Decreased elastic fibers and increased proteoglycans in the ligamentum flavum of patients with lumbar spinal canal stenosis.

Elastic fibers and proteoglycans are major components of the extracellular matrix and their changes have been reported in some pathological conditions. Further, recent studies have indicated that some glycosaminoglycans and proteoglycans inhibit elastic fiber assembly. The purpose of this study was to investigate changes of the elastic fibers and proteoglycans in the ligamentum flavum and analyze their relationships to thickening of the ligamentum flavum from lumbar spinal canal stenosis (LSCS). Ligamentum flavum samples were collected from 20 patients with LSCS (thickened flavum group) and 10 patients with lumbar disc herniation (non-thickened flavum group) as a control. Elastica-Masson staining and alcian blue staining were used to compare the relationship between the changes in the elastic fibers and proteoglycans. Gene and protein expressions of the elastic fibers and proteoglycans were analyzed by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Histological changes indicated that proteoglycans mainly increased on the dorsal side of the ligamentum flavum in accordance with the decreased elastic fibers in the thickened flavum group. The gene and protein expressions of fibrillin-2 and DANCE were significantly lower and decorin, lumican, osteoglycin, and versican were significantly higher in the thickened flavum group. Our study shows that elastic fibers decrease and proteoglycans increase in the thickened ligamentum flavum. Decreased gene expression of elastogenesis and disrupted elastic fiber assembly caused by increased proteoglycans may lead to a loss of elasticity in the thickened ligamentum flavum. Decreased elasticity may cause buckling of the tissue, which leads to thickening of the ligamentum flavum. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1241-1247, 2016.

Chondrogenic and fibrotic process in the ligamentum flavum of patients with lumbar spinal canal stenosis.

STUDY DESIGN: A histological, biological, and immunohisto-chemical study of human lumbar ligamentum flavum. OBJECTIVE: To analyze changes in the hypertrophied ligamentum flavum and clarify their etiology. SUMMARY OF BACKGROUND DATA: Hypertrophy of the ligamentum flavum has been considered a major contributor to the development of lumbar spinal canal stenosis (LSCS). Although previous studies have reported some factors related to ligamentum flavum hypertrophy, its etiology is still unclear. METHODS: Ligamentum flavum samples were collected from 20 patients with LSCS (LSCS group) and 10 patients with lumbar disc herniation (LDH group) as a control. The thickness of the ligamentum flavum was measured histologically. The amounts of elastic fibers and proteoglycans were assessed by Elastica-Masson staining and alcian blue staining, respectively. Gene and protein expressions related to fibrosis, inflammation, and chondrogenesis were analyzed by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. The total genes of the 2 groups were compared by DNA microarray analysis. RESULTS: The ligamentum flavum was significantly thicker in the LSCS group, which had a smaller amount of elastic fibers and a larger amount of proteoglycans. The gene expression related to fibrosis was significantly higher in the LSCS group; however, the immunoreactivities of collagen types I and III were weaker on the dorsal side of the ligamentum flavum in the LSCS group. The gene expression related to chondrogenesis and proteoglycan synthesis was significantly higher in the LSCS group. There was no significant difference in the gene expression related to inflammation between the 2 groups. CONCLUSION: Synthesis of the collagenous fibers and degradation of the elastic and collagenous fibers are both accelerated in the ligamentum flavum of patient with LSCS, which may be the reason for hypertrophy of the tissue. In addition, chondrogenesis and proteoglycan synthesis may have critical roles in the pathogenesis of the ligamentum flavum hypertrophy. LEVEL OF EVIDENCE: 5.

Joint immobilization induced hypoxic and inflammatory conditions in rat knee joints.

The purpose of this study was to examine the hypoxic and inflammatory conditions after immobilization in the joint capsule of rat knees. The unilateral knee joints of adult male rats were immobilized with an internal fixator (Im group) for 1 day, 3 days, and 1, 2, 4, 8, and 16 weeks. Sham-operated animals had holes drilled in the femur and tibia and screws inserted without a plate (control group). The number of cells and blood vessels in the capsule were histologically examined. The hypoxic condition in the capsule was histologically examined with a Hypoxyprobe™-1. The gene expressions related to the hypoxic (hypoxia inducible factor-1α, vascular endothelial growth factor, and fibroblast growth factor 2) and inflammatory conditions [interleukin-6 (IL-6), IL-1α, IL-1ß, tumor necrosis factor-α, and tumor necrosis factor-ß] were evaluated by quantitative reverse transcription polymerase chain reaction. The number of cells was unchanged at 1 day in the two groups; however, the number significantly increased at 3 days in the Im group. The number of blood vessels in the Im group gradually decreased. Strong immunostaining of Hypoxyprobe™-1 around the blood vessels was observed in the Im group. The gene expressions of hypoxia inducible factor-1α and fibroblast growth factor 2 were significantly higher in the Im group compared with those in the control group. The gene expressions of IL-6, IL-1α, IL-1ß, and tumor necrosis factor-ß were significantly higher in the Im group compared with those in the control group. These data indicated that joint immobilization induced hypoxic and inflammatory conditions in the joint capsule, which might be an initiating factor for joint contracture.

