Influence of osteopontin silencing on survival and migration of lung cancer cells.

BACKGROUND AND PURPOSE: Osteopontin (OPN) is a multifunctional protein overexpressed in many cancers and is involved in tumor progression and metastasis. In lung cancer, elevated OPN expression is associated with an unfavorable prognosis. Therefore, inhibition of OPN is an attractive approach for improving survival. MATERIALS AND METHODS: We used siRNA to specifically downregulate OPN expression in A549 lung cancer cells. OPN silencing was evaluated with quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for mRNA levels and with Western blotting for protein levels. Effects on cell proliferation were measured by cell counting. The influence on tumor cell migration was detected using a modified Boyden chamber. Changes in cell cycle distribution were assessed by flow cytometry. Using the colony formation assay, we determined changes in radiosensitivity. RESULTS: A specific and effective downregulation of OPN expression was detected in both RNA and protein levels. Cell proliferation and cell migration were significantly reduced by OPN silencing after 24 h and the effects were further increased by the addition of irradiation. The cell cycle distribution showed a reduction in S phase and an increase in cells arrested in both G(0)/G(1) and G(2)/M phases. Specific enhancement of radiosensitivity was clearly shown after OPN knockdown. CONCLUSION: The combination of OPN silencing and irradiation showed a synergistic effect leading to reduced cell survival.

Single-stage revision of peri-prosthetic infection following total elbow replacement.

This study reviews the predisposing features, the clinical, and laboratory findings at the time of diagnosis and the results of single-stage revision of prosthetic replacement of the elbow for infection. Deep infection occurred in six of 305 (1.9%) primary total elbow replacements. The mean follow-up after revision was 6.8 years (6 months to 16 years) and the mean age at the time of revision was 62.7 years (56 to 74). All six cases with infection had rheumatoid arthritis and had received steroid therapy. The infective organism was Staphylococcus aureus. Four of the six elbows had a developed radiolucency around one component or the other. Successful single-stage exchange arthroplasty was carried out with antibiotic-loaded cement in five of the six cases. In one, the revision prosthesis had to be removed following recurrence of the infection. The functional result was good in three elbows, fair in one, poor in one and fair in the resection arthroplasty.

One-stage exchange shoulder arthroplasty for peri-prosthetic infection.

There are few reports in the literature of the diagnosis and treatment of the infected shoulder arthroplasty. Most deal with resection arthroplasty and two-stage exchange surgery. We present our results of one-stage exchange operation as treatment for the infected shoulder arthroplasty. Our group comprised 16 patients (ten men, six women) with 16 infected arthroplasties. By the time of follow-up, two patients had died (mean 5.8 years), two could not be located and three had already undergone revision surgery. Nine patients were thus available for clinical examination and assessment. The infections were largely caused by staphylococci, Propionibacterium species and streptococci. Two were early infections (within three months of surgery) and 14 were late infections. The mean follow-up was 5.8 years (13 months to 13.25 years) when the mean Constant-Murley score was 33.6 points and the mean University College of Los Angeles score 18.3 points. Further revision was performed in three patients. One sustained a peri-prosthetic humeral fracture, another developed an acromial pseudarthrosis after transacromial surgery and the third suffered recurrent dislocations. No patient had a recurrence of infection. A one-stage exchange procedure using antibiotic-loaded bone cement eradicated infection in all our patients and we suggest that such a procedure is at least as successful as either a resection arthroplasty or a two-stage exchange in the management of the infected shoulder arthroplasty.

Is "aseptic" loosening of the prosthetic cup after total hip replacement due to nonculturable bacterial pathogens in patients with low-grade infection?

