Clinical, tomographic, and postmortem aspects of a rare congenital Dicephalus Monauchenos Iniodymus in a Nelore calf produced in vitro from Brazil: case report / Aspectos clínicos, tomográficos e pós-morte de um raro Dicephalus Monauchenos Iniodymus congênito em um bezerro Nelore produzido in vitro no Brasil: relato de caso

The aim of this work was to report the occurrence of dicephalus iniodymus monauchenos in a Nellore newborn. A three-days old calf, from in vitro production, with duplication of the head and a history of cesarean birth was attended. On physical examination, the dicephalus, iniodymus and monauchenos, which were almost the same size and shape, had four eyes and four ears. Computed tomography showed the presence of two skulls fused with a common occipital foramen, two nasopharynxes, oropharynxes with the presence of a cleft lip and a cleft palate in the right head, which continued in a single esophagus and a single trachea. At necropsy, the presence of duplication of the cerebrum and cerebellum was observed, with union of the parts in the region of the trapezoid body of the brainstem and continued as a single spinal cord. This study characterizes the clinical, tomographic, and necropsy findings of a dicephalus Nelore neonate.(AU)

O objetivo deste trabalho foi relatar a ocorrência de Dicephalus Iniodymus Monauchenos em um neonato da raça Nelore de produção in vitro. Foi atendida uma fêmea bovina, de três dias de idade, com duplicação das cabeças e histórico de nascimento por meio de cesariana. No exame físico, observou-se a dicefalia, Iniodymus e Monauchenos, apresentando quatro olhos e quatro orelhas. Na tomografia computadorizada, constatou-se a presença de dois crânios fundidos com um forame occipital comum, duas nasofaringes, orofaringes com presença de lábio leporino e fenda palatina na cabeça direita, que continuavam em um único esôfago e em uma única traqueia. Na necropsia, observou-se a presença de duplicação do encéfalo e cerebelo, com união das partes na região do corpo trapezoide do tronco encefálico, que continuavam como uma única medula espinhal. Este estudo caracteriza os achados clínicos, tomográficos e de necropsia de um neonato Nelore dicefálico.(AU)

First occurrence of Arnold Chiari type II malformation and associated abnormalities in a Gir calf produced in vitro from Brazil - case report / [Primeira ocorrência de malformação de Arnold Chiari tipo II e anormalidades associadas em uma bezerra Gir de produção in vitro no Brasil - relato de caso]

This study characterized the clinical, radiological, ultrasound, and necroscopic findings of a case of Arnold-Chiari type II malformation in a Gir breed calf from Brazil. The animal was hospitalized at sixty days of age, in permanent sternal recumbency, cutaneous appendix at the 4th lumbar vertebra and kyphoscoliosis of the caudal and lumbosacral thoracic spine. Radiographic examination of the spine and skull revealed spina bifida and suspected occipital hypoplasia. Upon examination of myelography with an injection of lumbar and atlantooccipital contrast, it was possible to visualize the meningocele at the 4th lumbar vertebra region and findings at the rhombencephalon level of increased regional pressure with failure to fill the contrast in the posterior fossa, in the presence of clear demarcation of the circumvolutions of the cerebral cortex and the subarachnoid space of the cervical spinal cord. Ultrasonographic examination of the cerebellum showed an insinuation of the cerebellar worm through the foramen magnum. The animal did not show changes in complete blood count, biochemical series, and cerebrospinal fluid and was negative for Pestivirus. There was a worsening of the clinical conditions and the animal died. This malformation of unknown etiology must be studied as a differential diagnosis of the nervous system disorders.(AU)

Este estudo caracterizou os achados clínicos, radiológicos, ultrassonográficos e necroscópicos de um caso de malformação de Arnold-Chiari tipo II em uma bezerra Gir no Brasil. O animal foi hospilatizado aos 60 dias de idade, apresentando decúbito esternal permanente, apêndice cutâneo na altura da quarta vértebra lombar e cifoescoliose da coluna vertebral torácica caudal e lombossacra. Ao exame radiográfico da coluna e do crânio, foram observadas espinha bífida e suspeita de hipoplasia occipital. Ao exame de mielografia com injeção de contraste lombar e atlanto-occipital, foi possivel visualizar a meningocele na altura da quarta vértebra lombar e achados em nível rombencefálico de aumento da pressão regional com falha de preenchimento do contraste na fossa posterior, na presença de nítida demarcação das circunvoluções do córtex cerebral e do espaço subaracnoide da medula espinhal cervical. Ao exame ultrassonográfico do cerebelo, foi observada insinuação do verme cerebelar através do forame magno. O animal não apresentou alterações em hemograma completo, série bioquímica e fluido cérebro-espinhal e foi negativo para Pestivirus. Houve uma piora do quadro clínico e o animal morreu. Essa malformação de etiologia desconhecida deve ser estudada como um diagnóstico diferencial.(AU)

