Numerous musculoskeletal disorders are caused by thickened ligament, tendon stiffness, or fibrosis of joint capsule. Relaxin, a peptide hormone, can exert collagenolytic effect on ligamentous and fibrotic tissues. We hypothesized that local injection of relaxin could be used to treat entrapment neuropathy and adhesive capsulitis. Because hormonal effect depends on the receptor of the hormone on the target cell, it is important to confirm the presence of such hormonal receptor at the target tissue before the hormone therapy is initiated. The aim of this study was to determine whether there were relaxin receptors in the ligament, tendon, and joint capsular tissues of rats and to identify the distribution of relaxin receptors in these tissues. Transverse carpal ligaments (TCLs), inguinal ligaments, anterior cruciate ligaments (ACLs), Achilles tendons, and shoulder joint capsules were obtained from male Wistar rats. Western blot analysis was used to identify relaxin receptor isoforms RXFP1 and RXFP2. The distribution of relaxin receptors was determined by immunohistochemical staining. The RXFP1 isoform was found in all tissues examined. The RXFP2 isoform was present in all tissues but the TCLs. Its expression in ACLs tissues was relatively weak compared to that in other tissues. Our results revealed that RXFP1 and RXFP2 were distributed in distinctly different patterns according to the type of tissue (vascular endothelial cells, fibroblast-like cells) they were identified.
Animals ; Blotting, Western ; *Gene Expression Regulation ; Immunohistochemistry ; Ligaments/*metabolism ; Male ; Rats ; Rats, Wistar ; Receptors, G-Protein-Coupled/*genetics/metabolism ; Receptors, Peptide/*genetics/metabolism ; Shoulder Joint/*metabolism ; Tendons/*metabolism

OBJECTIVETo investigate electrophysiology of cardiocytes in ligament of Marshall.

METHODSThe single cardiocytes obtained from ligament of Marshall were direct observed under inverted microscope. The cardiocyte action potential and current density of I(Na), I(Ca), L, I(to), I(K) and I(K1) were researched by whole-cell patch-clamp techniques.

RESULTSThere were two different cardiomyocytes in ligament of Marshall, one was rod shape, the other was short-rectangle shape. The short-rectangle myocyte was short and thick; the rod myocyte was long and thin. The short-rectangle myocyte was more than rod myocyte. The length/width rate of short-rectangle myocyte was less than that of rod myocyte (2.99 +/- 0.95 vs 12.05 +/- 2.41, P < 0.01). The action potential of ligament myocytes was similar to fast responsive cells. The action potential amplitude (APA) and duration (APD) of short-rectangle cells were less than those in rod cells. APA (mV), APD(50) (ms) and APD(90) (ms) were respectively 80.02 +/- 3.68 vs 91.72 +/- 7.56, 69.62 +/- 6.33 vs 83.14 +/- 3.66 and 107.55 +/- 4.25 vs 144.00 +/- 5.15, P < 0.05. The ion current density of I(Na), I(Ca), L, I(to), I(K1) was different between the two kind cells.

CONCLUSIONSThere are two different cardiocytes in ligament of Marshall. The action potential and ion current density of I(Na), I(Ca), L, I(to), I(K1) are different between the two kind cardiocytes.

Action Potentials ; Animals ; Dogs ; Electrophysiology ; Ion Channel Gating ; Ligaments, Articular ; metabolism ; Male ; Myocytes, Cardiac ; metabolism ; physiology ; Patch-Clamp Techniques

