The effect of extracellular acidosis on the behaviour of mesenchymal stem cells in vitro.

The stem cell fraction of a cell population is finely tuned by stimuli from the external microenvironment. Among these stimuli, a decrease of extracellular pH (pHe) may occur in a variety of physiological and pathological conditions, including hypoxia and inflammation. In this study, by using bone marrow stem cells and dental pulp stem cells, we provided evidence that extracellular acidosis endows the maintenance of stemness in mesenchymal cells. Indeed, continuous exposure for 21 d to low pHe (6.5-6.8) conditions impaired the osteogenic differentiation of both cell types. Moreover, the exposure to low pHe, for 1 and up to 7 d, induced the expression of stemness-related genes and proteins, drove cells to reside in the quiescent G0 alert state and enhanced their ability to form floating spheres. The pre-conditioning with extracellular acidosis for 7 d did not affect the differentiation potential of dental pulp stem cells since, when the cells were cultured again at physiological pHe, their multilineage potential was almost unmodified. Our data provided evidence of the role of extracellular acidosis as a modulator of the stemness of mesenchymal cells. This condition is commonly found both in systemic and local bone conditions, hence underlining the relevance of this phenomenon for a better comprehension of bone healing and regeneration.

Analysis of dihydrofolate reductase and reduced folate carrier gene status in relation to methotrexate resistance in osteosarcoma cells.

BACKGROUND: To evaluate the impact of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes on methotrexate (MTX) resistance in osteosarcoma cells in relation to retinoblastoma (RB1) gene status. MATERIALS AND METHODS: A series of human osteosarcoma cell lines-either sensitive or resistant to MTX-and 16 osteosarcoma tumour samples were used in this study. RESULTS: In U-2OS MTX-resistant variants, and in other RB1-positive cell lines, MTX resistance was associated with increased levels of DHFR and with a slight decrease of RFC gene expression. In Saos-2 MTX-resistant variants, and in another RB1-negative cell line, development of MTX resistance was associated with a decrease in expression of RFC, without any significant involvement of DHFR. In osteosarcoma clinical samples, amplification of the DHFR gene at clinical onset appeared to be more frequent in RB1-positive compared with RB1-negative tumours. CONCLUSIONS: Amplification of the DHFR gene may occur more frequently in the presence of RB1-mediated negative regulation of its activity and can be present at clinical onset in osteosarcoma patients. Simultaneous evaluation of RFC, DHFR and RB1 gene status at the time of diagnosis may become the basis for the identification of potentially MTX-unresponsive osteosarcoma patients, who could benefit from treatment protocols with alternative antifolate drugs.

Cytokine production in the infrapatellar fat pad: another source of cytokines in knee synovial fluids.

BACKGROUND: Recent studies have shown that adipose tissue is an endocrine organ that releases various cytokines. OBJECTIVE: To investigate the production of growth factors and proinflammatory cytokines in infrapatellar fat pad specimens. METHODS: Infrapatellar fat pad tissues were obtained from patients during knee surgery. Protein levels of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), tumour necrosis factor (TNF)alpha, and interleukin (IL)6 in homogenised tissues were measured by an enzyme immunoassay. Gene expressions for those cytokines were examined by reverse transcription-polymerase chain reaction (RT-PCR). Localisation of bFGF and VEGF was evaluated by immunohistochemistry and in situ hybridisation. RESULTS: Infrapatellar fat pads were found to contain various protein levels of bFGF, VEGF, TNF alpha, and IL6. Further, gene expressions for these cytokines were detected by RT-PCR. Immunohistochemistry and in situ hybridisation showed that the expressions of both bFGF and VEGF were localised in immature adipocytes, interstitial undifferentiated mesenchymal cells, and vascular endothelial cells. CONCLUSION: The production of bFGF, VEGF, TNF alpha, and IL6 in the infrapatellar fat pad was demonstrated. Although synovial cells and articular chondrocytes are thought to be primary sources of cytokines found in knee synovial fluids, the results suggest that they may also originate from this fat pad.