Estudo anatomorfológico, radiográfico e tomográfico de Schistosomus reflexus em ovino da raça Dorper: relato de caso / Anatomorphological, radiographic and tomographic studies of Schistosomus reflexus in Dorper breed sheep: case report

O Schistosomus reflexus (SR) é uma anomalia congênita fatal e rara, primariamente observada em ruminantes, a qual apresenta feto com dorsoflexão da coluna vertebral, exposição das vísceras abdominais e torácicas e escoliose. Foi realizada uma cesariana de ovelha da raça Dorper, em propriedade da região de Botucatu-SP. Pelas características gerais e achados de necropsia, tratava-se de um caso de SR. A radiografia revelou acentuado desvio ventrodorsal do segmento da coluna vertebral toracolombar e deformidades das costelas. Na tomografia computadorizada, observou-se presença da integridade dos ossos e órgãos, exceto ausência de uma vértebra lombar. Não havia dados na literatura de diagnóstico por imagem de SR em ovinos, portanto procedeu-se, de forma inédita, a exames radiográfico e tomográfico do feto.

Schistosomus reflexus (SR) is a rare and fatal congenital anomaly, primarily observed in ruminants, presenting fetus with dorsiflexion of the vertebral column, exposure of thoracic and visceral organs, and scoliosis. A caesarian was performed on a Dorper sheep on a farm in Botucatu-SP and according to the general characteristics and findings observed in the necropsy of the fetus it was an SR case. The radiography indicated a marked ventro-dorsal deviation of the thoracolumbar column segment and deformity of the ribs. On the computed tomography the integrity of the bones and organs were detected, except for the absence of a lumbar vertebra. No data were available in the literature for SR diagnosis in sheep by image, so radiographic and tomographic exams of the fetus were done.

Livin and Bcl-2 expression in high-grade osteosarcoma.

AIMS: The evaluation of prognosis in patients with osteosarcoma is limited to clinical parameters. Although numerous molecular markers have been studied, none are currently in routine clinical use. The aim of this study was to determine if Livin and Bcl-2, acting as antiapoptotic proteins through different mechanisms, are expressed in osteosarcoma, and whether they can be used as prognostic markers in human osteosarcoma. METHODS: Tumor specimens of 29 patients with high-grade central osteosarcoma, with complete clinical follow-up for a minimum of 5 years, were studied. The localization and distribution of Livin and Bcl-2 were investigated using immunohistochemistry. Results were correlated with the histological response to chemotherapy, 5-year disease-free and 5-year overall survival. RESULTS: Bcl-2 was expressed only in the cytoplasm of 16/29 cases and there was no statistically significant correlation between expression and any of the studied parameters. Livin was detected in 17/29 cases, in the cytoplasm of all 17 and in the nucleus of only 3 cases. Nuclear expression was significantly correlated with a decreased overall survival (P < 0.0002) compared with those patients without nuclear expression. CONCLUSIONS: The results of this study indicate that Bc1-2 and Livin are highly expressed in osteosarcoma cells and that possibly, the evaluation of nuclear Livin expression might be a useful prognostic marker in osteosarcoma.

Fusion of the wrist in rheumatoid arthritis: a clinical and functional evaluation of two surgical techniques.

We retrospectively compared wrist arthrodesis using the Mannerfelt technique in 19 or an AO-plate in 23 patients with long-standing rheumatoid arthritis. The mean follow-up was for 76 months. Compared with the Mannerfelt fusion group, patients in the AO-plate group reported greater satisfaction with their wrist function (74% vs 37%, p = 0.015). Complications were reported in six wrists in the AO-plate group and two wrists in the Mannerfelt fusion group (p = 0.258). At final follow-up, 95% of patients (41) reported either no pain or only mild pain. There was improvement in flexion of the finger joints in both groups but no significant improvement in the extension lag in either group. Both methods relieve pain and improve function. Overall, the activities of daily living scores and the patients' subjective assessment of outcome tended to be higher in the AO-plate group than in the Mannerfelt fusion group, although the difference was not statistically significant. Similarly, although more postoperative complications occurred in the AO-plate group, the difference between the two groups was not statistically significant.

