Abstract: Growth arrest in the epiphyseal plate during childhood often causes both periarticular deformities and limb length discrepancy, leading to compartmental osteoarthrosis and gait disturbance or spinal disorders, respectively. Distraction osteogenesis using external fixators with hinge systems appears to be useful for the simultaneous correction of deformity and shortening. In this paper, we evaluated cases of lower limbs with periarticular deformities and limb length discrepancy after epiphyseal plate injury that has been treated by distraction osteogenesis using external fixators. This is the first report regarding the outcomes of distraction osteogenesis for a group of patients having deformity and limb length discrepancy due to traumatic arrest of the physis. Successful outcomes may promise the use of this method as the first choice for the treatment of growth disorders after the arrest of the epiphyseal plate in the lower limbs. However, treatment under 20 years of age may provide a better outcome with a lower incidence of complications.

Abstract: Pycnodysostosis is a rare hereditary disease, characterized by systemic bone sclerosis. The most important orthopedic problem in this condition is the recurrent pathological fracture of long bones. In this paper, the surgical results for fractures of six limbs (three femurs and three tibias) in five cases of pycnodysostosis are reported. Five limbs achieved fracture union and union is developing in one tibia after intramedullary nail (IM) nailing or Ilizarov external fixation (IEF), although fracture line tends to persist for longer periods of time. One femoral fracture was treated by IM nailing, and one femoral and one tibial fracture were treated by IEF leading to final bone union. One femoral and one tibial fracture were initially treated by IEF, and were treated by IM nailing after re-fracture. One tibial fracture was initially treated by IEF leading to a failure of union, and was converted to IM nailing. All cases are able to walk; one case requires a single crutch. Infection was noted in two limbs after IM nailing following IEF. Fixation with IM nail was effective in preventing re-fracture as well as in alignment correction. Although the surgical technique is more difficult, IM nailing in the initial surgery may be a better choice for achieving successful union while reducing the risk of re-fracture or infection.

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Abstract: A 72-year-old woman with periprosthetic femoral fracture after cementless total hip arthroplasty (THA) underwent external fixation using the Ilizarov method. Although open reduction and internal fixation with a condylar plate system were initially attempted, deep infection with methicillin-resistant Staphylococcus aureus at the fracture site occurred 2 weeks postoperatively. Six weeks after removal of the plating system, the fracture was stabilized with external fixation using the Ilizarov method and went on to successful fusion at 3 months. To our knowledge, this is the first report in which Ilizarov external fixation has been used for periprosthetic femoral fracture after THA. Although this is a rare situation, where periprosthetic fracture and infection coexist, Ilizarov external fixation is a safe and reliable method for periprosthetic femoral fracture with infection.

Abstract: A case of deformity and shortening after post-traumatic growth arrest treated using the Taylor Spatial Frame (Smith & Nephew, Tennessee, USA) is presented. This is the first report showing the application of the frame for post-traumatic deformity in the distal femur, and successful outcomes promise utilization of the frame even for correction of severe deformity in the distal femur.

Abstract: The process of fracture healing involves a number of regenerative mechanisms underlying the skeletal systems, and bone morphogenetic protein (BMP) has been believed to be a key molecule during the reaction. Recent investigations showed that several members of BMPs are induced and activated by the impact of fracture, and play important roles via BMP receptors/Smads pathways. BMPs are newly synthesized by callus-forming cells near the fracture site, and form a "BMP-network" at the fracture callus. The "BMP-network" contributes to the regulation of callus formation, and the network contains noggin, sonic hedgehog (Shh), hepatocyte growth factor (HGF), and vascular endothelial cell growth factor (VEGF). Potential roles of BMPs during fracture repair open the way towards the formulation of new therapeutic strategies for promising fracture management. Recent molecular technologies with use of recombinant BMP and gene therapy will lead to the development of less-invasive and more successful fracture treatment.

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Abstract: Paget's disease is a localized bone disorder marked by increased turnover usually associated with a deformity of the affected bone. Distraction osteogenesis may be a useful method for correcting the deformity. A 57-year-old woman with Paget's disease had a 3 cm limb-length discrepancy with a 47 degrees procurvatum deformity of the ipsilateral femur. Distraction osteogenesis using a Taylor Spatial Frame was performed, leading to complete correction of the procurvatum deformity and limb-length discrepancy. After improvement of the limb-length discrepancy, the patient felt more comfortable than she had before surgery with less low back pain. This result suggests distraction osteogenesis may be appropriate for the treatment of some severe, complex deformities of the lower limbs in patients with Paget's disease.

Abstract: This study presents the clinical and radiographic outcomes of 6 feet (4 patients) with relapsed idiopathic clubfoot that were treated with a combination of subtalar release and the Ilizarov method. The mean patient age at the time of the surgery was 7.4 years (range, 4.5-10.5 years), and the mean follow-up was 5.1 years (range, 2.0-7.3 years). All cases achieved a plantigrade foot, better walking ability, and parental satisfaction with the result. Ankle joint range of motion increased from a mean of 17 degrees (range, 10-30 degrees) preoperatively to 45 degrees (range, 35-65 degrees) at final follow-up. The talocalcaneal angle improved from a mean of 26 degrees (range, 15-34 degrees) preoperatively to 55 degrees (range, 47-65 degrees) at follow-up. The mean tibiocalcaneal angle improved from 95 degrees (range, 87-115 degrees) preoperatively to 80 degrees (77-83 degrees) at follow-up, whereas the talometatarsal angle improved from a preoperative mean of -19 degrees (range, -35 to -10 degrees) to 3.5 degrees (range, -5 to 7 degrees) at follow-up. Recurrence was observed in only 1 foot with forefoot adductus, caused by a pin tract infection and early fixator removal. These cases suggest the Ilizarov method combined with subtalar release are beneficial for the treatment of relapsed idiopathic clubfoot.

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Abstract: In the current study, we investigated whether the systemic administration of alendronate, a third-generation bisphosphonate, suppressed the loosening of screws at the bone-screw interface. We systemically administered alendronate to rats fitted with external fixators. External fixators with two half pins were applied to the right femurs of rats, and alendronate was administrated once a week during a 5-week postoperative period. Radiographic, histologic, and immunohistochemical findings subsequently were analyzed. Treatment with alendronate reduced the width of the fibrous loosening membrane and the number of osteoclasts at the bone-screw interface. These findings indicate that systemic treatment with alendronate exerts an inhibitory effect on local bone resorption at the bone-screw interface.

Abstract: Leptin has been suggested to mediate a variety of actions, including bone development, via its ubiquitously expressed receptor (Ob-Rb). In this study, we investigated the role of leptin in endochondral ossification at the growth plate. The growth plates of wild-type and ob/ob mice were analyzed. Effects of leptin on chondrocyte gene expression, cell cycle, apoptosis and matrix mineralization were assessed using primary chondrocyte culture and the ATDC5 cell differentiation culture system. Immunohistochemistry and in situ hybridization showed that leptin was localized in prehypertrophic chondrocytes in normal mice and that Ob-Rb was localized in hypertrophic chondrocytes in normal and ob/ob mice. Growth plates of ob/ob mice were more fragile than those of wild-type mice in a mechanical test and were broken easily at the chondro-osseous junction. The growth plates of ob/ob mice showed disturbed columnar structure, decreased type X collagen expression, less organized collagen fibril arrangement, increased apoptosis and premature mineralization. Leptin administration in ob/ob mice led to an increase in femoral and humeral lengths and decrease in the proportional length of the calcified hypertrophic zone to the whole hypertrophic zone. In primary chondrocyte culture, the matrix mineralization in ob/ob chondrocytes was stronger than that of wild-type mice; this mineralization in both types of mice was abolished by the addition of exogenous leptin (10 ng/ml). During ATDC5 cell differentiation culture, exogenous leptin at a concentration of 1-10 ng/ml (equivalent to the normal serum concentration of leptin) altered type X collagen mRNA expression and suppressed apoptosis, cell growth and matrix calcification. In conclusion, we demonstrated that leptin modulates several events associated with terminal differentiation of chondrocytes. Our finding that the growth plates of ob/ob mice were fragile implies a disturbance in the differentiation/maturation process of growth plates due to depletion of leptin signaling in ob/ob mice. These findings suggest that peripheral leptin signaling plays an essential role in endochondral ossification at the growth plate.

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Abstract: Osteoclastogenesis is a key event of the cellular reaction in prosthetic loosening. Immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) were used to study the localization and expression of receptor activator of nuclear factor kappa B ligand (RANKL), a potent factor for osteoclastogenesis in the membranous tissue formed around loosened prosthetic joint implants. RANKL was identified in a wide variety of cells appearing in this membranous tissue. At least three types of RANKL-positive cells were identified, including prolyl 4-hydroxylase (PH)-positive fibroblast lineage cells, CD68 cells, and tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multi-nucleated macrophage lineage cells. Tumor necrosis factor (TNF)-alpha-converting enzyme (TACE) was colocalized with RANKL in these cells, suggesting the in-situ release of this factor. RT-PCR confirmed the actual expression of the RANKL and TACE genes in the tissues around the loosened implant. These observational findings indicate the possible synthesis of RANKL by fibroblast and macrophage lineage cells, and suggest the in-situ involvement of RANKL in both osteoclastogenesis and osteoclastic bone resorptive events occurring in prosthetic joint loosening.