OBJECTIVE: To investigate the effects of Toll like receptors on the osteogenesis of human pe-riodontal ligament stem cells (hPDLSCs) and probable molecular mechanism. METHODS: Real-time PCR and flow cytometry were applied to test the expression of TLRs in hPDLSCs and the positive cell percentage of TLR. hPDLSCs were cultured in osteogenic medium for 7 to 14 days with different TLR agonists at various concentrations . The effect of different TLR on osteogenic differentiation of hPDLSCs was evaluated by alizarin red S staining, alkaline phosphatase (ALP) staining and ALP activity assay. Western blotting was used to analyze the phosphorylation levels of extracellular regulated protein kinases (ERK), c-Jun N-terminal protein kinase (JNK), P38, AKT and expression of Runx2 an osteogenic related gene after treatment with TLR agonists, compared with the effect of inhibitors of mitogen activated protein kinase (MAPK) or protein kinase B (PKB or AKT) on Runx2 expression of hPDLSCs cultured in osteogenic medium. RESULTS: Higher expressions of TLR1,3,4,6 were found in hPDLSCs through real-time PCR. Positive cell percentage of TLR was determined by flow cytometry and described as TLR1: 2.82%±0.68%; TLR2: 1.26%±0.09%; TLR3: 13.23%±2.05%; TLR4: 3.64%±0.79%; TLR6: 3.21%±1.64%, whose tendency was comparable to their mRNA expression in hPDLSCs. Most TLR ligands had no effect on the ALP staining, activity and mineralization of hPDLSCs at lower concentration except for 0.1 mg/L PolyI:C could induce the osteogenic ability of hPDLSCs. On the contrary, Higher concentration of TLR ligands (PolyI:C: 10 mg/L, LPS: 10 mg/L , Pam3CSK4: 1 mg/L, FSL-1: 50 μg/L) had obviously inhibitory effect on osteogenic differentiation of hPDLSCs. Activation of TLR using higher concentration of TLR ligands could downregulate the phosphorylation levels of ERK, P38, JNK and AKT, and also reduced the expression of Runx2, compared with the untreated control. The inhibitors of MAPK (U0126, SP600125,SB203580) and inhibitor of AKT (perifosine) could also inhibit Runx2 expression. CONCLUSION Higher concentration of TLR ligands could inhibit osteogenic differentiation of hPDLSCs. This inhibitory effect seemed to be related to decreased phosphorylation of MAPK and AKT.
Cell Differentiation ; Cells, Cultured ; Humans ; Ligaments ; Mitogen-Activated Protein Kinases/metabolism* ; Osteogenesis ; Periodontal Ligament/metabolism* ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism* ; Stem Cells ; Toll-Like Receptors/metabolism*

BACKGROUNDPelvic organ prolapse (POP) is a major health problem in adult women that involves many factors. No proteomic analysis has been conducted exclusively in POP patients. This study aimed to identify the differential expression of proteins that may be involved in POP by proteomic analysis.

METHODSSamples of the uterosacral ligament (USL) were collected from five POP patients and five non-POP patients matched according to age, parity, and menopausal status and analyzed using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the mRNA expression of proteins that showed differential expression in the proteomic analyses.

RESULTSProteins differentially expressed between POP and non-POP patients were detected. Eight proteins that were down-regulated in the POP group were identified by MALDI-TOF-MS. These proteins included electron transfer flavoprotein, apolipoprotein A-I, actin, transgelin, cofilin-1, cyclophilin A, myosin, and galectin-1, and their expression was verified by qRT-PCR.

CONCLUSIONUsing comparative proteomics, we identified eight differentially expressed proteins (including four cytoskeleton proteins and three proteins related to apoptosis) in the USL that may be involved in apoptosis associated with the tissue effects in POP pathophysiology.