Metastatic bone disease: a 36-year single centre trend-analysis of patients admitted to a tertiary orthopaedic surgical department.

BACKGROUND AND OBJECTIVES: The treatment and outcome of primary malignant bone tumours has changed with the advances in diagnostic and treatment modalities. A trend-analysis on a large cohort of patients with metastatic bone disease was performed. METHODS: A retrospective chart review of all cases with metastatic bone disease admitted to a single tertiary orthopaedic referral centre, registered with the Vienna Bone and Soft Tissue Tumour Registry between 1968 and 2003 was conducted. For trend-analysis of frequency, survival, primary site, treatment methods, and others, the 36-year study duration was divided into four periods. RESULTS: The study identified 601 females and 580 males (mean: 60 years) with metastatic bone disease. The most common metastases were secondary to breast cancer (n = 275; 23%) and renal cell carcinoma (n = 242; 21%) and the majority were located in the femur (n = 332; 28%) and spine (n = 348; 29%). Overall, the proportion of patients who underwent surgery decreased. At follow-up, 887 (75%) patients were verified to have died of their disease. CONCLUSIONS: Over the 36-year period, the frequency of bone metastases has increased at our centre. Although survival increased over time, the difference was not significant; this was most likely attributable to the seriousness of cases referred to our tertiary care centre.

Toll-like receptors and chondrocytes: the lipopolysaccharide-induced decrease in cartilage matrix synthesis is dependent on the presence of toll-like receptor 4 and antagonized by bone morphogenetic protein 7.

OBJECTIVE: To assess the presence of Toll-like receptors (TLRs) 1-9 in human articular cartilage, and to investigate the effects of lipopolysaccharide (LPS)-induced activation of TLR-4 on biosynthetic activity and matrix production by human articular chondrocytes. METHODS: TLRs 1-9 were assessed in human articular cartilage by reverse transcription-polymerase chain reaction (RT-PCR); TLR-4 was also analyzed by Western blotting and immunohistochemistry. Articular chondrocytes were isolated from human donors and from wild-type or TLR-4(-/-) mice. Chondrocyte monolayer cultures were incubated with interleukin-1beta (IL-1beta) and LPS in the absence or presence of bone morphogenetic protein 7 (BMP-7) and IL-1 receptor antagonist (IL-1Ra). Neosynthesis of sulfated glycosaminoglycans (sGAG) was measured by (35)S-sulfate incorporation. Endogenous gene expression of cartilage markers as well as IL-1beta was examined using RT-PCR. The involvement of p38 kinase or p44/42 kinase (ERK-1/2) in LPS-mediated TLR-4 signaling was investigated by immunoblotting, RT-PCR, and sGAG synthesis. RESULTS: TLRs 1-9 were found on the messenger RNA (mRNA) level in human articular chondrocytes. The presence of TLR-4 was also observed on the protein level. In murine and human articular chondrocytes, but not in chondrocytes derived from TLR-4(-/-) mice, stimulation with LPS resulted in a decrease in total proteoglycan synthesis. IL-1beta mRNA expression was increased by TLR-4 activation, whereas expression of aggrecan and type II collagen was significantly decreased. The presence of BMP-7 and IL-1Ra antagonized the anti-anabolic effects of LPS. Blocking of p38, but not ERK-1/2, resulted in inhibition of both LPS-mediated IL-1beta gene expression and the negative effects of LPS on matrix biosynthesis. CONCLUSION: These data demonstrate the presence of TLRs in human articular cartilage. The suppressive effects of LPS on cartilage biosynthetic activity are dependent on the presence of TLR-4, are governed, at least in part, by an up-regulation of IL-1beta, and are mediated by p38 kinase. These in vitro data indicate an anti-anabolic effect of TLR-4 in articular chondrocytes that may hamper cartilage repair in various joint diseases.

[Endoprosthesis management of the extremities of children after resection of primary malignant bone tumors]. / Die endoprothetische Versorgung an den Extremitäten bei Kindern nach Resektion primär maligner Knochentumoren.