Decrease in serum nucleotide pyrophosphatase activity in ankylosing spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is a prototype of a group of rheumatic diseases referred to as spondyloarthropathy. AS patients show marked ectopic ossification in the spine, occasionally resulting in so-called bamboo spine. Although a strong association with HLA-B27 has been reported, its aetiology remains undetermined. Another rheumatic disease, ossification of the posterior longitudinal ligament of the spine (OPLL), demonstrates ectopic ossification of the spinal ligaments very similar to that of AS. Recently, nucleotide pyrophosphatase (NPPS) was implicated in the aetiology of OPLL: an Npps mutation was found to cause OPLL in mice, and an association between a polymorphism of the human NPPS gene and OPLL was identified. The clinical similarities between AS and OPLL led us to hypothesize that NPPS may also be implicated in the aetiology of AS. To elucidate the role of NPPS in the pathogenesis of AS, we examined serum NPPS activity and the possible association of the NPPS gene with AS. METHODS: Forty-four Japanese patients with AS, 43 patients with OPLL, and age- and sex-matched normal volunteers took part in this study. We determined serum NPPS activity using high-performance liquid chromatography and examined the association between AS and NPPS using single nucleotide polymorphisms (SNPs) of the NPPS gene. RESULTS: Serum NPPS activity in AS patients was significantly decreased compared with the controls (P < 0.0001). However, there was no association between AS and NPPS gene SNPs. CONCLUSION: NPPS is implicated in the pathogenesis of AS.

Identification of EWS/FLI-1 transcripts in giant-cell tumor of bone.

The EWS/FLI-1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the pathogenesis of Ewing's sarcoma. We demonstrate the presence of EWS/FLI-1 hybrid transcripts also in giant-cell tumor, a bone neoplasm featuring intermediate characteristics between benign and malignant lesions. Chimeric products were detected by semi-nested PCR after 2 cycles of amplification in 13/15 cases of giant-cell tumor, and their presence was confirmed by Southern and Western blots and fluorescence in situ hybridization. Moreover, 3/8 primary cultures of giant-cell tumor showed the same type of hybrid transcript observed in the original tumor sample. Sequencing of PCR products confirmed the presence of EWS and FLI-1 sequences in these products. Detection of EWS/FLI-1 fusion transcripts in giant-cell tumor of bone provides a model for the study of the transforming mechanisms of the EWS/FLI-1 fusion gene in mesenchymal tumors.

Mechanical stress induces the expression of high molecular mass heat shock protein in human chondrocytic cell line CS-OKB.

OBJECTIVE: Mechanical stress is an important regulator of chondrocyte function, but it is unknown how chondrocytes respond to mechanical stress. This study was performed to clarify the underlying mechanisms in human chondrocytes. DESIGN: Using a Flexercell strain unit (25% maximal elongation, 0.05 Hz-cyclic manner, and 48 h), mechanical stimulation was applied to confluent CS-OKB cells, human chondrocytic cells. To analyze transcriptional changes in response to mechanical stress, differential display reverse transcription-polymerase chain reaction (DDRT-PCR) and Northern blot analysis were performed. RESULTS: Among several differentially displayed fragments, one fragment (927 bp) tentatively named as SIC (Stress-Induced Chondrocytic) 1 was isolated from the human chondrocytic cell line and identified as one of the high molecular mass heat shock proteins. CONCLUSION: Mechanical stress induces the expression of a high molecular mass heat shock protein corresponding to SIC 1 in human chondrocytic cells. SIC 1 may play an important role in the mechanical stress-responded metabolism of human chondrocytes.

Murine model for skeletal metastases of Ewing's sarcoma.

Ewing's sarcoma shows a strong tendency to metastasize to the lungs or the skeleton, or both. A peculiar feature of the secondary involvement of bone with this tumor is that it may also appear in the absence of clinically evident lung metastases, both at clinical presentation and during the course of the disease. Although osseous metastases are critically relevant for prognosis, the pathogenesis of this peculiar feature of Ewing's sarcoma is poorly understood, partly due to the lack of appropriate experimental in vivo models. We show that the intravenous injection of TC-71 Ewing's sarcoma cells into athymic 4-5-week-old Crl/nu/nu (CD1) BR mice reproducibly colonizes specific sites of the skeleton in addition to the lungs and lymph nodes. The distribution and the morphologic appearance of these experimental bone metastases mimic the pattern of skeletal involvement observed in humans. This experimental model of bone metastasis of Ewing's sarcoma may be the basis for future studies aimed at understanding the pathophysiology and treatment of Ewing's sarcoma.

MRI findings in intramuscular lipomas.