BACKGROUND: Loosening of the prosthetic cup is the limiting factor in the service life of total hip prostheses (THPs). Despite effective culture methods, the detection of low-grade infection in patients with loose implants still presents a challenge. It is crucial to distinguish between "aseptic" loosening and loosening due to periprosthetic infection, so that appropriate treatment can be administered. We investigated whether aseptic loosening of the acetabular components of THPs is due to unrecognized infection. METHODS: From October through December 2002, a total of 24 patients with acetabular cup loosening were investigated. Only patients without clinical signs of infection and with negative results of bacteriologic culture of synovial fluid (obtained by preoperative aspiration) were included in the study. Intraoperative biopsy samples obtained from the neocapsule and synovia (e.g., the interface membrane) were examined by means of routine culture methods and by polymerase chain reaction (PCR) for detection of 16S ribosomal RNA (rRNA). Control subjects included 9 patients undergoing primary hip arthroplasty. RESULTS: C-reactive protein levels and erythrocyte sedimentation rates were slightly elevated in the group with loosening, compared with the control group, but the difference was not statistically significant. PCR and routine culture showed no microorganisms in either group, with the exception of 1 patient in the loosening group. CONCLUSIONS: PCR for detection of 16S rRNA in tissue specimens obtained from hip joints is not superior to routine bacteriologic culture techniques for detection of low-grade infections. However, these results demonstrate that the loosening of cups in THPs do not usually result from nonculturable periprosthetic infection, if the microbiological processing is adequate.

Polyetheretherketone--cytotoxicity and mutagenicity in vitro.

The results of the incubation of polyetheretherketone (PEEK) fibre material with seven different genotype variants of salmonella bacterium showed with and without an external metabolic activation system (S9) with no mutagenic or cytotoxic activity of the test material. In the so-called "plate incorporation test" in which the PEEK raw material is finely cut and applied direct to the agar plate without the addition of solvent there was, as expected, no evidence of cytotoxic or mutagenic effects. In the HPRT test there was a significant increase in the number of mutants per dish, both after addition of N-acetylaminofluorene and N-methyl-N'-nitro-N-nitrosoguanidine (with and without an external metabolic activation system = +S9), but not after treatment of the cells with PEEK-DMSO-eluate. This means that the PEEK material under study did not release any substances that cause V79 cells to mutate. The investigation of the toxic reaction on the material under study revealed that the number of surviving colonies per 10(5) surviving cells lay within the range of or below the solvent control even in the presence of high PEEK concentrations (5.0 microg/ml). Therefore, in summary, the study produced no evidence of cell damage caused by PEEK.

Cytotoxicity and mutagenicity of Kevlar: an in vitro evaluation.

Toxicity and mutagenicity of Kevlar 49 (PPPT; poly-para-phenylene-terephthalamide) was tested in six strains of Salmonella typhimurium (Ames test; TA97, TA98, TA100, TA102, TA1535, TA1537) with and without an external metabolic activation system (S9), as well as in a mammalian cell mutagenesis assay using V79 Chinese hamster cells. For the Ames test, liquid preincubation, which is considered particularly sensitive, was used. The cells were incubated for 24 h at a temperature of 37 degrees C either directly with Kevlar49 or with ethanol- or chloroform-extracted Kevlar49. The experiments were performed at least twice. The Ames test with six different Salmonella typhimurium strains featuring either base pair substitution or frameshift mutations revealed no cytotoxic or mutagenic activity of Kevlar49. In the mammalian cell mutagenesis assay, using 8-azaguanine (AG) as a selective agent, Kevlar49 was also devoid of cytotoxic or mutagenic activity. Both tests have to be regarded as an initial exploratory screening due to the chosen testing conditions and should be supplemented by tests at different temperatures.

In vitro toxicity and mutagenicity of CoCrMo and Ti6Al wear particles.

Contact of wear particles with body fluids can result in widespread dissemination of extractable constituents from joint implants. The aim of this in vitro study is to clarify whether there is a mutagenic and/or carcinogenic risk from CoCrMo and Ti6Al wear particles. Particles of a representative size were produced by fretting; toxicity and mutagenicity were investigated using the salmonella/microsome test according to AMES and the V79-HGPRT Test (Chinese Hamster Fibroblasts). To obtain the greatest possible elution of all constituents the metallic wear particles were extracted with dimethylsulfoxyd and water and the resulting eluates mixed together. Neither the bacterial assay nor the mammalian cell system after repeated test series under standardised conditions produced evidence of a toxic or mutagenic effect in the concentration range under study. It is therefore not to be expected that CoCrMo or Ti6Al alloys initiate carcinogenesis in the human organism.

