Functional outcome and quality of life following resection of the proximal humerus performed for musculoskeletal tumors and reconstruction done by four different methods.

INTRODUCTION: The proximal humerus is a frequent site for both primary and secondary bone tumors. Several options are currently available to reconstruct the resected humerus, but there is no consensus regarding optimal reconstruction. The aim of this retrospective study was to compare the functional outcome, complications and patient compliance following four different types of reconstructive techniques. MATERIAL AND METHODS: The authors performed 90 proximal humerus resections due to primary and secondary bone tumors over the past 21 years. Four different procedures were performed for reconstruction following the resection: fibula autograft transplantation, osteoarticular allograft implantation, modular tumor endoprosthesis (hemiarthroplasty) and reconstruction of the defect with a reverse shoulder prosthesis-allograft composite. A retrospective analysis of the complications and patient's physical status was performed. Functional outcome and life quality was evaluated by using the MSTS and SF-36 scores. RESULTS: The best range of motion was observed following arthroplasty with a reverse shoulder prosthesis-homograft composite followed by a fibula autograft reconstruction. Revision surgery was required due to major complications most frequently in the osteoarticular allograft group, followed by the reverse shoulder prosthesis-allograft composite group, the autologous fibula transplantation group; the tumor endoprosthesis hemiarthroplasty group had superior results regarding revision surgery (40, 25, 24 and 14% respectively). MSTS was 84% on average for the reverse shoulder prosthesis-allograft composite group, 70% for the autologous fibula group, 67% for the anatomical hemiarthroplasty group and 64% for the osteoartricular allograft group. Using the SF-36 questionnaire for assessment no significant differences were found between the four groups regarding quality of life. DISCUSSION: Based on the results of our study the best functional performance (range of motion and patient compliance) was achieved in the a reverse prosthesis-allograft combination group-in cases where the axillary nerve could be spared. The use of an osteoarticular allograft resulted in unsatisfying functional results and high complication rates, therefore we do not recommend it as a reconstructive method following resection of the proximal humerus due to either primary or metastatic bone tumors. Young patients who have good life expectancy but a small humerus or intramedullar cavity reconstruction by implantation of a fibula autograft is a good option. For patients with a poor prognosis (i.g. bone metastases) or in cases where the axillary nerve must be sacrificed, hemiarthroplasty using a tumor endoprosthesis was found to have acceptable results with a low complication rate. According to the MSTS and SF-36 functional scoring systems patients compliance was nearly identical following all four types of reconstruction techniques; the underlying cause may be the complexity of the shoulder girdle. However, we recommend the implantation of a reverse shoulder prosthesis-allograft whenever indication is appropriate, as it has been demonstrated to provide excellent functional outcomes, especially in young adults.

Transplantation of human mesenchymal stem cells in a non-autogenous setting for bone regeneration in a rabbit critical-size defect model.

Human mesenchymal stem cells (hMSC) represent an attractive cell population for tissue engineering purposes. Furthermore, hMSC are described as immune privileged, and non-autogenous application seems possible. The current study examines the regeneration potential of hMSC after xenogenic transplantation compared with autogenous rabbit MSC in a critical-size bone defect. After isolation, hMSC and rabbit MSC were seeded on calcium-deficient hydroxyapatite (CDHA) and transplanted into a radial critical-size defect of New Zealand white rabbits. Defects were filled with a CDHA scaffold seeded with autogenous rabbit MSC, CDHA seeded with xenogenic hMSC or unseeded CDHA. An empty defect served as control group. Animals were sacrificed after 3 months. Evaluation was performed using radiography, micro-computed tomography (micro-CT) and histology. In addition, a non-destructive four-point-bending test was performed in order to evaluate biomechanical stiffness. While autogenous MSC seeded on CDHA led to increased healing of critical-size bone defects from radiological (micro-CT; p = 0.009) and histological (p = 0.048) perspectives compared with unloaded CDHA, it was not possible to demonstrate analogous effects for the xenogenic transplantation of hMSC. The xenogenic treatment group displayed inferior results in all parameters compared with the autogenous MSC treatment group (histology p = 0.041; micro-CT p = 0.006; biomechanical testing p = 0.017). Nevertheless, no local or systemic inflammatory response resulting from xenogenic transplantation was observed. While previous papers suggest the use of non-autogenous hMSC cells for tissue engineering purposes, the present results show inferior clinical results from transplantation of hMSC in a xenogenic setting compared with autogenous MSC.

