IKKß in postnatal perichondrium remotely controls endochondral ossification of the growth plate through downregulation of MCP-5.

IκB kinase ß (IKKß) is a catalytic subunit of the IKK complex, which activates nuclear factor-κB (NF-κB). Although its role in osteoclastogenesis is well established, the role of IKKß in bone formation is poorly understood. Here, we report that conditional knockout of Ikkß in limb bud mesenchymal cells results in the upregulation of monocyte chemoattractant protein-5 (MCP-5) in the perichondrium, which in turn inhibits the growth of longitudinal bone by compromising chondrocyte hypertrophy and increasing the apoptosis of chondrocytes within the growth plate. Contrary to expectations, IKKß in cells of chondrocyte or osteoblast lineage was dispensable for bone growth. On the other hand, ex vivo experiments confirmed the role of MCP-5 in the growth of longitudinal bone. Furthermore, an in vitro study demonstrated that the action of IKKß on MCP-5 is cell autonomous. Collectively, our results provide evidence for a previously unrecognized role of IKKß in the regulation of the growth plate that is mediated through stimulation-independent downregulation of MCP-5 in the perichondrium.

Characteristics of flexed knee gait and functional outcome of a patient who underwent knee reconstruction with a hingeless prosthesis for bone tumor resection: a case report with gait analysis and comparison with healthy subjects.

We report on a patient after knee reconstruction for osteosarcoma in the distal femur using a hingeless prosthesis K-MAX KNEE system K-5 who walked without ipsilateral knee extension in the latter half of the stance phase (flexed knee gait). We evaluated the patient using three-dimensional gait analysis and isokinetic knee strength measurement, and compared the patient with five healthy subjects. The Musculoskeletal Tumor Society (MSTS) score was also used for evaluation. The patient kept his operated knee flexed during mid stance. The maximal ankle plantarflexion internal moment was lower on the ipsilateral side than on the contralateral side, and lower than in the healthy subjects. The negative ankle power during the stance phase was generally stronger on the ipsilateral side than on the contralateral side, and also in the healthy subjects. Unusual contralateral hip flexion occurred after the initial contact, indicating increased joint load on the ipsilateral ankle and the contralateral hip. The ratios of the peak knee extension/flexion torque were 0.7 on the ipsilateral side, 1.9 on the contralateral side, and 1.7 in the healthy subjects. The MSTS score of the patient was 23/30 (76.6%). Flexed knee gait might account for the reduction of ipsilateral hip flexion and ankle plantarflexion moment during the late stance phase. These results suggest the importance of focusing more on the ipsilateral ankle joint and the contralateral hip joint to maintain the function of the entire limb joints of the patients with flexed knee gait.

Characteristics of flexed knee gait and functional outcome of a patient who underwent knee reconstruction with a hingeless prosthesis for bone tumor resection: a case report with gait analysis and comparison with healthy subjects.

We report on a patient after knee reconstruction for osteosarcoma in the distal femur using a hingeless prosthesis K-MAX KNEE system K-5 who walked without ipsilateral knee extension in the latter half of the stance phase (flexed knee gait). We evaluated the patient using three-dimensional gait analysis and isokinetic knee strength measurement, and compared the patient with five healthy subjects. The Musculoskeletal Tumor Society (MSTS) score was also used for evaluation. The patient kept his operated knee flexed during mid stance. The maximal ankle plantarflexion internal moment was lower on the ipsilateral side than on the contralateral side, and lower than in the healthy subjects. The negative ankle power during the stance phase was generally stronger on the ipsilateral side than on the contralateral side, and also in the healthy subjects. Unusual contralateral hip flexion occurred after the initial contact, indicating increased joint load on the ipsilateral ankle and the contralateral hip. The ratios of the peak knee extension/flexion torque were 0.7 on the ipsilateral side, 1.9 on the contralateral side, and 1.7 in the healthy subjects. The MSTS score of the patient was 23/30 (76.6%). Flexed knee gait might account for the reduction of ipsilateral hip flexion and ankle plantarflexion moment during the late stance phase. These results suggest the importance of focusing more on the ipsilateral ankle joint and the contralateral hip joint to maintain the function of the entire limb joints of the patients with flexed knee gait.