Comparison of articular cartilage images assessed by high-frequency ultrasound microscope and scanning acoustic microscope.

PURPOSE: The purpose of this study was to compare images of a newly developed high-frequency ultrasound imaging system (HFUIS) and scanning acoustic microscope (SAM) and to calculate their Pearson product moment correlations with a view to applying HFUIS for clinical use. METHODS: Cylindrical cartilage-bone complexes from adult male Sprague-Dawley rats were obtained. The specimens were immersed in normal saline and scanned by HFUIS. Intensity by HFUIS was normalised by reflection from a steel plate at the same distance. After the scanning, specimens were fixed with paraformaldehyde, decalcified and embedded in paraffin. Thinly sliced tissues were prepared for SAM evaluation. After the scanning, three layers of articular cartilage (superficial, middle and deep) were independently evaluated and their relationships calculated. RESULTS: The superficial and deep layers indicated high relative intensity, whereas the middle layer showed nonhomogeneous relative intensity by HFUIS. A high relative intensity by HFUIS and high sound speed area by SAM had strong correlations (Pearson product moment correlation, superficial layer 0.704, middle layer 0.731). CONCLUSIONS: HFUIS produced high-resolution images of the articular cartilage and its intensity was strongly correlated with sound speed by SAM.

Distribution of type A and B synoviocytes in the adhesive and shortened synovial membrane during immobilization of the knee joint in rats.

Joint immobilization is commonly used for the treatment of joint injuries and diseases, but it also causes unfavorable outcomes such as joint contracture. The purpose of this study was to examine the morphological changes of the synovial membrane that is suspected as a cause of joint contracture, and localization of type A (macrophage-like) and type B (fibroblast-like) synoviocytes in the capsule after joint immobilization. Male Sprague-Dawley rats were used in this study. Unilateral knee joints were rigidly immobilized at 150 degrees of flexion with internal fixators for 3 days, 1, 2, 4, 8, and 16 weeks (7 rats/each immobilized group), while 42 rats were sham-operated. Sagittal sections of 5 mum were prepared from the medial midcondylar region of the knee joints and assessed with histological, histomorphometric, and immunohistochemical methods. Adhesions were observed both in the anterior and posterior synovial membranes in the immobilized group after 2 weeks. In the adhesion area, the cells were mainly composed of type A synoviocytes that were positive for CD68 and type B synoviocytes positive for prolyl 4-hydroxylase subunit beta. The length of synovial membrane in the immobilized group was significantly shorter than that in the control group after 2 and 4 weeks. After 8 weeks, the adhesion area in the immobilized group became fibrous and hypocellular. The staining intensity of hyaluronic acid-binding protein was increased after 16 weeks. Adhesion and shortening of the synovial membrane and the structural changes of the adhesion area may contribute to the development of joint contracture.

Non-neuronal regulation and repertoire of cholinergic receptors in organs.

Many studies on the cholinergic pathway have indicated that cholinergic receptors, which are widely expressed in various cells, play an important role in all body organs. In this review, we present the concept that cholinergic responses are regulated through a neuronal or non-neuronal mechanism. The neuronal mechanism is a system in which acetylcholine binds to cholinergic receptors on target cells through the nerves. In the non-neuronal mechanism, acetylcholine, produced by neighboring cells in an autocrine/paracrine manner, binds to cholinergic receptors on target cells. Both mechanisms subsequently lead to physiological and pathophysiological responses. We also investigated the subunits/subtypes of cholinergic receptors on target cells, physiological and pathophysiological responses of the organs via cholinergic receptors, and extracellular factors that alter the subtypes/subunits of cholinergic receptors. Collectively, this concept will elucidate how cholinergic responses occur and will help us conduct further experiments to develop new therapeutic agents.

Intra-articular injection of hyaluronan diminishes loss of chondrocytes in a rat immobilized-knee model.