VEGF producing bone marrow stromal cells (BMSC) enhance vascularization and resorption of a natural coral bone substitute.

Bone graft substitutes often exhibit poor bone regeneration in large defects because of inadequate vascularization. Studies have shown that if blood supply is compromised, application of osteogenic factors alone could not induce successful healing. This study was to evaluate the effects of vascular endothelial growth factor, which combined with a coralline scaffold, on vascularization, scaffold resorption and osteogenesis in a rabbit radius critical size defect model. The scaffold was either coated with a control-plasmid DNA (group 1), coated with VEGF-plasmid DNA (group 2), loaded with mesenchymal stem cells (BMSC) transfected with control plasmid (group 3) or with both stem cells and the VEGF plasmid (group 4). X-rays were taken every 4 weeks up to week 16, when animals were euthanized. The volume of new bone was measured by mu-CT scans and blood vessels were counted after anti-CD31 staining of endothelial cells. The results from the solitary VEGF- and VEGF-transfected cells (groups 2 and 4) demonstrated significantly enhanced vascularization, osteogenesis and resorption of the carrier when compared to the control group. The highest degree of osteogenesis was found when the carrier was loaded with BMSC (group 3), whereas VEGF-transfected cells led to the highest vascularization and fastest resorption of the bone substitute. Additionally, VEGF-transfected BMSC led to a more homogenous vascularization of the defect. The results indicate that VEGF can be a helpful factor to improve healing in large bone defects, in which bone substitutes will otherwise not be vascularized and replaced by fresh bone.

Comparison of the degenerative changes in weight-bearing joints following cementing or grafting techniques in giant cell tumour patients: medium-term results.

The aim of this retrospective study was to compare and assess the effect of bone grafting and cementing techniques--two common applications used in the treatment of subchondral giant cell tumours of bone (GCTs)--on the development of degenerative changes in the weight-bearing joints of the lower extremity. Eighty patients were included in this follow-up study, 44 of whom underwent curettage followed by bone grafting, and 36 who had curettage followed by cementation. At the 24-month post-operative examination, significantly less degenerative change was found in patients with bone cement than in those with bone grafting. At the 50-month and later (range: 50-148 months) post-operative examination, however, no significant differences were found between the two groups, indicating that there was a significant acceleration of degenerative changes in the cemented group after the 24-month follow-up.

Functional outcome and life quality after endoprosthetic reconstruction following malignant tumours around the knee.

Between 1993 and 2002, we treated 43 patients with malignant musculoskeletal tumours of the knee region. All patients had partial resection of the femur or tibia together with endoprosthetic replacement. We were able to follow-up 23 patients with an average follow-up of 30 (12-97) months. Complications occurred in ten cases, of which one was a case of local recurrence. Most of the patients were completely satisfied with their condition, with a decreased walking distance as the only notable restriction. There was no correlation between the functional outcome and life quality assessment and the type of the implanted prosthesis, length of resected bone and type of resection. However, patients with tumours in the distal femur had significantly better functional and life quality outcome than those with a proximal tibial tumour.

p53 is involved in the p120E4F-mediated growth arrest.

Control of cell growth and division by the p53 tumor suppressor protein requires its abilities to transactivate and repress specific target genes and to associate in complex with other proteins. Here we demonstrate that p53 binds to the E1A-regulated transcription factor p120E4F, a transcriptional repressor of the adenovirus E4 promoter. The interaction involves carboxy-terminal half of p120E4F and sequences located at the end of the sequence-specific DNA-binding domain of p53. Ectopic expression of p120E4F leads to a block of cell proliferation in several human and murine cell lines and this effect requires the association with wild-type (wt) p53. Although p120E4F can also bind to mutant p53, the growth suppression induced by overexpression of the protein is severely reduced in a cell line that contains mutant p53. These data suggest that p120E4F may represent an important element within the complex network of p53 checkpoint functions.

45T-1, an established cell line with characteristics of Sertoli cells, forms organized aggregates in vitro after exposure to tumor necrosis factor alpha.