Identification of AFAP1L1 as a prognostic marker for spindle cell sarcomas.

Spindle cell sarcomas consist of tumors with different biological features, of which distant metastasis is the most ominous sign for a poor prognosis. However, metastasis is difficult to predict on the basis of current histopathological analyses. We have identified actin filament-associated protein 1-like 1 (AFAP1L1) as a candidate for a metastasis-predicting marker from the gene expression profiles of 65 spindle cell sarcomas. A multivariate analysis determined that AFAP1L1 was an independent factor for predicting the occurrence of distant metastasis (P=0.0001), which was further confirmed in another set of 41 tumors by a quantitative mRNA expression analysis. Immunohistochemical staining using paraffin-embedded tumor tissues revealed that the metastasis-free rate was significantly better in tumors negative for AFAP1L1 (P=0.0093 by log-rank test). Knocking down the AFAP1L1 gene in sarcoma cells resulted in inhibition of the cell invasion, and forced expression of AFAP1L1 in immortalized human mesenchymal stem cells induced anchorage-independent growth and increased cell invasiveness with high activity levels of matrix metallopeptidase. Furthermore, tumor growth in vivo was accelerated in AFAP1L1-transduced sarcoma cell lines. These results suggest that AFAP1L1 has a role in the progression of spindle cell sarcomas and is a prognostic biomarker.

Mediastinal synovial sarcoma.

Synovial sarcoma of the mediastinum is a rare neoplasm that has overlapping histological and immunophenotypic features with other tumors in the differential diagnosis. We describe a case of this disease. The tumor was located at the right side of the pericardium, where an FDG-PET scan showed an uptake. It was resected, a resection which was complicated by the necessity of partially resecting the pericaridium and right middle lobe which were invaded by the tumor. The doubling time of the main tumor was 11.8 days. The margin of the resected specimen was tumor-free both macro- and microscopically. Reverse transcription-PCR confirmed the diagnosis of synovial sarcoma. The patient rejected chemotherapy or radiation therapy, and had recurrent tumors only one month after the operation. Finally, she opted to have only palliative care and died 79 days after the operation.

Activation of the non-canonical Dvl-Rac1-JNK pathway by Frizzled homologue 10 in human synovial sarcoma.

We previously reported that Frizzled homologue 10 (FZD10), a member of the Wnt signal receptor family, was highly and specifically upregulated in synovial sarcoma and played critical roles in its cell survival and growth. We here report a possible molecular mechanism of the FZD10 signaling in synovial sarcoma cells. We found a significant enhancement of phosphorylation of the Dishevelled (Dvl)2/Dvl3 complex as well as activation of the Rac1-JNK cascade in synovial sarcoma cells in which FZD10 was overexpressed. Activation of the FZD10-Dvls-Rac1 pathway induced lamellipodia formation and enhanced anchorage-independent cell growth cells. FZD10 overexpression also caused the destruction of the actin cytoskeleton structure, probably through the downregulation of the RhoA activity. Our results have strongly implied that FZD10 transactivation causes the activation of the non-canonical Dvl-Rac1-JNK pathway and plays critical roles in the development/progression of synovial sarcomas.

PGE2 signal via EP2 receptors evoked by a selective agonist enhances regeneration of injured articular cartilage.