[Carpo-metacarpal dislocation injuries]. / Karpometakarpale Luxationsverletzungen.

Carpometacarpal dislocations are rare. In most cases, the dislocation is caused by a violent injury, e.g. the crash of a motorcyclist or a fall from great height. A considerable swelling of the back of the hand may mask the characteristic lump at the root of the hand. The diagnosis can be easily missed due to serious associated injuries. The lesion is also often overlooked in the routine X-ray diagnostic. Lateral and oblique views are important for the recognition of the true extent of the lesion. Anatomical reposition is difficult if the dislocation is not fresh and open reposition is necessary. Between 1990 and 1999, altogether 16 patients who had suffered a carpometacarpal dislocation were treated. One patient had a rare dislocation of the III. to V. joints. Simultaneous dislocation of all five carpometacarpal joints was seen in three cases. The treatment mostly consists of closed reduction and temporary Kirschner wire transfixation. Fourteen patients were controlled (clinical examination and X-ray control) after an average follow-up time of three years. In addition, the DASH questionnaire was used. After an average time of 3 years the patients showed in 64% of the cases excellent and good results (eight excellent, one good). The subjective function of the hand was moderate in 29% (four patients), in one patient the subjective function was poor (7%). Five patients had a diminished range of motion of the hand. The average points in the DASH questionnaire were 24. We recommend as therapy of choice after primary closed repositioning, primary surgery by means of short Kirschner wires introduced from distal-dorsal to proximal-palmar. In cases of impossible closed reduction or in cases with local additional injuries, open treatment for fixation, is indicated.

Allocation of nonbirefringent wear debris: darkfield illumination associated with PIXE microanalysis reveals cobalt deposition in mineralized bone matrix adjacent to CoCr implants.

Abrasive joint replacement material that accumulates in the tissue induces reciprocal effects between prosthesis material and organism. Since the limitations of brightfield and polarized light microscopy for foreign body analysis are well known, a method was applied that ensures the detailed histological assessment of nonbirefringent particles in periprosthetic soft and hard tissue. Cemented and cementless interface regions of five selected autopsy hip implant cases (2 x Endo-Modell Mark III, LINK, 1 x St. Georg Mark II, LINK, Germany; 2 x Spongiosa Metal II, ESKA, Germany) were viewed under darkfield illumination and subsequently analyzed with proton-induced X-ray emission (PIXE). Eight autopsy cases without implants served as controls. Using darkfield illumination technique, metallic particles became visible as luminous points under the microscope. The majority of particles in the samples from the cemented cases were degradation products of radiopaque bone cement. There was minimal evidence of metallic alloy particles in the soft tissues. However, a considerable quantity of heavy metal cobalt (Co) was found in the periprosthetic mineralized bone tissue, which was not observed in the controls. The periprosthetic concentration of cobalt ranged from 38 to 413 ppm. The findings demonstrate a correlation between cobalt concentration, time since implantation, and distance from the implant. Darkfield microscopy associated with PIXE enables a detailed histological assessment of metal particles in the tissue. In an effort to optimize biomechanics, implant design and implantation techniques, the contamination of soft and hard tissue with heavy metal degradation products deserves similar attention in terms of alloy assortment.

[Polyethylene abrasion: cause or consequence of an endoprosthesis loosening? Investigations of firm and loosened hip implants]. / Polyethylen-Abrieb: Ursache oder Folge einer Endoprothesenlockerung? Untersuchungen an festen und gelockerten Hüftendoprothesen.