In the testis TNF is produced by germinal cells. The putative role of tumor necrosis factor alpha (TNF) in development and differentiation was investigated in 45T-1 mouse cell cultures, a cell line with characteristic markers of Sertoli cells, established from transgenic mouse families expressing the polyoma large T antigen in their testes. Exposure to TNF elicited a gradual assembly of the cells of the monolayer into highly organized spheroids. The first morphological sign of the changes was detected one week after TNF treatment by anti-desmin immunostaining which showed the formation of foci in the culture consisting of several hundred cells connected by an increasing number of cell contacts. Between days 10-20 the cells formed large ovoid or vermiform aggregates covered by several layers of flat, elongated cells. These cells extended septae into the inner mass of the spheroids consisting of loosely arranged, large polygonal or palisadic cells. The spheroids were surrounded by radially arranged elongated cells covered by small blebs. TNF treatment upregulated laminin expression in 45T-1 cell cultures, which is known to induce formation of cord-like structures by Sertoli cells in vitro. Coculturing 45T-1 cells with immortalized germinal cells or TNF-producing HeLa cells also lead to the formation of spheroids. These observations suggest that TNF production of germinal cells might contribute to the organization/differentiation of Sertoli cells.

Local non-synaptic modulation of aldosterone production by catecholamines and ATP in rat: implications for a direct neuronal fine tuning.

In addition to hypophyseal control, steroid synthesis and secretion in the adrenal cortex is also under direct local neural modulation. We obtained morphological and neurochemical evidence that a substantial proportion of the noradrenergic nerve endings lie in close proximity to zona glomerulosa cells without making synaptic contact, thus providing evidence for a direct local modulatory role of catecholamines in steroid secretion. These noradrenergic neurones, like other noradrenergic neurones in the central nervous system, are able to take up dopamine (DA), convert it partly into noradrenaline (NA) and to release both NA and DA together with the co-transmitter ATP when neuronal activity drives them to do so. These catecholamines and ATP may reach zona glomerulosa cells via diffusion in a paracrine way and modulate the synthesis of aldosterone. The presence of ecto-Ca-ATPases, enzymes that may terminate the effect of ATP, was demonstrated around the nerve profiles indicating that not only ATP but its metabolites (ADP, AMP, adenosine) can also influence the production of aldosterone. These data strongly support the possibility of a paracrine, non-synaptic modulatory role of catecholamines and ATP in the regulation of adrenocortical steroid secretion.

Cholinergic regulation of the rat adrenal zona glomerulosa.

Using histochemical and immunocytochemical methods, cholinergic nerve fibres were demonstrated in the rat adrenal cortex, primarily in the capsule and zona glomerulosa, and in the medulla. Some terminated among the glomerulosa cells or around blood vessels. Occasional fibres were also seen in the fasciculata, ending in islets of chromaffin tissue without ramifications on cortical cells. To clarify the role of cholinergic innervation, a microvolume perifusion system was used to study steroid production by the rat adrenal capsule-glomerulosa. Acetylcholine (ACh) itself had no reproducible effects; however, since variable amounts of endogenous ACh were present, the actions of antagonists were also studied. The M1 muscarinic receptor antagonist pirenzepine (10 and 100 microM) stimulated aldosterone secretion. This stimulation was abolished by co-incubation with carbachol, the M1 agonist McN A-343 and by atropine. We found that the action of pirenzepine was blocked by nifedipine (Ca2+ channel blocker), suggesting that pirenzepine (through release of endogenous ACh) provides an acute stimulus by enhancing Ca2+ inflow. Hemicholine, a choline uptake blocker, reduced the stimulatory effect of pirenzepine on steroid secretion, confirming that stimulation was of neural origin. Neither the non-selective muscarinic receptor antagonist atropine, the selective M1-M3 muscarinic receptor antagonist 4-DAMP, nor the selective M2 muscarinic receptor antagonist methoctramine influenced aldosterone output. Receptor-binding studies revealed the existence of M3 receptors in capsule-glomerulosa homogenates. We conclude that pirenzepine acts on presynaptic M1 autoreceptors to increase spontaneous ACh release from varicose axon terminals that lie in close proximity to the glomerulosa cells. In turn ACh may thus stimulate steroidogenesis acutely through M3 receptors. These results support the concept of a direct cholinergic influence on zona glomerulosa function in the rat.

Interactions between ouabain, atrial natriuretic peptide, angiotensin-II and potassium: effects on rat zona glomerulosa aldosterone production.

