FGF-2 signaling induces downregulation of TAZ protein in osteoblastic MC3T3-E1 cells.

Transcriptional coactivator with PDZ-binding motif (TAZ) protein is a coactivator of Runx2 and corepressor of PPARgamma. It also induces differentiation of mesenchymal cells into osteoblasts. In this study, we found that FGF-2, which inhibits bone mineralization and stimulates cell proliferation, reduced the TAZ protein expression level in osteoblast-like cells, MC3T3-E1. This reduction was recovered by removing FGF-2 from the culture medium, which also restored the osteoblastic features of MC3T3-E1 cells. Furthermore, FGF-2-induced reduction of TAZ is blocked by a SAPK/JNK-specific inhibitor. These findings suggest that the expression of TAZ protein is involved in osteoblast proliferation and differentiation. This may help elucidate the discrepancies in the effect of FGF-2 and contribute to the understanding of FGF/FGFR-associated craniosynostosis syndrome etiology and treatment.

Degree of mineralization-related collagen crosslinking in the femoral neck cancellous bone in cases of hip fracture and controls.

Based on the present definition of osteoporosis, both bone density and quality are important factors in the determination of bone strength. Collagen crosslinking is a determinant of bone quality. Cross-links can form enzymatically by the action of lysyl oxidase or non-enzymatically, resulting in advanced glycation end products. Collagen crosslinking is affected by tissue maturation as well as the degree of mineralization. Homocysteine and vitamin B6 (pyridoxal) are also regulatory factors of collagen crosslinking. We elucidate the relationship between the degree of mineralization and collagen cross-links in cancellous bone from hip fracture cases. We also determined plasma levels of homocysteine and pyridoxal. Twenty-five female intracapsular hip fracture cases (78 +/- 6 years) and 25 age-matched postmortem controls (77 +/- 6 years) were included in this study. Collagen crosslinking was analyzed after each bone specimen was fractionated into low (1.7-2.0 g/ml) and high (>2.0 g/ml) density fractions. The content of enzymatic (immature reducible and mature nonreducible cross-links) and nonenzymatic cross-link (pentosidine) were determined. In the controls, there was no difference in total enzymatic cross-links between low and high density bone, while pentosidine content was significantly higher in high density bone. In the fracture cases, not only reduced enzymatic cross-links in high density bone and increased pentosidine in both low and high density bone, but also higher plasma homocysteine and lower pyridoxal levels were evident compared with the controls. These results indicate that detrimental crosslinking in both low and high mineralized bone result in impaired bone quality in osteoporotic patients.

The "ligamentization" process in human anterior cruciate ligament reconstruction with autogenous patellar and hamstring tendons: a biochemical study.

BACKGROUND: There is little information documenting whether the phenomenon of "ligamentization," as proposed by Amiel, occurs in the human anterior cruciate ligament after clinically effective reconstruction. To clarify this point, we analyzed biochemical differences between the native anterior cruciate ligament; the patellar, semitendinosus, and gracilis tendons; and anterior cruciate ligaments reconstructed with autografts. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: Fifty patients who underwent arthroscopically assisted anterior cruciate ligament reconstruction using either semitendinosus and gracilis tendon or bone-patellar tendon-bone autografts were selected for the study. Samples of grafted tissue were collected during arthroscopy and quantitatively analyzed for collagen content and the amount of reducible and nonreducible crosslinks at 4 to 6 postoperative months in patients with semitendinosus and gracilis tendon grafts and at 11 to 13 months in all patients with semitendinosus and gracilis tendon or bone-patellar tendon-bone grafts. RESULTS: The total collagen content and nonreducible/reducible crosslink ratios increased significantly during the postoperative period (P < .05). The dihydroxylysinonorleucine/hydroxylysinonorleucine ratio was 3.11 +/- 0.56 in the native anterior cruciate ligament, 1.21 +/- 0.47 in the patellar tendon, and 3.59 +/- 1.58 in the anterior cruciate ligaments reconstructed with bone-patellar tendon-bone autografts 1 year after surgery. The dihydroxylysinonorleucine/hydroxylysinonorleucine ratio in both semitendinosus and gracilis tendons was less than 1.0. However, in anterior cruciate ligaments reconstructed with semitendinosus and gracilis tendon autografts, it was 2.34 +/- 0.98 at 4 to 6 months and 3.43 +/- 1.61 at 11 to 13 months after the operation. CONCLUSIONS: After anterior cruciate ligament reconstruction with autografts, biochemical characteristics of the graft resembled those of the native anterior cruciate ligament. These findings suggest that, regarding the amount of collagen crosslinks and their architecture, the phenomenon of ligamentization occurs in the successfully reconstructed human anterior cruciate ligament within 1 year after operation.