AIM: Periprosthetic tissue was analysed by the combination of different investigation techniques without destruction. The localisation and geometry of polyethylene abrasion particles were determined quantitatively to differentiate between abrasion due to function and abrasion due to implant loosening. Non-polyethylene particles from implant components which contaminate the tissue were micro-analytically measured. The results will help us to understand loosening mechanisms and thus lead to implant optimisations. METHOD: A non-destructive particle analysis using highly sensitive proton-induced X-ray emission (PIXE) was developed to achieve a better histological allocation. Five autopsy cases with firmly fitting hip endoprosthesis (2 x Endo-Modell Mark III, 1 x St. Georg Mark II, LINK, Germany; 2 x Spongiosa Metal II, ESKA, Germany) were prepared as ground tissue specimens. Wear investigations were accomplished with a combined application of different microscopic techniques and microanalysis. The abrasion due to implant loosening was histologically evaluated on 293 loosened cup implants (St. Georg Mark II, LINK, Germany). RESULTS: Wear particles are heterogeneously distributed in the soft tissue. In cases of cemented prostheses, cement particles are dominating whereas metal particles could rarely be detected. The concentration of the alloy constituent cobalt (Co) is increased in the mineralised bone tissue. The measured co-depositions depend on the localisation and/or lifetime of an implant. Functional polyethylene (PE) abrasion needs to be differentiated from PE abrasion of another genesis (loosening, impingement) morphologically and by different tissue reactions. CONCLUSION: In the past a reduction of abrasion was targeted primarily by the optimisation of the bearing surfaces and tribology. The interpretation of our findings indicates that different mechanisms of origin in terms of tissue contamination with wear debris and the alloy should be included in the improvement of implants or implantation techniques.

[Chronic cobalt poisoning in endoprosthetic replacement]. / Chronische Kobaltvergiftung in der Endoprothetik. Ein Fallbericht.

This article outlines symptoms and therapeutic options in chronic cobalt poisoning including a case report in which metallosis caused by a ceramic-metal articular pairing led to almost complete loss of sight and hearing after revision of a total hip prosthesis. At primary revision the firmly incorporated stem was left in place. For a better offset only the head was exchanged from a ceramic to a metal model which articulated with a socket containing a ceramic inlay. Postoperatively, movement of the hip joint became increasingly uncomfortable and painful. After 2 years, the patient started complaining about increasing impairment of his eyesight followed by a gradual loss of hearing. In a second revision, examination of the explanted material showed almost complete deterioration of the metal femoral head and a partially fractured ceramic inlay with extensive contamination of the bone and surrounding soft tissue by metal debris. At the time of revision increasing concentrations of the alloy elements cobalt, chromium, and molybdenum were measured in the serum and liquor. The concentration of cobalt, in particular, was remarkably high. Treatment options in cases with chronic cobalt poisoning include chelation therapy with EDTA or BAL/DMPS.

Dissimilar action of two cyclic adenosine-monophosphate analogues on the sodium current in intact rat papillary muscle.

In intact papillary muscles from rat we have found with the loose-patch-clamp technique an increase of the fast cardiac sodium current (INa+) by isoproterenol (ISO). In this study we have tested two membrane permeable analogues of the intracellular second messenger cyclic adenosine-monophosphate (cAMP) to investigate the intracellular pathway: 8-Br-cAMP (50 microM) and the newer developed Sp-5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole- 3',5'-cyclic-monophosphorothioate (5,6-DCl-cBiMPS, 20 microM). The availability of INa+ was determined with test pulses to +/- 0 mV every 3.5 seconds after 2.5-second conditioning between -130 mV and -50 mV and a holding potential at the resting potential of the cell under examination, and after wash-in of either compound. The peak currents were fit to a Boltzmann equation, and expressed by the maximal attainable current INa+,max, the mid-point potential V1/2, and a steepness parameter alpha. Values are given by mean +/- SEM. 8-Br-cAMP showed a significant shift of the availability curve in the hyperpolarized direction (V1/2 = -82 +/- 2 mV vs -86 +/- 2 mV, n = 5, P < 0.05) with only minor changes of INa+,max and alpha. In contrast, 5,6-DCl-cBiMPS had no significant effect on V1/2 but increased INa+,max by 8% +/- 2% versus control (n = 5, P < 0.05). In an intact muscle preparation we have found that 5,6-DCl-cBiMPS has a similar effect as that observed with the beta-adrenergic agonist ISO (100 nM), whereas 8-Br-cAMP exhibited a dissimilar action.(ABSTRACT TRUNCATED AT 250 WORDS)