The effects of ouabain, atrial natriuretic peptide, angiotensin-II and potassium on aldosterone production by collagenase dispersed rat zona glomerulosa cells were studied. A-II and 10(-4) M ouabain-induced increases in aldosterone production was inhibited by 10(-9) M ANP at all potassium concentrations examined. 10(-4) M ouabain inhibited the A-II induced increase in aldosterone production at all potassium concentrations. The degree of this inhibition was smaller at higher potassium levels. Ouabain enhanced the inhibitory effect of ANP on A-II-induced aldosterone synthesis at all potassium concentrations. Interactions between A-II, ANP, ouabain and potassium may be of physiological significance in the regulation of aldosterone secretion.

[Ultrasound-guided fine-needle aspiration biopsy of focal liver lesions]. / Gócos májelváltozások ultrahangvezérelt vékonytü aspirációs citológiája.

The ultrasound guided percutaneous fine needle biopsy (US-FNAB) of focal lesions in the liver is indispensable in many clinical situations. During the last 12 years, 657 US-FNAB were performed on patients with suspected neoplastic involvement of the liver with 22-gauge Chiba needles at our department. US-FNAB was performed mostly with the "free hand" technique. Sufficient material for cytologic analysis was obtained in 84% of the cases. The biopsies confirmed malignancy in 39.3%, including 9% primary hepatocellular carcinoma, 8% of the cases were suspect for malignancy, and in 36.7% were diagnosed benign lesion. 233 cases were confirmed histologically and with other follow up methods. The sensitivity rate was 91%, and specificity was 100%. There was no false positive diagnosis and no noteworthy complications were observed. US-FNAB is a highly reliable, safe, inexpensive and easy diagnostic procedure. On the basis of our experience, we recommend US-FNAB as a routine, first level procedure for the diagnosis of focal liver diseases.

ATP and [3H]noradrenaline release and the presence of ecto-Ca(2+)-ATPases in the capsule-glomerulosa fraction of the rat adrenal gland.

Both [3H]noradrenaline ([3H]NA) and ATP were released in response to supramaximal electric field stimulation in superfused rat adrenal capsule-glomerulosa preparations. The voltage-dependent potassium channel blocker 4-aminopyridine enhanced, while the ATP-sensitive potassium channel blocker glibenclamide failed to affect the stimulation-evoked release of [3H]NA. The selective alpha 2-adrenoceptor antagonist CH-38083 enhanced the evoked release of [3H]NA while the P2 receptor agonist ATP and alpha, beta-methylene-ATP failed to affect it. Neither the adenosine A1 receptor agonist N6-cyclopentyl-adenosine (CPA) nor the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) influenced the stimulation-evoked [3H]NA release. The data showed that ATP was released from capsule-glomerulosa preparations in response to field stimulation together with but independently from [3H]NA, and that the local noradrenergic varicose axon terminals are not equipped with purinoceptors sensitive to ATP and/or adenosine. High concentrations of ATP also stimulated steroid hormone secretion in vitro, and thus may have a physiological role in this tissue. The presence of ecto-Ca(2+)-ATPases, enzymes able to terminate the effect of ATP, was demonstrated around the nerve profiles at the border of the capsule and zona glomerulosa tissue.

TNF inhibits myogenesis and downregulates the expression of myogenic regulatory factors myoD and myogenin.

The presence of TNF and other inflammatory cytokines and their receptors is detected during embryonic development, but our knowledge about the role of these proteins in differentiation and development is very limited. TNF modulates the synthesis and activity of a number of transcriptional proteins that regulate the activity of tissue specific genes, therefore it may play a role in normal development. Since its synthesis is upregulated by stress and infections, it may also participate in the induction of pathological developmental processes and malformation. We investigated the effect of TNF in an in vitro differentiation system using C2 myoblasts. This inflammatory cytokine exerted a positive effect on the early steps of the process: it enhanced the proliferation and aggregation of myoblast cells. In contrast, TNF strongly inhibited the expression of those myogenic transcription factors (myoD and myogenin), which are known to be responsible for upregulated activity of muscle specific genes (like the genes of the myofilament proteins), and blocked the synthesis of mRNAs of myogenic differentiation markers (like skeletal alpha-actin, myosin heavy and light chains). As a result, these cells did not synthesize myofilament proteins and the organization of myofilaments did not take place in TNF-treated myoblasts.

Production of ouabain by rat adrenocortical cells.