[Correlation between degree of mineralization and collagen cross-links as determinants of bone quality].

Little is known about the correlation between degree of mineralization and collagen properties (enzymatic and non-enzymatic cross-links) in bone. In the patients with femoral neck fracture, not only the mineral embrittlement but also the qualitative changes in collagen cross-links were observed in both low and high mineralized bone fractions. These results suggest that this trend towards an increased loss of collagen quality may have led to accelerated increase of fragility in osteoporosis.

Use of a biphasic graft constructed with chondrocytes overlying a beta-tricalcium phosphate block in the treatment of rabbit osteochondral defects.

To evaluate the ability of a biphasic construct to repair osteochondral defects in articular cartilage, plugs made of chondrocytes in collagen gel overlying a resorbable porous beta-tricalcium phosphate (TCP) block were implanted into defects in rabbit knees. The repair tissue was evaluated at 8, 12, and 30 weeks. Eight weeks after implantation of the biphasic construct, histologic examination showed hyaline-like cartilage formation that was positive for safranin O and type II collagen. At 12 weeks, most of the beta-TCP was replaced by bone, with a small amount remaining in the underlying cartilage. In the cell-seeded layer, the newly formed middle and deep cartilage adjacent to the subchondral bone stained with safranin O, but no staining was observed in the superficial layer. In addition, cell morphology was distinctly different from the deep levels of the reparative cartilage, with hypertrophic cells at the bottom of the cartilaginous layer. At 30 weeks, beta-TCP had completely resorbed and a tidemark was observed in some areas. In contrast, controls (defects filled with a beta-TCP block alone) showed no cartilage formation but instead had subchondral bone formation. These findings indicate that beta-TCP-supported chondrocytes in collagen gel can partially repair isolated articular cartilage osteochondral defects.

Radial head dislocation with acute plastic bowing of the ulna.

Five radial head dislocations with acute plastic bowing of the ulna in patients aged 6-12 years were reviewed. Closed reduction was successful in two, and open reduction was required in three patients in whom treatment was started more than 2 weeks after injury. In one child who presented 2 months after injury, realignment by osteotomy of the ulna as well as open reduction of the radial head was necessary. Follow-up evaluations at 6-24 months revealed good clinical outcomes in all patients. Awareness of this type of radial head dislocation is important to avoid delays in diagnosis and treatment.

Solitary eosinophilic granuloma of the sternum.

We report a rare case of eosinophilic granuloma of the sternum in a 25-year-old woman, who presented with anterior chest pain and a tender mass over the sternum. Total-body bone scintigraphy and computed tomography scanning of the thorax revealed an isolated lytic lesion of the manubrium. An open biopsy showed the typical histologic appearance of an eosinophilic granuloma. Surgical curettage of the solitary lesion was performed, and the sternal defect was filled with a bone replacement material. At the 2-year follow-up, no local recurrence was found, and the patient was in good health.

Analysis for the major contributor of collagenase to the primary cleavage of type II collagens in cartilage degradation.