OBJECTIVE: The effect of the prostaglandin E2 (PGE2) signal through prostaglandin E receptor 2 (EP2) receptors on the repair of injured articular cartilage was investigated using a selective agonist for EP2. METHODS: Chondral and osteochondral defects were prepared on the rabbit femoral concave in both knee joints, and gelatin containing polylactic-co-glycolic acid microspheres conjugated with or without the EP2 agonist was placed nearby. Animals were sacrificed at 4 or 12 weeks post-operation, and regenerated cartilage tissues and subchondral structure remodeling were evaluated by histological scoring. The quality of regenerated tissues was also evaluated by the immunohistochemical staining of EP2, type II collagen, and proliferating cell nuclear antigen (PCNA). As an evaluation of side effects, the inflammatory reaction of the synovial membrane was analyzed based on histology and the mRNA expression of matrix metalloproteinase3 (MMP3), tissue inhibitor of metalloproteinase 3 (TIMP3), and interleukin-1 beta (IL-1 beta). Also, the activity of MMP3 and the amount of tumor necrosis factor-alpha (TNF-alpha) and C-reactive protein in joint fluid were measured. RESULTS: In both models, the EP2 agonist enhanced the regeneration of the type II collagen-positive tissues containing EP2- and PCNA-positive chondrocytes, and the histological scale of regenerated tissue and subchondral bone was better than that of on the control side, particularly at 12 weeks post-operation. No inflammatory reaction in the synovial membrane was observed, and no induction of pro-inflammatory cytokines was found in joint fluid. CONCLUSION: Selective stimulation of the PGE2 signal through EP2 receptors by a specific agonist promoted regeneration of cartilage tissues with a physiological osteochondral boundary, suggesting the potential usefulness of this small molecule for the treatment of injured articular cartilages.

Cell therapy for avascular osteonecrosis of femoral head.

Avascular osteonecrosis of femoral head causes severe musculoskeletal disability. There is not standard treatment to cure avascular osteonecrosis. Recently, cell therapy using bone marrow stromal cells has begun for this disease.

Expression of the cadherin-11 gene is a discriminative factor between articular and growth plate chondrocytes.

OBJECTIVE: Calcification of hypertrophic chondrocytes is the final step in the differentiation of growth plates, although the precise mechanism is not known. We have established two growth plate-derived chondrocyte cell lines, MMR14 and MMR17, from p53-/- mice (Nakamata T, Aoyama T, Okamoto T, Hosaka T, Nishijo K, Nakayama T, et al. In vitro demonstration of cell-to-cell interaction in growth plate cartilage using chondrocytes established from p53-/- mice. J Bone Miner Res 2003;18:97-107). Prolonged in vitro culture produced calcified nodules in MMR14, but not in MMR17. Factors responsible for the difference in calcification between the two cell lines may also be involved in the physiological calcification in growth plate. DESIGN: Gene expression profiles of MMR14 and MMR17 were compared using a cDNA microarray to identify candidate genes involved in the calcification process. RESULTS: Forty-five genes were identified as upregulated in MMR14, including the cadherin-11 (Cdh-11) gene. The expression of Cdh-11 in MMR14 was detected in cell-cell junctions, while no expression was observed in MMR17. Primary cultured chondrocytes from growth plate (GC) also expressed the Cdh-11, and the staining of Cdh-11 was observed in the late hypertrophic zone of growth plate. Cell aggregation assays showed that chondrocytes required Ca2+ to form nodules, and knockdown of the Cdh-11 gene expression using short interfering RNA inhibited the formation of calcified nodules in MMR14. The introduction of Cdh-11 into MMR17 failed to produce calcified nodules indicating that Cdh-11 is one, but not the sole, factor responsible for the production of calcified nodules. CONCLUSION: Although the physiological role is still unclear, Cdh-11 is a discriminative factor between articular and growth plate chondrocytes.

Partial hemiarthroplasty for the treatment of osteonecrosis of the femoral head. An experimental study in the dog.

The use of a composite osteochondral device for simulating partial hemiarthroplasty was examined. The device was composed of a polyvinyl alcohol hydrogel and a titanium fibre mesh, acting as artificial cartilage and as porous artificial bone, respectively. The titanium fibre mesh was designed to act as an interface material, allowing firm attachment to both the polyvinyl alcohol gel (through injection moulding) and the femoral joint surface (through bony ingrowth). We implanted 22 of these devices into canine femoral heads. Histological findings from the acetabular cartilage and synovial membrane, as well as the attachment of the prosthesis to bone, were examined up until one year after operation. No marked pathological changes were found and firm attachment of the device to the underlying bone was confirmed. The main potential application for this device is for partial surface replacement of the femoral head after osteonecrosis. Other applications could include articular resurfacing and the replacement of intervertebral discs.

