Involvement of Notch1/Hes signaling pathway in ankylosing spondylitis.

We aimed to investigate the role of Notch1/Hes signaling pathway in the pathogenesis of abnormal ossification of hip ligament in patients with ankylosing spondylitis (AS). 22 AS patients scheduled for artificial hip arthroplasty were randomly chosen as AS group. As controls, we used 4 patients diagnosed with transcervical fracture who underwent hip replacement surgery. Notch1 and Hes mRNA expressions were detected by real-time fluorescent quantitative polymerase chain reaction (RFQ-PCR). Immunohistochemistry (IHC) was used to detect Notch1 and Hes protein expression. Correlation analyses of Notch-l and Hes with AS-related clinical factors were conducted with spearman's correlation analysis and partial correlation analysis. RFQ-PCR results showed significant differences in Notch1 and Hes mRNA expressions between AS group and the control group (all P<0.05). IHC analysis further indicated positive nuclear signals of Notch1 and Hes protein, indicating functional activation of the Notch1 and Hes pathways. Semi-quantitative IHC showed a higher Notch1 and Hes expression levels in AS group compared to the control group (all P<0.05). Correlation analysis suggested that Hes protein expression was positively associated with the clinical course of the disease in AS patients. In conclusion, Notch1 and Hes overexpression was clearly detected in hip joint ligaments of AS patients, Hes protein expression was associated with the clinical course of AS. Taken together, we suggest that signaling pathways mediated by Notch1-Hes may contribute to ligament ossification of hip joints in AS patients.

Are myofibroblasts activated in idiopathic carpal tunnel syndrome? an immunohistochemical study.

OBJECTIVES: This study aims to investigate whether myofibroblasts participate in the fibrotic process of idiopathic carpal tunnel syndrome (CTS). PATIENTS AND METHODS: Forty patients (12 males, 28 females; median age 50.85 ± 11.2 years; range 30 to 71 years) who were operated in our clinic between March 2010 and August 2010 were included in the study. Twenty-five idiopathic CTS patients were assigned to the study group, and 15 trauma patients were assigned to the control group. Samples were taken from both transverse carpal ligament and subsynovial connective tissue (SSCT) of participants and they were analyzed by immunohistochemical method. Four immunohistochemical markers were used to analyze myofibroblast existence and vascular wall thickness (alpha smooth muscle actin [α-SMA]), collagen type IV antibodies, and T (CD3) and B (CD20) lymphocytes. RESULTS: The existence of myofibroblasts (α-SMA) in SSCT of patients who were in early phase of idiopathic CTS was shown through the positive reaction of their antibodies with fibroblasts. A significantly increased reaction of α-SMA and collagen antibodies in vascular structure of SSCT demonstrated increased vascular wall thickness and vascularity in the study group (p<0.01). No significant difference was detected between the two groups in terms of T and B lymphocyte antibody reaction (inflammation). CONCLUSION: The findings of this study indicate a potential for myofibroblasts to be activated during the early phase of the disease and contribute to the onset of disease. Further studies with larger sample sizes would be of great assistance in determining the role of myofibroblasts in idiopathic CTS.

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.

Analysis of estrogen binding sites of the posterior ligament of the human TMJ.

BACKGROUND: Conflicting results have been reported regarding the presence or absence of estrogen-binding sites (EBS) in the human temporomandibular joint (TMJ). The possible role of female sex hormones in the pathophysiology of internal derangement of the TMJ has been suggested to explain the prevalence of TMJ symptoms in female patients. PATIENTS AND METHODS: Posterior bilaminar tissue excised during TMJ articular disc repositioning and posterior ligament repair was taken from 28 patients (26 female, 2 male) for evaluation. Cryosections were stained using a monoclonal antibody (Mab) against EBS. To ensure efficacy of the antibody staining procedure, an internal positive control consisting of human breast tissue previously proven EBS-positive was used. No asymptomatic control TMJ tissue was available for our study. RESULTS: None (0%) of 28 TMJ tissue specimens showed nuclear-staining positive for the presence of EBS in the posterior bilaminar tissue of the TMJ. However, estrogen-binding sites associated with probable inflammatory cells were observed. Our results are consistent with the probability of positives as high as 0.1234 using a 95% confidence interval. CONCLUSIONS: The lack of EBS of the posterior ligament of the TMJ suggests that the role of estrogen contributing to internal derangement of the TMJ appears not to be significant.

The inferior glenohumeral ligament: a correlative investigation.