Degradation of type II collagen is a central process in cartilage destruction seen in osteoarthritis and rheumatoid arthritis. Primary cleavage of type II collagen at the collagenase site is rate-limiting and is, therefore, a critical step for its degradation. The major contributor to this cleavage was identified in three isozymes of collagenase in human cartilage. Primary cultured human chondrocytes were used for the study. The production of collagenase-1 was major in total production for three isozymes of collagenase after stimulations with any concentration of tumor necrosis factor-alpha and/or interleukin-1 at 48 and 72 h, comprising 98% or greater of the total collagenase. When the production of collagenase-1 was specifically suppressed by the transfection with duplexes of 21-nucleotide small interfering ribonucleic acid into the cells, the activity of type II collagen cleavage was linearly decreased at neutral pH after activation. The relative contribution of collagenase-1 to the primary cleavage of type II collagen was determined to be 85%-93%. These findings suggest that collagenase-1 is a major contributor to the primary cleavage of type II collagens in human cartilage and is a potential therapeutic target for osteoarthritis and rheumatoid arthritis.

No mixing of granulocytes and other lymphocytes in the inflamed joints of parabiosis mice with collagen-induced arthritis: possible in situ generation.

Collagen-induced arthritis was evoked by an injection of lipopolysaccharide and anti-type II collagen antibody in mice. In parallel with the onset of arthritis, granulocytes with large light scatter and a Mac-1(+) Gr-1(+) phenotype expanded in the joints of these mice. Lymphocytes with a CD3(-) B220(+) phenotype (i.e. B220(+) B cells) were the major population among lymphocyte subsets in the joints, irrespective of disease. To determine the origin of these leucocyte populations in the joints and other organs, parabiotic experiments using CBF(1)Ly5.1 and CBF(1)Ly5.2 mice were conducted in mice with and without collagen-induced arthritis. As expected, leucocyte populations in the liver and spleen became a half-and-half mixture of their own cells and partner cells (e.g. approximately 45% of Ly5.1(+) cells in Ly5.2(+) partner mice). However, such a mixture was extremely delayed in the joints and bone marrow, even in mice with arthritis. These results suggest that, because circulatory blood is not exchanged in the joints, granulocytes and other lymphocytes are generated in situ in the inflamed joints of mice with collagen-induced arthritis or are possibly supplied by the bone marrow. It is of interest that granulocytes in the joints expanded, even without a supply from another site, namely, the synovium.

Expression analysis of three isoforms of hyaluronan synthase and hyaluronidase in the synovium of knees in osteoarthritis and rheumatoid arthritis by quantitative real-time reverse transcriptase polymerase chain reaction.

Hyaluronan is a major molecule in joint fluid and plays a crucial role in joint motion and the maintenance of joint homeostasis. The concentration and average molecular weight of hyaluronan in the joint fluids are reduced in osteoarthritis and rheumatoid arthritis. To elucidate the underlying mechanism, we analyzed the message expression of three isoforms of hyaluronan synthase and hyaluronidase from knee synovium, using real-time reverse transcriptase polymerase chain reaction. Synovia were obtained from 17 patients with osteoarthritis, 14 patients with rheumatoid arthritis, and 20 healthy control donors. The message expression of hyaluronan synthase-1 and -2 in the synovium of both types of arthritis was significantly less than in the control synovium, whereas that of hyaluronidase-2 in the synovium of both arthritides was significantly greater than in the control synovium. The decreased expression of the messages for hyaluronan synthase-1 and -2 and/or the increased expression of the message for hyaluronidase-2 may be reflected in the reduced concentration and decreased average molecular weight of hyaluronan in the joint fluids of patients with osteoarthritis and rheumatoid arthritis.

Intensity-related differences in collagen post-translational modification in MC3T3-E1 osteoblasts after exposure to low- and high-intensity pulsed ultrasound.

