Development of a 3D-immunofluorescence analysis for sensory nerve endings in human ligaments.

BACKGROUND: The analysis of ligamentous mechanoreceptors is difficult due to a high amount of unclassifiable mechanoreceptors, which result from incomplete visualization through limited microscopic techniques. NEW METHOD: The method was developed using dorsal intercarpal ligaments and dorsal regions of the scapholunate interosseous ligament from human cadaver wrists. Consecutive 70 µm thick cryosections were stained with immunofluorescence markers for protein S100B, neurotrophin receptor p75 (p75), protein gene product 9.5 (PGP 9.5) and 4',6-diamidino-2-phenylindole (DAPI). 3D images of sensory nerve endings were obtained using a confocal laser scanning microscope. Experimental point spread functions (PSF) were used to deconvolve images. Sensory nerve endings were localised in each section plane and classified according to Freeman and Wyke. Finally, confocal data was visualized as 3D-images. RESULTS: The method produced excellent image quality, revealing detailed three-dimensional structures. The created 3D-model of sensory nerve endings could be analyzed in all three dimensions, augmenting visualization of the form and immunoreactive pattern of sensory nerve endings. Deconvolution with experimentally measured PSFs aided in enhancing image quality. COMPARISON WITH EXISTING METHODS: Using a triple immunofluorescent staining method allows to visualize the structure of sensory nerve endings more precisely than techniques with serial analysis of different monostaining of neural markers. Imaging in three dimensions enhances morphologic details, which are limited in 2D-microscopy. CONCLUSION: 3D-triple immunofluorescence produces high quality visualization of mechanoreceptors, thereby improving their analysis. As an elaborate technique, it is ideal for defined research questions concerning the microstructure of sensory nerve endings.

The effect of exercise on the protein profile of rat knee joint intra- and extra-articular ligaments.

Injuries to the intra-articular anterior cruciate ligament (ACL) and the extra-articular medial collateral ligament (MCL) result in significant knee joint instability, pain, and immobility. Moderate endurance-type exercise can increase ligament strength but little is known on the effect of short-term regular bouts of high-intensity exercise on the extracellular matrix (ECM) structure of knee ligaments. Therefore, this study aimed to identify the effect of short-term regular bouts high exercise on the proteome of the rat ACL and MCL using mass spectrometry. Sprague-Dawley male rats (n = 6) were split into control and exercise groups, and subjected to high-intensity training for four 4 weeks followed by proteomic analyses of the ACL and MCL. Knee joint health status was assessed using OARSI and a validated histological scoring system. Histopathological analyses demonstrated no significant changes in either in cruciate, collateral ligaments, or cartilage between the control and exercised knee joints. However, significant proteins were found to be more abundant in the exercised ACL compared to ACL control group but not between the exercised MCL and control MCL groups. The significant abundant proteins in ACL exercise groups were mostly cytoskeletal, ribosomal and enzymes with several abundant matrisomal proteins such as collagen proteins and proteoglycans being found in this group. In conclusion, our results indicate that short-term regular bouts of high-intensity exercise have an impact on the intra-articular ACL but not extra-articular MCL ECM protein expression.

Paediatric knee anterolateral capsule does not contain a distinct ligament: analysis of histology, immunohistochemistry and gene expression.