The inferior glenohumeral ligament (IGHL) was investigated by correlating the biomechanical properties, biochemical composition, and histologic morphology of its 3 anatomic regions (superior band, anterior axillary pouch, and posterior axillary pouch) in 8 human cadaveric shoulders. The overall biochemical composition of the IGHL appeared similar to other ligaments, with average water content of 80.9 +/- 2.5%, collagen content of 80.0 +/- 9.2%, and crosslinks of 0.715 +/- 0.13 mol/mol collagen. The proteoglycan content was highest in the superior band (2.73 +/- 0.7 mg/g dry weight) and may, in part, explain its viscoelastic behavior. Histologic analysis demonstrated longitudinally organized fiber bundles that were more uniform in the mid-substance but more interwoven and less uniformly oriented near the insertion sites. The superior band had the most pronounced fiber bundle interweaving, while crimping was more evident in the anterior axillary pouch. Elastin was identified in each of the regions. Tensile testing demonstrated a trend toward higher ultimate tensile stress (16.9 +/- 7.9 MPa) and tensile modulus (130.3 +/- 47.9 MPa) in the superior band compared to the axillary pouch. The mean ultimate tensile strain of the IGHL was 16.8 +/- 4.6%. These complex IGHL properties may help to explain its unique functions in stabilizing the shoulder in different arm positions and at different rates of loading, including the failure patterns seen clinically, as in Bankart lesions (insertion site) versus capsular stretching (ligament substance).

Do facet joint capsular ligaments contain estrogen receptors? Application to pathogenesis of degenerative spondylolisthesis.

Studies showing that degenerative spondylolisthesis is 4 to 5 times more common in females than in males have suggested that hormonal influences account for this gender difference. Estrogen has been shown to play a role in other instabilities, such as those of the anterior cruciate ligament and the shoulder capsular ligaments, and estrogen receptors have been identified in these tissues. We wanted to assess facet joint capsular ligaments for the presence of such receptors. Accordingly, we collected facet joint capsular ligaments from 14 consecutive patients undergoing lumbar spinal fusion. Tissue sample analysis was performed by immunohistology using prediluted estrogen monoclonal antibody (Vantana) and automated immunostaining on a Vantana instrument. None of the specimens analyzed contained estrogen receptors. We conclude that, though degenerative spondylolisthesis may have hormonal influences, estrogen seems not to play a direct role in its development.

Fibrocartilage in the transverse ligament of the human atlas.

STUDY DESIGN: Immunohistochemical investigation. OBJECTIVE: To determine whether molecules typical of articular cartilage are present in the transverse ligament and whether the ligament may be a target for an autoimmune response in rheumatoid arthritis. SUMMARY OF BACKGROUND DATA: In chronic rheumatoid arthritis there is often a marked instability of the atlantoaxial complex, and the transverse ligament can show degenerative changes that compromise its mechanical function. In some rheumatoid patients there can be an autoimmune response to cartilage link protein, aggrecan, and Type II collagen. METHODS: Transverse ligaments were removed from 13 cadavers and fixed in 90% methanol. Cryosections were immunolabeled with antibodies against proteoglycans (aggrecan, link protein, and versican), glycosaminoglycans (chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and keratan sulfate), and collagens (Types I, II, III, and VI). RESULTS: Labeling for aggrecan and link protein was characteristic of the fibrocartilages, but versican was only detected in the fibrous regions. Equally, Types I, III, and VI collagens and keratan, dermatan, and chondroitin-4-sulfates were found throughout the ligament, but labeling for Type II collagen and chondroitin-6-sulfate was restricted to the fibrocartilages. CONCLUSION: The presence of molecules typical of articular cartilage (aggrecan, link protein, and Type II collagen) in the transverse ligament explains why it can be a target for destruction in rheumatoid arthritis and also suggests that it is subject to constant compression against the dens rather than only at the extremes of movement.

Identification of sex hormone receptors in human and rabbit ligaments of the knee by reverse transcription-polymerase chain reaction: evidence that receptors are present in tissue from both male and female subjects.

Gender-related factors have been attributed to observed differences in the rate of injury to ligaments (e.g., anterior cruciate ligament) between male and female subjects. These differences may be a result of unique regulatory mechanisms within the tissue in response to the sex hormones estrogen and progesterone. These hormones, when bound to specific intracellular receptors (estrogen receptor and progesterone receptor, respectively), modulate gene expression within hormone-responsive tissue. The purpose of this study was to evaluate the expression of the estrogen and progesterone receptors in ligament tissue from male and female rabbits and humans by the sensitive molecular technique of reverse transcription-polymerase chain reaction. Total RNA was extracted from human anterior cruciate ligament tissue and from medial cruciate ligament, anterior cruciate ligament, patellar tendon, and synovium tissue of the New Zealand White rabbit by the newly developed TRIspin method. The total RNA was reverse transcribed and analyzed by polymerase chain reaction to assess the expression of estrogen and progesterone receptors. Our results demonstrate that estrogen and progesterone receptor transcripts are expressed in ligament tissue of male and female rabbits and humans and that alterations in receptor expression occur in ligaments during pregnancy. In the human samples, only a small percentage of the estrogen receptor appeared to be a nonfunctional mRNA splice variant, and the predominant form contained the estrogen-binding domain.

pO2 measurement in an experimental model of patellar tendon autograft pro-anterior cruciate ligament.