Role of uppermost superficial surface layer of articular cartilage in the lubrication mechanism of joints.

The uppermost superficial surface layer of articular cartilage, the 'lamina splendens' which provides a very low friction lubrication surface in articular joints, was investigated using atomic force microscopy (AFM). Complementary specimens were also observed under SEM at -10 degrees C without dehydration or sputter ion coating. Fresh adult pig osteochondral specimens were prepared from the patellas of pig knee joints and digested with the enzymes, hyaluronidase, chondroitinase ABC and alkaline protease. Friction coefficients between a pyrex glass plate and the osteochondral specimens digested by enzymes as well as natural (undigested) specimens were measured, using a thrust collar apparatus. Normal saline, hyaluronic acid (HA) and a mixture of albumin, globulin, HA (AGH) were used as lubrication media. The surface irregularities usually observed in SEM studies were not apparent under AFM. The articular cartilage surface was resistant to hyaluronidase and also to chondroitinase ABC, but a fibrous structure was exhibited in alkaline protease enzymes-digested specimens. AFM analysis revealed that the thickness of the uppermost superficial surface layer of articular cartilage was between 800 nm and 2 microm in adult pig articular cartilage. The coefficient of friction (c.f.) was significantly higher in chondroitinase ABC and alkaline protease enzymes digested specimens. Generally, in normal saline lubrication medium, c.f. was higher in comparison to HA and AGH lubrication media. The role of the uppermost, superficial surface layer of articular cartilage in the lubrication mechanism of joints is discussed.

Development of polyvinyl alcohol-hydrogel (PVA-H) shields with a high water content for tendon injury repair.

The main problem in tendon repair is adhesion formation between the tendon and surrounding tissue. To prevent this, we have developed adhesion preventive shields using polyvinyl alcohol hydrogel (PVA-H) with 90% water content. This implant experiment used the deep flexor tendon of the 3rd toe of the domestic fowl. Injured tendons shielded with PVA-H healed within about 3 weeks without adhesion to the surrounding tissues. Neither breakage of the PVA-H shield itself nor infection or degeneration in the surrounding tissue was observed. These results show that tendon is capable of intrinsic repair, and was able to regenerate using synovial nutrition through the PVA-H. The high water content of PVA-H may be clinically useful and applicable to adhesion preventive shields for tendon repair.

Definitive intraoperative very high-dose radiotherapy for localized osteosarcoma in the extremities.

PURPOSE: To evaluate the outcome and adverse effects in patients with osteosarcoma treated with very high-dose definitive intraoperative radiotherapy (IORT), with the intention of saving the affected limb. METHODS AND MATERIALS: Thirty-nine patients with osteosarcoma in their extremities were treated with definitive IORT. The irradiation field included the tumor plus an adequate wide margin and excluded the major vessels and nerves. Forty-five to 80 Gy of electrons or X-rays were delivered. The median follow-up of the surviving patients was 124 months. RESULTS: The cause-specific and relapse-free 5-year survival rate was 50% and 43%, respectively. Distant metastasis developed in 23 patients; 19 died and 4 were alive for >10 years. Nine local recurrences were found 4-29 months after IORT in the affected limb. No radiation-induced skin reaction or nerve palsy was observed in the patients treated with X-rays. Experiments using phantoms also confirmed that the scatter dose was below the toxic level in the IORT setting with X-rays. CONCLUSIONS: Very high-dose definitive IORT combined with preventive nailing and chemotherapy appeared to be a promising quality-of-life-oriented alternative to treating patients with osteosarcomas in the extremities, although the problem of recurrences from the surrounding unirradiated soft tissue remains to be solved.