OBJECTIVES: The presence of a discrete ligament within the knee anterolateral capsule (ALC) is controversial. Tendons and ligaments have typical collagens, ultrastructure, transcription factors and proteins. However, these characteristics have not been investigated in paediatric ALC. The purpose of this study was to characterise the paediatric ALC in terms of tissue ultrastructure and cellular expression of ligament markers scleraxis (SCX)-a basic helix-loop-helix transcription factor-and the downstream transmembrane glycoprotein tenomodulin (TNMD), as compared with the paediatric lateral collateral ligament (LCL) and paediatric quadriceps tendon (QT). We hypothesised that, in comparison to the LCL and QT, the ALC would possess poor collagen orientation and reduced SCX and TNMD expression. METHODS: 15 paediatric ALCs (age 6.3±3.3 years), 5 paediatric LCLs (age 3.4±1.3 years) and 5 paediatric QTs (age 2.0±1.2 years) from fresh cadaveric knees were used in this study. Fresh-frozen samples from each region were cryosectioned and then stained with H&E to evaluate collagen alignment and cell morphology. Expression of SCX and TNMD was determined by gene expression analysis and immunohistochemistry. RESULTS: The histological sections of the paediatric LCL and QT showed well-organised, dense collagenous tissue fibres with elongated fibroblasts, while the ALC showed more random collagen orientation without clear cellular directionality. The aspect ratio of cells in the ALC was significantly lower than that of the LCL and QT (p<0.0001 and p<0.0001, respectively). The normalised distribution curve of the inclination angles of the nuclei in the ALC was more broadly distributed than that of the LCL or QT, indicating random cell alignment in the ALC. SCX immunostaining was apparent in the paediatric LCL within regions of aligned fibres, while the comparatively disorganised structure of the ALC was negative for SCX. The paediatric LCL also stained positive for TNMD, while the ALC was only sparsely positive for this tendon/ligament cell-surface molecule. Relative gene expression of SCX and TNMD were higher in the LCL and QT than in the ALC. CONCLUSION: In this study, a distinct ligament could not be discerned in the ALC based on histology, immunohistochemistry and gene expression analysis. LEVEL OF EVIDENCE: Controlled laboratory study.

Profiling and Bioinformatics Analysis of Differentially Expressed circRNAs in Spinal Ligament Tissues of Patients with Ankylosing Spondylitis.

Recent studies have reported that circular RNAs (circRNAs) play a crucial regulatory role in a variety of human diseases. However, the roles of circRNAs in ankylosing spondylitis (AS) remain unclear. In this study, we conducted circRNA expression profiling of the spinal ligament tissues of patients with AS by RNA sequencing (RNA-seq) and analyzed the potential functions of differentially expressed circRNA by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses to investigate the potential mechanisms associated with AS. The results showed that a total of 1,172 circRNAs were detected in the spinal ligament tissue samples, of which 123 circRNAs were significantly differentially expressed by a fold change ≥ 1.5 and p value < 0.05. Among these, 57 circRNAs were upregulated, and 66 were downregulated. GO and KEGG analyses demonstrated that the differentially expressed circRNAs were mainly involved in the regulation of biological processes of peptidyl-serine phosphorylation and human immune system that may be related to AS. In addition, the circRNA/miRNA interaction networks were established to predict the potential roles of differentially expressed circRNAs by bioinformatics analysis. Taken together, these results revealed the expression profiles of circRNAs and the potential functions of the differentially expressed circRNAs in the spinal ligament tissue of patients with AS, which may provide new clues for understanding the mechanisms associated with AS, and proceed to identify novel potential molecular targets for the diagnoses and treatment of AS.

RAGE-dependent NF-kB inflammation processes in the capsule of frozen shoulders.

BACKGROUND: The etiology of frozen shoulder (FS) remains uncertain. Advanced glycation end-products (AGEs) cause the cross-linking and stabilization of collagen and are increased in FS. The purpose of this study was to elucidate the pathogenesis of FS by evaluating the receptor of AGE (RAGE)-dependent pathways. METHODS: Tissue samples of the coracohumeral ligament (CHL) and anterior inferior glenohumeral ligament (IGHL) were collected from 33 patients with FS, with severe stiffness, and 25 with rotator cuff tears (RCTs) as controls. Gene expression levels of RAGE, high-mobility group box 1 (HMGB1), Toll-like receptor 2 (TLR2), TLR4, nuclear factor-kappa B (NF-kB), and cytokines were evaluated using a quantitative real-time polymerase chain reaction. The immunoreactivities of carboxymethyllysine (CML), pentosidine, and RAGE were also evaluated. CML and pentosidine were further evaluated using high-performance liquid chromatography. RESULTS: Gene expression levels of RAGE, HMGB1, TLR2, TLR4, and NF-kB were significantly greater in the CHLs and IGHLs from the FS group than in those from the RCT group. Immunoreactivities of RAGE and CML were stronger in the CHLs and IGHLs from the FS group than in those from the RCT group. Pentosidine was weakly immunostained in the CHLs and IGHLs from the FS group. CML using high-performance liquid chromatography was significantly greater in the CHLs and IGHLs from the FS group than in those from the RCT group. CONCLUSIONS: AGEs and HMGB1 might play important roles in the pathogenesis of FS by binding to RAGE and activating NF-kB signaling pathways. Suppression of these pathways could be a treatment option for FS.