Actins ; metabolism ; Aged ; Apolipoprotein A-I ; metabolism ; Cyclophilin A ; metabolism ; Cytoskeleton ; metabolism ; Female ; Flavoproteins ; metabolism ; Galectin 1 ; metabolism ; Humans ; Ligaments ; metabolism ; Microfilament Proteins ; metabolism ; Middle Aged ; Muscle Proteins ; metabolism ; Myosins ; metabolism ; Pelvic Organ Prolapse ; metabolism ; Postmenopause ; metabolism ; Proteomics ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; Sacrum ; metabolism ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Uterus ; metabolism

The osteoarthritis is being emphasized in South Korea becoming an aged society. It is desirable to use a model that reflects human disease when using an animal model to understand the pathophysiology of osteoarthritis and verify the effective treatment materials. Because naturally occuring osteoarthritis is rare in rodent models, chemical or surgical methods are used to induce diseases. While these methods have the advantages of consistent occurrence and rapid progress of osteoarthritis, it is controversial about whether experimentally-induced osteoarthritis progresses in the same pathophysiology of human disease. The model using injection of chemical materials such as collagenase or monoiodoaceatate in joint space has been widely used. Each method leads to joint damage by chemical joint instability with destruction of articular connective tissue and cartilage cell apoptosis with inhibition of cell metabolism. Anterior cruciate ligament resection model, meniscus resection model, collateral ligament resection model, menisco-tibia ligament resection model and etc. are used as surgical models. These days, it tends to be used menisco-tibia ligament resection model more. It is required to observe the joint damage as well as induction of pain, recently. This review considers how to induce osteoarthritis of knee model used widely, usage of the pathogenesis studies, advantages and disadvantages.
Animals* ; Anterior Cruciate Ligament ; Apoptosis ; Cartilage ; Cartilage, Articular ; Collagenases ; Collateral Ligaments ; Connective Tissue ; Humans ; Joint Instability ; Joints ; Korea ; Ligaments ; Metabolism ; Models, Anatomic ; Models, Animal* ; Models, Chemical ; Osteoarthritis* ; Osteoarthritis, Knee ; Rodentia

We present a case of perivascular epithelioid cell tumors (PEComas) in the abdominal cavity at the falciform ligament. A 30-yr-old Korean man visited to hospital for the evaluation of a growing, palpable abdominal mass. He had felt the mass growing over 6 months. There was no family or personal history of tuberous sclerosis. The resected specimen showed a mass of 8.0x7.0x5.5 cm in size. Histological examination showed sheets of spindle-to-epithelioid cells with clear-to-eosinophilic cytoplasm. Immunohistochemically, tumor cells were positive for HMB-4 (gp100) and smooth muscle actin. They were also positive for the S-100, which is a marker of neurogenic and melanocytic tumors. Patient was treated with radical resection of tumor without any adjuvant therapy. He is well and on follow-up visits without tumor recurrence.
Abdominal Neoplasms/*diagnosis/pathology/surgery ; Actins/metabolism ; Adult ; Antigens, Neoplasm/metabolism ; Humans ; *Ligaments/pathology ; Male ; Neoplasm Proteins/metabolism ; Perivascular Epithelioid Cell Neoplasms/*diagnosis/pathology/surgery ; S100 Proteins/metabolism ; Tomography, X-Ray Computed

BACKGROUNDCervical spondylotic myelopathy (CSM), in part, results from degeneration of the posterior longitudinal ligament (PLL), which mechanically compresses the spinal cord. Much research was done on the ossification of PLL, but not concerning the non-ossifying degeneration of cervical PLL. The degeneration of cervical PLL may be related to inflammation. The aim of this study was to elucidate the pathological features of the PLL and the role of cyclooxygenase 2 (COX-2) in the degeneration of the PLL in CSM.

METHODSA total of 23 PLL specimens were collected during surgery from patients with CSM for the histological and immunohistochemical (type II collagen and Ki-67) study. For the control group 14 cervical PLL autopsy specimens were investigated in the same manner. mRNA expression of COX-2 was quantitatively measured by real-time reverse transcription-polymerase chain reaction (RT-PCR) from 18 PLL specimens of patients with CSM and 18 PLL specimens of autopsy cases. Immunohistochemistry was used to evaluate the cellular location of COX-2 in PLL.