Development of the shields for tendon injury repair using polyvinyl alcohol--hydrogel ( PVA-H).

In recent years, marked advances have been made in repair techniques for tendon injury, but the treatment of finger flexor tendon injury is still one of the most difficult and important problems in the orthopedic field. The main problem in tendon repair is adhesion between the tendon and surrounding tissue. To prevent this adhesion and achieve tendon union, we developed adhesion preventive shields for tendon repair using polyvinyl alcohol hydrogel ( PVA-H) with 90% water content, and carried out an implant experiment using the deep flexor tendon of the third toe of domestic fowl. Injured tendons shielded with PVA-H showed union at about 3 weeks after the operation without adhesion to the surrounding tissue and good function such as gliding and range of motion. Neither breakage of the PVA-H shield itself nor infection, nor degeneration in the surrounding tissue were observed. These results confirmed that the tendon itself has repair ability, and the tendon is regenerated by synovial nutrition through PVA-H. High water content PVA-H may have clinically potential and be applicable to adhesion-preventive shields for tendon repair. However, re-rupture was observed, probably due to accidental tendon injury at an early period after the operation. In some cases, tendon immobilization methods to prevent re-rupture might be necessary.

Study on the lubrication mechanism of natural joints by confocal laser scanning microscopy.

Natural joints have an excellent lubricating function, but the detailed mechanism is still unclear. To clarify this lubricating mechanism, we observed the behavior of the cartilage surface under the physiological loading condition with confocal laser scanning microscopy in normal and osteoarthritis (OA) cartilage rabbit specimens. Even with a considerable loading condition, in both natural and OA cartilage, the fluid pool area coexisted with the direct contact area. In the junction from the direct contact area to the fluid area, there was a third area with a liquid-crystal arrangement. In OA cartilage, these areas were generally irregular and small. These results suggest that a lubrication system in the fluid phase, such as squeeze film lubrication, might work under severe pressure in normal cartilage, and hyaluronic acid macromolecules in the synovial fluid might form a liquid-crystal structure and support pressure on the cartilage surface, whereas these systems did not affect the OA cartilage.

Translin binds to the sequences adjacent to the breakpoints of the TLS and CHOP genes in liposarcomas with translocation t(12;6).

Myxoid and round-cell liposarcomas share the translocation t(12;16)(q13;p11) creating the TLS-CHOP fusion gene as a common genetic alteration. We previously reported several unique characteristics of genomic sequences around the breakpoints in the TLS and CHOP loci, and among them was the presence of consensus recognition motifs of Translin, a protein that associates with chromosomal translocations of lymphoid neoplasms. We further extended our search for Translin binding motifs in sequences adjacent to breakpoints and investigated whether Translin binds to these sequences in vitro by mobility-shift assay. Computer-assisted search found sequences highly homologous (>70%) with Translin binding motifs adjacent to the breakpoints in 10 out of 11 liposarcomas with the TLS-CHOP fusion genes. All of 13 oligonucleotides corresponding to the putative binding sequences in these cases bind to Hela cell extract and also recombinant Translin protein, although the binding affinity of each motif showed considerable differences. The DNA-protein complex formation was inhibited by non-labeled competitor or anti-Translin antibody, suggesting the specificity of the complex formation. Considering the high incidence and specific binding property, the presence of Translin binding motif may be one of the important determinants for the location of breakpoints in the TLS and CHOP genes in liposarcomas.

Intra-operative radiation therapy for osteosarcoma in the extremities.

The outcome following intra-operative radiation therapy in the treatment of osteosarcoma in the extremity in 33 patients was evaluated for oncological and functional results. Local recurrence occurred in seven cases, six of which were in a non-irradiated region, indicating inappropriate planning of the radiation field. Twenty-one patients underwent either prosthetic replacement (14) or amputation (7). Irradiated tumours were left in situ in the remaining 12 patients. In this latter group no degenerative joint changes were observed radiologically. Twenty-six patients experienced local complications, of which fracture of the irradiated bone was the most significant. Associated intramedullary nailing showed encouraging results in preventing fracture. Although IORT is effective for the local control of osteosarcoma in extremities, critical patient selection and improvements of treatment protocol are required in order to obtain a satisfactory outcome.