OBJECTIVE: To investigate the spectrum of magnetic resonance (MR) findings of intramuscular lipoma. DESIGN AND PATIENTS: A retrospective review of 17 consecutive cases of intramuscular lipoma examined with MR imaging was undertaken. Features assessed included the size and margin of the mass; the homogeneity of the contents, including the presence or absence of intermingled muscle fibers; whether the mass was uninodular or multinodular; and the presence of linear structures between and within the tumor nodules. Three well-differentiated liposarcomas and one dedifferentiated liposarcoma associated with lipoma-like components were also studied to allow a comparison of the benign and malignant lesions. RESULTS: The diameter of the intramuscular lipomas varied from less than 3 cm to more than 10 cm. Ten of the intramuscular lipomas were homogeneous but the remaining seven were inhomogeneous with intermingled muscle fibers within the mass. The intramuscular lipomas were well defined in 12 cases, and infiltrative in five. In one case the margin of the lesion showed prominent infiltration of the surrounding muscle tissue. Of the 17 cases of intramuscular lipoma, 15 were composed of a single nodule, whereas three of four cases of liposarcoma were composed of multinodular masses. CONCLUSION: The MR findings of intramuscular lipoma varied from a small, single and homogeneous mass identical to ordinary (superficial) lipoma, to a large, inhomogeneous lesion with an infiltrative margin. The presence of infiltrative margins and intermingled muscle fibers in intramuscular lipoma indicates a benign lesion rather than malignancy. In addition, uninodularity of the mass is helpful in differentiating intramuscular lipoma from well-differentiated liposarcoma.

Redundancy of autocrine loops in human osteosarcoma cells.

With the aim of identifying innovative therapeutic strategies for osteosarcoma patients who are refractory to conventional chemotherapy, we analyzed the in vitro effects of the blockage of autocrine circuits. Since the insulin-like growth factor-I receptor (IGF-IR)-mediated loop is relevant to the growth of osteosarcoma, we analyzed the activity of the IGF-IR-blocking antibody alphaIR3 in both sensitive and multidrug-resistant osteosarcoma cell lines. Only limited effects, however, were observed, suggesting the simultaneous existence of other autocrine circuits. Indeed, in a representative panel of 12 human osteosarcoma cell lines, in addition to the IGF-IR-mediated circuit, we demonstrated also a loop mediated by epidermal growth factor receptor as well as the presence of nerve growth factor, low-affinity nerve growth factor receptor as well as tyrosine receptor kinase A in the great majority of osteosarcomas. Therapies based on the inhibition of single circuits may have only limited effects in osteosarcoma, whereas the use of suramin, a drug which, besides other activities, non-selectively interferes with the binding of growth factors to their receptors, appears as a promising alternative, in both sensitive and drug-resistant osteosarcoma cells.

Characterization of a newly established human chondrosarcoma cell line, CS-OKB.

A clonal cell line, CS-OKB, was derived from a human chondrosarcoma and characterized by cytogenetic study, immunocytochemical staining, and reverse transcriptase polymerase chain reaction (RT-PCR). Chromosomal abnormalities characteristic of malignant cartilaginous neoplasms were identified. CS-OKB cells were intensely stained with anti-type II collagen and anti-keratan sulphate antibodies. RT-PCR indicated that CS-OKB transcribes cartilage-specific genes such as type II, X procollagen, and aggrecan. This human chondrosarcoma cell line is stable and expresses well-differentiated chondrocyte-specific genes. It synthesizes well-differentiated chondrocyte-specific molecules in uncoated plastic dishes. CS-OKB may be useful for studies of human chondrocytes and in characterizing human chondrosarcomas.

Pubic osteolysis mimicking a malignant lesion: report a case with a fracture dislocation of the sacroiliac joint.

We report the case of a 67-year-old woman who presented to our institution with osteoblastic and osteolytic lesions of the pubis and fracture dislocation of the left sacroiliac joint. She had presented 7 months earlier to an outside institution with a left gluteal mass, which had been biopsied and had been believed to represent malignancy. A second biopsy at our institution showed no evidence of malignancy and was felt to represent fracture healing. A diagnosis of public osteolysis was made based on the radiographic and histologic findings. A follow-up radiograph 6 years after presentation revealed healing of the lesions, confirming its benignity.

Use of preoperative autologous blood donations and erythropoietin for treatment of giant cell tumor of the ischium.

A 24-year-old man with an osteolytic lesion of the ischium was referred to the authors' institution. Computed tomography and magnetic resonance imaging studies showed that the lesion extended to and involved the subchondral bone of the acetabulum. Histologic examination of the biopsy specimen revealed giant cell tumor of bone. Following the biopsy, autologous blood was collected 4 times with recombinant human erythropoietin treatment definitive surgery was performed. Three weeks after the biopsy, the lesion was curetted and bone cementation was performed. The total blood loss during surgery was 3100 ml, which was replaced successfully with stored autologous blood without the need for homologous blood transfusion. The authors believe that without the erythropoietin treatment, autologous blood could have been collected only 3 times instead of 4 times, and the patient would have needed homologous blood.

Lumboperitoneal shunt for cauda equina syndrome in ankylosing spondylitis.

