularization, among them extracellular matrix (ECM)-rich tissues such as tendons, ligaments and cartilage. The dense ECM of ligaments is particularly challenging to achieve a completely non-immunogenic ECM void of any cells. Here, the methods for decellularization adapted to ligamentous tissue of the iliotibial band (ITB) are presented along with cell isolation and several recolonization techniques using allogenic ITB-derived fibroblasts or mesenchymal stromal cells (MSCs).

Ultrastructural study of the neural microcircuits in the sensory epithelium of the paratympanic organ of the chicken.

The paratympanic organ (PTO) is a sensory organ located in the medial wall of the tympanic cavity of birds. The organ looks like a small tapering vesicle, and is equipped with a sensory epithelium formed by supporting cells (SCs) and Type II hair cells (Type II-HCs). The function of the PTO has not yet been precisely defined. The prevailing current hypothesis is that the PTO assesses the air pressure exerted on the external surface of the tympanic membrane. The PTO could may thus function as a barometer and, in flying birds, also as an altimeter. The afferent synapses of the PTO of chicken were described in detail in a previous paper. Reciprocal synapses between efferent nerve endings (ENEs) and the HCs were also observed, suggesting the existence of local microcircuits. The aim of this work was to provide a more detailed ultrastructural description of these microcircuits in the PTO of chicken. We observed for the first time: (1) reciprocal synapses between the HCs and the afferent nerve endings (ANEs); (2) presence of two distinct types of ENEs; (3) reciprocal synapses between the HCs and both types of ENEs. Overall, these results indicate that a complex processing of the incoming sensory signals may occur in the PTO. This thus suggests that the PTO may perform more complex functions than those supposed until now. We hypothesize that the PTO could have a role in the low-frequency sound perception.

Reconstruction of Ligament and Tendon Defects Using Cell Technologies.

We studied the possibility of restoring the integrity of the Achilles tendon in rabbits using autologous multipotent stromal cells. Collagen or gelatin sponges populated with cells were placed in a resorbable Vicryl mesh tube and this tissue-engineered construct was introduced into a defect of the middle part of the Achilles tendon. In 4 months, histological analysis showed complete regeneration of the tendon with the formation of parallel collagen fibers, spindle-shaped tenocytes, and newly formed vessels.

Three-dimensional visualisation of soft biological structures by X-ray computed micro-tomography.

Whereas the two-dimensional (2D) visualisation of biological samples is routine, three-dimensional (3D) imaging remains a time-consuming and relatively specialised pursuit. Current commonly adopted techniques for characterising the 3D structure of non-calcified tissues and biomaterials include optical and electron microscopy of serial sections and sectioned block faces, and the visualisation of intact samples by confocal microscopy or electron tomography. As an alternative to these approaches, X-ray computed micro-tomography (microCT) can both rapidly image the internal 3D structure of macroscopic volumes at sub-micron resolutions and visualise dynamic changes in living tissues at a microsecond scale. In this Commentary, we discuss the history and current capabilities of microCT. To that end, we present four case studies to illustrate the ability of microCT to visualise and quantify: (1) pressure-induced changes in the internal structure of unstained rat arteries, (2) the differential morphology of stained collagen fascicles in tendon and ligament, (3) the development of Vanessa cardui chrysalises, and (4) the distribution of cells within a tissue-engineering construct. Future developments in detector design and the use of synchrotron X-ray sources might enable real-time 3D imaging of dynamically remodelling biological samples.

Precision Pulse Capsulotomy: Preclinical Safety and Performance of a New Capsulotomy Technology.