Migrating Myofibroblastic Iliotibial Band-Derived Fibroblasts Represent a Promising Cell Source for Ligament Reconstruction.

The iliotibial band (ITB) is a suitable scaffold for anterior cruciate ligament (ACL) reconstruction, providing a sufficient mechanical resistance to loading. Hence, ITB-derived fibroblasts attract interest for ligament tissue engineering but have so far not been characterized. This present study aimed at characterizing ITB fibroblasts before, during, and after emigration from cadaveric ITB explants to decipher the emigration behavior and to utilize their migratory capacity for seeding biomaterials. ITB and, for comparison, ACL tissues were assessed for the content of alpha smooth muscle actin (αSMA) expressing fibroblasts and degeneration. The cell survival and αSMA expression were monitored in explants used for cell isolation, monolayer, self-assembled ITB spheroids, and spheroids seeded in polyglycolic acid (PGA) scaffolds. The protein expression profile of targets typically expressed by ligamentocytes (collagen types I-III, elastin, lubricin, decorin, aggrecan, fibronectin, tenascin C, CD44, ß1-integrins, vimentin, F-actin, αSMA, and vascular endothelial growth factor A [VEGFA]) was compared between ITB and ACL fibroblasts. A donor- and age-dependent differing percentage of αSMA positive cells could be detected, which was similar in ITB and ACL tissues despite the grade of degeneration being significantly higher in the ACL due to harvesting them from OA knees. ITB fibroblasts survived for several months in an explant culture, continuously forming monolayers with VEGFA and an increased αSMA expression. They shared their expression profile with ACL fibroblasts. αSMA decreased during the monolayer to spheroid/scaffold transition. Using self-assembled spheroids, the migratory capacity of reversible myofibroblastic ITB cells can be utilized for colonizing biomaterials for ACL tissue engineering and to support ligament healing.

Gene expression in hip soft tissues in incipient canine hip dysplasia and osteoarthritis.

Canine hip dysplasia and developmental dysplasia of the human hip share demographic, phenotypic, and clinical features including the predisposition to develop osteoarthritis in affected joints. To support the results of genetic mapping studies for CHD and its concomitant osteoarthritis with functional information, we performed RNA-seq on hip capsule and teres ligament of affected and unaffected dogs. RNA seq showed that expressed genes segregated according age, capsule or ligament, and hip phenotype. Expression of HHIP, DACT2, and WIF1 was significantly higher in capsule from control hips than dysplastic hips indicating a disruption of the hedgehog signaling pathway. Expression of SPON 1, a key component of the WNT pathway, was increased significantly in both dysplastic capsule and ligament while FBN2 and EMILIN3 were significantly increased in dysplastic capsule. Of genes associated with human hip osteoarthritis, expression of ACAN, IGF1, CILP2, COL11A1, COL8A1, and HAPLN was increased significantly in dysplastic capsule. The significant increase in expression of PLA2F, TNFRSF, TMEM, and IGFBP in dysplastic capsule indicated an injury response. Gene set enrichment analysis revealed that genes involved in extracellular matrix structure, epithelial to mesenchymal transition, myogenesis, growth factor signaling, cancer and immune pathways were enriched in dysplastic capsule. For teres ligament from dysplastic joints, genes in retinoic signaling pathways and those encoding extracellular matrix molecules, but not proteoglycans, were enriched. Hip tissues respond to abnormal mechanics early in dysplastic hip development and these pathways present targets for intervention in the early synovitis and capsulitis secondary to canine and human hip dysplasia. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:313-324, 2019.