RESULTSA distinct amount of fibrotic area, chondrometaplastic tissue and calcification were found in the PLL of the patient group, compared with the control group. Type II collagen was apparent around chondrometaplastic cells. Ki-67 positive reaction was less than 5%. A COX-2 positive reaction was found in 9 of the patient specimens (39.1%) in which the COX-2 was released from vascular endothelial cells in the PLL. However, such reactions were not found in the control group. Real-time PCR showed that the mRNA expression level of COX-2 in the patient group was significantly higher than that in the control group (P < 0.01).

CONCLUSIONSChondrometaplastic tissue producing type II collagen was identified as the most predominant pathological feature in the degenerative PLL. The higher expression of COX-2 might be related to degeneration of the PLL in CSM.

Adult ; Aged ; Aged, 80 and over ; Cervical Vertebrae ; enzymology ; pathology ; Collagen Type II ; metabolism ; Cyclooxygenase 2 ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; Ki-67 Antigen ; metabolism ; Longitudinal Ligaments ; metabolism ; Middle Aged ; Reverse Transcriptase Polymerase Chain Reaction ; Spinal Cord Compression ; enzymology ; Spondylosis ; enzymology

Bone marrow mesenchymal stem cells (BMSCs) can be directed to differentiate into a variety of cell types depending on their micro-environment. In this study, rat BMSCs were co-cultured with rat ligament fibroblasts during different time courses. The mRNA expressions of type I, type III collagens and tenascin-C were measured by real time RT-PCR, and the corresponding protein levels of type I and type III collagens by radioimmunoassay. Results show that the mRNA expressions of type I and type III collagens in the BMSCs were 2 times up-regulated after a 6-day co-culture, and the relative mRNA expressions of type I and type III collagens were 3.9 +/- 0.2 and 1.9 +/- 0.2, while they were 1.9 +/- 0.3 and 0.8 +/- 0.1 in the control groups, respectively. The protein syntheses of these two collagens were also increased after a 12-day co-culture; the type I and type III collagens synthesis were 13.6 +/- 1.3 ng/microg and 5.9 +/- 0.5 ng/microg in co-culture groups and 12.4 +/- 0.8 ng/microg and 5.0 +/- 0.4 ng/microg in their control groups, respectively. Likewise, there was a 2 times enhancement in tenascin-C mRNA expression after the 12-day co-culture (0.07 +/- 0.02 by control group and 0.14 +/- 0.02 by co-culture group, P < 0. 05). These data suggest that the presence of the ligament fibroblast promotes the syntheses of type I and the III collagens and tenascin-C in the rat BMSC.
Animals ; Bone Marrow Cells ; cytology ; Cells, Cultured ; Coculture Techniques ; Collagen Type I ; metabolism ; Collagen Type III ; metabolism ; Fibroblasts ; cytology ; Ligaments, Articular ; cytology ; Mesenchymal Stromal Cells ; cytology ; RNA, Messenger ; metabolism ; Rats ; Tenascin ; metabolism

OBJECTIVETo culture fibroblast cells from the knee ligaments and to study the biological characteristics of these cells.

METHODSCells of the anterior cruciate ligament (ACL) and the medial collateral ligament (MCL) from New Zealand white rabbit were cultured in vitro. Cellular growth and expression of the collagen were analyzed. Moreover, an in vitro wound closure model was established and the healing of the ACL and the MCL cells was compared.

RESULTSMaximal growth for all these cells were obtained with Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, but RPMI 1640 and Ham's F12 media were not suitable to maintain these cells. Morphology of both ACL and MCL cells from New Zealand white rabbit was alike in vitro, but the MCL cells grew faster than the ACL cells. Both cell types produced similar amount of collagen in culture, but the ratio of collage type I to type III produced by ACL cells was higher than that produced by MCL cells. Wound closure assay showed that at 36 hours after injury, cell-free zones created in the ACL cultures were occupied partially by the ACL cells; in contrast, the wounded zone in the MCL cultures was almost completely covered by the cells.