Cauda equina syndrome is a rare complication in the late stage of ankylosing spondylitis, for which approximately 60 cases have been reported in the literature. The cause of the syndrome is unclear, and there is no effective treatment. Recently lumboperitoneal shunt was reported to have been effective in two patients. In our study, we performed lumboperitoneal shunt in a patient and evaluated the condition after the operation compared with that preoperatively. Some alleviation of neurologic symptoms was observed for 6 months after operation. Histopathologic examination of the dural diverticulum revealed a residual change after old inflammation. Lumboperitoneal shunt was an effective surgical treatment for cauda equina syndrome in this patient with ankylosing spondylitis, but its effects were not extreme. Arachnoiditis is suggested to be involved in the pathogenesis of cauda equina syndrome.

Analysis of the presence of osteocalcin, S-100 protein, and proliferating cell nuclear antigen in cells of various types of osteosarcomas.

Osteosarcomas are characterized by different histologic subtypes that are composed of heterogeneous tumor cells. Although the histological origin of the malignant cells is unknown, it has been speculated that osteoblasts lead to the malignant cells. In the current study, the osteosarcoma cells in 27 lesions were assessed by means of immunohistochemical staining for osteocalcin (OC), S-100 protein (S-100) and proliferating cell nuclear antigen (PCNA). PCNA labeling indices were the highest in osteoblastic and stromal areas, and significantly lower in chondroblastic areas (p < 0.01). Cells that were positive for both PCNA and OC were abundant in osteoblastic and stromal areas, while cells that were positive for both PCNA and S-100 were rarely observed. These results were almost similar for conventional, parosteal and periosteal osteosarcomas. In contrast, OC reactivity was poor in fibroblastic osteosarcoma, in osteosarcoma with giant cells, and in telangiectatic osteosarcoma. Pulmonary metastatic osteosarcoma lesions weakly expressed OC (p < 0.01), but showed high values for the PCNA labeling indices. In conclusion, immunohistochemical staining for OC, S-100, and PCNA are useful to analyze the proliferating cells in osteosarcomas. The main proliferating cells in most osteosarcomas are mature osteoblast-like cells. OC-negative tumor cells predominate in some of osteosarcoma subtypes, and these tumors therefore probably represent a distinct osteosarcoma variant. OC expression in pulmonary metastatic lesions may be suppressed.

The identity of proliferating cells in bone tumors with cartilaginous components: evaluation by double-immunohistochemical staining using proliferating cell nuclear antigen and S-100 protein.

S-100 protein (S-100) appears to be a marker for bone tumors of cartilaginous origin. Any analyses of proliferative activity in S-100-positive tumor cells, however, has not yet been presented. This study assessed the proliferative activity of those cells by means of a double-immunohistochemical staining method using proliferating cell nuclear antigen (PCNA) and S-100. The most intense reactivity for S-100 was found in the well-differentiated chondrocytes of enchondromas, osteochondromas, and osteosarcomas. On the contrary, the more immature the tumor cells were, the more intensely positive they were for PCNA. In parosteal chondrosarcoma, exceptionally, PCNA-positive as well as S-100-positive cells were abundant, suggesting that these proliferating cells produced S-100. In periosteal osteosarcoma, however, the proliferating cells labeled by PCNA revealed little reactivity for S-100. This immunohistochemical method is potentially useful to know the identity and origin of proliferating cells and may sometimes be diagnostic for bone tumors containing cartilaginous elements.

Periosteal osteosarcoma and parosteal chondrosarcoma evaluated by double immunohistochemical staining. Report of 2 cases.

Differentiation of periosteal osteosarcoma and parosteal (periosteal) chondrosarcoma by conventional histology may be difficult. One case each of clinically and histologically proven periosteal osteosarcoma and parosteal chondrosarcoma were evaluated by a double-immunohistochemical staining method using proliferating cell nuclear antigen (PCNA) and S-100 protein (S-100). Conventional histology showed proliferation of both osteoblastic and chondroblastic cells in the periosteal osteosarcoma, while there was a growth of only chondroblastic tumor cells in the parosteal chondrosarcoma. Immunohistochemical studies indicated that the nuclei of chondroblastic cells recognized by S-100 were PCNA-negative, while osteoblastic stromal cells were PCNA-positive in the periosteal osteosarcoma. In contrast, chondroblastic cells in the parosteal chondrosarcoma were both S-100- and PCNA-positive. Our findings suggest that periosteal osteosarcoma is characterized by the proliferation of osteoblastic stromal cells, whereas parosteal chondrosarcoma is characterized by the proliferation of chondroblastic cells. This method of double immunohistochemical staining, using PCNA and S-100, may be useful in differentiating these chondroblastic tumors.