Effects of controlled abnormal joint movement on the molecular biological response in intra-articular tissues during the acute phase of anterior cruciate ligament injury in a rat model.

BACKGROUND: The anterior cruciate ligament (ACL) is responsible for braking forward movement of the tibia relative to the femur and for tibial rotation. After ACL injury, this braking performance deteriorates, inducing abnormal joint movement. The purpose of this study was to clarify the effects of controlled abnormal joint movement on the molecular biological response in intra-articular tissues during the acute phase of ACL injury. METHODS: Eighty-four mature Wistar male rats were randomly assigned to a controlled abnormal movement (CAM) group, an ACL-transection (ACL-T) group, a sham-operated group, or an intact group. The ACL was completely transected at its midportion in the ACL-T and CAM groups, and a nylon suture was used to control abnormal tibial translation in the CAM group. The sham-operated group underwent skin and joint capsule incisions and tibial drilling without ACL transection. Animals were not restricted activity until sacrifice 1, 3, or 5 days after surgery for histological and gene expression assessments. Acute-phase inflammation requires an important balance between degenerative and biosynthetic processes and is controlled by the activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Both types of gene were analyzed in this study. RESULTS: The ACL-T and CAM groups exhibited cleavage of the ACL at all time points. However, for the CAM group, the gap in the ligament stump was extremely small, and fibroblast proliferation was observed around the stump. Relative to the ACL-T group, the CAM group demonstrated significantly lower expression of MMP-13 mRNA and a lower MMP-13/TIMP-1 ratio on days 1 and 5 in the ACL, the medial meniscus and the lateral meniscus. The expression of TIMP-1 mRNA was not significantly different between the ACL-T and CAM groups. CONCLUSIONS: The study results suggested that controlling abnormal movement inhibited the inflammatory reaction in intra-articular tissues after ACL injury. This reaction was down-regulated in intra-articular tissues in the CAM group. Abnormal joint control caused prolonged inflammation and inhibited remodeling during the acute phase of ACL rupture.

Comparative proteome analysis of the capsule from patients with frozen shoulder.

BACKGROUND: The etiology of frozen shoulder (FS) is unclear. Accordingly, this study used a label-free quantitative shotgun proteomic approach to elucidate the pathogenesis of FS based on protein expression levels. METHODS: Tissue samples from the rotator interval (RI), middle glenohumeral ligament (MGHL), and anterior-inferior glenohumeral ligament (IGHL) were collected from 12 FSs with severe stiffness and 7 shoulders with a rotator cuff tear (RCT) as controls. Protein mixtures were digested and analyzed by nano-liquid chromatography/electrospray ionization-tandem mass spectrometry. Relative protein expression levels were calculated by the signal intensity of identified peptide ions on mass spectra. Differentially expressed proteins between FS and RCT samples were evaluated by a gene enrichment analysis using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. RESULTS: We identified 1594 proteins, 1358 of which were expressed in all 6 tissue groups. We detected more upregulated proteins in the upper (RI and MGHL) FS groups and the lower (IGHL) RCT group than in the comparative groups, respectively. Various proteins with functions in tissue repair, collagen metabolism and fibrillation, cell-cell and cell-matrix adhesion, blood coagulation, and the immune response were expressed more highly in the RI and MGHL FS groups than in the RCT group. Proteins with functions in phagocytosis, glutathione metabolism, retinoid metabolism, and cholesterol metabolism were expressed more highly in the IGHL RCT group than in the FS group. CONCLUSIONS: The pathophysiology of FS differs between the upper and lower parts of the joint capsule. Different treatment strategies for FS may be appropriate, depending on the location.