In recent years, significant progress has been made in the treatment of primary malignant bone tumors. The surgeon is confronted with increasing demand for limb-sparing surgery by patients and parents alike. In the skeletally immature, resection of the growing physis will ultimately result in leg length discrepancies and functional impairment. An adequate reconstruction therefore calls for the compensation of the expected growth deficit to achieve symmetry and an optimized function of the extremity. Between 1975 and 2000, 55 patients (24 female, 31 male) were referred and treated at our institution. All patients were not older than 10 years (4.2-10, mean: 7.9 years). Histological diagnosis was Ewing's tumors ( n=19) and osteosarcoma ( n=36). Localization in the extremities was predominantly in the lower extremity with 63.6% around the knee joint (distal femur and proximal tibia). Surgical treatment was performed in all patients. Endoprosthetic reconstruction was performed in 34 patients (lower extremity n=30, 29 Howmedica Modular Reconstruction System, HMRS, 1 Pafford prosthesis; upper extremity n=4, 4 HMRS); in another case of humeral tumor involvement a resection replantation procedure was carried out. The other patients received a rotationplasty ( n=7), a biologic reconstruction ( n=6), resection replantation of the humerus ( n=1), no reconstruction ( n=3), or had to be amputated ( n=4). After a mean follow-up of 114 months (24-256 months) 11 patients had died. Even in young children endoprosthetic reconstruction with relatively early mobilization and high quality of life can be a very successful method for limb salvage. Optimal reconstructive results following resection of primary malignant bone tumors in children are reached when indications and limitations of each method are carefully considered on an individual basis taking into account the patients' expectations.

Dura resection in combination with en bloc spondylectomy for primary malignant tumors of the spine.

STUDY DESIGN: Resection of a primary malignant tumor of the bone with wide surgical margins has become the golden standard in oncological surgery. In the case of a spinal tumor with tumor invasion of the spinal canal, a wide resection could necessitate resection and subsequent replacement of part of the dura. SUMMARY OF BACKGROUND DATA: Dura replacement is a consequent surgical step in the treatment of primary malignant tumors of the spine. METHODS: We present the case of a 27-year-old male with a tumor of the dorsal elements of D6. After paraplegic symptoms, a laminectomy of D5 to D7 was performed and histologic examination revealed the presence of an osteosarcoma. After neoadjuvant polychemotherapy, a wide resection of the dorsal elements from D4 to D7 combined with a resection of the dorsal part of the dura was planned. Replacement of the dura was performed with Lyodura (B. Braun Melsungen AG, Melsungen, Germany). The spine was stabilized with an ISOLA instrumentation (Depuy International Ltd., Leeds, England). For wound closure, a pedicled latissimus dorsi flap and split skin graft were necessary. In a second step, ventral spondylodesis with Texas Scottish Rite Hospital instrumentation (Sofamor Danek, Memphis, TN) was performed. RESULTS: One hundred sixteen months after surgery the patient is alive without evidence of disease, is pain free, and has returned to his profession and life without any restrictions.

[Diagnostic imaging in hip prostheses]. / Bildgebung bei Hüftprothesen.

Hip arthroplasty has become a common and still increasing procedure for the treatment of osteoarthritis, advanced head necrosis, post-inflammatory arthritis or rheumatoid arthritis. Radiography is the most important imaging modality for monitoring the normal, asymptomatic hip arthroplasty. Radiographs are obtained at the end of a surgical treatment, to exclude complications like fracture or component misplacement. In the follow-up radiographs are used for the diagnosis of loosening and infection of the hip arthroplasty as well as soft tissue ossification. Together with the history and clinical information, the analysis of morphological findings allows to find the grade of loosening. MRI has been advocated in the diagnosis of infection, in particular in the localisation of soft tissue involvement. Imaging, especially by radiographs, is used for the evaluation of the normal and complicated follow-up of hip arthroplasty.

Total vertebrectomy for primary malignant tumours of the spine.