CONCLUSIONSAlthough the ACL cells and the MCL cells from New Zealand white rabbit show similar appearance in morphology in culture, the cellular growth and the biochemical synthesis of collagen as well as the healing in vitro were significantly different. These differences in intrinsic properties of the two types of cells in vitro might contribute to the differential healing potentials of these ligaments in vivo.

Animals ; Anterior Cruciate Ligament ; cytology ; Cell Division ; physiology ; Cells, Cultured ; Collagen ; metabolism ; Collateral Ligaments ; cytology ; Culture Media ; Female ; Fibroblasts ; physiology ; Male ; Rabbits ; Sensitivity and Specificity

BACKGROUNDAlthough various systemic and local factors such as abnormal carbohydrate or calcium metabolism, aging, and hormonal disturbances have been suggested as causes of ossification of the posterior longitudinal ligament (OPLL), the etiology of OPLL is not fully understood. The purpose of this study was to investigate whether bone morphogenetic protein (BMP)-2 is a candidate gene to modify the susceptibility of OPLL and the mechanism of signal transduction in ossification.

METHODSA total of 420 OPLL patients and 506 age- and sex-matched controls were studied. The complete coding sequence of the human BMP-2 gene was analyzed using polymerase chain reaction (PCR) and direct sequencing. All single nucleotide polymorphisms (SNPs) were detected and genotyped. BMP-2 expression vectors containing positive polymorphisms were constructed and transfected into the C3H10T1/2 cells. The expression of BMP-2 and the Smad signal pathway in positive cell clones were detected by Western blotting. The alkaline phosphatase (ALP) activity was determined using quantitative detection kits.

RESULTSThe frequencies for the 109T > G and 570A > T polymorphisms were different between the case and control groups. The "TG" genotype in 109T > G polymorphism is associated with the occurrence of OPLL, the frequency of the "G" allele is significantly higher in patients with OPLL than in control subjects (P < 0.001). The "AT" genotype in 570A > T polymorphism is associated with the occurrence of OPLL, the frequency of the "T" allele is significantly higher in patients with OPLL than in control subjects (P = 0.005). Western blotting analysis revealed that the expression of P-Smad1/5/8 protein transfected by wild-type or mutant expression vectors were significantly higher than control groups (P < 0.05), but there was no statistical difference in each experimental group (P > 0.05). The expression of Smad4 protein transfected by wild-type or mutant expression vectors was significantly higher than control groups (P < 0.05). The expression of Smad4 protein transfected by pcDNA3.1-BMP2 (109G) and pcDNA3.1-BMP2 (109G, 570T) was significantly higher than the other experimental groups (P < 0.05). The increase in ALP activity has been detected in pcDNA3.1-BMP2 (109G) and pcDNA3.1-BMP2 (109G, 570T) transfected cells up to 4 weeks after stable transfection. Activity of ALP was (30.56 ± 0.46) nmol×min(-1)×mg(-1) protein and (29.62 ± 0.68) nmol×min(-1)×mg(-1) protein, respectively. This was statistically different compared with the other experimental groups (P < 0.05).

CONCLUSIONSBMP-2 is the predisposing gene of OPLL. The "TG" genotype in the 109T > G and the "AT" genotype in the 570A > T polymorphisms are associated with the occurrence of OPLL. The 109T > G polymorphism in exon-2 of the BMP-2 gene is positively associated with the level of Smad4 protein expression and the activity of ALP. The Smad mediated signaling pathway plays an important role during the pathological process of OPLL induced by SNPs of BMP-2 gene.

Adult ; Aged ; Bone Morphogenetic Protein 2 ; genetics ; Cells, Cultured ; Female ; Humans ; In Situ Hybridization ; Longitudinal Ligaments ; metabolism ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; genetics ; Signal Transduction ; genetics ; physiology ; Smad Proteins ; metabolism ; Spine ; metabolism