Abstract: Bone morphogenetic proteins (BMPs), inducers of ectopic bone formation in vivo, are present in a number of osteosarcomas. BMPs are responsible for reactive bone formation, including periosteal reactions by normal osteoblasts, rather than production of tumorous osteoid by tumor cells. Osteosarcomas producing BMPs contain less-differentiated mesenchymal cells, resulting in a poorer prognosis for those patients. BMPs are also expressed in malignant fibrous histiocytomas (MFHs) of bone and dedifferentiated chondrosarcomas exhibiting undifferentiated features. However, BMPs in MFH do not show any osteoinductive activity in vivo, suggesting that those BMPs may be inactive forms and have additional functions unrelated to bone formation. Among benign bone tumors, BMPs are expressed in osteoid osteomas or osteoblastomas and effect reactive bone formation such as a surrounding sclerosis. BMPs and a BMP receptor (BMPRIB) are also detected in the cartilage cap in osteochondroma, suggesting that BMP signaling via BMPRIB might be involved in the pathogenesis of osteochondroma. Clinically, BMPs have utility as diagnostic and prognostic markers for characterizing the stage of differentiation of mesenchymal cells and mesenchymal tumors, and they may be of value in predicting the prognosis of sarcoma patients. This article reviews the accumulated information on BMPs in bone tumors, including the most recent findings, and discusses the biological and clinical significance of BMPs in bone tumors.

Abstract: This report from five hospitals in Japan describes the results of correcting adult tibial deformities using external fixation. There were 49 patients with 59 lower limb deformities, with trauma being the most common cause of the deformity. Varus angulation was the most common deformity, and the most common magnitude was 11 degrees -30 degrees. Twenty-two patients had a leg-length discrepancy. The aim of the correction was to normalize both the mechanical axis and the inclination of the knee and ankle joints. In 63% of the patients corrections were performed gradually during bone lengthening or acutely after bone lengthening. Altogether, 71% of the patients were completely corrected, and no leg-length discrepancies remained after correction in 47%. Complications were encountered in 22 patients, about half of which were pin tract infections, 28% refractures, and the remainder delayed consolidation or fixator failure. There were no neurological or circulatory complications. The average fixation duration was 9 months. The average hospital charges were 3,740,000 yen in bilateral correction patients and 1,940,000 yen in unilateral correction patients. External fixation can correct not only the mechanical axis and joint inclination but also leg-length discrepancy simultaneously.

Abstract: Localization and expression of mRNAs for sonic hedgehog (Shh) at a fracture site in the early phase postfracture were investigated by in situ hybridization and reverse transcription and polymerase chain reaction (RT-PCR). A closed fracture was made in the midshaft of the right tibia of 5-week-old ICR mice, and fractured sites were harvested prefracture (day 0) and on days 2 and 12. In situ hybridization revealed that transcripts for Shh were not detected on day 0, but they were detected in proliferating callus-forming cells in the periosteum and the surrounding tissue, and in the medullary cavity prior to apparent new cartilage and bone formation. Gli 1 (a signaling mediator for Shh) and bone morphogenetic protein-4 transcripts were colocalized with those for Shh transcripts on day 2. The RT-PCR showed that Shh mRNA was detected in the PCR product from day 2, but not from days 0 and 12. These findings are the first description about the activation of Shh gene in the early postfracture reaction.

Abstract: STUDY DESIGN: Various amounts of static mechanical load were applied to mouse intervertebral discs in organ cultures. The apoptosis then was examined using nick end labeling. Two mitogen-activated protein kinase (MAPK) inhibitors were added to the medium. OBJECTIVES: To establish an experimental model for detecting factors regulating chondrocyte apoptosis induced by mechanical stress, and to determine the role of MAPK and p38 in the stress-induced apoptotic pathway of endplate chondrocytes. SUMMARY OF BACKGROUND DATA: The cause of degenerative change in the cartilaginous endplate (CEP) remains unclear. The authors' previous findings using a mouse model suggested that apoptosis in the cartilaginous endplate may play a role in intervertebral disc degeneration, and that mechanical stress may induce apoptosis. If apoptosis of endplate chondrocytes is involved in the cascade of intervertebral disc degeneration, then how apoptosis is induced by mechanical stress should be important in preventing disc degeneration. However, the mechanism of apoptosis induced by mechanical stress remains unclear. METHODS: Mouse coccygeal discs were harvested and organ cultured. Various static compression loads (0, 0.2, 0.4, 0.8, and 1.0 MPa) were applied on intervertebral discs placed in culture bottles for 24 hours. Paraffin-embedded sections of the harvested discs were stained using Safranin-O and the nick end labeling procedure. The apoptotic cells were counted in the cartilaginous endplate and junctional anulus fibrosus of each intervertebral disc. In addition, U0126 (MAPK inhibitor) and SB202190 (p38 inhibitor) were added to the culture medium to determine their regulatory roles in the apoptosis of endplate chondrocytes induced by mechanical load. RESULTS: Histologically, loaded discs became bulged, and the disc space became narrow. Apoptosis was absent in discs without load, but was particularly noticeable in loaded discs (load weight, 1.0 MPa). The number of apoptotic cells increased depending on the weight of the load. The two MAPK inhibitors significantly increased the number of apoptotic cells. CONCLUSIONS: Chondrocyte apoptosis was induced using a static mechanical load especially in the cartilaginous endplate in an organ culture. Apoptosis occurred similarly to previous findings using an in vivo model. This culture system thus reflected the apoptosis demonstrated in vivo. Because biologically active reagents such as MAPK inhibitors can be simply added to culture media, this system may be a useful method for detecting factors that influence apoptosis induced by mechanical stress. Both MAPK inhibitors increased the occurrence of apoptosis. This suggests that these two MAPKs can counteract the apoptotic pathway induced by mechanical stress.

Abstract: OBJECTIVES: To examine the localization of bone morphogenetic protein (BMP)-2 mRNA and protein in human osteoarthritic (OA) articular cartilage and osteophyte. DESIGN: Five normal, four growing and 14 OA human cartilage samples, graded histomorphologically by Mankin Score, were studied by in situ hybridization and immunohistochemistry for the expression of BMP-2. RESULTS: BMP-2 mRNA was present in chondrocytes in neonatal growing articular cartilage, but was scarcely present in normal adult articular cartilage. In OA articular cartilage, BMP-2 mRNA and protein were detected in both clustering and individual chondrocytes in moderately or severely damaged OA cartilage. In moderately damaged OA cartilage, BMP-2 mRNA was localized in both upper and middle zone chondrocytes, but was not detected in deep layer chondrocytes. In severely damaged OA cartilage, cellular localization of BMP-2 mRNA was extended to the deep zone. In the area of osteophyte formation, BMP-2 mRNA was intensely localized in fibroblastic mesenchymal cells, fibrochondrocytes, chondrocytes and osteoblasts in newly formed osteophytic tissue. The pattern of BMP-2/4 immunolocalization was associated with that of mRNA localization. CONCLUSIONS: BMP-2 mRNA and BMP-2/4 were detected in cells appearing in OA tissues. BMP-2 was localized in cells of degenerating cartilage as well as osteophytic tissue. Given the negative localization of BMP-2 in normal adult articular cartilage, BMP-2 might be involved in the regenerating and anabolic activities of OA cells, which respond to cartilage damage occurring in osteoarthritis.

Abstract: We investigated the effects of treatment with anti-parathyroid hormone-related protein (1-34) monoclonal murine antibody (anti-PTHrP MoAb) on apoptosis and the differentiation of chondrosarcoma HTB-94 cells. Treatment with anti-PTHrP MoAb accelerated apoptosis of HTB-94 cells in a dose-dependent manner, and anti-PTHrP MoAb also promoted the chondrogenic differentiation of HTB-94 cells. The induction of apoptosis by anti-PTHrP MoAb via imbalance of Bcl-2/Bax ratio and activation of caspase-3 may provide a mechanistic explanation for its potential antitumor effects. Our results suggest the possibility that anti-PTHrP MoAb may be beneficial as a new treatment for chondrosarcoma.