[Multiple exostoses].

Multiple exostoses is a hereditary disease characterized by multiple osteocartilagenous tumors, of which the histological structures are similar to those of normal epiphyses. Genetic linkage has identified three different loci for this disease: EXT1 on 8q, EXT2 on 11p, and EXT3 on 19p. The EXT1 and EXT2 genes were recently isolated and mutation analyses have been performed in a number of patients with different ethnic backgrounds. The data indicate that mutations of these genes occurred in broad regions of each gene, and the loss-of-function mutations were predominant, although there were some missense mutations that may create functionally defective protein. Tumor cells were shown to be homozygous for the mutant allele, which is consistent with the concept of these genes as tumor suppressor genes. Recent progress for the functional analyses has disclosed that these genes encode the protein with glycosyltransferase activity and regulate the diffusion of Hedgehog protein, which is the key molecule for the skeletal development. Further analyses of these genes may provide us with the knowledge for the development of epiphyses, and may open the new research field for the regeneration of epiphyses.

High incidence of SV40-like sequences detection in tumour and peripheral blood cells of Japanese osteosarcoma patients.

Recent studies have revealed the evidence for the significance of SV40 genome in human malignancies. In this paper, the presence of SV40-like sequences was investigated in 54 Japanese osteosarcomas in which mutations of the retinoblastoma (Rb), p53, MDM2, and CDK4 genes had been already analysed. Using polymerase chain reaction and Southern hybridization, SV40-like sequences were detected in 25 cases (46.3%). In most cases, only a part of SV40 genome was detected, and the regulatory region containing enhancer sequences was most frequently found (21/54, 38.9%). There was no apparent relationship between the presence of SV40-like sequences and tumour suppressor genes mutations in each tumour. The SV40-like sequences were also detected in peripheral blood cells of substantial proportion of the patients (43.3%), whereas the incidence was much lower (4.7%) in normal healthy controls. This difference is statistically highly significant (P < 0.0001), suggesting that the presence of SV40-like sequences, even if only a part, may play some roles to predispose individuals to osteosarcoma.

Association of the human NPPS gene with ossification of the posterior longitudinal ligament of the spine (OPLL).

OPLL (ossification of the posterior longitudinal ligament of the spine) is a common form of human myelopathy with a prevalence of as much as 4% in a variety of ethnic groups. To clarify the genetic factors that predispose to OPLL, we have studied ttw (tiptoe walking), a mouse model that presents ectopic ossification of the spinal ligaments similar to OPLL and have found that the ttw phenotype is caused by the nonsense mutation of the gene encoding nucleotide pyrophosphatase (NPPS), a membrane-bound glycoprotein thought to produce inorganic pyrophosphate, a major inhibitor of calcification and mineralization. To investigate a possible role of NPPS in the etiology of OPLL, we have examined its genetic variations in OPLL patients. A total of 323 OPLL patients was screened by means of polymerase chain reaction/single-strand conformation polymorphism analysis covering all the exons and their surrounding introns, plus about 1.5-kb of the promoter region. We identified ten nucleotide variations in the NPPS gene; five of the alterations caused amino-acid substitutions, and two of them were found specifically in OPLL patients. Subsequently, we performed an association study using these variations and found a significant association of an allele, viz., a deletion of T at a position 11 nucleotides upstream from the splice acceptor site of intron 20 (IVS20-11delT), with OPLL; the proportion of the individuals having this deletion was significantly higher (P = 0.0029) in OPLL patients than in controls, indicating that those who have this variation may be more susceptible to the abnormal ossification of the spinal ligaments. Thus, our study suggests that NPPS plays an important role in the etiology of human OPLL.