PURPOSE: To assess the preclinical safety and performance of a new precision pulse capsulotomy (PPC) method. DESIGN: Human cadaver eye studies and surgical, slit-lamp, and histopathologic evaluation in a consecutive series of 20 live rabbits. PARTICIPANTS: Human cadaver eyes and New Zealand white rabbits. METHODS: Precision pulse capsulotomy uses a highly focused, fast, multipulse, low-energy discharge to produce a perfectly round anterior capsulotomy instantaneously and simultaneously along all 360°. Capsulotomies are performed using a disposable handpiece with a soft collapsible tip and circular nitinol cutting element. Miyake-Apple imaging and scanning electron microscopy (SEM) of PPC were conducted in human cadaver eyes. Surgical, postoperative slit-lamp, and histopathologic assessments of PPC were performed in 20 live rabbits and were compared with manual continuous curvilinear capsulorrhexis (CCC) in the fellow eye. Anterior chamber (AC) thermocouple temperature measurements were evaluated in a subset of rabbit eyes. MAIN OUTCOME MEASURES: Capsulotomy edge circularity, SEM morphologic features and zonular movement with PPC in human cadaver eyes. Anterior chamber temperature during PPC and grading of ocular inflammation, corneal endothelial damage, anterior capsular opacification (ACO), and posterior capsular opacification (PCO). RESULTS: Miyake-Apple imaging showed minimal zonular stress, and thermocouple measurements demonstrated negligible AC temperature changes during PPC. Precision pulse capsulotomy produced round, complete capsulotomies in all 20 rabbit eyes, leading to successful in-the-bag intraocular lens (IOL) implantation. Slit-lamp examinations at 3 days and 1, 2, and 4 weeks after surgery showed no significant differences between PPC and CCC in corneal edema, AC inflammatory reaction, capsular fibrosis, ACO, and PCO. Postmortem studies showed no difference in the corneal endothelium between PPC and CCC eyes. All IOLs were well centered in PPC eyes, and histopathologic analysis showed no greater inflammatory infiltrates. CONCLUSIONS: Precision pulse capsulotomy is a new method to automate consistent creation of a perfectly circular anterior capsulotomy with a disposable handheld instrument that can be used in the normal phacoemulsification surgical sequence. Compared with CCC in fellow rabbit eyes, PPC was equally safe and showed no greater zonular stress compared with CCC in human cadaver eyes. Human cadaver eye SEM showed a much smoother capsulotomy edge compared to those produced by femtosecond laser.

Histo-mechanical properties of the swine cardinal and uterosacral ligaments.

The focus of this study was to determine the structural and mechanical properties of two major ligaments that support the uterus, cervix, and vagina: the cardinal ligament (CL) and the uterosacral ligament (USL). The adult swine was selected as animal model. Histological analysis was performed on longitudinal and cross sections of CL and USL specimens using Masson׳s trichrome and Verhoeff-van Giesson staining methods. Scanning electron microscopy was employed to visualize the through-thickness organization of the collagen fibers. Quasi-static uniaxial tests were conducted on specimens that were harvested from the CL/USL complex of a single swine. Dense connective tissue with a high content of elastin and collagen fibers was observed in the USL. Loose connective tissue with a considerable amount of smooth muscle cells and ground substance was detected in both the CL and USL. Collagen fibers, smooth muscle cells, blood vessels, and nerve fibers were arranged primarily in the plane of the ligaments. The USL was significantly stronger than the CL with higher ultimate stress and tangent modulus of the linear region of the stress-strain curve. Knowledge about the mechanical properties of the CL and USL will aid in the design of novel mesh materials, stretching routines, and surgical procedures for pelvic floor disorders.

Lymphatic Stomata in the Adult Human Pulmonary Ligament.

BACKGROUND: Lymphatic stomata are small lymphatic openings in the serosal membrane that communicate with the serosal cavity. Although these stomata have primarily been studied in experimental mammals, little is known concerning the presence and properties of lymphatic stomata in the adult human pleura. Thus, adult human pleurae were examined for the presence or absence of lymphatic stomata. METHODS AND RESULTS: A total of 26 pulmonary ligaments (13 left and 13 right) were obtained from 15 adult human autopsy cases and examined using electron and light microscopy. The microscopic studies revealed the presence of apertures fringed with D2-40-positive, CD31-positive, and cytokeratin-negative endothelial cells directly communicating with submesothelial lymphatics in all of the pulmonary ligaments. The apertures' sizes and densities varied from case to case according to the serial tissue section. The medians of these aperture sizes ranged from 2.25 to 8.75 µm in the left pulmonary ligaments and from 2.50 to 12.50 µm in the right pulmonary ligaments. The densities of the apertures ranged from 2 to 9 per mm(2) in the left pulmonary ligaments and from 2 to 18 per mm(2) in the right pulmonary ligaments. However, no significant differences were found regarding the aperture size (p=0.359) and density (p=0.438) between the left and the right pulmonary ligaments. CONCLUSIONS: Our study revealed that apertures exhibit structural adequacy as lymphatic stomata on the surface of the pulmonary ligament, thereby providing evidence that lymphatic stomata are present in the adult human pleura.

Do cells contribute to tendon and ligament biomechanics?