Fine mapping of human PI 3-kinase associated p85 alpha transcripts in the YAC contig surrounding the spinal muscular atrophy gene.

During a search for transcribed sequences within the gene region for autosomal recessive spinal muscular atrophy (SMA), two cDNA clones were isolated from a human fetal brain and an adult spinal cord cDNA library, respectively, by use of the cosmid LA96B (LAS96). The clones sized 950 bp and 1733 bp detect a 7.7-kb transcript in all tested human tissues. An additional transcript of 6.6 kb is detectable in brain and kidney, and faintly in skeletal muscle and liver. Using comparative human Northern blot analysis, the isolated LA96B cDNA clones could be identified as parts of the 3' untranslated region from the phosphatidylinositol 3 (PI3)-kinase associated p85 alpha transcripts; these were unknown up to now. The 5' end of the gene was mapped to YAC-EFTA:A, whereas the 3' end was localized within the distal overlapping YAC 85 flanking the SMA candidate gene region.

[Imaging of alloplastic ligament implant. An in vivo and in vitro study exemplified by Kevlar]. / Zur bildgebenden Darstellung alloplastischen Bandmaterials. Eine In-vivo und In-vitro-Untersuchung am Beispiel Kevlar.

Neither native X-ray nor CT or NMR allow to evaluate intraarticular implantation results of Kevlar -49 directly. In animal trials, the course of an artificial ligament may only be presumed from connective tissue ingrowth. Although soft tissue structure appears much better in NMR than in CT, direct proof of ligament continuity is still impossible. As soon as the connective tissue becomes continuous, it appears clearly and allows indirect evaluation of the prosthesis, as integrity can be judged by its shape like in natural cruciate ligament. Anatomic preparations show that connective tissue fills up the small space between the two cords of a Kevlar -49 two bundle prosthesis eight weeks after implantation, so that imaging systems show only one intraarticular bundle.

Mapping of a human rRNA gene in the YAC contig surrounding the SMA candidate gene.

Using the yeast artificial chromosome (YAC) 116 flanking the autosomal recessive spinal muscular atrophy (SMA) gene region, we have screened a human fetal brain cDNA library and isolated the cDNA clone 14-3/9 with an insert size of 2.5 kb. The cDNA clone could be identified as part of the human rRNA gene coding for 28S rRNA with a total size of 5025 bp. The human 28S rRNA is involved in the organization of the 60S ribosomal subparticle and is arranged in a 13-kb pre-rRNA transcription unit that occurs in tandem repeat clusters. Multiple copies of the rRNA gene have been mapped by pulsed field blot hybridization in the YAC contig between YAC 66 and YAC 116, which encompasses the SMA candidate gene, and additionally in the distally localized YAC 153.

[Pathologic humeral fracture in secondary osteosarcoma of the humerus: a rare complication of osteodystrophia deformans Paget]. / Pathologische Oberarmfraktur beim sekundären Osteosarkom des Humerus: eine seltene Komplikation der Osteodystrophia deformans Paget.

Pathological fracture in histologically proven post-Paget osteosarcoma of the humerus is a rare complication. Due to individual requests as well as age and comorbidity, a course of primary palliative treatment was chosen in the present case. Survival time after diagnosis was 9 months and the patient died of a tumor-independent disease. Even in combined treatment, consisting of surgery and (neo-)adjuvant radio-/chemotherapy, prognosis of osteosarcomas secondary to Paget's disease remains very disappointing. Therefore, in treatment of this highly lethal tumor the patient's individual requests and personal situation often require more consideration than in many other malignancies.