Abstract: The bone morphogenetic protein (BMP) family consists of a large number of members and has diverse biological activities during development. Various tissues express pleural BMP family members, which seem to cooperatively regulate developmental events. Here, multiple BMP signals were inactivated in chondrocytes to clarify the function of BMPs during skeletogenesis. To obtain tissue-specific inactivation, Noggin gene (Nog) was overexpressed in cartilage under the control of a2(XI) collagen gene (Collla2) promoter/enhancer sequences. The resultant transgenic mice lacked most of their cartilaginous components, suggesting that cartilage does not develop without BMP signals. These effects seem to be mediated through down-regulation of Sox9 expression. Conversely, specific BMP signals were activated in the skeleton by targeted expression of Bmp4 in cartilage and the resultant phenotype was compared with that of transgenic mice expressing growth and differentiation factor-5 (GDF-5), another BMP family member. Overactivity of Bmp4 in the skeleton caused an increase of cartilage production and enhanced chondrocyte differentiation, as GDF5 expression did, but it did not disturb joint formation as GDF5 did. During skeletogenesis, unique roles of each BMP may reside in the regulation of joint development. Together with the common effect on the cartilage overproduction by Bmp4 and GDF5 overactivation, loss of cartilage by inactivation of multiple BMPs in Noggin transgenic mice indicates that signals for cartilage production are reinforced by multiple BMPs exclusively. These conclusions may account for the reason why multiple BMPs are coexpressed in cartilage.

Abstract: OBJECTIVE: To investigate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) at sites of joint destruction in rheumatoid arthritis (RA) and to correlate it with the production of matrix metalloproteinases (MMPs). METHODS: Reverse transcription-polymerase chain reaction was performed to study the existence of EMMPRIN in synovial tissue derived from RA and osteoarthritis (OA) patients. In situ hybridization with a human complementary DNA specific for EMMPRIN and immunohistochemistry were performed to characterize the EMMPRIN-expressing cells at sites of joint destruction, including bone. Northern blot analysis was performed to detect the level of expression of EMMPRIN messenger RNA (mRNA) in synovial tissue. The production of MMP-1 and MMP-3 by synovial tissue from RA patients was examined by enzyme-linked immunosorbent assay. RESULTS: Expression of EMMPRIN mRNA was detected in synovium from 9 of 11 patients with RA and 1 of 5 patients with OA. The presence of mRNA encoding EMMPRIN was recognized in the invasive synovium at sites of joint destruction in RA but not OA. Fibroblast-like synovial cells and granulocytes were demonstrated to express EMMPRIN mRNA. MMP-1 and MMP-3 production by synovial tissue was correlated with levels of expression of EMMPRIN mRNA, as detected by Northern blotting. CONCLUSION: The expression of EMMPRIN stimulates the production of MMP-1 and MMP-3 in the synovial tissue of affected joints in RA. The results of this study suggest that EMMPRIN may be one of the important factors in progressive joint destruction in RA.

Abstract: Calcium hydroxyapatite ceramics (CHA) are nontoxic materials, provoke little reaction from tissues, and by virtue of these properties represent a good starting point for creating bone substitutes. Although several porous CHAs have been used clinically, there have been few reports that CHA is fully replaced by newly formed bone, which may be due to its structure and the limited connectivity between pores. We recently developed a fully interconnected porous CHA (IP-CHA) by adopting a "foam-gel" technique. Structural analysis by scanning electron microscopy revealed that IP-CHA had spherical pores of uniform size that were interconnected by window-like holes. The surface of the wall structure was smooth, and hydroxyapatite particles were bound tightly to one another. Most of the interpore connections of IP-CHA ranged from 10 to 80 microm in diameter (average, 40 microm). When the cylindrical IP-CHA (diameter, 6 mm; height, 15 mm) was implanted into a rabbit femoral condyle, bone, and bone marrow with abundant vessels formed deep in the pores through the interpore connections. Within a period of 6 weeks, new bone had formed and penetrated to a distance of 3 mm from the surface of the IP-CHA implant. Furthermore, a compression test at 9 weeks revealed that the implanted IP-CHA steadily increased in strength to more than double the value of the initial test. These results indicate that the IP-CHA may have clinical utility as a superior bone substitute.

Abstract: Farber's disease is a rare autosomal recessive disorder caused by a deficiency of acid ceramidase activity whose symptoms include hoarseness, subcutaneous nodules, and painful swollen and contracted joints. This case report focuses on hand abnormalities and surgical treatment of hand disorders in Farber's disease. A 9-year-old girl had occasional painful locking of the metacarpophalangeal joints of the middle fingers and severe tenderness of the dorsal aspect of the wrists. Resection of several nodules within the metacarpophalangeal joint and of a nodule that was firmly attached to the extensor pollicis longus tendon beneath the extensor retinaculum relieved pain and enabled the patient to perform daily activities.

Abstract: Localization and expression of cartilage-derived morphogenetic protein-1 in tissues of torn rotator cuff tendons were examined by in situ hybridization and immunohistochemical analysis. Histologic findings of torn rotator cuff tendons showed that active cells synthesizing the alpha-1 chain of collagen Type I messenger ribonucleic acid were localized predominantly in the torn edge and in the bursa side rather than in the joint side, and scarcely localized in a site distant from the torn edge. Cartilage-derived morphogenetic protein-1 had a similar distribution as the alpha-1 chain of collagen Type I. The current findings provide the first observational evidence that cartilage-derived morphogenetic protein-1 was activated specifically at the site of the torn rotator cuff tendon. The current findings suggest that the cells in the torn rotator cuffs are capable of synthesizing cartilage-derived morphogenetic protein-1, one of the known essential factors for tendon formation.

Abstract: Abnormalities of the epiphyseal growth plate that occur in collagen-induced arthritis (CIA) were studied. CIA was induced in 6-week-old Lewis rats by immunization with type II collagen. Radiographic examination revealed the early closure of the epiphyseal growth plate with growth retardation of the femur and tibia. Histological evaluation confirmed the early closure of the epiphyseal growth plate accompanied by decreased intensity of safranin-O staining indicating decreased amounts of proteoglycans in the extracellular matrix (ECM) of the cartilage. Immunohistochemical methods showed that the number of chondrocytes expressing matrix metalloproteinase (MMP)-3 and/or vascular endothelial growth factor (VEGF) increased in the growth plates of CIA rats. This study confirmed that disturbances of long bone growth with early closure of the epiphyseal growth plates occur in CIA. There appeared to be overexpression of MMP-3, which may be involved with proteoglycan degradation. Additionally, VEGF, which is associated with cartilage ossification and angiogenesis, might also play a role in this event. Further clarification of the mechanism of the growth disturbance in CIA may yield clinical benefits, especially in prevention of the premature closure of growth plate that is seen in juvenile rheumatoid arthritis and other diseases.

Abstract: OBJECT: Little is known about the molecular mechanisms underlying the process of spondylosis. The authors determined the extent of genetic localization of major regulators of chondrogenesis such as Indian hedgehog (Ihh) and parathyroid hormone (PTH)-related peptide (PTHrP) and their receptors during the development of spondylosis in their previously established experimental mouse model. METHODS: Experimental spondylosis was induced in 5-week-old ICR mice. The cervical spines were chronologically harvested, and histological sections were prepared. Messenger (m) RNA for PTHrP, Ihh, PTH receptor (PTHR; a receptor for PTHrP), patched (Ptc; a receptor for Ihh), bone morphogenetic protein (BMP)-6, and collagen type X (COL10; a marker for mature chondrocyte) was localized in the tissue sections by performing in situ hybridization. In the early stage, mRNA for COL10, Ihh, and BMP-6 was absent; however, mRNA for PTHrP, PTHR, and Ptc was detected in the anterior margin of the cervical discs. In the late stage, evidence of COL10 mRNA began to be detected, and transcripts for Ihh, PTHrP, and BMP-6 were localized in hypertrophic chondrocytes adjacent to the bone-forming area in osteophyte. Messenger RNA for Ptc and PTHR continued to localize at this stage. In control mice, expression of these genes was absent. CONCLUSIONS: The localization of PTHrP, Ihh, BMP-6, and the receptors PTHR and Ptc demonstrated in the present experimental model indicates the possible involvement of molecular signaling by PTHrP (through the PTHR), Ihh (through the Ptc), and BMP-6 in the regulation of chondrocyte maturation leading to endochondral ossification in spondylosis.

Abstract: Localization and expression of cartilage-derived morphogenetic protein (CDMP)-1 in tissues at the site of ossification of the ligamentum flavum (OLF) were examined by immunohistochemistry and in situ hybridization. The CDMP-1 protein and messenger ribonucleic acid (mRNA) were localized in spindle-shaped cells and chondrocytes in the OLF tissues. CDMP-1 was not detected in cells in non-ossified sites. These data indicate that CDMP-1 is locally activated and localized in spindle-shaped cells and chondrocytes at the site of OLE. Given the previously reported promoting action of CDMP-1 for chondrogenesis, the current results suggest that CDMP-1 may be involved in the progression of OLF, leading to the narrowing of spinal canal and thus causing severe clinical manifestations.