Topographical Anatomy of the Distal Ulna Attachment of the Radioulnar Ligament.

PURPOSE: The deep component of the distal radioulnar ligament provides translational stability and rotational guidance to the forearm. However, controversy exists regarding the importance of this structure as well as the nature of its attachment to the distal ulna. We aimed to evaluate the topographic anatomy of the distal ulna attachment of both the superficial and the deep components of the radioulnar ligament and to assess the relationship between its internal and its external morphometry. METHODS: Thirteen human distal ulnae attached by ulnar part of the distal radioulnar ligament were scanned using micro-computed tomography and reconstructed in 3 dimensions. In addition, the distal radioulnar ligaments were examined under polarized light microscopy to determine the histological characteristics of collagen contained within the ligaments. RESULTS: The deep limbs have broad marginal insertions at the fovea, whereas the superficial limbs have a circular and condensed insertion to the ulnar styloid. The center of the deep limb was separated from the base of the ulnar styloid by a mean of 2.0 ± 0.76 mm, and this distance was positively correlated with the width of the ulnar styloid. The mean distance between the center of the ulnar head and the center of the fovea was 2.4 ± 0.58 mm. The proportion of collagen type I was lower in the deep limb than in the superficial limb. CONCLUSIONS: This new observation of the footprint of the radioulnar ligament in the distal ulna indicates that the deep limb may serve as an internal capsular ligament of the distal radioulnar joint, whereas the superficial limb as the external ligament. CLINICAL RELEVANCE: Knowledge of the topographic anatomy of the radioulnar ligament's attachment to the distal ulna may provide a better understanding of distal radioulnar ligament-related pathologies.

Collagen Organization in Facet Capsular Ligaments Varies With Spinal Region and With Ligament Deformation.

The spinal facet capsular ligament (FCL) is primarily comprised of heterogeneous arrangements of collagen fibers. This complex fibrous structure and its evolution under loading play a critical role in determining the mechanical behavior of the FCL. A lack of analytical tools to characterize the spatial anisotropy and heterogeneity of the FCL's microstructure has limited the current understanding of its structure-function relationships. Here, the collagen organization was characterized using spatial correlation analysis of the FCL's optically obtained fiber orientation field. FCLs from the cervical and lumbar spinal regions were characterized in terms of their structure, as was the reorganization of collagen in stretched cervical FCLs. Higher degrees of intra- and intersample heterogeneity were found in cervical FCLs than in lumbar specimens. In the cervical FCLs, heterogeneity was manifested in the form of curvy patterns formed by collections of collagen fibers or fiber bundles. Tensile stretch, a common injury mechanism for the cervical FCL, significantly increased the spatial correlation length in the stretch direction, indicating an elongation of the observed structural features. Finally, an affine estimation for the change of correlation length under loading was performed which gave predictions very similar to the actual values. These findings provide structural insights for multiscale mechanical analyses of the FCLs from various spinal regions and also suggest methods for quantitative characterization of complex tissue patterns.

Distinct dysregulation of the small leucine-rich repeat protein family in osteoarthritic acetabular labrum compared to articular cartilage.