Primary malignant tumours should be resected with wide margins. This may be difficult to apply to lesions of the spine. We undertook total vertebrectomy on seven patients, four males and three females with a mean age at operation of 26.5 years (6.3 to 45.8). The mean follow-up was 52.3 months. Histological examination revealed a Ewing's sarcoma in two patients and osteosarcoma, leiomyosarcoma, spindle-cell sarcoma, chondrosarcoma and malignant schwannoma in one each. In five patients, histological examination showed that a wide resection had been achieved. At follow-up there was no infection and a permanent neurological deficit was only seen in those patients in whom the surgical procedure had required resection of nerve roots. Despite the high demands placed on the surgeon and anaesthetist and the length of postoperative care we consider total vertebrectomy to be an appropriate procedure for the operative treatment of primary malignant lesions of the spine.

Spaceflight effects on cultured embryonic chick bone cells.

A model calcifying system of primary osteoblast cell cultures derived from normal embryonic chicken calvaria has been flown aboard the shuttle, Endeavour, during the National Aeronautics and Space Administration (NASA) mission STS-59 (April 9-20, 1994) to characterize unloading and other spaceflight effects on the bone cells. Aliquots of cells (approximately 7 x 10(6)) grown in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) were mixed with microcarrier beads, inoculated into cartridge culture units of artificial hollow fiber capillaries, and carried on the shuttle. To promote cell differentiation, cartridge media were supplemented with 12.5 microg/ml ascorbate and 10 mM beta-glycerophosphate for varying time periods before and during flight. Four cartridges contained cells from 17-day-old embryos grown for 5 days in the presence of ascorbate prior to launch (defined as flight cells committed to the osteoblastic lineage) and four cartridges supported cells from 14-day-old embryos grown for 10 days with ascorbate before launch (uncommitted flight cells). Eight cartridges prepared in the same manner were maintained under normal gravity throughout the flight (control cells) and four additional identical cartridges under normal gravity were terminated on the day of launch (basal cells). From shuttle launch to landing, all cartridges were contained in closed hardware units maintaining 5% CO2, 37 degrees C, and media delivery at a rate of approximately 1.5 ml/6 h. During day 3 and day 5 of flight, duplicate aliquots of conditioned media and accumulated cell products were collected in both the flight and the control hardware units. At the mission end, comparisons among flight, basal, and control samples were made in cell metabolism, gene expression for type I collagen and osteocalcin, and ultrastructure. Both committed and uncommitted flight cells were metabolically active, as measured by glucose uptake and lactate production, at approximately the same statistical levels as control counterparts. Flight cells elaborated a less extensive extracellular matrix, evidenced by a reduced collagen gene expression and collagen protein appearance compared with controls. Osteocalcin was expressed by all cells, a result indicating progressive differentiation of both flight and control osteoblasts, but its message levels also were reduced in flight cells compared with ground samples. This finding suggested that osteoblasts subjected to flight followed a slower progression toward a differentiated function. The summary of data indicates that spaceflight, including microgravity exposure, demonstrably affects bone cells by down-regulating type I collagen and osteocalcin gene expression and thereby inhibiting expression of the osteogenic phenotype notably by committed osteoblasts. The information is important for insight into the response of bone cells to changes of gravity and of force in general.

Chondrogenic potential of skeletal cell populations: selective growth of chondrocytes and their morphogenesis and development in vitro.

Most vertebrate embryonic and post-embryonic skeletal tissue formation occurs through the endochondral process in which cartilage serves a transitory role as the anlage for the bone structure. The differentiation of chondrocytes during this process in vivo is characterized by progressive morphological changes associated with the hypertrophy of these cells and is defined by biochemical changes that result in the mineralization of the extracellular matrix. The mechanisms, which, like those in vivo, promote both chondrogenesis in presumptive skeletal cell populations and endochondral progression of chondrogenic cells, may be examined in vitro. The work presented here describes mechanisms by which cells within presumptive skeletal cell populations become restricted to a chondrogenic lineage as studied within cell populations derived from 12-day-old chicken embryo calvarial tissue. It is found that a major factor associated with selection of chondrogenic cells is the elimination of growth within serum-containing medium. Chondrogenesis within these cell populations appears to be the result of permissive conditions which select for chondrogenic proliferation over osteogenic cell proliferation. Data suggest that chondrocyte cultures produce autocrine factors that promote their own survival or proliferation. The conditions for promoting cell growth, hypertrophy, and extracellular matrix mineralization of embryonic chicken chondrocytes in vitro include ascorbic acid supplementation and the presence of an organic phosphate source. The differentiation of hypertrophic chondrocytes in vitro is associated with a 10-15-fold increase in alkaline phosphatase enzyme activity and deposition of mineral within the extracellular matrix. Temporal studies of the biochemical changes coincident with development of hypertrophy in vitro demonstrate that proteoglycan synthesis decreases 4-fold whereas type X collagen synthesis increases 10-fold within the same period. Ultrastructural examination reveals cellular and extracellular morphology similar to that of hypertrophic cells in vivo with chondrocytes embedded in a well formed extracellular matrix of randomly distributed collagen fibrils and proteoglycan. Mineral deposition is seen in the interterritorial regions of the matrix between the cells and is apatitic in nature. These characteristics of chondrogenic growth and development are very similar in vivo and in vitro and they suggest that studies of chondrogenesis in vitro may provide a valuable model for the process in vivo.