Joint immobilization is a useful and common treatment modality in orthopedics. However, it also causes unfavorable outcome such as articular cartilage degeneration. Intra-articular injection of hyaluronan has been accepted as a treatment of osteoarthritis, but its effects on immobilized joint remain to be clarified. Hyaluronan is a polysaccharide, distributed ubiquitously in various tissues. In this study, we examined the effect of hyaluronan on the articular cartilage in immobilized joints. The unilateral knee joints of adult male rats were immobilized at 150 degrees in flexion with an internal plate and screws for 1, 2, 4, 6, 8, 12, or 16 weeks (n = 84). Hyaluronan or saline (50 microl/each injection) was administered intra-articularly on the day of surgery and once a week. The articular cartilage from the medial midcondylar region of the knee was obtained, and divided into non-contact, contact and transitional areas (between the non-contact and the contact areas). In each area, a degree of degeneration was evaluated by histomorphometric grading, and measurements of thickness and number of chondrocytes. Histological grading scores in the hyaluronan group were smaller at 12 and 16 weeks compared with those in the saline group. The thickness of the articular cartilage increased in the transitional area in both groups. The number of chondrocytes in the contact and transitional areas gradually decreased, but their number in the hyaluronan group was greater at 12 and 16 weeks compared with that in the saline group. Hyaluronan showed chondroprotective effects on the articular cartilage in a rat immobilized-knee model.

[Multivariate analysis of factors affecting the status of wearing removable partial dentures].

PURPOSE: The purpose of this retrospective cohort study was to investigate factors that affected the status of wearing removable partial dentures (RPDs) using a multivariate analysis. METHODS: One hundred and sixty-one patients were treated with definitive dentures that were delivered by dental students under teacher supervision at Tohoku University Hospital between 1996 and 2001. Of the 161 patients, 67 patients who agreed to undergo a follow-up examination participated in this study. The subjects were 18 men and 49 women with a mean age of 66.0+/-9.5 years. They were re-examined 5 years after treatment. The status of wearing the RPDs was categorized into three groups, i.e: successful, wearing their original RPDs constantly for 5 years; replaced, wearing re-fabricated RPDs within 5 years, and failed; interrupted wearing the RPDs within 5 years. Factors that affected the status were also examined. The analyzed variables in this study were sex and age of subjects, previous experience in wearing RPDs, location of RPDs and edentulous area, number of occluding pairs, number of missing teeth, placement of RPDs on opposite jaw, denture base type, number of abutment teeth, type of clasp, and number of rests. A stepwise multiple logistic regression analysis was used to determine factors affecting the status of wearing RPDs. RESULTS: The response rate was 41.6%. Of the 90 RPDs, 55 RPDs were regarded as successful, 21 as replaced, and 14 as failed. Statistically significant correlations were found between denture usage and the age of subjects, the location of edentulous area, number of occluding pairs, and number of rests (p<0.05). CONCLUSION: The results suggest the importance of considering the significant factors mentioned above for treatment planning and designing of RPDs.

Expression of type I collagen in the capsule of a contracture knee in a rat model.

Contracture is a very common complication in daily examination and a fibrotic change of a capsule is suggested to be a one of the main causes. Type I collagen is a major component of a synovial capsule and also has been implicated in tissue elasticity of other organs. We immobilized the knee joints of 66 rats in 150 degrees of flexion using a plastic plate and metal screws. Sham operated knee joints had holes drilled and screws inserted but none of them were plated. The expression patterns of type I collagen were characterized using in situ hybridization and immunohistochemistry. The in situ hybridization demonstrated that the mRNA of type I collagen decreased rapidly after immobilization. However, the immunoreactivity of the capsule was not changed in the immobilized and the control groups at any time points. Other processes might be considered to evaluate the contracture capsule.

Osteoblasts and osteocytes express MMP2 and -8 and TIMP1, -2, and -3 along with extracellular matrix molecules during appositional bone formation.

Our previous studies suggested that a part of bone extracellular matrix (ECM) molecules are degraded and remodeled during embryonic bone formation. In contrast, little is known about ECM remodeling in postnatal appositional bone formation. The present study was designed to investigate expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during experimentally initiated appositional bone formation in rats. Expressions of ECM molecules, MMPs, and TIMPs were examined using in situ hybridization. Osteoblasts and osteocytes expressed MMP2 and -8, TIMP1, -2, and -3, as well as type I collagen, osteopontin, and osteocalcin in the course of the appositional bone formation, while they showed few transcripts of MMP13. The results indicated that while osteoblasts and osteocytes in the apposed bone produce ECM molecules, they degrade ECM molecules with MMPs and regulate the degradation by inhibiting the activity of MMPs using TIMPs. Osteoblasts and osteocytes may reorganize the ECM composition to mature the bone matrix in appositional bone formation.