[Endoprosthetic surgery of the elbow]. / Endoprothetik des Ellenbogens.

Artificial joint replacement has found a firm place in orthopaedic surgery since its first introduction in the late 1960s. While the initial fixed bearing implants tended to progress to early loosening, the development of so-called "sloppy joints" has seen a major advance in the survival and success rate of this arthroplasty. The surgical approach and technique have also been modified in such a way as to allow a complete ventral release of a flexion contracture, while at the same time preserving the integrity of the extensor mechanism. In this way, the improvement of the biomechanics of the implant combined with diligent surgical technique have enhanced this procedure dramatically. Between 1978 and 1999, 305 GSB 3-type prosthesis were implanted, with the underlying pathologies being rheumatoid arthritis (77%), posttraumatic arthritis (21%) and degenerative arthritis (2%). The range of motion could be significantly improved from the pre-operative state for extension as well as flexion. This was even more obvious in the rheumatoid than in the posttraumatic situation. The survival rate for this type of implant was 90% at 10 years, with the implants after rheumatoid surgery faring somewhat better than those of the posttraumatics. Alternative treatment options, such as resection arthroplasty, distraction arthroplasty, or arthrodesis, are nowadays employed only in rare cases where a previous infection, personal preference or an inability by the patient to co-operate in a rehabilitation program have to be considered. The overall functional results of these types of treatments seem to be inferior to that of elbow arthroplasty. Therefore, this procedure is recommended not only for advanced degenerative and rheumatoid pathologies, but also in cases of posttraumatic arthrosis.

[In vitro toxicity and mutagenicity of CoCrMo and TiAl wear particles]. / In-vitro-Toxizität und Mutagenität von CoCrMound TiAl-Abriebpartikeln.

Contact of wear particles with body fluids can result in widespread dissemination of extractable constituents from joint implants. The aim of this in vitro study is to clarify whether there is a mutagenic and/or carcinogenic risk from Co(28)Cr(6)Mo and Ti(6)Al(4)V wear particles. Particles of a representative size were produced by fretting; toxicity and mutagenicity were investigated using the Ames Salmonella/microsome test and the V79-HGPRT Test (Chinese hamster fibroblasts). To obtain the greatest possible elution of all constituents, the metallic wear particles were extracted with DMSO and water and the resulting eluates mixed together. After repeated test series under standardized conditions, neither the bacterial nor the mammalian cell assays produced evidence of toxic or mutagenic effects in the concentration range under study. It is therefore not to be expected that CoCrMo or TiAl alloys initiate carcinogenesis in the human organism.

[One-stage revision of shoulder arthroplasty in the case of periprosthetic infection]. / Einzeitiger Schulterendoprothesenwechsel bei periprothetischer Infektion.

AIM: Infection of shoulder arthroplasties is rare, but represents a potentially devastating complication. The aim of this work is to show the value of various diagnostic procedures, specify causative pathogens and present the results of one-stage revised patients. METHOD: We performed a retrospective analysis of our 16 consecutive patients with an infected shoulder arthroplasty. RESULTS: In 13 of 16 cases a causative pathogen could be established preoperatively. Staphylococcus and Propioni spp. dominated. Only 9 patients could be followed up because two died, two were lost and three patients were revised because of non-infectious complications. The follow-up time was 5.8 years (13 months-13.25 years). The Constant-Murley score was 33.6 of 100. Eradication of infection was achieved in all patients. In the follow-up time no reinfection has occurred. CONCLUSION: Culturing of the preoperative joint fluid aspirate and the determination of CRP provide an early diagnosing of shoulder arthroplasty infection. One-stage revision arthroplasty with radical debridement and application of antibiotics to the bone cement provide an accurate therapy of periprosthetic shoulder infection.