Our aim was to identify which adrenocortical cells produce ouabain and how it is regulated in vitro. With the help of an ouabain radioimmunoassay developed in our laboratory, we found that ouabain is produced both by zona glomerulosa and fasciculata cells. Our results were confirmed by reverse-phase HPLC. ACTH increased ouabain production in both cell types. Angiotensin-II, as well as changes in the potassium concentration of the incubation medium, affected ouabain production only in the zona glomerulosa.

Viscosity of rat adrenocortical lipids in different functional states: morphological characteristics.

Impaired adrenocortical steroidogenic activity is often concomitant with morphologically and physiologically altered lipids in the cells of the adrenal cortex. The physical state of these lipid droplets and the morphological characteristics of crystal-shaped bodies were studied in different functional states of adrenocortical cells. In the perinatal period when steroidogenesis is suppressed by a negative feedback mechanism, crystal-shaped bodies (i.e. rectangular, electron-lucent formations, either alone or in clusters, surrounded by lysosomal matrix or in close proximity of lysosomes) were frequently observed in the inner zona fasciculata and zona reticularis. In experimentally suppressed adrenocortical activity, following the administration of dexamethasone, aminoglutethimide or cycloheximide, almost identical crystal-shaped bodies were frequently observed in adrenocortical cells. These crystal-shaped bodies appear to be cholesterol, as revealed by the digitonin reaction at the electron microscopic level. Following stimulation of the zona fasciculata by ACTH treatment for 14 days, a marked increase in the fluidity of the lipid droplets was observed in the thermotropic phase transitions with the polarizing microscope. In contrast, following aminoglutethimide treatment, the fluidity of the lipid droplets decreased. The thermotropic phase transitions of normal and neoplastic human adrenal cells, namely adrenocortical tumours causing Conn's or Cushing's syndrome, were also investigated. When hormone biosynthesis was enhanced, the appearance of birefringence and multiple phase transitions of lipid droplets was demonstrable in the low-temperature range.

Effects of joining peptide (1-18) and histamine on dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) production of human adrenocortical cells in vitro.

Joining peptide 1-18 (JP 1-18), added alone in concentrations of 10(-13)-10(-7) M to collagenase-dispersed human adrenocortical cells, did not affect the basal production of corticosterone, cortisol, aldosterone, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS). JP 1-18 potentiated the ACTH-stimulated production of steroids. When administered in combination with histamine (10(-8)-10(-3) M), JP 1-18 (10(-8) or 10(-10) M), enhanced the synthesis of DHEA and DHEAS. JP 1-18, together with histamine, may play a role in the regulation of DHEA and DHEAS production.

Dopamine is taken up from the circulation by, and released from, local noradrenergic varicose axon terminals in zona glomerulosa of the rat: a neurochemical and immunocytochemical study.

The effect of supramaximal electric field stimulation on [3H]dopamine (DA) release by rat adrenal capsule-glomerulosa preparations was studied using a micro-volume perfusion system. When the tissues were preloaded with [3H]DA, a considerable amount of [3H]DA and [3H]noradrenaline (NA) were released in response to field stimuli. Reserpinization, calcium removal or tetrodotoxin blocking of Na+ influx all completely inhibited the stimulation-evoked release of DA/NA, indicating that the radioactivity released is of neuronal and vesicular origin. In the adrenal cortex, a substantial proportion of tyrosine hydroxylase and dopamine-beta-hydroxylase immunoreactive nerve fibres and varicosities were observed around the zona glomerulosa. DA-containing nerves were not seen in the adrenal cortex; however, the same immunocytochemical procedures clearly demonstrated dopaminergic nerve cells and fibres in the substantia nigra and the striatum respectively, and cells of the adrenal medulla. Like the NA release from noradrenergic varicosities in the zona glomerulosa, the DA release from noradrenergic endings is not subject to negative feedback modulation through DA2 receptors since apomorphine, a DA2-receptor agonist, and sulpiride, a selective DA2-receptor antagonist, failed to affect the release. After in-vivo i.v. administration of [3H]DA, the glomerulosa content of DA and NA and the in-vitro release of [3H]DA and [3H]NA of zona glomerulosa both increased, indicating that the local varicose axon terminals were able to accumulate DA from the circulation, convert it into NA and release it in response to neural activity. This local arrangement of noradrenergic axon terminals, able to take up DA from the circulation and release it or convert it into NA, provides the possibility of a fine tuning of local circulation and aldosterone synthesis in the zona glomerulosa.