Osteoblast cytoskeletal modulation in response to mechanical strain in vitro.

The structural integrity of microfilaments has been shown to be necessary for the signal transduction of mechanical stimuli within osteoblasts. Qualitative and quantitative changes within the cytoskeleton of osteoblasts may therefore be crucial components of the signal transduction processes of these cells in response to mechanical stimulation. Avian osteoblasts were strained with a device that deforms a flexible, cell-laden membrane at a defined frequency and intensity in a uniform biaxial manner. We examined the effects of mechanical strain on the accumulation of protein and the expression of the major cytoskeletal elements and specific integrin-binding (arginine-glycine-aspartic acid) proteins of these cells. Mechanical strain increased the level of total extracellular matrix-accumulated fibronectin by approximately 150% and decreased that of osteopontin by approximately 60% but had no quantifiable effect on the accumulation of beta1 integrin subunit or collagen type I. An examination of the major elements of the cytoskeleton demonstrated that neither the level of actin nor that of the intermediate filament protein vimentin changed; however, the amount of tubulin decreased by approximately 75% and the amount of vinculin, a major protein of focal adhesion complexes, increased by approximately 250%. An analysis of protein synthesis by two-dimensional gel electrophoresis of [35S]methionine-labeled cytoskeletal proteins demonstrated that the changes in the accumulation of vinculin and tubulin resulted from their altered synthesis. Messenger RNA analysis confirmed that the changes in accumulation and protein synthesis observed for vinculin, fibronectin, and osteopontin were controlled at a pretranslational level. Immunofluorescent microscopy demonstrated that mechanical strain led to increased formation and thickening of actin stress fibers, with a commensurate dissociation in microtubules and a clear increase in levels of vinculin at the peripheral edges of the cells. In conclusion, the elevated rate of synthesis and the increased accumulation of vinculin and fibronectin, as well as the increase in the number and size of stress fibers and focal adhesion complexes, suggest that mechanical strain leads to a coordinated change both in the cytoskeleton and in extracellular matrix proteins that will facilitate tighter adhesion of an osteoblast to its extracellular matrix.

Signal transduction of mechanical stimuli is dependent on microfilament integrity: identification of osteopontin as a mechanically induced gene in osteoblasts.

Mechanical perturbation has been shown to modulate a wide variety of changes in second message signals and patterns of gene expression in osteoblasts. Embryonic chick osteoblasts were subjected to a dynamic spatially uniform biaxial strain (1.3% applied strain) at 0.25 Hz for a single 2-h period, and osteopontin (OPN), an Arg-Gly-Asp (RGD)-containing protein, was shown to be a mechanoresponsive gene. Expression of opn mRNA reached a maximal 4-fold increase 9 h after the end of the mechanical perturbation that was not inhibited by cycloheximide, thus demonstrating that mechanoinduction of opn expression is a primary response through the activation of pre-existing transcriptional factors. The signal transduction pathways, which mediated the increased expression of opn in response to mechanical stimuli, were shown to be dependent on the activation of a tyrosine kinase(s) and protein kinase A (PKA) or a PKA-like kinase. Selective inhibition of protein kinase C (PKC) had no effect on the mechanoinduction of osteopontin even though opn has been demonstrated to be an early response gene to phorbol 12-myristate 13-acetate (PMA) stimulation. Mechanotransduction was dependent on microfilament integrity since cytochalasin-D blocked the up-regulation of the opn expression; however, microfilament disruption had no effect on the PMA induction of the gene. The microtubule component of the cytoskeleton was not related to the mechanism of signal transduction involved in controlling opn expression in response to mechanical stimulation since colchicine did not block opn expression. Mechanical stimulus was shown to activate focal adhesion kinase (FAK), which specifically became associated with the cytoskeleton after mechanical perturbation, and its association with the cytoskeleton was dependent on tyrosine kinase activity. In conclusion, these results demonstrate that the signal transduction pathway for mechanical activation of opn is uniquely dependent on the structural integrity of the microfilament component of the cytoskeleton. In contrast, the PKC pathway, which also activates this gene in osteoblasts, acts independently of the cytoskeleton in the transduction of its activity.