OBJECTIVE: Articular cartilage is well studied in osteoarthritis (OA). However, the role of supporting structures, such as the acetabular labrum, a sealing structure surrounding the hip joint, has been investigated much less. We recently showed that fibrochondrocytic labrum cells are metabolically active. This study was undertaken to investigate hip OA­associated changes in human acetabular labrum cells. METHODS: Microarray analysis was performed to compare OA labrum cells to healthy labrum cells cultured in a 3-dimensional alginate bead system. Data were analyzed by cluster analysis using gene set enrichment analysis software and by gene list analysis using PANTHER gene family tools. Selected candidates were validated by quantitative polymerase chain reaction analysis on labrum and cartilage samples and by immunohistochemistry. The functional impacts of the genes identified were investigated by in vitro stimulation experiments in labrum cells. RESULTS: Pathway analysis revealed increased cytokine and chemokine signaling in OA labrum cells, whereas reduced extracellular matrix interactions and transforming growth factor ß signaling were observed. Several genes were significantly differentially expressed in OA compared to healthy labrum. We specifically focused on 3 small leucine-rich repeat proteins (SLRPs), osteomodulin, osteoglycin, and asporin, that appeared to be distinctly regulated in OA labrum compared to OA cartilage. SLRPs were strongly down-regulated in OA labrum but up-regulated in OA articular chondrocytes. Moreover, in vitro stimulation with osteomodulin increased aggrecan expression in OA labrum cells. CONCLUSION: OA labrum fibrochondrocytes have several features similar to OA chondrocytes. However, SLRP expression seems to be differentially influenced by degeneration in OA labrum compared to cartilage, suggesting a specific role for this supporting structure in OA. The functional impact of SLRPs on labrum cells makes them interesting targets for further studies in hip OA.

The molecular composition of the extracellular matrix of the human iliolumbar ligament.

BACKGROUND CONTEXT: The human iliolumbar ligament connects the transverse process of L5 to the iliac crest and contributes to lumbosacral stability and has been associated with low back pain. However, different opinions exist regarding the functional relevance of the ligament. PURPOSE: In the present study, we analyze the regional molecular composition of the ligament extracellular matrix. STUDY DESIGN: Special attention is given to the attachment sites, to determine whether the ligament is subjected to a certain mechanical environment. METHODS: Iliolumbar ligament samples, extending from one enthesis to the other, were removed from 11 cadavers and fixed in methanol. Cryosections were immunolabeled with a panel of antibodies directed against collagens, glycosaminoglycans, proteoglycans, matrix proteins, and neurofilament. RESULTS: The mid-substance of the ligament labeled for all the molecules normally found in dense fibrous connective tissue including types I, III, and VI collagen, versican, dermatan -, chondroitin 4 -, and keratan sulfate. However, both entheses were fibrocartilaginous and labeled for type II collagen, aggrecan, and chondroitin 6- sulfate. A common feature was fat between the fiber bundles near the entheses. Occasionally this fat contained nerve fibers. CONCLUSIONS: The existence of fibrocartilaginous entheses suggests that the insertion sites of the ligament are subject to both tensile and compressive loading-probably because of insertional angle changes between ligament and bone during loading. Our findings support the suggestion that the iliolumbar ligament might play an important role in the stabilization of the lumbosacral junction.

Carpal tunnel syndrome is associated with high fibrinogen and fibrinogen deposits.

BACKGROUND: Idiopathic carpal tunnel syndrome (ICTS) is a common entrapment neuropathy. Some cases of ICTS are linked to mutations of the transthyretin gene, whereas others are associated with systemic amyloidosis. The majority of ICTS cases are of unknown etiology. OBJECTIVE: To study molecular mechanisms of ICTS development. METHODS: A total of 71 ICTS patients and 68 control subjects were included in the study. The fibrinogen level was determined before surgery and its deposition in the transversal carpal ligament (TCL) was detected by immunohistochemistry, Western blot, and mass spectrometry. Fibrinogen interaction with other proteins was studied by immunoprecipitation assay. RESULTS: Plasma levels of the proinflammatory and hemostatic protein fibrinogen are elevated in ICTS patients. Other measured systemic inflammatory markers were not affected, and local inflammatory responses in TCL were absent. ICTS patients have shorter bleeding times, probably because of the elevated plasma levels of fibrinogen. Polymorphisms of the fibrinogen B promoter region were previously associated with increased plasma fibrinogen, but this association was not observed among patients with ICTS. Interestingly, we detected fibrinogen deposits in the TCL, whereas transcriptional activity of the fibrinogen genes was low. Amyloidogenic proteins, including transthyretin and α-synuclein, were also found in the TCL, whereas their local transcriptional activity was rather high. Finally, we demonstrated that fibrinogen interacts with transthyretin and α-synuclein in TCL lysates. CONCLUSION: Our data indicate that fibrinogen and other aggregation-prone proteins have potentially important roles in the pathogenesis of ICTS.