Low-intensity pulsed ultrasound (LIPUS) has distinct effects on biologic mineralization at intensities of <100 mW/cm2. Intensity-dependent differences in the pattern of accelerated mineralization may be due to different alterations in regulation of collagenous matrix formation. However, little is known about the influence of LIPUS on collagen metabolism in the context of mineralization processes. Therefore, we attempted to evaluate differential effects of two intensities of pulsed ultrasound (30 vs. 120 mW/cm2) on collagen post-translational modification and mineralization in osteoblastic MC3T3-E1 cells. Murine osteoblastic MC3T3-E1 cells were exposed to pulsed ultrasound (1.5-MHz, 200-ms burst sine wave at 1.0-kHz frequency, either 30 or 120 mW/cm2 SATA, for 20 min/day from Day 14 to Day 35 postconfluence). Expression patterns of lysyl oxidase (LO), procollagen-lysine, 2-oxyglutarate, 5-dioxigenase 1 (PLOD1, LH1), and 2 (PLOD2, LH2) was examined using quantitative PCR. Quantitative analysis of reducible immature cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible mature cross-links (pyridinoline and deoxypyridinoline) as well as analysis of the maturation of immature to mature cross-links were performed. Exposure to 30 mW/cm2 LIPUS upregulated LH2 mRNA expression and enzyme activity compared to controls. It was associated with increased relative amounts of telopeptidyl hydroxylysine (Hyl)-derived cross-links beginning on Day 14, upregulated LO mRNA expression, increased total reducible and nonreducible cross-links, and increased ratios of newly formed nonreducible to reducible cross-links. Similarities in the pattern of cross-link formation and calcium deposition in matrices between 30 mW/cm2 LIPUS-treated MC3T3-E1 cultures and bone suggest that 30 mW/cm2 LIPUS may promote the maturation of collagenous matrix as a scaffold for calcification. In contrast, exposure to 120 mW/cm2 ultrasound increased calcium accumulation compared to control at Day 35, but increases were delayed until Day 25. No differences in the extent and pattern of cross-links were observed compared to controls. These results suggest that the promotion of mineralization induced by 120 mW/cm2 may be attributed to other factors involved in mineralization process rather than cross-link pattern. Our results demonstrated the existence of differential effects of lower versus higher intensities of ultrasound on mineralization processes in vitro.

Bone formation and bioresorption after implantation of injectable beta-tricalcium phosphate granules-hyaluronate complex in rabbit bone defects.

The objective of this study was to evaluate the effects of a complex of beta-tricalcium phosphate (beta-TCP) granules and 3.5% hyaluronate (beta-TCP granules-HY complex) compared with a beta-TCP block, in terms of osteoconductivity and biodegradability, to determine whether this complex would be a good candidate for bone void filler. Both materials were implanted into cavities drilled in rabbit femoral condyles. New bone formation and mineral apposition rate were evaluated to analyze osteoconductivity, whereas residual beta-TCP within the defects and tartrate-resistant acid phosphatase (TRAP) cellular activity were studied for beta-TCP resorption. The results show that both the beta-TCP block and the beta-TCP granules-HY complex support bone ingrowth; however, bioresorption was rapid for beta-TCP granules-HY but weak for beta-TCP block. This biodegradation mechanism was considered to be a cell-mediated disintegration by numerous TRAP-positive giant cells. The time lag between the peak value of TRAP-positive giant cell population and that of new bone formation rate suggests that a coupling-like phenomenon could be occurring in the beta-TCP-filled bone defects. In addition, beta-TCP granules-HY complex, which is an injectable, pastelike material, has similar osteoconductive properties to beta-TCP block. Thus, this complex may be useful as a bone filler in clinical application.

Tendon suspension sling arthroplasty for degenerative arthritis of the thumb trapeziometacarpal joint: long-term follow-up.