Abstract: We report three cases of spinal osteoblastoma with ossification of the ligamentum flavum (OLF) adjacent to the tumor. The patients in this report, all young adults, had no symptoms except for back pain. Computed tomography (CT) demonstrated a typical radiolucent nidus in the spinal pedicle/lamina with a dense sclerotic rim. In addition, ectopic bone formation at the insertion point of the ligamentum flavum adjacent to the tumor was clearly illustrated. Magnetic resonance imaging (MRI) revealed the tumor and surrounding inflammatory responses, but OLF was not detected clearly. Histological examination revealed endochondral ossification of the ligamentum flavum that is quite unusual for normal young adults. Immunohistochemical assays in one case demonstrated that bone morphogenetic protein (BMP)-2/4 was expressed in the osteoblastic tumor cells. This case raises the possibility that BMPs secreted from the tumor cells triggered ectopic ossification in the spinal ligament.

Abstract: Calcifying tendinitis of rotator cuff tendons is a common and painful condition caused by ectopic calcification in humans. To examine the involvement of osteopontin (OPN), a potent regulator of calcium deposition on connective tissues, localization and expression of OPN protein and messenger (m)RNA were investigated in human tissue samples of calcified rotator cuff tendons. Immunohistochemistry demonstrated that OPN was localized in cells surrounding the calcified area. OPN was localized in two distinct cell types, i.e., fibroblast-like cells negative for CD68 and tartrate-resistant acid phosphatase (TRAP) and multinucleated macrophages positive for CD68 and TRAP. In situ hybridization revealed that the mRNA expression of OPN in these cells coincided with the immunohistochemistry results, and these results were supported by reverse transcriptase polymerase chain reaction analysis using human OPN-specific oligonucleotides. Cells located away from the calcified area did not express OPN. The present findings indicate the involvement of OPN in the process of calcification of rotator cuff tendons and suggest that OPN plays a role in such painful disorders through the actions of at least two cell types.

Abstract: OBJECT: Because little is known about the molecular mechanisms underlying the process of spondylosis, the authors examined the extent of genetic localization of several members of bone morphogenetic protein (BMP) and BMP receptors in chondrogenesis during the process of inducing spondylosis in their previously established experimental mice model. METHODS: Experimental spondylosis was induced in 5-week-old ICR mice. The cervical spine was harvested chronologically, and histological sections were prepared. Messenger RNA for BMP-4, growth and differentiation (GDF)-5, BMP-6, and BMP receptors (ALK-3, -6, and BMP-RII) was localized in the tissue sections by in situ hybridization. In the early stage, BMP-4-derived mRNA was localized mainly in cells in the anterior margin of the cervical discs, together with ALK-6 and BMP-RII mRNA. No GDF-5 and BMP-6 mRNA was detected at this stage. In the late stage, cells positive for BMP-4 decreased, whereas GDF-5 and BMP-6 mRNA were localized in cells undergoing chondrogenesis. The ALK-3 mRNA began to appear in this stage, as did ALK-6 and BMP-RII. CONCLUSIONS: The localization of transcripts for BMP-4, -6, and GDF-5 as well as BMP receptors shown during the present experimental model indicate the possible involvement of molecular signaling by these BMPs in the chondrogenic progress in spondylosis.

Abstract: OBJECTIVE: We previously described abnormalities in the bone marrow of patients with rheumatoid arthritis (RA), but were able to shed little light on the pathogenic roles of inflammatory cytokines and proteinases in joint destruction in the subchondral region in RA. This is the first report to describe the co-localization of cytokines and proteinases in this area. METHODS: Decalcified paraffin-embedded sections from 10 patients with RA and five patients with osteoarthritis (OA) were examined for the immunolocalization of cathepsins B, K and L and the localization of messenger RNAs for interleukin 1beta (IL-1beta), tumour necrosis factor alpha (TNF-alpha) and matrix metalloproteinase 9 (MMP-9). The cells were double-stained with anti-CD68 or anti-prolyl 4-hydroxylase (PH) antibody. RESULTS: An immunohistochemical study confirmed the expression of cathepsins B and L by CD68-positive mononuclear cells at the sites of significant cartilage and bone erosion from the subchondral region in all RA specimens. Osteoclast-like cells showed intense staining for cathepsin K and MMP-9. Osteoblast-like cells strongly expressed MMP-9. Analysis of serial sections revealed that expression of the IL-1beta and TNF-alpha genes occurred near that of the cathepsins and MMP-9 in the subchondral region. CONCLUSION: We conclude that inflammatory cytokines and tissue-damaging proteinases play important roles in joint destruction in the subchondral region in RA.

Abstract: Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (Ihh), and patched (Ptc; a receptor for Ihh) were immunolocalized in tissue undergoing endochondral ossification in the human. PTHrP, Ihh, and Ptc were immunolocalized in prehypertrophic and hypertrophic chondrocytes in mature cartilage matrix. PTHrP and Ptc were immunostained in proliferating chondrocytes and perichondrial cells, whereas Ihh was not. PTHrP, Ihh, and Ptc showed positive immunostaining in osteoblasts in the bone-forming area. In the bone resorption site, PTHrP was immunolocalized in osteoclasts, whereas Ihh and Ptc were not. The present findings indicated that PTHrP, Ihh, and Ptc were associated with the process of endochondral ossification, and suggested the possible involvement of Ihh and PTHrP signaling in the regulation of proliferation and hypertrophy of chondrocytes in human chondrogenesis.

Abstract: In iliac bone marrow the absolute number of mononuclear cells (MNCs) was increased in RA patients compared with the non-RA controls. In CD8 positive cell and myeloid cell fractions, significant differences were recognized between RA patients and non-RA controls. The presence of abnormal myeloid lineage cells in epiphyseal bone marrow adjacent to joints affected with severe RA was shown. Stroma cell lines from RA bone marrow with nursing activity were established and shown to play a pivotal role in the pathogenesis in RA bone marrow. Histologic study also shows that subchondral region expressing tissue-damaging proteinases plays an important role in joint destruction in RA.

Abstract: STUDY DESIGN: Apoptosis in cervical intervertebral disc cells and cartilaginous endplate cells was examined by the nick end labeling (TUNEL) technique during the process of natural aging and in a mouse experimental spondylosis model. OBJECTIVES: To determine the role of apoptosis in aging and degeneration of intervertebral discs by monitoring chronologic changes in the quantity and localization of apoptotic cells. SUMMARY OF BACKGROUND DATA: Apoptosis occurs within human intervertebral discs, but little is known about the pathologic significance of this process. On the other hand, the cartilaginous endplate is known to decrease in thickness and to disappear with aging and degeneration. The cause of this age-related change remains unclear. METHODS: A mouse spondylosis model was prepared via surgical resection of the posterior spinal element in 12 mice to examine the experimentally induced spondylosis process. Eighteen naturally aged mice were also used to examine the influence of aging. Paraffin-embedded midsagittal sections of the cervical spine were obtained 2, 3, 6, and 12 months after surgery in the spondylosis model and in the age-matched naturally aged mice, as well as in 4-week-old and 18-month-old naturally aged mice. Sections were stained with hematoxylin and eosin, safranin-O, and the TUNEL procedure. The number of apoptotic cells and vital cells were counted in the cartilaginous endplate of the intervertebral disc excluding the growth cartilage, and the degree of disappearance of the cartilaginous endplate was evaluated. RESULTS: Apoptosis, particularly noticeable in the cartilaginous endplate, increased with age and resulted in a marked decrease in cell density. Subsequently, the structure of the cartilaginous endplate began to disappear. Apoptosis was more evident and the structure of the cartilaginous endplate began to disappear more rapidly in the surgically treated group than in the naturally aged group. CONCLUSIONS: TUNEL-positive cells in the cartilaginous endplate increased with age, with destruction of the cartilaginous endplate after apoptosis (TUNEL-positive cell death). The application of the spondylosis model increased the incidence of apoptosis preceding the development of spondylosis. This suggests that apoptosis plays a role in the age-related changes seen in the cartilaginous endplate of the intervertebral disc and in the experimentally induced spondylosis process.

Abstract: This study describes the distributions of bone morphogenetic protein (BMP)-2 as well as mRNAs for BMP receptor type IB (BMPRIB). collagen types II (Col II) and III (Col III) in a growing "cartilage cap" of osteochondroma. In situ hybridization and immunohistochemical study were performed using histological sections obtained during surgery. BMP-2 was detected in mesenchymal cells in the outer fibrous layer and chondrocytes in the inner cartilaginous matrix, positive for Col III and Col II, respectively. BMPRIB mRNA was distributed in chondrocytes. This is the first study to provide observational evidence of the involvement of BMP-2 signaling in the pathogenesis of cartilage cap of osteochondroma. and suggests the role of BMP-2 in the growth of cartilage cap in osteochondroma.