Comparative transcriptional analysis of three human ligaments with distinct biomechanical properties.

One major aim of regenerative medicine targeting the musculoskeletal system is to provide complementary and/or alternative therapeutic approaches to current surgical therapies, often involving the removal and prosthetic substitution of damaged tissues such as ligaments. For these approaches to be successful, detailed information regarding the cellular and molecular composition of different musculoskeletal tissues is required. Ligaments have often been considered homogeneous tissues with common biomechanical properties. However, advances in tissue engineering research have highlighted the functional relevance of the organisational and compositional differences between ligament types, especially in those with higher risks of injury. The aim of this study was to provide information concerning the relative expression levels of a subset of key genes (including extracellular matrix components, transcription factors and growth factors) that confer functional identity to ligaments. We compared the transcriptomes of three representative human ligaments subjected to different biomechanical demands: the anterior cruciate ligament (ACL); the ligamentum teres of the hip (LT); and the iliofemoral ligament (IL). We revealed significant differences in the expression of type I collagen, elastin, fibromodulin, biglycan, transforming growth factor ß1, transforming growth interacting factor 1, hypoxia-inducible factor 1-alpha and transforming growth factor ß-induced gene between the IL and the other two ligaments. Thus, considerable molecular heterogeneity can exist between anatomically distinct ligaments with differing biomechanical demands. However, the LT and ACL were found to show remarkable molecular homology, suggesting common functional properties. This finding provides experimental support for the proposed role of the LT as a hip joint stabiliser in humans.

Benefit of doxycycline treatment on articular disability caused by dialysis related amyloidosis.

Abstract Doxycycline inhibits amyloid formation in vitro and its therapeutic efficacy is under evaluation in clinical trials for different protein conformational diseases, including prion diseases, Alzheimer's disease and transthyretin amyloidosis. In patients on chronic hemodialysis, a persistently high concentration of ß2-microglobulin causes a form of amyloidosis (dialysis-related amyloidosis, DRA) localized in bones and ligaments. Since doxycycline inhibits ß2-microglobulin fibrillogenesis in vitro and accumulates in bones, DRA represents an ideal form of amyloidosis where doxycycline may reach a therapeutic concentration at the site of amyloid deposition. Three patients on long-term dialysis with severe articular impairment and uncontrollable pain due to DRA were treated with 100 mg of doxycycline daily. Pharmacokinetics and safety of treatment were conducted. Plasmatic levels of the drug reached a plateau after one week (1.1-2.3 µg/ml). Treatment was well tolerated in two patients for a year, while one was suspended after 5 months due to mild esophagitis. Treatment was associated with a significant reduction in articular pain and with a significant and measurable improvement in passive and active movements in all cases, despite the persistence of unchanged amyloid deposits measured by magnetic resonance imaging.

Estrogen receptors in hip joint capsule and ligamentum capitis femoris of babies with developmental dysplasia of the hip.

OBJECTIVE: The aim of this study was to detect the incidence of estrogen receptors in human hip joint capsule and ligamentum teres. METHODS: The study included biopsies of the ligamentum capitis femoris (LCF) and hip joint capsule from 15 patients undergoing hip surgery for developmental dysplasia of the hip (DDH) and from the control hips of 15 cases of intrauterine fetal death. Mean age was 10.3 (range: 6 to 18) months at the time of surgery. Full-thickness 1x1 cm anterior capsule and LCF portions were taken as biopsy specimens. An immunohistochemical study using monoclonal antibody against estrogen receptors was performed to identify the rate of target estrogen cells in the hip joint capsule and LCF. RESULTS: Estrogen receptor (ER) staining rates were 1.6±0.2% for the LCF and 1.3±0.2% for the hip joint capsule in the control groups, and 2.5±0.3% for the LCF and 2.0±0.3% for the hip joint capsule in the DDH groups. Estrogen receptor staining rates in the LCF and hip joint capsule control groups were significantly lower than that in the DDH groups (p<0.001). In both groups, ER rates were significantly lower in the hip joint capsule than in the LCF (p<0.01). CONCLUSION: The high rate of ERs in the LCF and hip joint capsule appears to support the effect of estrogen in the etiology of the DDH.