INTRODUCTION: Acellular scaffolds are increasingly used for the surgical repair of tendon injury and ligament tears. Despite this increased use, very little data exist directly comparing acellular scaffolds and their native counterparts. Such a comparison would help establish the effectiveness of the acellularization procedure of human tissues. Furthermore, such a comparison would help estimate the influence of cells in ligament and tendon stability and give insight into the effects of acellularization on collagen. MATERIAL AND METHODS: Eighteen human iliotibial tract samples were obtained from nine body donors. Nine samples were acellularized with sodium dodecyl sulphate (SDS), while nine counterparts from the same donors remained in the native condition. The ends of all samples were plastinated to minimize material slippage. Their water content was adjusted to 69%, using the osmotic stress technique to exclude water content-related alterations of the mechanical properties. Uniaxial tensile testing was performed to obtain the elastic modulus, ultimate stress and maximum strain. The effectiveness of the acellularization procedure was histologically verified by means of a DNA assay. RESULTS: The histology samples showed a complete removal of the cells, an extensive, yet incomplete removal of the DNA content and alterations to the extracellular collagen. Tensile properties of the tract samples such as elastic modulus and ultimate stress were unaffected by acellularization with the exception of maximum strain. DISCUSSION: The data indicate that cells influence the mechanical properties of ligaments and tendons in vitro to a negligible extent. Moreover, acellularization with SDS alters material properties to a minor extent, indicating that this method provides a biomechanical match in ligament and tendon reconstruction. However, the given protocol insufficiently removes DNA. This may increase the potential for transplant rejection when acellular tract scaffolds are used in soft tissue repair. Further research will help optimize the SDS-protocol for clinical application.

Structure, physiology, and biochemistry of collagens.

Tendons and ligaments are connective tissues that guide motion, share loads, and transmit forces in a manner that is unique to each as well as the anatomical site and biomechanical stresses to which they are subjected. Collagens are the major molecular components of both tendons and ligaments. The hierarchical structure of tendon and its functional properties are determined by the collagens present, as well as their supramolecular organization. There are 28 different types of collagen that assemble into a variety of supramolecular structures. The assembly of specific supramolecular structures is dependent on the interaction with other matrix molecules as well as the cellular elements. Multiple suprastructural assemblies are integrated to form the functional tendon/ligament. This chapter begins with a discussion of collagen molecules. This is followed by a definition of the supramolecular structures assembled by different collagen types. The general principles involved in the assembly of collagen-containing suprastructures are presented focusing on the regulation of tendon collagen fibrillogenesis. Finally, site-specific differences are discussed. While generalizations can be made, differences exist between different tendons as well as between tendons and ligaments. Compositional differences will impact structure that in turn will determine functional differences. Elucidation of the unique physiology and pathophysiology of different tendons and ligaments will require an appreciation of the role compositional differences have on collagen suprastructural assembly, tissue organization, and function.

A thermomechanical framework for reconciling the effects of ultraviolet radiation exposure time and wavelength on connective tissue elasticity.

Augmentation of the mechanical properties of connective tissue using ultraviolet (UV) radiation-by targeting collagen cross-linking in the tissue at predetermined UV exposure time [Formula: see text] and wavelength [Formula: see text]-has been proposed as a therapeutic method for supporting the treatment for structural-related injuries and pathologies. However, the effects of [Formula: see text] and [Formula: see text] on the tissue elasticity, namely elastic modulus [Formula: see text] and modulus of resilience [Formula: see text], are not entirely clear. We present a thermomechanical framework to reconcile the [Formula: see text]- and [Formula: see text]-related effects on [Formula: see text] and [Formula: see text]. The framework addresses (1) an energy transfer model to describe the dependence of the absorbed UV photon energy, [Formula: see text], per unit mass of the tissue on [Formula: see text] and [Formula: see text], (2) an intervening thermodynamic shear-related parameter, [Formula: see text], to quantify the extent of UV-induced cross-linking in the tissue, (3) a threshold model for the [Formula: see text] versus [Formula: see text] relationship, characterized by   [Formula: see text]-the critical [Formula: see text] underpinning the association of [Formula: see text] with [Formula: see text]-and (4) the role of [Formula: see text] in the tissue elasticity. We hypothesized that [Formula: see text] regulates [Formula: see text] (UV-stiffening hypothesis) and [Formula: see text] (UV-resilience hypothesis). The framework was evaluated with the support from data derived from tensile testing on isolated ligament fascicles, treated with two levels of [Formula: see text] (365 and 254 nm) and three levels of [Formula: see text] (15, 30 and 60 min). Predictions from the energy transfer model corroborated the findings from a two-factor analysis of variance of the effects of [Formula: see text] and [Formula: see text] treatments. Student's t test revealed positive change in [Formula: see text] and [Formula: see text] with increases in [Formula: see text]-the findings lend support to the hypotheses, implicating the implicit dependence of UV-induced cross-links on [Formula: see text] and [Formula: see text] for directing tissue stiffness and resilience. From a practical perspective, the study is a step in the direction to establish a UV irradiation treatment protocol for effective control of exogenous cross-linking in connective tissues.

Ultrastructure and innervation of thumb carpometacarpal ligaments in surgical patients with osteoarthritis.