Abstract: To determine the involvement of cathepsins G and L in the mechanism of spontaneous resorption of herniated intervertebral discs, localization of these cathepsins in this process was examined immunohistochemically using a rat model of autologous transplantation of coccygeal discs. Rat coccygeal discs were resected and autotransplanted into the subcutaneous space of the skin of the back. Paraffin-embedded sections of intervertebral disc tissue, harvested at various post-transplantational periods, were immunohistochemically stained with antibodies for cathepsin G, cathepsin L, MMP-1, MMP-3 and ED-2. The number of positive cells was counted in each part of the transplanted discs. Immunolocalization of cathepsins G and L in various types of disc cells was first observed early in the post-transplantation period. From two days after the operation, histology showed invasion by granulation tissue, with many macrophages, in all sections. Subsequently, the number of macrophages in granulation tissue was observed to increase, along with a gradual increase in the percentage of cells positive for MMP-1 and MMP-3. In addition to the ability of cathepsins G and L to degrade major extracellular matrix components of intervertebral discs, cathepsin G is capable of activating latent pro-MMPs. The up-regulation of cathepsins G and L in the intervertebral disc tissue in this spontaneous resorption model suggests that these proteinases may be involved in degradation of extracellular matrix, leading to the natural resorption of herniated discs.

Abstract: STUDY DESIGN: Age-related fluctuations in insulin-like growth factor-I dependent proteoglycan synthesis in rat intervertebral disc cells were investigated. OBJECTIVES: The purpose of this study was to determine whether synthetic responses to insulin-like growth factor-I decline with age and to explore the possibility that an age-related increase in the expression of insulin-like growth factor binding proteins suppresses matrix synthesis in intervertebral disc cells. SUMMARY AND BACKGROUND DATA: Several studies have reported that the responsiveness of chondrocytes to insulin-like growth factor-I decreases with age and furthermore that this phenomenon may be related to increased expression of insulin-like growth factor binding proteins by chondrocytes. MATERIALS AND METHODS: Nucleus pulposus tissue and cells were obtained from the coccygeal vertebrae of 8-week-old, 40-week-old, and 120-week-old rats. Age-related changes in the expression of insulin-like growth factor-I and its receptor were assessed together with insulin-like growth factor-I dependent proteoglycan synthesis by the cultured nucleus pulposus cells. Also, western blot analysis of insulin-like growth factor binding protein-1 was carried out, and further examination was performed of insulin-like growth factor-I signal transduction through tyrosine phosphorylation of insulin receptor substrate-1, which is a signal transducer of insulin-like growth factor-I. RESULTS: Semiquantitative reverse transcription polymerase chain reaction analysis indicated that the expression of insulin-like growth factor-I receptor in 120-week cells decreased clearly in comparison with the cells of younger animals. By contrast, insulin-like growth factor-I dependent proteoglycan synthesis decreased with age, and the sharpest decline of synthesis was found between 8-week and 40-week cells, although the level of insulin-like growth factor-I/insulin-like growth factor-I receptor gene expression was maintained in 40-week-old animals. Consistent with the results of proteoglycan synthesis, the expression of phosphorylated insulin receptor substrate-1 decreased with age. Thus, the expression of insulin-like growth factor binding protein-1 and proteoglycan synthesis was investigated by use of Long R3 insulin-like growth factor-I, which was not influenced by insulin-like growth factor binding proteins. Insulin-like growth factor binding protein-1 was strongly expressed in 40-week cells in comparison with the expression in 8-week cells. Furthermore, proteoglycan synthesis in 40-week cells supplemented with Long R3 insulin-like growth factor-I was upregulated in comparison with that in 40-week cells supplemented with insulin-like growth factor-I. CONCLUSION: The present findings indicate that the age-related decline in insulin-like growth factor-I dependent proteoglycan synthesis in nucleus pulposus is caused, at least in part, by the increase in insulin-like growth factor binding proteins at the early stages of aging, and further suggest that a loss of proteoglycan synthesis during the late stages of aging is caused by the downregulation of insulin-like growth factor-I receptor in addition to an increase in insulin-like growth factor binding proteins.

Abstract: STUDY DESIGN: Localization of cathepsins D, K, and L in degenerated intervertebral discs was examined by immunohistochemistry. OBJECTIVES: To determine the involvement of cathepsins in the pathomechanism of intervertebral disc degeneration by monitoring the immunolocalization of cathepsins in degenerated intervertebral disc tissue. SUMMARY OF BACKGROUND DATA: Cathepsins D, K, and L are enzymes that contribute to the matrix destruction seen in the articular cartilage affected by osteoarthritis and rheumatoid arthritis. However, little is known about the contribution of these cathepsins to intervertebral disc degeneration. METHODS: Paraffin-embedded sections of degenerated intervertebral disc tissue collected at the time of surgery (13 discs from 12 patients) were immunohistochemically stained with antibodies for cathepsins D, K, and L. For further characterization of the stained cells, immunohistochemical detection of CD68 and TRAP staining were performed. RESULTS: Hematoxylin and eosin staining revealed obvious signs of degeneration in all sections. Cathepsins D and L were immunolocalized in disc fibrochondrocytes at various sites exhibiting degeneration. Cathepsins K were found in tartrate-resistant acid phosphatase-positive multinucleated cells, in particular near the cleft within the cartilaginous endplate. However, few cells were positive for these cathepsins in anulus fibrosus that maintained the lamellar structure of collagen fibers. CONCLUSIONS: Marked expression of cathepsins D and L was observed at the site of degeneration. Cathepsins D and K localized in tartrate-resistant acid phosphatase-positive multinucleated cells existed at the cleft between the cartilaginous endplate and vertebral body. The site-specific localization of these cathepsins suggests the association of these proteinases with endplate separation and disorganization of the anulus fibrosus in degenerative spinal disorders.

Abstract: We report two similar, but unrelated, patients with congenital bilateral partial deficiencies of the tibia and fibula associated with intact feet. In both patients, the tibia and fibula were absent on initial radiographs, while the femur and the tarsal bones were well developed and there was bilateral teratologic dislocation of the hips. Ultrasound and magnetic resonance imaging (MRI) studies suggested the presence of cartilaginous remnants of the tibia and fibula. There were multidirectional instabilities in the knees and ankles. The clinical and radiological features of these cases are distinct from those of congenital longitudinal deficiency of the tibia, in which the fibula is always preserved, and from longitudinal deficiency of the fibula, in which the tibia is present and the foot is usually involved. We suggest that the bilateral partial deficiencies of the tibia and fibula associated with the intact foot and teratologic dislocation of the hips is a single-entity disorder, possibly categorized as an intercalary transverse deficiency of the lower limb.

Abstract: OBJECTIVE: Numerous cytokines are expressed in lesions of synovial hyperplasia of patients with rheumatoid arthritis (RA), and their pathophysiological contributions have been the subject of speculation. These genes are regulated by the transcription factor NFkappaB which in turn is activated by tumour necrosis factor-alpha (TNF-alpha) and cytokines. In this study we examined the inhibition of the production of pro-inflammatory cytokines, adhesion molecule and matrix metalloproteinase (MMP) from synovial tissue of patients with RA by the introduction of synthetic double-stranded DNA with high affinity for the NFkappaB binding site. METHOD: NFkappaB decoy oligonucleotides (ODN) were introduced with the aid of the haemagglutinating virus of Japan (HVJ)-liposome method into synovial tissue or synovial cells derived from patients with RA. The levels of interleukin-1beta (IL-1beta), IL-6, TNF-alpha, intercellular adhesion molecule-1 (ICAM-1) and MMP-1 were determined by means of enzyme-linked immunosorbent assay (ELISA) and Northern blotting analysis. A cell counting kit was used to study the effect of NFkappaB decoy ODN on synovial cell proliferation. RESULTS: The production of these mediators was significantly inhibited by the introduction of NFkappaB decoy ODN compared with the effect of scrambled decoy ODN. Transfection of NFkappaB decoy ODN resulted in a significant inhibition of synovial cell proliferation as compared with that of scrambled decoy ODN. CONCLUSION: The results demonstrated in this study suggest the potential usefulness of NFkappaB decoy ODN for gene therapy of inflammatory synovitis of RA.

Abstract: Nine patients with achondroplasia and one patient with Apert syndrome underwent the surgical lengthening of both humerus and simultaneous correction of both associated bone deformity. An unilateral external fixator was applied to the lateral aspect of the humerus with four half-pins and percutaneous predrilling osteotomy was performed at the apex of flexion deformity of the bone. During the waiting period before distraction, the flexion deformity of the distal humerus was corrected using an additional external fixator. Slow gradual distraction was subsequently carried out at a rate of 0. 25 mm every 6 hours. The average lengthening was 8 cm (range 7.5 to 9 cm), the overall treatment time 312 days (range 192 to 406 days), and the average healing index 39.0 days/cm. The average correction of the elbow flexion deformity was 20 degrees. We believe this treatment is useful to improve the function of the arms and the activity of daily living for the patients with bilateral short humeri.