Immunohistochemical composition of the human lunotriquetral interosseous ligament.

PURPOSE: The human lunotriquetral ligament (LTL) is a functionally important intrinsic hand ligament, which is assumedly subjected to insertion angle changes at the entheses during movement. To clarify whether the current model of the ligament's mechanical environment is reflected in its structural composition, we determined the regional distribution of extracellular matrix-related antigens. METHODS: The extracellular matrix was immunohistochemically investigated in 12 LTLs from both wrists of 6 human donors (Mean age: 60 y). RESULTS: The dorsal, proximal, and volar portions of the ligament immunolabeled for type I, III collagen and versican. Both entheses labeled strongly for type II collagen, aggrecan, and link protein and were distinctly cartilaginous. The ligament midsubstance was positive for collagen II in 30%, for aggrecan in 40%, and for keratocan and lumican in 100% of specimens. In contrast, keratocan and lumican were absent from the fibrocartilaginous entheses and the articular cartilage. Ligament insertion at a carpal bone occurs either directly through fibrocartilage or indirectly through a bilayered configuration of fibrocartilage and hyaline-like cartilage. The hyaline-like cartilage is continuous with the neighboring articular cartilage. CONCLUSIONS: The LTL has an extracellular matrix comparable with that of ligaments experiencing a combination of tensile and shear/compressive load at the attachment sites. All regions of the LTL exhibit fibrocartilaginous entheses; purely fibrous attachment sites are rare. The ligament midsubstance shows a more fibrous phenotype than the entheses and expresses keratocan and lumican, which previously have not been recorded in any human hand ligament.

Innervation patterns of thumb trapeziometacarpal joint ligaments.

PURPOSE: The human thumb trapeziometacarpal (TM) joint is a unique articulation that allows stability during pinch and grip and great degrees of mobility. Because the saddle-shaped articulating surfaces of the TM joint are inherently unstable, joint congruity depends on the action of restraining ligaments and periarticular muscles. From other joints, it is known that proprioceptive and neuromuscular joint stability depend on afferent information from nerve endings within ligaments. We hypothesize that the TM joint ligaments may similarly be innervated, indicating a possible proprioceptive function of the joint. METHODS: We harvested 5 TM joint ligaments in entirety from 10 fresh-frozen cadaver hands with no or only minor signs of osteoarthritis and suture-marked them for proximal-distal orientation. The ligaments harvested were the dorsal radial, dorsal central, posterior oblique, ulnar collateral, and anterior oblique ligaments. After paraffin-sectioning, we stained the ligaments using a triple-antibody immunofluorescent technique and analyzed them using immunofluorescence microscopy. RESULTS: Using the triple-stain technique, mechanoreceptors could be classified as Pacinian corpuscles, Ruffini endings, or Golgi-like endings. The 3 dorsal ligaments had significantly more nerve endings than the 2 volar ligaments. Most of the nerve endings were close to the bony attachments and significantly closer (P = .010) to the metacarpal insertion of each ligament. The anterior oblique ligament had little to no innervation in any of the specimens analyzed. DISCUSSION: The TM joint ligaments had an abundance of nerve endings in the dorsal ligaments but little to no innervation in the anterior oblique ligament. The Ruffini ending was the predominant mechanoreceptor type, with a greater density in the mobile metacarpal portion of each ligament. CLINICAL RELEVANCE: Presence of mechanoreceptors in the dorsal TM joint ligaments infers a proprioceptive function of these ligaments in addition to their biomechanical importance in TM joint stability.