Twenty-two tendon suspension sling arthroplasties for thumb trapeziometacarpal osteoarthritis were reviewed. Age at operation ranged from 53 to 74 years (average, 65 years); 20 of the 22 patients were women. The average follow-up period was 101 months (range, 63-139 months). At the final follow-up, 18 of 22 thumbs were painless. Grip and side pinch measurements averaged 98% and 79% of unaffected opposite sides, respectively. Range of motion at the trapeziometacarpal joint improved, and the average shortening of the trapezial space was 6.4 mm. Function was improved in all cases. The long-term results show that tendon suspension sling arthroplasty is a useful surgical procedure and an attractive alternative to existing tendon interposition arthroplasty with ligament reconstruction.

Old rupture of the triceps tendon with unique pathology: a case report.

We report the case of a 23-year-old man who presented 4 years after a triceps tendon rupture. A cord-like tissue, palpable and visible at full elbow flexion, was found to sublux to the lateral aspect of the elbow. Repair of the tendon has been achieved utilizing an artificial polyester mesh to augment the repair and permit early mobilization of the elbow. Although this patient regained excellent motion and function, awareness of this type of injury is necessary, and careful examination is needed at the time of the initial presentation.

Autoimmune reaction to type II collagen and cartilage degeneration in MRL/Mp- lpr/lpr mouse.

The early phase of cartilage degeneration was immunohistochemically examined in order to clarify the importance of autoimmune reaction against type II collagen in MRL/Mp- lpr/lpr (MRL/ l) mouse in an experimental model of rheumatoid arthritis (RA). Anti-type II collagen antibodies were detectable in 3-week-old mice and preceded the appearance of rheumatoid factors. Furthermore, mesenchymal cells and tartrate-resistant acid phosphatase-positive cells began to accumulate remarkably in the periphysis, a fibrochondro-osseous area in the bone marrow vicinity. The numbers of these cells increased with mice age, together with serum levels of anti-type II collagen antibodies. Immunostaining of the periphysis revealed expression of type II collagen, IgG, C3, Mac-3, MHC class II antigen Ia, and cathepsin-L. Osteoclast-like cells and macrophage infiltration into the lesion area were confirmed by transmission electron microscopy. The results indicate that cartilage degeneration in MRL/ l mouse may originate in the periphysis and progress via a pathway independent of synovial invasion.

Sequence-specific silencing of MT1-MMP expression suppresses tumor cell migration and invasion: importance of MT1-MMP as a therapeutic target for invasive tumors.

Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) has been believed a key enzyme in tumor invasion, because it is expressed in a variety of malignant human tumors, and overexpression of the enzyme enhances the ability of cellular invasiveness. However, it has not necessarily been clarified whether the endogenously expressed MT1-MMP in human tumors plays a critical role in their invasiveness. We used RNA silencing technology to downregulate the endogenous MT1-MMP expression in human tumor cells (fibrosarcoma HT1080 and gastric carcinoma MKN-28 cell lines), and evaluated the effect on the invasion of a reconstituted basement membrane (Matrigel). Transfection of a double-stranded RNA targeted to the MT1-MMP gene decreased the level of the enzyme to less than 10-20% without affecting production of other MMPs. According to the degree of silencing, activation of proMMP-2 was inhibited. CD44 shedding was also inhibited, but only in part. Decreased MT1-MMP levels were also reflected in reduced cell motility on hyaluronan (HA) and invasion in Matrigel. Thus, specific downregulation of MT1-MMP expression was sufficient to cause significant inhibition of the migration and invasion of tumor cells, even though other MMPs continued to be expressed.

Effect of hyper- and microgravity on collagen post-translational controls of MC3T3-E1 osteoblasts.