Abstract: Cathepsins D, K, and L were immunolocalized in tissue undergoing endochondral ossification in the human. Cathepsins D, K, and L were localized in osteoclasts and chondroclasts attached to bone matrix and cartilage matrix, respectively. Cathepsins D and L were immunostained in chondrocytes. Immunolocalization of cathepsin D was limited to hypertrophic chondrocytes adjacent to the osteochondral junction. In contrast, cathepsin L was immunolocalized in both proliferating and hypertrophic chondrocytes. In the bone marrow space, cathepsins D, K, and L were localized in multinucleated cells. Cathepsin D was diffusely detected in mononuclear bone marrow cells which were negative for cathepsins K and L. The present findings indicated that cathepsins K, D, and L were associated with the process of endochondral ossification in the human, and suggested that these cathepsins share roles in bone and cartilage turnover in the human.

Abstract: OBJECTIVES: Calcified tendinitis of the shoulder joint is a common painful condition. Resorption of the calcium deposits is one of the key events in the pathogenesis of this disease. The aim of this study was to examine whether the multinucleated giant cells that appear in this condition have osteoclast phenotypes. METHODS: Immunohistochemical and RNA in situ hybridization analysis of cathepsin K, a marker for osteoclasts, was performed in human surgical samples. RESULTS: The multinucleated cells located near the calcium deposits were positive for cathepsin K protein and mRNA. Reverse transcription-polymerase chain reaction using human cathepsin K-specific oligonucleotide primers confirmed that synthesis of cathepsin K mRNA occurs in the tissues of calcified rotator cuffs. CONCLUSION: The multinucleated giant cells which appear in the resorption area of calcium deposits in calcified tendinitis have the osteoclast phenotype.

Abstract: We have recently developed a technique of per-cutaneous multidrilling osteotomy for limb lengthening and deformity correction. The bone is drilled percutaneously, using a special drill guide, and osteotomy is accomplished by connecting the multiple drill holes with a small chisel. The bone segments are subjected to slow progressive distraction with an external fixation device. We have lengthened 33 limbs in 22 patients with congenital or post-traumatic limb shortening and/or bone deformities. All the patients underwent the proposed lengthening and/or correction of the bone deformities through a single-treatment procedure. None of the lengthened segments resulted in nonunion. This technique can prevent undesirable bone cracks and preserve soft tissue around the osteotomy site, and is also applicable to other fields of orthopedic surgery.

Abstract: BACKGROUND: The purpose of this study was to assess the usefulness of bone scintigraphy in predicting progressive collapse of the femoral head after transtrochanteric rotational osteotomy for the treatment of osteonecrosis of the femoral head. METHODS: We studied thirty-three hips in thirty patients with osteonecrosis of the femoral head who had undergone transtrochanteric rotational osteotomy. There were twenty male and ten female patients, with a mean age of 34.4 years at the time of the operation. The mean duration of follow-up was 10.0 years. According to the staging system of Ficat and Arlet, there were nineteen stage-2 hips and fourteen stage-3 hips at the time of the operation. Conventional anteroposterior and lateral radiographs were assessed. In addition, bone scans were performed at three weeks after the operation to predict the outcome with regard to the rotated femoral head. On the basis of the location of low scan activity within the femoral head, the scintigraphic findings were classified into one of two categories: type A if there was no low scan activity in the weight-bearing area of the femoral head or type B if low scan activity occupied the entire weight-bearing area. Six hips with collapse were studied histologically. RESULTS: Postoperative scintiscans revealed sixteen type-A hips and seventeen type-B hips. Of the type-A hips, only three exhibited progressive collapse of the femoral head after the osteotomy, whereas fourteen of the type-B hips exhibited progressive collapse. A significant association was found between the postoperative scintigraphic findings and the final radiographic result (p < 0.01). CONCLUSIONS: Bone scintiscans made three weeks after transtrochanteric rotational osteotomy were useful for predicting the final clinical result.

Abstract: We investigated the pathology of femoral head collapse following transtrochanteric anterior rotational osteotomy. Six femoral heads were obtained during total hip arthroplasty some 2-12 years after osteotomy. In all cases, the preoperatively necrotic lesions exhibited mostly osteonecrosis with accumulation of bone marrow cell debris and trabecular bone with empty lacunae, although repair tissue such as granulation tissue and appositional bone formation were observed in limited areas in some cases. In the transposed intact articular surface of the femoral head, osteoarthritic changes such as fissure penetration to the subchondral bone and osteophyte formation were commonly observed. In newly created subchondral areas at weight-bearing sites, trabecular thickness and the number of trabecular bones had decreased, with few osteoblasts, osteoclasts, and osteocytes being present, resulting in a coarse lamellar structure of the trabecular bone. These findings suggest that transposed areas in cases of failure consist mostly of low-turnover osteoporotic lesions which could cause collapse of the femoral head.

Abstract: In situ hybridization histochemistry is the sole tool available for detecting the localization and expression of specific RNA on histological sections under various in vivo conditions. For this paper, we examined the effect of microwave exposure on the time needed for decalcification of skeletal tissues and on the preservation of sensitivity for hybridization signals. Our data show that the use of microwave decalcification reduces the decalcification period while preserving intense hybridization signals for mouse alpha1 chain of procollagen type I mRNA in osteogenic cells in bone. The use of microwave treatment to decalcify skeletal tissues may prevent delay in obtaining experimental results or the loss of signals during in situ hybridization.

Abstract: Cartilage provides the template for endochondral ossification and is crucial for determining the length and width of the skeleton. Transgenic mice with targeted expression of recombinant cartilage-derived morphogenetic protein-1 (CDMP-1), a member of the bone morphogenetic protein family, were created to investigate the role of CDMP-1 in skeletal formation. The mice exhibited chondrodysplasia with expanded cartilage, which consists of the enlarged hypertrophic zone and the reduced proliferating chondrocyte zone. Histologically, CDMP-1 increased the number of chondroprogenitor cells and accelerated chondrocyte differentiation to hypertrophy. Expression of CDMP-1 in the notochord inhibited vertebral body formation by blocking migration of sclerotome cells to the notochord. These results indicate that CDMP-1 antagonizes the ventralization signals from the notochord. Our study suggests a molecular mechanism by which CDMP-1 regulates the formation, growth, and differentiation of the skeletal elements.

Abstract: Bone lengthening with osteotomy and gradual distraction was achieved in 57 rats, and the effect of mechanical tension-stress on gene expression of bone morphogenetic proteins (BMPs) was investigated by in situ hybridization and Northern blot analysis using probes of BMP-2, BMP-4, BMP-6, BMP-7, and growth/differentiation factor (GDF)-5. There was a lag phase for 7 days after femoral osteotomy until gradual distraction was carried out for 21 days at a rate of 0. 25 mm/12 h using a small external fixator. The signals of the above BMPs mRNA were not detected in the intact rat bone but they were induced after osteotomy except those for BMP-7. By 4 days after osteotomy, BMP-2 and BMP-4 mRNAs were detected in chondrogenic precursor cells in the subperiosteal immature callus. BMP-6 and GDF-5 mRNA were detected in more differentiated cells in chondroid bone. By 7 days after osteotomy, cartilaginous external callus and bony endosteal callus were formed. Meanwhile, the signals of BMP-2 and BMP-4 mRNAs declined to preoperative levels, whereas the signals of BMP-6 and GDF-5 mRNAs were rather elevated. As distraction was started, the callus elongated and eventually separated into proximal and distal segments forming a fibrous interzone in the middle. Expression of BMP-2 and BMP-4 mRNAs was markedly induced at this stage. Their signals were detected widely among chondrogenic and osteogenic cells and their precursor cells sustaining mechanical tension-stress at the fibrous interzone. BMP-6 and GDF-5 mRNAs were detected exclusively in chondrogenic cells at both ends of the fibrous interzone, where endochondral ossification occurred. But neither mRNA was detected in terminally differentiated hypertrophic chondrocytes. As distraction advanced, the cartilage was progressively resorbed from both ends and new bone was formed directly by intramembranous ossification. There was no new cartilage formation in the advanced stage of distraction. The signals of BMP-6 and GDF-5 mRNA declined by this stage, while those of BMP-2 and BMP-4 were maintained at high level for as long as distraction was continued. After completion of distraction, the fibrous interzone fused and the lengthened segment was consolidated. BMP-2, BMP-4, BMP-6, nor GDF-5 was expressed at this stage. The signals of BMP-7 were not detected throughout the experiment. The present results suggest that excellent and uninterrupted bone formation during distraction osteogenesis owes to enhanced expression of BMP-2 and BMP-4 genes by mechanical tension-stress. Abundant gene products of BMP-2 and BMP-4 could induce in situ bone formation by paracrine and autocrine mechanisms.