BACKGROUND: The complex configuration of the thumb carpometacarpal (CMC-1) joint relies on musculotendinous and ligamentous support for precise circumduction. Ligament innervation contributes to joint stability and proprioception. Evidence suggests abnormal ligament innervation is associated with osteoarthritis (OA) in large joints; however, little is known about CMC-1 ligament innervation characteristics in patients with OA. We studied the dorsal radial ligament (DRL) and the anterior oblique ligament (AOL), ligaments with a reported divergent presence of mechanoreceptors in nonosteoarthritic joints. QUESTIONS/PURPOSES: This study's purposes were (1) to examine the ultrastructural architecture of CMC-1 ligaments in surgical patients with OA; (2) to describe innervation, specifically looking at mechanoreceptors, of these ligaments using immunohistochemical techniques and compare the AOL and DRL in terms of innervation; and (3) to determine whether there is a correlation between age and mechanoreceptor density. METHODS: The AOL and DRL were harvested from 11 patients with OA during trapeziectomy (10 women, one man; mean age, 67 years). The 22 ligaments were sectioned in paraffin and analyzed using immunoflourescent triple staining microscopy. RESULTS: In contrast to the organized collagen bundles of the DRL, the AOL appeared to be composed of disorganized connective tissue with few collagen fibers and little innervation. Mechanoreceptors were identified in CMC-1 ligaments of all patients with OA. The DRL was significantly more innervated than the AOL. There was no significant correlation between innervation of the DRL and AOL and patient age. CONCLUSIONS: The dense collagen structure and rich innervation of the DRL in patients with OA suggest that the DRL has an important proprioceptive and stabilizing role. CLINICAL RELEVANCE: Ligament innervation may correlate with proprioceptive and neuromuscular changes in OA pathophysiology and consequently support further investigation of innervation in disease prevention and treatment strategies.

Nonselective assembly of fibrillin 1 and fibrillin 2 in the rodent ocular zonule and in cultured cells: implications for Marfan syndrome.

PURPOSE: Fibrillins are the major constituent of tissue microfibrils, which form the ocular zonule. In Marfan syndrome (MFS), FBN1 mutations lead to ectopia lentis. The goal of this work was to investigate zonule composition and formation in fibrillin-deficient and wild-type mice. METHODS: Immunofluorescence staining of eyes from wild-type, Fbn1-deficient, and Fbn2-deficient mice, as well as other species, was performed using monospecific fibrillin 1 and fibrillin 2 antibodies. The zonule of Fbn1-deficient and Fbn2-deficient mice was studied by electron microscopy. Microfibril formation in vitro was evaluated by immunofluorescence microscopy of cultured nonpigmented ciliary epithelial cells and fibroblasts. RESULTS: A zonule was present in both Fbn1-deficient and Fbn2-deficient mouse eyes. Immunofluorescence demonstrated that the zonule of Fbn1-deficient mice, wild-type mice, rats, and hamsters contained fibrillin 2. The zonule of Fbn2(-/-) mice contained fibrillin 1. Fibrillin 1 and fibrillin 2 colocalized in microfibrils formed in human nonpigmented ciliary epithelium cultures. Like fibrillin 1, fibrillin 2 microfibril assembly was fibronectin dependent and initiated by cell surface punctate deposits that elongated to form microfibrils. CONCLUSIONS: These data suggest that fibrillin 1 assembly and fibrillin 2 assembly share similar mechanisms. Microfibril composition depends substantially on the local levels of fibrillin isoforms and is not highly selective in regard to the isoform. This raises the intriguing possibility that the zonule could be strengthened in MFS by inducing fibrillin 2 expression in ciliary epithelium. The presence of fibrillin 2 in the murine zonule and an intact zonule in Fbn1-knockout mice may limit the utility of rodent models for studying ectopia lentis in MFS.

New insights into mutable collagenous tissue: correlations between the microstructure and mechanical state of a sea-urchin ligament.

The mutable collagenous tissue (MCT) of echinoderms has the ability to undergo rapid and reversible changes in passive mechanical properties that are initiated and modulated by the nervous system. Since the mechanism of MCT mutability is poorly understood, the aim of this work was to provide a detailed morphological analysis of a typical mutable collagenous structure in its different mechanical states. The model studied was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was characterized in different functional states mimicking MCT mutability. Transmission electron microscopy, histochemistry, cryo-scanning electron microscopy, focused ion beam/scanning electron microscopy, and field emission gun-environmental scanning electron microscopy were used to visualize CDLs at the micro- and nano-scales. This investigation has revealed previously unreported differences in both extracellular and cellular constituents, expanding the current knowledge of the relationship between the organization of the CDL and its mechanical state. Scanning electron microscopies in particular provided a three-dimensional overview of CDL architecture at the micro- and nano-scales, and clarified the micro-organization of the ECM components that are involved in mutability. Further evidence that the juxtaligamental cells are the effectors of these changes in mechanical properties was provided by a correlation between their cytology and the tensile state of the CDLs.