UNLABELLED: We attempted to study the effects of microgravity (by clinostat) and hypergravity (using centrifugation) on collagen metabolism using murine MC3T3-E1 osteoblasts, especially focusing on collagen cross-link formation. We found that altered gravitational load affected the post-translational modification of collagen, particularly the collagen maturation pathway, through altered expression of enzymes involved in cross-link formation. INTRODUCTION: Gravitational loading plays important roles in the stimulation of differentiated osteoblast function and in the maintenance of skeletal tissues, whereas microgravity seems to result in osteopenia caused by impaired osteoblast differentiation. The aim of our study was to clarify the effects of altered gravitational environments on collagen metabolism, particularly the relationship between post-translational collagen quality and enzymes involved in cross-link formation, using murine osteoblastic MC3T3-E1 cells. MATERIALS AND METHODS: Cells were cultured under vector-averaged microgravity (1 x 10(-3) g) using a clinostat or under conventional centrifugation techniques to generate hypergravity (20 g and 40 g) for 72 h. We then examined the expression patterns of lysyl oxidase and the two lysyl hydroxylase isoforms telopeptidyl lysyl hydroxylase (TLH; procollagen-lysine, 2-oxyglutarate, 5-dioxigenase 2 [PLOD2]) and helical lysyl hydroxylase (HLH; [PLOD1]) by quantitative real time polymerase chain reaction (PCR) analysis. Quantitative analysis of reducible immature (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible mature (pyridinoline and deoxypyridinoline) cross-links, and maturation rate analysis of immature to mature cross-links by conventional metabolic labeling using tritium lysine were also performed. RESULTS: Hypergravity upregulated both TLH mRNA expression and enzyme activity compared with stationary cultures, whereas microgravity stimulated both HLH mRNA expression and enzyme activity. These results were consistent with increased relative occupancy rates of telopeptidyl hydroxylysine-derived cross-links and helical hydroxylysine-derived forms observed under hypergravity and microgravity, respectively. Hypergravity stimulated not only lysyl oxidase mRNA expression but also increased enzyme activity and the sum of immature and mature cross-links. Furthermore, the conversion rate of immature cross-links to mature compounds was markedly increased under hypergravity but decreased under microgravity. CONCLUSION: Altered gravitational loading may affect the post-translational modification of collagen through altered expression of enzymes involved in cross-link formation. These observations may be important in elucidating the mechanisms of osteopenia during space flight.

In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH.

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on the expression of the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)(2)D(3) was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 microg/kg of 1,25(OH)(2)D(3) inhibited the PTH-induced RANKL mRNA expression, but 0.5 microg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)(2)D(3) dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)(2)D(3) suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)(2)D(3) stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling.

Vitamin D and bone.

It is now well established that supraphysiological doses of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] stimulate bone resorption. Recent studies have established that osteoblasts/stromal cells express receptor activator of NF-kappaB ligand (RANKL) in response to several bone-resorbing factors including 1alpha,25(OH)(2)D(3) to support osteoclast differentiation from their precursors. Osteoclast precursors which express receptor activator of NF-kappaB (RANK) recognize RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of macrophage-colony stimulating factor (M-CSF). Osteoprotegerin (OPG) acts as a decoy receptor for RANKL. We also found that daily oral administration of 1alpha,25(OH)(2)D(3) for 14 days to normocalcemic thyroparathyroidectomized (TPTX) rats constantly infused with parathyroid hormone (PTH) inhibited the PTH-induced expression of RANKL and cathepsin K mRNA in bone. The inhibitory effect of 1alpha,25(OH)(2)D(3) on the PTH-induced expression of RANKL mRNA occurred only with physiological doses of the vitamin. Supraphysiological doses of 1alpha,25(OH)(2)D(3) increased serum Ca and expression of RANKL in vivo in the presence of PTH. These results suggest that the bone-resorbing activity of vitamin D does not occur at physiological dose levels in vivo. A certain range of physiological doses of 1alpha,25(OH)(2)D(3) rather suppress the PTH-induced bone resorption in vivo, supporting the concept that 1alpha,25(OH)(2)D(3) or its derivatives are useful for the treatment of various metabolic bone diseases such as osteoporosis and secondary hyperparathyroidism.