Abstract: OBJECTIVE: In both rheumatoid arthritis and collagen-induced arthritis (CIA), the nuclear factor kappaB (NF-kappaB) transcription factor plays a pivotal role in the coordinated transactivation of many cytokines related to pathogenesis. This study investigated whether synthetic double-stranded DNA that show a high affinity for NF-kappaB could be introduced in vivo as "decoy" cis elements to bind the transcription factor and block the activation of such proinflammatory cytokine genes as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha), and thus suppress the severity of joint destruction. METHODS: NF-kappaB decoy oligonucleotides (ODN) were introduced by an intraarticular injection into the bilateral hind ankle joints of CIA rats using the hemagglutinating virus of Japan (HVJ)-liposome method. Joint destruction was evaluated by histology and radiography. IL-1 and TNFalpha levels were assessed by enzyme-linked immunosorbent assay and Northern blot analysis. RESULTS: Using the HVJ-liposome method, the presence of fluorescein isothiocyanate-labeled ODN in the synovium was confirmed until 28 days after intraarticular injection. In vivo transfection of NF-kappaB decoy ODN by an intraarticular injection into CIA rats decreased the severity of hind-paw swelling. Histologic and radiographic studies showed a marked suppression of joint destruction treated by NF-kappaB decoy ODN transfection. This treatment method also suppressed the production of IL-1 and TNFalpha in the synovium of arthritic joints. CONCLUSION: The present results demonstrate that administration of NF-kappaB decoy ODN in arthritic joints of rats with CIA led to an amelioration of arthritis. These findings suggest that intraarticular transfection of NF-kappaB decoy ODN may provide a useful therapeutic approach for the treatment of inflammatory arthritis.

Abstract: Six knees in three patients with Ellis-van Creveld syndrome were treated with lateral soft tissue release and corrective osteotomy of the tibia at 10 years of age on average. The main feature was valgus deformity with lateral dislocation of the patella. All patellae were reduced. The valgus deformity improved from 35 degrees (range, 48 degrees-20 degrees) to 17 degrees (range, 35 degrees-5 degrees) of the femorotibial angle (FTA) on average, although the FTA in five of six knees was < 5 degrees after surgery. There was one recurrent case and one transient peroneal nerve palsy. The reason for undercorrection was a depression of the lateral tibial plateau. The deformity of the articular surface is the most important problem in correcting the valgus deformity of the knee in this syndrome.

Abstract: The process of cartilage-to-bone transition (CBT) is a key event for the achievement of rigid bone healing during fracture repair. Since mineralization of cartilaginous matrix is a prerequisite for the initiation of CBT, the genetic localization of mineralization-related bone matrix proteins in CBT was examined in this study. An in situ hybridization method used on decalcified sections with digoxigenin-11-UTP labelled probes identified the cellular localizations of these genes in CBT. Cessation of osteonectin mRNA together with induction of osteopontin mRNA in chondrocyte maturation was observed during the process of CBT in the fracture callus on day 12 after fracture; osteocalcin mRNA was absent in chondrocytes of the CBT area. Induction of osteopontin mRNA in maturated chondrocytes was followed by the expression of mRNAs for osteonectin, osteopontin and osteocalcin in osteogenic cells in the ossification front of CBT. The data suggest that the switch from osteonectin to osteopontin mRNA expression in chondrocyte maturation is one of the key events during CBT. Transcriptional disorders of the expression of these molecules may be linked to the failure of fracture repair, i.e. delayed or prevented hypertrophic osteosynthesis.

Abstract: Distraction osteogenesis is a recently advanced principle of bone lengthening in which a bone separated by osteotomy is subjected to slow progressive distraction using an external fixation device. Appropriate mechanical tension-stress is believed not to break the callus but rather to stimulate osteogenesis. To study the molecular features of this process, the expression and localization of the mRNAs encoding osteopontin (OPN), osteocalcin (OC), matrix Gla protein (MGP), osteonectin (ON), and collagen type I and I during distraction osteogenesis were examined by in situ hybridization and Northern blot analysis. The process can be divided into three distinct phases: the lag phase for 7 days between osteotomy and the beginning of distraction, the distraction phase for 21 days, and the consolidation phase for several weeks. The histologic and molecular events taking place during the lag phase were similar to those observed in fracture healing. The osteotomy site was surrounded by external callus consisting of hyaline cartilage. As distraction started at the rate of 0.25 mm/12 h, the cartilaginous callus was elongated, deformed, and eventually separated into proximal and distal segments. The chondrocytes were stretched along the tension vector and became fibroblast-like in shape. Although morphologically these cells were distinguishable from osteogenic cells, they expressed OPN, OC, and alkaline phosphatase mRNAs. As distraction advanced, the cartilaginous callus was progressively replaced by bony callus by endochondral ossification and thereafter new bone was formed directly by intramembranous ossification. OPN mRNA was detected in preosteoblasts and osteoblasts at the boundary between fibrous tissue and new bone. ON, MGP, and OC mRNAs appeared early in the differentiation stage. The variety of cell types expressing mRNA encoding bone matrix proteins in distraction osteogenesis was much greater than that detected in the embryonic bone formation and fracture healing process. Moreover, the levels of OPN, ON, MGP, and OC mRNA expression markedly increased during the distraction phase. These results suggested that mechanical tension-stress modulates cell shape and phenotype, and stimulates the expression of the mRNA for bone matrix proteins.

Abstract: STUDY DESIGN: Cross-sectional study of cervical involvement in rheumatoid arthritis. OBJECTIVES: To clarify the correlation between the deterioration of cervical lesions and the systemic progression of rheumatoid arthritis. SUMMARY OF BACKGROUND DATA: The natural course of cervical lesions varies. To date, no systemic parameter has been clarified to predict the progression. METHODS: One hundred seventy-three patients with rheumatoid arthritis participated in this study. The authors studied the progression of cervical lesions and investigated the relation between the types of cervical subluxation at the end of study and the following four variables: the serum level of C-reactive protein, the number of joints with erosion, carpal height ratio, and disease subset (least erosive subset, more erosive subset, and mutilating disease subset). RESULTS: Of the 173 patients, 55 already had cervical subluxation before entering the study. During the follow-up period, 44 patients deteriorated radiographically, and 77 (45%) had cervical involvement, including involvement of upper cervical lesions in 65 patients, upper lesions combined with subaxial subluxation in 10, and subaxial subluxation alone in 2. The upper cervical subluxation progressed in the order of anterior atlantoaxial subluxation, atlantoaxial subluxation combined with vertical subluxation, and vertical subluxation alone. Deterioration of upper cervical lesion and occurrence of subaxial subluxation were closely correlated with an elevation of serum C-reactive protein level, an increase in the number of joints with erosion, and a decrease in the carpal height ratio. The incidence of cervical involvement and the extent of deterioration were different among the disease subsets. CONCLUSIONS: The serum level of C-reactive protein, the number of joints with erosion, and the carpal height ratio correlated closely with the extent of the cervical subluxation. The average C-reactive protein values during the follow-up period correlated with progression of the cervical lesions. The classification of rheumatoid disease subset was useful for predicting the terminal feature of the cervical lesions.

Abstract: We describe a case of rheumatoid arthritis (RA) with collapse of the L3 lumbar vertebra for which surgery was performed. The pathogenesis of lumbar lesions affected by RA is discussed and the literature reviewed.

Abstract: Focal fibrocartilaginous dysplasia (FFCD) is a rare condition causing tibia vara in childhood. It is characterized by progressive tibia vara in young children with a characteristic radiographic lesion. This paper is thought to be the first to describe FFCD exhibiting florid periosteal reaction at the time of presentation with a subtle faint osteolytic lesion in the diametaphysis of the proximal tibia.

Abstract: The modulatory effects of interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha on bone morphogenetic protein (BMP)-2- and -4-induced alkaline phosphatase (ALP) activity were examined in cultures of mouse MC3T3-E1 osteoblastic cells. Both BMP-2 and -4 significantly induced ALP in these cells. IL-1 beta alone had no effect on ALP activity, but it significantly enhanced BMP-2- and -4-induced ALP activity. TNF-alpha suppressed the induction of ALP by BMP-2 or -4. The results suggest that the action of BMP on osteogenic differentiation may be regulated by such immuno/inflammatory cytokines as IL-1 beta and TNF-alpha.

Abstract: A monoclonal antibody that reacts with murine and human bone morphogenetic protein-4 (BMP-4) has been developed using recombinant BMP-4 as an immunogen. The antibody that bound most tightly to recombinant murine (rm)BMP-4 was selected, subcloned, and characterized. The specificity of the antibody was confirmed using Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The antibody reacts with murine and human BMP-4 in both the reduced and nonreduced condition; however, this antibody shows cross-reactivity with neither human BMP-2 nor TGF-beta 1. Thus, the produced antibody could recognize the disulfide-linked dimeric structure of bioactive BMP-4, regardless of the species. Immunocytochemical study using this antibody successfully shows the cytosolic localization of BMP-4 in osteoinductive cells; i.e., BFO and Saos-2 in which the level of mRNA for BMP-4 was proved to be constitutively high by Northern blot analysis. In addition, the antibody could demonstrate the presence of BMP-4 in developmental bone formation in the alveolar bone of rat embryo by immunohistochemistry. The antibody could be used for a more sensitive approach for quantitative analysis of BMP-4.