Endothelial eicosanoid metabolism and signal transduction during exposure to oxygen radicals injury.

Several physiological agonists that induce elevation of cytosolic free calcium (Ca2+)-levels act via receptor coupled G-proteins, involving activation of phospholipase C (PLC) and hydrolysis of phosphatidylinositol 4,5-bisphosphate. Activation of the inositol signal transduction pathway that precedes Ca2+ ion mobilization is a well accepted signaling pathway in endothelial cell eicosanoid synthesis. This study was designed to examine possible involvement of phosphoinositides in the effects of oxygen free radicals on Ca2+ liberation and eicosanoid synthesis in human umbilical venous endothelial cells (HUVEC). Hydrogen peroxide (H2O2) was chosen as oxygen radicals generating agent. Stimulation of HUVEC with H2O2 (0.1 mmol/l) led to significant rises in inositol phosphate and diacylglycerol (DAG) levels within 300 seconds and an inhibition of Ca2+ release from internal stores. Eicosanoid formation was detectable despite unchanged levels of cytosolic free Ca2+ and no detectable activation of membrane associated phospholipase A2 (PLA2). This suggests that eicosanoid formation may be mediated through the activation of a Ca2+ independent, cytosolic 40 kDa PLA2 isoenzyme and that DAG could serve as an alternative source for arachidonic acid and seems to sensitize a cytosolic PLA2.

Developmental restriction of embryonic calvarial cell populations as characterized by their in vitro potential for chondrogenic differentiation.

The mechanism(s) by which the cells within the calvaria tissue are restricted into the osteogenic versus the chondrogenic lineage during intramembranous bone formation were examined. Cells were obtained from 12-day chicken embryo calvariae after tissue condensation, but before extensive osteogenic differentiation, and from 17-day embryo calvariae when osteogenesis is well progressed. Only cell populations from the younger embryos showed chondrogenic differentiation as characterized by the expression of collagen type II. The chondrocytes underwent a temporal progression of maturation and endochondral development, demonstrated by the expression of collagen type II B transcript and expression of collagen type X mRNA. Cell populations from both ages of embryos showed progressive osteogenic differentiation, based on the expression of osteopontin, bone sialoprotein, and osteocalcin mRNAs. Analysis using lineage markers for either chondrocytes or osteoblasts demonstrated that when the younger embryonic cultures were grown in conditions that were permissive for chondrogenesis, the number of chondrogenic cells increased from approximately 15 to approximately 50% of the population, while the number of osteogenic cells remained almost constant at approximately 35-40%. Pulse labeling of the cultures with BrdU showed selective labeling of the chondrogenic cells in comparison with the osteogenic cells. These data indicate that the developmental restriction of skeletal cells of the calvaria is not a result of positive selection for osteogenic differentiation but a negative selection against the progressive growth of chondrogenic cells in the absence of a permissive or inductive environment. These results further demonstrate that while extrinsic environmental factors can modulate the lineage progression of skeletal cells within the calvariae, there is a progressive restriction during embryogenesis in the number of cells within the calvaria with a chondrogenic potential. Finally, these data suggest that the loss of cells with chondrogenic potential from the calvaria may be related to the progressive limitation of the reparative capacity of the cranial bones.

Variable hormone responsiveness of osteoblast populations isolated at different stages of embryogenesis and its relationship to the osteogenic lineage.