Thirty-four sheep were submitted to surgery substituting the native ACL with the central third of the patellar tendon, ten enter this study. The purpose was to find a possible relationship between tissue pO2 and healing processes considering also the biomechanical and histomorphological aspects of the grafts. Four of them were sacrificed under general anaesthesia after 6 months, and six after 1 year in order to perform tissue pO2 measurement and an analysis of microvessel density on specimens of the normal ACL and the graft. Our data showed higher pO2 values of the autografts after 6 months. After 1 year the data was comparable to those of native ACL. This was confirmed by a microvessel count of the histological specimens and the data was in relationship to biomechanical and histomorphological analysis. Tissue pO2 can be observed and recorded in "in vivo" ACL, and patellar tendon used as graft, with no injury to their integrity. The monitoring system might be considered as an experimental tool for indirect controls of the anterior cruciate substitutes.

The intrinsic and extrinsic ligaments of the wrist. A correlation of collagen typing and histologic appearance.

20 fresh frozen human wrist specimens were dissected. Gross and histological examination and biochemical evaluation were performed on the intrinsic ligaments (scapho-lunate and luno-triquetral) and extrinsic ligaments (radio-scapho-capitate and radio-lunate). All ligaments were observed to have longitudinal collagen bundles. The intrinsic ligaments had large amounts of fibrocartilage near insertions and no elastin, while the extrinsic ligaments had little fibrocartilage and sparse amounts of elastin. The intrinsic ligaments were noted to have significantly more collagen Type 3 (41%) than the extrinsic ligaments (19%). These findings suggest that structural differences between the intrinsic and extrinsic ligaments of the wrist may in part account for the biomechanical observations that the intrinsic ligaments are stronger and elongate further prior to failure than the extrinsic ligaments.

Abnormal joint mechanics and the proteoglycan composition of normal and healing rabbit medial collateral ligament.

OBJECTIVE: This study investigated the influence that abnormal joint mechanics may have upon the biochemical composition of the joint's own soft tissue holding elements. DESIGN: The investigation used an animal model of ligament injury, the rabbit medial collateral ligament (MCL). The proteoglycan component of the ligament extracellular matrix was extracted, purified and characterized. INTERVENTIONS: The experimental groups consisted of: a) a control group consisting of the MCL from both right and left knees of six animals that had not undergone surgery; b) a group (healing gap injury) of six MCL from right knees in which a segment of tissue had been excised from the anterior cruciate and the MCL of the right knee 3 wk prior to sacrifice; and c) a third group (contralateral gap injury) comprised of the MCL from the six left knees of the same gap injury animals. OUTCOME MEASURES: The MCL water content, total proteoglycan content, hexose and hexuronate-containing proteoglycan and proteoglycan electrophoretic mobility were determined for each group studied. RESULTS: The healing gap injury MCL was found to have a higher water content, a higher total proteoglycan content and a higher proportion of aggregating proteoglycan than MCL from control animals. The nonaggregating proteoglycan fraction from the contralateral MCL (group 3) had a greater electrophoretic mobility and probably, therefore, a smaller molecular weight than that found in the MCL from the same knee of control animals. CONCLUSIONS: Since MCL healing took place in an abnormal mechanical environment, these results suggest that joint biomechanics may be an important factor in mediating connective tissue proteoglycan composition.

Immunoreactive neuropeptides in nerves in ligamentous tissue. An experimental neuroimmunohistochemical study.

A search for neuropeptide nerves in the healing of the experimentally ruptured medial collateral ligament (MCL) of the rabbit knee used specific antisera to the neuropeptides Substance P, calcitonin gene-related peptide (CGRP), and galanin. Sutured and unsutured MCLs were studied four and 14 weeks postoperatively. Both fluorescent thin nerve strands and small dotlike nerve terminals were regularly seen in the healing zone and in the adjacent normal ligamentous tissue, suggesting innervation of such structures by neuropeptide nerves. All three neuropeptides were more abundant in sutured ligaments than in unsutured ligaments, which may suggest beneficial effects of the apposition of the torn ligament ends on local nerve regeneration. Active involvement of the neural elements in the healing process was also suggested by kinetic studies showing a decrease in Substance P and CGRP staining as well as an increase in galanin staining during the study period. These changes in the periphery parallel the reactive changes earlier described in the dorsal root ganglion and dorsal horn cells occurring after a peripheral nerve injury. This may depend on the antidromic transport to the periphery of neuropeptides synthesized in the central nervous system. This experimental neuroimmunohistochemical mapping study and the known effects of neuropeptides on blood vessels, macrophages, and fibroblasts should stimulate further work on the role of innervation in ligamentous healing.