Abstract: We studied the effects of transforming growth factor-beta and basic fibroblast growth factor on the regulation of proliferation and osteochondrogenic differentiation of periosteum-derived cells, which have the potential to differentiate into bone and hypertrophic cartilage in vitro. Histological observation revealed that transforming growth factor-beta stimulated chondrogenesis of periosteum-derived cells while basic fibroblast growth factor stimulated proliferation of fibroblast-like cells and inhibited osteochondrogenic differentiation. Immunohistochemical studies revealed that basic fibroblast growth factor inhibited the expression of osteocalcin. Transforming growth factor-beta enhanced uronic acid content but decreased DNA content, alkaline phosphatase activity, and calcium content. In contrast, basic fibroblast growth factor enhanced DNA content but decreased alkaline phosphatase activity, calcium content, and uronic acid content. In addition, transforming growth factor-beta shortened the time-course of gene expression of type-X collagen whereas basic fibroblast growth factor inhibited the gene expression. These results indicate that transforming growth factor-beta stimulates osteochondrogenic differentiation of periosteum-derived cells but inhibits proliferation. They also indicate that basic fibroblast growth factor stimulates proliferation of periosteum-derived cells but inhibits osteochondrogenic differentiation.

Abstract: BACKGROUND: Shionogi carcinoma 115 (SC115) is an androgen-dependent medullary carcinoma with a compact cell pattern. When SC115 tumors grow in androgen-depleted hosts, spindle-shaped and round cells with abundant cytoplasm develop. These cells originate from the SC115 cells (Kitamura et al., Cancer Res 1979;39:4717; Terada et al., Lab Invest 1987;57:186). EXPERIMENTAL DESIGN: We investigated whether these spindle-shaped and round cells expressed mRNAs of noncollagenous connective tissue proteins, which are expressed by normal spindle-shaped cells and normal chondrocytes in developing bones. The expression and localization of osteonectin (OSN), osteopontin (OSP), matrix Gla protein (MGP), and osteocalcin (OSC) were determined by Northern blotting and in situ hybridization. RESULTS: No mRNA signals of these proteins were detectable in SC115 medullary carcinoma cells growing in DS mice that had been castrated but received injections of testosterone propionate. However, when the injection of TP was stopped, spindle-shaped and round cells with abundant cytoplasm developed. The former expressed OSN and OSP signals, and the latter OSN, OSP, and MGP signals. CONCLUSIONS: Transcripts of OSN, OSP, and MGP were expressed by some SC115-derived cells during the differentiation events that occurred after androgen removal. These results provide molecular biologic evidence that a tumor of epithelial origin can progress along the connective tissue differentiation pathway.

Abstract: Periosteal sunburst spiculation is a peculiar radiographic feature of osteosarcoma, and it represents a reactive ossification resulting from the action of normal osteoblasts rather than tumor cells. Because bone morphogenetic protein is known to be a potent inducer of ectopic bone formation, the authors hypothesized that bone morphogenetic protein may be involved in the pathogenesis of such reactive bone formation in osteosarcoma. Chinese hamster ovary cells transfected with the bone morphogenetic protein-4 gene were injected into the femurs of athymic nude mice to form experimental bone tumors producing bone morphogenetic protein. Two weeks after intramedullary injection, new bone formation was observed radiographically and histologically within the extraosseous portions of the tumors. This showed a close resemblance to sunburst spiculation in human osteosarcomas. In contrast, the control nontransformed Chinese hamster ovary tumors showed no extraosseous bone formation. Because the induced bone was composed of multiple parallel spicules similar to those found in human bone morphogenetic protein-producing osteosarcomas, these findings suggest that periosteal sunburst spiculation may be the result of bone morphogenetic protein production by osteosarcoma cells.

Abstract: Temporal and spatial distribution of a gene encoding murine bone morphogenetic protein 4 (mBMP-4) during fracture repair were investigated in mice by RT-PCR and in situ hybridization. For in situ hybridization, fractured ribs and surrounding tissues were decalcified and hybridized with a mBMP-4-specific complementary RNA probe labeled with digoxigenin-11 UTP. mBMP-4 messenger RNA (mRNA) was not detected in ribs without fracture, whereas it was detected only in the early phase of fracture from 12 to 72 h after the onset of fracture before new cartilage or bone formation. The mBMP-4 mRNAs were present in cells distributed in three distinct regions, namely, the proliferating periosteum, the medullary cavity, and the muscles near the fracture site. These BMP-4-positive cells did not express bone gla protein mRNA, which is a marker of the mature osteogenic cell. RT-PCR also showed a transient increase in the level of BMP-4 mRNA in the early phase of fracture repair. The findings provide us with some new information. (1) The BMP-4 gene is produced by less differentiated osteoprogenitor cells, not by differentiated osteoblasts. (2) The BMP-4 gene is enhanced by the impact of fracture and localized in callus-forming tissue before callus formation. Together with the activities of BMP-4, as was previously described, our results suggest that newly produced BMP-4 gene product is one of the local contributing factors in callus formation in the early phase of fracture healing.

Abstract: The effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) on osteochondrogenesis were examined in high-density cultures of periosteum-derived cells, which have the potential to differentiate into bone and hypertrophic cartilage in vitro. Proliferation of these cells was inhibited by treatment with rhBMP-2. The time course for alkaline phosphatase (ALP) expression was shortened and the mineralization of the culture was increased by supplementation with rhBMP-2. These stimulatory effects of rhBMP-2 were observed at doses of 10-100 ng/m. Bone Gla protein (BGP) was immunocytochemically detectable earlier in the culture treated with rhBMP-2, and the BGP-positive layer of the rhBMP-2-treated cultures was thicker than that of the control cultures. On the other hand, there was no difference in uronic acid content or the time course of alpha 1(II) collagen mRNA expression between the rhBMP-2-treated and the control cultures. These results indicate that rhBMP-2 shortens the time course of osteogenesis and increases the amount of bone formation, whereas chondrogenesis remains unaffected.

Abstract: Heterogeneity in the expression of three members of non-collagenous matrix proteins in osteogenic and chondrogenic development in vivo were investigated by in situ hybridization. Sections of several skeletal tissues from mice at various stages of development were hybridized with digoxigenin-labeled complementary RNA probes encoding osteonectin (Osn), osteopontin (Osp) and osteocalcin (Osc). In calvariae and mandibulae, Osn messenger RNA (mRNA) was detected in cells in pre-osseous and osseous tissues before mineralization. Osp mRNA was found in cells attached to the mineralized bone matrix together with Osn mRNA followed by the expression Osc mRNA. In long bones, mRNAs for Osn, Osp and Osc were sequentially expressed with bone development from primary spongiosa to diaphyseal bone. In growth cartilage, Osn mRNA was observed in chondrocytes in non-mineralized cartilage, whereas Osp mRNA was detected in hypertrophic chondrocytes in mineralized cartilage matrix with a characteristic switch in expression. Osc mRNA was not detected in any chondrocytes. These results indicate that osteogenic differentiation in bone development in vivo is characterized by the sequential expression of these three genes, and suggest that these genes are expressed differentially and specifically, in association with extra-cellular matrix mineralization.

Abstract: Chick periosteum-derived mesenchymal cells have been reported to exhibit both osteogenic and chondrogenic potentials in high cell density culture conditions. Using this culture system, the effects of transforming growth factor-beta (TGF beta) on proliferation and differentiation of periosteal mesenchymal cells were studied. Supplementation with TGF beta 1 at doses of 0.3-1.0 ng/ml shortened the time course of chondrogenesis and increased the amount of cartilage formed in the lower part of the culture. On the other hand, the amount of bone formed in the upper part of the culture decreased with TGF beta treatment, whereas the time course of osteogenesis remained unaffected.

Abstract: The developmental potential of periosteum-derived cells was clonally assessed with an agar gel culture system. Morphologically, two types of colonies were predominantly observed. By immunocytochemical observation with antibodies against aggrecan or bone Gla protein, one type of colony was judged to be chondrogenic, and the other osteogenic. By chronological observation, each type of colony did not convert to the other. Supplementation with transforming growth factor (TGF)-beta 1 shortened the time course of chondrogenesis and also increased colony forming efficiency of chondrogenic colonies. On the other hand, colony forming efficiency of osteogenic colonies decreased with TGF-beta 1 treatment, whereas the time course of osteogenesis remained unaffected. These observations suggest that there are both committed osteoprogenitor and chondroprogenitor cells present in the periosteal cell population, and TGF-beta 1 stimulates proliferation and differentiation of chondrogenic cell population by its targeted action.