A variable response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] hormone treatment was observed for osteoblast cell populations isolated from 12- and 17-day-old embryonic chick calvariae. The younger embryonic cell population showed 2- and 5-fold inductions of osteocalcin and osteopontin gene expression, respectively, and a 25% inhibition of collagen gene expression when treated with 1,25-(OH)2D3. In contrast, these same genes all displayed approximately 80% inhibition of their expression when the older embryonic cell populations were treated with hormone. The hormone response was related to the appearance of the vitamin D3 receptor (VDR) and the developmental state of teh two cell populations by assessing the numbers of cells that were immunologically labeled for two osteoblast lineage, stage-specific surface makers (alkaline phosphatase and SB-5, an osteocyte marker) and the VDR. Using the sequence of marker presentation, with VDR appearing first, followed by alkaline phosphatase and then SB-5, models were tested using logistic regression analysis to validate this order of marker presentation and establish that the two embryonic ages of the cell populations represent discrete stages of their lineage. This analysis indicated that 1,25-(OH)2D3 treatment progressed the 12-day-old embryo cell populations along their lineage and that the hormone promoted the appearance of its own receptor (P < 0.001) However, the appearance of the VDR does not appear to be a determinant in the variable responses of the different embryonic aged cell populations to the hormone. These data quantitatively establish the unique nature of osteoblast cell populations within their lineage progression for cells isolated from embryos of different ages, such that cell populations isolated from younger embryos are comprised of primarily presumptive or immature osteoblasts, whereas cells isolated from older embryos are comprised of mature osteoblasts. These data also demonstrate that the genomic effects of 1,25-(OH)2D3 are dependent on the developmental stage of the osteoblast lineage, and the stimulatory actions of the hormone are targeted to immature osteoblasts, whereas the effect of the hormone on mature osteoblasts is inhibitory.

[Hallux rigidus operated on by Keller and Brandes method: radiological parameters of success and prognosis]. / Hallux rigidus operiert nach Keller und Brandes: Radiologische Erfolgs- und Prognoseparameter.

PURPOSE: The aim of this study was to evaluate the pre- and postoperative radiographic findings of hallux rigidus treated with Keller and Brandes arthroplasty to determine the radiographic outcome and to identify a prognostic marker. METHODS: 83 patients with a total of 121 cases of hallux rigidus operated using Keller and Brandes arthroplasty were followed up (mean 9.7 y). A comparison of the pre- and postoperative radiographs, the clinical and subjective findings was predicated on a five point scale: 1. percentage of proximal phalanx resected ( < 33%, 33-50%, > 50%), 2. joint space, 3. ratio of the length of the first and second metatarsals, 4. first intermetatarsal angle, and 5. hallux valgus angle. RESULTS: In the patient group which had 33-50% of the proximal phalanx excised (n = 67. 55%) the highest patient satisfaction was observed (96%). If resection of the proximal phalanx exceeded 50% (n = 13. 11%), non physiologic dorsiflexion of the toe occurred and patients were dissatisfied (62%). Excision of less than 33% of the hallux (n = 41. 34%) was associated with a recurrent hallux rigidus. No other evaluated radiological parameter proved to be of significance. CONCLUSION: The most important radiological parameter in the evaluation of the outcome of Keller and Brandes arthroplasty as a surgical treatment method for hallux rigidus was the percentage of the proximal phalanx which had ben excised.

Fibronectin gene expression, synthesis and accumulation during in vitro differentiation of chicken osteoblasts.

A well-defined chicken osteoblast culture system(18) has been used to examine fibronectin (FN) mRNA levels, synthesis, and accumulation during in vitro differentiation and matrix mineralization. Immunofluorescent staining of cells after 6 or 18 days in culture revealed that FN was initially associated with the cell surface and in partial coalignment with cytoskeletal elements while at the latter time most FN was associated with the extracellular matrix as a ubiquitous fibrillar network. Western blot analysis of total cell-associated proteins also detected FN at all culture times. However, when results were normalized to cellular DNA, FN levels increased until 12-16 and remained relatively constant thereafter. Similarly, FN synthesis as measured by [35S]-methionine labeling, and immunoprecipitation was greatest in early cultures (culture day 3) and then declined such that synthesis decreased 60% at day 18 and 94% after 24-31 days. FN mRNA levels as measured by Northern blot analysis were well correlated with FN synthesis. These results clearly show that FN is made by primary osteoblasts during their in vitro maturation. In contrast to other osteoblast markers such as alkaline phosphatase, osteocalcin, and osteopontin, whose expression increases as cells differentiate, FN accumulates in the matrix during periods of early cell growth and attachment and then remains proportional to cell number. Results with FN differ from those obtained with collagen which continues to accumulate in the extracellular matrix during osteoblast maturation. These results are consistent with FN being important for the initial attachment of early osteoblasts or osteoblast precursors to the pericellular matrix.